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Sample records for expression cellular distribution

  1. Expression and Cellular Distribution of Ubiquitin in Response to Injury in the Developing Spinal Cord of Monodelphis domestica

    PubMed Central

    Noor, Natassya M.; Møllgård, Kjeld; Wheaton, Benjamin J.; Steer, David L.; Truettner, Jessie S.; Dziegielewska, Katarzyna M.; Dietrich, W. Dalton; Smith, A. Ian; Saunders, Norman R.

    2013-01-01

    Ubiquitin, an 8.5 kDa protein associated with the proteasome degradation pathway has been recently identified as differentially expressed in segment of cord caudal to site of injury in developing spinal cord. Here we describe ubiquitin expression and cellular distribution in spinal cord up to postnatal day P35 in control opossums (Monodelphis domestica) and in response to complete spinal transection (T10) at P7, when axonal growth through site of injury occurs, and P28 when this is no longer possible. Cords were collected 1 or 7 days after injury, with age-matched controls and segments rostral to lesion were studied. Following spinal injury ubiquitin levels (western blotting) appeared reduced compared to controls especially one day after injury at P28. In contrast, after injury mRNA expression (qRT-PCR) was slightly increased at P7 but decreased at P28. Changes in isoelectric point of separated ubiquitin indicated possible post-translational modifications. Cellular distribution demonstrated a developmental shift between earliest (P8) and latest (P35) ages examined, from a predominantly cytoplasmic immunoreactivity to a nuclear expression; staining level and shift to nuclear staining was more pronounced following injury, except 7 days after transection at P28. After injury at P7 immunostaining increased in neurons and additionally in oligodendrocytes at P28. Mass spectrometry showed two ubiquitin bands; the heavier was identified as a fusion product, likely to be an ubiquitin precursor. Apparent changes in ubiquitin expression and cellular distribution in development and response to spinal injury suggest an intricate regulatory system that modulates these responses which, when better understood, may lead to potential therapeutic targets. PMID:23626776

  2. Functional expression and cellular distribution of diastrophic dysplasia sulfate transporter (DTDST) gene mutations in HEK cells.

    PubMed

    Karniski, Lawrence P

    2004-10-01

    Defects in sulfate transport in chondrocytes lead to undersulfation of the cartilage extracellular matrix proteoglycans. Mutations in the diastrophic dysplasia sulfate transporter (DTDST) gene have been linked to four chondrodysplasias of varying severity. To characterize disease-causing mutations of DTDST, we expressed DTDST-mediated sulfate transport in mammalian HEK-293 cells and determined that the wild-type protein is glycosylated and localized to the cell plasma membrane. Four mutations, A715V, C653S, Q454P and R279W, stimulated sulfate transport at rates only 39-62% of wild-type DTDST. These four mutations were expressed on the plasma membrane of the cell, but the amount of expressed protein was reduced when compared with wild-type DTDST. The Q454P mutant is unique in that it is not properly glycosylated in HEK cells. There was no difference in sulfate transport activity between cells transfected with either the DeltaV340 or the G678V mutations and control HEK cells. Furthermore, the G678V mutation is not expressed along the plasma membrane, but is trapped within the cytoplasm. When comparing the sulfate transport capacity of each DTDST mutation with the chondrodysplasia in which it has been identified, we find that individuals with severe achondrogenesis 1B phenotype have null mutations on both DTDST alleles. Heterozygotes for both a null mutation and a partial-function mutation result in either atelosteogenesis type 2 or DTD, whereas the milder, recessive multiple epiphyseal dysplasia phenotype is homozygous for partial-function mutations. In contrast to previous studies in Xenopus laevis oocytes, we find a strong correlation between the severity of the phenotype and the level of residual transport function in mammalian cells. PMID:15294877

  3. Expression and cellular distribution of zona pellucida glycoproteins in canine oocytes before and after in vitro maturation.

    PubMed

    Kempisty, Bartosz; Piotrowska, Hanna; Bukowska, Dorota; Woźna, Magdalena; Ciesiółka, Sylwia; Wojtanowicz-Markiewicz, Katarzyna; Włodarczyk, Renata; Jopek, Karol; Jeseta, Michal; Bruska, Małgorzata; Nowicki, Michał; Jaśkowski, Jędrzej M; Brüssow, Klaus-Peter; Zabel, Maciej

    2015-12-01

    This study was aimed at investigating zona pellucida glycoproteins (ZP) ZP2, ZP3 mRNA expression as well as ZP3, ZP4 (ZPB) protein distribution before and after in vitro maturation (IVM) in canine oocytes. The cumulus-oocyte complexes (COCs) were recovered from 27 anoestrous mongrel bitches and matured for 72 h in TCM199 medium. The canine COCs were analysed before and after IVM. Using real-time quantitative polymerase chain reaction (RQ-PCR), both groups of oocytes were analysed for detection of ZP2 and ZP3 mRNA profiles as well as using confocal microscopic analysis for observation of ZP3 and ZP4 protein distribution. In post-IVM canine oocytes an increase in transcript content of ZP2 and ZP3 genes as well as a decrease in ZP3 and ZP4 protein levels were observed when compared with pre-IVM oocytes. Moreover, the ZP4 protein before IVM was significantly distributed in the peripheral area of cytoplasm, whereas after IVM it was localized rather than in the entire cytoplasm. In contrast, the ZP3 protein was found both before and after IVM was distributed in the peripheral area of the cytoplasm. In conclusion, we suggest that the expression of ZP2 and ZP3 genes is associated with the maturation stage of canine oocytes, as higher mRNAs levels were found after IVM. However, a decreased expression of ZP3 and ZP4 proteins after IVM suggests maturation-dependent down-regulation of these protein translations, which may result in disturbed fertilization.

  4. Expression and cellular distribution of transient receptor potential vanilloid 4 in cortical tubers of the tuberous sclerosis complex.

    PubMed

    Chen, Xin; Yang, Meihua; Sun, Feiji; Liang, Chao; Wei, Yujia; Wang, Lukang; Yue, Jiong; Chen, Bing; Li, Song; Liu, Shiyong; Yang, Hui

    2016-04-01

    Cortical tubers in patients with tuberous sclerosis complex (TSC) are highly associated with intractable epilepsy. Recent evidence has shown that transient receptor potential vanilloid 4 (TRPV4) has direct effects on both neurons and glial cells. To understand the role of TRPV4 in pathogenesis of cortical tubers, we investigated the expression patterns of TRPV4 in cortical tubers of TSC compared with normal control cortex (CTX). We found that TRPV4 was clearly up-regulated in cortical tubers at the protein levels. Immunostaining indicated that TRPV4 was specially distributed in abnormal cells, including dysplastic neurons (DNs) and giant cells (GCs). In addition, double immunofluorescent staining revealed that TRPV4 was localized on neurofilament proteins (NF200) positive neurons and glial fibrillary acidic portein (GFAP) positive reactive astrocytes. Moreover, TRPV4 co-localized with both glutamatergic and GABAergic neurons. Furthermore, protein levels of protein kinase C (PKC), but not protein kinase A (PKA), the important upstream factors of the TRPV4, were significantly increased in cortical tubers. Taken together, the overexpression and distribution patterns of TRPV4 may be linked with the intractable epilepsy caused by TSC. PMID:26874068

  5. Cellular distributions of monocarboxylate transporters: a review.

    PubMed

    Iwanaga, Toshihiko; Kishimoto, Ayuko

    2015-01-01

    Lactate and ketone bodies play important roles as alternative energy substrates, especially in conditions with a decreased utility of glucose. Short-chain fatty acids (acetate, propionate, and butyrate), produced by bacterial fermentation, supply most of the energy substrates in ruminants such as the cow and sheep. These monocarboxylates are transfered through the plasma membrane by proton-coupled monocarboxylate transporters (MCTs) and sodium-coupled MCTs (SMCTs). To reveal the metabolism and functional significance of monocarboxylates, the cellular localization of MCTs and SMCTs together with the expressed intensities holds great importance. This paper reviews the immunohistochemical localization of SMCTs and major MCT subtypes throughout the mammalian body. MCTs and SMCTs display a selective membrane-bound localization with porality. In contrast to the limited expression of SMCTs in the intestine and kidney, MCTs display a broader distribution pattern than GLUTs. The brain, kidney, placenta, and male genital tract express multiple subtypes of the MCT family. Determination of the cellular localization of MCTs is most controversial in the brain, possibly due to regional differences and the transcriptional modification of MCT proteins. Information on the localization of MCTs and SMCTs aids in understanding the nutrient absorption and metabolism throughout the mammalian body. In some cases, the body may use monocarboxylates as signal molecules, like hormones. PMID:26522146

  6. Cellular Automata Simulation for Wealth Distribution

    NASA Astrophysics Data System (ADS)

    Lo, Shih-Ching

    2009-08-01

    Wealth distribution of a country is a complicate system. A model, which is based on the Epstein & Axtell's "Sugars cape" model, is presented in Netlogo. The model considers the income, age, working opportunity and salary as control variables. There are still other variables should be considered while an artificial society is established. In this study, a more complicate cellular automata model for wealth distribution model is proposed. The effects of social welfare, tax, economical investment and inheritance are considered and simulated. According to the cellular automata simulation for wealth distribution, we will have a deep insight of financial policy of the government.

  7. Enhanced expression of cyclo-oxygenase isoenzyme 2 (COX-2) in asthmatic airways and its cellular distribution in aspirin-sensitive asthma

    PubMed Central

    Sousa, A.; Pfister, R.; Christie, P. E.; Lane, S. J.; Nasser, S. M.; Schmitz-Schumann, M.; Lee, T. H.

    1997-01-01

    BACKGROUND: There are two isoforms of cyclo-oxygenase (COX), namely COX- 1 and COX-2. COX-1 is constitutively expressed in most tissues and in blood platelets. The metabolites derived from COX-1 are probably involved in cellular housekeeping functions. COX-2 is expressed only following cellular activation by inflammatory stimuli and is thought to be involved in inflammation. METHODS: The expression of COX-1 and COX-2 isoenzymes has been studied in the bronchial mucosa of 10 normal and 18 asthmatic subjects, 11 of whom had aspirin-sensitive asthma (ASA) and seven had non-aspirin-sensitive asthma (NASA) RESULTS: There was a significant fourfold and 14-fold increase, respectively, in the epithelial and submucosal cellular expression of COX-2, but not of COX- 1, in asthmatic patients. There was no significant difference in the total number of cells staining for either COX-1 or COX-2 between subjects with ASA and NASA, but the number and percentage of mast cells that expressed COX-2 was significantly increased sixfold and twofold, respectively, in individuals with ASA. There was a mean fourfold increase in the percentage of COX-2 expressing cells that were mast cells in subjects with ASA and the number of eosinophils expressing COX- 2 was increased 2.5-fold in these subjects. CONCLUSION: COX-2-derived metabolites may play an essential part in the inflammatory processes present in asthmatic airways and development of drugs targeted at this isoenzyme may have therapeutic potential in the treatment of asthma. Mast cells and eosinophils may also have a central role in the pathology of aspirin-sensitive asthma. 


 PMID:9487340

  8. Expression of Cellular Oncogenes in Human Malignancies

    NASA Astrophysics Data System (ADS)

    Slamon, Dennis J.; Dekernion, Jean B.; Verma, Inder M.; Cline, Martin J.

    1984-04-01

    Cellular oncogenes have been implicated in the induction of malignant transformation in some model systems in vitro and may be related to malignancies in vivo in some vertebrate species. This article describes a study of the expression of 15 cellular oncogenes in fresh human tumors from 54 patients, representing 20 different tumor types. More than one cellular oncogene was transcriptionally active in all of the tumors examined. In 14 patients it was possible to study normal and malignant tissue from the same organ. In many of these patients, the transcriptional activity of certain oncogenes was greater in the malignant than the normal tissue. The cellular fes (feline sarcoma) oncogene, not previously known to be transcribed in mammalian tissue, was found to be active in lung and hematopoietic malignancies.

  9. Predicting cellular growth from gene expression signatures.

    PubMed

    Airoldi, Edoardo M; Huttenhower, Curtis; Gresham, David; Lu, Charles; Caudy, Amy A; Dunham, Maitreya J; Broach, James R; Botstein, David; Troyanskaya, Olga G

    2009-01-01

    Maintaining balanced growth in a changing environment is a fundamental systems-level challenge for cellular physiology, particularly in microorganisms. While the complete set of regulatory and functional pathways supporting growth and cellular proliferation are not yet known, portions of them are well understood. In particular, cellular proliferation is governed by mechanisms that are highly conserved from unicellular to multicellular organisms, and the disruption of these processes in metazoans is a major factor in the development of cancer. In this paper, we develop statistical methodology to identify quantitative aspects of the regulatory mechanisms underlying cellular proliferation in Saccharomyces cerevisiae. We find that the expression levels of a small set of genes can be exploited to predict the instantaneous growth rate of any cellular culture with high accuracy. The predictions obtained in this fashion are robust to changing biological conditions, experimental methods, and technological platforms. The proposed model is also effective in predicting growth rates for the related yeast Saccharomyces bayanus and the highly diverged yeast Schizosaccharomyces pombe, suggesting that the underlying regulatory signature is conserved across a wide range of unicellular evolution. We investigate the biological significance of the gene expression signature that the predictions are based upon from multiple perspectives: by perturbing the regulatory network through the Ras/PKA pathway, observing strong upregulation of growth rate even in the absence of appropriate nutrients, and discovering putative transcription factor binding sites, observing enrichment in growth-correlated genes. More broadly, the proposed methodology enables biological insights about growth at an instantaneous time scale, inaccessible by direct experimental methods. Data and tools enabling others to apply our methods are available at http://function.princeton.edu/growthrate.

  10. Cellular Distribution of NDRG1 Protein in the Rat Kidney and Brain During Normal Postnatal Development

    PubMed Central

    Wakisaka, Yoshinobu; Furuta, Akiko; Masuda, Katsuaki; Morikawa, Wataru; Kuwano, Michihiko; Iwaki, Toru

    2003-01-01

    N-myc downregulated gene 1 (NDRG1) is a 43-kD protein whose mRNA is induced by DNA damage, hypoxia, or prolonged elevation of intracellular calcium. Although NDRG1 is also upregulated during cell differentiation, there are few studies on NDRG1 expression during postnatal development. Here we investigated the expression and cellular distribution of NDRG1 protein in rat kidney and brain during postnatal development. Immunohistochemical analysis revealed that the cellular localization of NDRG1 protein in the kidney changed from the proximal convoluted tubules to the collecting ducts between postnatal days 10 and 20. In the brain, a change in cellular expression was also found from the hippocampal pyramidal neurons to the astrocytes in the gray matter during the same postnatal period. These alterations in the cellular distribution of NDRG1 were associated with shifts in the molecular assembly on Western blots. Under non-reduced conditions, the main NDRG1 band was found only around 215 kD in both kidney and brain during the early postnatal stage. After postnatal day 10, the immunoreactive bands shifted to 43 kD in the kidney and 129 kD in the brain. These changes in the cellular distribution and state of assembly may correlate with the functional maturation of both organs. PMID:14566023

  11. Mapping organism expression levels at cellular resolution in developing Drosophila

    NASA Astrophysics Data System (ADS)

    Knowles, David W.; Keranen, Soile; Biggin, Mark D.; Sudar, Damir

    2002-05-01

    The development of an animal embryo is orchestrated by a network of genetically determined, temporal and spatial gene expression patterns that determine the animals final form. To understand such networks, we are developing novel quantitative optical imaging techniques to map gene expression levels at cellular and sub-cellular resolution within pregastrula Drosophila. Embryos at different stages of development are labeled for total DNA and specific gene products using different fluorophors and imaged in 3D with confocal microscopy. Innovative steps have been made which allow the DNA-image to be automatically segmented to produce a morphological mask of the individual nuclear boundaries. For each stage of development an average morphology is chosen to which images from different embryo are compared. The morphological mask is then used to quantify gene-product on a per nuclei basis. What results is an atlas of the relative amount of the specific gene product expressed within the nucleus of every cell in the embryo at the various stages of development. We are creating a quantitative database of transcription factor and target gene expression patterns in wild-type and factor mutant embryos with single cell resolution. Our goal is to uncover the rules determining how patterns of gene expression are generated.

  12. A single-cell bioluminescence imaging system for monitoring cellular gene expression in a plant body.

    PubMed

    Muranaka, Tomoaki; Kubota, Saya; Oyama, Tokitaka

    2013-12-01

    Gene expression is a fundamental cellular process and expression dynamics are of great interest in life science. We succeeded in monitoring cellular gene expression in a duckweed plant, Lemna gibba, using bioluminescent reporters. Using particle bombardment, epidermal and mesophyll cells were transfected with the luciferase gene (luc+) under the control of a constitutive [Cauliflower mosaic virus 35S (CaMV35S)] and a rhythmic [Arabidopsis thaliana CIRCADIAN CLOCK ASSOCIATED 1 (AtCCA1)] promoter. Bioluminescence images were captured using an EM-CCD (electron multiply charged couple device) camera. Luminescent spots of the transfected cells in the plant body were quantitatively measured at the single-cell level. Luminescence intensities varied over a 1,000-fold range among CaMV35S::luc+-transfected cells in the same plant body and showed a log-normal-like frequency distribution. We monitored cellular gene expression under light-dark conditions by capturing bioluminescence images every hour. Luminescence traces of ≥50 individual cells in a frond were successfully obtained in each monitoring procedure. Rhythmic and constitutive luminescence behaviors were observed in cells transfected with AtCCA1::luc+ and CaMV35S::luc+, respectively. Diurnal rhythms were observed in every AtCCA1::luc+-introduced cell with traceable luminescence, and slight differences were detected in their rhythmic waveforms. Thus the single-cell bioluminescence monitoring system was useful for the characterization of cellular gene expression in a plant body. PMID:24058151

  13. A single-cell bioluminescence imaging system for monitoring cellular gene expression in a plant body.

    PubMed

    Muranaka, Tomoaki; Kubota, Saya; Oyama, Tokitaka

    2013-12-01

    Gene expression is a fundamental cellular process and expression dynamics are of great interest in life science. We succeeded in monitoring cellular gene expression in a duckweed plant, Lemna gibba, using bioluminescent reporters. Using particle bombardment, epidermal and mesophyll cells were transfected with the luciferase gene (luc+) under the control of a constitutive [Cauliflower mosaic virus 35S (CaMV35S)] and a rhythmic [Arabidopsis thaliana CIRCADIAN CLOCK ASSOCIATED 1 (AtCCA1)] promoter. Bioluminescence images were captured using an EM-CCD (electron multiply charged couple device) camera. Luminescent spots of the transfected cells in the plant body were quantitatively measured at the single-cell level. Luminescence intensities varied over a 1,000-fold range among CaMV35S::luc+-transfected cells in the same plant body and showed a log-normal-like frequency distribution. We monitored cellular gene expression under light-dark conditions by capturing bioluminescence images every hour. Luminescence traces of ≥50 individual cells in a frond were successfully obtained in each monitoring procedure. Rhythmic and constitutive luminescence behaviors were observed in cells transfected with AtCCA1::luc+ and CaMV35S::luc+, respectively. Diurnal rhythms were observed in every AtCCA1::luc+-introduced cell with traceable luminescence, and slight differences were detected in their rhythmic waveforms. Thus the single-cell bioluminescence monitoring system was useful for the characterization of cellular gene expression in a plant body.

  14. Cellular distribution of injected PLGA-nanoparticles in the liver.

    PubMed

    Park, Jin-Kyu; Utsumi, Teruo; Seo, Young-Eun; Deng, Yang; Satoh, Ayano; Saltzman, William Mark; Iwakiri, Yasuko

    2016-07-01

    The cellular fate of nanoparticles in the liver is not fully understood. Because the effectiveness and safety of nanoparticles in liver therapy depends on targeting nanoparticles to the right cell populations, this study aimed to determine a relative distribution of PLGA-nanoparticles (sizes 271±1.4 nm) among liver cells in vivo. We found that Kupffer cells were the major cells that took up nanoparticles, followed by liver sinusoidal endothelial cells and hepatic stellate cells. Nanoparticles were found in only 7% of hepatocytes. Depletion of Kupffer cells by clodronate liposomes increased nanoparticle retention in liver sinusoidal endothelial cells and hepatic stellate cells, but not in hepatocytes. It is importantly suggested that studies of drug-loaded nanoparticle delivery to the liver have to demonstrate not only uptake of nanoparticles by the target cell type but also non-uptake by other cell types to assess their effect as well as ensure their safety.

  15. Cellular uptake and in vivo distribution of polyhistidine peptides.

    PubMed

    Iwasaki, Takashi; Tokuda, Yoshihisa; Kotake, Ayaka; Okada, Hiroyuki; Takeda, Shuji; Kawano, Tsuyoshi; Nakayama, Yuji

    2015-07-28

    Cell-penetrating peptides (CPPs) are arginine/lysine-rich sequences, and they are effectively internalized into cells. In this process, positive charge is crucial. In the present study, we found polyhistidine peptides (PHPs), as the novel CPP, which are efficiently internalized into cells in a positive charge-independent manner. Interestingly, cellular uptake of the PHPs increased as the chain length increased, reaching a maximum uptake at H16 (HHHHHHHHHHHHHHHH-NH2). This H16 peptide showed up to 14.6-fold higher cell-penetrating capacity against HT1080 human fibrosarcoma cells relative to a major CPP, the octa-arginine (RRRRRRRR-NH2) peptide. Cellular uptake of the H16 peptide is mainly due to macropinocytosis and most of the H16 peptide localizes in the lysosome and Golgi apparatus. However, a cytoplasmic pro-apoptotic domain (KLAKLAKKLAKLAK-NH2) conjugated to the H16 peptide showed cytotoxic effects. This indicates that a proportion of the H16 peptide escapes from the macropinosome to the cytoplasm. In a protein transduction study, green fluorescence protein fused to the H16 peptide (GFP-H16) was purified by Ni-NTA chromatography, detected using an anti-His-tag antibody and internalized into cells. This serial process reveals that H16 functions as a His-tag and protein transduction domain. Furthermore, in vivo distribution analysis showed that the H16 peptide accumulates immediately in tumor tissue and is retained up to 132h following injection into the tumor (HT1080 human fibrosarcoma)-bearing mice. This is the first observation of a His-polymer being internalize into cells efficiently. The findings suggest that PHPs are novel CPPs. In particular, the H16 peptide represents a promising drug delivery carrier candidate in medical and biotechnological fields.

  16. The Effect of Gravity Fields on Cellular Gene Expression

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, Millie

    1999-01-01

    Early theoretical analysis predicted that microgravity effects on the isolated cell would be minuscule at the subcellular level; however, these speculations have not proven true in the real world. Astronauts experience a significant bone and muscle loss in as little as 2 weeks of spaceflight and changes are seen at the cellular level soon after exposure to microgravity. Changes in biological systems may be primarily due to the lack of gravity and the resulting loss of mechanical stress on tissues and cells. Recent ground and flight studies examining the effects of gravity or mechanical stress on cells demonstrate marked changes in gene expression when relatively small changes in mechanical forces or gravity fields were made. Several immediate early genes (IEG) like c-fos and c-myc are induced by mechanical stimulation within minutes. In contrast, several investigators report that the absence of mechanical forces during space flight result in decreased sera response element (SRE) activity and attenuation of expression of IEGs such as c-fos, c-jun and cox-2 mRNAs. Clearly, these early changes in gene expression may have long term consequences on mechanically sensitive cells. In our early studies on STS-56, we reported four major changes in the osteoblast; 1) prostaglandin synthesis in flight, 2) changes in cellular morphology, 3) altered actin cytoskeleton and 4) reduced osteoblast growth after four days exposure to microgravity. Initially, it was believed that changes in fibronectin (FN) RNA, FN protein synthesis or subsequent FN matrix formation might account for the changes in cytoskeleton and/ or reduction of growth. However our recent studies on Biorack (STS-76, STS-81 and STS-84), using ground and in-flight 1-G controls, demonstrated that fibronectin synthesis and matrix formation were normal in microgravity. In addition, in our most recent Biorack paper, our laboratory has documented that relative protein synthesis and mRNA synthesis are not changed after 24

  17. Cellular proteomes have broad distributions of protein stability.

    PubMed

    Ghosh, Kingshuk; Dill, Ken

    2010-12-15

    Biological cells are extremely sensitive to temperature. What is the mechanism? We compute the thermal stabilities of the whole proteomes of Escherichia coli, yeast, and Caenorhabditis elegans using an analytical model and an extensive database of stabilities of individual proteins. Our results support the hypothesis that a cell's thermal sensitivities arise from the collective instability of its proteins. This model shows a denaturation catastrophe at temperatures of 49-55°C, roughly the thermal death point of mesophiles. Cells live on the edge of a proteostasis catastrophe. According to the model, it is not that the average protein is problematic; it is the tail of the distribution. About 650 of E. coli's 4300 proteins are less than 4 kcal mol(-1) stable to denaturation. And upshifting by only 4° from 37° to 41°C is estimated to destabilize an average protein by nearly 20%. This model also treats effects of denaturants, osmolytes, and other physical stressors. In addition, it predicts the dependence of cellular growth rates on temperature. This approach may be useful for studying physical forces in biological evolution and the role of climate change on biology.

  18. Hyperglycosylated HCG expression in pregnancy: cellular origin and clinical applications.

    PubMed

    Kovalevskaya, G; Kakuma, T; Schlatterer, J; O'Connor, J F

    2007-01-01

    Employing a monoclonal antibody (B152) specific for a carbohydrate epitope found on a choriocarcinoma derived hCG, it was discovered that a similar hCG isoform is expressed during early pregnancy. This form differs from later pregnancy hCG in carbohydrate moieties. Profiling of these two hCG isoforms throughout pregnancy utilized two IRMA's: B152-B207 ("hyperglycosylated hCG"-specific assay) and B109-B108 (an IRMA for standard intact hCG isoforms in the WHO hCG reference preparation). The WHO hCG standard was used in both assays. Values were presented as a ratio of hCG isoform concentrations (B152/B109 ratio). In early pregnancy urine concentrations of B152 hCG were significantly higher in normal pregnancy (NP) compared to early pregnancy loss (EPL). Matched serum-urine samples from the first and third trimesters revealed that the B152 hCG form is predominant in both serum and urine in the first trimester compared with the third trimester. The proportion of the B152 hCG (HhCG) form is higher in urine than in matched serum. There was a significant difference in the B152/B109 ratio between days 5 and 20 from time of embryo transfer in normally developing pregnancy versus EPL in the urine of IVF patients. In spontaneous abortion (SA) the level of B109 hCG remained higher in NP compared with SA. However, the B152/B109 ratio declined with gestational age faster in SA than in NP suggesting perhaps a different loss mechanism in SA versus EPL. The cellular origin of the different hCG glycoforms was identified by assay of cell media from cytotrophoblasts (CTBs) and syncytiotrophoblasts (STBs). Isolated CTBs expressed predominantly HhCG. The level of expression was the highest in the first trimester. STBs were the source of the less glycosylated B109 hCG isoform. Analysis of hCG glycoforms during early pregnancy can distinguish pregnancies that will fail from those that will proceed normally. Since the B152 assay does not effectively discriminate between intact HhCG and free

  19. Establishment and expression of cellular polarity in fucoid zygotes.

    PubMed Central

    Kropf, D L

    1992-01-01

    . Approximately halfway through the first cell cycle, the latent polarity is expressed morphologically in the form of rhizoid growth. Elongation is by tip growth and does not appear to be fundamentally different from tip growth in other organisms. The zygote always divides perpendicular to the growth axis, and this is controlled by the microtubule cytoskeleton. Two microtubule-organizing centers on the nuclear envelope rotate such that they align with the growth axis. They then serve as spindle poles during mitosis. Cytokinesis bisects the axial spindle, resulting in a transverse crosswall. Although the chronology of cellular events associated with polarity is by now rather detailed, causal mechanisms remain obscure. Images PMID:1620068

  20. Alternative splicing, expression and cellular localization of Calneuron-1 in the rat and human brain.

    PubMed

    Hradsky, Johannes; Bernstein, Hans-Gert; Marunde, Monika; Mikhaylova, Marina; Kreutz, Michael R

    2015-10-01

    Calneuron-1 and -2 are members of the neuronal calcium-binding protein family (nCaBP). They are transmembrane Calmodulin-like EF-hand Ca(2+)-sensors, and a function in the control of Golgi-to-plasma membrane vesicle trafficking has been assigned to both proteins. In this paper, we describe the distribution of Calneuron-1 in rat and human brains. We show that Calneuron-1 is ubiquitously expressed in all brain regions examined. The protein is most abundant in Purkinje cells of the cerebellum and principal neurons of the cortex and limbic brain whereas no expression in glial cells is apparent. In addition, we identify two novel splice isoforms of Calneuron-1 with extended N-termini. These isoforms are particular abundant in the cerebellum. Taken together, these data set grounds for a better understanding of the cellular function of Calneurons.

  1. Cellular Cholesterol Distribution Influences Proteolytic Release of the LRP-1 Ectodomain

    PubMed Central

    Dekky, Bassil; Wahart, Amandine; Sartelet, Hervé; Féré, Michaël; Angiboust, Jean-François; Dedieu, Stéphane; Piot, Olivier; Devy, Jérôme; Emonard, Hervé

    2016-01-01

    Low-density lipoprotein receptor-related protein-1 (LRP-1) is a multifunctional matricellular receptor composed of a large ligand-binding subunit (515-kDa α-chain) associated with a short trans-membrane subunit (85-kDa β-chain). LRP-1, which exhibits both endocytosis and cell signaling properties, plays a key role in tumor invasion by regulating the activity of proteinases such as matrix metalloproteinases (MMPs). LRP-1 is shed at the cell surface by proteinases such as membrane-type 1 MMP (MT1-MMP) and a disintegrin and metalloproteinase-12 (ADAM-12). Here, we show by using biophysical, biochemical, and cellular imaging approaches that efficient extraction of cell cholesterol and increased LRP-1 shedding occur in MDA-MB-231 breast cancer cells but not in MDA-MB-435 cells. Our data show that cholesterol is differently distributed in both cell lines; predominantly intracellularly for MDA-MB-231 cells and at the plasma membrane for MDA-MB-435 cells. This study highlights the relationship between the rate and cellular distribution of cholesterol and its impact on LRP-1 shedding modulation. Altogether, our data strongly suggest that the increase of LRP-1 shedding upon cholesterol depletion induces a higher accessibility of the sheddase substrate, i.e., LRP-1, at the cell surface rather than an increase of expression of the enzyme. PMID:26903870

  2. Cellular Cholesterol Distribution Influences Proteolytic Release of the LRP-1 Ectodomain.

    PubMed

    Dekky, Bassil; Wahart, Amandine; Sartelet, Hervé; Féré, Michaël; Angiboust, Jean-François; Dedieu, Stéphane; Piot, Olivier; Devy, Jérôme; Emonard, Hervé

    2016-01-01

    Low-density lipoprotein receptor-related protein-1 (LRP-1) is a multifunctional matricellular receptor composed of a large ligand-binding subunit (515-kDa α-chain) associated with a short trans-membrane subunit (85-kDa β-chain). LRP-1, which exhibits both endocytosis and cell signaling properties, plays a key role in tumor invasion by regulating the activity of proteinases such as matrix metalloproteinases (MMPs). LRP-1 is shed at the cell surface by proteinases such as membrane-type 1 MMP (MT1-MMP) and a disintegrin and metalloproteinase-12 (ADAM-12). Here, we show by using biophysical, biochemical, and cellular imaging approaches that efficient extraction of cell cholesterol and increased LRP-1 shedding occur in MDA-MB-231 breast cancer cells but not in MDA-MB-435 cells. Our data show that cholesterol is differently distributed in both cell lines; predominantly intracellularly for MDA-MB-231 cells and at the plasma membrane for MDA-MB-435 cells. This study highlights the relationship between the rate and cellular distribution of cholesterol and its impact on LRP-1 shedding modulation. Altogether, our data strongly suggest that the increase of LRP-1 shedding upon cholesterol depletion induces a higher accessibility of the sheddase substrate, i.e., LRP-1, at the cell surface rather than an increase of expression of the enzyme. PMID:26903870

  3. Cellular Cholesterol Distribution Influences Proteolytic Release of the LRP-1 Ectodomain.

    PubMed

    Dekky, Bassil; Wahart, Amandine; Sartelet, Hervé; Féré, Michaël; Angiboust, Jean-François; Dedieu, Stéphane; Piot, Olivier; Devy, Jérôme; Emonard, Hervé

    2016-01-01

    Low-density lipoprotein receptor-related protein-1 (LRP-1) is a multifunctional matricellular receptor composed of a large ligand-binding subunit (515-kDa α-chain) associated with a short trans-membrane subunit (85-kDa β-chain). LRP-1, which exhibits both endocytosis and cell signaling properties, plays a key role in tumor invasion by regulating the activity of proteinases such as matrix metalloproteinases (MMPs). LRP-1 is shed at the cell surface by proteinases such as membrane-type 1 MMP (MT1-MMP) and a disintegrin and metalloproteinase-12 (ADAM-12). Here, we show by using biophysical, biochemical, and cellular imaging approaches that efficient extraction of cell cholesterol and increased LRP-1 shedding occur in MDA-MB-231 breast cancer cells but not in MDA-MB-435 cells. Our data show that cholesterol is differently distributed in both cell lines; predominantly intracellularly for MDA-MB-231 cells and at the plasma membrane for MDA-MB-435 cells. This study highlights the relationship between the rate and cellular distribution of cholesterol and its impact on LRP-1 shedding modulation. Altogether, our data strongly suggest that the increase of LRP-1 shedding upon cholesterol depletion induces a higher accessibility of the sheddase substrate, i.e., LRP-1, at the cell surface rather than an increase of expression of the enzyme.

  4. Cloning, expression, cellular distribution, and role in chemotaxis of a C5a receptor in rainbow trout: the first identification of a C5a receptor in a nonmammalian species

    USGS Publications Warehouse

    Boshra, Hani; Li, Jun; Peters, Rodney; Hansen, John; Matlapudi, Anjan; Sunyer, J. Oriol

    2004-01-01

    C3a, C4a, and C5a anaphylatoxins generated during complement activation play a key role in inflammation. C5a is the most potent of the three anaphylatoxins in eliciting biological responses. The effects of C5a are mediated by its binding to C5a receptor (C5aR, CD88). To date, C5aR has only been identified and cloned in mammalian species, and its evolutionary history remains ill-defined. To gain insights into the evolution, conserved structural domains, and functions of C5aR, we have cloned and characterized a C5aR in rainbow trout, a teleost fish. The isolated cDNA encoded a 350-aa protein that showed the highest sequence similarity to C5aR from other species. Genomic analysis revealed the presence of one continuous exon encoding the entire open reading frame. Northern blot analysis showed significant expression of the trout C5a receptor (TC5aR) message in PBLs and kidney. Flow cytometric analysis showed that two Abs generated against two different areas of the extracellular N-terminal region of TC5aR positively stained the same leukocyte populations from PBLs. B lymphocytes and granulocytes comprised the majority of cells recognized by the anti-TC5aR. More importantly, these Abs inhibited chemotaxis of PBLs toward a chemoattractant fraction purified from complement-activated trout serum. Our data suggest that the split between C5aR and C3aR from a common ancestral molecule occurred before the emergence of teleost fish. Moreover, we demonstrate that the overall structure of C5aR as well as its role in chemotaxis have remained conserved for >300 million years.

  5. Multi-fractal analysis for vehicle distribution based on cellular automation model

    NASA Astrophysics Data System (ADS)

    Zhang, Yong; Li, Shi-Gao

    2015-09-01

    It is well known that traffic flow presents multi-fractal characteristics at time scales. The aim of this study is to test its multi-fractality at spacial scales. The vehicular cellular automation (CA) model is chosen as a tool to get vehicle positions on a single lane road. First, multi-fractal of vehicle distribution is checked, and multi-fractal spectrums are plotted. Second, analysis results show that the width of a multi-fractal spectrum expresses the ratio of the maximum to minimum densities, and the height difference between the left and right vertexes represents the relative size between the numbers of sections with the maximum and minimum densities. Finally, the effects of the random deceleration probability and the average density on homogeneity of vehicle distribution are analyzed. The results show that random deceleration increases the ratio of the maximum to minimum densities, and deceases the relative size between the numbers of sections with the maximum and minimum densities, when the global density is limited to a specific range. Therefore, the multi-fractal spectrum can be used to quantify the homogeneity of spacial distribution of traffic flow.

  6. Mutations in MCT8 in patients with Allan-Herndon-Dudley-syndrome affecting its cellular distribution.

    PubMed

    Kersseboom, Simone; Kremers, Gert-Jan; Friesema, Edith C H; Visser, W Edward; Klootwijk, Wim; Peeters, Robin P; Visser, Theo J

    2013-05-01

    Monocarboxylate transporter 8 (MCT8) is a thyroid hormone (TH)-specific transporter. Mutations in the MCT8 gene are associated with Allan-Herndon-Dudley Syndrome (AHDS), consisting of severe psychomotor retardation and disturbed TH parameters. To study the functional consequences of different MCT8 mutations in detail, we combined functional analysis in different cell types with live-cell imaging of the cellular distribution of seven mutations that we identified in patients with AHDS. We used two cell models to study the mutations in vitro: 1) transiently transfected COS1 and JEG3 cells, and 2) stably transfected Flp-in 293 cells expressing a MCT8-cyan fluorescent protein construct. All seven mutants were expressed at the protein level and showed a defect in T3 and T4 transport in uptake and metabolism studies. Three mutants (G282C, P537L, and G558D) had residual uptake activity in Flp-in 293 and COS1 cells, but not in JEG3 cells. Four mutants (G221R, P321L, D453V, P537L) were expressed at the plasma membrane. The mobility in the plasma membrane of P537L was similar to WT, but the mobility of P321L was altered. The other mutants studied (insV236, G282C, G558D) were predominantly localized in the endoplasmic reticulum. In essence, loss of function by MCT8 mutations can be divided in two groups: mutations that result in partial or complete loss of transport activity (G221R, P321L, D453V, P537L) and mutations that mainly disturb protein expression and trafficking (insV236, G282C, G558D). The cell type-dependent results suggest that MCT8 mutations in AHDS patients may have tissue-specific effects on TH transport probably caused by tissue-specific expression of yet unknown MCT8-interacting proteins. PMID:23550058

  7. Tenomodulin Expression in the Periodontal Ligament Enhances Cellular Adhesion

    PubMed Central

    Komiyama, Yuske; Ohba, Shinsuke; Shimohata, Nobuyuki; Nakajima, Keiji; Hojo, Hironori; Yano, Fumiko; Takato, Tsuyoshi; Docheva, Denitsa; Shukunami, Chisa; Hiraki, Yuji; Chung, Ung-il

    2013-01-01

    Tenomodulin (Tnmd) is a type II transmembrane protein characteristically expressed in dense connective tissues such as tendons and ligaments. Its expression in the periodontal ligament (PDL) has also been demonstrated, though the timing and function remain unclear. We investigated the expression of Tnmd during murine tooth eruption and explored its biological functions in vitro. Tnmd expression was related to the time of eruption when occlusal force was transferred to the teeth and surrounding tissues. Tnmd overexpression enhanced cell adhesion in NIH3T3 and human PDL cells. In addition, Tnmd-knockout fibroblasts showed decreased cell adhesion. In the extracellular portions of Tnmd, the BRICHOS domain or CS region was found to be responsible for Tnmd-mediated enhancement of cell adhesion. These results suggest that Tnmd acts on the maturation or maintenance of the PDL by positively regulating cell adhesion via its BRICHOS domain. PMID:23593173

  8. Analysis of Rheb in the cellular slime mold Dictyostelium discoideum: cellular localization, spatial expression and overexpression.

    PubMed

    Swer, Pynskhem Bok; Bhadoriya, Pooja; Saran, Shweta

    2014-03-01

    Dictyostelium discoideum encodes a single Rheb protein showing sequence similarity to human homologues of Rheb. The DdRheb protein shares 52 percent identity and 100 percent similarity with the human Rheb1 protein. Fluorescence of Rheb yellow fluorescent protein fusion was detected in the D. discoideum cytoplasm. Reverse transcription-polymerase chain reaction and whole-mount in situ hybridization analyses showed that rheb is expressed at all stages of development and in prestalk cells in the multicellular structures developed. When the expression of rheb as a fusion with lacZ was driven under its own promoter, the beta-galactosidase activity was seen in the prestalk cells. D. discoideum overexpressing Rheb shows an increase in the size of the cell. Treatment of the overexpressing Rheb cells with rapamycin confirms its involvement in the TOR signalling pathway.

  9. Analysis of Rheb in the cellular slime mold Dictyostelium discoideum: cellular localization, spatial expression and overexpression.

    PubMed

    Swer, Pynskhem Bok; Bhadoriya, Pooja; Saran, Shweta

    2014-03-01

    Dictyostelium discoideum encodes a single Rheb protein showing sequence similarity to human homologues of Rheb. The DdRheb protein shares 52 percent identity and 100 percent similarity with the human Rheb1 protein. Fluorescence of Rheb yellow fluorescent protein fusion was detected in the D. discoideum cytoplasm. Reverse transcription-polymerase chain reaction and whole-mount in situ hybridization analyses showed that rheb is expressed at all stages of development and in prestalk cells in the multicellular structures developed. When the expression of rheb as a fusion with lacZ was driven under its own promoter, the beta-galactosidase activity was seen in the prestalk cells. D. discoideum overexpressing Rheb shows an increase in the size of the cell. Treatment of the overexpressing Rheb cells with rapamycin confirms its involvement in the TOR signalling pathway. PMID:24499792

  10. The expression of AIB1 correlates with cellular proliferation in human prolactinomas.

    PubMed

    Carretero, José; Blanco, Enrique J; Carretero, Manuel; Carretero-Hernández, Marta; García-Barrado, Ma José; Iglesias-Osma, Ma Carmen; Burks, Deborah Jane; Font de Mora, Jaime

    2013-05-01

    Estrogens as well as certain growth factors strongly influence the development and growth of prolactinomas. However, the molecular mechanisms by which extracellular factors trigger prolactinomas are not well known. Amplified in breast cancer 1 (AIB1), also known as steroid receptor co-activator 3 (SRC-3), belongs to the p160/SRC family of nuclear receptor co-activators and is a major co-activator of the estrogen receptor. Here, we report that the estrogen receptor coactivator AIB1 is overexpressed in human prolactinomas and correlates with the detection of aromatase and estrogen receptor α (ERα). Of the 87 pituitary tumors evaluated in women, 56%, corresponding to hyperoprolactinemic women, contained an enriched population of prolactin-positive cells and hence were further classified as prolactinomas. All prolactinomas stained positive for both ERα and AIB1. Moreover, AIB1 sub-cellular distribution was indicative of the cell-cycle status of tumors; the nuclear expression of AIB1 was correlated with proliferative markers whereas the cytoplasmic localization of AIB1 coincided with active caspase-3. Thus, our results demonstrate for the first time that AIB1 is expressed in prolactinomas and suggest its participation in the regulation of proliferation and apoptosis of tumoral cells. Because aromatase expression is also enhanced in these prolactinomas and it is involved in the local production of estradiol, both mechanisms, ER-AIB1 and aromatase could be related.

  11. Cellular distribution and localisation of iron in adult rat brain ( substantia nigra)

    NASA Astrophysics Data System (ADS)

    Meinecke, Ch.; Morawski, M.; Reinert, T.; Arendt, T.; Butz, T.

    2006-08-01

    Iron appears to be one of the main factors in the metal induced neurodegeneration. Quantitative information on cellular, sub-cellular and cell specific distributions of iron is therefore important to assess. The investigations reported here were carried out on a brain from an adult rat. Therefore, 6 μm thick embedded, unstained brain sections containing the midbrain (substantia nigra, SN) were analysed. Particle induced X-ray emission (PIXE) using a focussed proton beam (beam - diameter app. 1 μm) was performed to determine the quantitative iron content on a cellular and sub-cellular level. The integral analysis shows that the iron content in the SN pars reticulata is twice as high than in the SN pars compacta. The analysis of the iron content on the cellular level revealed no remarkable differences between glia cells and neurons. This is in contrast to other studies using staining techniques.

  12. Prion search and cellular prion protein expression in stranded dolphins.

    PubMed

    Di Guardo, G; Cocumelli, C; Meoli, R; Barbaro, K; Terracciano, G; Di Francesco, C E; Mazzariol, S; Eleni, C

    2012-01-01

    The recent description of a prion disease (PD) case in a free-ranging bottlenose dolphin (Tursiops truncatus) prompted us to carry out an extensive search for the disease-associated isoform (PrPSc) of the cellular prion protein (PrPC) in the brain and in a range of lymphoid tissues from 23 striped dolphins (Stenella coeruleoalba), 5 bottlenose dolphins and 2 Risso s dolphins (Grampus griseus) found stranded between 2007 and 2012 along the Italian coastline. Three striped dolphins and one bottlenose dolphin showed microscopic lesions of encephalitis, with no evidence of spongiform brain lesions being detected in any of the 30 free-ranging cetaceans investigated herein. Nevertheless, we could still observe a prominent PrPC immunoreactivity in the brain as well as in lymphoid tissues from these dolphins. Although immunohistochemical and Western blot investigations yielded negative results for PrPSc deposition in all tissues from the dolphins under study, the reported occurrence of a spontaneous PD case in a wild dolphin is an intriguing issue and a matter of concern for both prion biology and intra/inter-species transmissibility, as well as for cetacean conservation medicine. PMID:23034277

  13. Comparison of cellular distribution of LH receptors and steroidogenic enzymes in the porcine ovary.

    PubMed

    Meduri, G; Vu Hai, M T; Jolivet, A; Takemori, S; Kominami, S; Driancourt, M A; Milgrom, E

    1996-03-01

    Previous studies have shown a heterogeneous expression of LH receptors in various structures of the porcine ovary. Specially striking was the existence in the preovulatory follicle of inner layers of theca interna cells devoid of LH receptor and the confinement in the corpus luteum of the LH receptor to the external cellular layers. In the present study, we have compared the steroidogenic capabilities of LH receptor-positive and -negative cells using immunocytochemistry for side-chain cleavage P450, 3 beta-hydroxysteroid-dehydrogenase, 17 alpha-hydroxylase P450 and aromatase P450. We have also examined, using the same methods, the evolution of the various cell types after ovulation and during the development of the corpus luteum. In preovulatory follicles the inner layers of theca cells which were not labelled with anti-LH receptor antibodies appeared to express the steroidogenic enzymes in a way similar to that of the outer LH receptor-positive cell layers. Ovulation per se did not change the distribution of LH receptors (present in the outer luteal cells and in the granulosa) or of steroidogenic enzymes. However, 48 h after follicular rupture there as a marked decrease in overall labelling with anti-LH receptor antibody, and especially a disappearance of immunostaining in the luteal cells of granulosa origin. In the mid-luteal phase (6 days after ovulation), the receptor content seemed to increase in the peripheral luteal cells derived from the theca but the receptor did not reappear in the granulosa-derived luteal cells. Thus the down-regulation of LH receptor appeared to be reversible in the external thecal layers but irreversible in the granulosa cells. Furthermore, the distribution of the various steroidogenic enzymes in the corpora lutea delineated granulosa-derived from theca-derived cells and showed that only the external layers of the latter expressed the LH receptor. These results showed the existence in the preovulatory follicle of two theca interna

  14. Epstein-Barr virus transcription factor Zta acts through distal regulatory elements to directly control cellular gene expression.

    PubMed

    Ramasubramanyan, Sharada; Osborn, Kay; Al-Mohammad, Rajaei; Naranjo Perez-Fernandez, Ijiel B; Zuo, Jianmin; Balan, Nicolae; Godfrey, Anja; Patel, Harshil; Peters, Gordon; Rowe, Martin; Jenner, Richard G; Sinclair, Alison J

    2015-04-20

    Lytic replication of the human gamma herpes virus Epstein-Barr virus (EBV) is an essential prerequisite for the spread of the virus. Differential regulation of a limited number of cellular genes has been reported in B-cells during the viral lytic replication cycle. We asked whether a viral bZIP transcription factor, Zta (BZLF1, ZEBRA, EB1), drives some of these changes. Using genome-wide chromatin immunoprecipitation coupled to next-generation DNA sequencing (ChIP-seq) we established a map of Zta interactions across the human genome. Using sensitive transcriptome analyses we identified 2263 cellular genes whose expression is significantly changed during the EBV lytic replication cycle. Zta binds 278 of the regulated genes and the distribution of binding sites shows that Zta binds mostly to sites that are distal to transcription start sites. This differs from the prevailing view that Zta activates viral genes by binding exclusively at promoter elements. We show that a synthetic Zta binding element confers Zta regulation at a distance and that distal Zta binding sites from cellular genes can confer Zta-mediated regulation on a heterologous promoter. This leads us to propose that Zta directly reprograms the expression of cellular genes through distal elements. PMID:25779048

  15. Epstein–Barr virus transcription factor Zta acts through distal regulatory elements to directly control cellular gene expression

    PubMed Central

    Ramasubramanyan, Sharada; Osborn, Kay; Al-Mohammad, Rajaei; Naranjo Perez-Fernandez, Ijiel B.; Zuo, Jianmin; Balan, Nicolae; Godfrey, Anja; Patel, Harshil; Peters, Gordon; Rowe, Martin; Jenner, Richard G.; Sinclair, Alison J.

    2015-01-01

    Lytic replication of the human gamma herpes virus Epstein-Barr virus (EBV) is an essential prerequisite for the spread of the virus. Differential regulation of a limited number of cellular genes has been reported in B-cells during the viral lytic replication cycle. We asked whether a viral bZIP transcription factor, Zta (BZLF1, ZEBRA, EB1), drives some of these changes. Using genome-wide chromatin immunoprecipitation coupled to next-generation DNA sequencing (ChIP-seq) we established a map of Zta interactions across the human genome. Using sensitive transcriptome analyses we identified 2263 cellular genes whose expression is significantly changed during the EBV lytic replication cycle. Zta binds 278 of the regulated genes and the distribution of binding sites shows that Zta binds mostly to sites that are distal to transcription start sites. This differs from the prevailing view that Zta activates viral genes by binding exclusively at promoter elements. We show that a synthetic Zta binding element confers Zta regulation at a distance and that distal Zta binding sites from cellular genes can confer Zta-mediated regulation on a heterologous promoter. This leads us to propose that Zta directly reprograms the expression of cellular genes through distal elements. PMID:25779048

  16. microRNA Expression and Biogenesis in Cellular Response to Ionizing Radiation

    PubMed Central

    Mao, Aihong; Liu, Yang; Di, Cuixia; Sun, Chao

    2014-01-01

    Increasing evidence demonstrates that the expression levels of microRNAs (miRNAs) significantly change upon ionizing radiation (IR) and play a critical role in cellular response to IR. Although several radiation responsive miRNAs and their targets have been identified, little is known about how miRNAs expression and biogenesis is regulated by IR-caused DNA damage response (DDR). Hence, in this review, we summarize miRNA expression and biogenesis in cellular response to IR and mainly elucidate the regulatory mechanisms of miRNA expression and biogenesis from different aspects including ataxia-telangiectasia mutated (ATM) kinase, p53/p63/p73 family and other potential factors. Furthermore, we focus on ΔNp73, which might be a potential regulator of miRNA expression and biogenesis in cellular response to IR. miRNAs could effectively activate the IR-induced DDR and modulate the radiation response and cellular radiosensitivity, which have an important potential clinical application. Therefore, thoroughly understanding the regulatory mechanisms of miRNAs expression and biogenesis in radiation response will provide new insights for clinical cancer radiotherapy. PMID:24905898

  17. Regulation of myokine expression: Role of exercise and cellular stress.

    PubMed

    Ost, Mario; Coleman, Verena; Kasch, Juliane; Klaus, Susanne

    2016-09-01

    Exercise training is well known to improve physical fitness and to combat chronic diseases and aging related disorders. Part of this is thought to be mediated by myokines, muscle derived secretory proteins (mainly cytokines) that elicit auto/paracrine but also endocrine effects on organs such as liver, adipose tissue, and bone. Today, several hundred potential myokines have been identified most of them not exclusive to muscle cells. Strenuous exercise is associated with increased production of free radicals and reactive oxidant species (ROS) as well as endoplasmic reticulum (ER)-stress which at an excessive level can lead to muscle damage and cell death. On the other hand, transient elevations in oxidative and ER-stress are thought to be necessary for adaptive improvements by regular exercise through a hormesis action termed mitohormesis since mitochondria are essential for the generation of energy and tightly connected to ER- and oxidative stress. Exercise induced myokines have been identified by various in vivo and in vitro approaches and accumulating evidence suggests that ROS and ER-stress linked pathways are involved in myokine induction. For example, interleukin (IL)-6, the prototypic exercise myokine is also induced by oxidative and ER-stress. Exercise induced expression of some myokines such as irisin and meteorin-like is linked to the transcription factor PGC-1α and apparently not related to ER-stress whereas typical ER-stress induced cytokines such as FGF-21 and GDF-15 are not exercise myokines under normal physiological conditions. Recent technological advances have led to the identification of numerous potential new myokines but for most of them regulation by oxidative and ER-stress still needs to be unraveled.

  18. Spinophilin expression determines cellular growth, cancer stemness and 5-flourouracil resistance in colorectal cancer

    PubMed Central

    Schwarzenbacher, Daniela; Deutsch, Alexander; Perakis, Samantha; Ling, Hui; Ivan, Cristina; Calin, George Adrian; Rinner, Beate; Gerger, Armin; Pichler, Martin

    2014-01-01

    The putative tumor suppressor gene spinophilin has been involved in cancer progression in several types of cancer. In this study, we explored the prognostic value of spinophilin expression in 162 colon adenocarcinoma patients. In addition, we generated stably expressing spinophilin-directed shRNA CRC cell lines and studied the influence of spinophilin expression on cellular phenotypes and molecular interactions. We independently confirmed that low spinophilin expression levels are associated with poor prognosis in CRC patients (p = 0.038). A reduction of spinophilin levels in p53 wild-type HCT116 and p53-mutated Caco-2 cells led to increased cellular growth rates and anchorage-independent growth (p<0.05). At molecular level, reduced spinophilin levels increased the expression of the transcription factor E2F-1. In addition, we observed an increased formation of tumor spheres, increased number of CD133 positive cells and an increased resistance to 5-flourouracil (p<0.05). Finally, treatment with the de-methylating agent 5-aza-dC increased spinophilin expression in CRC cells (p<0.05), corroborated by a correlation of spinophilin expression and extent of methylated CpG sites in the gene promoter region (p<0.001). In conclusion, gain of aggressive biological properties of CRC cells including cellular growth, cancer stem cell features and 5-flourouracil resistance partly explains the role of spinophilin in CRC. PMID:25261368

  19. Glucose Oxidase Induces Cellular Senescence in Immortal Renal Cells through ILK by Downregulating Klotho Gene Expression

    PubMed Central

    Troyano-Suárez, Nuria; del Nogal-Avila, María; Mora, Inés; Sosa, Patricia; López-Ongil, Susana; Rodriguez-Puyol, Diego; Olmos, Gemma; Ruíz-Torres, María Piedad

    2015-01-01

    Cellular senescence can be prematurely induced by oxidative stress involved in aging. In this work, we were searching for novel intermediaries in oxidative stress-induced senescence, focusing our interest on integrin-linked kinase (ILK), a scaffold protein at cell-extracellular matrix (ECM) adhesion sites, and on the Klotho gene. Cultured renal cells were treated with glucose oxidase (GOx) for long time periods. GOx induced senescence, increasing senescence associated β-galactosidase activity and the expression of p16. In parallel, GOx increased ILK protein expression and activity. Ectopic overexpression of ILK in cells increased p16 expression, even in the absence of GOx, whereas downregulation of ILK inhibited the increase in p16 due to oxidative stress. Additionally, GOx reduced Klotho gene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition. We demonstrated a direct link between ILK and Klotho since silencing ILK expression in cells and mice increases Klotho expression and reduces p53 and p16 expression in renal cortex. In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reduces Klotho expression. We hereby present ILK as a novel downregulator of Klotho gene expression. PMID:26583057

  20. Aiolos transcription factor controls cell death in T cells by regulating Bcl-2 expression and its cellular localization.

    PubMed Central

    Romero, F; Martínez-A, C; Camonis, J; Rebollo, A

    1999-01-01

    We searched for proteins that interact with Ras in interleukin (IL)-2-stimulated or IL-2-deprived cells, and found that the transcription factor Aiolos interacts with Ras. The Ras-Aiolos interaction was confirmed in vitro and in vivo by co-immunoprecipitation. Indirect immunofluorescence shows that IL-2 controls the cellular distribution of Aiolos and induces its tyrosine phosphorylation, required for dissociation from Ras. We also identified functional Aiolos-binding sites in the Bcl-2 promoter, which are able to activate the luciferase reporter gene. Mutation of Aiolos-binding sites within the Bcl-2 promoter inhibits transactivation of the reporter gene luciferase, suggesting direct control of Bcl-2 expression by Aiolos. Co-transfection experiments confirm that Aiolos induces Bcl-2 expression and prevents apoptosis in IL-2-deprived cells. We propose a model for the regulation of Bcl-2 expression via Aiolos. PMID:10369681

  1. Degradation of plasma membrane phosphatidylcholine appears not to affect the cellular cholesterol distribution.

    PubMed

    Pörn, M I; Ares, M P; Slotte, J P

    1993-08-01

    To clarify the role of possible cholesterol/phosphatidylcholine interactions in cellular cholesterol distribution, we have used a phosphatidylcholine-specific phospholipase C from Bacillus cereus to degrade the cell surface phosphatidylcholine of cultured human fibroblasts. Of cellular phosphatidylcholine, approximately 15% was susceptible to degradation by the phospholipase. In spite of the dramatic redistribution of cellular cholesterol that can be observed after sphingomyelin depletion, the degradation of cell surface phosphatidylcholine did not affect the distribution of cholesterol in fibroblasts. In cholesterol-depleted cells as well as in cholesterol-loaded cells, the size of the cell surface cholesterol pool (susceptible to cholesterol oxidase) remained unchanged after phosphatidylcholine degradation. The rate of cholesterol esterification with [3H]oleic acid and the rate of [3H]cholesterol efflux from fibroblasts to high density lipoproteins also remained unchanged after degradation of plasma membrane phosphatidylcholine. An increase in the level of [3H]cholesterol efflux to high density lipoproteins was observed after degradation of plasma membrane sphingomyelin with exogenous sphingomyelinase, in-contrast to earlier reports, where no such effect was observed. The results suggest that interactions between cholesterol and phosphatidylcholine in the fibroblast plasma membranes are less important than cholesterol/sphingomyelin interactions for the asymmetric distribution of cellular cholesterol.

  2. A signature microRNA expression profile for the cellular response to thermal stress

    NASA Astrophysics Data System (ADS)

    Wilmink, Gerald J.; Roth, Caleb C.; Ketchum, Norma; Ibey, Bennett L.; Waterworth, Angela; Suarez, Maria; Roach, William P.

    2009-02-01

    Recently, an extensive layer of intra-cellular signals was discovered that was previously undetected by genetic radar. It is now known that this layer consists primarily of a class of short noncoding RNA species that are referred to as microRNAs (miRNAs). MiRNAs regulate protein synthesis at the post-transcriptional level, and studies have shown that they are involved in many fundamental cellular processes. In this study, we hypothesized that miRNAs may be involved in cellular stress response mechanisms, and that cells exposed to thermal stress may exhibit a signature miRNA expression profile indicative of their functional involvement in such mechanisms. To test our hypothesis, human dermal fibroblasts were exposed to an established hyperthermic protocol, and the ensuing miRNA expression levels were evaluated 4 hr post-exposure using microRNA microarray gene chips. The microarray data shows that 123 miRNAs were differentially expressed in cells exposed to thermal stress. We collectively refer to these miRNAs as thermalregulated microRNAs (TRMs). Since miRNA research is in its infancy, it is interesting to note that only 27 of the 123 TRMs are currently annotated in the Sanger miRNA registry. Prior to publication, we plan to submit the remaining novel 96 miRNA gene sequences for proper naming. Computational and thermodynamic modeling algorithms were employed to identify putative mRNA targets for the TRMs, and these studies predict that TRMs regulate the mRNA expression of various proteins that are involved in the cellular stress response. Future empirical studies will be conducted to validate these theoretical predictions, and to further examine the specific role that TRMs play in the cellular stress response.

  3. Contaminant loading in remote Arctic lakes affects cellular stress-related proteins expression in feral charr.

    USGS Publications Warehouse

    Wiseman, Steve; Jorgensen, Even H.; Maule, Alec G.; Vijayan, Mathilakath M.

    2011-01-01

    The remote Arctic lakes on Bjornoya Island, Norway, offer a unique opportunity to study possible affect of lifelong contaminant exposure in wild populations of landlocked Arctic charr (Salvelinus alpinus). This is because Lake Ellasjoen has persistent organic pollutant (POP) levels that are significantly greater than in the nearby Lake Oyangen. We examined whether this differential contaminant loading was reflected in the expression of protein markers of exposure and effect in the native fish. We assessed the expressions of cellular stress markers, including cytochrome P4501A (Cyp1A), heat shock protein 70 (hsp70), and glucocorticoid receptor (GR) in feral charr from the two lakes. The average polychlorinated biphenyl (PCB) load in the charr liver from Ellasjoen was approximately 25-fold higher than in individuals from Oyangen. Liver Cyp1A protein expression was significantly higher in individuals from Ellasjoen compared with Oyangen, confirming differential PCB exposure. There was no significant difference in hsp70 protein expression in charr liver between the two lakes. However, brain hsp70 protein expression was significantly elevated in charr from Ellasjoen compared with Oyangen. Also, liver GR protein expression was significantly higher in the Ellasjoen charr compared with Oyangen charr. Taken together, our results suggest changes to cellular stress-related protein expression as a possible adaptation to chronic-contaminant exposure in feral charr in the Norwegian high-Arctic.

  4. Imaging Self-assembly Dependent Spatial Distribution of Small Molecules in Cellular Environment

    PubMed Central

    Gao, Yuan; Kuang, Yi; Du, Xuewen; Zhou, Jie; Chandran, Preethi; Horkay, Ferenc; Xu, Bing

    2014-01-01

    Self-assembly of small molecules, as a more common phenomenon than one previously thought, can be either beneficial or detrimental to cells. Despite its profound biological implications, how the self-assembly of small molecules behave in cellular environment is largely unknown and barely explored. This work studies four fluorescent molecules that consist of the same peptidic backbone (e.g., Phe-Phe-Lys) and enzyme trigger (e.g., a phosphotyrosine residue), but bear different fluorophores on the side chain of the lysine residue of the peptidic motif. These molecules, however, exhibit different ability of self-assembly before and after enzymatic transformation (e.g., dephosphorylation). Fluorescent imaging reveals that self-assembly directly affects the distribution of these small molecules in cellular environment. Moreover, cell viability tests suggest that the states and the location of the molecular assemblies in the cellular environment control the phenotypes of the cells. For example, the molecular nanofibers of one of the small molecules apparently stabilize actin filaments and alleviate the insult of an F-actin toxin (e.g., latrunculin A). Combining fluorescent imaging and enzyme-instructed self-assembly of small peptidic molecules, this work not only demonstrates that self-assembly as a key factor for dictating the spatial distribution of small molecules in cellular environment. In addition, it illustrates a useful approach, based on enzyme-instructed self-assembly of small molecules, to modulate spatiotemporal profiles of small molecules in cellular environment, which allows the use of the emergent properties of small molecules to control the fate of cells. PMID:24266765

  5. Expression of virus-encoded proteinases: functional and structural similarities with cellular enzymes.

    PubMed Central

    Dougherty, W G; Semler, B L

    1993-01-01

    Many viruses express their genome, or part of their genome, initially as a polyprotein precursor that undergoes proteolytic processing. Molecular genetic analyses of viral gene expression have revealed that many of these processing events are mediated by virus-encoded proteinases. Biochemical activity studies and structural analyses of these viral enzymes reveal that they have remarkable similarities to cellular proteinases. However, the viral proteinases have evolved unique features that permit them to function in a cellular environment. In this article, the current status of plant and animal virus proteinases is described along with their role in the viral replication cycle. The reactions catalyzed by viral proteinases are not simple enzyme-substrate interactions; rather, the processing steps are highly regulated, are coordinated with other viral processes, and frequently involve the participation of other factors. Images PMID:8302216

  6. Posttranscriptional regulation of cellular gene expression by the c-myc oncogene

    SciTech Connect

    Prendergast, G.C.; Cole, M.D. . Dept. of Biology)

    1989-01-01

    The c-myc oncogene has been implicated in the development of many different cancers, yet the mechanism by which the c-myc protein alters cellular growth control has proven elusive. The authors used a cDNA hybridization difference assay to isolate two genes, mr1 and mr2, that were constitutively expressed (i.e., deregulated) in rodent fibroblast cell lines immortalized by transfection of a viral promoter-linked c-myc gene. Both cDNAs were serum inducible in quiescent G/sub o/ fibroblasts, suggesting that they are functionally related to cellular proliferative processes. Although there were significant differences in cytoplasmic mRNA levels between myc-immortalized and control cells, the rates of transcription and mRNA turnover of both genes were similar, suggesting that c-myc regulates mr1 and mr2 expression by some nuclear posttranscriptional mechanism. Their results provide evidence that c-myc can rapidly modulate cellular gene expression and suggest that c-myc may function in gene regulation at the level of RNA export, splicing, or nuclear RNA turnover.

  7. Cellular oncogene expression following exposure of mice to {gamma}-rays

    SciTech Connect

    Anderson, A.; Woloschak, G.E.

    1991-06-12

    We examined the effects of total body exposure of BCF1 mice to {gamma}-rays (300 cGy) in modulating expression of cellular oncogenes in both gut and liver tissues. We selected specific cellular oncogenes (c-fos, c-myc, c-src, and c-H-ras), based on their normal expression in liver and gut tissues from untreated mice. As early as 5 min. following whole body exposure of BCF1 mice to {gamma}-rays we detected induction of mRNA specific for c-src and c-H-ras in both liver and gut tissues. c-fos RNA was slightly decreased in accumulation in gut but was unaffected in liver tissue from irradiated mice relative to untreated controls. c-myc mRNA accumulation was unaffected in all tissues examined. These experiments document that modulation of cellular oncogene expression can occur as an early event in tissues following irradiation and suggest that this modulation may play a role in radiation-induced carcinogenesis.

  8. Distinct 5′ UTRs regulate XIAP expression under normal growth conditions and during cellular stress

    PubMed Central

    Riley, Alura; Jordan, Lindsay E.; Holcik, Martin

    2010-01-01

    X-chromosome linked inhibitor of apoptosis, XIAP, is cellular caspase inhibitor and a key regulator of apoptosis. We and others have previously shown that XIAP expression is regulated primarily at the level of protein synthesis; the 5′ untranslated region (UTR) of XIAP mRNA contains an Internal Ribosome Entry Site (IRES) that supports cap-independent expression of XIAP protein during conditions of pathophysiological stress, such as serum deprivation or gamma irradiation. Here, we show that XIAP is encoded by two distinct mRNAs that differ in their 5′ UTRs. We further show that the dominant, shorter, 5′ UTR promotes a basal level of XIAP expression under normal growth conditions. In contrast, the less abundant longer 5′ UTR contains an IRES and supports cap-independent translation during stress. Our data suggest that the combination of alternate regulatory regions and distinct translational initiation modes is critical in maintaining XIAP levels in response to cellular stress and may represent a general mechanism of cellular adaptation. PMID:20385593

  9. Myofibroblast Differentiation Modulates Keratocyte Crystallin Protein Expression, Concentration, and Cellular Light Scattering

    PubMed Central

    Brown, Donald; Pappa, Aglaia; Vasiliou, Vasilis

    2012-01-01

    Purpose. The purpose of this study was to determine whether myofibroblast differentiation altered keratocyte crystallin protein concentration and increased cellular light scattering. Methods. Serum-free cultured rabbit corneal keratocytes and TGFβ (5 ng/mL) induced myofibroblasts were harvested and counted and protein/RNA extracted. Expression of myofibroblast and keratocyte markers was determined by real-time PCR and Western blot analysis. The cell volume of calcein AM–loaded keratocytes and myofibroblasts was determined by using nonlinear optical microscopy. Cellular light scattering of transformed myofibroblasts expressing human keratocyte crystallins was measured by reflectance confocal microscopy. Results. Differentiated myofibroblasts showed a significant decrease in RNA levels for the keratocyte markers ALDH1A1, lumican, and keratocan and a significant increase in the myofibroblast marker α-smooth muscle actin. Volumetric and protein measurements showed that myofibroblast differentiation significantly increased cytoplasmic volume (293%; P < 0.001) and water-soluble and -insoluble protein content per cell (respectively, 442% and 431%; P < 0.002) compared to keratocytes. Western blot analysis showed that the level of ALDH1A1 protein per cell was similar between myofibroblasts and keratocytes, but was substantially reduced as a percentage of total water-soluble protein. Light scattering measurements showed that induced expression of corneal crystallins significantly decreased light scattering. Conclusions. These data suggest that myofibroblast differentiation leads to a marked increase in cell volume and dilution of corneal crystallins associated with an increase in cellular light scattering. PMID:22247459

  10. Cellular gene expression induced by parasite antigens and allergens in neonates from parasite-infected mothers.

    PubMed

    Soboslay, Peter T; Orlikowsky, Thorsten; Huang, Xiangsheng; Gille, Christian; Spring, Bärbel; Kocherscheidt, Lars; Agossou, Abram; Banla, Meba; Bonin, Michael; Köhler, Carsten

    2016-05-01

    Prenatal exposure to parasite antigens or allergens will influence the profile and strength of postnatal immune responses, such contact may tolerize and increase susceptibility to future infections or sensitize to environmental allergens. Exposure in utero to parasite antigens will distinctly alter cellular gene expression in newborns. Gene microarrays were applied to study gene expression in umbilical cord blood cell (UCBC) from parasite-exposed (Para-POS) and non-exposed (Para-NEG) neonates. UCBC were activated with antigens of helminth (Onchocerca volvulus), amoeba (Entamoeba histolytica) or allergens of mite (Dermatophagoides farinae). When UCBC from Para-POS and Para-NEG newborns were exposed to helminth antigens or allergens consistent differences occurred in the expression of genes encoding for MHC class I and II alleles, signal transducers of activation and transcription (STATs), cytokines, chemokines, immunoglobulin heavy and light chains, and molecules associated with immune regulation (SOCS, TLR, TGF), inflammation (TNF, CCR) and apoptosis (CASP). Expression of genes associated with innate immune responses were enhanced in Para-NEG, while in Para-POS, the expression of MHC class II and STAT genes was reduced. Within functional gene networks for cellular growth, proliferation and immune responses, Para-NEG neonates presented with significantly higher expression values than Para-POS. In Para-NEG newborns, the gene cluster and pathway analyses suggested that gene expression profiles may predispose for the development of immunological, hematological and dermatological disorders upon postnatal helminth parasite infection or allergen exposure. Thus, prenatal parasite contact will sensitize without generating aberrant inflammatory immune responses, and increased pro-inflammatory but decreased regulatory gene expression profiles will be present in those neonates lacking prenatal parasite antigen encounter. PMID:27062712

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-02-18

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

  13. Regulation of Cellular Caveolin-1 Protein Expression in Murine Macrophages by Microbial Products

    PubMed Central

    Lei, Mei G.; Tan, Xiaoyu; Qureshi, Nilofer; Morrison, David C.

    2005-01-01

    Previously, we reported that expression of caveolin-1 in elicited peritoneal mouse macrophages was up-regulated by remarkably low (1.0-pg/ml) concentrations of Escherichia coli O111 lipopolysaccharide (LPS). Here we report that increases in caveolin-1 expression are manifested by different types of LPS, LPS-mimetic taxol, and heat-killed E. coli and to a much lesser extent by zymosan, polysaccharide-peptidoglycan, and heat-killed Staphylococcus aureus. Rhodobacter sphaeroides lipid A (RsDPLA) could not induce caveolin-1 expression in macrophages. Interestingly, polymyxin B (5 μg/ml) and RsDPLA show only a limited capacity to inhibit LPS-induced caveolin-1 expression. These findings suggest that expression of caveolin-1 in response to LPS may only partially be dependent upon lipid A. Recombinant tumor necrosis factor alpha marginally induces caveolin-1, suggesting that the ability of LPS to regulate caveolin-1 is not mediated primarily through an autocrine/paracrine mechanism involving this cytokine. Under conditions in which cellular levels of caveolin-1 are profoundly induced, no significant changes in TLR4 expression are observed. Of interest, caveolin-1 appears to localize to two cellular compartments, one associated with lipid rafts and a second associated with TLR4. Gamma interferon treatment inhibits the induction of caveolin-1 by LPS in macrophages. Inhibition of the p38 kinase-dependent pathway, but not the extracellular signal-regulated kinase pathway, effectively reduced the ability of LPS to mediate caveolin-1 up-regulation. Lactacystin, a potent inhibitor of the proteasome pathway, significantly modulates LPS-independent caveolin-1 expression, and lactacystin inhibits LPS-triggered caveolin-1 responses. These studies suggest that caveolin-1 up-regulation in response to LPS is likely to be proteasome dependent and triggered through the p38 kinase pathway. PMID:16299308

  14. BMP2 Regulation of CXCL12 Cellular, Temporal, and Spatial Expression is Essential During Fracture Repair

    PubMed Central

    Myers, Timothy J; Longobardi, Lara; Willcockson, Helen; Temple, Joseph D; Tagliafierro, Lidia; Ye, Ping; Li, Tieshi; Esposito, Alessandra; Moats-Staats, Billie M; Spagnoli, Anna

    2016-01-01

    The cellular and humoral responses that orchestrate fracture healing are still elusive. Here we report that bone morphogenic protein 2 (BMP2)-dependent fracture healing occurs through a tight control of chemokine C-X-C motif-ligand-12 (CXCL12) cellular, spatial, and temporal expression. We found that the fracture repair process elicited an early site-specific response of CXCL12+-BMP2+ endosteal cells and osteocytes that was not present in unfractured bones and gradually decreased as healing progressed. Absence of a full complement of BMP2 in mesenchyme osteoprogenitors (BMP2cKO/+) prevented healing and led to a dysregulated temporal and cellular upregulation of CXCL12 expression associated with a deranged angiogenic response. Healing was rescued when BMP2cKO/+ mice were systemically treated with AMD3100, an antagonist of CXCR4 and agonist for CXCR7 both receptors for CXCL12. We further found that mesenchymal stromal cells (MSCs), capable of delivering BMP2 at the endosteal site, restored fracture healing when transplanted into BMP2cKO/+ mice by rectifying the CXCL12 expression pattern. Our in vitro studies showed that in isolated endosteal cells, BMP2, while inducing osteoblastic differentiation, stimulated expression of pericyte markers that was coupled with a decrease in CXCL12. Furthermore, in isolated BMP2cKO/cKO endosteal cells, high expression levels of CXCL12 inhibited osteoblastic differentiation that was restored by AMD3100 treatment or coculture with BMP2-expressing MSCs that led to an upregulation of pericyte markers while decreasing platelet endothelial cell adhesion molecule (PECAM). Taken together, our studies show that following fracture, a CXCL12+-BMP2+ perivascular cell population is recruited along the endosteum, then a timely increase of BMP2 leads to downregulation of CXCL12 that is essential to determine the fate of the CXCL12+-BMP2+ to osteogenesis while departing their supportive role to angiogenesis. Our findings have far

  15. Transient expression of protein tyrosine phosphatases encoded in Cotesia plutellae bracovirus inhibits insect cellular immune responses

    NASA Astrophysics Data System (ADS)

    Ibrahim, Ahmed M. A.; Kim, Yonggyun

    2008-01-01

    Several immunosuppressive factors are associated with parasitism of an endoparasitoid wasp, Cotesia plutellae, on the diamondback moth, Plutella xylostella. C. plutellae bracovirus (CpBV) encodes a large number of putative protein tyrosine phosphatases (PTPs), which may play a role in inhibiting host cellular immunity. To address this inhibitory hypothesis of CpBV-PTPs, we performed transient expression of individual CpBV-PTPs in hemocytes of the beet armyworm, Spodoptera exigua, and analyzed their cellular immune responses. Two different forms of CpBV-PTPs were chosen and cloned into a eukaryotic expression vector under the control of the p10 promoter of baculovirus: one with the normal cysteine active site (CpBV-PTP1) and the other with a mutated active site (CpBV-PTP5). The hemocytes transfected with CpBV-PTP1 significantly increased in PTP activity compared to control hemocytes, but those with CpBV-PTP5 exhibited a significant decrease in the PTP activity. All transfected hemocytes exhibited a significant reduction in both cell spreading and encapsulation activities compared to control hemocytes. Co-transfection of CpBV-PTP1 together with its double-stranded RNA reduced the messenger RNA (mRNA) level of CpBV-PTP1 and resulted in recovery of both hemocyte behaviors. This is the first report demonstrating that the polydnaviral PTPs can manipulate PTP activity of the hemocytes to interrupt cellular immune responses.

  16. Cellular distribution of transforming growth factor-beta 1 and procollagen types I, III, and IV transcripts in carbon tetrachloride-induced rat liver fibrosis.

    PubMed Central

    Nakatsukasa, H; Nagy, P; Evarts, R P; Hsia, C C; Marsden, E; Thorgeirsson, S S

    1990-01-01

    The cellular distribution and temporal expression of transcripts from transforming growth factor-beta 1 (TGF-beta 1) and procollagen alpha 1(I), alpha 1(III), and alpha 1(IV) genes were studied in carbon tetrachloride (CCl4)-induced rat liver fibrosis by using in situ hybridization technique. During the fibrotic process, TGF-beta 1 and procollagen genes were similarly and predominantly expressed in Desmin-positive perisinusoidal cells (e.g., fat-storing cells and myofibroblasts) and fibroblasts and their expression continued to be higher than those observed in control rats. These transcripts were also observed in inflammatory cells mainly granulocytes and macrophage-like cells at the early stages of liver fibrosis. The production of extracellular matrix along small blood vessels and fibrous septa coincided with the expression of these genes. Expression of TGF-beta 1 and procollagen genes were not detected in hepatocytes throughout the experiment. No significant differences in cellular distribution or time course of gene expression among procollagen alpha 1(I), alpha 1(III), and alpha 1(IV) were observed. Desmin-positive perisinusoidal cells and fibroblasts appeared to play the principal role in synthesis of collagens in CCl4-induced hepatic fibrosis. The simultaneous expression of TGF-beta 1 and procollagen genes in mesenchymal cells, including Desmin-positive perisinusoidal cells, during hepatic fibrosis suggests the possibility that TGF-beta 1 may have an important role in the production of fibrosis. Images PMID:1693377

  17. Three-dimensional morphology and gene expression in the Drosophila blastoderm at cellular resolution II: dynamics

    PubMed Central

    Keränen, Soile VE; Fowlkes, Charless C; Luengo Hendriks, Cris L; Sudar, Damir; Knowles, David W; Malik, Jitendra; Biggin, Mark D

    2006-01-01

    Background To accurately describe gene expression and computationally model animal transcriptional networks, it is essential to determine the changing locations of cells in developing embryos. Results Using automated image analysis methods, we provide the first quantitative description of temporal changes in morphology and gene expression at cellular resolution in whole embryos, using the Drosophila blastoderm as a model. Analyses based on both fixed and live embryos reveal complex, previously undetected three-dimensional changes in nuclear density patterns caused by nuclear movements prior to gastrulation. Gene expression patterns move, in part, with these changes in morphology, but additional spatial shifts in expression patterns are also seen, supporting a previously proposed model of pattern dynamics based on the induction and inhibition of gene expression. We show that mutations that disrupt either the anterior/posterior (a/p) or the dorsal/ventral (d/v) transcriptional cascades alter morphology and gene expression along both the a/p and d/v axes in a way suggesting that these two patterning systems interact via both transcriptional and morphological mechanisms. Conclusion Our work establishes a new strategy for measuring temporal changes in the locations of cells and gene expression patterns that uses fixed cell material and computational modeling. It also provides a coordinate framework for the blastoderm embryo that will allow increasingly accurate spatio-temporal modeling of both the transcriptional control network and morphogenesis. PMID:17184547

  18. Ebola Virion Attachment and Entry into Human Macrophages Profoundly Effects Early Cellular Gene Expression

    PubMed Central

    Feldmann, Friedericke; Buehler, Lukas K.; Kindrachuk, Jason; DeFilippis, Victor; da Silva Correia, Jean; Früh, Klaus; Kuhn, Jens H.; Burton, Dennis R.; Feldmann, Heinz

    2011-01-01

    Zaire ebolavirus (ZEBOV) infections are associated with high lethality in primates. ZEBOV primarily targets mononuclear phagocytes, which are activated upon infection and secrete mediators believed to trigger initial stages of pathogenesis. The characterization of the responses of target cells to ZEBOV infection may therefore not only further understanding of pathogenesis but also suggest possible points of therapeutic intervention. Gene expression profiles of primary human macrophages exposed to ZEBOV were determined using DNA microarrays and quantitative PCR to gain insight into the cellular response immediately after cell entry. Significant changes in mRNA concentrations encoding for 88 cellular proteins were observed. Most of these proteins have not yet been implicated in ZEBOV infection. Some, however, are inflammatory mediators known to be elevated during the acute phase of disease in the blood of ZEBOV-infected humans. Interestingly, the cellular response occurred within the first hour of Ebola virion exposure, i.e. prior to virus gene expression. This observation supports the hypothesis that virion binding or entry mediated by the spike glycoprotein (GP1,2) is the primary stimulus for an initial response. Indeed, ZEBOV virions, LPS, and virus-like particles consisting of only the ZEBOV matrix protein VP40 and GP1,2 (VLPVP40-GP) triggered comparable responses in macrophages, including pro-inflammatory and pro-apoptotic signals. In contrast, VLPVP40 (particles lacking GP1,2) caused an aberrant response. This suggests that GP1,2 binding to macrophages plays an important role in the immediate cellular response. PMID:22028943

  19. DGAT1-deficiency affects the cellular distribution of hepatic retinoid and attenuates the progression of CCl4-induced liver fibrosis

    PubMed Central

    Yuen, Jason J.; Lee, Seung-Ah; Jiang, Hongfeng; Brun, Pierre-Jacques

    2015-01-01

    Background Diacylglycerol O-acyltransferase 1 (DGAT1) catalyzes the final step of triglyceride synthesis, transferring an acyl group from acyl-CoA to diacylglycerol. DGAT1 also catalyzes the acyl-CoA-dependent formation of retinyl esters in vitro and in mouse intestine and skin. Although DGAT1 is expressed in both hepatocytes and hepatic stellate cells (HSCs), we reported genetic and nutritional studies that established that DGAT1 does not contribute to retinyl ester formation in the liver. Methods We now have explored in more depth the role(s) of DGAT1 in hepatic retinoid metabolism and storage. Results Our data show that DGAT1 affects the cellular distribution between hepatocytes and HSCs of stored and newly absorbed dietary retinol. For livers of Dgat1-deficient mice, a greater percentage of stored retinyl ester is present in HSCs at the expense of hepatocytes. This is also true for newly absorbed oral [3H]retinol. These differences are associated with significantly increased expression, by 2.8-fold, of cellular retinol-binding protein, type I (RBP1) in freshly isolated HSCs from Dgat1-deficient mice, raising the possibility that RBP1, which contributes to retinol uptake into cells and retinyl ester synthesis, accounts for the differences. We further show that the retinyl ester-containing lipid droplets in HSCs are affected in Dgat1-null mice, being fewer in number but, on average, larger than in wild type (WT) HSCs. Finally, we demonstrate that DGAT1 affects experimentally induced HSC activation in vivo but that this effect is independent of altered retinoic acid availability or effects on gene expression. Conclusions Our studies establish that DGAT1 has a role in hepatic retinoid storage and metabolism, but this does not involve direct actions of DGAT1 in retinyl ester synthesis. PMID:26151058

  20. Cellular localization of transforming growth factor-beta expression in bleomycin-induced pulmonary fibrosis.

    PubMed Central

    Zhang, K.; Flanders, K. C.; Phan, S. H.

    1995-01-01

    Bleomycin-induced pulmonary fibrosis is associated with increased lung transforming growth factor-beta (TGF-beta) gene expression, but cellular localization of the source of this expression has not been unequivocally established. In this study, lung fibrosis was induced in rats by endotracheal bleomycin injection on day 0 and, on selected days afterwards, lungs were harvested for in situ hybridization, immunohistochemical and histochemical analyses for TGF-beta 1 mRNA and protein expression, and cell identification. The results show that control lungs express essentially no detectable TGF-beta 1 mRNA or protein in the parenchyma. Before day 3 after bleomycin treatment, scattered bronchiolar epithelial cells, mononuclear cells, and eosinophils expressed elevated levels of TGF-beta 1. Between days 3 and 14, there was a major increase in the number of eosinophils, myofibroblasts, and fibroblasts strongly expressing TGF-beta 1 mRNA and protein. TGF-beta 1-producing cells were predominantly localized within areas of injury and active fibrosis. After day 14, the intensity and number of TGF-beta 1-expressing cells significantly declined and were predominantly found in fibroblasts in fibrotic areas. The expression of TGF-beta 1 protein was generally coincident with that for mRNA with the exception of bronchiolar epithelial cells in which strong protein expression was unaccompanied by a commensurate increase in mRNA. The study demonstrates that myofibroblasts, fibroblasts, and eosinophils represent the major sources of increased lung TGF-beta 1 expression in this model of pulmonary fibrosis. Images Figure 2 Figure 3 Figure 4 PMID:7543734

  1. GASTRIN-RELEASING PEPTIDE RECEPTOR IN BREAST CANCER MEDIATES CELLULAR MIGRATION AND INTERLEUKIN-8 EXPRESSION

    PubMed Central

    Chao, Celia; Ives, Kirk; Hellmich, Helen L.; Townsend, Courtney M.; Hellmich, Mark R.

    2015-01-01

    Background Breast cancers aberrantly express gastrin-releasing peptide (GRP) hormone and its cognate receptor, gastrin-releasing peptide receptor (GRP-R). Experimental evidence suggests that bombesin (BBS), the pharmacological homologue of GRP, promotes breast cancer growth and progression. The contribution of GRP-R to other poor prognostic indicators in breast cancer, such as the expression of the EGF-R family of growth factors, and hormone insensitivity is unknown. Materials and Methods Two estrogen receptor (ER)-negative breast cancer cell lines were used. MDA-MB-231 overexpress both EGFR and GRPR, whereas SK-BR-3 cells express EGF-R but lack GRP-R. Cellular proliferation was assessed by Coulter counter. Chemotactic migration was performed using Transwell chambers and the migrated cells were quantified. Northern blot and real-time PCR were used to evaluate if pro-angiogenic factor interleukin-8 (IL-8) mRNA expression. Results In MDA-MB-231 cells, GRP-R and EGF-R synergize to regulate cell migration, IL-8 expression, but not cell proliferation. In SK-BR-3 cells, ectopic expression of GRP-R was sufficient to increase migration and IL-8 mRNA. Conclusions These data suggest relevant roles for GRP-R in ER-negative breast cancer progression. Future mechanistic studies to define the molecular role of GRP-R in breast cancer metastasis provide novel targets for the treatment of ER-negative breast cancers. PMID:19631337

  2. Mechanisms of cellular distribution of psychotropic drugs. Significance for drug action and interactions.

    PubMed

    Daniel, Władysława A

    2003-02-01

    Distribution of a drug in the body is dependent on its permeation properties, the blood flow rates in various tissues, and on plasma and tissue uptake. The distribution of drugs in vivo is largely determined by uptake competitions between blood and tissues, as well as competitions among individual tissues. Basic lipophilic drugs are characterized by extensive accumulation in tissues, which leads to a high volume of distribution. Nonspecific binding to cellular membranes and uptake by acidic compartments (mainly lysosomes) are responsible for such a distribution pattern. Lysosomal trapping is an important mechanism of distribution of basic psychotropic drugs; however, the tissue distribution of the aliphatic-type phenothiazine neuroleptic promazine, tricyclic antidepressants (TADs) and selective serotonin reuptake inhibitors (SSRIs) depends more on phospholipid binding than on lysosomal trapping, whereas in the case thioridazine and perazine, lysosomal trapping is as important for the tissue uptake as is phospholipid binding. Neuroleptics and antidepressants mutually inhibit their lysosomal uptake. A decrease in the intralysosomal drug concentrations in vivo leads to a shift of the drug from organs abundant in lysosomes (lungs, liver and kidneys) to those poor in these organella, e.g., the heart, which may be of clinical importance (cardiotoxicity). The brain is not a homogenous organ, i.e., the phospholipid pattern and density of lysosomes vary in its different regions. Therefore, the contribution of the two mechanisms (lysosomal trapping and tissue binding) to total drug uptake is different in areas of the brain. Both lysosomal trapping and binding to cellular elements for psychotropics are higher in the grey matter and neurons than in the white matter and astrocytes, respectively. Lysosomal trapping and distribution interactions of psychotropics take place mainly in neurons. A decrease (via a distributive interaction) in the concentration of psychotropics in

  3. Investigating the Cellular Distribution and Interactions of HIV-1 Nucleocapsid Protein by Quantitative Fluorescence Microscopy

    PubMed Central

    Anton, Halina; Taha, Nedal; Boutant, Emmanuel; Richert, Ludovic; Khatter, Heena; Klaholz, Bruno; Rondé, Philippe; Réal, Eléonore; de Rocquigny, Hugues; Mély, Yves

    2015-01-01

    The nucleocapsid protein (NCp7) of the Human immunodeficiency virus type 1 (HIV-1) is a small basic protein containing two zinc fingers. About 2000 NCp7 molecules coat the genomic RNA in the HIV-1 virion. After infection of a target cell, the viral core enters into the cytoplasm, where NCp7 chaperones the reverse transcription of the genomic RNA into the proviral DNA. As a consequence of their much lower affinity for double-stranded DNA as compared to single-stranded RNAs, NCp7 molecules are thought to be released in the cytoplasm and the nucleus of infected cells in the late steps of reverse transcription. Yet, little is known on the cellular distribution of the released NCp7 molecules and on their possible interactions with cell components. Hence, the aim of this study was to identify potential cellular partners of NCp7 and to monitor its intracellular distribution and dynamics by means of confocal fluorescence microscopy, fluorescence lifetime imaging microscopy, fluorescence recovery after photobleaching, fluorescence correlation and cross-correlation spectroscopy, and raster imaging correlation spectroscopy. HeLa cells transfected with eGFP-labeled NCp7 were used as a model system. We found that NCp7-eGFP localizes mainly in the cytoplasm and the nucleoli, where it binds to cellular RNAs, and notably to ribosomal RNAs which are the most abundant. The binding of NCp7 to ribosomes was further substantiated by the intracellular co-diffusion of NCp7 with the ribosomal protein 26, a component of the large ribosomal subunit. Finally, gradient centrifugation experiments demonstrate a direct association of NCp7 with purified 80S ribosomes. Thus, our data suggest that NCp7 molecules released in newly infected cells may primarily bind to ribosomes, where they may exert a new potential role in HIV-1 infection. PMID:25723396

  4. Irradiation affects cellular properties and Eph receptor expression in human melanoma cells

    PubMed Central

    Mosch, Birgit; Pietzsch, Doreen; Pietzsch, Jens

    2012-01-01

    X-ray irradiation influences metastatic properties of tumor cells and, moreover, metastasis and cellular motility can be modified by members of the Eph receptor/ephrin family of receptor tyrosine kinases. We hypothesized that irradiation-induced changes in cellular properties relevant for metastasis in melanoma cells could be mediated by Eph receptor/ephrin signaling. In this pilot study, we analyzed one pre-metastatic (Mel-Juso) and three metastatic human melanoma (Mel-Juso-L3, A375, and A2058) cells lines and predominantly found anti-metastatic effects of X-ray irradiation with impaired cell growth, clonal growth and motility. Additionally, we observed an irradiation-induced increase in adhesion paralleled by a decrease in migration in Mel-Juso and Mel-Juso-L3 cells and, in part, also in A375 cells. We further demonstrate a decrease of EphA2 both in expression and activity at 7 d after irradiation paralleled by an upregulation of EphA3. Analyzing downstream signaling after irradiation, we detected decreased Src kinase phosphorylation, but unchanged focal adhesion kinase (FAK) phosphorylation, indicating, in part, irradiation-induced downregulation of signaling via the EphA2-Src-FAK axis in melanoma cells. However, to which extent this finding contributes to the modification of metastasis-relevant cellular properties remains to be elucidated. PMID:22568947

  5. GIM3E: Condition-specific Models of Cellular Metabolism Developed from Metabolomics and Expression Data

    SciTech Connect

    Schmidt, Brian; Ebrahim, Ali; Metz, Thomas O.; Adkins, Joshua N.; Palsson, Bernard O.; Hyduke, Daniel R.

    2013-11-15

    Motivation: Genome-scale metabolic models have been used extensively to investigate alterations in cellular metabolism. The accuracy of these models to represent cellular metabolism in specific conditions has been improved by constraining the model with omics data sources. However, few practical methods for integrating metabolomics data with other omics data sources into genome-scale models of metabolism have been reported. Results: GIMMME (Gene Inactivation Moderated by Metabolism, Metabolomics, and Expression) is an algorithm that enables the development of condition-specific models based on an objective function, transcriptomics, and intracellular metabolomics data. GIMMME establishes metabolite utilization requirements with metabolomics data, uses model-paired transcriptomics data to find experimentally supported solutions, and also provides calculations of the turnover (production / consumption) flux of metabolites. GIMMME was employed to investigate the effects of integrating additional omics datasets to create increasingly constrained solution spaces of Salmonella Typhimurium metabolism during growth in both rich and virulence media. This integration proved to be informative and resulted in a requirement of additional active reactions (12 in each case) or metabolites (26 or 29, respectively). The addition of constraints from transcriptomics also impacted the allowed solution space, and the cellular metabolites with turnover fluxes that were necessarily altered by the change in conditions increased from 118 to 271 of 1397. Availability: GIMMME has been implemented in Python and requires a COBRApy 0.2.x. The algorithm and sample data described here are freely available at: http://opencobra.sourceforge.net/

  6. Cellular distribution of AMPA receptor subunits and mGlu5 following acute and repeated administration of morphine or methamphetamine.

    PubMed

    Herrold, Amy A; Persons, Amanda L; Napier, T Celeste

    2013-08-01

    Ionotropic AMPA receptors (AMPAR) and metabotropic glutamate group I subtype 5 receptors (mGlu5) mediate neuronal and behavioral effects of abused drugs. mGlu5 stimulation increases expression of striatal-enriched tyrosine phosphatase isoform 61 (STEP61 ) which internalizes AMPARs. We determined the rat brain profile of these proteins using two different classes of abused drugs, opiates, and stimulants. STEP61 levels, and cellular distribution/expression of AMPAR subunits (GluA1, GluA2) and mGlu5, were evaluated via a protein cross-linking assay in medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and ventral pallidum (VP) harvested 1 day after acute, or fourteen days after repeated morphine (8 mg/kg) or methamphetamine (1 mg/kg) (treatments producing behavioral sensitization). Acute morphine decreased GluA1 and GluA2 surface expression in mPFC and GluA1 in NAc. Fourteen days after repeated morphine or methamphetamine, mGlu5 surface expression increased in VP. In mPFC, mGlu5 were unaltered; however, after methamphetamine, STEP61 levels decreased and GluA2 surface expression increased. Pre-treatment with a mGlu5-selective negative allosteric modulator, blocked methamphetamine-induced behavioral sensitization and changes in mPFC GluA2 and STEP61 . These data reveal (i) region-specific distinctions in glutamate receptor trafficking between acute and repeated treatments of morphine and methamphetamine, and (ii) that mGlu5 is necessary for methamphetamine-induced alterations in mPFC GluA2 and STEP61 .

  7. Spatial association of apoptosis-related gene expression and cellular death in clinical neuroblastoma.

    PubMed Central

    Hoehner, J. C.; Gestblom, C.; Olsen, L.; PÃ¥hlman, S.

    1997-01-01

    Several unique features of neuroblastoma (NB), including the capacity for spontaneous regression and maturation to benign pathology, suggest that genes that regulate cellular proliferation, survival and differentiation may be involved in directing clinical tumour aggressiveness. The in situ expression of Bcl-2, Rb, p21, p53 and Bax proteins, as well as the proliferation marker proliferating cell nuclear antigen (PCNA) were examined immunocytochemically in a selection of 38 stage- and outcome-identified NB tumours. Apoptotic cells were identified morphologically and by a DNA fragmentation labelling technique (TUNEL). Although the tumour cell density of Bcl-2, p53, Bax, PCNA and TUNEL positivity correlated with patient survival, a spatially organized expression pattern was further recognized in stroma-poor differentiating tumours. Immature tumour cells adjacent to thin fibrovascular stroma are proliferating, as evidenced by PCNA positivity, and often express Bcl-2. At increasing distance from this fibrovascular stroma, intermediately differentiated tumour cells express Rb, while with more advanced differentiation, proliferation ceases and Bcl-2 immunoreactivity is lost. The most differentiated tumour cells, which often express p53, and occasionally p21 and Bax, lie adjacent to TUNEL-positive, morphologically apoptotic cells. This spatial organization in favourable outcome NB tumours suggests that physiological regulation of differentiation and apoptosis may be involved in tumour regression. Images Figure 2 PMID:9099968

  8. Cellular localization of retinoic acid receptor-gamma expression in normal and neoplastic skin.

    PubMed Central

    Finzi, E.; Blake, M. J.; Celano, P.; Skouge, J.; Diwan, R.

    1992-01-01

    Retinoids profoundly affect the normal growth and differentiation of epithelial tissues. Retinoic acid receptor-gamma (RAR-gamma) is a member of a family of retinoid receptors, and has been shown to be expressed almost exclusively in skin. However, little is known about the cellular localization of this receptor in human skin. The authors studied the expression of RAR-gamma in normal skin and human skin tumors by Northern blot analysis and in situ hybridization. RAR-gamma mRNA was detected in normal skin as well as in cultures of neonatal keratinocytes. Using an oligonucleotide specific for the RAR-gamma cDNA isoform 1 (RAR-gamma 1), RAR-gamma 1 mRNA was localized to all layers of the epidermis, the outer root sheath of hair follicles, follicular hair bulbs, eccrine and sebaceous glands. Basal cell carcinoma constitutively expressed gamma-1 mRNA and one of seven squamous cell carcinomas showed loss of gamma-1 mRNA expression, relative to adjacent epithelium. By contrast, normal melanocytic nevi and tumor-associated lymphocytes expressed little or no RAR-gamma mRNA. These results suggest that RAR-gamma 1 may play an important role in the maintenance and differentiation of normal epidermis and skin appendages. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:1318641

  9. Developmental and Spatial Expression of sir2 Genes in the Cellular Slime Mold Dictyostelium discoideum.

    PubMed

    Katayama, Takahiro; Yasukawa, Hiro

    2008-01-01

    The cellular slime mold Dictyostelium discoideum grows as unicellular free-living amoebae in the presence of nutrients. Upon starvation, the amoebae aggregate and form multicellular structures that each consist of a stalk and spores. D. discoideum encodes at least four proteins (Sir2A, Sir2B, Sir2C, and Sir2D) homologous to human SIRT. RT-PCR and WISH analyses showed that the genes for Sir2A, Sir2C, and Sir2D were expressed at high levels in growing cells but at decreased levels in developing cells, whereas the gene encoding Sir2B was expressed in the prestalk-cell region in the developmental phase.

  10. GIM3E: condition-specific models of cellular metabolism developed from metabolomics and expression data

    PubMed Central

    Schmidt, Brian J.; Ebrahim, Ali; Metz, Thomas O.; Adkins, Joshua N.; Palsson, Bernhard Ø.; Hyduke, Daniel R.

    2013-01-01

    Motivation: Genome-scale metabolic models have been used extensively to investigate alterations in cellular metabolism. The accuracy of these models to represent cellular metabolism in specific conditions has been improved by constraining the model with omics data sources. However, few practical methods for integrating metabolomics data with other omics data sources into genome-scale models of metabolism have been developed. Results: GIM3E (Gene Inactivation Moderated by Metabolism, Metabolomics and Expression) is an algorithm that enables the development of condition-specific models based on an objective function, transcriptomics and cellular metabolomics data. GIM3E establishes metabolite use requirements with metabolomics data, uses model-paired transcriptomics data to find experimentally supported solutions and provides calculations of the turnover (production/consumption) flux of metabolites. GIM3E was used to investigate the effects of integrating additional omics datasets to create increasingly constrained solution spaces of Salmonella Typhimurium metabolism during growth in both rich and virulence media. This integration proved to be informative and resulted in a requirement of additional active reactions (12 in each case) or metabolites (26 or 29, respectively). The addition of constraints from transcriptomics also impacted the allowed solution space, and the cellular metabolites with turnover fluxes that were necessarily altered by the change in conditions increased from 118 to 271 of 1397. Availability: GIM3E has been implemented in Python and requires a COBRApy 0.2.x. The algorithm and sample data described here are freely available at: http://opencobra.sourceforge.net/ Contacts: brianjamesschmidt@gmail.com or hyduke@usu.edu Supplementary information: Supplementary information is available at Bioinformatics online. PMID:23975765

  11. 75 FR 72686 - Express Mail Open and Distribute and Priority Mail Open and Distribute

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-26

    ... September 17, 2010, the Postal Service published a proposed rule in the Federal Register (75 FR 56920-56922... 111 Express Mail Open and Distribute and Priority Mail Open and Distribute AGENCY: Postal Service.\\TM..., or Label 257S, on all Express Mail Open and Distribute containers. The Postal Service is...

  12. High expression of cellular retinol binding protein-1 in lung adenocarcinoma is associated with poor prognosis

    PubMed Central

    Doldo, Elena; Costanza, Gaetana; Ferlosio, Amedeo; Pompeo, Eugenio; Agostinelli, Sara; Bellezza, Guido; Mazzaglia, Donatella; Giunta, Alessandro; Sidoni, Angelo; Orlandi, Augusto

    2015-01-01

    Purpose Adenocarcinoma, the most common non-small cell lung cancer is a leading cause of death worldwide, with a low overall survival (OS) despite increasing attempts to achieve an early diagnosis and accomplish surgical and multimodality treatment strategies. Cellular retinol binding protein-1 (CRBP-1) regulates retinol bioavailability and cell differentiation, but its role in lung cancerogenesis remains uncertain. Experimental design CRBP-1 expression, clinical outcome and other prognostic factors were investigated in 167 lung adenocarcinoma patients. CRBP-1 expression was evaluated by immunohistochemistry of tissue microarray sections, gene copy number analysis and tumor methylation specific PCR. Effects of CRBP-1 expression on proliferation/apoptosis gene array, protein and transcripts were investigated in transfected A549 lung adenocarcinoma cells. Results CRBP-1High expression was observed in 62.3% of adenocarcinomas and correlated with increased tumor grade and reduced OS as an independent prognostic factor. CRBP-1 gene copy gain also associated with tumor CRBP-1High status and dedifferentiation. CRBP-1-transfected (CRBP-1+) A549 grew more than CRBP-1− A549 cells. At >1μM concentrations, all trans-retinoic acid and retinol reduced viability more in CRBP-1+ than in CRBP-1− A549 cells. CRBP-1+ A549 cells showed up-regulated RARα/ RXRα and proliferative and transcriptional genes including pAkt, pEGFR, pErk1/2, creb1 and c-jun, whereas RARβ and p53 were strongly down-regulated; pAkt/pErk/ pEGFR inhibitors counteracted proliferative advantage and increased RARα/RXRα, c-jun and CD44 expression in CRBP-1+ A549 cells. Conclusion CRBP-1High expression in lung adenocarcinoma correlated with increased tumor grade and reduced OS, likely through increased Akt/Erk/EGFR-mediated cell proliferation and differentiation. CRBP-1High expression can be considered an additional marker of poor prognosis in lung adenocarcinoma patients. PMID:26807202

  13. Pteromalus puparum venom impairs host cellular immune responses by decreasing expression of its scavenger receptor gene.

    PubMed

    Fang, Qi; Wang, Lei; Zhu, Yangkeng; Stanley, David W; Chen, Xuexin; Hu, Cui; Ye, Gongyin

    2011-11-01

    Insect host/parasitoid interactions are co-evolved systems in which host defenses are balanced by parasitoid mechanisms to disable or hide from host immune effectors. Although there is a rich literature on these systems, parasitoid immune-disabling mechanisms have not been fully elucidated. Here we report on a newly discovered immune-disabling mechanism in the Pieris rapae/Pteromalus puparum host/parasitoid system. Because venom injections and parasitization suppresses host phagocytosis, we turned attention to the P. rapae scavenger receptor (Pr-SR), posing the hypothesis that P. puparum venom suppresses expression of the host Pr-SR gene. To test our hypothesis, we cloned a full-length cDNA of the Pr-SR. Multiple sequences alignment showed the deduced amino acid sequence of Pr-SR is similar to scavenger receptors of other lepidopterans. Bacterial and bead injections induced Pr-SR mRNA and protein expression, which peaked at 4h post-bead injection. Venom injection inhibited Pr-SR expression. Pr-SR was specifically expressed in granulocytes compared to plasmatocytes. We localized the Pr-SR protein in cytoplasm and cellular membrane, with no evidence of secretion into host plasma. Double-strand RNA designed to Pr-SR mRNA silenced expression of Pr-SR and significantly impaired host phagocytosis and encapsulation reactions. Venom injections similarly silenced Pr-SR expression during the first 8h post-treatment, after which the silencing effects gradually abated. We infer from these findings that one mechanism of impairing P. rapae hemocytic immune reactions is by silencing expression of Pr-SR.

  14. Modeling time variant distributions of cellular lifespans: increases in circulating reticulocyte lifespans following double phlebotomies in sheep

    PubMed Central

    Freise, Kevin J.; Widness, John A.; Schmidt, Robert L.

    2009-01-01

    Many pharmacodynamic (PD) models of cellular response assume a single and time invariant lifespan of all cells, despite the existence of a true underlying distribution of cellular lifespans and known changes in the lifespan distributions with time. To account for these features of cellular populations, a time variant cellular lifespan distribution PD model was formulated and theoretical aspects of modeling cellular populations presented. The model extends prior work assuming time variant “point distributions” of cellular lifespans (Freise et al. J Pharmacokinet Pharmacodyn 34:519–547, 2007) and models assuming a time invariant lifespan distribution (Krzyzanski et al. J Pharmacokinet Pharmacodyn 33:125–166, 2006). The formulated time variant lifespan distribution model was fitted to endogenous plasma erythropoietin (EPO), reticulocyte, and red blood cell (RBC) concentrations in sheep phlebotomized on two occasions, 8 days apart. The time variant circulating reticulocyte lifespan was modeled as a truncated and scaled Weibull distribution, with the location parameter of the distribution non-parametrically represented by an end constrained quadratic spline function. The formulated time variant lifespan distribution model was compared to the identical time invariant distribution, time variant “point distribution”, and time invariant “point distribution” cellular lifespan models. Parameters of the time variant lifespan distribution model were well estimated with low standard errors. The mean circulating reticulocyte lifespan was estimated at 0.304 days, which rapidly increased over 3-fold following the first phlebotomy to a maximum of 1.03 days (P = 0.009). On average, the percentage of erythrocytes being released as reticulocytes maximally increased an estimated two-fold following the phlebotomies. The primary features of immature RBC physiology were captured by the model and gave results consistent with other estimates in sheep and humans. The

  15. Proteomic analysis of cellular protein expression profiles in response to grass carp reovirus infection.

    PubMed

    Xu, Dan; Song, Lang; Wang, Hao; Xu, Xiaoyan; Wang, Tu; Lu, Liqun

    2015-06-01

    Grass carp (Ctenopharyngodon idella) hemorrhagic disease, caused by grass carp reovirus (GCRV), is emerging as a serious problem in grass carp aquaculture. To better understand the molecular responses to GCRV infection, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization tandem mass spectroscopy were performed to investigate altered proteins in C. idella kidney (CIK) cells. Differentially expressed proteins in mock infected CIK cells and GCRV-infected CIK cells were compared. Twenty-three differentially expressed spots were identified (22 upregulated spots and 1 downregulated spot), which included cytoskeleton proteins, macromolecular biosynthesis-associated proteins, stress response proteins, signal transduction proteins, energy metabolism-associated proteins and ubiquitin proteasome pathway-associated proteins. Moreover, 10 of the corresponding genes of the differentially expressed proteins were quantified by real-time reverse transcription polymerase chain reaction to examine their transcriptional profiles. The T cell internal antigen 1 (TIA1) and Ras-GTPase-activating SH3-domain-binding protein1 (G3BP1) of the cellular stress granule pathway from grass carp C. idella (designated as CiTIA1 and CiG3BP1) were upregulated and downregulated during GCRV infection, respectively. The full-length cDNA of CiTIA1 was 2753 bp, with an open reading frame (ORF) of 1155bp, which encodes a putative 385-amino acid protein. The 2271 bp full-length cDNA of CiG3BP1 comprised an ORF of 1455 bp that encodes a putative 485-amino acid protein. Phylogenetic analysis revealed that the complete ORFs of CiTIA1 and CiG3BP1 were very similar to zebrafish and well-characterized mammalian homologs. The expressions of the cellular proteins CiTIA1 and CiG3BP1 in response to GCRV were validated by western blotting, which indicated that the GCRV should unlink TIA1 aggregation and stress granule formation. This study provides useful information on the proteomic

  16. Unveiling the mechanism of uptake and sub-cellular distribution of cerium oxide nanoparticles†

    PubMed Central

    Singh, Sanjay; Kumar, Amit; Karakoti, Ajay; Seal, Sudipta; Self, William T.

    2011-01-01

    Cerium oxide nanoparticles (CNPs) have been recently studied for their potent superoxide scavenging properties in both cell and animal model systems. Data from these model systems have shown that exposure of cells to CNPs results in the protection against reactive oxygen species (ROS). Despite these exciting findings, very little is known regarding the uptake or subcellular distribution of these nanomaterials inside cells. In this study we utilized fluorophore (carboxyfluorescein) conjugated cerium oxide NPs (CCNPs) to study the mechanism of uptake and to elucidate the subcellular localization of CNPs using a keratinocyte model system. We observed rapid uptake (within 3 h) of CCNPs that was governed by energy-dependent, clathrin-mediated and caveolae-mediated endocytic pathways. We found CCNPs co-localized with mitochondria, lysosomes and endoplasmic reticulum as well as being abundant in the cytoplasm and the nucleus. Given the radical scavenging properties of cerium oxide and the widespread cellular disposition we observed, CNPs likely act as cellular antioxidants in multiple compartments of the cell imparting protection against a variety of oxidant injuries. PMID:20697616

  17. Active Degradation Explains the Distribution of Nuclear Proteins during Cellular Senescence

    PubMed Central

    Giampieri, Enrico; De Cecco, Marco; Remondini, Daniel; Sedivy, John; Castellani, Gastone

    2015-01-01

    The amount of cellular proteins is a crucial parameter that is known to vary between cells as a function of the replicative passages, and can be important during physiological aging. The process of protein degradation is known to be performed by a series of enzymatic reactions, ranging from an initial step of protein ubiquitination to their final fragmentation by the proteasome. In this paper we propose a stochastic dynamical model of nuclear proteins concentration resulting from a balance between a constant production of proteins and their degradation by a cooperative enzymatic reaction. The predictions of this model are compared with experimental data obtained by fluorescence measurements of the amount of nuclear proteins in murine tail fibroblast (MTF) undergoing cellular senescence. Our model provides a three-parameter stationary distribution that is in good agreement with the experimental data even during the transition to the senescent state, where the nuclear protein concentration changes abruptly. The estimation of three parameters (cooperativity, saturation threshold, and maximal velocity of the reaction), and their evolution during replicative passages shows that only the maximal velocity varies significantly. Based on our modeling we speculate the reduction of functionality of the protein degradation mechanism as a possible competitive inhibition of the proteasome. PMID:26115222

  18. Cellular distribution of the new growth factor pleiotrophin (HB-GAM) mRNA in developing and adult rat tissues.

    PubMed

    Vanderwinden, J M; Mailleux, P; Schiffmann, S N; Vanderhaeghen, J J

    1992-09-01

    Pleiotrophin (PTN), also known as HB-GAM, belongs to an emerging cytokine family unrelated to other growth factors. We report here the first comprehensive study using in situ hybridization on the cellular distribution of this new heparin-binding growth factor mRNA in rat tissues. PTN mRNA was developmentally expressed in many--but not all--neuroectodermal and mesodermal lineages, whilst no PTN mRNA was detected in endoderm, ectoderm and trophoblast. PTN mRNA was found in the nervous system throughout development, with a post-natal peak of expression. In the adult nervous system, significant expression persisted in hippocampal CA1 pyramidal neurons and in cortical neurons, but also in different non-neuronal cells types in various locations (olfactory nerve, cerebellar astrocytes, pituicytes, Schwann cells surrounding the neurons in sensory ganglia). PTN mRNA was also found during development in the mesenchyme of lung, gut, kidney and reproductive tract, in bone and cartilage progenitors, in dental pulp, in myoblasts, and in several other sites. Expression was differently regulated in each location, but usually faded around birth. In the adult, PTN mRNA was still present in the meninges, the iris, the Leydig cells of the testis and in the uterus. PTN mRNA was also strongly expressed in the basal layers of the tongue epithelium, which is the only epithelium and ectodermal derivative to express PTN mRNA, and this only after birth. PTN is known to be a growth factor for perinatal brain neurons and a mitogen for fibroblasts in vitro. Recently, trophic effects on epithelial cells and a role as a tumour growth factor have been reported. The mechanisms of regulation and the functions of PTN are however still uncertain. Its expression pattern during development suggests important roles in growth and differentiation. Moreover, the presence of PTN mRNA in several adult tissues and the up-regulation of PTN mRNA expression in the gravid uterus indicate that PTN also has

  19. Immunohistochemical expression of EGFR in oral leukoplakia: Association with clinicopathological features and cellular proliferation

    PubMed Central

    Ribeiro, Daniela C.; Gleber-Netto, Frederico O.; Sousa, Sílvia F.; Bernardes, Vanessa F.; Guimarães-Abreu, Mauro H.N.

    2012-01-01

    Objectives: to investigate the immunoexpression of epidermal growth factor receptor (EGFR) in a sample of oral leukoplakias (OL) and to determine the receptor’s association with dysplasia, tobacco consumption, lesion site, and proliferation rate. Although EGFR should be overexpressed in some oral leukoplakias, the factors that may interfere with this expression and the influence of this receptor on epithelial proliferation have yet to be investigated. Study Design: Samples of oral leukoplakias (48) and of normal oral epithelium (10) were immunohistologically examined for expression of EGFR. Immunohistochemistry for Ki-67, and p27 were also performed in leukoplakias. EGFR expression was associated with clinical and pathological features. Results: EGFR was positive in 62.5% of the leukoplakias and 50% of normal oral epithelium. The number of EGFR positive OL located in high-risk sites was significantly higher than EGFR positive OL located in low-risk sites. Most of the p27 negative leukoplakias were EGFR positive, and the p27 index in the parabasal layer was diminished in the presence of dysplasia. Positivity for EGFR was not associated with dysplasia, tobacco exposure, or Ki-67. Conclusion: EGFR is expressed in leukoplakia regardless of dysplasia, but EGFR positivity should be more frequent in lesions sited in areas of high cancer risk. The association between EGFR and p27 may represent an important mechanism in the control of cellular proliferation and malignant progression of oral epithelium and therefore warrants further investigation. Key words:Oral leukoplakia, EGFR, p27, Ki-67, epithelial dysplasia. PMID:22322523

  20. Cellular Distribution and Subcellular Localization of Molecular Components of Vesicular Transmitter Release in Horizontal Cells of Rabbit Retina

    PubMed Central

    HIRANO, ARLENE A.; BRANDSTÄTTER, JOHANN H.; BRECHA, NICHOLAS C.

    2010-01-01

    The mechanism underlying transmitter release from retinal horizontal cells is poorly understood. We investigated the possibility of vesicular transmitter release from mammalian horizontal cells by examining the expression of synaptic proteins that participate in vesicular transmitter release at chemical synapses. Using immunocytochemistry, we evaluated the cellular and subcellular distribution of complexin I/II, syntaxin-1, and synapsin I in rabbit retina. Strong labeling for complexin I/II, proteins that regulate a late step in vesicular transmitter release, was found in both synaptic layers of the retina, and in somata of A- and B-type horizontal cells, of γ-aminobutyric acid (GABA)- and glycinergic amacrine cells, and of ganglion cells. Immunoelectron microscopy demonstrated the presence of complexin I/II in horizontal cell processes postsynaptic to rod and cone ribbon synapses. Syntaxin-1, a core protein of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex known to bind to complexin, and synapsin I, a synaptic vesicle-associated protein involved in the Ca2+-dependent recruitment of synaptic vesicles for transmitter release, were also present in the horizontal cells and their processes at photoreceptor synapses. Photoreceptors and bipolar cells did not express any of these proteins at their axon terminals. The presence of complexin I/II, syntaxin-1, and synapsin I in rabbit horizontal cell processes and tips suggests that a vesicular mechanism may underlie transmitter release from mammalian horizontal cells. PMID:15912504

  1. Developmental, regional, and cellular expression of SFT/UbcH5A and DMT1 mRNA in brain.

    PubMed

    Knutson, Mitchell; Menzies, Sharon; Connor, James; Wessling-Resnick, Marianne

    2004-06-01

    Brain iron has marked developmental, regional, and cellular distribution patterns. To characterize better the potential mechanisms for iron transport into and within the brain, we have analyzed expression patterns of two factors: divalent metal transporter 1 (DMT1) and stimulator of Fe transport (SFT). DMT1 is known to participate in brain iron uptake although functional information is lacking. Even less clear is the possible role of SFT, which is related to a member of the ubiquitin-conjugating E2 family UbcH5A, but previous studies have found SFT/Ubc5Ha mRNA expressed abundantly in mouse brain. Like DMT1, SFT function has been implicated in transferrin and nontransferrin-bound iron uptake. Comparative Northern analysis indicates that SFT/UbcH5A mRNA levels are threefold higher in 3-day-old mice than at later ages, whereas levels of DMT1 mRNA do not change. In situ analysis of neonatal mouse brain reveals prominent SFT/UbcH5A mRNA expression in epithelial and ependymal cells in the choroid plexus and neurons of the olfactory bulb, hippocampus, and cortex. Adult mouse brain expresses SFT/UbcH5A mRNA mainly in white matter of the cerebellum and pons. Using a multiple tissue expression (MTE) array containing 20 different human brain regions, the highest levels of both SFT/UbcH5A and DMT1 mRNA are detected in the corpus callosum and cerebellum. The significantly elevated levels of SFT/UbcH5A mRNA in the neonatal mouse and its localization to choroid plexus, a major site of brain iron acquisition, suggest that this factor may contribute to the rapid rate of brain iron uptake that occurs in the early postnatal period.

  2. Self-assembly of patterned nanoparticles on cellular membranes: effect of charge distribution.

    PubMed

    Li, Ye; Zhang, Xianren; Cao, Dapeng

    2013-06-01

    Nanoparticle-assisted drug delivery has been emerging as an active research area. Achieving high drug loading is only one facet of drug delivery issues; it is also important to investigate the effect of surface charge distribution on self-assembly of nanoparticles on cellular membranes. By considering the electrostatic distribution of patterned nanoparticles, we used dissipative particle dynamics simulations to investigate the self-assembly of pattern charged nanoparticles with five different surface charged patterns. It is found that both surface charged pattern and nanoparticle size significantly affect the self-assembly of nanoparticles on cellular membranes. Results indicate that 1/2 pattern charged small nanoparticles can self-assemble into dendritic structures, while those with a 1/4 pattern self-assemble into clusters. As the nanoparticle size increases, 1/2 pattern charged medium nanoparticles can self-assemble into linear structures, while those with a 1/4 pattern self-assemble into clusters. For very large nanoparticles, both 1/2 pattern and 1/4 pattern charged nanoparticles self-assemble into flaky structures with different connections. By considering the effects of surface charged pattern and nanoparticle size on self-assembly, we found that nanoparticle self-assembly requires a minimum effective charged area. When the local charged area of nanoparticles is less than the threshold, surface charge cannot induce nanoparticle self-assembly; that is, the surface charged pattern of a nanoparticle would determine effectively the self-assembly structure. It is expected that this work will provide guidance for nanoparticle-assisted drug delivery.

  3. High expression of epithelial cellular adhesion molecule in peritoneal metastasis of gastric cancer.

    PubMed

    Imano, Motohiro; Itoh, Tatsuki; Satou, Takao; Yasuda, Atsushi; Nishiki, Kohei; Kato, Hiroaki; Shiraishi, Osamu; Peng, Ying-Feng; Shinkai, Masayuki; Tsubaki, Masahiro; Yasuda, Takushi; Imamoto, Haruhiko; Nishida, Shozo; Takeyama, Yoshifumi; Furkawa, Hiroshi; Okuno, Kiyokata; Shiozaki, Hitoshi

    2013-12-01

    Intraperitoneally administrated epithelial cellular adhesion molecule (EpCAM) monoclonal antibody is a therapeutic agent in patients with malignant effusion in several types of carcinoma. However, the role of EpCAM in peritoneal metastasis (PM) lesions and primary lesions of gastric cancer (GC) is still unclear. Therefore, in this study, we investigated EpCAM expression in GC patients with PM. We investigated the expression of EpCAM in 35PM lesions and 104 biopsy samples as primary lesions. Immunohistochemical staining was performed using the Ventana Benchmark XT (Roche Diagnostics) system. EpCAM expression was evaluated by calculating the total immunostaining score, which is the product of the proportion score and the intensity score. Overexpression was defined as a total score greater than 4. All PM specimens showed overexpression of EpCAM, and GC cells in both the surface layer and the deep layer of the PM showed a high expression of EpCAM. Meanwhile, in the biopsy sample, the expression of EpCAM ranged from none to strong. The EpCAM score results for PM specimens and biopsy samples were 11.0 ± 2.0 and 6.9 ± 3.9, respectively. The difference between the scores was statistically significant (P < 0.05). The intraperitoneally administrated EpCAM antibody might have a anti-cancer effect in PM lesions of GC. Additionally, it can be assumed that only GC cells which express a high level of EpCAM might metastasize to the peritoneum.

  4. A Celiac Cellular Phenotype, with Altered LPP Sub-Cellular Distribution, Is Inducible in Controls by the Toxic Gliadin Peptide P31-43

    PubMed Central

    Nanayakkara, Merlin; Kosova, Roberta; Lania, Giuliana; Sarno, Marco; Gaito, Alessandra; Galatola, Martina; Greco, Luigi; Cuomo, Marialaura; Troncone, Riccardo; Auricchio, Salvatore

    2013-01-01

    Celiac disease (CD) is a frequent inflammatory intestinal disease, with a genetic background, caused by gliadin-containing food. Undigested gliadin peptides P31-43 and P57-68 induce innate and adaptive T cell-mediated immune responses, respectively. Alterations in the cell shape and actin cytoskeleton are present in celiac enterocytes, and gliadin peptides induce actin rearrangements in both the CD mucosa and cell lines. Cell shape is maintained by the actin cytoskeleton and focal adhesions, sites of membrane attachment to the extracellular matrix. The locus of the human Lipoma Preferred Partner (LPP) gene was identified as strongly associated with CD using genome-wide association studies (GWAS). The LPP protein plays an important role in focal adhesion architecture and acts as a transcription factor in the nucleus. In this study, we examined the hypothesis that a constitutive alteration of the cell shape and the cytoskeleton, involving LPP, occurs in a cell compartment far from the main inflammation site in CD fibroblasts from skin explants. We analyzed the cell shape, actin organization, focal adhesion number, focal adhesion proteins, LPP sub-cellular distribution and adhesion to fibronectin of fibroblasts obtained from CD patients on a Gluten-Free Diet (GFD) and controls, without and with treatment with A-gliadin peptide P31-43. We observed a “CD cellular phenotype” in these fibroblasts, characterized by an altered cell shape and actin organization, increased number of focal adhesions, and altered intracellular LPP protein distribution. The treatment of controls fibroblasts with gliadin peptide P31-43 mimics the CD cellular phenotype regarding the cell shape, adhesion capacity, focal adhesion number and LPP sub-cellular distribution, suggesting a close association between these alterations and CD pathogenesis. PMID:24278174

  5. Cellular defense system gene expression profiling of human whole blood: opportunities to predict health benefits in response to diet.

    PubMed

    Drew, Janice E

    2012-07-01

    Diet is a critical factor in the maintenance of human cellular defense systems, immunity, inflammation, redox regulation, metabolism, and DNA repair that ensure optimal health and reduce disease risk. Assessment of dietary modulation of cellular defense systems in humans has been limited due to difficulties in accessing target tissues. Notably, peripheral blood gene expression profiles associated with nonhematologic disease are detectable. Coupled with recent innovations in gene expression technologies, gene expression profiling of human blood to determine predictive markers associated with health status and dietary modulation is now a feasible prospect for nutrition scientists. This review focuses on cellular defense system gene expression profiling of human whole blood and the opportunities this presents, using recent technological advances, to predict health status and benefits conferred by diet. PMID:22797985

  6. Peripheral blood leukocytes of cows with subclinical endometritis show an altered cellular composition and gene expression.

    PubMed

    Düvel, Anna; Maaß, Janine; Heppelmann, Maike; Hussen, Jamal; Koy, Mirja; Piechotta, Marion; Sandra, Olivier; Smith, David G E; Sheldon, Iain Martin; Dieuzy-Labaye, Isabelle; Zieger, Peter; Schuberth, Hans Joachim

    2014-04-15

    Subclinical endometritis (SCE) is an important postpartum disease in dairy cows, but conventional cytobrush diagnosis often gives imprecise results. The aim of this study was to analyze disease-associated changes in peripheral blood as potential diagnostic parameters. Cellular subpopulations of blood leukocytes from cows with or without SCE (45-55 days postpartum) were flow-cytometrically quantified. Gene expression of whole blood leukocytes was assessed by PAXgene analysis. Subclinical endometritis cows showed significantly higher number of blood mononuclear cells and neutrophils. Among mononuclear cells, numbers of B-cells, NK-cells, and CD172a-positive monocytes were significantly elevated. Compared with non-SCE cows, blood leukocytes of SCE cows significantly expressed higher copy numbers of CXCL8, TNF, and IL12. To test whether circulating plasma factors are responsible for these changes, leukocytes, polymorphonuclear cells, and monocyte subpopulations (classical, intermediate, nonclassical) of healthy cows were stimulated with plasma of SCE and non-SCE cows. Although gene expression of whole leukocytes and polymorphonuclear cells remained unaltered, plasma from SCE animals significantly elevated expressed messenger RNA copy numbers of CXCL8, CXCL1, and IL1B in intermediate monocytes. In conclusion, elevated number of selected mononuclear subpopulations in peripheral blood and enhanced expression of distinct genes encoding for inflammatory mediators in blood leukocytes reflect the subclinical uterine inflammatory process in cows. Whether the observed changes in the periphery of SCE cows are the consequence of the uterine inflammatory process, or whether they affect the pathogenesis of the disease is currently unknown. PMID:24560452

  7. Vanadium as a modulator of cellular regulatory cascades and oncogene expression.

    PubMed

    Stern, A; Yin, X; Tsang, S S; Davison, A; Moon, J

    1993-01-01

    Vanadium, a trace metal in the environment and in biological systems, influences the behavior of enzymes, mimics and regulates growth factor activity, is a potential mutagenic and carcinogenic agent, and regulates gene expression. The diverse biological actions of vanadium result from its capacity to function as an oxyanion, oxycation, or prooxidant. Vanadium is found in water, rocks, and soils in low concentration and in relatively high concentrations in coal and oil deposits. Vanadium compounds at much higher concentrations than are typically ingested are being considered in the treatment of diabetes mellitus. The actions of insulin and vanadium on the insulin receptor are similar, but the mechanisms are not identical. Vanadium modulates growth-factor-mediated signal transduction pathways. Vanadium promotes cell transformation and diminishes cell adhesion. Consistent with its mitogenic action and its capacity to mimic mitogenic growth factors, vanadium stimulates expression of protooncogenes. In particular, oxygen-derived active species are involved in the expression of the jun protooncogene in the presence of vanadium. The unique cellular activity of vanadium makes it a tool of unparalleled potential for studying mechanisms of cell growth, differentiation, and metabolism.

  8. Mechanistic links between cellular trade-offs, gene expression, and growth.

    PubMed

    Weiße, Andrea Y; Oyarzún, Diego A; Danos, Vincent; Swain, Peter S

    2015-03-01

    Intracellular processes rarely work in isolation but continually interact with the rest of the cell. In microbes, for example, we now know that gene expression across the whole genome typically changes with growth rate. The mechanisms driving such global regulation, however, are not well understood. Here we consider three trade-offs that, because of limitations in levels of cellular energy, free ribosomes, and proteins, are faced by all living cells and we construct a mechanistic model that comprises these trade-offs. Our model couples gene expression with growth rate and growth rate with a growing population of cells. We show that the model recovers Monod's law for the growth of microbes and two other empirical relationships connecting growth rate to the mass fraction of ribosomes. Further, we can explain growth-related effects in dosage compensation by paralogs and predict host-circuit interactions in synthetic biology. Simulating competitions between strains, we find that the regulation of metabolic pathways may have evolved not to match expression of enzymes to levels of extracellular substrates in changing environments but rather to balance a trade-off between exploiting one type of nutrient over another. Although coarse-grained, the trade-offs that the model embodies are fundamental, and, as such, our modeling framework has potentially wide application, including in both biotechnology and medicine.

  9. Quality controls in cellular immunotherapies: rapid assessment of clinical grade dendritic cells by gene expression profiling.

    PubMed

    Castiello, Luciano; Sabatino, Marianna; Zhao, Yingdong; Tumaini, Barbara; Ren, Jiaqiang; Ping, Jin; Wang, Ena; Wood, Lauren V; Marincola, Francesco M; Puri, Raj K; Stroncek, David F

    2013-02-01

    Cell-based immunotherapies are among the most promising approaches for developing effective and targeted immune response. However, their clinical usefulness and the evaluation of their efficacy rely heavily on complex quality control assessment. Therefore, rapid systematic methods are urgently needed for the in-depth characterization of relevant factors affecting newly developed cell product consistency and the identification of reliable markers for quality control. Using dendritic cells (DCs) as a model, we present a strategy to comprehensively characterize manufactured cellular products in order to define factors affecting their variability, quality and function. After generating clinical grade human monocyte-derived mature DCs (mDCs), we tested by gene expression profiling the degrees of product consistency related to the manufacturing process and variability due to intra- and interdonor factors, and how each factor affects single gene variation. Then, by calculating for each gene an index of variation we selected candidate markers for identity testing, and defined a set of genes that may be useful comparability and potency markers. Subsequently, we confirmed the observed gene index of variation in a larger clinical data set. In conclusion, using high-throughput technology we developed a method for the characterization of cellular therapies and the discovery of novel candidate quality assurance markers.

  10. Disruption of a cystine transporter downregulates expression of genes involved in sulfur regulation and cellular respiration.

    PubMed

    Simpkins, Jessica A; Rickel, Kirby E; Madeo, Marianna; Ahlers, Bethany A; Carlisle, Gabriel B; Nelson, Heidi J; Cardillo, Andrew L; Weber, Emily A; Vitiello, Peter F; Pearce, David A; Vitiello, Seasson P

    2016-01-01

    Cystine and cysteine are important molecules for pathways such as redox signaling and regulation, and thus identifying cellular deficits upon deletion of the Saccharomyces cerevisiae cystine transporter Ers1p allows for a further understanding of cystine homeostasis. Previous complementation studies using the human ortholog suggest yeast Ers1p is a cystine transporter. Human CTNS encodes the protein Cystinosin, a cystine transporter that is embedded in the lysosomal membrane and facilitates the export of cystine from the lysosome. When CTNS is mutated, cystine transport is disrupted, leading to cystine accumulation, the diagnostic hallmark of the lysosomal storage disorder cystinosis. Here, we provide biochemical evidence for Ers1p-dependent cystine transport. However, the accumulation of intracellular cystine is not observed when the ERS1 gene is deleted from ers1-Δ yeast, supporting the existence of modifier genes that provide a mechanism in ers1-Δ yeast that prevents or corrects cystine accumulation. Upon comparison of the transcriptomes of isogenic ERS1+ and ers1-Δ strains of S. cerevisiae by DNA microarray followed by targeted qPCR, sixteen genes were identified as being differentially expressed between the two genotypes. Genes that encode proteins functioning in sulfur regulation, cellular respiration, and general transport were enriched in our screen, demonstrating pleiotropic effects of ers1-Δ. These results give insight into yeast cystine regulation and the multiple, seemingly distal, pathways that involve proper cystine recycling. PMID:27142334

  11. Disruption of a cystine transporter downregulates expression of genes involved in sulfur regulation and cellular respiration

    PubMed Central

    Simpkins, Jessica A.; Rickel, Kirby E.; Madeo, Marianna; Ahlers, Bethany A.; Carlisle, Gabriel B.; Nelson, Heidi J.; Cardillo, Andrew L.; Weber, Emily A.; Vitiello, Peter F.; Pearce, David A.

    2016-01-01

    ABSTRACT Cystine and cysteine are important molecules for pathways such as redox signaling and regulation, and thus identifying cellular deficits upon deletion of the Saccharomyces cerevisiae cystine transporter Ers1p allows for a further understanding of cystine homeostasis. Previous complementation studies using the human ortholog suggest yeast Ers1p is a cystine transporter. Human CTNS encodes the protein Cystinosin, a cystine transporter that is embedded in the lysosomal membrane and facilitates the export of cystine from the lysosome. When CTNS is mutated, cystine transport is disrupted, leading to cystine accumulation, the diagnostic hallmark of the lysosomal storage disorder cystinosis. Here, we provide biochemical evidence for Ers1p-dependent cystine transport. However, the accumulation of intracellular cystine is not observed when the ERS1 gene is deleted from ers1-Δ yeast, supporting the existence of modifier genes that provide a mechanism in ers1-Δ yeast that prevents or corrects cystine accumulation. Upon comparison of the transcriptomes of isogenic ERS1+ and ers1-Δ strains of S. cerevisiae by DNA microarray followed by targeted qPCR, sixteen genes were identified as being differentially expressed between the two genotypes. Genes that encode proteins functioning in sulfur regulation, cellular respiration, and general transport were enriched in our screen, demonstrating pleiotropic effects of ers1-Δ. These results give insight into yeast cystine regulation and the multiple, seemingly distal, pathways that involve proper cystine recycling. PMID:27142334

  12. A scale-invariant cellular-automata model for distributed seismicity

    NASA Technical Reports Server (NTRS)

    Barriere, Benoit; Turcotte, Donald L.

    1991-01-01

    In the standard cellular-automata model for a fault an element of stress is randomly added to a grid of boxes until a box has four elements, these are then redistributed to the adjacent boxes on the grid. The redistribution can result in one or more of these boxes having four or more elements in which case further redistributions are required. On the average added elements are lost from the edges of the grid. The model is modified so that the boxes have a scale-invariant distribution of sizes. The objective is to model a scale-invariant distribution of fault sizes. When a redistribution from a box occurs it is equivalent to a characteristic earthquake on the fault. A redistribution from a small box (a foreshock) can trigger an instability in a large box (the main shock). A redistribution from a large box always triggers many instabilities in the smaller boxes (aftershocks). The frequency-size statistics for both main shocks and aftershocks satisfy the Gutenberg-Richter relation with b = 0.835 for main shocks and b = 0.635 for aftershocks. Model foreshocks occur 28 percent of the time.

  13. Diacylglycerol levels modulate the cellular distribution of the nicotinic acetylcholine receptor.

    PubMed

    Kamerbeek, Constanza B; Mateos, Melina V; Vallés, Ana S; Pediconi, María F; Barrantes, Francisco J; Borroni, Virginia

    2016-05-01

    Diacylglycerol (DAG), a second messenger involved in different cell signaling cascades, activates protein kinase C (PKC) and D (PKD), among other kinases. The present work analyzes the effects resulting from the alteration of DAG levels on neuronal and muscle nicotinic acetylcholine receptor (AChR) distribution. We employ CHO-K1/A5 cells, expressing adult muscle-type AChR in a stable manner, and hippocampal neurons, which endogenously express various subtypes of neuronal AChR. CHO-K1/A5 cells treated with dioctanoylglycerol (DOG) for different periods showed augmented AChR cell surface levels at short incubation times (30min-4h) whereas at longer times (18h) the AChR was shifted to intracellular compartments. Similarly, in cultured hippocampal neurons surface AChR levels increased as a result of DOG incubation for 4h. Inhibition of endogenous DAG catabolism produced changes in AChR distribution similar to those induced by DOG treatment. Specific enzyme inhibitors and Western blot assays revealed that DAGs exert their effect on AChR distribution through the modulation of the activity of classical PKC (cPKC), novel PKC (nPKC) and PKD activity.

  14. Metalloproteinases and tissue inhibitor of metalloproteinases in mesothelial cells. Cellular differentiation influences expression.

    PubMed

    Marshall, B C; Santana, A; Xu, Q P; Petersen, M J; Campbell, E J; Hoidal, J R; Welgus, H G

    1993-04-01

    Mesothelial cells play a critical role in the remodeling process that follows serosal injury. Although mesothelial cells are known to synthesize a variety of extracellular matrix components including types I, III, and IV collagens, their potential to participate in matrix degradation has not been explored. We now report that human pleural and peritoneal mesothelial cells express interstitial collagenase, 72- and 92-kD gelatinases (type IV collagenases), and the counterregulatory tissue inhibitor of metalloproteinases (TIMP). Our initial characterization of the mesothelial cell metalloenzymes and TIMP has revealed: (a) they are likely identical to corresponding molecules secreted by other human cells; (b) they are secreted rather than stored in an intracellular pool; (c) a primary site of regulation occurs at a pretranslational level; (d) phorbol myristate acetate, via activation of protein kinase C, upregulates expression of collagenase, 92-kD gelatinase, and TIMP, but has no effect on expression of 72-kD gelatinase; and (e) lipopolysaccharide fails to upregulate the biosynthesis of either metalloproteinases or TIMP. Of particular interest is the observation that the state of cellular differentiation has a striking influence on the expression of metalloenzymes and TIMP, such that epitheloid cells display a more matrix-degradative phenotype (increased 92-kD gelatinase and decreased TIMP) than their fibroblastoid counterparts. We speculate that mesothelial cells directly participate in the extracellular matrix turnover that follows serosal injury via elaboration of metalloproteinases and TIMP. Additionally, the reactive cuboidal mesothelium which is characteristic of the early response to serosal injury may manifest a matrix-degenerative phenotype favoring normal repair rather than fibrosis.

  15. A Stable HeLa Cell Line That Inducibly Expresses Poliovirus 2Apro: Effects on Cellular and Viral Gene Expression

    PubMed Central

    Barco, Angel; Feduchi, Elena; Carrasco, Luis

    2000-01-01

    A HeLa cell clone (2A7d) that inducibly expresses the gene for poliovirus protease 2A (2Apro) under the control of tetracycline has been obtained. Synthesis of 2Apro induces severe morphological changes in 2A7d cells. One day after tetracycline removal, cells round up and a few hours later die. Poliovirus 2Apro cleaves both forms of initiation factor eIF4G, causing extensive inhibition of capped-mRNA translation a few hours after protease induction. Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, a selective inhibitor of 2Apro, prevents both eIF4G cleavage and inhibition of translation but not cellular death. Expression of 2Apro still allows both the replication of poliovirus and the translation of mRNAs containing a picornavirus leader sequence, while vaccinia virus replication is drastically inhibited. Translation of transfected capped mRNA is blocked in 2A7d-On cells, while luciferase synthesis from a mRNA bearing a picornavirus internal ribosome entry site (IRES) sequence is enhanced by the presence of 2Apro. Moreover, synthesis of 2Apro in 2A7d cells complements the translational defect of a poliovirus 2Apro-defective variant. These results show that poliovirus 2Apro expression mimics some phenotypical characteristics of poliovirus-infected cells, such as cell rounding, inhibition of protein synthesis and enhancement of IRES-driven translation. This cell line constitutes a useful tool to further analyze 2Apro functions, to complement poliovirus 2Apro mutants, and to test antiviral compounds. PMID:10666269

  16. FAT/CD36 expression alone is insufficient to enhance cellular uptake of oleate

    SciTech Connect

    Eyre, Nicholas S.; Cleland, Leslie G.; Mayrhofer, Graham

    2008-06-06

    Fatty acid translocase (FAT/CD36) is one of several proteins implicated in receptor-mediated uptake of long-chain fatty acids (LCFAs). We have tested whether levels of FAT/CD36 correlate with cellular oleic acid import, using a Tet-Off inducible transfected CHO cell line. Consistent with our previous findings, FAT/CD36 was enriched in lipid raft-derived detergent-resistant membranes (DRMs) that also contained caveolin-1, the marker protein of caveolae. Furthermore in transfected cells, plasma membrane FAT/CD36 co-localized extensively with the lipid raft-enriched ganglioside GM1, and partially with a caveolin-1-EGFP fusion protein. Nevertheless, even at high levels of expression, FAT/CD36 did not affect uptake of oleic acid. We propose that the ability of FAT/CD36 to mediate enhanced uptake of LCFAs is dependent on co-expression of other proteins or factors that are lacking in CHO cells.

  17. Cellular localization of human p53 expressed in the yeast Saccharomyces cerevisiae: effect of NLSI deletion.

    PubMed

    Abdelmoula-Souissi, Salma; Delahodde, Agnès; Bolotin-Fukuhara, Monique; Gargouri, Ali; Mokdad-Gargouri, Raja

    2011-07-01

    The tumor suppressor p53 plays a central role in the regulation of cellular growth and apoptosis. In Saccharomyces cerevisiae, over-expression of the human wtp53 leads to growth inhibition and cell death on minimal medium. In the present work, we showed that deletion of the nuclear localization signal (NLSI) of p53 restores the yeast growth. In this heterologous context, the level of p53∆NLSI was low and the protein mainly located in the cytoplasm while the wtp53 was observed in both the cytoplasmic and nuclear compartments. Interestingly, the wtp53 protein was observed in the mitochondria, whereas the p53∆NLSI protein failed to localize in mitochondria. Moreover, mitochondrial morphology defect and release of cytochrome c in the cytosol were noticed only in the yeast strain expressing the wtp53. In conclusion, our results provide evidence that the human wtp53 is active in S. cerevisiae probably through dependent and independent transcriptional mechanisms leading to cell death. The deletion of the NLSI sequence decreases p53 nuclear translocation as well as its mitochondrial localization and consequently its effect on yeast growth.

  18. Irradiation with heavy-ion particles changes the cellular distribution of human histone acetyltranferase HAT1

    SciTech Connect

    Lebel, E.A.; Tafrov, S.; Boukamp, P.

    2010-06-01

    Hat1 was the first histone acetyltransferase identified, however its biological function is still unclear. In this report, we show that the human Hat1 has two isoforms. Isoform a has 418 amino acids (aa) and is localized exclusively in the nuclear matrix of normal human keratinocytes (NHKs). Isoform b has 334 aa and is located in thecytoplasm, the nucleoplasm, attached to the chromatin and to the nuclear matrix. Immunohistochemical analyses revealed that the bulk of Hat1 is confined to the nucleus, with much lesser amounts in the cytoplasm. Cells undergoing mitotic division have an elevated amount of Hat1 compared to non-mitotic ones. NHKs exposed to hydrogen peroxide (H2O2) or to a beam of high mass and energy (HZE) ion particles expressed bright nuclear staining for Hat1, a phenotype that was not observed in NHKs exposed to &947;-rays. We established that the enhanced nuclear staining for Hat1 in response to these treatments is regulated by the PI3K and the MAPK signaling pathways. Our observations clearly implicate Hat1 in the cellular response assuring the survival of the treated cells.

  19. Distribution of the cellular prion protein in the central nervous system of the chicken.

    PubMed

    Atoji, Yasuro; Ishiguro, Naotaka

    2009-12-01

    The cellular prion protein (PrP), a cell membrane-bound glycoprotein mainly located in the dendrites and axons of the central nervous system (CNS), is responsible for transmissible spongiform encephalopathies in mammals. PrP genes are widely conserved in vertebrates. In birds, the presence of PrP mRNA has been confirmed in neurons of the chicken brain, but localization of the protein remains to be determined. In the present study, we demonstrated the regional distribution of PrP in the CNS of adult chickens by immunohistochemical staining with a monoclonal antibody that recognizes chicken PrP 161-164. Immunoreactivity was observed in the neuropil, but not in neuronal somata or glial cells. It was preferentially intense in the olfactory bulb, the dorsal thalamus, the hypothalamus, and most regions of the telencephalon. Immunostaining became less intense toward the brainstem, but many nuclei were immunoreactive. Among brainstem nuclei, moderate immunostaining was observed in the nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve, and substantia gelatinosa Rolandi. The cerebellar cortex was devoid of PrP immunoreactivity. The dorsal horn in the spinal cord was strongly immunoreactive. In situ hybridization with two probes of the C-terminal portion demonstrated localization of PrP mRNA in neurons of the brain and spinal cord. These findings suggest that PrP in the chicken CNS is localized in the dendrites and axons of neurons and that it is associated with certain sensory systems.

  20. Preparation of bufalin-loaded pluronic polyetherimide nanoparticles, cellular uptake, distribution, and effect on colorectal cancer.

    PubMed

    Hu, Qiang; Liang, Bo; Sun, Ying; Guo, Xiao-Ling; Bao, Yi-Jie; Xie, Dong-Hao; Zhou, Ming; Duan, You-Rong; Yin, Pei-Hao; Peng, Zhi-Hai

    2014-01-01

    A large number of studies have shown that bufalin can have a significant antitumor effect in a variety of tumors. However, because of toxicity, insolubility in water, fast metabolism, short half-life, and other shortcomings, its application is limited in cancer therapy. In this study, we explored the anti-metastatic role of bufalin-loaded pluronic polyetherimide nanoparticles on HCT116 colon cancer-bearing mice. Nanoparticle size, shape, drug loading, encapsulation efficiency, and in vitro drug release were studied. Also, cellular uptake of nanoparticles, in vivo tumor targeting, and tumor metastasis were studied. The nanoparticles had a particle size of about 60 nm and an encapsulation efficiency of 75.71%, by weight. The in vitro release data showed that free bufalin was released faster than bufalin-loaded pluronic polyetherimide nanoparticles, and almost 80% of free bufalin was released after 32 hours. Nanoparticles had an even size distribution, were stable, and had a slow release and a tumor-targeting effect. Bufalin-loaded pluronic polyetherimide nanoparticles can significantly inhibit the growth and metastasis of colorectal cancer.

  1. Distribution of elements in individual blood cells in metabolic disorders at the cellular level

    NASA Astrophysics Data System (ADS)

    Johansson, Erland; Lindh, Ulf

    1985-08-01

    In comparison with controls neutrophil granulocytes from Rheumatoid arthritis (RA), Infantile Neuronal Ceroid Lipofuscinosis (INCL), Chronic Lymphatic Leukemia (L) and Aplastic Anemia (AA) displayed significant alterations in essential and non-essential elements which might be interpreted as fingerprints of these deseases. The neutrophils from RA patients displayed alterations in the concentrations of iron, calcium, strontium, manganese, zinc and copper. INCL displayed alterations in the concentrations of iron and copper but in the INCL disease the iron concentration was about 2 times higher than in RA. In leukemia, aluminium was observed but not in the controls (< 0.5 μg/ g). The zinc concentration was lowered in leukemia. Aplastic anemia displayed alterations in zirconium, arsenic, molybdenum, iron and zinc. The platelets from RA, INCL, L and AA patients also displayed alterations in the elemental profiles. The platelets from AA patients displayed a unique elemental distribution of arsenic, zirconium and molybdenum. The elemental profiles of the thrombocytes and neutrophils might be used as a complement in the diagnosis of the examined diseases and in therapy the elemental profile might be used to monitor drugs at the cellular level.

  2. In silico analyses of dystrophin Dp40 cellular distribution, nuclear export signals and structure modeling

    PubMed Central

    Martínez-Herrera, Alejandro; Aragón, Jorge; Bermúdez-Cruz, Rosa Ma.; Bazán, Ma. Luisa; Soid-Raggi, Gabriela; Ceja, Víctor; Santos Coy-Arechavaleta, Andrea; Alemán, Víctor; Depardón, Francisco; Montañez, Cecilia

    2015-01-01

    Dystrophin Dp40 is the shortest protein encoded by the DMD (Duchenne muscular dystrophy) gene. This protein is unique since it lacks the C-terminal end of dystrophins. In this data article, we describe the subcellular localization, nuclear export signals and the three-dimensional structure modeling of putative Dp40 proteins using bioinformatics tools. The Dp40 wild type protein was predicted as a cytoplasmic protein while the Dp40n4 was predicted to be nuclear. Changes L93P and L170P are involved in the nuclear localization of Dp40n4 protein. A close analysis of Dp40 protein scored that amino acids 93LEQEHNNLV101 and 168LLLHDSIQI176 could function as NES sequences and the scores are lost in Dp40n4. In addition, the changes L93/170P modify the tertiary structure of putative Dp40 mutants. The analysis showed that changes of residues 93 and 170 from leucine to proline allow the nuclear localization of Dp40 proteins. The data described here are related to the research article entitled “EF-hand domains are involved in the differential cellular distribution of dystrophin Dp40” (J. Aragón et al. Neurosci. Lett. 600 (2015) 115–120) [1]. PMID:26217814

  3. Functional domains and sub-cellular distribution of the Hedgehog transducing protein Smoothened in Drosophila.

    PubMed

    Nakano, Y; Nystedt, S; Shivdasani, A A; Strutt, H; Thomas, C; Ingham, P W

    2004-06-01

    The Hedgehog signalling pathway is deployed repeatedly during normal animal development and its inappropriate activity is associated with various tumours in human. The serpentine protein Smoothened (Smo) is essential for cells to respond to the Hedeghog (Hh) signal; oncogenic forms of Smo have been isolated from human basal cell carcinomas. Despite similarities with ligand binding G-protein coupled receptors, the molecular basis of Smo activity and its regulation remains unclear. In non-responding cells, Smo is suppressed by the activity of another multipass membrane spanning protein Ptc, which acts as the Hh receptor. In Drosophila, binding of Hh to Ptc has been shown to cause an accumulation of phosphorylated Smo protein and a concomitant stabilisation of the activated form of the Ci transcription factor. Here, we identify domains essential for Smo activity and investigate the sub-cellular distribution of the wild type protein in vivo. We find that deletion of the amino terminus and the juxtamembrane region of the carboxy terminus of the protein result in the loss of normal Smo activity. Using Green Fluorescent Protein (GFP) and horseradish peroxidase fusion proteins we show that Smo accumulates in the plasma membrane of cells in which Ptc activity is abrogated by Hh but is targeted to the degradative pathway in cells where Ptc is active. We further demonstrate that Smo accumulation is likely to be a cause, rather than a consequence, of Hh signal transduction.

  4. Cellular distribution of copper to superoxide dismutase involves scaffolding by membranes

    PubMed Central

    Pope, Christopher R.; De Feo, Christopher J.; Unger, Vinzenz M.

    2013-01-01

    Efficient delivery of copper ions to specific intracellular targets requires copper chaperones that acquire metal cargo through unknown mechanisms. Here we demonstrate that the human and yeast copper chaperones (CCS) for superoxide dismutase 1 (SOD1), long thought to exclusively reside in the cytosol and mitochondrial intermembrane space, can engage negatively charged bilayers through a positively charged lipid-binding interface. The significance of this membrane-binding interface is established through SOD1 activity and genetic complementation studies in Saccharomyces cerevisiae, showing that recruitment of CCS to the membrane is required for activation of SOD1. Moreover, we show that a CCS:SOD1 complex binds to bilayers in vitro and that CCS can interact with human high affinity copper transporter 1. Shifting current paradigms, we propose that CCS-dependent copper acquisition and distribution largely occur at membrane interfaces and that this emerging role of the bilayer may reflect a general mechanistic aspect of cellular transition metal ion acquisition. PMID:24297923

  5. Skeletal Muscle Cellularity and Glycogen Distribution in the Hypermuscular Compact Mice

    PubMed Central

    Kocsis, T.; Baán, J.; Müller, G.; Mendler, L.; Dux, L.

    2014-01-01

    The TGF-beta member myostatin acts as a negative regulator of skeletal muscle mass. The Compact mice were selected for high protein content and hypermuscularity, and carry a naturally occurring 12-bp deletion in the propeptide region of the myostatin precursor. We aimed to investigate the cellular characteristics and the glycogen distribution of the Compact tibialis anterior (TA) muscle by quantitative histochemistry and spectrophotometry. We have found that the deficiency in myostatin resulted in significantly increased weight of the investigated hindlimb muscles compared to wild type. Although the average glycogen content of the individual fibers kept unchanged, the total amount of glycogen in the Compact TA muscle increased two-fold, which can be explained by the presence of more fibers in Compact compared to wild type muscle. Moreover, the ratio of the most glycolytic IIB fibers significantly increased in the Compact TA muscle, of which glycogen content was the highest among the fast fibers. In summary, myostatin deficiency caused elevated amount of glycogen in the TA muscle but did not increase the glycogen content of the individual fibers despite the marked glycolytic shift observed in Compact mice. PMID:25308840

  6. Oscillations in probability distributions for stochastic gene expression

    SciTech Connect

    Petrosyan, K. G. Hu, Chin-Kun

    2014-05-28

    The phenomenon of oscillations in probability distribution functions of number of components is found for a model of stochastic gene expression. It takes place in cases of low levels of molecules or strong intracellular noise. The oscillations distinguish between more probable even and less probable odd number of particles. The even-odd symmetry restores as the number of molecules increases with the probability distribution function tending to Poisson distribution. We discuss the possibility of observation of the phenomenon in gene, protein, and mRNA expression experiments.

  7. Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes

    PubMed Central

    Nakase, Ikuhiko; Kobayashi, Nahoko Bailey; Takatani-Nakase, Tomoka; Yoshida, Tetsuhiko

    2015-01-01

    Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. Endocytosis has been reported as a major pathway for the cellular uptake of exosomes; however, the detailed mechanisms of their cellular uptake are still unknown. Here, we demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. We also demonstrate that oncogenic K-Ras-expressing MIA PaCa-2 cells exhibit intensive macropinocytosis that actively transports extracellular exosomes into the cells compared with wild-type K-Ras-expressing BxPC-3 cells. Furthermore, encapsulation of the ribosome-inactivating protein saporin with EGF in exosomes using our simple electroporation method produces superior cytotoxicity via the enhanced cellular uptake of exosomes. Our findings contribute to the biological, pharmaceutical, and medical research fields in terms of understanding the macropinocytosis-mediated cellular uptake of exosomes with applications for exosomal delivery systems. PMID:26036864

  8. Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes.

    PubMed

    Nakase, Ikuhiko; Kobayashi, Nahoko Bailey; Takatani-Nakase, Tomoka; Yoshida, Tetsuhiko

    2015-06-03

    Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. Endocytosis has been reported as a major pathway for the cellular uptake of exosomes; however, the detailed mechanisms of their cellular uptake are still unknown. Here, we demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. We also demonstrate that oncogenic K-Ras-expressing MIA PaCa-2 cells exhibit intensive macropinocytosis that actively transports extracellular exosomes into the cells compared with wild-type K-Ras-expressing BxPC-3 cells. Furthermore, encapsulation of the ribosome-inactivating protein saporin with EGF in exosomes using our simple electroporation method produces superior cytotoxicity via the enhanced cellular uptake of exosomes. Our findings contribute to the biological, pharmaceutical, and medical research fields in terms of understanding the macropinocytosis-mediated cellular uptake of exosomes with applications for exosomal delivery systems.

  9. Acute changes in cellular zinc alters zinc uptake rates prior to zinc transporter gene expression in Jurkat cells.

    PubMed

    Holland, Tai C; Killilea, David W; Shenvi, Swapna V; King, Janet C

    2015-12-01

    A coordinated network of zinc transporters and binding proteins tightly regulate cellular zinc levels. Canonical responses to zinc availability are thought to be mediated by changes in gene expression of key zinc transporters. We investigated the temporal relationships of actual zinc uptake with patterns of gene expression in membrane-bound zinc transporters in the human immortalized T lymphocyte Jurkat cell line. Cellular zinc levels were elevated or reduced with exogenous zinc sulfate or N,N,N',N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), respectively. Excess zinc resulted in a rapid 44 % decrease in the rate of zinc uptake within 10 min. After 120 min, the expression of metallothionein (positive control) increased, as well as the zinc exporter, ZnT1; however, the expression of zinc importers did not change during this time period. Zinc chelation with TPEN resulted in a rapid twofold increase in the rate of zinc uptake within 10 min. After 120 min, the expression of ZnT1 decreased, while again the expression of zinc importers did not change. Overall, zinc transporter gene expression kinetics did not match actual changes in cellular zinc uptake with exogenous zinc or TPEN treatments. This suggests zinc transporter regulation may be the initial response to changes in zinc within Jurkat cells.

  10. Salinity and temperature variations reflecting on cellular PCNA, IGF-I and II expressions, body growth and muscle cellularity of a freshwater fish larvae.

    PubMed

    Martins, Y S; Melo, R M C; Campos-Junior, P H A; Santos, J C E; Luz, R K; Rizzo, E; Bazzoli, N

    2014-06-01

    The present study assessed the influence of salinity and temperature on body growth and on muscle cellularity of Lophiosilurus alexaxdri vitelinic larvae. Slightly salted environments negatively influenced body growth of freshwater fish larvae and we observed that those conditions notably act as an environmental influencer on muscle growth and on local expression of hypertrophia and hypeplasia markers (IGFs and PCNA). Furthermore, we could see that salinity tolerance for NaCl 4gl(-)(1) diminishes with increasing temperature, evidenced by variation in body and muscle growth, and by irregular morphology of the lateral skeletal muscle of larvae. We saw that an increase of both PCNA and autocrine IGF-II are correlated to an increase in fibre numbers and fibre diameter as the temperature increases and salinity diminishes. On the other hand, autocrine IGF-I follows the opposite way to the other biological parameters assessed, increasing as salinity increases and temperature diminishes, showing that this protein did not participate in muscle cellularity, but participating in molecular/cellular repair. Therefore, slightly salted environments may provide adverse conditions that cause some obstacles to somatic growth of this species, suggesting some osmotic expenditure with a salinity increment.

  11. Gene Expression Profile Changes and Cellular Responses to Bleomycin-Induced DNA Damage in Human Fibroblast Cells in Space

    NASA Technical Reports Server (NTRS)

    Lu, Tao; Zhang, Ye; Kidane, Yared; Feiveson, Alan; Stodieck, Louis; Karouia, Fathi; Rohde, Larry; Wu, Honglu

    2016-01-01

    Living organisms are constantly exposed to space radiation that consists of energetic protons and other heavier charged particles. In addition, DNA in space can be damaged by toxic chemicals or reactive oxygen species generated due to increased levels of environmental and psychological stresses. Understanding the impact of spaceflight factors, microgravity in particular, on cellular responses to DNA damage affects the accuracy of the radiation risk assessment for astronauts and the mutation rate in microorganisms. Although possible synergistic effects of space radiation and microgravity have been investigated since the early days of the human space program, the published results were mostly conflicting and inconsistent. To investigate the effects of spaceflight on cellular responses to DNA damage, confluent human fibroblast cells (AG1522) flown on the International Space Station (ISS) were treated with bleomycin for three hours in the true microgravity environment, which induced DNA damages including double-strand breaks (DSB). Damages in the DNA were quantified by immunofluorescence staining for ?-H2AX, which showed similar percentages of different types of stained cells between flight and ground. However, there was a slight shift in the distribution of the ?-H2AX foci number in the flown cells with countable foci. Comparison of the cells in confluent and in exponential growth conditions indicated that the proliferation rate between flight and the ground may be responsible for such a shift. A microarray analysis of gene expressions in response to bleomycin treatment was also performed. Comparison of the responsive pathways between the flown and ground cells showed similar responses with the p53 network being the top upstream regulator. Similar responses at the RNA level between different gravity conditions were also observed with a PCR array analysis containing a set of genes involved in DNA damage signaling; with BBC3, CDKN1A, PCNA and PPM1D being significantly

  12. Comparative expression and tissue distribution analyses of astacin-like squid metalloprotease in squid and cuttlefish.

    PubMed

    Kanzawa, Nobuyuki; Ogawa, Takuya; Asakura, Masanori; Okiyama, Keisuke; Honda, Michiyo; Tsuchiya, Takahide

    2008-01-01

    Astacin-like squid metalloprotease (ALSM) is a member of the astacin family of metalloproteases. In the present study, we investigated the expression and tissue distribution of ALSM in bigfin reef squid (Sepioteuthis lessoniana) and golden cuttlefish (Sepia esculenta). Myosin heavy chain hydrolysis tests showed ALSM-I-like activity in both species. We isolated partial cDNA clones showing high sequence similarity to ALSM-I and -III, suggesting that ALSM is common to squid and cuttlefish. Phylogenetic analysis showed that ALSMs are classified into two clades: ALSM-I forms one clade, and ALSM-II and -III form the other. ALSM was expressed in several tissues in bigfin reef squid, though expression was confined to the liver in cuttlefish. ALSMs are distributed in digestive organs but not in mantle muscle of squid and cuttlefish. Immunofluorescence analysis further showed that cellular localization of ALSM is evident not only in hepatic cells but also in pancreatic cells of bigfin reef squid. Thus, ALSM is commonly expressed in squid and cuttlefish, but its expression levels and distribution are distinct.

  13. MNK1 expression increases during cellular senescence and modulates the subcellular localization of hnRNP A1

    SciTech Connect

    Ziaei, Samira; Shimada, Naoko; Kucharavy, Herman; Hubbard, Karen

    2012-03-10

    Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is an RNA-binding protein that modulates splice site usage, polyadenylation, and cleavage efficiency. This protein has also been implicated in mRNA stability and transport from the nucleus. We have previously demonstrated that hnRNP A1 had diminished protein levels and showed cytoplasmic accumulation in senescent human diploid fibroblasts. Furthermore, we have shown that inhibition of p38 MAPK, a key regulator of cellular senescence, elevated hnRNP A1 protein levels and inhibited hnRNP A1 cytoplasmic localization. In this study, we have explored the possible involvement of MNK1, one of the downstream effector of p38 MAPK, in the regulation of hnRNP A1. We have demonstrated that pharmacological inhibition of MNK1 by CGP 57380 decreased the phosphorylation levels of hnRNP A1 in young and senescent fibroblast cells and blocked the cytoplasmic accumulation of hnRNP A1 in senescent cells. In addition, MNK1 formed a complex with hnRNP A1 in vivo. The expression levels of MNK1, phospho-MNK1, and phospho-eIF4E proteins were found to be elevated in senescent cells. These data suggest that MNK1 regulates the phosphorylation and the subcellular distribution of hnRNP A1 and that MNK1 may play a role in the induction of senescence. -- Highlights: Black-Right-Pointing-Pointer MNK1 and not MAPKAPK2 phosphorylates hnRNP A1. Black-Right-Pointing-Pointer MNK1 has elevated levels in senescent cells, this has not been reported previously. Black-Right-Pointing-Pointer MNK1 activity induces cytoplasmic accumulation of hnRNP A1 in senescent cells. Black-Right-Pointing-Pointer Altered cytoplasmic localization of hnRNP A1 may alter gene expression patterns. Black-Right-Pointing-Pointer Our studies may increase our understanding of RNA metabolism during cellular aging.

  14. Distribution of population-averaged observables in stochastic gene expression.

    PubMed

    Bhattacharyya, Bhaswati; Kalay, Ziya

    2014-01-01

    Observation of phenotypic diversity in a population of genetically identical cells is often linked to the stochastic nature of chemical reactions involved in gene regulatory networks. We investigate the distribution of population-averaged gene expression levels as a function of population, or sample, size for several stochastic gene expression models to find out to what extent population-averaged quantities reflect the underlying mechanism of gene expression. We consider three basic gene regulation networks corresponding to transcription with and without gene state switching and translation. Using analytical expressions for the probability generating function of observables and large deviation theory, we calculate the distribution and first two moments of the population-averaged mRNA and protein levels as a function of model parameters, population size, and number of measurements contained in a data set. We validate our results using stochastic simulations also report exact results on the asymptotic properties of population averages which show qualitative differences among different models. PMID:24580265

  15. p16 Expression Is Lost in Severely Atypical Cellular Blue Nevi and Melanoma Compared to Conventional, Mildly, and Moderately Atypical Cellular Blue Nevi.

    PubMed

    Chang, Laura M; Cassarino, David S

    2014-01-01

    Background. Significant decreases in p16 expression have been shown to occur in melanoma compared to Spitz tumors, and loss of p16 staining has been found to correlate with melanoma tumor progression. However, comparison of p16 between atypical cellular blue nevi (CBN) and melanoma has not been reported previously. Methods. p16 immunohistochemical staining was evaluated in 14 atypical CBN, 8 conventional and atypical melanocytic nevi, and 16 melanomas, including 4 malignant CBN. p16 staining intensity was graded on a scale of 0-3 and the percentage of melanocytes stained with p16 was determined. Results. p16 staining was significantly higher in all CBN as a group when compared to melanomas (P = 0.001) and malignant CBN (P = 0.00008). Higher p16 expression was also seen in mildly (P = 0.0002) and moderately atypical (P = 0.02), but not severely atypical, CBN compared to melanomas. Conclusions. p16 immunohistochemical expression is higher in mildly and moderately atypical CBN compared to severely atypical CBN and melanomas. In conjunction with additional markers and histology, p16 staining may be useful in confirming the benign nature of these tumors, but is not useful in distinguishing severely atypical CBN from malignant cases, consistent with the overlapping histologic features between these tumors.

  16. miR-29 and miR-30 regulate B-Myb expression during cellular senescence

    PubMed Central

    Martinez, Ivan; Cazalla, Demian; Almstead, Laura L.; Steitz, Joan A.; DiMaio, Daniel

    2011-01-01

    Cellular senescence is a form of irreversible growth arrest and a major tumor suppressor mechanism. We show here that the miR-29 and miR-30 microRNA families are up-regulated during induced and replicative senescence and that up-regulation requires activation of the Rb pathway. Expression of a reporter construct containing the 3′UTR of the B-Myb oncogene is repressed during senescence, and repression is blocked by mutations in conserved miR-29 and miR-30 binding sites in the B-Myb 3′UTR. In proliferating cells, transfection of miR-29 and miR-30 represses a reporter construct containing the wild-type but not the mutant B-Myb 3′UTR, and repression of the mutant 3′UTR is reinstituted by compensatory mutations in miR-29 and miR-30 that restore binding to the mutant sites. miR-29 and miR-30 introduction also represses expression of endogenous B-Myb and inhibits cellular DNA synthesis. Finally, interference with miR-29 and miR-30 expression inhibits senescence. These findings demonstrate that miR-29 and miR-30 regulate B-Myb expression by binding to its 3′UTR and suggest that these microRNAs play an important role in Rb-driven cellular senescence. PMID:21187425

  17. Differential Control of Interleukin-6 mRNA Levels by Cellular Distribution of YB-1

    PubMed Central

    Kang, Sujin; Lee, Taeyun A.; Ra, Eun A.; Lee, Eunhye; Choi, Hyun jin; Lee, Sungwook; Park, Boyoun

    2014-01-01

    Cytokine production is essential for innate and adaptive immunity against microbial invaders and must be tightly controlled. Cytokine messenger RNA (mRNA) is in constant flux between the nucleus and the cytoplasm and in transcription, splicing, or decay; such processes must be tightly controlled. Here, we report a novel function of Y-box-binding protein 1 (YB-1) in modulating interleukin-6 (IL-6) mRNA levels in a cell type-specific manner. In lipopolysaccharide (LPS)-stimulated macrophages, YB-1 interacts with IL-6 mRNA and actively transports it to the extracellular space by YB-1-enriched vesicles, resulting in the proper maintenance of intracellular IL-6 mRNA levels. YB-1 secretion occurs in a cell type-specific manner. Whereas macrophages actively secret YB-1, dendritic cells maintain it predominantly in the cytoplasm even in response to LPS. Intracellular YB-1 has the distinct function of regulating IL-6 mRNA stability in dendritic cells. Moreover, because LPS differentially regulates the expression of histone deacetylase 6 (HDAC6) in macrophages and dendritic cells, this stimulus might control YB-1 acetylation differentially in both cell types. Taken together, these results suggest a unique feature of YB-1 in controlling intracellular IL-6 mRNA levels in a cell type-specific manner, thereby leading to functions that are dependent on the extracellular and intracellular distribution of YB-1. PMID:25398005

  18. Cellular Distribution and Functions of Wild-Type and Constitutively Activated Dictyostelium PakBV⃞

    PubMed Central

    de la Roche, Marc; Mahasneh, Amjad; Lee, Sheu-Fen; Rivero, Francisco; Côté, Graham P.

    2005-01-01

    Dictyostelium PakB, previously termed myosin I heavy chain kinase, is a member of the p21-activated kinase (PAK) family. Two-hybrid assays showed that PakB interacts with Dictyostelium Rac1a/b/c, RacA (a RhoBTB protein), RacB, RacC, and RacF1. Wild-type PakB displayed a cytosolic distribution with a modest enrichment at the leading edge of migrating cells and at macropinocytic and phagocytic cups, sites consistent with a role in activating myosin I. PakB fused at the N terminus to green fluorescent protein was proteolyzed in cells, resulting in removal of the catalytic domain. C-terminal truncated PakB and activated PakB lacking the p21-binding domain strongly localized to the cell cortex, to macropinocytic cups, to the posterior of migrating cells, and to the cleavage furrow of dividing cells. These data indicate that in its open, active state, the N terminus of PakB forms a tight association with cortical actin filaments. PakB-null cells displayed no significant behavioral defects, but cells expressing activated PakB were unable to complete cytokinesis when grown in suspension and exhibited increased rates of phagocytosis and pinocytosis. PMID:15509655

  19. Loss of Pnn expression results in mouse early embryonic lethality and cellular apoptosis through SRSF1-mediated alternative expression of Bcl-xS and ICAD.

    PubMed

    Leu, Steve; Lin, Yen-Ming; Wu, Chu-Han; Ouyang, Pin

    2012-07-01

    Pinin (Pnn), a serine/arginine-rich (SR)-related protein, has been shown to play multiple roles within eukaryotic cells including cell-cell adhesion, cell migration, regulation of gene transcription, mRNA export and alternative splicing. In this study, an attempt to generate mice homozygously deficient in Pnn failed because of early embryonic lethality. To evaluate the effects of loss of Pnn expression on cell survival, RNA interference experiments were performed in MCF-7 cells. Depletion of Pnn resulted in cellular apoptosis and nuclear condensation. In addition, nuclear speckles were disrupted, and expression levels of SR proteins were diminished. RT-PCR analysis showed that alternative splicing patterns of SRSF1 as well as of apoptosis-related genes Bcl-x and ICAD were altered, and expression levels of Bim isoforms were modulated in Pnn-depleted cells. Cellular apoptosis induced by Pnn depletion was rescued by overexpression of SRSF1, which also restored generation of Bcl-xL and functionless ICAD. Pnn expression is, therefore, essential for survival of mouse embryos and the breast carcinoma cell line MCF-7. Moreover, Pnn depletion, modulated by SRSF1, determines cellular apoptosis through activation of the expression of pro-apoptotic Bcl-xS transcripts. PMID:22454513

  20. Loss of Pnn expression results in mouse early embryonic lethality and cellular apoptosis through SRSF1-mediated alternative expression of Bcl-xS and ICAD.

    PubMed

    Leu, Steve; Lin, Yen-Ming; Wu, Chu-Han; Ouyang, Pin

    2012-07-01

    Pinin (Pnn), a serine/arginine-rich (SR)-related protein, has been shown to play multiple roles within eukaryotic cells including cell-cell adhesion, cell migration, regulation of gene transcription, mRNA export and alternative splicing. In this study, an attempt to generate mice homozygously deficient in Pnn failed because of early embryonic lethality. To evaluate the effects of loss of Pnn expression on cell survival, RNA interference experiments were performed in MCF-7 cells. Depletion of Pnn resulted in cellular apoptosis and nuclear condensation. In addition, nuclear speckles were disrupted, and expression levels of SR proteins were diminished. RT-PCR analysis showed that alternative splicing patterns of SRSF1 as well as of apoptosis-related genes Bcl-x and ICAD were altered, and expression levels of Bim isoforms were modulated in Pnn-depleted cells. Cellular apoptosis induced by Pnn depletion was rescued by overexpression of SRSF1, which also restored generation of Bcl-xL and functionless ICAD. Pnn expression is, therefore, essential for survival of mouse embryos and the breast carcinoma cell line MCF-7. Moreover, Pnn depletion, modulated by SRSF1, determines cellular apoptosis through activation of the expression of pro-apoptotic Bcl-xS transcripts.

  1. Distribution of dipeptide repeat proteins in cellular models and C9orf72 mutation cases suggests link to transcriptional silencing.

    PubMed

    Schludi, Martin H; May, Stephanie; Grässer, Friedrich A; Rentzsch, Kristin; Kremmer, Elisabeth; Küpper, Clemens; Klopstock, Thomas; Arzberger, Thomas; Edbauer, Dieter

    2015-10-01

    A massive expansion of a GGGGCC repeat upstream of the C9orf72 coding region is the most common known cause of amyotrophic lateral sclerosis and frontotemporal dementia. Despite its intronic localization and lack of a canonical start codon, both strands are translated into aggregating dipeptide repeat (DPR) proteins: poly-GA, poly-GP, poly-GR, poly-PR and poly-PA. To address conflicting findings on the predominant toxicity of the different DPR species in model systems, we compared the expression pattern of the DPR proteins in rat primary neurons and postmortem brain and spinal cord of C9orf72 mutation patients. Only poly-GA overexpression closely mimicked the p62-positive neuronal cytoplasmic inclusions commonly observed for all DPR proteins in patients. In contrast, overexpressed poly-GR and poly-PR formed nucleolar p62-negative inclusions. In patients, most of the less common neuronal intranuclear DPR inclusions were para-nucleolar and p62 positive. Neuronal nucleoli in C9orf72 cases showed normal size and morphology regardless of the presence of poly-GR and poly-PR inclusions arguing against widespread nucleolar stress, reported in cellular models. Colocalization of para-nucleolar DPR inclusions with heterochromatin and a marker of transcriptional repression (H3K9me2) indicates a link to gene transcription. In contrast, we detected numerous intranuclear DPR inclusions not associated with nucleolar structures in ependymal and subependymal cells. In patients, neuronal inclusions of poly-GR, poly-GP and the poly-GA interacting protein Unc119 were less abundant than poly-GA inclusions, but showed similar regional and subcellular distribution. Regardless of neurodegeneration, all inclusions were most abundant in neocortex, hippocampus and thalamus, with few inclusions in brain stem and spinal cord. In the granular cell layer of the cerebellum, poly-GA and Unc119 inclusions were significantly more abundant in cases with FTLD than in cases with MND and FTLD/MND. Poly

  2. Measuring Cellular-scale Nutrient Distribution in Algal Biofilms with Synchrotron Confocal Infrared Microspectroscopy

    SciTech Connect

    J Murdock; W Dodds; J Reffner; D Wetzel

    2011-12-31

    The microscope and infrared spectrometer are two of the most useful tools for the study of biological materials, and their combined analytical power far exceeds the sum of the two. Performing molecular spectroscopy through a microscope superimposes chemical information onto the physical microstructure obtained from the optical microscope when visible and infrared information are collected under the same conditions. The instrument developments that enable current infrared microspectroscopic studies began with the introduction of the first research-grade infrared microscope, patented in 1989 (1). By 1993, published reports using this method to determine macroalgae (seaweed) cell-wall composition appeared (2-4). Since these initial reports, the use of infrared microspectroscopy (IMS) in microalgal (single cells or groups of cells) research has grown. Primarily, cultured algae have been used to hone IMS methodology and evaluate its capabilities in algal research (5-8). Studies involving natural, mixed species assemblages, which can utilize the spatial resolution potential of this technique fully are rare (9-11). For instance, in a recent review of IMS microalgal ecological research (12), only 3 of the 29 peer-reviewed publications investigated natural algal assemblages. Both thermal and synchrotron infrared sources provide a resolution capable of measuring individual algae in mixed species assemblages, and each has its advantages. For example, thermal source IMS is more accessible, allowing more samples to be analyzed than synchrotron IMS. However, synchrotron IMS with confocal masking provides superior resolution, which can be critical in isolating small or contiguous cells. Algal ecology is the study of the interaction between algae and their environment. Infrared microspectroscopy addresses a major logistical problem in this field, obtaining species-specific cellular biochemical information from natural, mixed-species assemblages (11,12). Benthic (bottom

  3. Interaction of HSP20 with a viral RdRp changes its sub-cellular localization and distribution pattern in plants

    PubMed Central

    Li, Jing; Xiang, Cong-Ying; Yang, Jian; Chen, Jian-Ping; Zhang, Heng-Mu

    2015-01-01

    Small heat shock proteins (sHSPs) perform a fundamental role in protecting cells against a wide array of stresses but their biological function during viral infection remains unknown. Rice stripe virus (RSV) causes a severe disease of rice in Eastern Asia. OsHSP20 and its homologue (NbHSP20) were used as baits in yeast two-hybrid (YTH) assays to screen an RSV cDNA library and were found to interact with the viral RNA-dependent RNA polymerase (RdRp) of RSV. Interactions were confirmed by pull-down and BiFC assays. Further analysis showed that the N-terminus (residues 1–296) of the RdRp was crucial for the interaction between the HSP20s and viral RdRp and responsible for the alteration of the sub-cellular localization and distribution pattern of HSP20s in protoplasts of rice and epidermal cells of Nicotiana benthamiana. This is the first report that a plant virus or a viral protein alters the expression pattern or sub-cellular distribution of sHSPs. PMID:26359114

  4. SELDI-TOF analysis of glioblastoma cyst fluid is an approach for assessing cellular protein expression

    PubMed Central

    Hoelscher, Martin; Richter, Nina; Melle, Christian; von Eggeling, Ferdinand; Schaenzer, Anne; Nestler, Ulf

    2013-01-01

    Objectives: In about 10% of glioblastoma patients, preoperative MRI discloses the presence of tumor cysts. Whereas the impact of cystic appearance on prognosis has been discussed extensively, only little is known about the tumor cyst fluid. In this study, we tested the feasibility of the surface enhanced laser desorption ionization time of flight (SELDI-TOF) technique to detect cyst fluid proteins. Methods: Cyst fluid was collected from 21 glioblastoma patients for SELDI-TOF analysis and compared to control cerebrospinal fluids from 15 patients with spinal stenosis. Resulting protein peaks with significant differences between groups were further described, using the molecular weight in an internet search of protein databases and publications. Two potential cyst fluid proteins, basigin and ferritin light chain, were selected for immunohistological detection in the histologic slides of the patients, metallothionein (MT) served as negative control. Results: As supposed from the results of the SELDI-TOF analysis, basigin and ferritin were detected immunohistochemically in the cyst wall, whereas MT was more equally distributed between the cyst wall and the surrounding tumor tissue. Median survival time of the patients was 20 months (range 2 to 102 months) and correlated with age, but not with expression of the three proteins. Discussion: The SELDI-TOF approach reveals a number of proteins, potentially present in glioblastoma cyst fluid. Identification of these proteins in tumor cells may help understand the pathogenetic pathways and the prognostic value of cystic changes. PMID:24225180

  5. Characterization of a multiple endogenously expressed adenosine triphosphate-binding cassette transporters using nuclear and cellular membrane affinity chromatography columns.

    PubMed

    Habicht, K-L; Singh, N S; Khadeer, M A; Shimmo, R; Wainer, I W; Moaddel, R

    2014-04-25

    Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP-Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN-229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN-229)) and (CMAC(LN-229)), respectively. Pgp, MRP1 and BCRP transporters co-immobilized on both columns were characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs. 3.7μM), verapamil (0.6 vs. 0.7μM) and prazosin (0.099 vs. 0.033μM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of eight compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN-229) column and decreased it (-5%) on the NMAC(LN-229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences.

  6. Characterization of a multiple endogenously expressed Adenosine triphosphate-Binding Cassette transporters using nuclear and cellular membrane affinity chromatography columns

    PubMed Central

    Khadeer, M.A.; Shimmo, R.; Wainer, I.W.; Moaddel, R.

    2014-01-01

    Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN229)) and (CMAC(LN229)), respectively. Pgp, MRP1and BCRP transporters co-immobilized on both columns was characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs 3.7μM), verapamil (0.6 vs 0.7μM) and prazosin (0.099 vs 0.033μM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of 8 compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN229) column and decreased it (−5%) on the NMAC(LN229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences. PMID:24642394

  7. Epigallocatechin-3-gallate affects the growth of LNCaP cells via membrane fluidity and distribution of cellular zinc*

    PubMed Central

    Yang, Jun-guo; Yu, Hai-ning; Sun, Shi-li; Zhang, Lan-cui; He, Guo-qing; Das, Undurti N.; Ruan, Hui; Shen, Sheng-rong

    2009-01-01

    Objective: To evaluate effects of epigallocatechin-3-gallate (EGCG) on the viability, membrane properties, and zinc distribution, with and without the presence of Zn2+, in human prostate carcinoma LNCaP cells. Methods: We examined changes in cellular morphology and membrane fluidity of LNCaP cells, distribution of cellular zinc, and the incorporated portion of EGCG after treatments with EGCG, Zn2+, and EGCG+Zn2+. Results: We observed an alteration in cellular morphology and a decrease in membrane fluidity of LNCaP cells after treatment with EGCG or Zn2+. The proportion of EGCG incorporated into liposomes treated with the mixture of EGCG and Zn2+ at the ratio of 1:1 was 90.57%, which was significantly higher than that treated with EGCG alone (30.33%). Electron spin resonance (ESR) studies and determination of fatty acids showed that the effects of EGCG on the membrane fluidity of LNCaP were decreased by Zn2+. EGCG accelerated the accumulation of zinc in the mitochondria and cytosol as observed by atomic absorption spectrometer. Conclusion: These results show that EGCG interacted with cell membrane, decreased the membrane fluidity of LNCaP cells, and accelerated zinc accumulation in the mitochondria and cytosol, which could be the mechanism by which EGCG inhibits proliferation of LNCaP cells. In addition, high concentrations of Zn2+ could attenuate the actions elicited by EGCG. PMID:19489106

  8. 75 FR 14076 - Express Mail Open and Distribute and Priority Mail Open and Distribute Changes and Updates

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-24

    ..., the Postal Service published a Federal Register proposed rule (75 FR 4741-4742) inviting comments on a... 111 Express Mail Open and Distribute and Priority Mail Open and Distribute Changes and Updates AGENCY... Express Mail Open and Distribute and Priority Mail Open and Distribute to improve efficiencies...

  9. [Cellular distribution and behavior of metallothionein in mammalian cells following exposure to silver nanoparticles and silver ions].

    PubMed

    Miyayama, Takamitsu; Arai, Yuta; Suzuki, Noriyuki; Hirano, Seishiro

    2014-01-01

    Silver nanoparticles (AgNPs) are commercially used mainly as antibacterial reagents in wound dressing and deodorant powders. However, the mechanisms underlying Ag toxicity in mammals are not fully understood. In the present study, we assessed cellular distribution and toxicity of AgNPs and AgNO3 in mouse macrophage cell line (J774.1) and those of AgNO3 in human bronchial epithelial cell line (BEAS-2B) focusing on behavior of metallothionein (MT). J774.1 cells were exposed to 0-100 μg Ag/mL AgNPs or AgNO3 and BEAS-2B cells were exposed to 0-100 μM AgNO3 for 24 h. The cytotoxicity was assayed by a modified MTT method. The cellular concentration and distribution of Ag were evaluated by inductively coupled plasma-mass spectorometry (ICP-MS) and laser scanning microscopy. Distribution of Ag to MT and other proteins was determined using HPLC-ICP-MS. Most AgNPs were found in lysosomes in J774.1 at 3 h after post exposure. Ag was distributed to high molecular weight proteins in AgNPs-exposed cells, while most Ag was bound to MT in AgNO3-exposed cells. In AgNO3-exposed BEAS-2B cells cellular Ag concentration and Ag-bound MT (Ag-MT) were sharply increased up to 3 h and then decreased. ROS production appeared to cause relocation of MT-bound Ag to mitochondria, which evoked inhibition of electron transport chain. AgNPs were sequestered by high-molecular weight proteins rather than MT, probably because they were taken up by lysosomes before induction of MT.

  10. Human immunodeficiency virus type 1 Tat increases the expression of cleavage and polyadenylation specificity factor 73-kilodalton subunit modulating cellular and viral expression.

    PubMed

    Calzado, Marco A; Sancho, Rocío; Muñoz, Eduardo

    2004-07-01

    The human immunodeficiency virus type 1 (HIV-1) Tat protein, which is essential for HIV gene expression and viral replication, is known to mediate pleiotropic effects on various cell functions. For instance, Tat protein is able to regulate the rate of transcription of host cellular genes and to interact with the signaling machinery, leading to cellular dysfunction. To study the effect that HIV-1 Tat exerts on the host cell, we identified several genes that were up- or down-regulated in tat-expressing cell lines by using the differential display method. HIV-1 Tat specifically increases the expression of the cleavage and polyadenylation specificity factor (CPSF) 73-kDa subunit (CPSF3) without affecting the expression of the 160- and 100-kDa subunits of the CPSF complex. This complex comprises four subunits and has a key function in the 3'-end processing of pre-mRNAs by a coordinated interaction with other factors. CPSF3 overexpression experiments and knockdown of the endogenous CPSF3 by mRNA interference have shown that this subunit of the complex is an important regulatory protein for both viral and cellular gene expression. In addition to the known CPSF3 function in RNA polyadenylation, we also present evidence that this protein exerts transcriptional activities by repressing the mdm2 gene promoter. Thus, HIV-1-Tat up-regulation of CPSF3 could represent a novel mechanism by which this virus increases mRNA processing, causing an increase in both cell and viral gene expression.

  11. Human Immunodeficiency Virus Type 1 Tat Increases the Expression of Cleavage and Polyadenylation Specificity Factor 73-Kilodalton Subunit Modulating Cellular and Viral Expression

    PubMed Central

    Calzado, Marco A.; Sancho, Rocío; Muñoz, Eduardo

    2004-01-01

    The human immunodeficiency virus type 1 (HIV-1) Tat protein, which is essential for HIV gene expression and viral replication, is known to mediate pleiotropic effects on various cell functions. For instance, Tat protein is able to regulate the rate of transcription of host cellular genes and to interact with the signaling machinery, leading to cellular dysfunction. To study the effect that HIV-1 Tat exerts on the host cell, we identified several genes that were up- or down-regulated in tat-expressing cell lines by using the differential display method. HIV-1 Tat specifically increases the expression of the cleavage and polyadenylation specificity factor (CPSF) 73-kDa subunit (CPSF3) without affecting the expression of the 160- and 100-kDa subunits of the CPSF complex. This complex comprises four subunits and has a key function in the 3′-end processing of pre-mRNAs by a coordinated interaction with other factors. CPSF3 overexpression experiments and knockdown of the endogenous CPSF3 by mRNA interference have shown that this subunit of the complex is an important regulatory protein for both viral and cellular gene expression. In addition to the known CPSF3 function in RNA polyadenylation, we also present evidence that this protein exerts transcriptional activities by repressing the mdm2 gene promoter. Thus, HIV-1-Tat up-regulation of CPSF3 could represent a novel mechanism by which this virus increases mRNA processing, causing an increase in both cell and viral gene expression. PMID:15194760

  12. The anti‑dengue virus properties of statins may be associated with alterations in the cellular antiviral profile expression.

    PubMed

    Bryan-Marrugo, Owen Lloyd; Arellanos-Soto, Daniel; Rojas-Martinez, Augusto; Barrera-Saldaña, Hugo; Ramos-Jimenez, Javier; Vidaltamayo, Roman; Rivas-Estilla, Ana María

    2016-09-01

    Dengue virus (DENV) susceptibility to cholesterol depleting treatments has been previously reported. There are numerous questions regarding how DENV seizes cellular machinery and cholesterol to improve viral production and the effect of cholesterol sequestering agents on the cellular antiviral response. The aim of the present study was to evaluate the mechanisms involved in the negative regulation of DENV replication induced by agents that diminish intracellular cholesterol levels. Cholesterol synthesis was pharmacologically (fluvastatin, atorvastatin, lovastatin, pravastatin and simvastatin treatment) and genetically (HMGCR‑RNAi) inhibited, in uninfected and DENV2‑infected hepatoma Huh‑7 cells. The cholesterol levels, DENV titer and cellular antiviral expression profile were evaluated. A reduction in the DENV titer, measured as plaque forming units, was observed in DENV‑infected cells following 48 h treatment with 10 µM fluvastatin, 10 µM atorvastatin, 20 µM lovastatin and 20 µM simvastatin, which achieved 70, 70, 65 and 55% DENV2 inhibition, respectively, compared with the untreated cells. In addition, the cytopathic effect was reduced in the statin‑treated DENV‑infected cells. Statins simultaneously reduced cholesterol levels at 48 h, with the exception of DENV2 infected cells. Genetic inhibition of cholesterol synthesis was performed using RNA interference for 3‑hydroxy‑3‑methylglutaryl‑CoA reductase (HMGCR‑siRNA), which indicated a slight reduction in DENV2 titer at 48 h post‑infection, however, with no significant reduction in cholesterol levels. In addition, DENV2 infection was observed to augment the intracellular cholesterol levels in all experimental conditions. Comparison between the cellular antiviral response triggered by DENV2 infection, statin treatment and HMGCR‑siRNA in infected, uninfected, treated and untreated Huh7 cells, showed different expression profiles for the antiviral genes evaluated. All

  13. Quantitative in situ hybridization for the study of gene expression at the regional and cellular levels.

    PubMed

    Le Moine, Catherine

    2003-08-01

    Quantitative in situ hybridization allows measurement of mRNA level modifications in a variety of experimental conditions. This analysis may be performed both at the regional anatomical and cellular levels by densitometry, neuronal counting and silver grain measurements. PMID:18428577

  14. Adipose depots differ in cellularity, adipokines produced, gene expression, and cell systems

    PubMed Central

    Dodson, Michael V; Du, Min; Wang, Songbo; Bergen, Werner G; Fernyhough-Culver, Melinda; Basu, Urmila; Poulos, Sylvia P; Hausman, Gary J

    2014-01-01

    The race to manage the health concerns related to excess fat deposition has spawned a proliferation of clinical and basic research efforts to understand variables including dietary uptake, metabolism, and lipid deposition by adipocytes. A full appreciation of these variables must also include a depot-specific understanding of content and location in order to elucidate mechanisms governing cellular development and regulation of fat deposition. Because adipose tissue depots contain various cell types, differences in the cellularity among and within adipose depots are presently being documented to ascertain functional differences. This has led to the possibility of there being, within any one adipose depot, cellular distinctions that essentially result in adipose depots within depots. The papers comprising this issue will underscore numerous differences in cellularity (development, histogenesis, growth, metabolic function, regulation) of different adipose depots. Such information is useful in deciphering adipose depot involvement both in normal physiology and in pathology. Obesity, diabetes, metabolic syndrome, carcass composition of meat animals, performance of elite athletes, physiology/pathophysiology of aging, and numerous other diseases might be altered with a greater understanding of adipose depots and the cells that comprise them—including stem cells—during initial development and subsequent periods of normal/abnormal growth into senescence. Once thought to be dormant and innocuous, the adipocyte is emerging as a dynamic and influential cell and research will continue to identify complex physiologic regulation of processes involved in adipose depot physiology. PMID:26317047

  15. Expression and Distribution Pattern of Aquaporin 4, 5 and 11 in Retinas of 15 Different Species.

    PubMed

    Amann, Barbara; Kleinwort, Kristina J H; Hirmer, Sieglinde; Sekundo, Walter; Kremmer, Elisabeth; Hauck, Stefanie M; Deeg, Cornelia A

    2016-01-01

    Aquaporins (AQPs) are small integral membrane proteins with 13 members in mammals and are essential for water transport across membranes. They are found in many different tissues and cells. Currently, there are conflicting results regarding retinal aquaporin expression and subcellular localization between genome and protein analyses and among various species. AQP4, 7, 9 and 11 were described in the retina of men; whereas AQP6, 8 and 10 were earlier identified in rat retinas and AQP4, 5 and 11 in horses. Since there is a lack of knowledge regarding AQP expression on protein level in retinas of different animal models, we decided to analyze retinal cellular expression of AQP4, 5 and 11 in situ with immunohistochemistry. AQP4 was detected in all 15 explored species, AQP5 and AQP11 in 14 out of 15. Interestingly, AQP4 was unambiguously expressed in Muller glial cells, whereas AQP5 was differentially allocated among the species analyzed. AQP11 expression was Muller glial cell-specific in 50% of the animals, whereas in the others, AQP11 was detected in ganglion cell layer and at photoreceptor outer segments. Our data indicate a disparity in aquaporin distribution in retinas of various animals, especially for AQP5 and 11. PMID:27438827

  16. Expression and Distribution Pattern of Aquaporin 4, 5 and 11 in Retinas of 15 Different Species

    PubMed Central

    Amann, Barbara; Kleinwort, Kristina J. H.; Hirmer, Sieglinde; Sekundo, Walter; Kremmer, Elisabeth; Hauck, Stefanie M.; Deeg, Cornelia A.

    2016-01-01

    Aquaporins (AQPs) are small integral membrane proteins with 13 members in mammals and are essential for water transport across membranes. They are found in many different tissues and cells. Currently, there are conflicting results regarding retinal aquaporin expression and subcellular localization between genome and protein analyses and among various species. AQP4, 7, 9 and 11 were described in the retina of men; whereas AQP6, 8 and 10 were earlier identified in rat retinas and AQP4, 5 and 11 in horses. Since there is a lack of knowledge regarding AQP expression on protein level in retinas of different animal models, we decided to analyze retinal cellular expression of AQP4, 5 and 11 in situ with immunohistochemistry. AQP4 was detected in all 15 explored species, AQP5 and AQP11 in 14 out of 15. Interestingly, AQP4 was unambiguously expressed in Muller glial cells, whereas AQP5 was differentially allocated among the species analyzed. AQP11 expression was Muller glial cell-specific in 50% of the animals, whereas in the others, AQP11 was detected in ganglion cell layer and at photoreceptor outer segments. Our data indicate a disparity in aquaporin distribution in retinas of various animals, especially for AQP5 and 11. PMID:27438827

  17. Immunohistochemical Expression of Matrix Metalloproteinase-7 in Human Colorectal Adenomas Using Specified Automated Cellular Image Analysis System: A Clinicopathological Study

    PubMed Central

    Qasim, Ban J.; Ali, Hussam H.; Hussein, Alaa G.

    2013-01-01

    Background/Aim: To evaluate the immunohistochemical expression of matrix metalloproteinase-7 (MMP-7) in colorectal adenomas, and to correlate this expression with different clinicopathological parameters. Patients and Methods: The study was retrospectively designed. Thirty three paraffin blocks from patients with colorectal adenoma and 20 samples of non-tumerous colonic tissue taken as control group were included in the study. MMP-7 expression was assessed by immunohistochemistry method. The scoring of immunohistochemical staining was conducted utilizing a specified automated cellular image analysis system (Digimizer). Results: The frequency of positive immunohistochemical expression of MMP-7 was significantly higher in adenoma than control group (45.45% versus 10%) (P value < 0.001). Strong MMP-7 staining was mainly seen in adenoma cases (30.30%) in comparison with control (0%) the difference is significant (P < 0.001). The three digital parameters of MMP-7 immunohistochemical expression (Area (A), Number of objects (N), and intensity (I)) were significantly higher in adenoma than control. Mean (A and I) of MMP-7 showed a significant correlation with large sized adenoma (≥ 1cm) (P < 0.05), also a significant positive correlation of the three digital parameters (A, N, and I) of MMP-7 expression with villous configuration and severe dysplasia in colorectal adenoma had been identified (P < 0.05). Conclusion: MMP-7 plays an important role in the growth and malignant conversion of colorectal adenomas as it is more likely to be expressed in advanced colorectal adenomatous polyps with large size, severe dysplasia and villous histology. The use of automated cellular image analysis system (Digmizer) to quantify immunohistochemical staining yields more consistent assay results, converts semi-quantitative assay to a truly quantitative assay, and improves assay objectivity and reproducibility. PMID:23319034

  18. High-throughput study of alpha-synuclein expression in yeast using microfluidics for control of local cellular microenvironment

    PubMed Central

    Rosa, Patrícia; Tenreiro, Sandra; Chu, Virginia; Outeiro, Tiago F.; Conde, João Pedro

    2012-01-01

    Microfluidics is an emerging technology which allows the miniaturization, integration, and automation of fluid handling processes. Microfluidic systems offer low sample consumption, significantly reduced processing time, and the prospect of massive parallelization. A microfluidic platform was developed for the control of the soluble cellular microenvironment of Saccharomyces cerevisiae cells, which enabled high-throughput monitoring of the controlled expression of alpha-synuclein (aSyn), a protein involved in Parkinson’s disease. Y-shaped structures were fabricated using particle desorption mass spectrometry-based soft-lithography techniques to generate biomolecular gradients along a microchannel. Cell traps integrated along the microchannel allowed the positioning and monitoring of cells in precise locations, where different, well-controlled chemical environments were established. S. cerevisiae cells genetically engineered to encode the fusion protein aSyn-GFP (green fluorescent protein) under the control of GAL1, a galactose inducible promoter, were loaded in the microfluidic structure. A galactose concentration gradient was established in the channel and a time-dependent aSyn-GFP expression was obtained as a function of the positioning of cells along the galactose gradient. Our results demonstrate the applicability of this microfluidic platform to the spatiotemporal control of cellular microenvironment and open a range of possibilities for the study of cellular processes based on single-cell analysis. PMID:22662094

  19. Single-cell expression analyses during cellular reprogramming reveal an early stochastic and a late hierarchic phase.

    PubMed

    Buganim, Yosef; Faddah, Dina A; Cheng, Albert W; Itskovich, Elena; Markoulaki, Styliani; Ganz, Kibibi; Klemm, Sandy L; van Oudenaarden, Alexander; Jaenisch, Rudolf

    2012-09-14

    During cellular reprogramming, only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. We utilized two gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. Expression of Esrrb, Utf1, Lin28, and Dppa2 is a better predictor for cells to progress into iPSCs than expression of the previously suggested reprogramming markers Fbxo15, Fgf4, and Oct4. Stochastic gene expression early in reprogramming is followed by a late hierarchical phase with Sox2 being the upstream factor in a gene expression hierarchy. Finally, downstream factors derived from the late phase, which do not include Oct4, Sox2, Klf4, c-Myc, and Nanog, can activate the pluripotency circuitry. PMID:22980981

  20. Single-cell gene expression analyses of cellular reprogramming reveal a stochastic early and hierarchic late phase

    PubMed Central

    Buganim, Yosef; Faddah, Dina A.; Cheng, Albert W.; Itskovich, Elena; Markoulaki, Styliani; Ganz, Kibibi; Klemm, Sandy L.; van Oudenaarden, Alexander; Jaenisch, Rudolf

    2012-01-01

    During cellular reprogramming only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. We utilized two gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. Expression of Esrrb, Utf1, Lin28, and Dppa2 is a better predictor for cells to progress into iPSCs than expression of Fbxo15, Fgf4, and Oct4 previously suggested to be reprogramming markers. Stochastic gene expression early in reprogramming is followed by a late hierarchical phase with Sox2 being the upstream factor in a gene expression hierarchy. Finally, downstream factors derived from the late phase, which do not include Oct4, Sox2, Klf4, c-Myc and Nanog, can activate the pluripotency circuitry. PMID:22980981

  1. Driving mechanisms of passive and active transport across cellular membranes as the mechanisms of cell metabolism and development as well as the mechanisms of cellular distance reactions on hormonal expression and the immune response.

    PubMed

    Ponisovskiy, M R

    2011-01-01

    The article presents mechanisms of cell metabolism, cell development, cell activity, and maintenance of cellular stability. The literature is reviewed from the point of view of these concepts. The balance between anabolic and catabolic processes induces chemical potentials in the extracellular and intracellular media. The chemical potentials of these media are defined as the driving forces of both passive and active transport of substances across cellular membranes. The driving forces of substance transport across cellular membranes as in cellular metabolism and in immune responses and hormonal expressions are considered in the biochemical and biophysical models, reflecting the mechanisms for maintenance of stability of the internal medium and internal energy of an organism. The interactions of passive transport and active transport of substances across cellular walls promote cell proliferation, as well as the mechanism of cellular capacitors, promoting remote reactions across distance for hormonal expression and immune responses. The offered concept of cellular capacitors has given the possibility to explain the mechanism of remote responses of cells to new situations, resulting in the appearance of additional agents. The biophysical model develops an explanation of some cellular functions: cellular membrane action have been identified with capacitor action, based on the similarity of the structures and as well as on similarity of biophysical properties of electric data that confirm the action of the compound-specific interactions of cells within an organism, promoting hormonal expressions and immune responses to stabilize the thermodynamic system of an organism. Comparison of a cellular membrane action to a capacitor has given the possibility for the explanations of exocytosis and endocytosis mechanisms, internalization of the receptor-ligand complex, selection as a receptor reaction to a ligand by immune responses or hormonal effects, reflecting cellular

  2. Myocardial Gene Transfer: Routes and Devices for Regulation of Transgene Expression by Modulation of Cellular Permeability

    PubMed Central

    Katz, Michael G.; Bridges, Charles R.

    2013-01-01

    Abstract Heart diseases are major causes of morbidity and mortality in Western society. Gene therapy approaches are becoming promising therapeutic modalities to improve underlying molecular processes affecting failing cardiomyocytes. Numerous cardiac clinical gene therapy trials have yet to demonstrate strong positive results and advantages over current pharmacotherapy. The success of gene therapy depends largely on the creation of a reliable and efficient delivery method. The establishment of such a system is determined by its ability to overcome the existing biological barriers, including cellular uptake and intracellular trafficking as well as modulation of cellular permeability. In this article, we describe a variety of physical and mechanical methods, based on the transient disruption of the cell membrane, which are applied in nonviral gene transfer. In addition, we focus on the use of different physiological techniques and devices and pharmacological agents to enhance endothelial permeability. Development of these methods will undoubtedly help solve major problems facing gene therapy. PMID:23427834

  3. Identification of driving network of cellular differentiation from single sample time course gene expression data

    NASA Astrophysics Data System (ADS)

    Chen, Ye; Wolanyk, Nathaniel; Ilker, Tunc; Gao, Shouguo; Wang, Xujing

    Methods developed based on bifurcation theory have demonstrated their potential in driving network identification for complex human diseases, including the work by Chen, et al. Recently bifurcation theory has been successfully applied to model cellular differentiation. However, there one often faces a technical challenge in driving network prediction: time course cellular differentiation study often only contains one sample at each time point, while driving network prediction typically require multiple samples at each time point to infer the variation and interaction structures of candidate genes for the driving network. In this study, we investigate several methods to identify both the critical time point and the driving network through examination of how each time point affects the autocorrelation and phase locking. We apply these methods to a high-throughput sequencing (RNA-Seq) dataset of 42 subsets of thymocytes and mature peripheral T cells at multiple time points during their differentiation (GSE48138 from GEO). We compare the predicted driving genes with known transcription regulators of cellular differentiation. We will discuss the advantages and limitations of our proposed methods, as well as potential further improvements of our methods.

  4. Cellular and Laminar Expression of Dab-1 during the Postnatal Critical Period in Cat Visual Cortex and the Effects of Dark Rearing

    PubMed Central

    Kiser, Paul J.; Liu, Zijing; Wilt, Steven D.; Mower, George D.

    2011-01-01

    This study describes postnatal critical period changes in cellular and laminar expression of Dab-1, a gene shown to play a role in controlling neuronal positioning during embryonic brain development, in cat visual cortex and the effects of dark rearing (DR). At 1 week, there is dense cellular staining which is uniform across cortical layers and very light neuropil staining. At the peak of the critical period (5 weeks), dense cell staining is largely restricted to large pyramidal cells of deep layer III and layer V, there is faint cell body staining throughout all cortical layers, neuropil staining is markedly increased and uniform in layers III to VI. This dramatic change in laminar and cellular labeling is independent of visual input, since immunostaining is similar in 5 week DR cats. By 10 weeks, the mature laminar and cellular staining pattern is established and the major subsequent change is a further reduction in the density of cellular staining in all cortical layers. Neuropil staining is pronounced and uniform across cortical layers. These developmental changes are altered by DR. Quantification by cell counts indicated that age and DR interact such that differences in cellular expression are opposite in direction between 5 week old and 20 week old cats. This bidirectional regulation of cellular expression is the same in all cortical laminae. The bidirectional regulation of cellular expression matches the effects of age and DR on physiological plasticity during the critical period as assessed by ocular dominance shifts in response to monocular deprivation. PMID:21303666

  5. JC virus induces altered patterns of cellular gene expression: Interferon-inducible genes as major transcriptional targets

    SciTech Connect

    Verma, Saguna; Ziegler, Katja; Ananthula, Praveen; Co, Juliene K.G.; Frisque, Richard J.; Yanagihara, Richard; Nerurkar, Vivek R. . E-mail: nerurkar@pbrc.hawaii.edu

    2006-02-20

    Human polyomavirus JC (JCV) infects 80% of the population worldwide. Primary infection, typically occurring during childhood, is asymptomatic in immunocompetent individuals and results in lifelong latency and persistent infection. However, among the severely immunocompromised, JCV may cause a fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Virus-host interactions influencing persistence and pathogenicity are not well understood, although significant regulation of JCV activity is thought to occur at the level of transcription. Regulation of the JCV early and late promoters during the lytic cycle is a complex event that requires participation of both viral and cellular factors. We have used cDNA microarray technology to analyze global alterations in gene expression in JCV-permissive primary human fetal glial cells (PHFG). Expression of more than 400 cellular genes was altered, including many that influence cell proliferation, cell communication and interferon (IFN)-mediated host defense responses. Genes in the latter category included signal transducer and activator of transcription 1 (STAT1), interferon stimulating gene 56 (ISG56), myxovirus resistance 1 (MxA), 2'5'-oligoadenylate synthetase (OAS), and cig5. The expression of these genes was further confirmed in JCV-infected PHFG cells and the human glioblastoma cell line U87MG to ensure the specificity of JCV in inducing this strong antiviral response. Results obtained by real-time RT-PCR and Western blot analyses supported the microarray data and provide temporal information related to virus-induced changes in the IFN response pathway. Our data indicate that the induction of an antiviral response may be one of the cellular factors regulating/controlling JCV replication in immunocompetent hosts and therefore constraining the development of PML.

  6. Inhibition of Glutathione Production Induces Macrophage CD36 Expression and Enhances Cellular-oxidized Low Density Lipoprotein (oxLDL) Uptake.

    PubMed

    Yang, Xiaoxiao; Yao, Hui; Chen, Yuanli; Sun, Lei; Li, Yan; Ma, Xingzhe; Duan, Shengzhong; Li, Xiaoju; Xiang, Rong; Han, Jihong; Duan, Yajun

    2015-09-01

    The glutathione (GSH)-dependent antioxidant system has been demonstrated to inhibit atherosclerosis. Macrophage CD36 uptakes oxidized low density lipoprotein (oxLDL) thereby facilitating foam cell formation and development of atherosclerosis. It remains unknown if GSH can influence macrophage CD36 expression and cellular oxLDL uptake directly. Herein we report that treatment of macrophages with l-buthionine-S,R-sulfoximine (BSO) decreased cellular GSH production and ratios of GSH to glutathione disulfide (GSH/GSSG) while increasing production of reactive oxygen species. Associated with decreased GSH levels, macrophage CD36 expression was increased, which resulted in enhanced cellular oxLDL uptake. In contrast, N-acetyl cysteine and antioxidant enzyme (catalase or superoxide dismutase) blocked BSO-induced CD36 expression as well as oxLDL uptake. In vivo, administration of mice with BSO increased CD36 expression in peritoneal macrophages and kidneys. BSO had no effect on CD36 mRNA expression and promoter activity but still induced CD36 protein expression in macrophages lacking peroxisome proliferator-activated receptor γ expression, suggesting it induced CD36 expression at the translational level. Indeed, we determined that BSO enhanced CD36 translational efficiency. Taken together, our study demonstrates that cellular GSH levels and GSH/GSSG status can regulate macrophage CD36 expression and cellular oxLDL uptake and demonstrate an important anti-atherogenic function of the GSH-dependent antioxidant system by providing a novel molecular mechanism.

  7. Inhibition of Glutathione Production Induces Macrophage CD36 Expression and Enhances Cellular-oxidized Low Density Lipoprotein (oxLDL) Uptake*

    PubMed Central

    Yang, Xiaoxiao; Yao, Hui; Chen, Yuanli; Sun, Lei; Li, Yan; Ma, Xingzhe; Duan, Shengzhong; Li, Xiaoju; Xiang, Rong; Han, Jihong; Duan, Yajun

    2015-01-01

    The glutathione (GSH)-dependent antioxidant system has been demonstrated to inhibit atherosclerosis. Macrophage CD36 uptakes oxidized low density lipoprotein (oxLDL) thereby facilitating foam cell formation and development of atherosclerosis. It remains unknown if GSH can influence macrophage CD36 expression and cellular oxLDL uptake directly. Herein we report that treatment of macrophages with l-buthionine-S,R-sulfoximine (BSO) decreased cellular GSH production and ratios of GSH to glutathione disulfide (GSH/GSSG) while increasing production of reactive oxygen species. Associated with decreased GSH levels, macrophage CD36 expression was increased, which resulted in enhanced cellular oxLDL uptake. In contrast, N-acetyl cysteine and antioxidant enzyme (catalase or superoxide dismutase) blocked BSO-induced CD36 expression as well as oxLDL uptake. In vivo, administration of mice with BSO increased CD36 expression in peritoneal macrophages and kidneys. BSO had no effect on CD36 mRNA expression and promoter activity but still induced CD36 protein expression in macrophages lacking peroxisome proliferator-activated receptor γ expression, suggesting it induced CD36 expression at the translational level. Indeed, we determined that BSO enhanced CD36 translational efficiency. Taken together, our study demonstrates that cellular GSH levels and GSH/GSSG status can regulate macrophage CD36 expression and cellular oxLDL uptake and demonstrate an important anti-atherogenic function of the GSH-dependent antioxidant system by providing a novel molecular mechanism. PMID:26187465

  8. Dystrophin Distribution and Expression in Human and Experimental Temporal Lobe Epilepsy

    PubMed Central

    Hendriksen, Ruben G. F.; Schipper, Sandra; Hoogland, Govert; Schijns, Olaf E. M. G.; Dings, Jim T. A.; Aalbers, Marlien W.; Vles, Johan S. H.

    2016-01-01

    Objective: Dystrophin is part of a protein complex that connects the cytoskeleton to the extracellular matrix. In addition to its role in muscle tissue, it functions as an anchoring protein within the central nervous system such as in hippocampus and cerebellum. Its presence in the latter regions is illustrated by the cognitive problems seen in Duchenne Muscular Dystrophy (DMD). Since epilepsy is also supposed to constitute a comorbidity of DMD, it is hypothesized that dystrophin plays a role in neuronal excitability. Here, we aimed to study brain dystrophin distribution and expression in both, human and experimental temporal lobe epilepsy (TLE). Method: Regional and cellular dystrophin distribution was evaluated in both human and rat hippocampi and in rat cerebellar tissue by immunofluorescent colocalization with neuronal (NeuN and calbindin) and glial (GFAP) markers. In addition, hippocampal dystrophin levels were estimated by Western blot analysis in biopsies from TLE patients, post-mortem controls, amygdala kindled (AK)-, and control rats. Results: Dystrophin was expressed in all hippocampal pyramidal subfields and in the molecular-, Purkinje-, and granular cell layer of the cerebellum. In these regions it colocalized with GFAP, suggesting expression in astrocytes such as Bergmann glia (BG) and velate protoplasmic astrocytes. In rat hippocampus and cerebellum there were neither differences in dystrophin positive cell types, nor in the regional dystrophin distribution between AK and control animals. Quantitatively, hippocampal full-length dystrophin (Dp427) levels were about 60% higher in human TLE patients than in post-mortem controls (p < 0.05), whereas the level of the shorter Dp71 isoform did not differ. In contrast, AK animals showed similar dystrophin levels as controls. Conclusion: Dystrophin is ubiquitously expressed by astrocytes in the human and rat hippocampus and in the rat cerebellum. Hippocampal full-length dystrophin (Dp427) levels are upregulated

  9. 75 FR 4741 - Express Mail Open and Distribute and Priority Mail Open and Distribute Changes and Updates

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-29

    ... 111 Express Mail Open and Distribute and Priority Mail Open and Distribute Changes and Updates AGENCY... to reflect changes and updates for Express Mail Open and Distribute and Priority Mail Open and... address of the commenter, may be sent to: MailingStandards@usps.gov , with a subject line of ``Open...

  10. Acute effects of pregabalin on the function and cellular distribution of Ca(V)2.1 in HEK293t cells.

    PubMed

    Weissmann, Carina; Di Guilmi, Mariano N; Urbano, Francisco J; Uchitel, Osvaldo D

    2013-01-01

    We established a cell model to study the acute effects of pregabalin (PGB), a drug widely used in epilepsy and neuropathic pain, on voltage gated Ca(V)2.1 (P/Q-type) calcium channels function and distribution at the membrane level. HEK293t cells were transfected with plasmids coding for all subunits of the Ca(V)2.1 channel. We used a α1 fused to an eGFP tag to follow its distribution in time and at different experimental conditions. The expressed channel was functional as shown by the presence of barium-mediated, calcium currents of transfected cells measured by 'whole-cell voltage-clamp' recordings, showing a maximum current peak in the I-V curve at +20 mV. The GFP fluorescent signal was confined to the periphery of the cells. Incubation with 500 μM PGB, that binds α2δ subunits, for 30 min induced changes in localization of the fluorescent subunits as measured by fluorescent time lapse microscopy. These changes correlated with a reversible reduction of barium currents through Ca(V)2.1 calcium channels under the same conditions. However, no changes in the cellular distribution of the subunits were visualized for cells either expressing another membrane associated protein or after exposure of the Ca(V)2.1 channels to isoleucine, another α2δ ligand. Together these results show strong evidence for an acute effect of PGB on Ca(V)2.1 calcium channels' currents and distribution and suggest that internalization of Ca(V)2.1 channels might be a mechanism of PGB action. PMID:23063705

  11. Subcellular distribution of small interfering RNA: directed delivery through RNA polymerase III expression cassettes and localization by in situ hybridization.

    PubMed

    Paul, Cynthia P

    2005-01-01

    Reduction in the expression of specific genes through small interfering RNAs (siRNAs) is dependent on the colocalization of siRNAs with other components of the RNA interference (RNAi) pathways within the cell. The expression of siRNAs within cells from cassettes that are derived from genes transcribed by RNA polymerase III (pol III) and provide for selective subcellular distribution of their products can be used to direct siRNAs to the cellular pathways. Expression from the human U6 promoter, resulting in siRNA accumulation in the nucleus, is effective in reducing gene expression, whereas cytoplasmic and nucleolar localization of the siRNA when expressed from the 5S or 7 SL promoters is not effective. The distribution of siRNA within the cell is determined by fluorescence in situ hybridization. Although the long uninterrupted duplex of siRNA makes it difficult to detect with DNA oligonucleotide probes, labeled oligonucleotide probes with 2'-O-methyl RNA backbones provide the stability needed for a strong signal. These methods contribute to studies of the interconnected cellular RNAi pathways and are useful in adapting RNAi as a tool to determine gene function and develop RNA-based therapeutics. PMID:15644179

  12. Cellular Mechanisms of Tissue Fibrosis. 2. Contributory pathways leading to myocardial fibrosis: moving beyond collagen expression

    PubMed Central

    Goldsmith, Edie C.; Bradshaw, Amy D.

    2013-01-01

    While the term “fibrosis” can be misleading in terms of the complex patterns and processes of myocardial extracellular matrix (ECM) remodeling, fibrillar collagen accumulation is a common consequence of relevant pathophysiological stimuli, such as pressure overload (PO) and myocardial infarction (MI). Fibrillar collagen accumulation in both PO and MI is predicated on a number of diverse cellular and extracellular events, which include changes in fibroblast phenotype (transdifferentiation), posttranslational processing and assembly, and finally, degradation. The expansion of a population of transformed fibroblasts/myofibroblasts is a significant cellular event with respect to ECM remodeling in both PO and MI. The concept that this cellular expansion within the myocardial ECM may be due, at least in part, to endothelial-mesenchymal transformation and thereby not dissimilar to events observed in cancer progression holds intriguing future possibilities. Studies regarding determinants of procollagen processing, such as procollagen C-endopeptidase enhancer (PCOLCE), and collagen assembly, such as the secreted protein acidic and rich in cysteine (SPARC), have identified potential new targets for modifying the fibrotic response in both PO and MI. Finally, the transmembrane matrix metalloproteinases, such as MMP-14, underscore the diversity and complexity of this ECM proteolytic family as this protease can degrade the ECM as well as induce a profibrotic response. The growing recognition that the myocardial ECM is a dynamic entity containing a diversity of matricellular and nonstructural proteins as well as proteases and that the fibrillar collagens can change in structure and content in a rapid temporal fashion has opened up new avenues for modulating what was once considered an irreversible event - myocardial fibrosis. PMID:23174564

  13. Derepression of hTERT gene expression promotes escape from oncogene-induced cellular senescence.

    PubMed

    Patel, Priyanka L; Suram, Anitha; Mirani, Neena; Bischof, Oliver; Herbig, Utz

    2016-08-23

    Oncogene-induced senescence (OIS) is a critical tumor-suppressing mechanism that restrains cancer progression at premalignant stages, in part by causing telomere dysfunction. Currently it is unknown whether this proliferative arrest presents a stable and therefore irreversible barrier to cancer progression. Here we demonstrate that cells frequently escape OIS induced by oncogenic H-Ras and B-Raf, after a prolonged period in the senescence arrested state. Cells that had escaped senescence displayed high oncogene expression levels, retained functional DNA damage responses, and acquired chromatin changes that promoted c-Myc-dependent expression of the human telomerase reverse transcriptase gene (hTERT). Telomerase was able to resolve existing telomeric DNA damage response foci and suppressed formation of new ones that were generated as a consequence of DNA replication stress and oncogenic signals. Inhibition of MAP kinase signaling, suppressing c-Myc expression, or inhibiting telomerase activity, caused telomere dysfunction and proliferative defects in cells that had escaped senescence, whereas ectopic expression of hTERT facilitated OIS escape. In human early neoplastic skin and breast tissue, hTERT expression was detected in cells that displayed features of senescence, suggesting that reactivation of telomerase expression in senescent cells is an early event during cancer progression in humans. Together, our data demonstrate that cells arrested in OIS retain the potential to escape senescence by mechanisms that involve derepression of hTERT expression. PMID:27503890

  14. Derepression of hTERT gene expression promotes escape from oncogene-induced cellular senescence

    PubMed Central

    Patel, Priyanka L.; Suram, Anitha; Mirani, Neena; Bischof, Oliver; Herbig, Utz

    2016-01-01

    Oncogene-induced senescence (OIS) is a critical tumor-suppressing mechanism that restrains cancer progression at premalignant stages, in part by causing telomere dysfunction. Currently it is unknown whether this proliferative arrest presents a stable and therefore irreversible barrier to cancer progression. Here we demonstrate that cells frequently escape OIS induced by oncogenic H-Ras and B-Raf, after a prolonged period in the senescence arrested state. Cells that had escaped senescence displayed high oncogene expression levels, retained functional DNA damage responses, and acquired chromatin changes that promoted c-Myc–dependent expression of the human telomerase reverse transcriptase gene (hTERT). Telomerase was able to resolve existing telomeric DNA damage response foci and suppressed formation of new ones that were generated as a consequence of DNA replication stress and oncogenic signals. Inhibition of MAP kinase signaling, suppressing c-Myc expression, or inhibiting telomerase activity, caused telomere dysfunction and proliferative defects in cells that had escaped senescence, whereas ectopic expression of hTERT facilitated OIS escape. In human early neoplastic skin and breast tissue, hTERT expression was detected in cells that displayed features of senescence, suggesting that reactivation of telomerase expression in senescent cells is an early event during cancer progression in humans. Together, our data demonstrate that cells arrested in OIS retain the potential to escape senescence by mechanisms that involve derepression of hTERT expression. PMID:27503890

  15. Expression and cellular localizaion of melatonin-synthesizing enzymes in rat and human salivary glands.

    PubMed

    Shimozuma, Masashi; Tokuyama, Reiko; Tatehara, Seiko; Umeki, Hirochika; Ide, Shinji; Mishima, Kenji; Saito, Ichiro; Satomura, Kazuhito

    2011-04-01

    Melatonin, discovered in 1958, is secreted by the pineal gland primarily during the night. Its secretion is controlled by the light/dark cycle of the environment. Melatonin is also produced in and secreted by various extrapineal organs, tissues and cells and its synthesizing enzyme arylalkylamine N-acetyltransferase (AANAT) is expressed in various extrapineal organs, tissues and cells. Recently, it was reported that melatonin is present in saliva, but it is not certain where melatonin was synthesized and whether it was secreted into saliva and what function it may have in saliva. The present study was performed to investigate where melatonin was synthesized and whether it was secreted by salivary glands into saliva. We performed immunohistochemical analysis of the expression of AANAT in rat parotid, submandibular and sublingual glands and the expression of both AANAT and hydroxyindole-O-methyltransferase (HIOMT) in human submandibular glands. We evaluated the expression of AANAT and HIOMT mRNA in rat submandibular glands by quantitative reverse transcription-polymerase chain reaction. As a result, we observed expression of AANAT in epithelial cells of striated ducts in rat salivary glands and expression of AANAT, HIOMT and melatonin in epithelial cells of striated ducts in human submandibular glands. In addition, we also confirmed the expression of the most potent melatonin receptor, melatonin 1a receptor, in rat buccal mucosa. Our findings suggest that melatonin might be produced and secreted by salivary glands directly into saliva and that it might play some physiological role in the oral cavity.

  16. Expression of transferrin receptors on mitogen-stimulated human peripheral blood lymphocytes: relation to cellular activation and related metabolic events.

    PubMed Central

    Galbraith, R M; Galbraith, G M

    1981-01-01

    Mitogen-activated normal human peripheral blood lymphocytes bind transferrin to specific membrane receptors. In this study, lymphocytes stimulated with phytohaemagglutinin for 0-66 hr were examined to determine the relation of this phenomenon to cellular activation and related metabolic events. Transferrin receptors were first detected at 20-24 hr. This event was consistently preceded by RNA and protein turnover which commenced during the first 6 hr of culture, whereas initiation of DNA synthesis was detected concurrently with the appearance of receptors or slightly later (24-30 hr). Exposure of cells to inhibitors of RNA and protein synthesis early during culture (at 0 or 24 hr) prevented the expression of transferrin receptors, but also caused generalized metabolic failure, and abrogated cellular activation. In contrast, later addition of these agents at 48 hr did not interfere significantly with the process of activation, but did suppress the terminal increase in receptor-bearing cells observed during the final 18 hr in control cultures lacking inhibitor. After deliberate thermal stripping of receptors from activated cells, the reappearance of membrance binding sites which normally occurred within 30 min, was also blocked by cycloheximide, puromycin and actinomycin D. However, similar inhibition of DNA which was induced by hydroxyurea had much less effect upon both the initial appearance of receptors and their reappearance after ligand-induced depletion. These results demonstrate that the appearance of transferrin receptors upon human lymphocytes is dependent upon cellular activation and requires synthesis of protein and RNA. PMID:6172372

  17. First cellular approach of the effects of global warming on groundwater organisms: a study of the HSP70 gene expression.

    PubMed

    Colson-Proch, Céline; Morales, Anne; Hervant, Frédéric; Konecny, Lara; Moulin, Colette; Douady, Christophe J

    2010-05-01

    Whereas the consequences of global warming at population or community levels are well documented, studies at the cellular level are still scarce. The study of the physiological or metabolic effects of such small increases in temperature (between +2 degrees C and +6 degrees C) is difficult because they are below the amplitude of the daily or seasonal thermal variations occurring in most environments. In contrast, subterranean biotopes are highly thermally buffered (+/-1 degrees C within a year), and underground water organisms could thus be particularly well suited to characterise cellular responses of global warming. To this purpose, we studied genes encoding chaperone proteins of the HSP70 family in amphipod crustaceans belonging to the ubiquitous subterranean genus Niphargus. An HSP70 sequence was identified in eight populations of two complexes of species of the Niphargus genus (Niphargus rhenorhodanensis and Niphargus virei complexes). Expression profiles were determined for one of these by reverse transcription and quantitative polymerase chain reaction, confirming the inducible nature of this gene. An increase in temperature of 2 degrees C seemed to be without effect on N. rhenorhodanensis physiology, whereas a heat shock of +6 degrees C represented an important thermal stress for these individuals. Thus, this study shows that although Niphargus individuals do not undergo any daily or seasonal thermal variations in underground water, they display an inducible HSP70 heat shock response. This controlled laboratory-based physiological experiment constitutes a first step towards field investigations of the cellular consequences of global warming on subterranean organisms.

  18. Tricyclic pyrone compounds prevent aggregation and reverse cellular phenotypes caused by expression of mutant huntingtin protein in striatal neurons

    PubMed Central

    Trushina, Eugenia; Rana, Sandeep; McMurray, Cynthia T; Hua, Duy H

    2009-01-01

    Background Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion mutation in the coding region of a novel gene. The mechanism of HD is unknown. Most data suggest that polyglutamine-mediated aggregation associated with expression of mutant huntingtin protein (mhtt) contributes to the pathology. However, recent studies have identified early cellular dysfunctions that preclude aggregate formation. Suppression of aggregation is accepted as one of the markers of successful therapeutic approaches. Previously, we demonstrated that tricyclic pyrone (TP) compounds efficiently inhibited formation of amyloid-β (Aβ) aggregates in cell and mouse models representing Alzheimer's Disease (AD). In the present study, we aimed to determine whether TP compounds could prevent aggregation and restore early cellular defects in primary embryonic striatal neurons from animal model representing HD. Results TP compounds effectively inhibit aggregation caused by mhtt in neurons and glial cells. Treatment with TP compounds also alleviated cholesterol accumulation and restored clathrin-independent endocytosis in HD neurons. Conclusion We have found that TP compounds not only blocked mhtt-induced aggregation, but also alleviated early cellular dysfunctions that preclude aggregate formation. Our data suggest TP molecules may be used as lead compounds for prevention or treatment of multiple neurodegenerative diseases including HD and AD. PMID:19586540

  19. Expression of cellular FLICE inhibitory proteins (cFLIP) in normal and traumatic murine and human cerebral cortex

    PubMed Central

    Hainsworth, Atticus H; Bermpohl, Daniela; Webb, Tania E; Darwish, Ribal; Fiskum, Gary; Qiu, Jianhua; McCarthy, Deirdre; Moskowitz, Michael A; Whalen, Michael J

    2005-01-01

    Cellular Fas-associated death domain-like interleukin-1-beta converting enzyme (FLICE) inhibitory proteins (cFLIPs) are endogenous caspase homologues that inhibit programmed cell death. We hypothesized that cFLIPs are differentially expressed in response to traumatic brain injury (TBI). cFLIP-alpha and cFLIP-delta mRNA were expressed in normal mouse brain—specifically cFLIP-delta (but not cFLIP-alpha) protein was robustly expressed. After controlled cortical impact (CCI), cFLIP-alpha expression increased initially then decreased to control levels at 12 h, increasing again at 24–72 h (P<0.05). cFLIP-delta expression was decreased in brain homogenates by 12 h after CCI, then increased again at 24 to 72 h (P<0.05). cFLIP-delta immunostaining was markedly reduced in injured cortex, but not hippocampus, at 3 to 72 h after CCI. In cortex, reduced cFLIP-delta staining was found in TUNEL-positive cells, but in hippocampus TUNEL-positive cells expressed cFLIP-delta immunoreactivity. cFLIP-delta was increased in a subset of reactive astrocytes in pericontusional cortex and hippocampus at 48 to 72 h. Low levels of both cFLIP isoforms were detected in human cortical tissue with no TBI, from four patients undergoing brain surgery for epilepsy and <24 h post mortem from three patients without CNS pathologic assessment. In cortical tissue surgically removed <18 h after severe TBI (n=3), cFLIP-alpha expression was increased relative to epilepsy controls (P<0.05) but not relative to post-mortem controls. The data suggest differential spatial and temporal regulation of cFLIP-alpha and cFLIP-delta expression that may influence the magnitude of cell death and further implicate programmed mechanisms of cell death after TBI. PMID:15815586

  20. The emerging role of pseudogene expressed non-coding RNAs in cellular functions

    PubMed Central

    Groen, Jessica N.; Capraro, David; Morris, Kevin V.

    2014-01-01

    A paradigm shift is sweeping modern day molecular biology following the realisation that large amounts of “junk” DNA”, thought initially to be evolutionary remnants, may actually be functional. Several recent studies support a functional role for pseudogene-expressed non-coding RNAs in regulating their protein-coding counterparts. Several hundreds of pseudogenes have been reported as transcribed into RNA in a large variety of tissues and tumours. Most studies have focused on pseudogenes expressed in the sense direction, but some reports suggest that pseudogenes can also be transcribed as antisense RNAs (asRNAs). A few examples of key regulatory genes, such as PTEN and OCT4, have in fact been reported to be under the regulation of pseudogene-expressed asRNAs. Here, we review what are known about pseudogene expressed non-coding RNA mediated gene regulation and their roles in the control of epigenetic states. PMID:24842102

  1. New water-soluble ruthenium(II) cytotoxic complex: biological activity and cellular distribution.

    PubMed

    Morais, Tânia S; Santos, Filipa C; Jorge, Tiago F; Côrte-Real, Leonor; Madeira, Paulo J Amorim; Marques, Fernanda; Robalo, M Paula; Matos, António; Santos, Isabel; Garcia, M Helena

    2014-01-01

    A novel water soluble organometallic compound, [RuCp(mTPPMSNa)(2,2'-bipy)][CF3SO3] (TM85, where Cp=η(5)-cyclopentadienyl, mTPPMS=diphenylphosphane-benzene-3-sulfonate and 2,2'-bipy=2,2'-bipyridine) is presented herein. Studies of interactions with relevant proteins were performed to understand the behavior and mode of action of this complex in the biological environment. Electrochemical and fluorescence studies showed that TM85 strongly binds to albumin. Studies carried out to study the formation of TM85 which adducts with ubiquitin and cytochrome c were performed by electrospray ionization mass spectrometry (ESI-MS). Antitumor activity was evaluated against a variety of human cancer cell lines, namely A2780, A2780cisR, MCF7, MDAMB231, HT29, PC3 and V79 non-tumorigenic cells and compared with the reference drug cisplatin. TM85 cytotoxic effect was reduced in the presence of endocytosis modulators at low temperatures, suggesting an energy-dependent mechanism consistent with endocytosis. Ultrastructural analysis by transmission electron microscopy (TEM) revealed that TM85 targets the endomembranar system disrupting the Golgi and also affects the mitochondria. Disruption of plasma membrane observed by flow cytometry could lead to cellular damage and cell death. On the whole, the biological activity evaluated herein combined with the water solubility property suggests that complex TM85 could be a promising anticancer agent. PMID:24145065

  2. A Digital Framework to Build, Visualize and Analyze a Gene Expression Atlas with Cellular Resolution in Zebrafish Early Embryogenesis

    PubMed Central

    Castro-González, Carlos; Luengo-Oroz, Miguel A.; Duloquin, Louise; Savy, Thierry; Rizzi, Barbara; Desnoulez, Sophie; Doursat, René; Kergosien, Yannick L.; Ledesma-Carbayo, María J.; Bourgine, Paul

    2014-01-01

    A gene expression atlas is an essential resource to quantify and understand the multiscale processes of embryogenesis in time and space. The automated reconstruction of a prototypic 4D atlas for vertebrate early embryos, using multicolor fluorescence in situ hybridization with nuclear counterstain, requires dedicated computational strategies. To this goal, we designed an original methodological framework implemented in a software tool called Match-IT. With only minimal human supervision, our system is able to gather gene expression patterns observed in different analyzed embryos with phenotypic variability and map them onto a series of common 3D templates over time, creating a 4D atlas. This framework was used to construct an atlas composed of 6 gene expression templates from a cohort of zebrafish early embryos spanning 6 developmental stages from 4 to 6.3 hpf (hours post fertilization). They included 53 specimens, 181,415 detected cell nuclei and the segmentation of 98 gene expression patterns observed in 3D for 9 different genes. In addition, an interactive visualization software, Atlas-IT, was developed to inspect, supervise and analyze the atlas. Match-IT and Atlas-IT, including user manuals, representative datasets and video tutorials, are publicly and freely available online. We also propose computational methods and tools for the quantitative assessment of the gene expression templates at the cellular scale, with the identification, visualization and analysis of coexpression patterns, synexpression groups and their dynamics through developmental stages. PMID:24945246

  3. Cellular distribution of vascular endothelial growth factor A (VEGFA) and B (VEGFB) and VEGF receptors 1 and 2 in focal cortical dysplasia type IIB

    PubMed Central

    Boer, Karin; Troost, Dirk; Spliet, Wim G. M.; van Rijen, Peter C.; Gorter, Jan A.

    2008-01-01

    Members of the vascular endothelial growth factor (VEGF) family are key signaling proteins in the induction and regulation of angiogenesis, both during development and in pathological conditions. However, signaling mediated through VEGF family proteins and their receptors has recently been shown to have direct effects on neurons and glial cells. In the present study, we immunocytochemically investigated the expression and cellular distribution of VEGFA, VEGFB, and their associated receptors (VEGFR-1 and VEGFR-2) in focal cortical dysplasia (FCD) type IIB from patients with medically intractable epilepsy. Histologically normal temporal cortex and perilesional regions displayed neuronal immunoreactivity (IR) for VEGFA, VEGFB, and VEGF receptors (VEGFR-1 and VEGFR-2), mainly in pyramidal neurons. Weak IR was observed in blood vessels and there was no notable glial IR within the grey and white matter. In all FCD specimens, VEGFA, VEGFB, and both VEGF receptors were highly expressed in dysplastic neurons. IR in astroglial and balloon cells was observed for VEGFA and its receptors. VEGFR-1 displayed strong endothelial staining in FCD. Double-labeling also showed expression of VEGFA, VEGFB and VEGFR-1 in cells of the microglia/macrophage lineage. The neuronal expression of both VEGFA and VEGFB, together with their specific receptors in FCD, suggests autocrine/paracrine effects on dysplastic neurons. These autocrine/paracrine effects could play a role in the development of FCD, preventing the death of abnormal neuronal cells. In addition, the expression of VEGFA and its receptors in glial cells within the dysplastic cortex indicates that VEGF-mediated signaling could contribute to astroglial activation and associated inflammatory reactions. PMID:18317782

  4. Cellular localization of proenkephalin mRNA in rat brain: gene expression in the caudate-putamen and cerebellar cortex.

    PubMed Central

    Shivers, B D; Harlan, R E; Romano, G J; Howells, R D; Pfaff, D W

    1986-01-01

    The cellular locations of proenkephalin mRNA have been determined for the caudate-putamen and cerebellar cortex of the rat brain by in situ hybridization. In the caudate-putamen, more than half of the neurons express the proenkephalin gene. Morphologically, they are medium-sized cells that may represent projection neurons. In the cerebellar cortex, proenkephalin mRNA is present in a subpopulation of neurons in the granule layer that appear to be Golgi cells--i.e., inhibitory interneurons. The presence of [Met]enkephalin, a pentapeptide derived from proenkephalin, in these two brain areas is consistent with a synthetic role for this mRNA and implicates proenkephalin gene expression in the control of motor function. Images PMID:3461484

  5. Human Cortical Neural Stem Cells Expressing Insulin-Like Growth Factor-I: A Novel Cellular Therapy for Alzheimer's Disease.

    PubMed

    McGinley, Lisa M; Sims, Erika; Lunn, J Simon; Kashlan, Osama N; Chen, Kevin S; Bruno, Elizabeth S; Pacut, Crystal M; Hazel, Tom; Johe, Karl; Sakowski, Stacey A; Feldman, Eva L

    2016-03-01

    Alzheimer's disease (AD) is the most prevalent age-related neurodegenerative disorder and a leading cause of dementia. Current treatment fails to modify underlying disease pathologies and very little progress has been made to develop effective drug treatments. Cellular therapies impact disease by multiple mechanisms, providing increased efficacy compared with traditional single-target approaches. In amyotrophic lateral sclerosis, we have shown that transplanted spinal neural stem cells (NSCs) integrate into the spinal cord, form synapses with the host, improve inflammation, and reduce disease-associated pathologies. Our current goal is to develop a similar "best in class" cellular therapy for AD. Here, we characterize a novel human cortex-derived NSC line modified to express insulin-like growth factor-I (IGF-I), HK532-IGF-I. Because IGF-I promotes neurogenesis and synaptogenesis in vivo, this enhanced NSC line offers additional environmental enrichment, enhanced neuroprotection, and a multifaceted approach to treating complex AD pathologies. We show that autocrine IGF-I production does not impact the cell secretome or normal cellular functions, including proliferation, migration, or maintenance of progenitor status. However, HK532-IGF-I cells preferentially differentiate into gamma-aminobutyric acid-ergic neurons, a subtype dysregulated in AD; produce increased vascular endothelial growth factor levels; and display an increased neuroprotective capacity in vitro. We also demonstrate that HK532-IGF-I cells survive peri-hippocampal transplantation in a murine AD model and exhibit long-term persistence in targeted brain areas. In conclusion, we believe that harnessing the benefits of cellular and IGF-I therapies together will provide the optimal therapeutic benefit to patients, and our findings support further preclinical development of HK532-IGF-I cells into a disease-modifying intervention for AD.

  6. Predominant Expression of Hybrid N-Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N-Glycans

    PubMed Central

    Hall, M. Kristen; Weidner, Douglas A.; Zhu, Yong; Dayal, Sahil; Whitman, Austin A.; Schwalbe, Ruth A.

    2016-01-01

    Glycosylation modulates growth, maintenance, and stress signaling processes. Consequently, altered N-glycosylation is associated with reduced fitness and disease. Therefore, expanding our understanding of N-glycans in altering biological processes is of utmost interest. Herein, clustered regularly interspaced short palindromic repeats/caspase9 (CRISPR/Cas9) technology was employed to engineer a glycosylation mutant Chinese Hamster Ovary (CHO) cell line, K16, which expresses predominantly hybrid type N-glycans. This newly engineered cell line enabled us to compare N-glycan effects on cellular properties of hybrid type N-glycans, to the well-established Pro−5 and Lec1 cell lines, which express complex and oligomannose types of N-glycans, respectively. Lectin binding studies revealed the predominant N-glycan expressed in K16 is hybrid type. Cell dissociation and migration assays demonstrated the greatest strength of cell–cell adhesion and fastest migratory rates for oligomannose N-glycans, and these properties decreased as oligomannose type were converted to hybrid type, and further decreased upon conversion to complex type. Next, we examined the roles of three general types of N-glycans on ectopic expression of E-cadherin, a cell–cell adhesion protein. Microscopy revealed more functional E-cadherin at the cell–cell border when N-glycans were oligomannose and these levels decreased as the oligomannose N-glycans were processed to hybrid and then to complex. Thus, we provide evidence that all three general types of N-glycans impact plasma membrane architecture and cellular properties. PMID:27304954

  7. Expression of Arabidopsis FCS-Like Zinc finger genes is differentially regulated by sugars, cellular energy level, and abiotic stress

    PubMed Central

    Jamsheer K, Muhammed; Laxmi, Ashverya

    2015-01-01

    Cellular energy status is an important regulator of plant growth, development, and stress mitigation. Environmental stresses ultimately lead to energy deficit in the cell which activates the SNF1-RELATED KINASE 1 (SnRK1) signaling cascade which eventually triggering a massive reprogramming of transcription to enable the plant to survive under low-energy conditions. The role of Arabidopsis thaliana FCS-Like Zinc finger (FLZ) gene family in energy and stress signaling is recently come to highlight after their interaction with kinase subunits of SnRK1 were identified. In a detailed expression analysis in different sugars, energy starvation, and replenishment series, we identified that the expression of most of the FLZ genes is differentially modulated by cellular energy level. It was found that FLZ gene family contains genes which are both positively and negatively regulated by energy deficit as well as energy-rich conditions. Genetic and pharmacological studies identified the role of HEXOKINASE 1- dependent and energy signaling pathways in the sugar-induced expression of FLZ genes. Further, these genes were also found to be highly responsive to different stresses as well as abscisic acid. In over-expression of kinase subunit of SnRK1, FLZ genes were found to be differentially regulated in accordance with their response toward energy fluctuation suggesting that these genes may work downstream to the established SnRK1 signaling under low-energy stress. Taken together, the present study provides a conceptual framework for further studies related to SnRK1-FLZ interaction in relation to sugar and energy signaling and stress response. PMID:26442059

  8. Function and expression of a novel rat salt-tolerant protein: evidence of a role in cellular sodium metabolism.

    PubMed

    Tsuji, E; Tsuji, Y; Sasaguri, M; Arakawa, K

    1998-09-01

    Higher dietary salt intake in humans is associated with higher BP, but the BP response to NaCl, so-called salt sensitivity, is heterogeneous among individuals. It has been postulated that modifications in cellular cation metabolism may be related to salt sensitivity in mammalian hypertension. The authors have isolated a novel rat complementary DNA, called salt-tolerant protein (STP), that can functionally complement Saccharomyces cervisiae HAL1, which improves salt tolerance by modulating the cation transport system. On high-salt (8% NaCl) diets, both Dahl salt-sensitive and salt-resistant rats displayed an elevated BP and increased STP mRNA expression. Immunohistochemistry using an anti-rat STP antibody demonstrated the presence of STP immunoreactivity in the proximal tubules. In cells that transiently expressed STP, the intracellular [Na+]/[K+] ratio was higher than that in control cells. STP contains predicted coiled-coil and Src homology 3 domains, and shows a partially high degree of nucleotide identity to human thyroid-hormone receptor interacting protein. These results suggest that STP may play an important role in salt sensitivity through cellular sodium metabolism by mediating signal transduction and a hormone-dependent transcription mechanism. PMID:9727364

  9. Resistance to Degradation and Cellular Distribution are Important Features for the Antitumor Activity of Gomesin

    PubMed Central

    Buri, Marcus V.; Domingues, Tatiana M.; Paredes-Gamero, Edgar J.; Casaes-Rodrigues, Rafael L.; Rodrigues, Elaine Guadelupe; Miranda, Antonio

    2013-01-01

    Many reports have shown that antimicrobial peptides exhibit anticancer abilities. Gomesin (Gm) exhibits potent cytotoxic activity against cancer cells by a membrane pore formation induced after well-orchestrated intracellular mechanisms. In this report, the replacements of the Cys by Ser or Thr, and the use D-amino acids in the Gm structure were done to investigate the importance of the resistance to degradation of the molecule with its cytotoxicity. [Thr2,6,11,15]-Gm, and [Ser2,6,11,15]-Gm exhibits low cytotoxicity, and low resistance to degradation, and after 24 h are present in localized area near to the membrane. Conversely, the use of D-amino acids in the analogue [D-Thr2,6,11,15]-D-Gm confers resistance to degradation, increases its potency, and maintained this peptide spread in the cytosol similarly to what happens with Gm. Replacements of Cys by Thr and Gln by L- or D-Pro ([D-Thr2,6,11,15, Pro9]-D-Gm, and [Thr2,6,11,15, D-Pro9]-Gm), which induced a similar β-hairpin conformation, also increase their resistance to degradation, and cytotoxicity, but after 24 h they are not present spread in the cytosol, exhibiting lower cytotoxicity in comparison to Gm. Additionally, chloroquine, a lysosomal enzyme inhibitor potentiated the effect of the peptides. Furthermore, the binding and internalization of peptides was determined, but a direct correlation among these factors was not observed. However, cholesterol ablation, which increase fluidity of cellular membrane, also increase cytotoxicity and internalization of peptides. β-hairpin spatial conformation, and intracellular localization/target, and the capability of entry are important properties of gomesin cytotoxicity. PMID:24312251

  10. In Absence of the Cellular Prion Protein, Alterations in Copper Metabolism and Copper-Dependent Oxidase Activity Affect Iron Distribution

    PubMed Central

    Gasperini, Lisa; Meneghetti, Elisa; Legname, Giuseppe; Benetti, Federico

    2016-01-01

    Essential elements as copper and iron modulate a wide range of physiological functions. Their metabolism is strictly regulated by cellular pathways, since dysregulation of metal homeostasis is responsible for many detrimental effects. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and prion diseases are characterized by alterations of metal ions. These neurodegenerative maladies involve proteins that bind metals and mediate their metabolism through not well-defined mechanisms. Prion protein, for instance, interacts with divalent cations via multiple metal-binding sites and it modulates several metal-dependent physiological functions, such as S-nitrosylation of NMDA receptors. In this work we focused on the effect of prion protein absence on copper and iron metabolism during development and adulthood. In particular, we investigated copper and iron functional values in serum and several organs such as liver, spleen, total brain and isolated hippocampus. Our results show that iron content is diminished in prion protein-null mouse serum, while it accumulates in liver and spleen. Our data suggest that these alterations can be due to impairments in copper-dependent cerulopalsmin activity which is known to affect iron mobilization. In prion protein-null mouse total brain and hippocampus, metal ion content shows a fluctuating trend, suggesting the presence of homeostatic compensatory mechanisms. However, copper and iron functional values are likely altered also in these two organs, as indicated by the modulation of metal-binding protein expression levels. Altogether, these results reveal that the absence of the cellular prion protein impairs copper metabolism and copper-dependent oxidase activity, with ensuing alteration of iron mobilization from cellular storage compartments. PMID:27729845

  11. Expression of Cellular Components in Granulomatous Inflammatory Response in Piaractus mesopotamicus Model

    PubMed Central

    Manrique, Wilson Gómez; da Silva Claudiano, Gustavo; de Castro, Marcello Pardi; Petrillo, Thalita Regina; Figueiredo, Mayra Araguaia Pereira; de Andrade Belo, Marco Antonio; Berdeal, María Isabel Quiroga; de Moraes, Julieta Engracia Rodini; de Moraes, Flávio Ruas

    2015-01-01

    The present study aimed to describe and characterize the cellular components during the evolution of chronic granulomatous inflammation in the teleost fish pacus (P. mesopotamicus) induced by Bacillus Calmette-Guerin (BCG), using S-100, iNOS and cytokeratin antibodies. 50 fish (120±5.0 g) were anesthetized and 45 inoculated with 20 μL (40 mg/mL) (2.0 x 106 CFU/mg) and five inoculated with saline (0,65%) into muscle tissue in the laterodorsal region. To evaluate the inflammatory process, nine fish inoculated with BCG and one control were sampled in five periods: 3rd, 7th, 14th, 21st and 33rd days post-inoculation (DPI). Immunohistochemical examination showed that the marking with anti-S-100 protein and anti-iNOS antibodies was weak, with a diffuse pattern, between the third and seventh DPI. From the 14th to the 33rd day, the marking became stronger and marked the cytoplasm of the macrophages. Positivity for cytokeratin was initially observed in the 14th DPI, and the stronger immunostaining in the 33rd day, period in which the epithelioid cells were more evident and the granuloma was fully formed. Also after the 14th day, a certain degree of cellular organization was observed, due to the arrangement of the macrophages around the inoculated material, with little evidence of edema. The arrangement of the macrophages around the inoculum, the fibroblasts, the lymphocytes and, in most cases, the presence of melanomacrophages formed the granuloma and kept the inoculum isolated in the 33rd DPI. The present study suggested that the granulomatous experimental model using teleost fish P. mesopotamicus presented a similar response to those observed in mammals, confirming its importance for studies of chronic inflammatory reaction. PMID:25811875

  12. Expression and cellular localization of human hyaluronidase-2 in articular chondrocytes and cultured cell lines

    PubMed Central

    Chow, G.; Knudson, C. B.; Knudson, W.

    2011-01-01

    Summary Objective There is debate whether hyaluronan (HA) can be enzymatically degraded within the extracellular matrix of cartilage and other tissues or whether its catabolism occurs strictly within the lysosomal compartment of chondrocytes and other cell types. Previous studies have suggested that one of the lysosomal hyaluronidases (hyaluronidase-2) can be expressed as a functionally-active glycosyl phosphatidylinositol-linked protein at the surface of mammalian cells. If this form of hyaluronidase expression occurs in chondrocytes, this could represent a possible mechanism for extracellular HA cleavage. Thus, which hyaluronidases are expressed and where was the objective of this study. Methods mRNA for hyaluronidases was quantified by reverse transcription-polymerase chain reaction (RT-PCR) and enzymatic activity by HA zymograms. Recombinant forms of hyaluronidase-2 were generated and expressed in model cell lines. A peptide-specific polyclonal antiserum was prepared to localize endogenous human hyaluronidase-2 in human articular chondrocytes. Results Hyaluronidase-2 is the principal mRNA transcript expressed by primary human articular chondrocytes as well as various model cell lines. Recombinant hyaluronidase-2, containing N-terminal or C-terminal epitope tags, was strictly localized intracellularly and not released by treatment with a phosphatidylinositol-specific phospholipase. Endogenous hyaluronidase-2 expressed by human chondrocytes as well as HeLa cells could only be detected following detergent permeabilization of the plasma membranes. Conclusions These data suggest that on chondrocytes and other cell types examined, hyaluronidase-2 is not present or functional at the external plasma membrane. Thus, local turnover of HA is dependent on receptor-mediated endocytosis and delivery to low pH intracellular organelles for its complete degradation. PMID:16600643

  13. Cellular distribution of the neutral amino acid transporter subtype ASCT2 in mouse brain.

    PubMed

    Gliddon, Catherine M; Shao, Zongjun; LeMaistre, Jillian L; Anderson, Christopher M

    2009-01-01

    ASCT2 is an ASC (alanine-, serine-, cysteine-preferring) neutral amino acid exchanger that may regulate CNS function by transporting amino acid substrates including L-serine, L-cysteine, L-glutamine, L-glutamate and D-serine. Despite the potentially important role of ASCT2 in influencing metabolic and signaling functions of these amino acids in brain, there has been little description of its distribution in brain tissue. We employed a commercially available human ASCT2 antibody in immunohistochemistry studies in adult mouse brain and found a wide regional distribution for ASCT2 that was limited to dendrites labeled by anti-microtubule-associated protein-2 in cortex, hippocampus and striatum. No ASCT2 immunoreactivity was observed in areas labeled by antibodies against a neuronal cell body marker (NeuN), or either of the astrocyte markers, glial fibrillary acidic protein or S100beta. In cerebellum both Purkinje cell bodies and dendrites were positive for ASCT2 immunoreactivity. In support of a dendritic localization for ASCT2 in cortex, low affinity (K(T) > 1 mM), Na(+)-dependent D-serine and L-glutamine uptake characteristic of ASCT2-mediated transport was observed in P2 synaptosomal preparations. These results suggest that ASCT2 may be an important neuronal neutral amino acid transporter and highlight a discrepancy between findings of astrocyte ASCT2 function in tissue culture and brain in situ.

  14. Cellular distribution of calmodulin and calmodulin-binding proteins in Vicia faba L

    NASA Technical Reports Server (NTRS)

    Ling, V.; Assmann, S. M.

    1992-01-01

    The distribution of calmodulin (CaM) and CaM-binding proteins within Vicia faba was investigated. Both CaM and CaM-binding proteins were found to be differentially distributed among organs, tissues, and protoplast types. CaM levels, on a per protein basis, were found to be the highest in leaf epidermis, containing 3-fold higher levels of CaM than in total leaf. Similarly, guard cell and epidermal cell protoplasts were also found to have higher levels of CaM than mesophyll cell protoplasts. 125I-CaM blot overlay assays were performed to qualitatively examine CaM-binding proteins in these protoplast types as well as in whole tissues and organs. CaM-binding proteins with Mr 52,000, 78,000, and 115,000 were common in all metabolically active plant parts. Unique CaM-binding protein bands were detected in guard cell protoplasts (Mr 39,000, 88,000), stems (Mr 45,000, 60,000, 64,000), and roots (Mr 62,000), suggesting the presence of specialized CaM-dependent processes in these cells and organs.

  15. Gene expression profiling bovine ovarian follicular and luteal cells provides insight into cellular identities and functions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    After ovulation, somatic cells of the ovarian follicle (theca and granulosa cells) become the small and large luteal cells of the corpus luteum. Aside from known cell type-specific receptors and steroidogenic enzymes, little is known about the differences in the gene expression profiles of these fou...

  16. Cellular retinol-binding protein-1 is transiently expressed in granulation tissue fibroblasts and differentially expressed in fibroblasts cultured from different organs.

    PubMed Central

    Xu, G.; Redard, M.; Gabbiani, G.; Neuville, P.

    1997-01-01

    We have reported that cellular retinol-binding protein-1 (CRBP-1) is transiently expressed by arterial smooth muscle cells during experimental intimal repair (P. Neuville, A. Geinoz, G. Benzonana, M. Redard, F. Gabbiani, P. Ropraz, G. Gabbiani: Am J Pathol 1997, 150:509-521). We have examined here the expression of CRBP-1 during wound healing after a full-thickness rat skin wound. CRBP-1 was transiently expressed by a significant proportion of fibroblastic cells including myofibroblasts. Expression started 4 days after wounding, reached a maximum at 12 days, and persisted up to 30 days when a scar was formed. After wound closure, most CRBP-1-containing fibroblastic cells underwent apoptosis. We have further investigated CRBP-1 expression in rat fibroblasts cultured from different organs. CRBP-1 was abundant in lung and heart fibroblasts and was detected in decreasing amounts in muscle, tendon, subcutaneous tissue, and granulation tissue fibroblasts. Dermis fibroblasts contained no detectable levels of CRBP-1. All-trans retinoic acid and transforming growth factor-beta1 inhibited cell proliferation and increased CRBP-1 expression in fibroblastic populations except dermis fibroblasts. We demonstrate that during granulation tissue formation a subpopulation of fibroblastic cells express CRBP-1 de novo. We also demonstrate that CRBP-1 expression by fibroblasts is regulated in vitro by retinoic acid and transforming growth factor-beta1. Our results suggest that CRBP-1 and possibly retinoic acid play a role in the evolution of granulation tissue. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 7 PMID:9403724

  17. Effect of passage number on cellular response DNA-damaging agents: cell survival and gene expression

    SciTech Connect

    Chang-Liu, Chin-Mei; Wolschak, G.E.

    1996-03-01

    The effect of different passage numbers on plating efficiency, doubling time, cell growth, and radiation sensitivity was assessed in Syrian hamster embryo (SHE) cells. Changes in gene expression after UV or {gamma}-ray irradiation at different passage numbers were also examined. The SHE cells were maintained in culture medium for up to 64 passages. Cells were exposed to {sup 60}Co {gamma} rays or 254-m UV radiation. Differential display of cDNAs and Northern blots were used for the study of gene expression. With increasing passage number, SHE cells demonstrated decreased doubling time, increased plating efficiency, and a decreased yield in the number of cells per plate. Between passages 41 and 48 a ``crisis`` period was evident during which time cell growth in high serum (20%) was no longer optimal, and serum concentrations were reduced (to 10%) to maintain cell growth. Sensitivity to ionizing radiation was no different between early- and intermediate-passage cells. However, after UV exposure at low passages (passage 3), confluent cells were more sensitive to the killing effects of UV than were log-phase cells. At intermediate passages (passages 43, 48), confluent cells were slightly more radioresistant- than were log-phase cells. By passage 64, however, both confluent and log-phase cells showed similar patterns of UV sensitivity. Expression of {gamma}-actin, PCNA, and p53 transcripts did not change following UV exposure. p53 mRNA was induced following {gamma}-ray exposure of the intermediate (passage 45) epithelial cells. Differential display, however, revealed changes in expression of several transcripts following exposure to ionizing and ultraviolet radiations. The observed differences in radiation sensitivity associated with increasing passage number may be influenced by radiation-induced gene expression. We are conducting experiments to identify these genes.

  18. Effect of solid distribution on elastic properties of open-cell cellular solids using numerical and experimental methods.

    PubMed

    Zargarian, A; Esfahanian, M; Kadkhodapour, J; Ziaei-Rad, S

    2014-09-01

    Effect of solid distribution between edges and vertices of three-dimensional cellular solid with an open-cell structure was investigated both numerically and experimentally. Finite element analysis (FEA) with continuum elements and appropriate periodic boundary condition was employed to calculate the elastic properties of cellular solids using tetrakaidecahedral (Kelvin) unit cell. Relative densities between 0.01 and 0.1 and various values of solid fractions were considered. In order to validate the numerical model, three scaffolds with the relative density of 0.08, but different amounts of solid in vertices, were fabricated via 3-D printing technique. Good agreement was observed between numerical simulation and experimental results. Results of numerical simulation showed that, at low relative densities (<0.03), Young׳s modulus increased by shifting materials away from edges to vertices at first and then decreased after reaching a critical point. However, for the high values of relative density, Young׳s modulus increased monotonically. Mechanisms of such a behavior were discussed in detail. Results also indicated that Poisson׳s ratio decreased by increasing relative density and solid fraction in vertices. By fitting a curve to the data obtained from the numerical simulation and considering the relative density and solid fraction in vertices, empirical relations were derived for Young׳s modulus and Poisson׳s ratio. PMID:24956160

  19. Lipase genes in Mucor circinelloides: identification, sub-cellular location, phylogenetic analysis and expression profiling during growth and lipid accumulation.

    PubMed

    Zan, Xinyi; Tang, Xin; Chu, Linfang; Zhao, Lina; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Song, Yuanda

    2016-10-01

    Lipases or triacylglycerol hydrolases are widely spread in nature and are particularly common in the microbial world. The filamentous fungus Mucor circinelloides is a potential lipase producer, as it grows well in triacylglycerol-contained culture media. So far only one lipase from M. circinelloides has been characterized, while the majority of lipases remain unknown in this fungus. In the present study, 47 potential lipase genes in M. circinelloides WJ11 and 30 potential lipase genes in M. circinelloides CBS 277.49 were identified by extensive bioinformatics analysis. An overview of these lipases is presented, including several characteristics, sub-cellular location, phylogenetic analysis and expression profiling of the lipase genes during growth and lipid accumulation. All of these proteins contained the consensus sequence for a classical lipase (GXSXG motif) and were divided into four types including α/β-hydrolase_1, α/β-hydrolase_3, class_3 and GDSL lipase (GDSL) based on gene annotations. Phylogenetic analyses revealed that class_3 family and α/β-hydrolase_3 family were the conserved lipase family in M. circinelloides. Additionally, some lipases also contained a typical acyltransferase motif of H-(X) 4-D, and these lipases may play a dual role in lipid metabolism, catalyzing both lipid hydrolysis and transacylation reactions. The differential expression of all lipase genes were confirmed by quantitative real-time PCR, and the expression profiling were analyzed to predict the possible biological roles of these lipase genes in lipid metabolism in M. circinelloides. We preliminarily hypothesized that lipases may be involved in triacylglycerol degradation, phospholipid synthesis and beta-oxidation. Moreover, the results of sub-cellular localization, the presence of signal peptide and transcriptional analyses of lipase genes indicated that four lipase in WJ11 most likely belong to extracellular lipases with a signal peptide. These findings provide a platform

  20. Changes in the expression of the human adenine nucleotide translocase isoforms condition cellular metabolic/proliferative status

    PubMed Central

    Mampel, Teresa; Viñas, Octavi

    2016-01-01

    Human cells express four mitochondrial adenine nucleotide translocase (hANT) isoforms that are tissue-specific and developmentally regulated. hANT1 is mainly expressed in terminally differentiated muscle cells; hANT2 is growth-regulated and is upregulated in highly glycolytic and proliferative cells; and hANT3 is considered to be ubiquitous and non-specifically regulated. Here, we studied how the expression of hANT isoforms is regulated by proliferation and in response to metabolic stimuli, and examined the metabolic consequences of their silencing and overexpression. In HeLa and HepG2 cells, expression of hANT3 was upregulated by shifting metabolism towards oxidation or by slowed growth associated with contact inhibition or growth-factor deprivation, indicating that hANT3 expression is highly regulated. Under these conditions, changes in hANT2 mRNA expression were not observed in either HeLa or HepG2 cells, whereas in SGBS preadipocytes (which, unlike HeLa and HepG2 cells, are growth-arrest-sensitive cells), hANT2 mRNA levels decreased. Additionally, overexpression of hANT2 promoted cell growth and glycolysis, whereas silencing of hANT3 decreased cellular ATP levels, limited cell growth and induced a stress-like response. Thus, cancer cells require both hANT2 and hANT3, depending on their proliferation status: hANT2 when proliferation rates are high, and hANT3 when proliferation slows. PMID:26842067

  1. Cellular and subcellular distributions of delta opioid receptor activation sites in the ventral oral pontine tegmentum of the cat.

    PubMed

    Alvira-Botero, Maria Ximena; Garzón, Miguel

    2006-12-01

    The ventral division of the reticular oral pontine nucleus (vRPO) is a pontine tegmentum region critically involved in REM sleep generation. Previous reports of morphine microinjections in the cat pontine tegmentum have shown that opioid receptor activation in this region modulates REM sleep. Even though opiate administration has marked effects on sleep-wake cycle architecture, the distribution of opioid receptors in vRPO has only been partially described. Using an antiserum directed against delta opioid receptor (DOR), to which morphine binds, in the present study, we use (1) light microscopy to determine DOR cellular distribution in the rostral pontine tegmentum and (2) electron microscopy to determine DOR subcellular distribution in the cat vRPO. In the dorsal pons, DOR immunoreactivity was evenly distributed throughout the neuropil of the reticular formation and was particularly intense in the parabrachial nuclei and locus coeruleus; the ventral and central areas of the RPO and locus coeruleus complex were especially rich in DOR-labeled somata. Within the vRPO, DOR was localized mainly in the cytoplasm and on plasma membranes of medium to large dendrites (47.8% of DOR-labeled profiles), which received both symmetric and asymmetric synaptic contacts mainly from non-labeled (82% of total inputs) axon terminals. Less frequently, DOR was distributed presynaptically in axon terminals (19% of DOR-labeled profiles). Our results suggest that DOR activation in vRPO regulates REM sleep occurrence by modulating postsynaptic responses to both excitatory and inhibitory afferents. DOR activation in vRPO could have, however, an additional role in direct modulation of neurotransmitter release from axon terminals.

  2. Cellular bioenergetics changes in magnocellular neurons may affect copeptin expression in the late phase of sepsis.

    PubMed

    Oliveira-Pelegrin, Gabriela R; Basso, Paulo J; Rocha, Maria José A

    2014-02-15

    We investigated whether inflammatory mediators during cecal ligation and puncture (CLP)-induced sepsis may diminish copeptin expression in magnocellular neurons, thus affecting arginine-vasopressin (AVP) synthesis. The transcript abundance of IL-1β, IL-1R1, iNOS and HIF-1α was continuously elevated. IL-1β, iNOS and cytochrome c protein levels progressively increased until 24h. Immunostaining for these proteins was higher at 6 and 24h, as also seen in the annexin-V assay, while copeptin was continuously decreased. This suggests that increased IL-1β and NO levels may cause significant bioenergetics changes in magnocellular neurons, affecting copeptin expression and compromising AVP synthesis and secretion in the late phase of sepsis.

  3. Distinctive expression of extracellular matrix molecules at mRNA and protein levels during formation of cellular and acellular cementum in the rat.

    PubMed

    Sasano, Y; Maruya, Y; Sato, H; Zhu, J X; Takahashi, I; Mizoguchi, I; Kagayama, M

    2001-02-01

    Little is known about differential expression of extracellular matrices secreted by cementoblasts between cellular and acellular cementum. We hypothesize that cementoblasts lining acellular cementum express extracellular matrix genes differently from those lining cellular cementum, thereby forming two distinct types of extracellular matrices. To test this hypothesis, we investigated spatial and temporal gene expression of selected extracellular matrix molecules, that is type I collagen, bone sialoprotein, osteocalcin and osteopontin, during formation of both cellular and acellular cementum using in situ hybridization. In addition, their extracellularly deposited and accumulated proteins were examined immunohistochemically. The mRNA transcripts of pro-alpha1 (I) collagen were primarily localized in cementoblasts of cellular cementum and cementocytes, while those of bone sialoprotein were predominantly seen in cementoblasts lining acellular cementum. In contrast, osteocalcin was expressed by both types of cementoblasts and cementocytes and so was osteopontin but only transiently. Our immunohistochemical examination revealed that translated proteins were localized extracellularly where the genes had been expressed intracellularly. The present study demonstrated the distinctive expression of genes and proteins of the extracellular matrix molecules between cellular and acellular cementum. PMID:11432645

  4. The EHV-1 UL4 protein that tempers viral gene expression interacts with cellular transcription factors.

    PubMed

    Zhang, Yunfei; Charvat, Robert A; Kim, Seong K; O'Callaghan, Dennis J

    2014-01-20

    The UL4 gene is conserved within the genome of defective interfering particles of equine herpesvirus type 1 (EHV-1) that mediate persistent infection. Here, we show that the UL4 protein inhibits EHV-1 reporter gene expression by decreasing the level of transcribed mRNA. The UL4 protein did not bind any gene class of EHV-1 promoters in electromobility or chromatin immunoprecipitation assays, but directly interacted with the TATA box-binding protein (TBP) and the carboxy-terminal domain of RNA polymerase II both in vitro (GST-pulldown assays) and in infected cells (coimmunoprecipitation analyses). Microarray analyses of the expression of the 78 EHV-1 genes revealed that viral late genes important for virion assembly displayed enhanced expression in cells infected with UL4-null virus as compared to wild-type or UL4-restored EHV-1. Quantitative PCR analyses showed that viral DNA replication was not retarded in cells infected with the UL4-null virus as compared to wild-type EHV-1. PMID:24418534

  5. Analytic expression for in-field scattered light distribution

    NASA Astrophysics Data System (ADS)

    Peterson, Gary L.

    2004-01-01

    Light that is scattered from lenses and mirrors in an optical system produces a halo of stray light around bright objects within the field of view. The angular distribution of scattered light from any one component is usually described by the Harvey model. This paper presents analytic expressions for the scattered irradiance at a focal plane from optical components that scatter light in accordance with the Harvey model. It is found that the irradiance is independent of the location of an optical element within the system, provided the element is not located at or near an intermediate image plane. It is also found that the irradiance has little or no dependence on the size of the element.

  6. Cellular fibronectin 1 promotes VEGF-C expression, lymphangiogenesis and lymph node metastasis associated with human oral squamous cell carcinoma.

    PubMed

    Morita, Yoshihiro; Hata, Kenji; Nakanishi, Masako; Omata, Tetsuji; Morita, Nobuo; Yura, Yoshiaki; Nishimura, Riko; Yoneda, Toshiyuki

    2015-10-01

    Lymph node metastasis (LNM) is associated with poor survival in patients with oral squamous cell carcinoma (OSCC). Vascular endothelial growth factor-C (VEGF-C) is thought to be responsible for increased lymphangiogenesis and LNM. Understanding of the mechanism by which VEGF-C expression is regulated in OSCC is thus important to design logic therapeutic interventions. We showed that inoculation of the SAS human OSCC cells expressing the venus GFP (V-SAS cells) into the tongue in nude mice developed LNM. V-SAS cells in LNM were isolated by FACS and re-inoculated into the tongue. This procedure was repeated eight times, establishing V-SAS-LM8 cells. Differential metastasis PCR array between the parental V-SAS and V-SAS-LM8 was performed to identify a molecule responsible for lymphangiogenesis and LNM. Fibronectin 1 (FN1) expression was elevated in V-SAS-LM8 cells compared to V-SAS-cells. V-SAS-LM8 tongue tumor showed increased expression of FN1 and VEGF-C, and promoted lymphangiogenesis and LNM compared with V-SAS tumor. Further, phosphorylation of focal adhesion kinase (FAK), a main downstream signaling molecule of FN1, was up-regulated, and epithelial-mesenchymal transition (EMT) was promoted in V-SAS-LM8 cells. Silencing of FN1 by shRNA in V-SAS-LM8 cells decreased FAK phosphorylation, VEGF-C expression and inhibited lymphangiogenesis and LNM. EMT was also reversed. The FAK phosphorylation inhibitor PF573228 also decreased VEGF-C expression and reversed EMT in V-SAS-LM8 cells. Finally, we detected intense FN1 expression in some clinical specimens obtained from OSCC patients with LNM. These results demonstrate that elevated expression of cellular FN1 and following activation of FAK lead to increased VEGF-C expression, lymphangiogenesis and LNM and promoted EMT in SAS human OSCC cells and suggest that FN1-phosphorylated FAK signaling cascade is a potential therapeutic target in the treatment of LNM in OSCC. PMID:26319373

  7. First cellular approach of the effects of global warming on groundwater organisms: a study of the HSP70 gene expression

    PubMed Central

    Morales, Anne; Hervant, Frédéric; Konecny, Lara; Moulin, Colette; Douady, Christophe J.

    2009-01-01

    Whereas the consequences of global warming at population or community levels are well documented, studies at the cellular level are still scarce. The study of the physiological or metabolic effects of such small increases in temperature (between +2°C and +6°C) is difficult because they are below the amplitude of the daily or seasonal thermal variations occurring in most environments. In contrast, subterranean biotopes are highly thermally buffered (±1°C within a year), and underground water organisms could thus be particularly well suited to characterise cellular responses of global warming. To this purpose, we studied genes encoding chaperone proteins of the HSP70 family in amphipod crustaceans belonging to the ubiquitous subterranean genus Niphargus. An HSP70 sequence was identified in eight populations of two complexes of species of the Niphargus genus (Niphargus rhenorhodanensis and Niphargus virei complexes). Expression profiles were determined for one of these by reverse transcription and quantitative polymerase chain reaction, confirming the inducible nature of this gene. An increase in temperature of 2°C seemed to be without effect on N. rhenorhodanensis physiology, whereas a heat shock of +6°C represented an important thermal stress for these individuals. Thus, this study shows that although Niphargus individuals do not undergo any daily or seasonal thermal variations in underground water, they display an inducible HSP70 heat shock response. This controlled laboratory-based physiological experiment constitutes a first step towards field investigations of the cellular consequences of global warming on subterranean organisms. PMID:19777376

  8. Secretory proteins characteristic of environmental changes in cellular signal transduction: Expression in oral fluid

    NASA Astrophysics Data System (ADS)

    Mednieks, M. I.; Burke, J. C.; Sivakumar, T. P.; Hand, A. R.; Grindeland, R. E.

    2000-01-01

    Past studies have shown that both hypo- and hyper-gravity have significant consequences on a variety of tissues and organ systems. It is not known if the effects of environmental stimuli such as altered gravity are beneficial or detrimental, and if the effects can be prevented or reversed. Animal experiments from the Space Lab and Cosmos missions indicate that events that are mediated by cyclic AMP, such as cellular responses to catecholamine and peptide hormone action, are significantly altered in a number of tissues as a consequence of space flight. A secretory cyclic AMP-receptor protein (cARP), is present in saliva, and can serve as an indicator of individual responses to physiologic and environmental stress. Animal experiments have shown that the hypergravity component of space flight is a significant stress factor. In humans, cARP levels in each individual are constant under normal conditions, but elevated after acute stress. Additionally, the levels of cARP in secreted saliva can be compared to those in gingival crevicular fluid (GCF), which reflects the protein composition of serum. The ratio of cARP in saliva to that in GCF can be used as a measure of basal compared to hyper-or hypo-gravity values. An ultimate goal is to test hyper and zero G responses in human saliva to determine if cARP is a suitable index of acute and chronic stress. A miniaturized test kit for saliva collection has been designed. Samples can be collected and stored till analyses are carried out that will distinguish the effects of increased gravity from those of one and zero G. Such tests can serve as an individualized monitoring system for physiologic responses either in space or on earth. .

  9. Expression of a mutant human fibrillin allele upon a normal human or murine genetic background recapitulates a Marfan cellular phenotype.

    PubMed Central

    Eldadah, Z A; Brenn, T; Furthmayr, H; Dietz, H C

    1995-01-01

    The Marfan syndrome (MFS) is a connective tissue disorder inherited as an autosomal dominant trait and caused by mutations in the gene encoding fibrillin, a 350-kD glycoprotein that multimerizes to form extracellular microfibrils. It has been unclear whether disease results from a relative deficiency of wild-type fibrillin; from a dominant-negative effect, in which mutant fibrillin monomers disrupt the function of the wild-type protein encoded by the normal allele; or from a dynamic and variable interplay between these two pathogenetic mechanisms. We have now addressed this issue in a cell culture system. A mutant fibrillin allele from a patient with severe MFS was expressed in normal human and murine fibroblasts by stable transfection. Immunohistochemical analysis of the resultant cell lines revealed markedly diminished fibrillin deposition and disorganized microfibrillar architecture. Pulse-chase studies demonstrated normal levels of fibrillin synthesis but substantially reduced deposition into the extracellular matrix. These data illustrate that expression of a mutant fibrillin allele, on a background of two normal alleles, is sufficient to disrupt normal microfibrillar assembly and reproduce the MFS cellular phenotype. This underscores the importance of the fibrillin amino-terminus in normal microfibrillar assembly and suggests that expression of the human extreme 5' fibrillin coding sequence may be sufficient, in isolation, to produce an animal model of MFS. Lastly, this substantiation of a dominant-negative effect offers mutant allele knockout as a potential strategy for gene therapy. Images PMID:7860770

  10. Systematic Analysis of Multiwalled Carbon Nanotube-Induced Cellular Signaling and Gene Expression in Human Small Airway Epithelial Cells

    PubMed Central

    Snyder-Talkington, Brandi N.

    2013-01-01

    Multiwalled carbon nanotubes (MWCNT) are one of the most commonly produced nanomaterials, and pulmonary exposure during production, use, and disposal is a concern for the developing nanotechnology field. The airway epithelium is the first line of defense against inhaled particles. In a mouse model, MWCNT were reported to reach the alveolar space of the lung after in vivo exposure, penetrate the epithelial lining, and result in inflammation and progressive fibrosis. This study sought to determine the cellular and gene expression changes in small airway epithelial cells (SAEC) after in vitro exposure to MWCNT in an effort to elucidate potential toxicity mechanisms and signaling pathways. A direct interaction between SAEC and MWCNT was confirmed by both internalization of MWCNT and interaction at the cell periphery. Following exposure, SAEC showed time-dependent increases in reactive oxygen species production, total protein phosphotyrosine and phosphothreonine levels, and migratory behavior. Analysis of gene and protein expression suggested altered regulation of multiple biomarkers of lung damage, carcinogenesis, and tumor progression, as well as genes involved in related signaling pathways. These results demonstrate that MWCNT exposure resulted in the activation of SAEC. Gene expression data derived from MWCNT exposure provide information that may be used to elucidate the underlying mode of action of MWCNT in the small airway and suggest potential prognostic gene signatures for risk assessment. PMID:23377615

  11. Expression in Arabidopsis and cellular localization reveal involvement of rice NRAMP, OsNRAMP1, in arsenic transport and tolerance.

    PubMed

    Tiwari, Manish; Sharma, Deepika; Dwivedi, Sanjay; Singh, Munna; Tripathi, Rudra Deo; Trivedi, Prabodh Kumar

    2014-01-01

    Irrigation of paddy fields to arsenic (As) containing groundwater leads to As accumulation in rice grains and causes serious health risk to the people worldwide. To reduce As intake via consumption of contaminated rice grain, identification of the mechanisms for As accumulation and detoxification in rice is a prerequisite. Herein, we report involvement of a member of rice NRAMP (Natural Resistance-Associated Macrophage Protein) transporter, OsNRAMP1, in As, in addition to cadmium (Cd), accumulation through expression in yeast and Arabidopsis. Expression of OsNRAMP1 in yeast mutant (fet3fet4) rescued iron (Fe) uptake and exhibited enhanced accumulation of As and Cd. Expression of OsNRAMP1 in Arabidopsis provided tolerance with enhanced As and Cd accumulation in root and shoot. Cellular localization revealed that OsNRAMP1 resides on plasma membrane of endodermis and pericycle cells and may assist in xylem loading for root to shoot mobilization. This is the first report demonstrating role of NRAMP in xylem mediated loading and enhanced accumulation of As and Cd in plants. We propose that genetic modification of OsNRAMP1 in rice might be helpful in developing rice with low As and Cd content in grain and minimize the risk of food chain contamination to these toxic metals. PMID:23700971

  12. Expression in Arabidopsis and cellular localization reveal involvement of rice NRAMP, OsNRAMP1, in arsenic transport and tolerance.

    PubMed

    Tiwari, Manish; Sharma, Deepika; Dwivedi, Sanjay; Singh, Munna; Tripathi, Rudra Deo; Trivedi, Prabodh Kumar

    2014-01-01

    Irrigation of paddy fields to arsenic (As) containing groundwater leads to As accumulation in rice grains and causes serious health risk to the people worldwide. To reduce As intake via consumption of contaminated rice grain, identification of the mechanisms for As accumulation and detoxification in rice is a prerequisite. Herein, we report involvement of a member of rice NRAMP (Natural Resistance-Associated Macrophage Protein) transporter, OsNRAMP1, in As, in addition to cadmium (Cd), accumulation through expression in yeast and Arabidopsis. Expression of OsNRAMP1 in yeast mutant (fet3fet4) rescued iron (Fe) uptake and exhibited enhanced accumulation of As and Cd. Expression of OsNRAMP1 in Arabidopsis provided tolerance with enhanced As and Cd accumulation in root and shoot. Cellular localization revealed that OsNRAMP1 resides on plasma membrane of endodermis and pericycle cells and may assist in xylem loading for root to shoot mobilization. This is the first report demonstrating role of NRAMP in xylem mediated loading and enhanced accumulation of As and Cd in plants. We propose that genetic modification of OsNRAMP1 in rice might be helpful in developing rice with low As and Cd content in grain and minimize the risk of food chain contamination to these toxic metals.

  13. Broad MICA/B Expression in the Small Bowel Mucosa: A Link between Cellular Stress and Celiac Disease

    PubMed Central

    Allegretti, Yessica L.; Bondar, Constanza; Guzman, Luciana; Cueto Rua, Eduardo; Chopita, Nestor; Fuertes, Mercedes; Zwirner, Norberto W.; Chirdo, Fernando G.

    2013-01-01

    The MICA/B genes (MHC class I chain related genes A and B) encode for non conventional class I HLA molecules which have no role in antigen presentation. MICA/B are up-regulated by different stress conditions such as heat-shock, oxidative stress, neoplasic transformation and viral infection. Particularly, MICA/B are expressed in enterocytes where they can mediate enterocyte apoptosis when recognised by the activating NKG2D receptor present on intraepithelial lymphocytes. This mechanism was suggested to play a major pathogenic role in active celiac disease (CD). Due to the importance of MICA/B in CD pathogenesis we studied their expression in duodenal tissue from CD patients. By immunofluorescence confocal microscopy and flow cytometry we established that MICA/B was mainly intracellularly located in enterocytes. In addition, we identified MICA/B+ T cells in both the intraepithelial and lamina propria compartments. We also found MICA/B+ B cells, plasma cells and some macrophages in the lamina propria. The pattern of MICA/B staining in mucosal tissue in severe enteropathy was similar to that found in in vitro models of cellular stress. In such models, MICA/B were located in stress granules that are associated to the oxidative and ER stress response observed in active CD enteropathy. Our results suggest that expression of MICA/B in the intestinal mucosa of CD patients is linked to disregulation of mucosa homeostasis in which the stress response plays an active role. PMID:24058482

  14. Broad MICA/B expression in the small bowel mucosa: a link between cellular stress and celiac disease.

    PubMed

    Allegretti, Yessica L; Bondar, Constanza; Guzman, Luciana; Cueto Rua, Eduardo; Chopita, Nestor; Fuertes, Mercedes; Zwirner, Norberto W; Chirdo, Fernando G

    2013-01-01

    The MICA/B genes (MHC class I chain related genes A and B) encode for non conventional class I HLA molecules which have no role in antigen presentation. MICA/B are up-regulated by different stress conditions such as heat-shock, oxidative stress, neoplasic transformation and viral infection. Particularly, MICA/B are expressed in enterocytes where they can mediate enterocyte apoptosis when recognised by the activating NKG2D receptor present on intraepithelial lymphocytes. This mechanism was suggested to play a major pathogenic role in active celiac disease (CD). Due to the importance of MICA/B in CD pathogenesis we studied their expression in duodenal tissue from CD patients. By immunofluorescence confocal microscopy and flow cytometry we established that MICA/B was mainly intracellularly located in enterocytes. In addition, we identified MICA/B(+) T cells in both the intraepithelial and lamina propria compartments. We also found MICA/B(+) B cells, plasma cells and some macrophages in the lamina propria. The pattern of MICA/B staining in mucosal tissue in severe enteropathy was similar to that found in in vitro models of cellular stress. In such models, MICA/B were located in stress granules that are associated to the oxidative and ER stress response observed in active CD enteropathy. Our results suggest that expression of MICA/B in the intestinal mucosa of CD patients is linked to disregulation of mucosa homeostasis in which the stress response plays an active role.

  15. Allelic expression mapping across cellular lineages to establish impact of non-coding SNPs

    PubMed Central

    Adoue, Veronique; Schiavi, Alicia; Light, Nicholas; Almlöf, Jonas Carlsson; Lundmark, Per; Ge, Bing; Kwan, Tony; Caron, Maxime; Rönnblom, Lars; Wang, Chuan; Chen, Shu-Huang; Goodall, Alison H; Cambien, Francois; Deloukas, Panos; Ouwehand, Willem H; Syvänen, Ann-Christine; Pastinen, Tomi

    2014-01-01

    Most complex disease-associated genetic variants are located in non-coding regions and are therefore thought to be regulatory in nature. Association mapping of differential allelic expression (AE) is a powerful method to identify SNPs with direct cis-regulatory impact (cis-rSNPs). We used AE mapping to identify cis-rSNPs regulating gene expression in 55 and 63 HapMap lymphoblastoid cell lines from a Caucasian and an African population, respectively, 70 fibroblast cell lines, and 188 purified monocyte samples and found 40–60% of these cis-rSNPs to be shared across cell types. We uncover a new class of cis-rSNPs, which disrupt footprint-derived de novo motifs that are predominantly bound by repressive factors and are implicated in disease susceptibility through overlaps with GWAS SNPs. Finally, we provide the proof-of-principle for a new approach for genome-wide functional validation of transcription factor–SNP interactions. By perturbing NFκB action in lymphoblasts, we identified 489 cis-regulated transcripts with altered AE after NFκB perturbation. Altogether, we perform a comprehensive analysis of cis-variation in four cell populations and provide new tools for the identification of functional variants associated to complex diseases. PMID:25326100

  16. Alternative splicing, a new target to block cellular gene expression by poliovirus 2A protease

    SciTech Connect

    Alvarez, Enrique; Castello, Alfredo; Carrasco, Luis; Izquierdo, Jose M.

    2011-10-14

    Highlights: {yields} Novel role for poliovirus 2A protease as splicing modulator. {yields} Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. {yields} Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A{sup pro} modulating the alternative splicing of pre-mRNAs. Expression of 2A{sup pro} potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A{sup pro} abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A{sup pro}, leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A{sup pro} on splicing is to selectively block the second catalytic step.

  17. Potential for cellular stress response to hepatic factor VIII expression from AAV vector

    PubMed Central

    Zolotukhin, Irene; Markusic, David M; Palaschak, Brett; Hoffman, Brad E; Srikanthan, Meera A; Herzog, Roland W

    2016-01-01

    Hemophilia A and B are coagulation disorders resulting from the loss of functional coagulation factor VIII (FVIII) or factor IX proteins, respectively. Gene therapy for hemophilia with adeno-associated virus vectors has shown efficacy in hemophilia B patients. Although hemophilia A patients are more prevalent, the development of therapeutic adeno-associated virus vectors has been impeded by the size of the F8 cDNA and impaired secretion of FVIII protein. Further, it has been reported that over-expression of the FVIII protein induces endoplasmic reticulum stress and activates the unfolded protein response pathway both in vitro and in hepatocytes in vivo, presumably due to retention of misfolded FVIII protein within the endoplasmic reticulum. Engineering of the F8 transgene, including removal of the B domain (BDD-FVIII) and codon optimization, now allows for the generation of adeno-associated virus vectors capable of expressing therapeutic levels of FVIII. Here we sought to determine if the risks of inducing the unfolded protein response in murine hepatocytes extend to adeno-associated virus gene transfer. Although our data show a mild activation of unfolded protein response markers following F8 gene delivery at a certain vector dose in C57BL/6 mice, it was not augmented upon further elevated dosing, did not induce liver pathology or apoptosis, and did not impact FVIII immunogenicity. PMID:27738644

  18. Computational evaluation of cellular metabolic costs successfully predicts genes whose expression is deleterious

    PubMed Central

    Wagner, Allon; Zarecki, Raphy; Reshef, Leah; Gochev, Camelia; Sorek, Rotem; Gophna, Uri; Ruppin, Eytan

    2013-01-01

    Gene suppression and overexpression are both fundamental tools in linking genotype to phenotype in model organisms. Computational methods have proven invaluable in studying and predicting the deleterious effects of gene deletions, and yet parallel computational methods for overexpression are still lacking. Here, we present Expression-Dependent Gene Effects (EDGE), an in silico method that can predict the deleterious effects resulting from overexpression of either native or foreign metabolic genes. We first test and validate EDGE’s predictive power in bacteria through a combination of small-scale growth experiments that we performed and analysis of extant large-scale datasets. Second, a broad cross-species analysis, ranging from microorganisms to multiple plant and human tissues, shows that genes that EDGE predicts to be deleterious when overexpressed are indeed typically down-regulated. This reflects a universal selection force keeping the expression of potentially deleterious genes in check. Third, EDGE-based analysis shows that cancer genetic reprogramming specifically suppresses genes whose overexpression impedes proliferation. The magnitude of this suppression is large enough to enable an almost perfect distinction between normal and cancerous tissues based solely on EDGE results. We expect EDGE to advance our understanding of human pathologies associated with up-regulation of particular transcripts and to facilitate the utilization of gene overexpression in metabolic engineering. PMID:24198337

  19. Cellular extract facilitates nuclear reprogramming by altering DNA methylation and pluripotency gene expression.

    PubMed

    Xiong, Xian-Rong; Lan, Dao-Liang; Li, Jian; Zi, Xiang-Dong; Ma, Li; Wang, Yong

    2014-06-01

    The functional reprogramming of a differentiated cell to a pluripotent state presents potential beneficial applications in disease mechanisms and regenerative medicine. Epigenetic modifications enable differentiated cells to perpetuate molecular memory to retain their identity. Therefore, the aim of this study was to investigate the reprogramming modification of yak fibroblast cells that were permeabilized and incubated in the extracts of mesenchymal stem cells derived from mice adipose tissue [adipose-derived stem cells (ADSCs)]. According to the results, the treatment of ADSC extracts promoted colony formation. Moreover, pluripotent gene expression was associated with the loss of repressive histone modifications and increased global demethylation. The genes Col1a1 and Col1a2, which are typically found in differentiated cells only, demonstrated decreased expression and increased methylation in the 5'-flanking regulatory regions. Moreover, yak fibroblast cells that were exposed to ADSC extracts resulted in significantly different eight-cell and blastocyst formation rates of cloned embryos compared with their untreated counterparts. This investigation provides the first evidence that nuclear reprogramming of yak fibroblast cells is modified after the ADSC extract treatment. This research also presents a methodology for studying the dedifferentiation of somatic cells that can potentially lead to an efficient way of reprogramming somatic cells toward a pluripotent state without genetic alteration. PMID:24738992

  20. Haplotype Structure and Expression Divergence at the Drosophila Cellular Immune Gene eater

    PubMed Central

    Juneja, Punita; Lazzaro, Brian P.

    2010-01-01

    The protein Eater plays an important role in microbial recognition and defensive phagocytosis in Drosophila melanogaster. We sequenced multiple alleles of the eater gene from an African and a North American population of D. melanogaster and found signatures of a partial selective sweep in North America that is localized around the second intron. This pattern is consistent with local adaptation to novel selective pressures during range expansion out of Africa. The North American sample is divided into two predominant haplotype groups, and the putatively selected haplotype is associated with a significantly higher gene expression level, suggesting that gene regulation is a possible target of selection. The eater alleles contain from 22 to 40 repeat units that are characterized by the presence of a cysteine-rich NIM motif. NIM repeats in the structural stalk of the protein exhibit concerted evolution as a function of physical location in the repeat array. Several NIM repeats within eater have previously been implicated in binding to microbial ligands, a function which in principle might subject them to special evolutionary pressures. However, we find no evidence of elevated positive selection on these pathogen-interacting units. Our study presents an instance where gene expression rather than protein structure is thought to drive the adaptive evolution of a pathogen recognition molecule in the immune system. PMID:20444883

  1. Expression kinetics of hepatic progenitor markers in cellular models of human liver development recapitulating hepatocyte and biliary cell fate commitment.

    PubMed

    Chaudhari, Pooja; Tian, Lipeng; Deshmukh, Abhijeet; Jang, Yoon-Young

    2016-09-01

    Due to the limitations of research using human embryos and the lack of a biological model of human liver development, the roles of the various markers associated with liver stem or progenitor cell potential in humans are largely speculative, and based on studies utilizing animal models and certain patient tissues. Human pluripotent stem cell-based in vitro multistage hepatic differentiation systems may serve as good surrogate models for mimicking normal human liver development, pathogenesis and injury/regeneration studies. Here, we describe the implications of various liver stem or progenitor cell markers and their bipotency (i.e. hepatocytic- and biliary-epithelial cell differentiation), based on the pluripotent stem cell-derived model of human liver development. Future studies using the human cellular model(s) of liver and biliary development will provide more human relevant biological and/or pathological roles of distinct markers expressed in heterogeneous liver stem/progenitor cell populations.

  2. High-resolution MALDI imaging mass spectrometry allows localization of peptide distributions at cellular length scales in pituitary tissue sections

    NASA Astrophysics Data System (ADS)

    Altelaar, A. F. Maarten; Taban, Ioana M.; McDonnell, Liam A.; Verhaert, Peter D. E. M.; de Lange, Robert P. J.; Adan, Roger A. H.; Mooi, Wolter J.; Heeren, Ron M. A.; Piersma, Sander R.

    2007-02-01

    Matrix assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) has been used to determine peptide distributions directly from rat, mouse and human pituitary tissue sections. Since these organs are small (102-103 [mu]m) the spatial resolution of IMS is a key issue in molecular imaging of pituitary tissue sections. Here we show that high-resolution IMS allows localization of neuropeptide distributions within different cell clusters of a single organ of a pituitary tissue section. The sample preparation protocol does not result in analyte redistribution and is therefore applicable to IMS experiments at cellular length scales. The stigmatic imaging mass spectrometer used in this study produces selected-ion-count images with pixel sizes of 500 nm and a resolving power of 4 [mu]m, yielding superior spatial detail compared to images obtained in microprobe imaging experiments. Furthermore, we show that with imaging mass spectrometry a distinction can be made between different mammalian tissue sections based on differences in the amino acid sequence of neuropeptides with the same function. This example demonstrates the power of IMS for label-free molecular imaging at relevant biological length scales.

  3. Characterization of Antibodies to Identify Cellular Expression of Dopamine Receptor 4.

    PubMed

    Deming, Janise D; Van Craenenbroeck, Kathleen; Eom, Yun Sung; Lee, Eun-Jin; Craft, Cheryl Mae

    2016-01-01

    The dopamine receptor D4 (DRD4) plays an important role in vision. In order to study the DRD4 expression in vivo, it is important to have antibodies that are specific for DRD4 for both immunoblot and immunohistochemical (IHC) applications. In this study, six antibodies raised against DRD4 peptides were tested in vitro, using transfected mammalian cells, and in vivo, using mouse retinas. Three Santa Cruz (SC) antibodies, D-16, N-20, and R-20, were successful in IHC of transfected DRD4; however, N-20 was the only one effective on immunoblot analysis in DRD4 transfected cells and IHC of mouse retinal sections, while R-20, 2B9, and Antibody Verify AAS63631C were non-specific or below detection.

  4. Cigarette smoke effects on TSPO and VDAC expression in a cellular lung cancer model.

    PubMed

    Gavish, Moshe; Cohen, Shiri; Nagler, Rafael

    2016-09-01

    As redox iron and copper ions are found in lung pleural fluid and parenchyma, we aimed to examine the effect of cigarette smoke (CS) alone and the combined effects of CS and redox metals, iron and copper ions, containing medium (saliva), on epithelial H1299 lung cancer cells. We also examined the expression levels of the anticarcinogenic and proapoptotic 18 kDa translocator protein (TSPO) and its closely associated protein voltage-dependent anion channel (VDAC). H1299 cells were subjected to western blot analysis using anti-TSPO and anti-VDAC antibodies. With the former, the 18 kDa band appeared as expected and a 72 kDa band also appeared. It may be assumed that in H1299 lung cancer cells, an additional form of TSPO protein appears as a four-unit tetrameric complex, which is affected by CS exposure. A significant decrease in the expression level of the 72 kDa protein occurred following only 60 min of CS exposure, whereas VDAC protein levels were increased following only 30 min of CS exposure. These results, together with our previous related studies, suggest a comprehensive two-arm novel paradigm for lung cancer induced by CS, and mediated by an altered TSPO protein, possibly resulting from both the 72 kDa TSPO degradation and redox metal ion-induced enhancement of free radical attack. We suggest that both of the most important proapoptotic and anticancer proteins, p53 and TSPO, are damaged by CS, paving the way for lung cancer initiation and progression.

  5. Centromere protein B of African green monkey cells: gene structure, cellular expression, and centromeric localization.

    PubMed Central

    Yoda, K; Nakamura, T; Masumoto, H; Suzuki, N; Kitagawa, K; Nakano, M; Shinjo, A; Okazaki, T

    1996-01-01

    Centromere protein B (CENP-B) is a centromeric DNA-binding protein which recognizes a 17-bp sequence (CENP-B box) in human and mouse centromeric satellite DNA. The African green monkey (AGM) is phylogenetically closer to humans than mice and is known to contain large amounts of alpha-satellite DNA, but there has been no report of CENP-B boxes or CENP-B in the centromere domains of its chromosomes. To elucidate the AGM CENP-B-CENP-B box interaction, we have analyzed the gene structure, expression, biochemical properties, and centromeric localization of its CENP-B. The amino acid sequence deduced from the cloned AGM CENP-B gene was established to be highly homologous to that of human and mouse CENP-B. In particular, the DNA binding and homodimer formation domains demonstrated 100% identity to their human and mouse counterparts. Immunoblotting and DNA mobility shift analyses revealed CENP-B to be expressed in AGM cell lines. As predicted from the gene structure, the AGM CENP-B in the cell extracts exhibited the same DNA binding specificity and homodimer forming activity as human CENP-B. By indirect immunofluorescent staining of AGM mitotic cells with anti-CENP-B antibodies, a centromere-specific localization of AGM CENP-B could be demonstrated. We also isolated AGM alpha-satellite DNA with a CENP-B box-like sequence with CENP-B affinity. These results not only prove that CENP-B functionally persists in AGM cells but also suggest that the AGM genome contains the recognition sequences for CENP-B (CENP-B boxes with the core recognition sequence or CENP-B box variants) in centromeric satellite DNA. PMID:8756674

  6. Physiological, cellular and biochemical thermal stress response of intertidal shrimps with different vertical distributions: Palaemon elegans and Palaemon serratus.

    PubMed

    Madeira, Diana; Mendonça, Vanessa; Dias, Marta; Roma, Joana; Costa, Pedro M; Larguinho, Miguel; Vinagre, Catarina; Diniz, Mário S

    2015-05-01

    The ability to cope with high temperature variations is a critical factor in intertidal communities. Two species of intertidal rocky shore shrimps (Palaemon sp.) with different vertical distributions were collected from the Portuguese coast in order to test if they were differentially sensitive to thermal stress. Three distinct levels of biological organization (organismal, biochemical, and cellular) were surveyed. The shrimp were exposed to a constant rate of temperature increase of 1°C x h(-1), starting at 20°C until reaching the CTMax (critical thermal maximum). During heat stress, two biomarkers of protein damage were quantified in the muscle via enzyme-linked immunosorbent assays: heat shock proteins HSP70 (hsp70/hsc70) and total ubiquitin. Muscle histopathological alterations caused by temperature were also evaluated. CTMax values were not significantly different between the congeners (P. elegans 33.4 ± 0.5 °C; P. serratus 33.0 ± 0.5 °C). Biomarker levels did not increase along the temperature trial, but P. elegans (higher intertidal) showed higher amounts of HSP70 and total ubiquitin than P. serratus (lower intertidal). HSP70 and total ubiquitin levels showed a positive significant correlation in both species, suggesting that their association is important in thermal tolerance. Histopathological observations of muscle tissue in P. serratus showed no gross alterations due to temperature but did show localized atrophy of muscle fibers at CTMax. In P. elegans, alterations occurred at a larger scale, showing multiple foci of atrophic muscular fascicles caused by necrotic or autolytic processes. In conclusion, Palaemon congeners displayed different responses to stress at a cellular level, with P. elegans having greater biomarker levels and histopathological alterations. PMID:25582544

  7. Effects of Selected Egyptian Honeys on the Cellular Ultrastructure and the Gene Expression Profile of Escherichia coli.

    PubMed

    Wasfi, Reham; Elkhatib, Walid F; Khairalla, Ahmed S

    2016-01-01

    The purpose of this study was to: (i) evaluate the antibacterial activities of three Egyptian honeys collected from different floral sources (namely, citrus, clover, and marjoram) against Escherichia coli; (ii) investigate the effects of these honeys on bacterial ultrastructure; and (iii) assess the anti-virulence potential of these honeys, by examining their impacts on the expression of eight selected genes (involved in biofilm formation, quorum sensing, and stress survival) in the test organism. The minimum inhibitory concentration (MIC) of the honey samples against E. coli ATCC 8739 were assessed by the broth microdilution assay in the presence and absence of catalase enzyme. Impacts of the honeys on the cellular ultrastructure and the expression profiles of the selected genes of E. coli were examined using transmission electron microscopy (TEM) and quantitative real-time polymerase chain reaction (qPCR) analysis, respectively. The susceptibility tests showed promising antibacterial activities of all the tested honeys against E. coli. This was supported by the TEM observations, which revealed "ghost" cells lacking DNA, in addition to cells with increased vacuoles, and/or with irregular shrunken cytoplasm. Among the tested honeys, marjoram exhibited the highest total antibacterial activity and the highest levels of peroxide-dependent activity. The qPCR analysis showed that all honey-treated cells share a similar overall pattern of gene expression, with a trend toward reduced expression of the virulence genes of interest. Our results indicate that some varieties of the Egyptian honey have the potential to be effective inhibitor and virulence modulator of E. coli via multiple molecular targets. PMID:26954570

  8. Reduction of Cellular Expression Levels Is a Common Feature of Functionally Affected Pendrin (SLC26A4) Protein Variants

    PubMed Central

    de Moraes, Vanessa C S; Bernardinelli, Emanuele; Zocal, Nathalia; Fernandez, Jhonathan A; Nofziger, Charity; Castilho, Arthur M; Sartorato, Edi L; Paulmichl, Markus; Dossena, Silvia

    2016-01-01

    Sequence alterations in the pendrin gene (SLC26A4) leading to functionally affected protein variants are frequently involved in the pathogenesis of syndromic and nonsyndromic deafness. Considering the high number of SLC26A4 sequence alterations reported to date, discriminating between functionally affected and unaffected pendrin protein variants is essential in contributing to determine the genetic cause of deafness in a given patient. In addition, identifying molecular features common to the functionally affected protein variants can be extremely useful to design future molecule-directed therapeutic approaches. Here we show the functional and molecular characterization of six previously uncharacterized pendrin protein variants found in a cohort of 58 Brazilian deaf patients. Two variants (p.T193I and p.L445W) were undetectable in the plasma membrane, completely retained in the endoplasmic reticulum and showed no transport function; four (p.P142L, p.G149R, p.C282Y and p.Q413R) showed reduced function and significant, although heterogeneous, expression levels in the plasma membrane. Importantly, total expression levels of all of the functionally affected protein variants were significantly reduced with respect to the wild-type and a fully functional variant (p.R776C), regardless of their subcellular localization. Interestingly, reduction of expression may also reduce the transport activity of variants with an intrinsic gain of function (p.Q413R). As reduction of overall cellular abundance was identified as a common molecular feature of pendrin variants with affected function, the identification of strategies to prevent reduction in expression levels may represent a crucial step of potential future therapeutic interventions aimed at restoring the transport activity of dysfunctional pendrin variants. PMID:26752218

  9. Effects of Selected Egyptian Honeys on the Cellular Ultrastructure and the Gene Expression Profile of Escherichia coli

    PubMed Central

    Elkhatib, Walid F.

    2016-01-01

    The purpose of this study was to: (i) evaluate the antibacterial activities of three Egyptian honeys collected from different floral sources (namely, citrus, clover, and marjoram) against Escherichia coli; (ii) investigate the effects of these honeys on bacterial ultrastructure; and (iii) assess the anti-virulence potential of these honeys, by examining their impacts on the expression of eight selected genes (involved in biofilm formation, quorum sensing, and stress survival) in the test organism. The minimum inhibitory concentration (MIC) of the honey samples against E. coli ATCC 8739 were assessed by the broth microdilution assay in the presence and absence of catalase enzyme. Impacts of the honeys on the cellular ultrastructure and the expression profiles of the selected genes of E. coli were examined using transmission electron microscopy (TEM) and quantitative real-time polymerase chain reaction (qPCR) analysis, respectively. The susceptibility tests showed promising antibacterial activities of all the tested honeys against E. coli. This was supported by the TEM observations, which revealed “ghost” cells lacking DNA, in addition to cells with increased vacuoles, and/or with irregular shrunken cytoplasm. Among the tested honeys, marjoram exhibited the highest total antibacterial activity and the highest levels of peroxide-dependent activity. The qPCR analysis showed that all honey-treated cells share a similar overall pattern of gene expression, with a trend toward reduced expression of the virulence genes of interest. Our results indicate that some varieties of the Egyptian honey have the potential to be effective inhibitor and virulence modulator of E. coli via multiple molecular targets. PMID:26954570

  10. Spaceflight effects on T lymphocyte distribution, function and gene expression

    PubMed Central

    Gridley, Daila S.; Slater, James M.; Luo-Owen, Xian; Rizvi, Asma; Chapes, Stephen K.; Stodieck, Louis S.; Ferguson, Virginia L.; Pecaut, Michael J.

    2009-01-01

    The immune system is highly sensitive to stressors present during spaceflight. The major emphasis of this study was on the T lymphocytes in C57BL/6NTac mice after return from a 13-day space shuttle mission (STS-118). Spleens and thymuses from flight animals (FLT) and ground controls similarly housed in animal enclosure modules (AEM) were evaluated within 3–6 h after landing. Phytohemagglutinin-induced splenocyte DNA synthesis was significantly reduced in FLT mice when based on both counts per minute and stimulation indexes (P < 0.05). Flow cytometry showed that CD3+ T and CD19+ B cell counts were low in spleens from the FLT group, whereas the number of NK1.1+ natural killer (NK) cells was increased (P < 0.01 for all three populations vs. AEM). The numerical changes resulted in a low percentage of T cells and high percentage of NK cells in FLT animals (P < 0.05). After activation of spleen cells with anti-CD3 monoclonal antibody, interleukin-2 (IL-2) was decreased, but IL-10, interferon-γ, and macrophage inflammatory protein-1α were increased in FLT mice (P < 0.05). Analysis of cancer-related genes in the thymus showed that the expression of 30 of 84 genes was significantly affected by flight (P < 0.05). Genes that differed from AEM controls by at least 1.5-fold were Birc5, Figf, Grb2, and Tert (upregulated) and Fos, Ifnb1, Itgb3, Mmp9, Myc, Pdgfb, S100a4, Thbs, and Tnf (downregulated). Collectively, the data show that T cell distribution, function, and gene expression are significantly modified shortly after return from the spaceflight environment. PMID:18988762

  11. Altered expression of key cellular gene products accompanies development of resistance to nitric oxide.

    PubMed

    Aguilar-Santelises, Miguel; Mozart, Marlene; Scuderi, Richard; Celsing, Fredrik

    2006-12-01

    NALM-6 is a pre-B leukemia cell line sensitive to exogenous nitric oxide (NO), which enters into apoptosis during 24 h of exposure to low doses of the NO donors SNAP (100 microM) or DETA-NO (250 microM). By culturing NALM-6 with repeated and increasing concentrations of SNAP, we obtained a variant (NALM-6R) that retains >95% viability and does not enter into apoptosis during 24 h culture in the presence of up to 500 microM SNAP or 750 microM DETA-NO. A power blot screen performed with 277 antibodies on cell lysates from NALM-6 and NALM-6R cultured without NO donors served to determine the altered constitutive expression of 19 proteins in NALM-6R. Proteins affected in the less sensitive cell line NALM6-R are involved in the regulation of apoptosis, the cell cycle, cell interactions, signal transduction, cell morphology, and cell motility. This model shows that repeated exposure of tumor cells to NO may either select NO-resistant cells or contribute to NO-sensitive conversion into NO-resistant cells. The identification of the proteins that are affected during this transition may help us to define the mechanisms that are involved in cell resistance to NO-cytotoxicity which often accompany clinical progression.

  12. Elevated Cellular PD1/PD-L1 Expression Confers Acquired Resistance to Cisplatin in Small Cell Lung Cancer Cells.

    PubMed

    Yan, Fei; Pang, Jiuxia; Peng, Yong; Molina, Julian R; Yang, Ping; Liu, Shujun

    2016-01-01

    Although small cell lung cancer (SCLC) is highly responsive to chemotherapies (e.g., cisplatin-etoposide doublet), virtually almost all responsive SCLC patients experience disease recurrence characterized by drug resistance. The mechanisms underlying cisplatin resistance remain elusive. Here we report that cell-intrinsic expression of PD1 and PD-L1, two immune checkpoints, is required for sustained expansion of SCLC cells under cisplatin selection. Indeed, PD1 and PD-L1 were expressed at a higher level in lung cancer cell lines, tumor tissues, and importantly, in SCLC cells resistant to cisplatin (H69R, H82R), when compared to respective controls. Genetic abrogation of PD1 and PD-L1 in H69R and H82R cells decreased their proliferation rate, and restored their sensitivity to cisplatin. Mechanistically, PD-L1 upregulation in H69R and H82R cells was attributed to the overexpression of DNA methyltransferase 1 (DNMT1) or receptor tyrosine kinase KIT, as knockdown of DNMT1 or KIT in H69R and H82R cells led to PD-L1 downregulation. Consequently, combined knockdown of PD-L1 with KIT or DNMT1 resulted in more pronounced inhibition of H69R and H82R cell growth. Thus, cell intrinsic PD1/PD-L1 signaling may be a predictor for poor efficacy of cisplatin treatment, and targeting the cellular PD1/PD-L1 axis may improve chemosensitization of aggressive SCLC.

  13. Prenatal ethanol consumption increases retinol and cellular retinol-binding protein expression in the rat fetal snout.

    PubMed

    Zachman, R D; Grummer, M A

    2001-08-01

    Fetal exposure to ethanol disrupts normal craniofacial development, resulting in characteristic features of fetal alcohol syndrome (FAS). One mechanism that could result in some anomalies of this syndrome is through ethanol disrupting the regulatory role played by vitamin A in fetal development, thereby inducing morphological alterations which manifest as FAS. This work begins to explore a possible interaction of ethanol with vitamin A in craniofacial development. Retinoid levels and the expression of cellular retinol-binding protein (CRBP) and retinoic acid receptor (RAR) mRNA were determined in snouts of 20-day fetuses exposed to ethanol throughout gestation, compared to controls. Snout retinol and retinyl palmitate levels were elevated in fetuses of ethanol-treated rats, but retinoic acid levels were unaffected. The expression of CRBP mRNA, as determined by Northern analysis, was greater in snouts of fetuses exposed to ethanol, but there was no change in RAR alpha, beta, gamma or retinoid X receptor beta mRNA. These results demonstrate that prenatal ethanol consumption can alter certain markers of vitamin A metabolism and function in the fetal snout. PMID:11509816

  14. Increased expression of fatty acid synthase provides a survival advantage to colorectal cancer cells via upregulation of cellular respiration.

    PubMed

    Zaytseva, Yekaterina Y; Harris, Jennifer W; Mitov, Mihail I; Kim, Ji Tae; Butterfield, D Allan; Lee, Eun Y; Weiss, Heidi L; Gao, Tianyan; Evers, B Mark

    2015-08-01

    Fatty acid synthase (FASN), a lipogenic enzyme, is upregulated in colorectal cancer (CRC). Increased de novo lipid synthesis is thought to be a metabolic adaptation of cancer cells that promotes survival and metastasis; however, the mechanisms for this phenomenon are not fully understood. We show that FASN plays a role in regulation of energy homeostasis by enhancing cellular respiration in CRC. We demonstrate that endogenously synthesized lipids fuel fatty acid oxidation, particularly during metabolic stress, and maintain energy homeostasis. Increased FASN expression is associated with a decrease in activation of energy-sensing pathways and accumulation of lipid droplets in CRC cells and orthotopic CRCs. Immunohistochemical evaluation demonstrated increased expression of FASN and p62, a marker of autophagy inhibition, in primary CRCs and liver metastases compared to matched normal colonic mucosa. Our findings indicate that overexpression of FASN plays a crucial role in maintaining energy homeostasis in CRC via increased oxidation of endogenously synthesized lipids. Importantly, activation of fatty acid oxidation and consequent downregulation of stress-response signaling pathways may be key adaptation mechanisms that mediate the effects of FASN on cancer cell survival and metastasis, providing a strong rationale for targeting this pathway in advanced CRC.

  15. Role of plasma membrane lipid composition on cellular homeostasis: learning from cell line models expressing fatty acid desaturases

    PubMed Central

    Jaureguiberry, María S.; Tricerri, M. Alejandra; Sanchez, Susana A.; Finarelli, Gabriela S.; Montanaro, Mauro A.; Prieto, Eduardo D.; Rimoldi, Omar J.

    2014-01-01

    Experimental evidence has suggested that plasma membrane (PM)-associated signaling and hence cell metabolism and viability depend on lipid composition and organization. The aim of the present work is to develop a cell model to study the endogenous polyunsaturated fatty acids (PUFAs) effect on PM properties and analyze its influence on cholesterol (Chol) homeostasis. We have previously shown that by using a cell line over-expressing stearoyl-CoA-desaturase, membrane composition and organization coordinate cellular pathways involved in Chol efflux and cell viability by different mechanisms. Now, we expanded our studies to a cell model over-expressing both Δ5 and Δ6 desaturases, which resulted in a permanently higher PUFA content in PM. Furthermore, this cell line showed increased PM fluidity, Chol storage, and mitochondrial activity. In addition, human apolipoprotein A-I-mediated Chol removal was less efficient in these cells than in the corresponding control. Taken together, our results suggested that the cell functionality is preserved by regulating PM organization and Chol exportation and homeostasis. PMID:24473084

  16. Arabidopsis thaliana expresses two functional isoforms of Arvp, a protein involved in the regulation of cellular lipid homeostasis.

    PubMed

    Forés, Oriol; Arró, Montserrat; Pahissa, Albert; Ferrero, Sergi; Germann, Melody; Stukey, Joseph; McDonough, Virginia; Nickels, Joseph T; Campos, Narciso; Ferrer, Albert

    2006-07-01

    Arv1p is involved in the regulation of cellular lipid homeostasis in the yeast Saccharomyces cerevisiae. Here, we report the characterization of the two Arabidopsis thaliana ARV genes and the encoded proteins, AtArv1p and AtArv2p. The functional identity of AtArv1p and AtArv2p was demonstrated by complementation of the thermosensitive phenotype of the arv1Delta yeast mutant strain YJN1756. Both A. thaliana proteins contain the bipartite Arv1 homology domain (AHD), which consists of an NH(2)-terminal cysteine-rich subdomain with a putative zinc-binding motif followed by a C-terminal subdomain of 33 amino acids. Removal of the cysteine-rich subdomain has no effect on Arvp activity, whereas the presence of the C-terminal subdomain of the AHD is critical for Arvp function. Localization experiments of AtArv1p and AtArv2p tagged with green fluorescent protein (GFP) and expressed in onion epidermal cells demonstrated that both proteins are exclusively targeted to the endoplasmic reticulum. Analysis of beta-glucuronidase (GUS) activity in transgenic A. thaliana plants carrying chimeric ARV1::GUS and ARV2::GUS genes showed that ARV gene promoters direct largely overlapping patterns of expression that are restricted to tissues in which cells are actively dividing or expanding. The results of this study support the notion that plants, yeast and mammals share common molecular mechanisms regulating intracellular lipid homeostasis.

  17. Expression of α(1)-adrenergic receptors in rat prefrontal cortex: cellular co-localization with 5-HT(2A) receptors.

    PubMed

    Santana, Noemí; Mengod, Guadalupe; Artigas, Francesc

    2013-06-01

    The prefrontal cortex (PFC) is involved in behavioural control and cognitive processes that are altered in schizophrenia. The brainstem monoaminergic systems control PFC function, yet the cells/networks involved are not fully known. Serotonin (5-HT) and norepinephrine (NE) increase PFC neuronal activity through the activation of α(1)-adrenergic receptors (α(1)ARs) and 5-HT(2A) receptors (5-HT(2A)Rs), respectively. Neurochemical and behavioural interactions between these receptors have been reported. Further, classical and atypical antipsychotic drugs share nm in vitro affinity for α(1)ARs while having preferential affinity for D(2) and 5-HT(2A)Rs, respectively. Using double in situ hybridization we examined the cellular expression of α(1)ARs in pyramidal (vGluT1-positive) and GABAergic (GAD(65/67)-positive) neurons in rat PFC and their co-localization with 5-HT(2A)Rs. α(1)ARs are expressed by a high proportion of pyramidal (59-85%) and GABAergic (52-79%) neurons. The expression in pyramidal neurons exhibited a dorsoventral gradient, with a lower percentage of α(1)AR-positive neurons in infralimbic cortex compared to anterior cingulate and prelimbic cortex. The expression of α(1A), α(1B) and α(1D) adrenergic receptors was segregated in different layers and subdivisions. In all them there is a high co-expression with 5-HT(2A)Rs (∼80%). These observations indicate that NE controls the activity of most PFC pyramidal neurons via α(1)ARs, either directly or indirectly, via GABAergic interneurons. Antipsychotic drugs can thus modulate the activity of PFC via α(1)AR blockade. The high co-expression with 5-HT(2A)Rs indicates a convergence of excitatory serotonergic and noradrenergic inputs onto the same neuronal populations. Moreover, atypical antipsychotics may exert a more powerful control of PFC function through the simultaneous blockade of α(1)ARs and 5-HT(2A)Rs.

  18. Limited but durable changes to cellular gene expression in a model of latent adenovirus infection are reflected in childhood leukemic cell lines

    PubMed Central

    Ornelles, D.A.; Gooding, L.R.; Dickherber, M.L.; Policard, M.; Garnett-Benson, C.

    2016-01-01

    Mucosal lymphocytes support latent infections of species C adenoviruses. Because infected lymphocytes resist re-infection with adenovirus, we sought to identify changes in cellular gene expression that could inhibit the infectious process. The expression of over 30,000 genes was evaluated by microarray in persistently infected B-and T-lymphocytic cells. BBS9, BNIP3, BTG3, CXADR, SLFN11 and SPARCL1 were the only genes differentially expressed between mock and infected B cells. Most of these genes are associated with oncogenesis or cancer progression. Histone deacetylase and DNA methyltransferase inhibitors released the repression of some of these genes. Cellular and viral gene expression was compared among leukemic cell lines following adenovirus infection. Childhood leukemic B-cell lines resist adenovirus infection and also show reduced expression of CXADR and SPARCL. Thus adenovirus induces limited changes to infected B-cell lines that are similar to changes observed in childhood leukemic cell lines. PMID:27085068

  19. Effect of DNA/liposome mixing ratio on the physicochemical characteristics, cellular uptake and intracellular trafficking of plasmid DNA/cationic liposome complexes and subsequent gene expression.

    PubMed

    Sakurai, F; Inoue, R; Nishino, Y; Okuda, A; Matsumoto, O; Taga, T; Yamashita, F; Takakura, Y; Hashida, M

    2000-05-15

    In order to identify the important factors involved in cationic liposome-mediated gene transfer, in vitro transfection efficiencies by plasmid DNA complexed with DOTMA/DOPE liposomes at different DNA/liposome mixing ratios were evaluated using four types of cultured cells with respect to their physicochemical properties. Significant changes were observed in the particle size and zeta potential of the complexes as well as in their structures, assessed by atomic force microscopy, which depended on the mixing ratio. In transfection experiments, except for RAW 264.7 cells (mouse macrophages), efficient gene expression was obtained in MBT-2 cells (mouse bladder tumor), NLH3T3 cells (mouse fibroblasts) and HUVEC (human umbilical vein endothelial cells) at an optimal ratio of 1:5, 1:7.5 or 1:5, respectively. On the other hand, cellular uptake of the [32P]DNA/liposome complexes increased in all cell types with an increase in the mixing ratio, which was not reflected by the transfection efficiency. The cellular damage determined by MTT assay was minimal even at the highest DNA/liposome ratio (1:10), indicating that the lower gene expression level at the higher ratio was not due to cytotoxicity induced by the complex. An ethidium bromide intercalation assay showed that the release of plasmid DNA from the complex, following the addition of negatively charged liposomes, was restricted as the mixing ratio increased. Furthermore, confocal microscopic studies using HUVEC showed that the 1:5 complexes exhibited a dispersed distribution in the cytoplasm whereas a punctuate intracellular distribution was observed for the 1:10 complexes. This suggests that there was a significant difference in intracellular trafficking, probably release from the endosomes or lysosomes, of the plasmid DNA/cationic liposome complexes between these mixing ratios. Taken together, these findings suggest that the DNA/liposome mixing ratio significantly affects the intracellular trafficking of plasmid DNA

  20. Expression and cellular localization of the transcription factor NeuroD1 in the developing and adult rat pineal gland.

    PubMed

    Castro, Analía E; Benitez, Sergio G; Farias Altamirano, Luz E; Savastano, Luis E; Patterson, Sean I; Muñoz, Estela M

    2015-05-01

    Circadian rhythms govern many aspects of mammalian physiology. The daily pattern of melatonin synthesis and secretion is one of the classic examples of circadian oscillations. It is mediated by a class of neuroendocrine cells known as pinealocytes which are not yet fully defined. An established method to evaluate functional and cytological characters is through the expression of lineage-specific transcriptional regulators. NeuroD1 is a basic helix-loop-helix transcription factor involved in the specification and maintenance of both endocrine and neuronal phenotypes. We have previously described developmental and adult regulation of NeuroD1 mRNA in the rodent pineal gland. However, the transcript levels were not influenced by the elimination of sympathetic input, suggesting that any rhythmicity of NeuroD1 might be found downstream of transcription. Here, we describe NeuroD1 protein expression and cellular localization in the rat pineal gland during development and the daily cycle. In embryonic and perinatal stages, protein expression follows the mRNA pattern and is predominantly nuclear. Thereafter, NeuroD1 is mostly found in pinealocyte nuclei in the early part of the night and in cytoplasm during the day, a rhythm maintained into adulthood. Additionally, nocturnal nuclear NeuroD1 levels are reduced after sympathetic disruption, an effect mimicked by the in vivo administration of α- and β-adrenoceptor blockers. NeuroD1 phosphorylation at two sites, Ser(274) and Ser(336) , associates with nuclear localization in pinealocytes. These data suggest that NeuroD1 influences pineal phenotype both during development and adulthood, in an autonomic and phosphorylation-dependent manner.

  1. Interaction of cadmium and zinc on accumulation and sub-cellular distribution in leaves of hyperaccumulator Potentilla griffithii.

    PubMed

    Qiu, Rong-Liang; Thangavel, Palaniswamy; Hu, Peng-Jie; Senthilkumar, Palaninaicker; Ying, Rong-Rong; Tang, Ye-Tao

    2011-02-28

    Potentilla griffithii Hook is a newly found hyperaccumulator plant capable of high tolerance and accumulation of Zn and Cd. We investigated the interactive effects between Cd and Zn on accumulation and vacuolar sequestration in P. griffithii. Stimulatory effect of growth was noted at 0.2 mM Cd and 1.25 and 2.5 mM Zn tested. Accumulation of Zn and Cd in roots, petioles and leaves were increased significantly with addition of these metals individually. However, the Zn supplement decreased root Cd accumulation but increased the concentration of Cd in petioles and leaves. The results from sub-cellular distribution showed that up to 94% and 70% of the total Zn and Cd in the leaves were present in the protoplasts, and more than 90% Cd and Zn in the protoplasts were localized in the vacuoles. Nearly, 88% and 85% of total Cd and Zn were extracted in the cell sap of the leaves suggesting that most of the Cd and Zn in the leaves were available in soluble form. The present results indicate that Zn supplement significantly enhanced the petiole accumulation of Cd and further vacuolar sequestration plays an important role in tolerance, detoxification and hyperaccumulation of these metals in P. griffithii. PMID:21211902

  2. Effects of nicotine on cellular proliferation, macromolecular synthesis and cell cycle phase distribution in human and murine cells

    SciTech Connect

    Konno, S.; Chiao, J.; Rossi, J.; Wang, C.H.; Wu, J.M.

    1986-05-01

    Addition of nicotine causes a dose- and time-dependent inhibition of cell growth in established human and murine cells. In the human promyelocytic HL-60 leukemic cells, 3 mM nicotine results in a 50% inhibition of cellular proliferation after 80 h. Nicotine was also found to affect the cell cycle distribution of HL-60 cells. Treatment with 4 mM nicotine for 20 h causes an increase in proportion of Gl-phase cells (from 49% to 57%) and a significant decrease in the proportion of S-phase cells (from 41% to 32%). These results suggest that nicotine causes cell arrest in the Gl-phase which may in part account for its effects on cell growth. To determine whether nicotine has a primary effect on the uptake/transport of macromolecular precursors into cells, HL-60 cells were treated with 2-6 mM nicotine for 30 h/sub 3/ at the end of which time cells were labeled with (/sup 3/H)thymidine, (/sup 3/H)uridine, (/sup 14/C)lysine and (/sup 35/S)methionine, the trichloroacetic acid (TCA) soluble and insoluble radioactivities from each of the labeling conditions were determined. These studies show that nicotine primarily affect the synthesis of proteins.

  3. Shroom3 functions downstream of planar cell polarity to regulate myosin II distribution and cellular organization during neural tube closure

    PubMed Central

    McGreevy, Erica M.; Vijayraghavan, Deepthi; Davidson, Lance A.; Hildebrand, Jeffrey D.

    2015-01-01

    ABSTRACT Neural tube closure is a critical developmental event that relies on actomyosin contractility to facilitate specific processes such as apical constriction, tissue bending, and directional cell rearrangements. These complicated processes require the coordinated activities of Rho-Kinase (Rock), to regulate cytoskeletal dynamics and actomyosin contractility, and the Planar Cell Polarity (PCP) pathway, to direct the polarized cellular behaviors that drive convergent extension (CE) movements. Here we investigate the role of Shroom3 as a direct linker between PCP and actomyosin contractility during mouse neural tube morphogenesis. In embryos, simultaneous depletion of Shroom3 and the PCP components Vangl2 or Wnt5a results in an increased liability to NTDs and CE failure. We further show that these pathways intersect at Dishevelled, as Shroom3 and Dishevelled 2 co-distribute and form a physical complex in cells. We observed that multiple components of the Shroom3 pathway are planar polarized along mediolateral cell junctions in the neural plate of E8.5 embryos in a Shroom3 and PCP-dependent manner. Finally, we demonstrate that Shroom3 mutant embryos exhibit defects in planar cell arrangement during neural tube closure, suggesting a role for Shroom3 activity in CE. These findings support a model in which the Shroom3 and PCP pathways interact to control CE and polarized bending of the neural plate and provide a clear illustration of the complex genetic basis of NTDs. PMID:25596276

  4. Both FA- and mPEG-conjugated chitosan nanoparticles for targeted cellular uptake and enhanced tumor tissue distribution

    NASA Astrophysics Data System (ADS)

    Hou, Zhenqing; Zhan, Chuanming; Jiang, Qiwei; Hu, Quan; Li, Le; Chang, Di; Yang, Xiangrui; Wang, Yixiao; Li, Yang; Ye, Shefang; Xie, Liya; Yi, Yunfeng; Zhang, Qiqing

    2011-10-01

    Both folic acid (FA)- and methoxypoly(ethylene glycol) (mPEG)-conjugated chitosan nanoparticles (NPs) had been designed for targeted and prolong anticancer drug delivery system. The chitosan NPs were prepared with combination of ionic gelation and chemical cross-linking method, followed by conjugation with both FA and mPEG, respectively. FA-mPEG-NPs were compared with either NPs or mPEG-/FA-NPs in terms of their size, targeting cellular efficiency and tumor tissue distribution. The specificity of the mPEG-FA-NPs targeting cancerous cells was demonstrated by comparative intracellular uptake of NPs and mPEG-/FA-NPs by human adenocarcinoma HeLa cells. Mitomycin C (MMC), as a model drug, was loaded to the mPEG-FA-NPs. Results show that the chitosan NPs presented a narrow-size distribution with an average diameter about 200 nm regardless of the type of functional group. In addition, MMC was easily loaded to the mPEG-FA-NPs with drug-loading content of 9.1%, and the drug releases were biphasic with an initial burst release, followed by a subsequent slower release. Laser confocal scanning imaging proved that both mPEG-FA-NPs and FA-NPs could greatly enhance uptake by HeLa cells. In vivo animal experiments, using a nude mice xenograft model, demonstrated that an increased amount of mPEG-FA-NPs or FA-NPs were accumulated in the tumor tissue relative to the mPEG-NPs or NPs alone. These results suggest that both FA- and mPEG-conjugated chitosan NPs are potentially prolonged drug delivery system for tumor cell-selective targeting treatments.

  5. Prediction of Metabolic Flux Distribution from Gene Expression Data Based on the Flux Minimization Principle

    PubMed Central

    Song, Hyun-Seob; Reifman, Jaques; Wallqvist, Anders

    2014-01-01

    Prediction of possible flux distributions in a metabolic network provides detailed phenotypic information that links metabolism to cellular physiology. To estimate metabolic steady-state fluxes, the most common approach is to solve a set of macroscopic mass balance equations subjected to stoichiometric constraints while attempting to optimize an assumed optimal objective function. This assumption is justifiable in specific cases but may be invalid when tested across different conditions, cell populations, or other organisms. With an aim to providing a more consistent and reliable prediction of flux distributions over a wide range of conditions, in this article we propose a framework that uses the flux minimization principle to predict active metabolic pathways from mRNA expression data. The proposed algorithm minimizes a weighted sum of flux magnitudes, while biomass production can be bounded to fit an ample range from very low to very high values according to the analyzed context. We have formulated the flux weights as a function of the corresponding enzyme reaction's gene expression value, enabling the creation of context-specific fluxes based on a generic metabolic network. In case studies of wild-type Saccharomyces cerevisiae, and wild-type and mutant Escherichia coli strains, our method achieved high prediction accuracy, as gauged by correlation coefficients and sums of squared error, with respect to the experimentally measured values. In contrast to other approaches, our method was able to provide quantitative predictions for both model organisms under a variety of conditions. Our approach requires no prior knowledge or assumption of a context-specific metabolic functionality and does not require trial-and-error parameter adjustments. Thus, our framework is of general applicability for modeling the transcription-dependent metabolism of bacteria and yeasts. PMID:25397773

  6. Dynamic alternations in cellular and molecular components during blossom-end rot development in tomatoes expressing sCAX1, a constitutively active Ca2+/H+ antiporter from Arabidopsis.

    PubMed

    Tonetto de Freitas, Sergio; Padda, Malkeet; Wu, Qingyu; Park, Sunghun; Mitcham, Elizabeth J

    2011-06-01

    Although calcium (Ca) concentration in cellular compartments has been suggested to be tightly regulated, Ca deficiency disorders such as blossom-end rot (BER) in tomato (Solanum lycopersicum) fruit may be induced by abnormal regulation of Ca partitioning and distribution in the cell. The objectives of this work were to analyze the effects of high expression of the constitutively functional Arabidopsis (Arabidopsis thaliana) Ca(2+)/H(+) exchanger (sCAX1) tonoplast protein in tomato fruit on cellular Ca partitioning and distribution, membrane integrity, and the transcriptional profile of genes potentially involved in BER development. Wild-type and sCAX1-expressing tomato plants were grown in a greenhouse. Wild-type plants did not develop BER, whereas sCAX1-expressing plants reached 100% BER incidence at 15 d after pollination. The sCAX1-expressing fruit pericarp had higher total tissue and water-soluble Ca concentrations, lower apoplastic and cytosolic Ca concentrations, higher membrane leakage, and Ca accumulation in the vacuole of sCAX1-expressing cells. Microarray analysis of healthy sCAX1-expressing fruit tissue indicated down-regulation of genes potentially involved in BER development, such as genes involved in membrane structure and repair and cytoskeleton metabolism, as well as up-regulation of genes that may have limited BER damage expansion, such as genes coding for heat shock proteins, glutathione S-transferases, and peroxidases. The results indicate that the high expression of the sCAX1 gene reduces cytosolic and apoplastic Ca concentrations, affecting plasma membrane structure and leading to BER symptom development in the fruit tissue. PMID:21464475

  7. Distribution of miRNA expression across human tissues.

    PubMed

    Ludwig, Nicole; Leidinger, Petra; Becker, Kurt; Backes, Christina; Fehlmann, Tobias; Pallasch, Christian; Rheinheimer, Steffi; Meder, Benjamin; Stähler, Cord; Meese, Eckart; Keller, Andreas

    2016-05-01

    We present a human miRNA tissue atlas by determining the abundance of 1997 miRNAs in 61 tissue biopsies of different organs from two individuals collected post-mortem. One thousand three hundred sixty-four miRNAs were discovered in at least one tissue, 143 were present in each tissue. To define the distribution of miRNAs, we utilized a tissue specificity index (TSI). The majority of miRNAs (82.9%) fell in a middle TSI range i.e. were neither specific for single tissues (TSI > 0.85) nor housekeeping miRNAs (TSI < 0.5). Nonetheless, we observed many different miRNAs and miRNA families that were predominantly expressed in certain tissues. Clustering of miRNA abundances revealed that tissues like several areas of the brain clustered together. Considering -3p and -5p mature forms we observed miR-150 with different tissue specificity. Analysis of additional lung and prostate biopsies indicated that inter-organism variability was significantly lower than inter-organ variability. Tissue-specific differences between the miRNA patterns appeared not to be significantly altered by storage as shown for heart and lung tissue. MiRNAs TSI values of human tissues were significantly (P = 10(-8)) correlated with those of rats; miRNAs that were highly abundant in certain human tissues were likewise abundant in according rat tissues. We implemented a web-based repository enabling scientists to access and browse the data (https://ccb-web.cs.uni-saarland.de/tissueatlas).

  8. Specificity of G alpha q and G alpha 11 gene expression in platelets and erythrocytes. Expressions of cellular differentiation and species differences.

    PubMed Central

    Johnson, G J; Leis, L A; Dunlop, P C

    1996-01-01

    G alpha q and G alpha 11, members of the Gq family of G-proteins, transduce signals from receptors to the beta isoenzymes of phosphatidyl-inositol-specific phospholipase C (PI-PLC). The receptor specificity of these alpha subunits is unknown. G alpha q and G alpha 11 are ubiquitously expressed in tissues; however, there have been conflicting reports of the presence or absence of G alpha 11 protein in haematopoietic cells. Platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors activate PI-PLC via G alpha q, but the role of G alpha 11 is uncertain. To define their roles in platelet activation we studied G alpha q and G alpha 11 gene expression by immunotransfer blotting and by reverse transcription of mRNA followed by PCR (RT-PCR) and direct sequencing. An antiserum specific for mouse G alpha 11 failed to identify G alpha 11 in dog or human platelets or in dog liver, a tissue known to contain G alpha 11. RT-PCR performed with gene-specific primers demonstrated G alpha q mRNA, but not G alpha 11 mRNA, in normal human and mouse platelets and in thromboxane-sensitive and thromboxane-insensitive dog platelets. Studies of mouse and dog liver and human retina confirmed that the cDNA, primers and probes used could amplify and recognize G alpha 11 in other tissues. However, species-specific oligonucleotide primers and probes were essential to demonstrate G alpha 11, but not G alpha q, mRNA. Compared with mouse cDNA, dog and human G alpha 11 cDNA had twice as many nucleotide substitutions (approx. 12% compared with approx. 6%) as G alpha q, G alpha q mRNA was also found in mature erythrocytes but G alpha 11 mRNA was not identified, whereas both G alpha q and G alpha 11 mRNAs were found in bone marrow stem cells. Therefore G alpha 11 gene expression in haematopoietic cells is linked with cellular differentiation. The lack of G alpha 11 indicates that signal transduction from platelet TXA2/PGH2 receptors to PI-PLC occurs via G alpha q, and that G alpha 11 deficiency is

  9. No Effect of the Transforming Growth Factor {beta}1 Promoter Polymorphism C-509T on TGFB1 Gene Expression, Protein Secretion, or Cellular Radiosensitivity

    SciTech Connect

    Reuther, Sebastian; Metzke, Elisabeth; Bonin, Michael; Petersen, Cordula; Dikomey, Ekkehard; Raabe, Annette

    2013-02-01

    Purpose: To study whether the promoter polymorphism (C-509T) affects transforming growth factor {beta}1 gene (TGFB1) expression, protein secretion, and/or cellular radiosensitivity for both human lymphocytes and fibroblasts. Methods and Materials: Experiments were performed with lymphocytes taken either from 124 breast cancer patients or 59 pairs of normal monozygotic twins. We used 15 normal human primary fibroblast strains as controls. The C-509T genotype was determined by polymerase chain reaction-restriction fragment length polymorphism or TaqMan single nucleotide polymorphism (SNP) genotyping assay. The cellular radiosensitivity of lymphocytes was measured by G0/1 assay and that of fibroblasts by colony assay. The amount of extracellular TGFB1 protein was determined by enzyme-linked immunosorbent assay, and TGFB1 expression was assessed via microarray analysis or reverse transcription-polymerase chain reaction. Results: The C-509T genotype was found not to be associated with cellular radiosensitivity, neither for lymphocytes (breast cancer patients, P=.811; healthy donors, P=.181) nor for fibroblasts (P=.589). Both TGFB1 expression and TGFB1 protein secretion showed considerable variation, which, however, did not depend on the C-509T genotype (protein secretion: P=.879; gene expression: lymphocytes, P=.134, fibroblasts, P=.605). There was also no general correlation between TGFB1 expression and cellular radiosensitivity (lymphocytes, P=.632; fibroblasts, P=.573). Conclusion: Our data indicate that any association between the SNP C-509T of TGFB1 and risk of normal tissue toxicity cannot be ascribed to a functional consequence of this SNP, either on the level of gene expression, protein secretion, or cellular radiosensitivity.

  10. A stable HeLa cell line that inducibly expresses poliovirus 2A(pro): effects on cellular and viral gene expression.

    PubMed

    Barco, A; Feduchi, E; Carrasco, L

    2000-03-01

    A HeLa cell clone (2A7d) that inducibly expresses the gene for poliovirus protease 2A (2A(pro)) under the control of tetracycline has been obtained. Synthesis of 2A(pro) induces severe morphological changes in 2A7d cells. One day after tetracycline removal, cells round up and a few hours later die. Poliovirus 2A(pro) cleaves both forms of initiation factor eIF4G, causing extensive inhibition of capped-mRNA translation a few hours after protease induction. Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, a selective inhibitor of 2A(pro), prevents both eIF4G cleavage and inhibition of translation but not cellular death. Expression of 2A(pro) still allows both the replication of poliovirus and the translation of mRNAs containing a picornavirus leader sequence, while vaccinia virus replication is drastically inhibited. Translation of transfected capped mRNA is blocked in 2A7d-On cells, while luciferase synthesis from a mRNA bearing a picornavirus internal ribosome entry site (IRES) sequence is enhanced by the presence of 2A(pro). Moreover, synthesis of 2A(pro) in 2A7d cells complements the translational defect of a poliovirus 2A(pro)-defective variant. These results show that poliovirus 2A(pro) expression mimics some phenotypical characteristics of poliovirus-infected cells, such as cell rounding, inhibition of protein synthesis and enhancement of IRES-driven translation. This cell line constitutes a useful tool to further analyze 2A(pro) functions, to complement poliovirus 2A(pro) mutants, and to test antiviral compounds.

  11. Elevated Cellular PD1/PD-L1 Expression Confers Acquired Resistance to Cisplatin in Small Cell Lung Cancer Cells

    PubMed Central

    Yan, Fei; Pang, Jiuxia; Peng, Yong; Molina, Julian R.; Yang, Ping; Liu, Shujun

    2016-01-01

    Although small cell lung cancer (SCLC) is highly responsive to chemotherapies (e.g., cisplatin-etoposide doublet), virtually almost all responsive SCLC patients experience disease recurrence characterized by drug resistance. The mechanisms underlying cisplatin resistance remain elusive. Here we report that cell-intrinsic expression of PD1 and PD-L1, two immune checkpoints, is required for sustained expansion of SCLC cells under cisplatin selection. Indeed, PD1 and PD-L1 were expressed at a higher level in lung cancer cell lines, tumor tissues, and importantly, in SCLC cells resistant to cisplatin (H69R, H82R), when compared to respective controls. Genetic abrogation of PD1 and PD-L1 in H69R and H82R cells decreased their proliferation rate, and restored their sensitivity to cisplatin. Mechanistically, PD-L1 upregulation in H69R and H82R cells was attributed to the overexpression of DNA methyltransferase 1 (DNMT1) or receptor tyrosine kinase KIT, as knockdown of DNMT1 or KIT in H69R and H82R cells led to PD-L1 downregulation. Consequently, combined knockdown of PD-L1 with KIT or DNMT1 resulted in more pronounced inhibition of H69R and H82R cell growth. Thus, cell intrinsic PD1/PD-L1 signaling may be a predictor for poor efficacy of cisplatin treatment, and targeting the cellular PD1/PD-L1 axis may improve chemosensitization of aggressive SCLC. PMID:27610620

  12. Elevated Cellular PD1/PD-L1 Expression Confers Acquired Resistance to Cisplatin in Small Cell Lung Cancer Cells.

    PubMed

    Yan, Fei; Pang, Jiuxia; Peng, Yong; Molina, Julian R; Yang, Ping; Liu, Shujun

    2016-01-01

    Although small cell lung cancer (SCLC) is highly responsive to chemotherapies (e.g., cisplatin-etoposide doublet), virtually almost all responsive SCLC patients experience disease recurrence characterized by drug resistance. The mechanisms underlying cisplatin resistance remain elusive. Here we report that cell-intrinsic expression of PD1 and PD-L1, two immune checkpoints, is required for sustained expansion of SCLC cells under cisplatin selection. Indeed, PD1 and PD-L1 were expressed at a higher level in lung cancer cell lines, tumor tissues, and importantly, in SCLC cells resistant to cisplatin (H69R, H82R), when compared to respective controls. Genetic abrogation of PD1 and PD-L1 in H69R and H82R cells decreased their proliferation rate, and restored their sensitivity to cisplatin. Mechanistically, PD-L1 upregulation in H69R and H82R cells was attributed to the overexpression of DNA methyltransferase 1 (DNMT1) or receptor tyrosine kinase KIT, as knockdown of DNMT1 or KIT in H69R and H82R cells led to PD-L1 downregulation. Consequently, combined knockdown of PD-L1 with KIT or DNMT1 resulted in more pronounced inhibition of H69R and H82R cell growth. Thus, cell intrinsic PD1/PD-L1 signaling may be a predictor for poor efficacy of cisplatin treatment, and targeting the cellular PD1/PD-L1 axis may improve chemosensitization of aggressive SCLC. PMID:27610620

  13. Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting.

    PubMed

    Fu, Glenn K; Wilhelmy, Julie; Stern, David; Fan, H Christina; Fodor, Stephen P A

    2014-03-18

    We present a new approach for the sensitive detection and accurate quantitation of messenger ribonucleic acid (mRNA) gene transcripts in single cells. First, the entire population of mRNAs is encoded with molecular barcodes during reverse transcription. After amplification of the gene targets of interest, molecular barcodes are counted by sequencing or scored on a simple hybridization detector to reveal the number of molecules in the starting sample. Since absolute quantities are measured, calibration to standards is unnecessary, and many of the relative quantitation challenges such as polymerase chain reaction (PCR) bias are avoided. We apply the method to gene expression analysis of minute sample quantities and demonstrate precise measurements with sensitivity down to sub single-cell levels. The method is an easy, single-tube, end point assay utilizing standard thermal cyclers and PCR reagents. Accurate and precise measurements are obtained without any need for cycle-to-cycle intensity-based real-time monitoring or physical partitioning into multiple reactions (e.g., digital PCR). Further, since all mRNA molecules are encoded with molecular barcodes, amplification can be used to generate more material for multiple measurements and technical replicates can be carried out on limited samples. The method is particularly useful for small sample quantities, such as single-cell experiments. Digital encoding of cellular content preserves true abundance levels and overcomes distortions introduced by amplification.

  14. Digital Encoding of Cellular mRNAs Enabling Precise and Absolute Gene Expression Measurement by Single-Molecule Counting

    PubMed Central

    2014-01-01

    We present a new approach for the sensitive detection and accurate quantitation of messenger ribonucleic acid (mRNA) gene transcripts in single cells. First, the entire population of mRNAs is encoded with molecular barcodes during reverse transcription. After amplification of the gene targets of interest, molecular barcodes are counted by sequencing or scored on a simple hybridization detector to reveal the number of molecules in the starting sample. Since absolute quantities are measured, calibration to standards is unnecessary, and many of the relative quantitation challenges such as polymerase chain reaction (PCR) bias are avoided. We apply the method to gene expression analysis of minute sample quantities and demonstrate precise measurements with sensitivity down to sub single-cell levels. The method is an easy, single-tube, end point assay utilizing standard thermal cyclers and PCR reagents. Accurate and precise measurements are obtained without any need for cycle-to-cycle intensity-based real-time monitoring or physical partitioning into multiple reactions (e.g., digital PCR). Further, since all mRNA molecules are encoded with molecular barcodes, amplification can be used to generate more material for multiple measurements and technical replicates can be carried out on limited samples. The method is particularly useful for small sample quantities, such as single-cell experiments. Digital encoding of cellular content preserves true abundance levels and overcomes distortions introduced by amplification. PMID:24579851

  15. Utero-placental cellular and nuclear expression of bradykinin B2 receptors in normal and preeclamptic pregnancies.

    PubMed

    Valdés, Gloria; Acuña, Stephanie; Munizaga, Alejandro; Soto, Gloria X; Figueroa, Carlos D

    2016-01-01

    The bradykinin type 2 receptor (B2R), main effector of the pleiotropic kallikrein-kinin system (KKS), has been localized in the key sites related to placentation in human, rat and guinea pig utero-placental units. The present study was directed to characterize the content, the cellular and subcellular localization of B2R in the villi and basal plate of placentas from normal and preeclamptic pregnancies by means of western blotting, immunohistochemistry and immunoelectron microscopy. The protein content of B2R was demonstrated in both placental zones. The villous placenta of normal and preeclamptic pregnancies expressed B2R in syncytiotrophoblast and fetal endothelium; the basal plate displayed B2R in extravillous trophoblasts and decidual cells. Lastly, immunogold electron microscopy revealed B2R in fetal endothelium, syncytiotrophoblast, extravillous cytotrophoblasts and decidual cells; in all cell types the receptor was mainly located in the cytosol and nucleus. The protein content of placental homogenates and the immunoreactivity in the different cells types did not differ between both study groups; however the abundance of nuclear immunogold B2R positive beads in extravillous trophoblasts was greater in the normal than in the preeclamptic placentas. The purpose of describing nuclear B2R in the utero-placental unit, and its increment in normal extravillous trophoblasts, is to stimulate the study of the functional pathways that may be relevant to understand the local role of the B2R in normal and preeclamptic gestation. PMID:26955769

  16. Potential toxic effect of trifloxystrobin on cellular microstructure, mRNA expression and antioxidant enzymes in Chlorella vulgaris.

    PubMed

    Shen, Yu-Feng; Liu, Lei; Gong, Yu-Xin; Zhu, Bin; Liu, Guang-Lu; Wang, Gao-Xue

    2014-05-01

    This study investigated the effects of trifloxystrobin that one strobilurin used widely in the world as an effective fungicidal agent to control Asian soybean rust on aquatic unicellular algae Chlorella vulgaris. We determined the potential toxic effect of trifloxystrobin on C. vulgaris, and found median inhibition concentration (IC(50)) value 255.58 (95% confidence interval, 207.81-330.29)μgL(-1). In addition, the algal cells were obviously depressed or shrunk at different concentrations by electron microscopy. In the study, a real-time polymerase chain reaction (PCR) assay showed changes in transcript abundances of three photosynthetic genes, psaB, psbC, and rbcL, and one energy gene, ATPs. The results showed that trifloxystrobin reduced the transcript abundances of the three genes and enhanced expression of ATPs after 48 and 96 h. The lowest abundances of psaB, psbC and rbcL transcripts in response to trifloxystrobin exposure were 58%, 79% and 60% of those of the control, respectively. For the potential toxic influences, trifloxystrobin could decrease the soluble protein and total antioxidant contents (T-AOC), and increase superoxide dismutase (SOD) and peroxidase (POD) activity with a gradual concentration-response relationship. Overall, the present study demonstrated that trifloxystrobin could affect the activities of antioxidant enzymes, disrupts photosynthesis in C. vulgaris, and damage cellular structure. PMID:24762415

  17. Utero-placental cellular and nuclear expression of bradykinin B2 receptors in normal and preeclamptic pregnancies.

    PubMed

    Valdés, Gloria; Acuña, Stephanie; Munizaga, Alejandro; Soto, Gloria X; Figueroa, Carlos D

    2016-01-01

    The bradykinin type 2 receptor (B2R), main effector of the pleiotropic kallikrein-kinin system (KKS), has been localized in the key sites related to placentation in human, rat and guinea pig utero-placental units. The present study was directed to characterize the content, the cellular and subcellular localization of B2R in the villi and basal plate of placentas from normal and preeclamptic pregnancies by means of western blotting, immunohistochemistry and immunoelectron microscopy. The protein content of B2R was demonstrated in both placental zones. The villous placenta of normal and preeclamptic pregnancies expressed B2R in syncytiotrophoblast and fetal endothelium; the basal plate displayed B2R in extravillous trophoblasts and decidual cells. Lastly, immunogold electron microscopy revealed B2R in fetal endothelium, syncytiotrophoblast, extravillous cytotrophoblasts and decidual cells; in all cell types the receptor was mainly located in the cytosol and nucleus. The protein content of placental homogenates and the immunoreactivity in the different cells types did not differ between both study groups; however the abundance of nuclear immunogold B2R positive beads in extravillous trophoblasts was greater in the normal than in the preeclamptic placentas. The purpose of describing nuclear B2R in the utero-placental unit, and its increment in normal extravillous trophoblasts, is to stimulate the study of the functional pathways that may be relevant to understand the local role of the B2R in normal and preeclamptic gestation.

  18. The use of cellular thermal shift assay (CETSA) to study Crizotinib resistance in ALK-expressing human cancers.

    PubMed

    Alshareef, Abdulraheem; Zhang, Hai-Feng; Huang, Yung-Hsing; Wu, Chengsheng; Zhang, Jing Dong; Wang, Peng; El-Sehemy, Ahmed; Fares, Mohamed; Lai, Raymond

    2016-01-01

    Various forms of oncogenic ALK proteins have been identified in various types of human cancers. While Crizotinib, an ALK inhibitor, has been found to be therapeutically useful against a subset of ALK(+) tumours, clinical resistance to this drug has been well recognized and the mechanism of this phenomenon is incompletely understood. Using the cellular thermal shift assay (CETSA), we measured the Crizotinib-ALK binding in a panel of ALK(+) cell lines, and correlated the findings with the ALK structure and its interactions with specific binding proteins. The Crizotinib IC50 significantly correlated with Crizotinib-ALK binding. The suboptimal Crizotinib-ALK binding in Crizotinib-resistant cells is not due to the cell-specific environment, since transfection of NPM-ALK into these cells revealed substantial Crizotinib-NPM-ALK binding. Interestingly, we found that the resistant cells expressed higher protein level of β-catenin and siRNA knockdown restored Crizotinib-ALK binding (correlated with a significant lowering of IC50). Computational analysis of the crystal structures suggests that β-catenin exerts steric hindrance to the Crizotinib-ALK binding. In conclusion, the Crizotinib-ALK binding measurable by CETSA is useful in predicting Crizotinib sensitivity, and Crizotinib-ALK binding is in turn dictated by the structure of ALK and some of its binding partners. PMID:27641368

  19. Alpha2-macroglobulin from an Atlantic shrimp: biochemical characterization, sub-cellular localization and gene expression upon fungal challenge.

    PubMed

    Perazzolo, Luciane Maria; Bachère, Evelyne; Rosa, Rafael Diego; Goncalves, Priscila; Andreatta, Edemar Roberto; Daffre, Sirlei; Barracco, Margherita Anna

    2011-12-01

    In this study, we report on the isolation and characterization of an alpha2-macroglobulin (α2M) from the plasma of the pink shrimp Farfantepenaeus paulensis, its sub-cellular localization and transcriptional changes after infection by fungi. The molecular mass of the α2M was estimated at 389 kDa by gel filtration and 197 kDa by SDS-PAGE, under reducing conditions, suggesting that α2M from F. paulensis consists of two identical sub-units, covalently linked by disulphide bonds. The N-terminal amino acid sequence of the α2M from F. paulensis was very similar to those of other penaeid shrimps, crayfish and lobster (70-90% identity) and to a less extent with that of freshwater prawn (40% identity). A monoclonal antibody raised against the Marsupenaeus japonicus α2M made it possible to demonstrate that α2M of F. paulensis is stored in the vesicles of the shrimp granular hemocytes (through immunogold assay). Quantitative real-time PCR (qPCR) analysis showed that α2M mRNA transcripts significantly increased 24 h after an experimental infection with the shrimp pathogen Fusarium solani and it returned to the basal levels at 48 h post-injection. This is the first report on a α2M characterization in an Atlantic penaeid species and its expression profile upon a fungal infection. PMID:21888978

  20. Stage specific gene expression and cellular localization of two isoforms of the serine hydroxymethyltransferase in the protozoan parasite Leishmania.

    PubMed

    Gagnon, Dominic; Foucher, Aude; Girard, Isabelle; Ouellette, Marc

    2006-11-01

    Serine hydroxymethyltransferase (SHMT) catalyses the reversible conversion of serine and tetrahydrofolate to glycine and methylene-tetrahydrofolate. The recent completion of the genome sequence of Leishmania major revealed the presence of two genes coding for two isoforms of this protein. In silico analysis showed that one isoform had an extension at its N-terminus and was predicted to localize to the mitochondrion. The situation is different in other kinetoplastid parasites with only one SHMT encoding gene in Trypanosoma cruzi and no SHMT encoding gene in Trypanosoma brucei. The two L. major SHMT genes were cloned in frame with the green fluorescent protein and the resulting fusion proteins showed differential localization: the short form (SHMT-S) was found in the cytosol while the long one (SHMT-L) was found in an organelle that has hallmarks of the parasite mitochondrion. Indeed, SHMT-L had a similar cellular fractionation pattern as the mitochondrial HSP60 as determined by digitonin fractionation. Both SHMT-S and SHMT-L genes were expressed preferentially in the amastigote stage of the parasite and the RNA levels of SHMT-L could be modulated by glycine, serine, and folate. Overexpression of SHMT-S increased resistance to the antifolate methotrexate and to a lower level to the inhibitor thiosemicarbazide in a rich folate containing medium. These findings suggest that folate metabolism is compartmentalised in Leishmania and that SHMT RNA levels are responsive to environmental conditions. PMID:16876889

  1. The use of cellular thermal shift assay (CETSA) to study Crizotinib resistance in ALK-expressing human cancers

    PubMed Central

    Alshareef, Abdulraheem; Zhang, Hai-Feng; Huang, Yung-Hsing; Wu, Chengsheng; Zhang, Jing Dong; Wang, Peng; El-Sehemy, Ahmed; Fares, Mohamed; Lai, Raymond

    2016-01-01

    Various forms of oncogenic ALK proteins have been identified in various types of human cancers. While Crizotinib, an ALK inhibitor, has been found to be therapeutically useful against a subset of ALK+ tumours, clinical resistance to this drug has been well recognized and the mechanism of this phenomenon is incompletely understood. Using the cellular thermal shift assay (CETSA), we measured the Crizotinib—ALK binding in a panel of ALK+ cell lines, and correlated the findings with the ALK structure and its interactions with specific binding proteins. The Crizotinib IC50 significantly correlated with Crizotinib—ALK binding. The suboptimal Crizotinib—ALK binding in Crizotinib-resistant cells is not due to the cell-specific environment, since transfection of NPM-ALK into these cells revealed substantial Crizotinib—NPM-ALK binding. Interestingly, we found that the resistant cells expressed higher protein level of β-catenin and siRNA knockdown restored Crizotinib—ALK binding (correlated with a significant lowering of IC50). Computational analysis of the crystal structures suggests that β-catenin exerts steric hindrance to the Crizotinib—ALK binding. In conclusion, the Crizotinib—ALK binding measurable by CETSA is useful in predicting Crizotinib sensitivity, and Crizotinib—ALK binding is in turn dictated by the structure of ALK and some of its binding partners. PMID:27641368

  2. Alpha2-macroglobulin from an Atlantic shrimp: biochemical characterization, sub-cellular localization and gene expression upon fungal challenge.

    PubMed

    Perazzolo, Luciane Maria; Bachère, Evelyne; Rosa, Rafael Diego; Goncalves, Priscila; Andreatta, Edemar Roberto; Daffre, Sirlei; Barracco, Margherita Anna

    2011-12-01

    In this study, we report on the isolation and characterization of an alpha2-macroglobulin (α2M) from the plasma of the pink shrimp Farfantepenaeus paulensis, its sub-cellular localization and transcriptional changes after infection by fungi. The molecular mass of the α2M was estimated at 389 kDa by gel filtration and 197 kDa by SDS-PAGE, under reducing conditions, suggesting that α2M from F. paulensis consists of two identical sub-units, covalently linked by disulphide bonds. The N-terminal amino acid sequence of the α2M from F. paulensis was very similar to those of other penaeid shrimps, crayfish and lobster (70-90% identity) and to a less extent with that of freshwater prawn (40% identity). A monoclonal antibody raised against the Marsupenaeus japonicus α2M made it possible to demonstrate that α2M of F. paulensis is stored in the vesicles of the shrimp granular hemocytes (through immunogold assay). Quantitative real-time PCR (qPCR) analysis showed that α2M mRNA transcripts significantly increased 24 h after an experimental infection with the shrimp pathogen Fusarium solani and it returned to the basal levels at 48 h post-injection. This is the first report on a α2M characterization in an Atlantic penaeid species and its expression profile upon a fungal infection.

  3. CD147 and AGR2 expression promote cellular proliferation and metastasis of head and neck squamous cell carcinoma

    SciTech Connect

    Sweeny, Larissa; Liu, Zhiyong; Bush, Benjamin D.; Hartman, Yolanda; Zhou, Tong; Rosenthal, Eben L.

    2012-08-15

    The signaling pathways facilitating metastasis of head and neck squamous cell carcinoma (HNSCC) cells are not fully understood. CD147 is a transmembrane glycoprotein known to induce cell migration and invasion. AGR2 is a secreted peptide also known to promote cell metastasis. Here we describe their importance in the migration and invasion of HNSCC cells (FADU and OSC-19) in vitro and in vivo. In vitro, knockdown of CD147 or AGR2 decreased cellular proliferation, migration and invasion. In vivo, knockdown of CD147 or AGR2 expression decreased primary tumor growth as well as regional and distant metastasis. -- Highlights: Black-Right-Pointing-Pointer We investigated AGR2 in head and neck squamous cell carcinoma for the first time. Black-Right-Pointing-Pointer We explored the relationship between AGR2 and CD147 for the first time. Black-Right-Pointing-Pointer AGR2 and CD147 appear to co-localize in head and squamous cell carcinoma samples. Black-Right-Pointing-Pointer Knockdown of both AGR2 and CD147 reduced migration and invasion in vitro. Black-Right-Pointing-Pointer Knockdown of both AGR2 and CD147 decreased metastasis in vivo.

  4. BRD4 Phosphorylation Regulates HPV E2-Mediated Viral Transcription, Origin Replication, and Cellular MMP-9 Expression.

    PubMed

    Wu, Shwu-Yuan; Nin, Dawn Sijin; Lee, A-Young; Simanski, Scott; Kodadek, Thomas; Chiang, Cheng-Ming

    2016-08-01

    Post-translational modification can modulate protein conformation and alter binding partner recruitment within gene regulatory regions. Here, we report that bromodomain-containing protein 4 (BRD4), a transcription co-factor and chromatin regulator, uses a phosphorylation-induced switch mechanism to recruit E2 protein encoded by cancer-associated human papillomavirus (HPV) to viral early gene and cellular matrix metalloproteinase-9 (MMP-9) promoters. Enhanced MMP-9 expression, induced upon keratinocyte differentiation, occurs via BRD4-dependent recruitment of active AP-1 and NF-κB to their target sequences. This is triggered by replacement of AP-1 family members JunB and JunD by c-Jun and by re-localization of NF-κB from the cytoplasm to the nucleus. In addition, BRD4 phosphorylation is critical for E2- and origin-dependent HPV DNA replication. A class of phospho-BRD4-targeting compounds, distinct from the BET bromodomain inhibitors, effectively blocks BRD4 phosphorylation-specific functions in transcription and factor recruitment. PMID:27477287

  5. Potential toxic effect of trifloxystrobin on cellular microstructure, mRNA expression and antioxidant enzymes in Chlorella vulgaris.

    PubMed

    Shen, Yu-Feng; Liu, Lei; Gong, Yu-Xin; Zhu, Bin; Liu, Guang-Lu; Wang, Gao-Xue

    2014-05-01

    This study investigated the effects of trifloxystrobin that one strobilurin used widely in the world as an effective fungicidal agent to control Asian soybean rust on aquatic unicellular algae Chlorella vulgaris. We determined the potential toxic effect of trifloxystrobin on C. vulgaris, and found median inhibition concentration (IC(50)) value 255.58 (95% confidence interval, 207.81-330.29)μgL(-1). In addition, the algal cells were obviously depressed or shrunk at different concentrations by electron microscopy. In the study, a real-time polymerase chain reaction (PCR) assay showed changes in transcript abundances of three photosynthetic genes, psaB, psbC, and rbcL, and one energy gene, ATPs. The results showed that trifloxystrobin reduced the transcript abundances of the three genes and enhanced expression of ATPs after 48 and 96 h. The lowest abundances of psaB, psbC and rbcL transcripts in response to trifloxystrobin exposure were 58%, 79% and 60% of those of the control, respectively. For the potential toxic influences, trifloxystrobin could decrease the soluble protein and total antioxidant contents (T-AOC), and increase superoxide dismutase (SOD) and peroxidase (POD) activity with a gradual concentration-response relationship. Overall, the present study demonstrated that trifloxystrobin could affect the activities of antioxidant enzymes, disrupts photosynthesis in C. vulgaris, and damage cellular structure.

  6. Differential expression proteomics of human colorectal cancer based on a syngeneic cellular model for the progression of adenoma to carcinoma.

    PubMed

    Roth, Udo; Razawi, Hanieh; Hommer, Julia; Engelmann, Katja; Schwientek, Tilo; Müller, Stefan; Baldus, Stephan E; Patsos, Georgios; Corfield, Anthony P; Paraskeva, Christos; Hanisch, Franz-Georg

    2010-01-01

    This is the first differential expression proteomics study on a human syngeneic cellular in vitro progression model of the colorectal adenoma-to-carcinoma sequence, the anchorage-dependent non-tumorigenic adenoma derived cell line AA/C1 and the derived anchorage-independent and tumorigenic carcinoma cell line AA/C1/SB10C. The study is based on quantitative 2-DE and is complemented by Western blot validation. Excluding redundancies due to proteolysis and post-translational modified isoforms of over 2000 protein spots, 13 proteins were revealed as regulated with statistical variance being within the 95th confidence level and were identified by peptide mass fingerprinting in MALDI MS. Progression-associated proteins belong to the functional complexes of anaerobic glycolysis/gluconeogenesis, steroid biosynthesis, prostaglandin biosynthesis, the regulation and maintenance of the cytoskeleton, protein biosynthesis and degradation, the regulation of apoptosis or other functions. Partial but significant overlap was revealed with previous proteomics and transcriptomics studies in colorectal carcinoma. Among upregulated proteins we identified 3-HMG-CoA synthase, protein phosphatase 1, prostaglandin E synthase 2, villin 1, annexin A1, triosephosphate isomerase, phosphoserine aminotransferase 1, fumarylacetoacetate hydrolase and pyrroline-5-carboxylate reductase 1 (PYCR1), while glucose-regulated protein 78, cathepsin D, lamin A/C and quinolate phosphoribosyltransferase were downregulated.

  7. The Cellular Localization of Human Cytomegalovirus Glycoprotein Expression Greatly Influences the Frequency and Functional Phenotype of Specific CD4+ T Cell Responses.

    PubMed

    Pachnio, Annette; Zuo, Jianmin; Ryan, Gordon B; Begum, Jusnara; Moss, Paul A H

    2015-10-15

    CMV infection is a significant cause of morbidity and mortality in immunocompromised individuals, and the development of a vaccine is of high priority. Glycoprotein B (gB) is a leading vaccine candidate but the glycoprotein H (gH) pentameric complex is now recognized as the major target for neutralizing Abs. However, little is known about the T cell immune response against gH and glycoprotein L (gL) and this is likely to be an important attribute for vaccine immunogenicity. In this study, we examine and contrast the magnitude and phenotype of the T cell immune response against gB, gH, and gL within healthy donors. gB-specific CD4(+) T cells were found in 95% of donors, and 29 epitopes were defined with gB-specific response sizes ranging from 0.02 to 2.88% of the CD4(+) T cell pool. In contrast, only 20% of donors exhibited a T cell response against gH or gL. Additionally, gB-specific CD4(+) T cells exhibited a more cytotoxic phenotype, with high levels of granzyme B expression. Glycoproteins were effectively presented following delivery to APCs but only gB-derived epitopes were presented following endogenous synthesis. gB expression was observed exclusively within vesicular structures colocalizing with HLA-DM whereas gH was distributed evenly throughout the cytoplasm. Grafting of the C-terminal domain from gB onto gH could not transfer this pattern of presentation. These results reveal that gB is a uniquely immunogenic CMV glycoprotein and this is likely to reflect its unique pattern of endogenous Ag presentation. Consideration may be required toward mechanisms that boost cellular immunity to gH and gL within future subunit vaccines.

  8. Ectopic AP4 expression induces cellular senescence via activation of p53 in long-term confluent retinal pigment epithelial cells.

    PubMed

    Wang, Yiping; Wong, Matthew Man-Kin; Zhang, Xiaojian; Chiu, Sung-Kay

    2015-11-15

    When cells are grown to confluence, cell-cell contact inhibition occurs and drives the cells to enter reversible quiescence rather than senescence. Confluent retinal pigment epithelial (RPE) cells exhibiting contact inhibition was used as a model in this study to examine the role of overexpression of transcription factor AP4, a highly expressed transcription factor in many types of cancer, in these cells during long-term culture. We generated stable inducible RPE cell clones expressing AP4 or AP4 without the DNA binding domain (DN-AP4) and observed that, when cultured for 24 days, RPE cells with a high level of AP4 exhibit a large, flattened morphology and even cease proliferating; these changes were not observed in DN-AP4-expressing cells or non-induced cells. In addition, AP4-expressing cells exhibited senescence-associated β-galactosidase activity and the senescence-associated secretory phenotype. We demonstrated that the induced cellular senescence was mediated by enhanced p53 expression and that AP4 regulates the p53 gene by binding directly to two of the three E-boxes present on the promoter of the p53 gene. Moreover, we showed that serum is essential for AP4 in inducing p53-associated cellular senescence. Collectively, we showed that overexpression of AP4 mediates cellular senescence involving in activation of p53 in long-term post-confluent RPE cells.

  9. Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies.

    PubMed

    Dhamrait, Sukhbir S; Maubaret, Cecilia; Pedersen-Bjergaard, Ulrik; Brull, David J; Gohlke, Peter; Payne, John R; World, Michael; Thorsteinsson, Birger; Humphries, Steve E; Montgomery, Hugh E

    2016-07-01

    Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin-angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations (healthy young UK men and Scandinavian diabetic patients) serum ACE (sACE) activity was significantly higher amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P < 0·01) whilst increasing ACE expression within a physiological range (<1·8-fold at 48 h; P < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role. PMID:27417115

  10. Mitochondrial uncoupling proteins regulate angiotensin‐converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies

    PubMed Central

    Maubaret, Cecilia; Pedersen‐Bjergaard, Ulrik; Brull, David J.; Gohlke, Peter; Payne, John R.; World, Michael; Thorsteinsson, Birger; Humphries, Steve E.; Montgomery, Hugh E.

    2015-01-01

    Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin‐converting enzyme (ACE) is the central component of endocrine and local tissue renin–angiotensin systems (RAS), which also regulate diverse aspects of whole‐body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations (healthy young UK men and Scandinavian diabetic patients) serum ACE (sACE) activity was significantly higher amongst UCP3‐55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P < 0·01) whilst increasing ACE expression within a physiological range (<1·8‐fold at 48 h; P < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role. PMID:27347560

  11. Robust modeling of differential gene expression data using normal/independent distributions: a Bayesian approach.

    PubMed

    Ganjali, Mojtaba; Baghfalaki, Taban; Berridge, Damon

    2015-01-01

    In this paper, the problem of identifying differentially expressed genes under different conditions using gene expression microarray data, in the presence of outliers, is discussed. For this purpose, the robust modeling of gene expression data using some powerful distributions known as normal/independent distributions is considered. These distributions include the Student's t and normal distributions which have been used previously, but also include extensions such as the slash, the contaminated normal and the Laplace distributions. The purpose of this paper is to identify differentially expressed genes by considering these distributional assumptions instead of the normal distribution. A Bayesian approach using the Markov Chain Monte Carlo method is adopted for parameter estimation. Two publicly available gene expression data sets are analyzed using the proposed approach. The use of the robust models for detecting differentially expressed genes is investigated. This investigation shows that the choice of model for differentiating gene expression data is very important. This is due to the small number of replicates for each gene and the existence of outlying data. Comparison of the performance of these models is made using different statistical criteria and the ROC curve. The method is illustrated using some simulation studies. We demonstrate the flexibility of these robust models in identifying differentially expressed genes. PMID:25910040

  12. Meta-analysis of expression of hepatic organic anion-transporting polypeptide (OATP) transporters in cellular systems relative to human liver tissue.

    PubMed

    Badée, Justine; Achour, Brahim; Rostami-Hodjegan, Amin; Galetin, Aleksandra

    2015-04-01

    Organic anion-transporting polypeptide (OATP)1B1, OATP1B3, and OATP2B1 transporters play an important role in hepatic drug disposition. Recently, an increasing number of studies have reported proteomic expression data for OATP transporters. However, systematic analysis and understanding of the actual differences in OATP expression between liver tissue and commonly used cellular systems is lacking. In the current study, meta-analysis was performed to assess the protein expression of OATP transporters reported in hepatocytes relative to liver tissue and to identify any potential correlations in transporter expression levels in the same individual. OATP1B1 was identified as the most abundant uptake transporter at 5.9 ± 8.3, 5.8 ± 3.3, and 4.2 ± 1.7 fmol/μg protein in liver tissue, sandwich-cultured human hepatocytes (SCHH), and cryopreserved suspended hepatocytes, respectively. The rank order in average expression in liver tissue and cellular systems was OATP1B1 > OATP1B3 ≈ OATP2B1. Abundance levels of the OATP transporters investigated were not significantly different between liver and cellular systems, with the exception of OATP2B1 expression in SCHH relative to liver tissue. Analysis of OATP1B1, OATP1B3, and OATP2B1 liver expression data in the same individuals (n = 86) identified weak (OATP1B1-OATP2B1) to moderately (OATP1B3-OATP2B1) significant correlations. A significant weak correlation was noted between OATP1B1 abundance and age of human donors, whereas expression of the OATPs investigated was independent of sex. Implications of the current analysis on the in vitro-in vivo extrapolation of transporter-mediated drug disposition using physiologically based pharmacokinetic models are discussed.

  13. Comparison of gene expression signatures of diamide, H2O2 and menadione exposed Aspergillus nidulans cultures – linking genome-wide transcriptional changes to cellular physiology

    PubMed Central

    Pócsi, István; Miskei, Márton; Karányi, Zsolt; Emri, Tamás; Ayoubi, Patricia; Pusztahelyi, Tünde; Balla, György; Prade, Rolf A

    2005-01-01

    Background In addition to their cytotoxic nature, reactive oxygen species (ROS) are also signal molecules in diverse cellular processes in eukaryotic organisms. Linking genome-wide transcriptional changes to cellular physiology in oxidative stress-exposed Aspergillus nidulans cultures provides the opportunity to estimate the sizes of peroxide (O22-), superoxide (O2•-) and glutathione/glutathione disulphide (GSH/GSSG) redox imbalance responses. Results Genome-wide transcriptional changes triggered by diamide, H2O2 and menadione in A. nidulans vegetative tissues were recorded using DNA microarrays containing 3533 unique PCR-amplified probes. Evaluation of LOESS-normalized data indicated that 2499 gene probes were affected by at least one stress-inducing agent. The stress induced by diamide and H2O2 were pulse-like, with recovery after 1 h exposure time while no recovery was observed with menadione. The distribution of stress-responsive gene probes among major physiological functional categories was approximately the same for each agent. The gene group sizes solely responsive to changes in intracellular O22-, O2•- concentrations or to GSH/GSSG redox imbalance were estimated at 7.7, 32.6 and 13.0 %, respectively. Gene groups responsive to diamide, H2O2 and menadione treatments and gene groups influenced by GSH/GSSG, O22- and O2•- were only partly overlapping with distinct enrichment profiles within functional categories. Changes in the GSH/GSSG redox state influenced expression of genes coding for PBS2 like MAPK kinase homologue, PSK2 kinase homologue, AtfA transcription factor, and many elements of ubiquitin tagging, cell division cycle regulators, translation machinery proteins, defense and stress proteins, transport proteins as well as many enzymes of the primary and secondary metabolisms. Meanwhile, a separate set of genes encoding transport proteins, CpcA and JlbA amino acid starvation-responsive transcription factors, and some elements of sexual development

  14. Insulin stimulates movement of sorting nexin 9 between cellular compartments: a putative role mediating cell surface receptor expression and insulin action.

    PubMed Central

    MaCaulay, S Lance; Stoichevska, Violet; Grusovin, Julian; Gough, Keith H; Castelli, Laura A; Ward, Colin W

    2003-01-01

    SNX9 (sorting nexin 9) is one member of a family of proteins implicated in protein trafficking. This family is characterized by a unique PX (Phox homology) domain that includes a proline-rich sequence and an upstream phospholipid binding domain. Many sorting nexins, including SNX9, also have a C-terminal coiled region. SNX9 additionally has an N-terminal SH3 (Src homology 3) domain. Here we have investigated the cellular localization of SNX9 and the potential role it plays in insulin action. SNX9 had a cytosolic and punctate distribution, consistent with endosomal and cytosolic localization, in 3T3L1 adipocytes. It was excluded from the nucleus. The SH3 domain was responsible, at least in part, for the membrane localization of SNX9, since expression of an SH3-domain-deleted GFP (green fluorescent protein)-SNX9 fusion protein in HEK293T cells rendered the protein cytosolic. Membrane localization may also be attributed in part to the PX domain, since in vitro phospholipid binding studies demonstrated SNX9 binding to polyphosphoinositides. Insulin induced movement of SNX9 to membrane fractions from the cytosol. A GST (glutathione S-transferase)-SNX9 fusion protein was associated with IGF1 (insulin-like growth factor 1) and insulin receptors in vitro. A GFP-SNX9 fusion protein, overexpressed in 3T3L1 adipocytes, co-immunoprecipitated with insulin receptors. Furthermore, overexpression of this GFP-SNX9 fusion protein in CHOT cells decreased insulin binding, consistent with a role for SNX9 in the trafficking of insulin receptors. Microinjection of 3T3L1 cells with an antibody against SNX9 inhibited stimulation by insulin of GLUT4 translocation. These results support the involvement of SNX9 in insulin action, via an influence on the processing/trafficking of insulin receptors. A secondary role in regulation of the cellular processing, transport and/or subcellular localization of GLUT4 is also suggested. PMID:12917015

  15. Expression of Senescence-Associated microRNAs and Target Genes in Cellular Aging and Modulation by Tocotrienol-Rich Fraction

    PubMed Central

    2014-01-01

    Emerging evidences highlight the implication of microRNAs as a posttranscriptional regulator in aging. Several senescence-associated microRNAs (SA-miRNAs) are found to be differentially expressed during cellular senescence. However, the role of dietary compounds on SA-miRNAs remains elusive. This study aimed to elucidate the modulatory role of tocotrienol-rich fraction (TRF) on SA-miRNAs (miR-20a, miR-24, miR-34a, miR-106a, and miR-449a) and established target genes of miR-34a (CCND1, CDK4, and SIRT1) during replicative senescence of human diploid fibroblasts (HDFs). Primary cultures of HDFs at young and senescent were incubated with TRF at 0.5 mg/mL. Taqman microRNA assay showed significant upregulation of miR-24 and miR-34a and downregulation of miR-20a and miR-449a in senescent HDFs (P < 0.05). TRF reduced miR-34a expression in senescent HDFs and increased miR-20a expression in young HDFs and increased miR-449a expression in both young and senescent HDFs. Our results also demonstrated that ectopic expression of miR-34a reduced the expression of CDK4 significantly (P < 0.05). TRF inhibited miR-34a expression thus relieved its inhibition on CDK4 gene expression. No significant change was observed on the expression of CCND1, SIRT1, and miR-34a upstream transcriptional regulator, TP53. In conclusion tocotrienol-rich fraction prevented cellular senescence of human diploid fibroblasts via modulation of SA-miRNAs and target genes expression. PMID:25132913

  16. The UL24 protein of herpes simplex virus 1 affects the sub-cellular distribution of viral glycoproteins involved in fusion

    SciTech Connect

    Ben Abdeljelil, Nawel; Rochette, Pierre-Alexandre; Pearson, Angela

    2013-09-15

    Mutations in UL24 of herpes simplex virus type 1 can lead to a syncytial phenotype. We hypothesized that UL24 affects the sub-cellular distribution of viral glycoproteins involved in fusion. In non-immortalized human foreskin fibroblasts (HFFs) we detected viral glycoproteins B (gB), gD, gH and gL present in extended blotches throughout the cytoplasm with limited nuclear membrane staining; however, in HFFs infected with a UL24-deficient virus (UL24X), staining for the viral glycoproteins appeared as long, thin streaks running across the cell. Interestingly, there was a decrease in co-localized staining of gB and gD with F-actin at late times in UL24X-infected HFFs. Treatment with chemical agents that perturbed the actin cytoskeleton hindered the formation of UL24X-induced syncytia in these cells. These data support a model whereby the UL24 syncytial phenotype results from a mislocalization of viral glycoproteins late in infection. - Highlights: • UL24 affects the sub-cellular distribution of viral glycoproteins required for fusion. • Sub-cellular distribution of viral glycoproteins varies in cell-type dependent manner. • Drugs targeting actin microfilaments affect formation of UL24-related syncytia in HFFs.

  17. 75 FR 56920 - Express Mail Open and Distribute and Priority Mail Open and Distribute

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-17

    ... guaranteed end of day product; and to add a five-pound minimum weight requirement for Express Mail Open and... windows, without loss of service, we are proposing to change the service commitment to end of day (11:59 p... commitments, we are proposing to establish a five-pound minimum weight requirement for all Express Mail...

  18. The expression of S100P increases and promotes cellular proliferation by increasing nuclear translocation of β-catenin in endometrial cancer.

    PubMed

    Guo, Luyan; Chen, Shuqin; Jiang, Hongye; Huang, Jiaming; Jin, Wenyan; Yao, Shuzhong

    2014-01-01

    There is increasing evidence suggesting that S100P has a significant role in cancer, and is associated with poor clinical outcomes. The expression of S100P mRNA and protein in endometrial cancer and normal endometrium tissues was detected by real-time quantitative RT-PCR and immunohistochemistry. Moreover, we reduced the expression of S100P in HEC-1A and Ishikawa endometrial cancer cell lines by siRNA transfection. Based on the reduced S100P mRNA expression, we measured the effects of S100P on cellular proliferation by the cell-counting kit-8. Nuclear β-catenin protein level was detected by western blotting. Cyclin D1 and c-myc mRNA expression regulated by β-catenin was detected by real-time quantitative RT-PCR. We found that the expression of S100P mRNA and protein increased in endometrial cancer tissues compared with the normal endometrium. Local S100P expression progressively increased from pathologic differenciation grade 1 to 3. After reducing the S100P expression, the cellular proliferation ability, nuclear β-catenin protein level, cyclin D1 and c-myc mRNA levels reduced. It indicated that S100P could promote cell proliferation by increasing nuclear translocation of β-catenin. The expression of S100P mRNA and protein in endometrial cancer significantly increased and is associated with pathologic differenciation grade. S100P may promote endometrial cell proliferation by increasing nuclear translocation of β-catenin.

  19. The Neurona at Home project: Simulating a large-scale cellular automata brain in a distributed computing environment

    NASA Astrophysics Data System (ADS)

    Acedo, L.; Villanueva-Oller, J.; Moraño, J. A.; Villanueva, R.-J.

    2013-01-01

    The Berkeley Open Infrastructure for Network Computing (BOINC) has become the standard open source solution for grid computing in the Internet. Volunteers use their computers to complete an small part of the task assigned by a dedicated server. We have developed a BOINC project called Neurona@Home whose objective is to simulate a cellular automata random network with, at least, one million neurons. We consider a cellular automata version of the integrate-and-fire model in which excitatory and inhibitory nodes can activate or deactivate neighbor nodes according to a set of probabilistic rules. Our aim is to determine the phase diagram of the model and its behaviour and to compare it with the electroencephalographic signals measured in real brains.

  20. Effects of 5-Fluorouracil in Nuclear and Cellular Morphology, Proliferation, Cell Cycle, Apoptosis, Cytoskeletal and Caveolar Distribution in Primary Cultures of Smooth Muscle Cells

    PubMed Central

    Filgueiras, Marcelo de Carvalho; Morrot, Alexandre; Soares, Pedro Marcos Gomes; Costa, Manoel Luis; Mermelstein, Cláudia

    2013-01-01

    Colon cancer is one of the most prevalent types of cancer in the world and is one of the leading causes of cancer death. The anti-metabolite 5- fluorouracil (5-FU) is widely used in the treatment of patients with colon cancer and other cancer types. 5-FU-based chemotherapy has been shown to be very efficient in the improvement of overall survival of the patients and for the eradication of the disease. Unfortunately, common side effects of 5-FU include severe alterations in the motility of the gastrointestinal tissues. Nevertheless, the molecular and cellular effects of 5-FU in smooth muscle cells are poorly understood. Primary smooth muscle cell cultures are an important tool for studies of the biological consequences of 5-FU at the cellular level. The avian gizzard is one of the most robust organs of smooth muscle cells. Here we studied the molecular and cellular effects of the chemotherapic drug 5-FU in a primary culture of chick gizzard smooth muscle cells. We found that treatment of smooth muscle cells with 5-FU inhibits cell proliferation by the arrest of cells in the G1 phase of cell cycle and induce apoptosis. 5-FU induced a decrease in the percentage of histone H3-positive cells. Treatment of cells with 5-FU induced changes in cellular and nuclear morphology, a decrease in the number of stress fibers and a major decrease in the number of caveolin-3 positive cells. Our results suggest that the disorganization of the actin cytoskeleton and the reduction of caveolin-3 expression could explain the alterations in contractility observed in patients treated with 5-FU. These findings might have an impact in the understanding of the cellular effects of 5-FU in smooth muscle tissues and might help the improvement of new therapeutic protocols for the treatment of colon cancer. PMID:23646193

  1. Effects of 5-fluorouracil in nuclear and cellular morphology, proliferation, cell cycle, apoptosis, cytoskeletal and caveolar distribution in primary cultures of smooth muscle cells.

    PubMed

    Filgueiras, Marcelo de Carvalho; Morrot, Alexandre; Soares, Pedro Marcos Gomes; Costa, Manoel Luis; Mermelstein, Cláudia

    2013-01-01

    Colon cancer is one of the most prevalent types of cancer in the world and is one of the leading causes of cancer death. The anti-metabolite 5- fluorouracil (5-FU) is widely used in the treatment of patients with colon cancer and other cancer types. 5-FU-based chemotherapy has been shown to be very efficient in the improvement of overall survival of the patients and for the eradication of the disease. Unfortunately, common side effects of 5-FU include severe alterations in the motility of the gastrointestinal tissues. Nevertheless, the molecular and cellular effects of 5-FU in smooth muscle cells are poorly understood. Primary smooth muscle cell cultures are an important tool for studies of the biological consequences of 5-FU at the cellular level. The avian gizzard is one of the most robust organs of smooth muscle cells. Here we studied the molecular and cellular effects of the chemotherapic drug 5-FU in a primary culture of chick gizzard smooth muscle cells. We found that treatment of smooth muscle cells with 5-FU inhibits cell proliferation by the arrest of cells in the G1 phase of cell cycle and induce apoptosis. 5-FU induced a decrease in the percentage of histone H3-positive cells. Treatment of cells with 5-FU induced changes in cellular and nuclear morphology, a decrease in the number of stress fibers and a major decrease in the number of caveolin-3 positive cells. Our results suggest that the disorganization of the actin cytoskeleton and the reduction of caveolin-3 expression could explain the alterations in contractility observed in patients treated with 5-FU. These findings might have an impact in the understanding of the cellular effects of 5-FU in smooth muscle tissues and might help the improvement of new therapeutic protocols for the treatment of colon cancer.

  2. The influence of metal speciation on the bioavailability and sub-cellular distribution of cadmium to the terrestrial isopod, Porcellio dilatatus.

    PubMed

    Calhôa, Carla Filipa; Monteiro, Marta S; Soares, Amadeu M V M; Mann, Reinier M

    2011-04-01

    Cadmium is a non-essential toxic metal that is able to bioaccumulate in both flora fauna and has the potential to biomagnify in some food chains. However, the form in which cadmium is presented to consumers can alter the bioavailability and possibly the internal distribution of assimilated Cd. Previous studies in our laboratory highlighted differences in Cd assimilation among isopods when they were provided with a plant-based food with either Cd biologically incorporated into plant tissue or superficially amended with ionic Cd(2+). Cd is known for its high affinity for sulphur ligands in cysteine residues which form the basis for metal-binding proteins such as metallothionein. This study compares Cd assimilation efficiency (AE) in Porcellio dilatatus fed with food amended with either cadmium cysteinate or cadmium nitrate in an examination of the influence of Cd speciation on metal bioavailability followed by an examination of the sub-cellular distribution using a centrifugal fractionation protocol. As hypothesized the AE of Cd among isopods fed with Cd(NO(3))(2) (64%, SE=5%) was higher than AE for isopods fed with Cd(Cys)(2) (20%, SE=3%). The sub-cellular distribution also depended on the Cd species provided. Those isopods fed Cd(Cys)(2) allocated significantly more Cd to the cell debris and organelles fractions at the expense of allocation to metal-rich granules (MRG). The significance of the difference in sub-cellular distribution with regard to toxicity is discussed. This paper demonstrates that the assimilation and internal detoxification of Cd is dependent on the chemical form of Cd presented to the isopod.

  3. Identification of Absorption, Distribution, Metabolism, and Excretion (ADME) Genes Relevant to Steatosis Using a Differential Gene Expression Approach

    EPA Science Inventory

    Absorption, distribution, metabolism, and excretion (ADME) parameters represent important connections between exposure to chemicals and the activation of molecular initiating events of Adverse Outcome Pathways (AOPs) in cellular, tissue, and organ level targets. ADME parameters u...

  4. Effects of fade distribution on a mobile satellite downlink and uplink performance in a frequency reuse cellular configuration

    NASA Technical Reports Server (NTRS)

    Boutin, Karl; Lecours, Michel; Pelletier, Marcel; Delisle, Gilles Y.

    1990-01-01

    In a mobile satellite system with a frequency reuse cellular configuration, significant co-channel interference can be experienced due to the antenna sidelobe level. The signal will be subjected not only to its own fading, but also to the effect of the varying degree of fading on co-channel interferer, and this interference will behave differently in the up and in the down link. This paper presents a quantitative evaluation of the combined effects of fades and co-channel interference on a mobile satellite link.

  5. Mechanisms of Nrf2/Keap1-Dependent Phase II Cytoprotective and Detoxifying Gene Expression and Potential Cellular Targets of Chemopreventive Isothiocyanates

    PubMed Central

    Das, Biswa Nath; Kim, Young-Woo

    2013-01-01

    Isothiocyanates (ITCs) are abundantly found in cruciferous vegetables. Epidemiological studies suggest that chronic consumption of cruciferous vegetables can lower the overall risk of cancer. Natural ITCs are key chemopreventive ingredients of cruciferous vegetables, and one of the prime chemopreventive mechanisms of natural isothiocyanates is the induction of Nrf2/ARE-dependent gene expression that plays a critical role in cellular defense against electrophiles and reactive oxygen species. In the present review, we first discuss the underlying mechanisms how natural ITCs affect the intracellular signaling kinase cascades to regulate the Keap1/Nrf2 activities, thereby inducing phase II cytoprotective and detoxifying enzymes. We also discuss the potential cellular protein targets to which natural ITCs are directly conjugated and how these events aid in the chemopreventive effects of natural ITCs. Finally, we discuss the posttranslational modifications of Keap1 and nucleocytoplasmic trafficking of Nrf2 in response to electrophiles and oxidants. PMID:23781297

  6. Mechanical models of the cellular cytoskeletal network for the analysis of intracellular mechanical properties and force distributions: a review.

    PubMed

    Chen, Ting-Jung; Wu, Chia-Ching; Su, Fong-Chin

    2012-12-01

    The cytoskeleton, which is the major mechanical component of cells, supports the cell body and regulates the cellular motility to assist the cell in performing its biological functions. Several cytoskeletal network models have been proposed to investigate the mechanical properties of cells. This review paper summarizes these models with a focus on the prestressed cable network, the semi-flexible chain network, the open-cell foam, the tensegrity, and the granular models. The components, material parameters, types of connection joints, tension conditions, and the advantages and disadvantages of each model are evaluated from a structural and biological point of view. The underlying mechanisms that are associated with the morphological changes of spreading cells are expected to be simulated using a cytoskeletal model; however, it is still paid less attention most likely due to the lack of a suitable cytoskeletal model that can accurately model the spreading process. In this review article, the established cytoskeletal models are hoped to provide useful information for the development of future cytoskeletal models with different degrees of cell attachment for the study of the mechanical mechanisms underlying the cellular behaviors in response to external stimulations. PMID:23062682

  7. Perfringolysin O Theta Toxin as a Tool to Monitor the Distribution and Inhomogeneity of Cholesterol in Cellular Membranes

    PubMed Central

    Maekawa, Masashi; Yang, Yanbo; Fairn, Gregory D.

    2016-01-01

    Cholesterol is an essential structural component of cellular membranes in eukaryotes. Cholesterol in the exofacial leaflet of the plasma membrane is thought to form membrane nanodomains with sphingolipids and specific proteins. Additionally, cholesterol is found in the intracellular membranes of endosomes and has crucial functions in membrane trafficking. Furthermore, cellular cholesterol homeostasis and regulation of de novo synthesis rely on transport via both vesicular and non-vesicular pathways. Thus, the ability to visualize and detect intracellular cholesterol, especially in the plasma membrane, is critical to understanding the complex biology associated with cholesterol and the nanodomains. Perfringolysin O (PFO) theta toxin is one of the toxins secreted by the anaerobic bacteria Clostridium perfringens and this toxin forms pores in the plasma membrane that causes cell lysis. It is well understood that PFO recognizes and binds to cholesterol in the exofacial leaflets of the plasma membrane, and domain 4 of PFO (D4) is sufficient for the binding of cholesterol. Recent studies have taken advantage of this high-affinity cholesterol-binding domain to create a variety of cholesterol biosensors by using a non-toxic PFO or the D4 in isolation. This review highlights the characteristics and usefulness of, and the principal findings related to, these PFO-derived cholesterol biosensors. PMID:27005662

  8. The effect of oil-water partition coefficient on the distribution and cellular uptake of liposome-encapsulated gold nanoparticles.

    PubMed

    Bao, Quan-Ying; Liu, Ai-Yun; Ma, Yu; Chen, Huan; Hong, Jin; Shen, Wen-Bin; Zhang, Can; Ding, Ya

    2016-10-01

    The shape, size, and surface features of nanoparticles greatly influence the structure and properties of resulting hybrid nanosystems. In this work, gold nanoparticles (GNPs) were modified via S-Au covalent bonding by glycol monomethyl ether thioctate with poly(ethylene glycol) methyl ether of different molecular weights (i.e., 350, 550, and 750Da). These modified GNPs (i.e., GNP350, GNP550, and GNP750) showed different oil-water partition coefficients (Kp), as detected using inductively coupled plasma-atomic emission spectroscopy. The different Kp values of the gold conjugates (i.e., 13.98, 2.11, and 0.036 for GNP350, GNP550, and GNP750, respectively) resulted in different conjugate localization within liposomes, as observed by transmission electron microscopy. In addition, the cellular uptake of hybrid liposomes co-encapsulating gold conjugates and Nile red was evaluated using intracellular fluorescence intensity. The results indicated that precise GNP localization in the hydrophilic or hydrophobic liposome cavity could be achieved by regulating the GNP oil-water partition coefficient via surface modification; such localization could further affect the properties and functions of hybrid liposomes, including their cellular uptake profiles. This study furthers the understanding not only of the interaction between liposomes and inorganic nanoparticles but also of adjusting liposome-gold hybrid nanostructure properties via the surface chemistry of gold materials. PMID:27400242

  9. Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts

    SciTech Connect

    Eom, Young Woo; Lee, Jong Eun; Yang, Mal Sook; Jang, In Keun; Kim, Hyo Eun; Lee, Doo Hoon; Kim, Young Jin; Park, Won Jin; Kong, Jee Hyun; Shim, Kwang Yong; Lee, Jong In; Kim, Hyun Soo

    2011-04-29

    Highlights: {yields} hASCs were differentiated into skeletal muscle cells by treatment with 5-azacytidine, FGF-2, and the supernatant of cultured hASCs. {yields} Dystrophin and MyHC were expressed in late differentiation step by treatment with the supernatant of cultured hASCs. {yields} hASCs expressing dystrophin and MyHC contributed to myotube formation during co-culture with mouse myoblast C2C12 cells. -- Abstract: Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of human adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.

  10. The Expression Pattern of the Pre-B Cell Receptor Components Correlates with Cellular Stage and Clinical Outcome in Acute Lymphoblastic Leukemia

    PubMed Central

    Chen, Dongfeng; Zheng, Junxiong; Gerasimcik, Natalija; Lagerstedt, Kristina; Sjögren, Helene; Abrahamsson, Jonas; Fogelstrand, Linda; Mårtensson, Inga-Lill

    2016-01-01

    Precursor-B cell receptor (pre-BCR) signaling represents a crucial checkpoint at the pre-B cell stage. Aberrant pre-BCR signaling is considered as a key factor for B-cell precursor acute lymphoblastic leukemia (BCP-ALL) development. BCP-ALL are believed to be arrested at the pre-BCR checkpoint independent of pre-BCR expression. However, the cellular stage at which BCP-ALL are arrested and whether this relates to expression of the pre-BCR components (IGHM, IGLL1 and VPREB1) is still unclear. Here, we show differential protein expression and copy number variation (CNV) patterns of the pre-BCR components in pediatric BCP-ALL. Moreover, analyzing six BCP-ALL data sets (n = 733), we demonstrate that TCF3-PBX1 ALL express high levels of IGHM, IGLL1 and VPREB1, and are arrested at the pre-B stage. By contrast, ETV6-RUNX1 ALL express low levels of IGHM or VPREB1, and are arrested at the pro-B stage. Irrespective of subtype, ALL with high levels of IGHM, IGLL1 and VPREB1 are arrested at the pre-B stage and correlate with good prognosis in high-risk pediatric BCP-ALL (n = 207). Our findings suggest that BCP-ALL are arrested at different cellular stages, which relates to the expression pattern of the pre-BCR components that could serve as prognostic markers for high-risk pediatric BCP-ALL patients. PMID:27611867

  11. The Expression Pattern of the Pre-B Cell Receptor Components Correlates with Cellular Stage and Clinical Outcome in Acute Lymphoblastic Leukemia.

    PubMed

    Chen, Dongfeng; Zheng, Junxiong; Gerasimcik, Natalija; Lagerstedt, Kristina; Sjögren, Helene; Abrahamsson, Jonas; Fogelstrand, Linda; Mårtensson, Inga-Lill

    2016-01-01

    Precursor-B cell receptor (pre-BCR) signaling represents a crucial checkpoint at the pre-B cell stage. Aberrant pre-BCR signaling is considered as a key factor for B-cell precursor acute lymphoblastic leukemia (BCP-ALL) development. BCP-ALL are believed to be arrested at the pre-BCR checkpoint independent of pre-BCR expression. However, the cellular stage at which BCP-ALL are arrested and whether this relates to expression of the pre-BCR components (IGHM, IGLL1 and VPREB1) is still unclear. Here, we show differential protein expression and copy number variation (CNV) patterns of the pre-BCR components in pediatric BCP-ALL. Moreover, analyzing six BCP-ALL data sets (n = 733), we demonstrate that TCF3-PBX1 ALL express high levels of IGHM, IGLL1 and VPREB1, and are arrested at the pre-B stage. By contrast, ETV6-RUNX1 ALL express low levels of IGHM or VPREB1, and are arrested at the pro-B stage. Irrespective of subtype, ALL with high levels of IGHM, IGLL1 and VPREB1 are arrested at the pre-B stage and correlate with good prognosis in high-risk pediatric BCP-ALL (n = 207). Our findings suggest that BCP-ALL are arrested at different cellular stages, which relates to the expression pattern of the pre-BCR components that could serve as prognostic markers for high-risk pediatric BCP-ALL patients. PMID:27611867

  12. Expression Profile of Human Fc Receptors in Mucosal Tissue: Implications for Antibody-Dependent Cellular Effector Functions Targeting HIV-1 Transmission

    PubMed Central

    Cheeseman, Hannah M.; Carias, Ann M.; Evans, Abbey B.; Olejniczak, Natalia J.; Ziprin, Paul; King, Deborah F. L.; Hope, Thomas J.; Shattock, Robin J.

    2016-01-01

    The majority of new Human Immunodeficiency Virus (HIV)-1 infections are acquired via sexual transmission at mucosal surfaces. Partial efficacy (31.2%) of the Thai RV144 HIV-1 vaccine trial has been correlated with Antibody-dependent Cellular Cytotoxicity (ADCC) mediated by non-neutralizing antibodies targeting the V1V2 region of the HIV-1 envelope. This has led to speculation that ADCC and other antibody-dependent cellular effector functions might provide an important defense against mucosal acquisition of HIV-1 infection. However, the ability of antibody-dependent cellular effector mechanisms to impact on early mucosal transmission events will depend on a variety of parameters including effector cell type, frequency, the class of Fc-Receptor (FcR) expressed, the number of FcR per cell and the glycoslyation pattern of the induced antibodies. In this study, we characterize and compare the frequency and phenotype of IgG (CD16 [FcγRIII], CD32 [FcγRII] and CD64 [FcγRI]) and IgA (CD89 [FcαR]) receptor expression on effector cells within male and female genital mucosal tissue, colorectal tissue and red blood cell-lysed whole blood. The frequency of FcR expression on CD14+ monocytic cells, myeloid dendritic cells and natural killer cells were similar across the three mucosal tissue compartments, but significantly lower when compared to the FcR expression profile of effector cells isolated from whole blood, with many cells negative for all FcRs. Of the three tissues tested, penile tissue had the highest percentage of FcR positive effector cells. Immunofluorescent staining was used to determine the location of CD14+, CD11c+ and CD56+ cells within the three mucosal tissues. We show that the majority of effector cells across the different mucosal locations reside within the subepithelial lamina propria. The potential implication of the observed FcR expression patterns on the effectiveness of FcR-dependent cellular effector functions to impact on the initial events in

  13. Global Exponential Stability of Almost Periodic Solution for Neutral-Type Cohen-Grossberg Shunting Inhibitory Cellular Neural Networks with Distributed Delays and Impulses

    PubMed Central

    Xu, Lijun; Jiang, Qi; Gu, Guodong

    2016-01-01

    A kind of neutral-type Cohen-Grossberg shunting inhibitory cellular neural networks with distributed delays and impulses is considered. Firstly, by using the theory of impulsive differential equations and the contracting mapping principle, the existence and uniqueness of the almost periodic solution for the above system are obtained. Secondly, by constructing a suitable Lyapunov functional, the global exponential stability of the unique almost periodic solution is also investigated. The work in this paper improves and extends some results in recent years. As an application, an example and numerical simulations are presented to demonstrate the feasibility and effectiveness of the main results. PMID:27190502

  14. Modulation of cellular iron metabolism by hydrogen peroxide. Effects of H2O2 on the expression and function of iron-responsive element-containing mRNAs in B6 fibroblasts.

    PubMed

    Caltagirone, A; Weiss, G; Pantopoulos, K

    2001-06-01

    Cellular iron uptake and storage are coordinately controlled by binding of iron-regulatory proteins (IRP), IRP1 and IRP2, to iron-responsive elements (IREs) within the mRNAs encoding transferrin receptor (TfR) and ferritin. Under conditions of iron starvation, both IRP1 and IRP2 bind with high affinity to cognate IREs, thus stabilizing TfR and inhibiting translation of ferritin mRNAs. The IRE/IRP regulatory system receives additional input by oxidative stress in the form of H(2)O(2) that leads to rapid activation of IRP1. Here we show that treating murine B6 fibroblasts with a pulse of 100 microm H(2)O(2) for 1 h is sufficient to alter critical parameters of iron homeostasis in a time-dependent manner. First, this stimulus inhibits ferritin synthesis for at least 8 h, leading to a significant (50%) reduction of cellular ferritin content. Second, treatment with H(2)O(2) induces a approximately 4-fold increase in TfR mRNA levels within 2-6 h, and subsequent accumulation of newly synthesized protein after 4 h. This is associated with a profound increase in the cell surface expression of TfR, enhanced binding to fluorescein-tagged transferrin, and stimulation of transferrin-mediated iron uptake into cells. Under these conditions, no significant alterations are observed in the levels of mitochondrial aconitase and the Divalent Metal Transporter DMT1, although both are encoded by two as yet lesser characterized IRE-containing mRNAs. Finally, H(2)O(2)-treated cells display an increased capacity to sequester (59)Fe in ferritin, despite a reduction in the ferritin pool, which results in a rearrangement of (59)Fe intracellular distribution. Our data suggest that H(2)O(2) regulates cellular iron acquisition and intracellular iron distribution by both IRP1-dependent and -independent mechanisms.

  15. Expression of human papilloma virus type 16 E5 protein in amelanotic melanoma cells regulates endo-cellular pH and restores tyrosinase activity

    PubMed Central

    Di Domenico, Fabio; Foppoli, Cesira; Blarzino, Carla; Perluigi, Marzia; Paolini, Francesca; Morici, Salvatrice; Coccia, Raffaella; Cini, Chiara; De Marco, Federico

    2009-01-01

    Background Melanin synthesis, the elective trait of melanocytes, is regulated by tyrosinase activity. In tyrosinase-positive amelanotic melanomas this rate limiting enzyme is inactive because of acidic endo-melanosomal pH. The E5 oncogene of the Human Papillomavirus Type 16 is a small transmembrane protein with a weak transforming activity and a role during the early steps of viral infections. E5 has been shown to interact with 16 kDa subunit C of the trans-membrane Vacuolar ATPase proton pump ultimately resulting in its functional suppressions. However, the cellular effects of such an interaction are still under debate. With this work we intended to explore whether the HPV16 E5 oncoprotein does indeed interact with the vacuolar ATPase proton pump once expressed in intact human cells and whether this interaction has functional consequences on cell metabolism and phenotype. Methods The expression of the HPV16-E5 oncoproteins was induced in two Tyrosinase-positive amelanotic melanomas (the cell lines FRM and M14) by a retroviral expression construct. Modulation of the intracellular pH was measured with Acridine orange and fluorescence microscopy. Expression of tyrosinase and its activity was followed by RT-PCR, Western Blot and enzyme assay. The anchorage-independence growth and the metabolic activity of E5 expressing cells were also monitored. Results We provide evidence that in the E5 expressing cells interaction between E5 and V-ATPase determines an increase of endo-cellular pH. The cellular alkalinisation in turn leads to the post-translational activation of tyrosinase, melanin synthesis and phenotype modulation. These effects are associated with an increased activation of tyrosine analogue anti-blastic drugs. Conclusion Once expressed within intact human cells the HPV16-E5 oncoprotein does actually interact with the vacuolar V-ATPase proton pump and this interaction induces a number of functional effects. In amelanotic melanomas these effects can modulate the

  16. Evaluation of cellular retinoic acid binding protein 2 gene expression through the retinoic acid pathway by co-incubation of Blastocystis ST-1 with HT29 cells in vitro.

    PubMed

    Liao, Chen-Chieh; Song, Eing-Ju; Chang, Tsuey-Yu; Lin, Wei-Chen; Liu, Hsiao-Sheng; Chen, Lih-Ren; Huang, Lynn L H; Shin, Jyh-Wei

    2016-05-01

    Blastocystis is a parasitic protist with a worldwide distribution that is commonly found in patients with colon and gastrointestinal pathological symptoms. Blastocystis infection has also commonly been reported in colorectal cancer and HIV/AIDS patients with gastrointestinal symptoms. To understand the pathway networks of gene regulation and the probable mechanisms influencing functions of HT-29 host cells in response to parasite infection, we examined the expression of 163 human oncogenes and kinases in human colon adenocarcinoma HT-29 cells co-incubated with Blastocystis by in-house cDNA microarray and PCR analysis. At least 10 genes were shown to be modified following Blastocystis co-incubation, including those with immunological, tumorigenesis, and antitumorigenesis functions. The expression of genes encoding cellular retinoic acid binding protein 2 (CRABP2) and proliferating cell nuclear antigen (PCNA) was markedly upregulated and downregulated, respectively. Reverse transcriptase-PCR validated the modified transcript expression of CRABP2 and other associated genes such as retinoic acid (RA)-related nuclear-receptor (RARα). Together, our data indicate that CRABP2, RARα, and PCNA expressions are involved in RA signaling regulatory networks that affect the growth, proliferation, and inflammation of HT-29 cells. PMID:26911149

  17. Adaptation to acetaminophen exposure elicits major changes in expression and distribution of the hepatic proteome.

    PubMed

    Eakins, R; Walsh, J; Randle, L; Jenkins, R E; Schuppe-Koistinen, I; Rowe, C; Starkey Lewis, P; Vasieva, O; Prats, N; Brillant, N; Auli, M; Bayliss, M; Webb, S; Rees, J A; Kitteringham, N R; Goldring, C E; Park, B K

    2015-01-01

    Acetaminophen overdose is the leading cause of acute liver failure. One dose of 10-15 g causes severe liver damage in humans, whereas repeated exposure to acetaminophen in humans and animal models results in autoprotection. Insight of this process is limited to select proteins implicated in acetaminophen toxicity and cellular defence. Here we investigate hepatic adaptation to acetaminophen toxicity from a whole proteome perspective, using quantitative mass spectrometry. In a rat model, we show the response to acetaminophen involves the expression of 30% of all proteins detected in the liver. Genetic ablation of a master regulator of cellular defence, NFE2L2, has little effect, suggesting redundancy in the regulation of adaptation. We show that adaptation to acetaminophen has a spatial component, involving a shift in regionalisation of CYP2E1, which may prevent toxicity thresholds being reached. These data reveal unexpected complexity and dynamic behaviour in the biological response to drug-induced liver injury. PMID:26607827

  18. Adaptation to acetaminophen exposure elicits major changes in expression and distribution of the hepatic proteome

    PubMed Central

    Eakins, R.; Walsh, J.; Randle, L.; Jenkins, R. E.; Schuppe-Koistinen, I.; Rowe, C.; Starkey Lewis, P.; Vasieva, O.; Prats, N.; Brillant, N.; Auli, M.; Bayliss, M.; Webb, S.; Rees, J. A.; Kitteringham, N. R.; Goldring, C. E.; Park, B. K.

    2015-01-01

    Acetaminophen overdose is the leading cause of acute liver failure. One dose of 10–15 g causes severe liver damage in humans, whereas repeated exposure to acetaminophen in humans and animal models results in autoprotection. Insight of this process is limited to select proteins implicated in acetaminophen toxicity and cellular defence. Here we investigate hepatic adaptation to acetaminophen toxicity from a whole proteome perspective, using quantitative mass spectrometry. In a rat model, we show the response to acetaminophen involves the expression of 30% of all proteins detected in the liver. Genetic ablation of a master regulator of cellular defence, NFE2L2, has little effect, suggesting redundancy in the regulation of adaptation. We show that adaptation to acetaminophen has a spatial component, involving a shift in regionalisation of CYP2E1, which may prevent toxicity thresholds being reached. These data reveal unexpected complexity and dynamic behaviour in the biological response to drug-induced liver injury. PMID:26607827

  19. Adaptation to acetaminophen exposure elicits major changes in expression and distribution of the hepatic proteome.

    PubMed

    Eakins, R; Walsh, J; Randle, L; Jenkins, R E; Schuppe-Koistinen, I; Rowe, C; Starkey Lewis, P; Vasieva, O; Prats, N; Brillant, N; Auli, M; Bayliss, M; Webb, S; Rees, J A; Kitteringham, N R; Goldring, C E; Park, B K

    2015-11-26

    Acetaminophen overdose is the leading cause of acute liver failure. One dose of 10-15 g causes severe liver damage in humans, whereas repeated exposure to acetaminophen in humans and animal models results in autoprotection. Insight of this process is limited to select proteins implicated in acetaminophen toxicity and cellular defence. Here we investigate hepatic adaptation to acetaminophen toxicity from a whole proteome perspective, using quantitative mass spectrometry. In a rat model, we show the response to acetaminophen involves the expression of 30% of all proteins detected in the liver. Genetic ablation of a master regulator of cellular defence, NFE2L2, has little effect, suggesting redundancy in the regulation of adaptation. We show that adaptation to acetaminophen has a spatial component, involving a shift in regionalisation of CYP2E1, which may prevent toxicity thresholds being reached. These data reveal unexpected complexity and dynamic behaviour in the biological response to drug-induced liver injury.

  20. Explicit expressions of the Pietra index for the generalized function for the size distribution of income

    NASA Astrophysics Data System (ADS)

    Sarabia, José María; Jordá, Vanesa

    2014-12-01

    The importance of the Pietra index in socioeconomic systems and econophysics has been highlighted by Eliazar and Sokolov (2010). In this paper, we obtain closed expressions for the Pietra index for the generalized function for the size of income proposed by McDonald (1984). This family is composed of three classes of distributions: the generalized gamma distribution (GG), the generalized beta of the first kind (GB1) and the generalized beta of the second kind (GB2). For the different distributions, we obtain closed and simple expressions of the Pietra index, which can be easily computed. We also obtain the Pietra index for other relevant income models including finite mixtures of distributions and the κ-generalized distribution (Clementi et al., 2008). Finally, two empirical applications with real income data are given.

  1. Expression of a cellular gene cloned in herpes simplex virus: rabbit beta-globin is regulated as an early viral gene in infected fibroblasts.

    PubMed Central

    Smiley, J R; Smibert, C; Everett, R D

    1987-01-01

    We constructed nondefective herpes simplex virus type 1 recombinants bearing the intact rabbit beta-globin gene inserted into the viral gene for thymidine kinase to study the expression of a cellular gene when it is present in the viral genome during lytic viral infections. The globin promoter was activated to high levels during productive infection of Vero cells, giving rise to properly spliced and processed cytoplasmic globin transcripts. Expression of globin RNA occurred with early kinetics, was not affected by blocking viral DNA replication, and was strongly inhibited by preventing viral immediate-early protein synthesis with cycloheximide. These results support the hypothesis that temporal control of herpes simplex virus early gene expression is accomplished by mechanisms that are not restricted to viral promoters. In addition, these data show that a cellular transcript can be correctly processed and can accumulate to high levels during viral infection; this indicates that the mechanisms of virally induced shutoff of host RNA accumulation and degradation of host mRNAs do not depend on sequence-specific differentiation between host and viral RNAs. These findings also suggest that herpesviruses have considerable potential as high-capacity gene transfer vectors for a variety of applications. Images PMID:3037101

  2. Expression of epithelial cellular adhesion molecule (Ep-CAM) in chronic (necro-)inflammatory liver diseases and hepatocellular carcinoma.

    PubMed

    Breuhahn, Kai; Baeuerle, Patrick A; Peters, Malte; Prang, Nadja; Töx, Ulrich; Köhne-Volland, Rudolph; Dries, Volker; Schirmacher, Peter; Leo, Eugen

    2006-01-01

    Epithelial cell adhesion molecule (Ep-CAM) is expressed in a several epithelial tissues and carcinomas, but not on mature hepatocytes. Here, we analysed the expression of Ep-CAM in 230 patients suffering from various liver diseases like chronic hepatitis B and C (HBV and HCV infection), chronic autoimmune hepatitis (AIH), chronic alcoholic liver disease (ALD), primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), hereditary hemochromatosis and dysplastic nodules (DNs) as well as hepatocellular carcinomas (HCCs) and cholangiocellular carcinomas (CCCs) by immunohistochemistry. De novo hepatocellular Ep-CAM expression was found in 75.9% of ALD (22/29), 63.6% of HCV (21/33) and 55.6% of each AIH and HBV cases (5/9 and 15/27, respectively). Lower Ep-CAM expression levels were observed for primary sclerosing liver diseases (PBC and PSC) with 25% (3/12) and 7.7% (1/13) of cases. Moreover, only 14.3% of HCCs (9/63) manifested expression, while all CCCs showed strong Ep-CAM expression (5/5). For DNs and hereditary hemochromatosis, Ep-CAM expression was found in 10 and 50% (3/30 and 2/4), respectively. In HBV and HCV, Ep-CAM expression correlated significantly with inflammatory activity as assessed by histological parameters and to the extent of fibrosis. In addition, for HCV also transaminase levels correlated significantly with Ep-CAM expression. Our results indicate that de novo Ep-CAM expression in hepatocytes is frequent in inflammatory liver diseases and is potentially linked to regenerative activity. CCCs and Ep-CAM positive HCCs may represent an attractive target group for Ep-CAM-directed immunotherapies, yet unwanted toxicity may limit the use of such strategies due to Ep-CAM expression in biliary epithelium and several chronic liver diseases such as HBV-and HCV-hepatitis.

  3. An array of Escherichia coli clones over-expressing essential proteins: A new strategy of identifying cellular targets of potent antibacterial compounds

    SciTech Connect

    Xu, H. Howard . E-mail: hxu3@calstatela.edu; Real, Lilian; Bailey, Melissa Wu

    2006-11-03

    With the advancement of high throughput screening, it has become easier and faster to discover hit compounds that inhibit proliferation of bacterial cells. However, development in technologies used to identify cellular targets of potent antibacterial inhibitors has lagged behind. Here, we describe a novel strategy of target identification for antibacterial inhibitors using an array of Escherichia coli clones each over-expressing one essential protein. In a proof-of-concept study, eight essential genes were cloned into pLex5BA vector under the control of an inducible promoter. Over-expression of target proteins was confirmed. For two clones, one over-expressing FabI and the other over-expressing MurA enzymes, the host cells became 17- and 139-fold more resistant to the specific inhibitors triclosan and phosphomycin, respectively, while the susceptibility of other clones towards these inhibitors remained unchanged after induction of gene expression. Target identification via target protein over-expression was demonstrated using both mixed clone and individual clone assay formats.

  4. Exact protein distributions for stochastic models of gene expression using partitioning of Poisson processes

    NASA Astrophysics Data System (ADS)

    Pendar, Hodjat; Platini, Thierry; Kulkarni, Rahul V.

    2013-04-01

    Stochasticity in gene expression gives rise to fluctuations in protein levels across a population of genetically identical cells. Such fluctuations can lead to phenotypic variation in clonal populations; hence, there is considerable interest in quantifying noise in gene expression using stochastic models. However, obtaining exact analytical results for protein distributions has been an intractable task for all but the simplest models. Here, we invoke the partitioning property of Poisson processes to develop a mapping that significantly simplifies the analysis of stochastic models of gene expression. The mapping leads to exact protein distributions using results for mRNA distributions in models with promoter-based regulation. Using this approach, we derive exact analytical results for steady-state and time-dependent distributions for the basic two-stage model of gene expression. Furthermore, we show how the mapping leads to exact protein distributions for extensions of the basic model that include the effects of posttranscriptional and posttranslational regulation. The approach developed in this work is widely applicable and can contribute to a quantitative understanding of stochasticity in gene expression and its regulation.

  5. Exact protein distributions for stochastic models of gene expression using partitioning of Poisson processes.

    PubMed

    Pendar, Hodjat; Platini, Thierry; Kulkarni, Rahul V

    2013-04-01

    Stochasticity in gene expression gives rise to fluctuations in protein levels across a population of genetically identical cells. Such fluctuations can lead to phenotypic variation in clonal populations; hence, there is considerable interest in quantifying noise in gene expression using stochastic models. However, obtaining exact analytical results for protein distributions has been an intractable task for all but the simplest models. Here, we invoke the partitioning property of Poisson processes to develop a mapping that significantly simplifies the analysis of stochastic models of gene expression. The mapping leads to exact protein distributions using results for mRNA distributions in models with promoter-based regulation. Using this approach, we derive exact analytical results for steady-state and time-dependent distributions for the basic two-stage model of gene expression. Furthermore, we show how the mapping leads to exact protein distributions for extensions of the basic model that include the effects of posttranscriptional and posttranslational regulation. The approach developed in this work is widely applicable and can contribute to a quantitative understanding of stochasticity in gene expression and its regulation.

  6. Delayed Correlation of mRNA and Protein Expression in Rapamycin-treated Cells and a Role for Ggc1 in Cellular Sensitivity to Rapamycin*

    PubMed Central

    Fournier, Marjorie L.; Paulson, Ariel; Pavelka, Norman; Mosley, Amber L.; Gaudenz, Karin; Bradford, William D.; Glynn, Earl; Li, Hua; Sardiu, Mihaela E.; Fleharty, Brian; Seidel, Christopher; Florens, Laurence; Washburn, Michael P.

    2010-01-01

    To identify new molecular targets of rapamycin, an anticancer and immunosuppressive drug, we analyzed temporal changes in yeast over 6 h in response to rapamycin at the transcriptome and proteome levels and integrated the expression patterns with functional profiling. We show that the integration of transcriptomics, proteomics, and functional data sets provides novel insights into the molecular mechanisms of rapamycin action. We first observed a temporal delay in the correlation of mRNA and protein expression where mRNA expression at 1 and 2 h correlated best with protein expression changes after 6 h of rapamycin treatment. This was especially the case for the inhibition of ribosome biogenesis and induction of heat shock and autophagy essential to promote the cellular sensitivity to rapamycin. However, increased levels of vacuolar protease could enhance resistance to rapamycin. Of the 85 proteins identified as statistically significantly changing in abundance, most of the proteins that decreased in abundance were correlated with a decrease in mRNA expression. However, of the 56 proteins increasing in abundance, 26 were not correlated with an increase in mRNA expression. These protein changes were correlated with unchanged or down-regulated mRNA expression. These proteins, involved in mitochondrial genome maintenance, endocytosis, or drug export, represent new candidates effecting rapamycin action whose expression might be post-transcriptionally or post-translationally regulated. We identified GGC1, a mitochondrial GTP/GDP carrier, as a new component of the rapamycin/target of rapamycin (TOR) signaling pathway. We determined that the protein product of GGC1 was stabilized in the presence of rapamycin, and the deletion of the GGC1 enhanced growth fitness in the presence of rapamycin. A dynamic mRNA expression analysis of Δggc1 and wild-type cells treated with rapamycin revealed a key role for Ggc1p in the regulation of ribosome biogenesis and cell cycle progression

  7. Molecular and cellular changes in skin and muscle during metamorphosis of Atlantic halibut (Hippoglossus hippoglossus) are accompanied by changes in deiodinases expression.

    PubMed

    Campinho, M A; Galay-Burgos, M; Silva, N; Costa, R A; Alves, R N; Sweeney, G E; Power, D M

    2012-11-01

    Flatfish metamorphosis is the most dramatic post-natal developmental event in teleosts. Thyroid hormones (TH), thyroxine (T4) and 3,3'-5'-triiodothyronine (T3) are the necessary and sufficient factors that induce and regulate flatfish metamorphosis. Most of the cellular and molecular action of TH is directed through the binding of T3 to thyroid nuclear receptors bound to promoters with consequent changes in the expression of target genes. The conversion of T4 to T3 and nuclear availability of T3 depends on the expression and activity of a family of 3 selenocysteine deiodinases that activate T4 into T3 or degrade T4 and T3. We have investigated the role of deiodinases in skin and muscle metamorphic changes in halibut. We show that, both at the whole body level and at the cellular level in muscle and skin of the Atlantic halibut (Hippoglossus hippoglossus) during metamorphosis, the coordination between activating (D2) and deactivating (D3) deiodinases expression is strongly correlated with the developmental TH-driven changes. The expression pattern of D2 and D3 in cells of both skin and muscle indicate that TH are necessary for the maintenance of larval metamorphic development and juvenile cell types in these tissues. No break in symmetry occurs in the expression of deiodinases and in metamorphic developmental changes occurring both in trunk skin and muscle. The findings that two of the major tissues in both larvae and juveniles maintain their symmetry throughout metamorphosis suggest that the asymmetric changes occurring during flatfish metamorphosis are restricted to the eye and head region.

  8. Expression, subcellular localization, and enzyme activity of a recombinant human extra-cellular superoxide dismutase in tobacco (Nicotiana benthamiana L.).

    PubMed

    Park, Ki Youl; Kim, Eun Yu; Lee, Weontae; Kim, Tae-Yoon; Kim, Woo Taek

    2016-03-01

    Human extracellular superoxide dismutase (hEC-SOD) is an enzyme that scavenges reactive oxygen species (ROS). Because of its antioxidant activity, hEC-SOD has been used as a therapeutic protein to treat skin disease and arthritis in mammalian systems. In this study, codon-optimized hEC-SOD was expressed in tobacco (Nicotiana benthamiana L.) via a plant-based transient protein expression system. Plant expression binary vectors containing full-length hEC-SOD (f-hEC-SOD) and modified hEC-SOD (m-hEC-SOD), in which the signal peptide and heparin-binding domain were deleted, were constructed for the cytosolic-, endoplasmic reticulum (ER)-, and chloroplast-localizations in tobacco leaf mesophyll cells. The results demonstrated that f-hEC-SOD was more efficiently expressed in the cytosolic fractions than in the ER or chloroplasts of tobacco cells. Our data further indicated that differently localized f-hEC-SOD and m-hEC-SOD displayed SOD enzyme activities, suggesting that the hEC-SODs expressed by plants may be functionally active. The f-hEC-SOD was expressed up to 3.8% of the total leaf soluble protein and the expression yield was calculated to be 313.7 μg f-hEC-SOD per g fresh weight of leaf. Overall, our results reveal that it was possible to express catalytically active hEC-SODs by means of a transient plant expression system in tobacco leaf cells. PMID:26611610

  9. Reconstructing and analysing cellular states, space and time from gene expression profiles of many cells and single cells.

    PubMed

    Francesconi, Mirko; Lehner, Ben

    2015-10-01

    Genome-wide gene expression profiling is a fast, cheap and standardised analysis that provides a high dimensional measurement of the state of a biological sample. In this review we describe computational methods that can be applied to identify and interpret sources of variance in gene expression in whole organisms, organs, tissues or single cells. This allows the identification of constituent cell types and states in complex mixtures, the reconstruction of temporal trajectories of development, differentiation and progression, and the reconstruction of spatial patterning. When applied to genetically variable samples, these methods allow the efficient investigation of how genetic variation influences gene expression and biological processes in space and time.

  10. Direct interaction of cellular hnRNP-F and NS1 of influenza A virus accelerates viral replication by modulation of viral transcriptional activity and host gene expression

    SciTech Connect

    Lee, Jun Han; Kim, Sung-Hak; Pascua, Philippe Noriel Q.; Song, Min-Suk; Baek, Yun Hee; Jin, Xun; Choi, Joong-Kook; Kim, Chul-Joong; Kim, Hyunggee; Choi, Young Ki

    2010-02-05

    To investigate novel NS1-interacting proteins, we conducted a yeast two-hybrid analysis, followed by co-immunoprecipitation assays. We identified heterogeneous nuclear ribonucleoprotein F (hnRNP-F) as a cellular protein interacting with NS1 during influenza A virus infection. Co-precipitation assays suggest that interaction between hnRNP-F and NS1 is a common and direct event among human or avian influenza viruses. NS1 and hnRNP-F co-localize in the nucleus of host cells, and the RNA-binding domain of NS1 directly interacts with the GY-rich region of hnRNP-F determined by GST pull-down assays with truncated proteins. Importantly, hnRNP-F expression levels in host cells indicate regulatory role on virus replication. hnRNP-F depletion by small interfering RNA (siRNA) shows 10- to 100-fold increases in virus titers corresponding to enhanced viral RNA polymerase activity. Our results delineate novel mechanism of action by which NS1 accelerates influenza virus replication by modulating normal cellular mRNA processes through direct interaction with cellular hnRNP-F protein.

  11. Human Cortical Neural Stem Cells Expressing Insulin-Like Growth Factor-I: A Novel Cellular Therapy for Alzheimer’s Disease

    PubMed Central

    McGinley, Lisa M.; Sims, Erika; Lunn, J. Simon; Kashlan, Osama N.; Chen, Kevin S.; Bruno, Elizabeth S.; Pacut, Crystal M.; Hazel, Tom; Johe, Karl; Sakowski, Stacey A.

    2016-01-01

    Alzheimer’s disease (AD) is the most prevalent age-related neurodegenerative disorder and a leading cause of dementia. Current treatment fails to modify underlying disease pathologies and very little progress has been made to develop effective drug treatments. Cellular therapies impact disease by multiple mechanisms, providing increased efficacy compared with traditional single-target approaches. In amyotrophic lateral sclerosis, we have shown that transplanted spinal neural stem cells (NSCs) integrate into the spinal cord, form synapses with the host, improve inflammation, and reduce disease-associated pathologies. Our current goal is to develop a similar “best in class” cellular therapy for AD. Here, we characterize a novel human cortex-derived NSC line modified to express insulin-like growth factor-I (IGF-I), HK532-IGF-I. Because IGF-I promotes neurogenesis and synaptogenesis in vivo, this enhanced NSC line offers additional environmental enrichment, enhanced neuroprotection, and a multifaceted approach to treating complex AD pathologies. We show that autocrine IGF-I production does not impact the cell secretome or normal cellular functions, including proliferation, migration, or maintenance of progenitor status. However, HK532-IGF-I cells preferentially differentiate into gamma-aminobutyric acid-ergic neurons, a subtype dysregulated in AD; produce increased vascular endothelial growth factor levels; and display an increased neuroprotective capacity in vitro. We also demonstrate that HK532-IGF-I cells survive peri-hippocampal transplantation in a murine AD model and exhibit long-term persistence in targeted brain areas. In conclusion, we believe that harnessing the benefits of cellular and IGF-I therapies together will provide the optimal therapeutic benefit to patients, and our findings support further preclinical development of HK532-IGF-I cells into a disease-modifying intervention for AD. Significance There is no cure for Alzheimer’s disease (AD) and

  12. Genome Organization and Gene Expression Shape the Transposable Element Distribution in the Drosophila melanogaster Euchromatin

    PubMed Central

    Fontanillas, Pierre; Hartl, Daniel L; Reuter, Max

    2007-01-01

    The distribution of transposable elements (TEs) in a genome reflects a balance between insertion rate and selection against new insertions. Understanding the distribution of TEs therefore provides insights into the forces shaping the organization of genomes. Past research has shown that TEs tend to accumulate in genomic regions with low gene density and low recombination rate. However, little is known about the factors modulating insertion rates across the genome and their evolutionary significance. One candidate factor is gene expression, which has been suggested to increase local insertion rate by rendering DNA more accessible. We test this hypothesis by comparing the TE density around germline- and soma-expressed genes in the euchromatin of Drosophila melanogaster. Because only insertions that occur in the germline are transmitted to the next generation, we predicted a higher density of TEs around germline-expressed genes than soma-expressed genes. We show that the rate of TE insertions is greater near germline- than soma-expressed genes. However, this effect is partly offset by stronger selection for genome compactness (against excess noncoding DNA) on germline-expressed genes. We also demonstrate that the local genome organization in clusters of coexpressed genes plays a fundamental role in the genomic distribution of TEs. Our analysis shows that—in addition to recombination rate—the distribution of TEs is shaped by the interaction of gene expression and genome organization. The important role of selection for compactness sheds a new light on the role of TEs in genome evolution. Instead of making genomes grow passively, TEs are controlled by the forces shaping genome compactness, most likely linked to the efficiency of gene expression or its complexity and possibly their interaction with mechanisms of TE silencing. PMID:18081425

  13. Copper changes the yield and cadmium/zinc accumulation and cellular distribution in the cadmium/zinc hyperaccumulator Sedum plumbizincicola.

    PubMed

    Li, Zhu; Wu, Longhua; Hu, Pengjie; Luo, Yongming; Christie, Peter

    2013-10-15

    Non-accumulated metals in mixed metal contaminated soils may affect hyperaccumulator growth and metal accumulation and thus remediation efficiency. Two hydroponics experiments were conducted to investigate the effects of copper (Cu) on cadmium (Cd) and zinc (Zn) accumulation by the Cd/Zn hyperaccumulator Sedum plumbizincicola, Cu toxicity and plant detoxification using chemical sequential extraction of metals, sub-cellular separation, micro synchrotron radiation based X-ray fluorescence, and transmission electron microscopy. Compared with the control (0.31 μM Cu), 5-50 μM Cu had no significant effect on Cd/Zn accumulation, but Cu at 200 μM induced root cell plasmolysis and disordered chloroplast structure. The plants held Cu in the roots and cell walls and complexed Cu in insoluble forms as their main detoxification mechanisms. Exposure to 200 μM Cu for 4 days inhibited plant Cd uptake and translocation but did not affect Zn concentrations in roots and stems. Moreover, unloading of Cd and Zn from stem to leaf was restrained compared to control plants, perhaps due to Cu accumulation in leaf veins. Copper may thus interfere with root Cd uptake and restrain Cd/Zn unloading to the leaves. Further investigation of how Cu affects plant metal uptake may help elucidate the Cd/Zn hyper-accumulating mechanisms of S. plumbizincicola. PMID:23959253

  14. Expression of a glutathione reductase from Brassica rapa subsp. pekinensis enhanced cellular redox homeostasis by modulating antioxidant proteins in Escherichia coli.

    PubMed

    Kim, Il-Sup; Shin, Sun-Young; Kim, Young-Saeng; Kim, Hyun-Young; Yoon, Ho-Sung

    2009-11-30

    Glutathione reductase (GR) is an enzyme that recycles a key cellular antioxidant molecule glutathione (GSH) from its oxidized form (GSSG) thus maintaining cellular redox homeostasis. A recombinant plasmid to overexpress a GR of Brassica rapa subsp. pekinensis (BrGR) in E. coli BL21 (DE3) was constructed using an expression vector pKM260. Expression of the introduced gene was confirmed by semiquantitative RT-PCR, immunoblotting and enzyme assays. Purification of the BrGR protein was performed by IMAC method and indicated that the BrGR was a dimmer. The BrGR required NADPH as a cofactor and specific activity was approximately 458 U. The BrGR-expressing E. coli cells showed increased GR activity and tolerance to H(2)O(2), menadione, and heavy metal (CdCl(2), ZnCl(2) and AlCl(2))-mediated growth inhibition. The ectopic expression of BrGR provoked the co-regulation of a variety of antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase. Consequently, the transformed cells showed decreased hydroperoxide levels when exposed to stressful conditions. A proteomic analysis demonstrated the higher level of induction of proteins involved in glycolysis, detoxification/oxidative stress response, protein folding, transport/binding proteins, cell envelope/porins, and protein translation and modification when exposed to H(2)O(2) stress. Taken together, these results indicate that the plant GR protein is functional in a cooperative way in the E. coli system to protect cells against oxidative stress.

  15. Blood cell gene expression associated with cellular stress defense is modulated by antioxidant-rich food in a randomised controlled clinical trial of male smokers

    PubMed Central

    2010-01-01

    Background Plant-based diets rich in fruit and vegetables can prevent development of several chronic age-related diseases. However, the mechanisms behind this protective effect are not elucidated. We have tested the hypothesis that intake of antioxidant-rich foods can affect groups of genes associated with cellular stress defence in human blood cells. Trial registration number: NCT00520819 http://clinicaltrials.gov. Methods In an 8-week dietary intervention study, 102 healthy male smokers were randomised to either a diet rich in various antioxidant-rich foods, a kiwifruit diet (three kiwifruits/d added to the regular diet) or a control group. Blood cell gene expression profiles were obtained from 10 randomly selected individuals of each group. Diet-induced changes on gene expression were compared to controls using a novel application of the gene set enrichment analysis (GSEA) on transcription profiles obtained using Affymetrix HG-U133-Plus 2.0 whole genome arrays. Results Changes were observed in the blood cell gene expression profiles in both intervention groups when compared to the control group. Groups of genes involved in regulation of cellular stress defence, such as DNA repair, apoptosis and hypoxia, were significantly upregulated (GSEA, FDR q-values < 5%) by both diets compared to the control group. Genes with common regulatory motifs for aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (AhR/ARNT) were upregulated by both interventions (FDR q-values < 5%). Plasma antioxidant biomarkers (polyphenols/carotenoids) increased in both groups. Conclusions The observed changes in the blood cell gene expression profiles suggest that the beneficial effects of a plant-based diet on human health may be mediated through optimization of defence processes. PMID:20846424

  16. Increased cortical expression of the zinc transporter SLC39A12 suggests a breakdown in zinc cellular homeostasis as part of the pathophysiology of schizophrenia

    PubMed Central

    Scarr, Elizabeth; Udawela, Madhara; Greenough, Mark A; Neo, Jaclyn; Suk Seo, Myoung; Money, Tammie T; Upadhyay, Aradhana; Bush, Ashley I; Everall, Ian P; Thomas, Elizabeth A; Dean, Brian

    2016-01-01

    Our expression microarray studies showed messenger RNA (mRNA) for solute carrier family 39 (zinc transporter), member 12 (SLC39A12) was higher in dorsolateral prefrontal cortex from subjects with schizophrenia (Sz) in comparison with controls. To better understand the significance of these data we ascertained whether SLC39A12 mRNA was altered in a number of cortical regions (Brodmann’s area (BA) 8, 9, 44) from subjects with Sz, in BA 9 from subjects with mood disorders and in rats treated with antipsychotic drugs. In addition, we determined whether inducing the expression of SLC39A12 resulted in an increased cellular zinc uptake. SLC39A12 variant 1 and 2 mRNA was measured using quantitative PCR. Zinc uptake was measured in CHO cells transfected with human SLC39A12 variant 1 and 2. In Sz, compared with controls, SLC39A12 variant 1 and 2 mRNA was higher in all cortical regions studied. The were no differences in levels of mRNA for either variant of SLC39A12 in BA 9 from subjects with mood disorders and levels of mRNA for Slc39a12 was not different in the cortex of rats treated with antipsychotic drugs. Finally, expressing both variants in CHO-K1 cells was associated with an increase in radioactive zinc uptake. As increased levels of murine Slc39a12 mRNA has been shown to correlate with increasing cellular zinc uptake, our data would be consistent with the possibility of a dysregulated zinc homeostasis in the cortex of subjects with schizophrenia due to altered expression of SLC39A12. PMID:27336053

  17. Increased cortical expression of the zinc transporter SLC39A12 suggests a breakdown in zinc cellular homeostasis as part of the pathophysiology of schizophrenia.

    PubMed

    Scarr, Elizabeth; Udawela, Madhara; Greenough, Mark A; Neo, Jaclyn; Suk Seo, Myoung; Money, Tammie T; Upadhyay, Aradhana; Bush, Ashley I; Everall, Ian P; Thomas, Elizabeth A; Dean, Brian

    2016-01-01

    Our expression microarray studies showed messenger RNA (mRNA) for solute carrier family 39 (zinc transporter), member 12 (SLC39A12) was higher in dorsolateral prefrontal cortex from subjects with schizophrenia (Sz) in comparison with controls. To better understand the significance of these data we ascertained whether SLC39A12 mRNA was altered in a number of cortical regions (Brodmann's area (BA) 8, 9, 44) from subjects with Sz, in BA 9 from subjects with mood disorders and in rats treated with antipsychotic drugs. In addition, we determined whether inducing the expression of SLC39A12 resulted in an increased cellular zinc uptake. SLC39A12 variant 1 and 2 mRNA was measured using quantitative PCR. Zinc uptake was measured in CHO cells transfected with human SLC39A12 variant 1 and 2. In Sz, compared with controls, SLC39A12 variant 1 and 2 mRNA was higher in all cortical regions studied. The were no differences in levels of mRNA for either variant of SLC39A12 in BA 9 from subjects with mood disorders and levels of mRNA for Slc39a12 was not different in the cortex of rats treated with antipsychotic drugs. Finally, expressing both variants in CHO-K1 cells was associated with an increase in radioactive zinc uptake. As increased levels of murine Slc39a12 mRNA has been shown to correlate with increasing cellular zinc uptake, our data would be consistent with the possibility of a dysregulated zinc homeostasis in the cortex of subjects with schizophrenia due to altered expression of SLC39A12. PMID:27336053

  18. Effect of arginine on cellular adhesion molecule expression and leukocyte transmigration in endothelial cells stimulated by biological fluid from surgical patients.

    PubMed

    Yeh, Chiu-Li; Hsu, Chun-Sen; Chen, Soul-Chin; Hou, Yu-Chen; Chiu, Wan-Chun; Yeh, Sung-Ling

    2007-07-01

    This study investigated the effect of different arginine (Arg) concentrations on adhesion molecule expression on endothelial cells (ECs) and leukocytes and the transendothelial migration of polymorphonuclear neutrophils (PMNs) through ECs stimulated by plasma or peritoneal drain fluid (PDF) from surgical patients. Human umbilical vein ECs (HUVECs) and PMNs from healthy subjects were treated with different concentrations (0, 50, 100, and 1000 micromol/L) of Arg for 24 h. After that, HUVECs were stimulated for 3 h with plasma or PDF from patients who underwent abdominal surgery, and PMNs were allowed to transmigrate through ECs for 2 h. The HUVEC expression of cellular adhesion molecules (CAMs) and integrin (CD11b) and the interleukin (IL) 8 receptor expression on PMNs were measured by flow cytometry. The PMNs transmigrating through ECs were also analyzed. The results showed that CAM and integrin expressions in PDF groups were higher than those in control groups. Among the PDF groups, IL-8 secretions from ECs and PMNs were lower with 100 and 1000 micromol/L Arg than with 0 and 50 micromol/L Arg, and this was consistent with the expression of the IL-8 receptor on PMNs. In addition, CAM expressions on ECs and CD11b expression on PMNs, as well as PMN transmigration, were lower with 100 and 1000 micromol/L Arg than with 0 and 50 micromol/L Arg. The HUVECs stimulated by plasma from surgical patients had similar effects on surface molecule expression as PDF; however, as shown in PDF stimulation, the effects were not so obvious. Inhibition of nitric oxide production results in high CAM and IL-8 expressions comparable with groups with low Arg administration. The results of this in vitro study suggest that ECs and PMNs were activated after patients' plasma or PDF stimulation. A low Arg concentration comparable with catabolic conditions resulted in higher adhesion molecule expression and greater transendothelial migration of neutrophils. Arginine administration at levels

  19. Differential expression of cellular microRNAs in HPV-11 transfected cells. An analysis by three different array platforms and qRT-PCR.

    PubMed

    Dreher, Anita; Rossing, Maria; Kaczkowski, Bogumil; Nielsen, Finn Cilius; Norrild, Bodil

    2010-12-17

    Human papillomavirus type 11 (HPV-11) infects the genital and the respiratory tract leading to condylomas and respiratory papillomatosis. HPV infections are restricted to epithelial tissue and the progression through the virus lifecycle is tightly coordinated to the differentiation of the host cell. The changes of cellular microRNAs by HPV-11 gene expression were investigated in a cell culture model of HaCaT cells transfected with HPV-11, with the goal of understanding which cellular processes were affected by the virus. Human microRNA profiling was conducted on three different array platform systems and because very few microRNAs (miR-663, -638, -149* and -92b*) were consistently found in all three array data sets we performed extensive statistical analyses of the array data and the qRT-PCR validation. We assume that the most reliable differentially expressed microRNAs are the ones identified by more than one array platform. We also show that TaqMan® qRT-PCR validation is of limited use for less abundant microRNAs.

  20. The effects of okra (Abelmoschus esculentus Linn.) on the cellular events associated with Alzheimer's disease in a stably expressed HFE neuroblastoma SH-SY5Y cell line.

    PubMed

    Mairuae, Nootchanat; Connor, James R; Lee, Sang Y; Cheepsunthorn, Poonlarp; Tongjaroenbuangam, Walaiporn

    2015-08-31

    It has been reported that persons carrying the H63D variant in their hemochromatosis (HFE) gene are at increased risk of Alzheimer's disease (AD). We investigated the possibility that okra (Abelmoschus esculentus) and quercetin could mitigate this risk factor by examining its effect on AD-associated cellular events in HFE stably expressing SH-SY5Y cells. Treatment of H63D HFE cells either with okra or quercetin significantly decreased reactive oxygen species (ROS), hydrogen peroxide (H2O2), and protein oxidation compared to untreated cells. The levels of tau phosphorylation at serine-199, serine-202, and serine-396 sites were also significantly decreased when cells were treated with okra. Exposure of the H63D and wild type (WT) cells to iron increased tau phosphorylation, but this response was decreased significantly when cells were treated with okra. The mechanism responsible for these changes appears to be related to decreased glycogen synthase kinase (GSK)-3β activity, an upstream signaling kinase of tau phosphorylation. We also established that okra treatment dramatically decreases intracellular iron levels in H63D cells compared to untreated cells. Our results provide important in vitro data on the effects of okra on various AD-associated cellular processes in H63D variant HFE cells. These results suggest okra may be beneficial in people expressing the H63D variant to reduce the risk of AD and other neurodegenerative diseases related to oxidative stress. Further in vivo studies would help confirm this.

  1. The effects of okra (Abelmoschus esculentus Linn.) on the cellular events associated with Alzheimer's disease in a stably expressed HFE neuroblastoma SH-SY5Y cell line.

    PubMed

    Mairuae, Nootchanat; Connor, James R; Lee, Sang Y; Cheepsunthorn, Poonlarp; Tongjaroenbuangam, Walaiporn

    2015-08-31

    It has been reported that persons carrying the H63D variant in their hemochromatosis (HFE) gene are at increased risk of Alzheimer's disease (AD). We investigated the possibility that okra (Abelmoschus esculentus) and quercetin could mitigate this risk factor by examining its effect on AD-associated cellular events in HFE stably expressing SH-SY5Y cells. Treatment of H63D HFE cells either with okra or quercetin significantly decreased reactive oxygen species (ROS), hydrogen peroxide (H2O2), and protein oxidation compared to untreated cells. The levels of tau phosphorylation at serine-199, serine-202, and serine-396 sites were also significantly decreased when cells were treated with okra. Exposure of the H63D and wild type (WT) cells to iron increased tau phosphorylation, but this response was decreased significantly when cells were treated with okra. The mechanism responsible for these changes appears to be related to decreased glycogen synthase kinase (GSK)-3β activity, an upstream signaling kinase of tau phosphorylation. We also established that okra treatment dramatically decreases intracellular iron levels in H63D cells compared to untreated cells. Our results provide important in vitro data on the effects of okra on various AD-associated cellular processes in H63D variant HFE cells. These results suggest okra may be beneficial in people expressing the H63D variant to reduce the risk of AD and other neurodegenerative diseases related to oxidative stress. Further in vivo studies would help confirm this. PMID:26170247

  2. Increased expression of SIRT2 is a novel marker of cellular senescence and is dependent on wild type p53 status.

    PubMed

    Anwar, Tarique; Khosla, Sanjeev; Ramakrishna, Gayatri

    2016-07-17

    Sirtuins (SIRT) belonging to the NAD+ dependent histone deacetylase III class of enzymes have emerged as master regulators of metabolism and longevity. However, their role in prevention of organismal aging and cellular senescence still remains controversial. In the present study, we now report upregulation of SIRT2 as a specific feature associated with stress induced premature senescence but not with either quiescence or cell death. Additionally, increase in SIRT2 expression was noted in different types of senescent conditions such as replicative and oncogene induced senescence using multiple cell lines. Induction of SIRT2 expression during senescence was dependent on p53 status as depletion of p53 by shRNA prevented its accumulation. Chromatin immunoprecipitation revealed the presence of p53 binding sites on the SIRT2 promoter suggesting its regulation by p53, which was also corroborated by the SEAP reporter assay. Overexpression or knockdown of SIRT2 had no effect on stress induced premature senescence, thereby indicating that SIRT2 increase is not a cause of senescence; rather it is an effect linked to senescence-associated changes. Overall, our results suggest SIRT2 as a promising marker of cellular senescence at least in cells with wild type p53 status. PMID:27229617

  3. Contrasting effects of insulin and cellular differentiation on expression of the novel insulin receptor substrate APS in skeletal muscle.

    PubMed

    Rea, Rustam; Gray, Samuel; Donnelly, Richard

    2005-11-01

    The novel insulin receptor substrate protein APS is highly expressed in insulin-sensitive tissues and plays an important role in insulin-mediated glucose uptake and GLUT4 translocation via the Cbl/CAP pathway. Tyrosine phosphorylation of APS leads to recruitment of c-Cbl and Crk, while overexpression of APS mutant inhibits GLUT4 translocation in response to insulin, but the regulation of APS expression in skeletal muscle has not been previously reported. L6 myoblasts were differentiated in 2% FBS and serum starved for 24h prior to stimulation for 24h with either insulin 1 microM (n=6), rosiglitazone 10 microM (n=6), resistin 500 nM (n=6) or the MAP kinase inhibitor PD098059 50 microM (n=6) for 30 min, followed by insulin 1 microM for 24h. Semi-quantitative real-time RT-PCR was used to determine the expression of APS mRNA relative to the control gene TF2D. APS expression was markedly upregulated by myoblast differentiation (0.55+/-0.08 versus 1.14+/-0.08, p=0.001), and this effect was augmented by addition of rosiglitazone 10 microM for 24h to the differentiated myotubes (1.50+/-0.09, p=0.025). Insulin caused a 3.1-fold decrease in APS mRNA expression (0.37+/-0.01 versus 1.14+/-0.08, p=0.001), an effect that was attenuated by the MAP kinase inhibitor PD098059 (0.80+/-0.03, p=0.001). Exposure to resistin produced a modest decrease (1.4-fold) in myotube expression of APS (0.8+/-0.09, p=0.025). In conclusion, this is the first study to show that exposure to insulin markedly reduces the expression of APS in skeletal muscle via a MAP kinase dependent pathway, whereas myocyte differentiation and rosiglitazone increase APS expression. Changes in APS expression may be important in the aetiology and therapeutic reversal of insulin resistance in skeletal muscle.

  4. Quantitative evaluation of berberine subcellular distribution and cellular accumulation in non-small cell lung cancer cells by UPLC-MS/MS.

    PubMed

    Yuan, Zhong-Wen; Leung, Elaine Lai-Han; Fan, Xing-Xing; Zhou, Hua; Ma, Wen-Zhe; Liu, Liang; Xie, Ying

    2015-11-01

    Berberine, an isoquinoline alkaloid, has been demonstrated to be a safe anti-cancer agent with multiple effects on mitochondria. Intracellular concentration and distribution around the targeting sites are determinants of efficacy, but subcellular distribution of berberine has not been fully elucidated yet, which relies on the sensitive and robustness assay. In this study, a sensitive and robust UPLC-MS/MS method has been developed and validated with optimized extraction solvents and detection conditions. Key factors such as the purity and integrity of isolated organelle fractions, and the effects of isolation procedures on the subcellular concentration of berberine were systemically evaluated. With the developed assay, we found that the intracellular accumulations of berberine in two gefitinib resistant NSCLC cell lines H1650 and H1975 were 2-3 folds higher than that of normal epithelial cells BEAS-2B. Moreover, significantly different subcellular distribution profiles in NSCLC cancer cells from that of BEAS-2B cells with a striking increase in content in most organelles may contribute to its selective cytotoxicity to cancer cells. Furthermore, a predominant accumulation of berberine was observed for the first time in microsomal fraction for all three cell lines. Therefore, this method could be used for quantitative evaluation of subcellular distribution and cellular accumulation of berberine and for further evaluation of the concentration-effects relationship.

  5. Spatial Distribution of Cellular Function: The Partitioning of Proteins between Mitochondria and the Nucleus in MCF7 Breast Cancer Cells

    PubMed Central

    Qattan, Amal T.; Radulovic, Marko; Crawford, Mark; Godovac-Zimmermann, Jasminka

    2014-01-01

    Concurrent proteomics analysis of the nuclei and mitochondria of MCF7 breast cancer cells identified 985 proteins (40% of all detected proteins) present in both organelles. Numerous proteins from all five complexes involved in oxidative phosphorylation (e.g., NDUFA5, NDUFB10, NDUFS1, NDUF2, SDHA, UQRB, UQRC2, UQCRH, COX5A, COX5B, MT-CO2, ATP5A1, ATP5B, ATP5H, etc.), from the TCA-cycle (DLST, IDH2, IDH3A, OGDH, SUCLAG2, etc.), and from glycolysis (ALDOA, ENO1, FBP1, GPI, PGK1, TALDO1, etc.) were distributed to both the nucleus and mitochondria. In contrast, proteins involved in nuclear/mitochondrial RNA processing/translation and Ras/Rab signaling showed different partitioning patterns. The identity of the OxPhos, TCA-cycle, and glycolysis proteins distributed to both the nucleus and mitochondria provides evidence for spatio-functional integration of these processes over the two different subcellular organelles. We suggest that there are unrecognized aspects of functional coordination between the nucleus and mitochondria, that integration of core functional processes via wide subcellular distribution of constituent proteins is a common characteristic of cells, and that subcellular spatial integration of function may be a vital aspect of cancer. PMID:23051583

  6. Changes in the distribution of plastids and endoplasmic reticulum during cellular differentiation in root caps of Zea mays

    NASA Technical Reports Server (NTRS)

    Moore, R.; McClelen, C. E.

    1985-01-01

    In calyptrogen cells of Zea mays, proplastids are distributed randomly throughout the cell, and the endoplasmic reticulum (ER) is distributed parallel to the cell walls. The differentiation of calyptrogen cells into columella statocytes is characterized by the following sequential events: (1) formation of ER complexes at the distal and proximal ends of the cell, (2) differentiation of proplastids into amyloplasts, (3) sedimentation of amyloplasts onto the distal ER complex, (4) breakdown of the distal ER complex and sedimentation of amyloplasts to the bottom of the cell, and (5) formation of sheets of ER parallel to the longitudinal cell walls. Columella statocytes located in the centre of the cap each possess 4530 +/- 780 micrometers2 of ER surface area, an increase of 670 per cent over that of calyptrogen cells. The differentiation of peripheral cells correlates positively with (1) the ER becoming arranged in concentric sheets, (2) amyloplasts and ER becoming randomly distributed, and (3) a 280 per cent increase in ER surface area over that of columella statocytes. These results are discussed relative to graviperception and mucilage secretion, which are functions of columella and peripheral cells, respectively.

  7. Cellular folate vitamer distribution during and after correction of vitamin B12 deficiency: a case for the methylfolate trap.

    PubMed

    Smulders, Y M; Smith, D E C; Kok, R M; Teerlink, T; Swinkels, D W; Stehouwer, C D A; Jakobs, C

    2006-03-01

    Haematological sequellae of vitamin B12 deficiency are attributed to disturbed DNA synthesis, but vitamin B12 itself plays no role in DNA biosynthesis. A proposed explanation for this is the methylfolate trap hypothesis. This hypothesis states that B12 deficiency impairs overall folate metabolism because 5-methyltetrahydrofolate (5MTHF) becomes metabolically trapped. This trap results from the fact that 5MTHF can neither be metabolised via the methionine synthase pathway, nor can it be reconverted to its precursor, methylenetetrahydrofolate. Other manifestations of the methylfolate trap include cellular folate loss because of shorter 5MTHF polyglutamate chains and global hypomethylation. The methylfolate trap has never been demonstrated in humans. We describe a patient with B12 deficiency who was homozygous for the common methylenetetrahydrofolate reductase (MTHFR) C677T mutation. We analysed red blood cell (RBC) folate vitamers and global DNA methylation by liquid chromatography (LC) in combination with tandem mass spectrometry, and 5MTHF polyglutamate length by LC-electrochemical detection. Compared to post-B12 supplementation values, homocysteine was higher (52.9 micromol/l vs. 16.8 micromol/l), RBC folate was lower (268.92 nmol/l vs. 501.2 nmol/l), the 5MTHF fraction of RBC folate was much higher (94.5% vs. 67.4%), polyglutamate chain length was shorter (more tetra- and pentaglutamates), and global DNA methylation was 22% lower. This is the first time that virtually all features of the methylfolate trap hypothesis have been demonstrated in a human with vitamin B12 deficiency. PMID:16445837

  8. Expression of the Human Endogenous Retrovirus HTDV/HERV-K Is Enhanced by Cellular Transcription Factor YY1

    PubMed Central

    Knössl, Michael; Löwer, Roswitha; Löwer, Johannes

    1999-01-01

    The human endogenous retrovirus HTDV/HERV-K, which resides in moderate copy numbers in the human genome, is expressed in a cell-type-specific manner, predominantly in teratocarcinoma cells. We have analyzed the regulatory potential of the 5′ enhancer of the HERV-K long terminal repeat. Protein extracts of HERV-K-expressing teratocarcinoma cell lines (GH and Tera2) and nonexpressing HeLa and HepG2 cells form different protein complexes on the enhancer sequence as detected by electrophoretic mobility shift assays (EMSA). Using competition EMSAs, DNase I footprinting, and supershift experiments, we localized the binding site of these complexes to a 20-bp sequence within the enhancer and showed that the transcription factor YY1 is one component of the HERV-K enhancer complex. Replacement of the YY1 binding site with unrelated sequences reduced expression of the luciferase gene as a reporter in transient-transfection assays. PMID:9882329

  9. Increased cellular apoptosis susceptibility (CSE1L/CAS) protein expression promotes protrusion extension and enhances migration of MCF-7 breast cancer cells

    SciTech Connect

    Tai, Cheng-Jeng; Shen, Shing-Chuan; Lee, Woan-Ruoh; Liao, Ching-Fong; Deng, Win-Ping; Chiou, Hung-Yi; Hsieh, Cheng-I; Tung, Jai-Nien; Chen, Ching-Shyang; Chiou, Jeng-Fong; Li, Li-Tzu; Lin, Chuang-Yu; Hsu, Chung-Huei; Jiang, Ming-Chung

    2010-10-15

    Microtubules are part of cell structures that play a role in regulating the migration of cancer cells. The cellular apoptosis susceptibility (CSE1L/CAS) protein is a microtubule-associated protein that is highly expressed in cancer. We report here that CSE1L regulates the association of {alpha}-tubulin with {beta}-tubulin and promotes the migration of MCF-7 breast cancer cells. CSE1L was associated with {alpha}-tubulin and {beta}-tubulin in GST (glutathione S-transferase) pull-down and immunoprecipitation assays. CSE1L-GFP (green fluorescence protein) fusion protein experiments showed that the N-terminal of CSE1L interacted with microtubules. Increased CSE1L expression resulted in decreased tyrosine phosphorylation of {alpha}-tubulin and {beta}-tubulin, increased {alpha}-tubulin and {beta}-tubulin association, and enhanced assembly of microtubules. Cell protrusions or pseudopodia are temporary extensions of the plasma membrane and are implicated in cancer cell migration and invasion. Increased CSE1L expression increased the extension of MCF-7 cell protrusions. In vitro migration assay showed that enhanced CSE1L expression increased the migration of MCF-7 cells. Our results indicate that CSE1L plays a role in regulating the extension of cell protrusions and promotes the migration of cancer cells.

  10. Cellular and molecular changes associated with competence acquisition during passion fruit somatic embryogenesis: ultrastructural characterization and analysis of SERK gene expression.

    PubMed

    Rocha, Diego Ismael; Pinto, Daniela Lopes Paim; Vieira, Lorena Melo; Tanaka, Francisco André Ossamu; Dornelas, Marcelo Carnier; Otoni, Wagner Campos

    2016-03-01

    The integration of cellular and molecular data is essential for understanding the mechanisms involved in the acquisition of competence by plant somatic cells and the cytological changes that underlie this process. In the present study, we investigated the dynamics and fate of Passiflora edulis Sims cotyledon explants that were committed to somatic embryogenesis by characterizing the associated ultrastructural events and analysing the expression of a putative P. edulis ortholog of the Somatic Embryogenesis Receptor-like Kinase (SERK) gene. Embryogenic calli were obtained from zygotic embryo explants cultured on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Callus formation was initiated by the division of cells derived from the protodermal and subprotodermal cells on the abaxial side of the cotyledons. The isodiametric protodermal cells of the cotyledon explants adopted a columnar shape and became meristematic at the onset of PeSERK expression, which was not initially detected in explant cells. Therefore, we propose that these changes represent the first observable steps towards the acquisition of a competent state within this regeneration system. PeSERK expression was limited to the early stages of somatic embryogenesis; the expression of this gene was confined to proembryogenic zones and was absent in the embryos after the globular stage. Our data also demonstrated that the dynamics of the mobilization of reserve compounds correlated with the differentiation of the embryogenic callus. PMID:26008651

  11. Functional characterization of calliphorid cell death genes and cellularization gene promoters for controlling gene expression and cell viability in early embryos.

    PubMed

    Edman, R M; Linger, R J; Belikoff, E J; Li, F; Sze, S-H; Tarone, A M; Scott, M J

    2015-02-01

    The New World screwworm fly, Cochliomyia hominivorax, and the Australian sheep blow fly, Lucilia cuprina, are major pests of livestock. The sterile insect technique was used to eradicate C. hominivorax from North and Central America. This involved area-wide releases of male and female flies that had been sterilized by radiation. Genetic systems have been developed for making 'male-only' strains that would improve the efficiency of genetic control of insect pests. One system involves induction of female lethality in embryos through activation of a pro-apoptotic gene by the tetracycline-dependent transactivator. Sex-specific expression is achieved using an intron from the transformer gene, which we previously isolated from several calliphorids. In the present study, we report the isolation of the promoters from the C. hominivorax slam and Lucilia sericata bnk cellularization genes and show that these promoters can drive expression of a GFP reporter gene in early embryos of transgenic L. cuprina. Additionally, we report the isolation of the L. sericata pro-apoptotic hid and rpr genes, identify conserved motifs in the encoded proteins and determine the relative expression of these genes at different stages of development. We show that widespread expression of the L. sericata pro-apoptotic genes was lethal in Drosophila melanogaster. The isolated gene promoters and pro-apoptotic genes could potentially be used to build transgenic embryonic sexing strains of calliphorid livestock pests.

  12. Improvement of the cellular quality of cryopreserved bovine blastocysts accompanied by enhancement of the ATP-binding cassette sub-family B member 1 expression.

    PubMed

    Mori, Miyuki; Kasa, Shojiro; Isozaki, Yoshihiro; Kamori, Tsugumitsu; Yamaguchi, Shoichiro; Ueda, Shuji; Kuwano, Toshio; Eguchi, Minako; Isayama, Keishiro; Nishimura, Shotaro; Tabata, Shoji; Yamauchi, Nobuhiko; Hattori, Masa-aki

    2013-01-01

    The ATP-binding cassette sub-family B member 1 (ABCB1) plays a critical role in maintaining the metabolic capability of cells as an efflux transporter that pumps xenobiotics out of cells. We investigated the effects of highly expressed ABCB1 on the development and viability of cryopreserved bovine embryos. The ABCB1 level in cultured bovine embryos was decreased during development to blastocyst-stage compared to germinal vesicle- and second metaphase-stage oocytes. When bovine embryos were cultured with forskolin and/or rifampicin, the ABCB1 level was significantly increased in blastocysts but embryo development was not significantly improved. After embryo cryopreservation, highly ABCB1-expressed blastocysts exhibited significant increases in viability and hatching rates. The high viability of the cryopreserved blastocysts was accompanied by a significant increase in cell proliferation during culture for 48 h. Thus, ABCB1 is expressed in bovine oocytes and embryos, and the cellular quality of bovine blastocysts is improved by the enhancement of ABCB1 expression. PMID:23164983

  13. A comparison of ovarian follicular and luteal cell gene expression profiles provides insight into cellular identities and functions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    After ovulation, somatic cells of the ovarian follicle (theca and granulosa cells) become the small and large luteal cells of the corpus luteum. Aside from known cell type-specific receptors and steroidogenic enzymes, little is known about the differences in the gene expression profiles of these fou...

  14. TITER AND PRODUCT AFFECTS THE DISTRIBUTION OF GENE EXPRESSION AFTER INTRAPUTAMINAL CONVECTION-ENHANCED DELIVERY

    PubMed Central

    Emborg, Marina E.; Hurley, Samuel A.; Joers, Valerie; Tromp, Do P.M.; Swanson, Christine R.; Ohshima-Hosoyama, Sachiko; Bondarenko, Viktorya; Cummisford, Kyle; Sonnemans, Marc; Hermening, Stephan; Blits, Bas; Alexander, Andrew L.

    2014-01-01

    Background Efficacy and safety of intracerebral gene therapy for brain disorders, like Parkinson’s disease, depends on appropriate distribution of gene expression. Objectives To assess if the distribution of gene expression is affected by vector titer and protein type. Methods Four adult macaque monkeys seronegative for adeno-associated virus 5 (AAV5) received in the right and left ventral postcommisural putamen 30μl inoculation of a high or low titer suspension of AAV5 encoding glial derived neurotrophic factor (GDNF) or green fluorescent protein (GFP). Inoculations were performed using convection enhanced delivery and intraoperative MRI (IMRI). Results IMRI confirmed targeting and infusion cloud irradiating from the catheter tip into surrounding area. Postmortem analysis six weeks after surgery revealed GFP and GDNF expression ipsilateral to the injection side that had a titer-dependent distribution. GFP and GDNF expression was also observed in fibers in the Substantia Nigra (SN) pars reticulata (pr), demonstrating anterograde transport. Few GFP-positive neurons were present in the SN pars compacta (pc), possibly by direct retrograde transport of the vector. GDNF was present in many SNpc and SNpr neurons. Conclusions After controlling for target and infusate volume, intracerebral distribution of gene product is affected by vector titer and product biology. PMID:24943657

  15. Collectivity, Distributivity, and the Interpretation of Plural Numerical Expressions in Child and Adult Language

    ERIC Educational Resources Information Center

    Syrett, Kristen; Musolino, Julien

    2013-01-01

    Sentences containing plural numerical expressions (e.g., "two boys") can give rise to two interpretations (collective and distributive), arising from the fact that their representation admits of a part-whole structure. We present the results of a series of experiments designed to explore children's understanding of this distinction…

  16. Identification of Differentially Expressed Genes in RNA-seq Data of Arabidopsis thaliana: A Compound Distribution Approach

    PubMed Central

    Anjum, Arfa; Jaggi, Seema; Lall, Shwetank; Bhowmik, Arpan; Rai, Anil

    2016-01-01

    Abstract Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product, which may be proteins. A gene is declared differentially expressed if an observed difference or change in read counts or expression levels between two experimental conditions is statistically significant. To identify differentially expressed genes between two conditions, it is important to find statistical distributional property of the data to approximate the nature of differential genes. In the present study, the focus is mainly to investigate the differential gene expression analysis for sequence data based on compound distribution model. This approach was applied in RNA-seq count data of Arabidopsis thaliana and it has been found that compound Poisson distribution is more appropriate to capture the variability as compared with Poisson distribution. Thus, fitting of appropriate distribution to gene expression data provides statistically sound cutoff values for identifying differentially expressed genes. PMID:26949988

  17. Effect of glutamine on cellular adhesion molecule expression and leukocyte transmigration in endothelial cells stimulated by plasma or peritoneal drain fluid from a surgical patient.

    PubMed

    Yeh, Chiu-Li; Hsu, Chun-Sen; Chen, Soul-Chin; Pai, Man-Hui; Yeh, Sung-Ling

    2006-03-01

    This study investigated the effect of glutamine (GLN) concentration on surface molecule expression on endothelial cells (ECs) and leukocytes and the transendothelial migration of polymorphonuclear neutrophils (PMNs) through ECs stimulated by plasma or peritoneal drain fluid (PDF) from a surgical patient. Human umbilical vein ECs (HUVECs) and PMNs from normal subjects were treated with different concentrations (0, 300, 600, and 1000 micromol/L) of GLN for 24 h. After that, HUVECs were stimulated for 3 h with plasma or PDF from a patient who had undergone abdominal surgery, and PMNs were allowed to transmigrate through ECs for 2 h. HUVEC surface expression of cell adhesion molecules and integrin (CD11b) and interleukin (IL) 8 receptor expression on PMNs were measured by flow cytometry. PMNs transmigrating through ECs were also analyzed. The results showed that cell adhesion molecule and integrin expressions in PDF groups were higher than those in control groups. Among the PDF groups, cellular adhesion molecule expressions on ECs and CD11b expression on PMNs were lower with 600 and 1000 micromol/L than with 300 micromol/L GLN. IL-8 secretions from ECs and PMNs were higher with 300 and 600 micromol/L than with 1000 micromol/L GLN, and this was consistent with the expression of the IL-8 receptor on PMNs. PMN transmigration was significantly higher with 300 micromol/L GLN than with the other GLN concentrations. HUVECs stimulated by plasma from surgical patient had the similar effects on surface molecule expression as PDF; however, the influences were not so obvious as shown in PDF stimulation. The results of this in vitro study suggest that ECs and PMNs were activated after patient's plasma or PDF stimulation. A low GLN concentration comparable to catabolic conditions resulted in higher adhesion molecule expression and greater transendothelial migration of neutrophils. GLN administration at levels similar to or higher than physiological concentrations reduced IL-8 and

  18. Wnt Signaling Alteration in the Spinal Cord of Amyotrophic Lateral Sclerosis Transgenic Mice: Special Focus on Frizzled-5 Cellular Expression Pattern

    PubMed Central

    González-Fernández, Carlos; Mancuso, Renzo; del Valle, Jaume; Navarro, Xavier; Rodríguez, Francisco Javier

    2016-01-01

    Background Amyotrophic lateral sclerosis is a chronic neurodegenerative disease characterized by progressive paralysis due to degeneration of motor neurons by unknown causes. Recent evidence shows that Wnt signaling is involved in neurodegenerative processes, including Amyotrophic Lateral Sclerosis. However, to date, little is known regarding the expression of Wnt signaling components in this fatal condition. In the present study we used transgenic SOD1G93A mice to evaluate the expression of several Wnt signaling components, with special focus on Frizzled-5 cellular expression alteration along disease progression. Findings Based on previous studies demonstrating the expression of Wnts and their transcriptional regulation during Amyotrophic lateral sclerosis development, we have analyzed the mRNA expression of several Wnt signaling components in the spinal cord of SOD1G93A transgenic mice at different stages of the disease by using real time quantitative PCR analysis. Strikingly, one of the molecules that seemed not to be altered at mRNA level, Frizzled-5, showed a clear up-regulation at late stages in neurons, as evidenced by immunofluorescence assays. Moreover, increased Frizzled-5 appears to correlate with a decrease in NeuN signal in these cells, suggesting a correlation between neuronal affectation and the increased expression of this receptor. Conclusions Our data suggest the involvement of Wnt signaling pathways in the pathophysiology of Amyotrophic Lateral Sclerosis and, more specifically, the implication of Frizzled-5 receptor in the response of neuronal cells against neurodegeneration. Nevertheless, further experimental studies are needed to shed light on the specific role of Frizzled-5 and the emerging but increasing Wnt family of proteins research field as a potential target for this neuropathology. PMID:27192435

  19. Expression and immunohistochemical distribution of duck plague virus glycoprotein gE in infected ducks.

    PubMed

    Chang, Hua; Cheng, Anchun; Wang, Mingshu; Xiang, Jun; Xie, Wei; Shen, Fuxiao; Jia, Renyong; Zhu, Dekang; Luo, Qihui; Zhou, Yi; Chen, Xiaoyue

    2011-03-01

    To determine the distribution of duck plague virus (DPV) gE protein in paraformaldehyde-fixed, paraffin-embedded tissues of experimentally DPV-infected ducks, an indirect immunoperoxidase assay was established to detect glycoprotein E (gE) protein for the first time. The rabbit anti-His-gE serum, raised against the recombinant His-gE fusion protein expressed in Escherichia coli BL21 (DE3), was prepared and purified. Western blotting and indirect immunofluorescence analysis showed that the anti-His-gE serum had a high level of reactivity and specificity and could be used as the first antibody for further experiments to study the distribution of DPV gE protein in DPV-infected tissues. A number of DPV gE proteins were distributed in the bursa of Fabricius, thymus, spleen, liver, esophagus, duodenum, jejunum, ileum, and kidney of DPV-infected ducks and a few DPV gE were distributed in the Harders glands, myocardium, cerebrum, and lung, whereas the gE was not seen in the skin, muscle, and pancreas. Moreover, DPV gE was expressed abundantly in the cytoplasm of lymphocytes, reticulum cells, macrophages, epithelial cells, and hepatocytes. The present study may be useful not only for describing the characteristics of gE expression and distribution in infected ducks but also for understanding the pathogenesis of DPV.

  20. Effect of passage number on cellular response to DNA-damaging agents: Cell survival and gene expression

    SciTech Connect

    Chang-Liu, C.M.; Woloschak, G.E.

    1997-08-01

    The effect of different passage numbers on plating efficiency, doubling time, cell growth, and radiation sensitivity was assessed in Syrian hamster embryo (SHE) cells. Changes in gene expression after UV or {gamma}-ray irradiation at different passage numbers were also examined. The SHE cells were maintained in culture medium for up to 64 passages. Cells were exposed to {sup 60}Co {gamma} rays or 254-nm UV radiation. Differential display of cDNAs and northern blots were used for the study of gene expression. With increasing passage number, SHE cells demonstrated decreased doubling time, increased plating efficiency, and a decreased yield in the number of cells per plate. Between passages 41 and 48 a crisis period was evident during which time cell growth in high serum was no longer optimal, and serum concentrations were reduced to maintain cell growth. Sensitivity to ionizing radiation was no different between early- and intermediate-passage cells. However, after UV exposure at low passages (passage 3), confluent cells were more sensitive to the killing effects of UV than were log-phase cells. At intermediate passages (passages 43, 48), confluent cells were slightly more radioresistant than were log-phase cells. By passage 64, however, both confluent and log-phase cells showed similar patterns of UV sensitivity. Expression of {gamma}-actin, PCNA, and p53 transcripts did not change following UV exposure. p53 mRNA was induced following {gamma}-ray exposure of the intermediate (passage 45) epithelial cells. The observed differences in radiation sensitivity associated with increasing passage number may be influenced by radiation-induced gene expression. The authors are conducted experiments to identify these genes.

  1. Type 1 ribosome-inactivating proteins from Phytolacca dioica L. leaves: differential seasonal and age expression, and cellular localization.

    PubMed

    Parente, Augusto; Conforto, Barbara; Di Maro, Antimo; Chambery, Angela; De Luca, Paolo; Bolognesi, Andrea; Iriti, Marcello; Faoro, Franco

    2008-11-01

    The expression of type 1 ribosome-inactivating proteins (RIPs) in Phytolacca dioica L. leaves was investigated. Fully expanded leaves of young P. dioica plants (up to 3 years old) expressed two novel RIPs, dioicin 1 and dioicin 2. The former was also found in developing leaves from adult P. dioica within about two and a half weeks after leaf development, and the latter continuously synthesized, with no seasonal or ontogenetic constraint. Fully expanded leaves from adult P. dioica expressed four RIPs (PD-Ls1-4) exhibiting seasonal variation. RIPs were localized in the extracellular space, in the vacuole and in the Golgi apparatus of mesophyll cells. Dioicin 1 and dioicin 2 showed rRNA N-beta-glycosidase activity and displayed the following properties, respectively: (1) Mr values of 30,047.00 and 29,910.00, (2) pIs of 8.74 and 9.37, and (3) IC(50) values of 19.74 (0.658 nM) and 6.85 ng/mL (0.229 nM). Furthermore, they showed adenine polynucleotide glycosylase activity and nicked pBR322 dsDNA. The amino acid sequence of dioicin 2 had 266 amino acid residues, and the highest percentage identity (81.6%) and similarity (84.6%) with PAP-II from Phytolacca americana, while its identity with other RIPs from Phytolaccaceae was around 40%.

  2. Expression of early and late cellular damage markers by ARPE-19 cells following prolonged treatment with UV-A radiation.

    PubMed

    Tringali, Giuseppe; Sampaolese, Beatrice; Clementi, Maria Elisabetta

    2016-10-01

    Pathological alterations to the retinal pigment epithelium underlie several eye diseases, which lead to visual impairment and even blindness. Exposure to ultraviolet (UV) radiation is associated with some skin and ocular pathologies; UV radiation may induce DNA breakdown and cause cellular damage through the production of reactive oxygen species (ROS), thus leading to programmed cell death. The present study aimed to investigate the production of ROS and the gene expression levels of anti‑ and proapoptotic proteins [B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X protein (Bax) and caspase‑3] in human retinal pigment epithelial cells (ARPE‑19) treated with UV‑A for 5 h consecutively. The results demonstrated that prolonged exposure to UV‑A induced: i) Cell death, the decrease in cell viability was time‑dependent and reached statistical significance after 3 h; ii) a significant and substantial increase in ROS levels that remained constant for the duration of the experiment, the levels were significantly increased after 1 h of exposure; iii) an activation of apoptotic genes (Bax and caspase‑3) after 1 h of treatment, which was accompanied by a decrease in the anti‑apoptotic gene Bcl‑2; and iv) a loss of apoptotic signals and a rapid decrease in cellular viability after 3 h of consecutive treatment. These processes may trigger necrosis, which was observed in the cells following treatment with UV‑A for 5 consecutive hours. In conclusion, the present study is the first, to the best of our knowledge, to provide in vitro evidence regarding the sequence of events that underlie the cellular damage induced by prolonged UV‑A radiation, starting from the first 30 min of treatment. UV‑A radiation resulted in the activation of apoptotic events, and subsequently led to irreversible cell necrosis. PMID:27573029

  3. Expression of early and late cellular damage markers by ARPE-19 cells following prolonged treatment with UV-A radiation.

    PubMed

    Tringali, Giuseppe; Sampaolese, Beatrice; Clementi, Maria Elisabetta

    2016-10-01

    Pathological alterations to the retinal pigment epithelium underlie several eye diseases, which lead to visual impairment and even blindness. Exposure to ultraviolet (UV) radiation is associated with some skin and ocular pathologies; UV radiation may induce DNA breakdown and cause cellular damage through the production of reactive oxygen species (ROS), thus leading to programmed cell death. The present study aimed to investigate the production of ROS and the gene expression levels of anti‑ and proapoptotic proteins [B‑cell lymphoma 2 (Bcl‑2), Bcl‑2‑associated X protein (Bax) and caspase‑3] in human retinal pigment epithelial cells (ARPE‑19) treated with UV‑A for 5 h consecutively. The results demonstrated that prolonged exposure to UV‑A induced: i) Cell death, the decrease in cell viability was time‑dependent and reached statistical significance after 3 h; ii) a significant and substantial increase in ROS levels that remained constant for the duration of the experiment, the levels were significantly increased after 1 h of exposure; iii) an activation of apoptotic genes (Bax and caspase‑3) after 1 h of treatment, which was accompanied by a decrease in the anti‑apoptotic gene Bcl‑2; and iv) a loss of apoptotic signals and a rapid decrease in cellular viability after 3 h of consecutive treatment. These processes may trigger necrosis, which was observed in the cells following treatment with UV‑A for 5 consecutive hours. In conclusion, the present study is the first, to the best of our knowledge, to provide in vitro evidence regarding the sequence of events that underlie the cellular damage induced by prolonged UV‑A radiation, starting from the first 30 min of treatment. UV‑A radiation resulted in the activation of apoptotic events, and subsequently led to irreversible cell necrosis.

  4. Immunohistochemical Expression of PCNA and CD34 in Colorectal Adenomas and Carcinomas Using Specified Automated Cellular Image Analysis System: A Clinicopathologic Study

    PubMed Central

    Qasim, Ban J.; Ali, Hussam H.; Hussein, Alaa G.

    2012-01-01

    Background/Aim: To evaluate the immunohistochemical expression of proliferating cell nuclear antigen (PCNA) and CD34 in colorectal adenomas and carcinomas, and to correlate this expression with different clinicopathologic parameters. Materials and Methods: The study was retrospectively designed. A total of 86 tissue samples, including 33 paraffin blocks from patients with colorectal adenomas, 33 paraffin blocks from patients with colorectal adenocarcinomas, and a control group of 20 samples of nontumerous colonic tissue, were included in the study. From each block, 3 sections of 5 ΅m thickness were taken, 1 section was stained with hematoxylin and eosin (H and E) and the other 2 sections were stained immunohistochemically for PCNA and CD34. Scoring of the immunohistochemical staining was performed using a specified automated cellular image analysis system (Digimizer). Results: PCNA expression was significantly increased in a sequence of normal mucosa–adenoma–carcinoma. It was significantly higher in adenomas ≥ 1 cm and those with severe dysplasia, and it showed a significant positive correlation with grade and lymph node involvement in colorectal carcinoma. CD34 showed significantly higher expression in carcinoma than adenoma and in adenoma than in the control group. CD34 expression showed a significant correlation with adenomas carrying severe dysplasia and large-sized adenomas (≥1cm). It was significantly correlated with tumor grade, lymphovascular invasion, and lymph node involvement in colorectal carcinoma. Conclusion: PCNA plays an important role in colorectal neoplastic progression and can be utilized as ancillary marker for the risk of malignant transformation in colorectal adenomas as it correlates with high grade dysplasia and size. Intratumoral quantification of the mean (A and N) of CD34 in colorectal carcinoma reflects the grade of tumors and can predict lymph node involvement and lymphovascular invasion, to make a useful additional prognostic

  5. Dual Role of the Saccharomyces cerevisiae TEA/ATTS Family Transcription Factor Tec1p in Regulation of Gene Expression and Cellular Development

    PubMed Central

    Köhler, Tim; Wesche, Stefanie; Taheri, Naimeh; Braus, Gerhard H.; Mösch, Hans-Ulrich

    2002-01-01

    In Saccharomyces cerevisiae, the transcription factors Tec1p and Ste12p are required for haploid invasive and diploid pseudohyphal growth. Tec1p and Ste12p have been postulated to regulate these developmental processes primarily by cooperative binding to filamentous and invasion-responsive elements (FREs), which are combined enhancer elements that consist of a Tec1p-binding site (TCS) and an Ste12p-binding site (PRE). They are present in the promoter regions of target genes, e.g., FLO11. Here, we show that Tec1p efficiently activates target gene expression and cellular development in the absence of Ste12p. We further demonstrate that TCS elements alone are sufficient to mediate Tec1p-driven gene expression by a mechanism termed TCS control that is operative even when Ste12p is absent. Mutational analysis of TEC1 revealed that TCS control, FLO11 expression, and haploid invasive growth require the C terminus of Tec1p. In contrast, the Ste12p-dependent FRE control mechanism is sufficiently executed by the N-terminal portion of Tec1p, which contains the TEA/ATTS DNA-binding domain. Our study suggests that regulation of haploid invasive and diploid pseudohyphal growth by Ste12p and Tec1p is not only executed by combinatorial control but involves additional control mechanisms in which Ste12p activates TEC1 expression via clustered PREs and where Tec1p regulates expression of target genes, e.g., FLO11, by TCS control. PMID:12455687

  6. Time course and cellular localization of interleukin-10 mRNA and protein expression in autoimmune inflammation of the rat central nervous system.

    PubMed Central

    Jander, S.; Pohl, J.; D'Urso, D.; Gillen, C.; Stoll, G.

    1998-01-01

    Experimental autoimmune encephalomyelitis of the Lewis rat is a T-cell-mediated autoimmune disease of the central nervous system characterized by a self-limiting monophasic course. In this study, we analyzed the expression of the anti-inflammatory cytokine interleukin (IL)-10 at the mRNA and protein level in experimental autoimmune encephalomyelitis actively induced with the encephalitogenic 68-86 peptide of guinea pig myelin basic protein. Semiquantitative reverse transcriptase-polymerase chain reaction revealed that IL-10 mRNA expression peaked during the acute phase of the disease at days 11 and 13. IL-10 mRNA was synchronously induced with mRNA for the proinflammatory cytokine interferon-gamma. Immunocytochemistry with a monoclonal antibody against rat IL-10 showed that the peak of IL-10 mRNA was accompanied by an abundant expression of IL-10 protein during the acute stage of the disease. Both in situ hybridization and double labeling immunocytochemistry in combination with confocal microscopy identified T cells, macrophages/microglia, and astrocytes as major cellular sources of IL-10 in vivo. The early peak of IL-10 production was unexpected in light of its well-documented anti-inflammatory properties. Additional studies are required to determine whether endogenous IL-10 contributes to rapid clinical remission typical for Lewis rat experimental autoimmune encephalomyelitis or if it plays other, yet undefined, roles in central nervous system autoimmunity. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9546358

  7. Metal-specific control of gene expression mediated by Bradyrhizobium japonicum Mur and Escherichia coli Fur is determined by the cellular context.

    PubMed

    Hohle, Thomas H; O'Brian, Mark R

    2016-07-01

    Bradyrhizobium japonicum Mur and Escherichia coli Fur are manganese- and iron-responsive transcriptional regulators, respectively, that belong to the same protein family. Here, we show that neither Mur nor Fur discriminate between Fe(2+) and Mn(2+) in vitro nor is there a metal preference for conferral of DNA-binding activity on the purified proteins. When expressed in E. coli, B. japonicum Mur responded to iron, but not manganese, as determined by in vivo promoter occupancy and transcriptional repression activity. Moreover, E. coli Fur activity was manganese-dependent in B. japonicum. Total and chelatable iron levels were higher in E. coli than in B. japonicum under identical growth conditions, and Mur responded to iron in a B. japonicum iron export mutant that accumulated high levels of the metal. However, elevated manganese content in E. coli did not confer activity on Fur or Mur, suggesting a regulatory pool of manganese in B. japonicum that is absent in E. coli. We conclude that the metal selectivity of Mur and Fur depends on the cellular context in which they function, not on intrinsic properties of the proteins. Also, the novel iron sensing mechanism found in the rhizobia may be an evolutionary adaptation to the cellular manganese status.

  8. Cellular senescence or EGFR signaling induces Interleukin 6 (IL-6) receptor expression controlled by mammalian target of rapamycin (mTOR)

    PubMed Central

    Garbers, Christoph; Kuck, Fabian; Aparicio-Siegmund, Samadhi; Konzak, Kirstin; Kessenbrock, Mareike; Sommerfeld, Annika; Häussinger, Dieter; Lang, Philipp A; Brenner, Dirk; Mak, Tak W.; Rose-John, Stefan; Essmann, Frank; Schulze-Osthoff, Klaus; Piekorz, Roland P; Scheller, Jürgen

    2013-01-01

    Interleukin 6 (IL-6) signaling plays a role in inflammation, cancer, and senescence. Here, we identified soluble IL-6 receptor (sIL-6R) as a member of the senescence-associated secretory phenotype (SASP). Senescence-associated sIL-6R upregulation was mediated by mammalian target of rapamycin (mTOR). sIL-6R was mainly generated by a disintegrin and metalloprotease 10 (ADAM10)-dependent ectodomain shedding to enable IL-6 trans-signaling. In vivo, heterozygous PTEN-knockout mice exhibited higher mTOR activity and increased sIL-6R levels. Moreover, aberrant EGF receptor (EGFR) activation triggered IL-6 synthesis. In analogy to senescence, EGFR-induced activation of mTOR also induced IL-6R expression and sIL-6R generation. Hence, mTOR activation reprograms IL-6 non-responder cells into IL-6 responder cells. Our data suggest that mTOR serves as a central molecular switch to facilitate cellular IL-6 classic and trans-signaling via IL-6R upregulation with direct implications for cellular senescence and tumor development. PMID:24047696

  9. Hydrogen sulfide donor NaHS induces death of alveolar epithelial L2 cells that is associated with cellular shrinkage, transgelin expression and myosin phosphorylation.

    PubMed

    Fujii, Yusuke; Funakoshi, Takeshi; Unuma, Kana; Noritake, Kanako; Aki, Toshihiko; Uemura, Koichi

    2016-01-01

    Hydrogen sulfide (H2S) is a highly toxic gaseous molecule that causes death to humans exposed to high concentrations. H2S is absorbed into the body through the alveolar epithelium and other tissues. The aim of this study is to evaluate the molecular mechanism underling acute lung injury caused by the inhalation of high concentrations of H2S. Rat lung epithelium-derived L2 cells were exposed to a H2S donor, NaHS, at concentrations of 2-4 mM for 1-6 hr. NaHS caused shrinkage and death of the cells without caspase activation. An actin-binding protein, transgelin, was identified as one of the NaHS-inducible proteins in the cells. NaHS increased myosin light chain (MLC) phosphorylation, indicating that actomyosin-mediated cellular contractility and/or motility could be increased after NaHS exposure. The administration of ML-7, a myosin light chain kinase (MLCK) inhibitor, accelerated cell death after NaHS exposure. Based on these data, we conclude that the increase in MLC phosphorylation in response to NaHS exposure is a cellular protective reaction against NaHS toxicity. Enhancements in smooth muscle cell properties such as transgelin expression and actomyosin-mediated contractility/motility might be involved in cell survival after NaHS exposure. PMID:27665774

  10. Low-temperature atmospheric plasma increases the expression of anti-aging genes of skin cells without causing cellular damages.

    PubMed

    Choi, Jeong-Hae; Lee, Hyun-Wook; Lee, Jae-Koo; Hong, Jin-woo; Kim, Gyoo-cheon

    2013-03-01

    Efforts to employ various types of plasma in the field of skin care have increased consistently because it can regulate many biochemical reactions that are normally unaffected by light-based therapy. One method for skin rejuvenation adopted a high-temperature plasma generator to remove skin epithelial cells. In this case, the catalyzing effects of the plasma were rarely used due to the high temperature. Hence, the benefits of the plasma were not magnified. Recently, many types of low-temperature plasma devices have been developed for medical applications but their detailed functions and working mechanisms are unclear. The present study examined the effect of low-temperature microwave plasma on skin cells. Treatment with low-temperature plasma increased the expression of anti-aging genes in skin cells, including collagen, fibronectin and vascular endothelial growth factor. Furthermore, the plasma treatment did not cause cell death, but only induced slight cell growth arrest at the G2 phase. Although the cells treated with low-temperature plasma showed moderate growth arrest, there were no signs of thermal or genetic damage of skin cells. Overall, this low-temperature microwave plasma device induces the expressions of some anti-aging-related genes in skin cells without causing damage.

  11. Heat exposure in female rats elicits abnormal fear expression and cellular changes in prefrontal cortex and hippocampus.

    PubMed

    Gruene, Tina M; Lipps, Jennifer; Rey, Colin D; Bouck, Anna; Shansky, Rebecca M

    2014-11-01

    Despite a twofold higher prevalence of fear-related disorders in women, the neurobiological factors that modulate and drive fear expression are rarely studied in female animals. Fear conditioning and extinction are useful tools for dissecting these mechanisms, and here we tested the effects of environmental manipulations - four days of exposure to 31°C temperatures in the animal housing facility - on fear learning and memory exclusively in female rats. We found that heat exposure disrupted freezing to tone during fear conditioning, and elicited enhanced freezing during extinction and extinction retrieval. We also performed immunohistochemistry for c-fos expression in the infralimbic (IL) and prelimbic (PL) regions of the prefrontal cortex during extinction retrieval, and found that heat exposure induced a switch from IL-dominated activity to PL-dominated activity. Finally, morphological analysis of spines in hippocampal CA3 neurons revealed an increase in spine head diameter in heat-exposed animals, which may partly underlie the persistent freezing observed in these animals. Together, our data show that heat exposure can induce changes at behavioral, physiological, and structural levels, and add to a woefully lacking body of literature on fear processes in female animals.

  12. Cellular expression of human centromere protein C demonstrates a cyclic behavior with highest abundance in the G1 phase.

    PubMed Central

    Knehr, M; Poppe, M; Schroeter, D; Eickelbaum, W; Finze, E M; Kiesewetter, U L; Enulescu, M; Arand, M; Paweletz, N

    1996-01-01

    Centromere proteins are localized within the centromere-kinetochore complex, which can be proven by means of immunofluorescence microscopy and immunoelectron microscopy. In consequence, their putative functions seem to be related exclusively to mitosis, namely to the interaction of the chromosomal kinetochores with spindle microtubules. However, electron microscopy using immune sera enriched with specific antibodies against human centromere protein C (CENP-C) showed that it occurs not only in mitosis but during the whole cell cycle. Therefore, we investigated the cell cycle-specific expression of CENP-C systematically on protein and mRNA levels applying HeLa cells synchronized in all cell cycle phases. Immunoblotting confirmed protein expression during the whole cell cycle and revealed an increase of CENP-C from the S phase through the G2 phase and mitosis to highest abundance in the G1 phase. Since this was rather surprising, we verified it by quantifying phase-specific mRNA levels of CENP-C, paralleled by the amplification of suitable internal standards, using the polymerase chain reaction. The results were in excellent agreement with abundant protein amounts and confirmed the cyclic behavior of CENP-C during the cell cycle. In consequence, we postulate that in addition to its role in mitosis, CENP-C has a further role in the G1 phase that may be related to cell cycle control. Images Fig. 1 Fig. 2 Fig. 4 PMID:8816782

  13. Effects of Salmonella enterica serovar Enteritidis on cellular recruitment and cytokine gene expression in caecum of vaccinated chickens.

    PubMed

    Carvajal, Bárbara González; Methner, Ulrich; Pieper, Jana; Berndt, Angela

    2008-10-01

    Although vaccination of poultry is a suitable method to limit human food borne gastroenteritis caused by Salmonella (S.), the immune mechanisms responsible for a longer lasting protection against Salmonella infection in birds are not completely understood. To reveal unique protection-related immune parameters, day-old chicks were vaccinated with a commercial live S. Enteritidis vaccine and challenged with wild-type S. Enteritidis 147N at day 56 of life. The bacterial cell count was determined in gut and liver, while the immune cell composition and cytokine gene expression patterns were analysed by immunohistochemistry and quantitative real-time RT-PCR in caecum samples. The presented data suggest that the vaccine-elicited immune protection against the Salmonella wild-type infection was rather related to the bacterial count in gut mucosa and liver than to the colonisation in gut lumen. The higher number of Salmonella wild-type organisms found in caecal wall and liver of the non-immunised compared to immunised birds after challenge correlated with a more pronounced gene expression rate for IL-8, LITAF, iNOS, IL-12 and IFN-gamma. In contrast, immunised birds exhibited higher amounts of CD8(+) T cells as well as IgA than the non-immunised chickens after S. Enteritidis 147N infection in caecum. The results demonstrated a distinctive immune reaction pattern of previously vaccinated compared to non-vaccinated chickens upon S. Enteritidis wild-type challenge.

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

    PubMed

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

    2013-08-01

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

  15. Adolescent Risperidone treatment alters protein expression associated with protein trafficking and cellular metabolism in the adult rat prefrontal cortex.

    PubMed

    Farrelly, Lorna A; Dicker, Patrick; Wynne, Kieran; English, Jane; Cagney, Gerard; Föcking, Melanie; Cotter, David R

    2014-06-01

    The prefrontal cortex (PFC) is associated with mental health illnesses including schizophrenia, depression, bipolar disorder, and autism spectrum disorders. It richly expresses neuroreceptors which are the target for antipsychotics. However, as the precise mechanism of action of antipsychotic medications is not known, proteomic studies of the effects of antipsychotic drugs on the brain are warranted. In the current study, we aimed to characterize protein expression in the adult rodent PFC (n = 5 per group) following low-dose treatment with Risperidone or saline in adolescence (postnatal days 34-47). The PFC was examined by triplicate 1 h runs of label-free LC-MS/MS. The raw mass spectral data were analyzed with the MaxQuant(TM) software. Statistical analysis was carried out using SAS® Version 9.1. Pathway and functional analysis was performed with IngenuityPathway Analysis and in the Database for Annotation, Visualization and Integrated Discovery (DAVID), respectively, the most implicated pathways were found to be related to mitochondrial function, protein trafficking, and the cytoskeleton. This report adds to the current repertoire of data available concerning the effects of antipsychotic drugs on the brain and sheds light on their biological mechanisms. The MS data have been deposited with the ProteomeXchange Consortium with dataset identifier PXD000480. PMID:24733778

  16. Cellular changes and cytokine expression in the ilea of gnotobiotic piglets resulting from peroral Salmonella typhimurium challenge.

    PubMed Central

    Trebichavský, I; Dlabac, V; Reháková, Z; Zahradnícková, M; Splíchal, I

    1997-01-01

    Two stable rough mutants of Salmonella spp. were studied as live peroral vaccines. The SF1591 mutant of S. typhimurium (Ra chemotype) protected germ-free piglets against subsequent infection with virulent smooth S. typhimurium LT2, whereas a deep-rough mutant of S. minnesota mR595 (Re chemotype) did not. We investigated cytokine and leukocyte profiles in the ilea of gnotobiotic piglets colonized for 1 week either with rough mutants alone or with rough mutants followed by S. typhimurium LT2. The ileal mucosae of piglets associated with strain SF1591 alone were not inflamed. Villi contained activated macrophages, and enterocytes expressed transforming growth factor beta (TGF-beta). Subsequent infection of piglets with S. typhimurium LT2 resulted in immigration of alphabeta T cells and immunoglobulin A (IgA) response. In contrast, the ileal mucosae of piglets associated with strain mR595 alone expressed heat shock proteins and inflammatory cytokines but not TGF-beta. Acellular villi contained numerous gammadelta T cells but no alphabeta T cells. After subsequent challenge with the LT2 strain, most piglets died of sepsis. Intestinal mucosae contained IgG but no IgA. These findings suggest the importance of cytokine signals in the regulation of intestinal responses against Salmonella infection. PMID:9393822

  17. Expression of RB C pocket fragments in HSF induces delayed cell cycle progression and sensitizes to apoptosis upon cellular stresses.

    PubMed

    Chung, Junah; Cho, Jae-We; Baek, Won-Ki; Suh, Seong-Il; Kwon, Taeg Kyu; Park, Jong-Wook; Suh, Min-Ho

    2002-08-01

    The retinoblastoma protein (RB) plays an important role in growth suppression through the formation of multiple protein complexes with its target proteins using A/B and C pockets. Even though the A/B and C pockets co-operate for growth suppression, the function of RB in growth arrest is inhibited by the coexpression of RB C fragments with full length RB in the absence of p53, which implies that C pocket fragments are likely to act as a dominant-negative inhibitor of RB function. In contrast, the loss of the RB functions in the presence of p53 triggers a cell cycle arrest or apoptosis by p53-dependent pathways. Thus, it still remains to be elucidated whether the expression of RB C pocket fragments in the presence of p53 induces delayed cell cycle progression and sensitizes cells to apoptosis through p53-dependent pathways. Our results show that the expression of RB C pocket fragments not only induces delayed cell cycle progression, which is mediated by the down-regulation of cyclin A, cyclin E, and E2F-1, but also sensitizes cells to apoptosis through p53-dependent pathways. PMID:12153616

  18. Different patterns of 5{alpha}-reductase expression, cellular distribution, and testosterone metabolism in human follicular dermal papilla cells

    SciTech Connect

    Liu, Shicheng Yamauchi, Hitoshi

    2008-04-18

    Androgens regulate hair growth, and 5{alpha}-reductase (5{alpha}R) plays a pivotal role in the action of androgens on target organs. To clarify the molecular mechanisms responsible for controlling hair growth, the present study presents evidence that the human follicular dermal papilla cells (DPCs) from either beard (bDPCs) or scalp hair (sDPCs) possess endogenous 5{alpha}R activity. Real-time RT-PCR revealed that the highest level of 5{alpha}R1 mRNA was found in bDPCs, followed by sDPCs, and a low but detectable level of 5{alpha}R1 mRNA was observed in fibroblasts. Minimally detectable levels of 5{alpha}R2 mRNA were found in all three cell types. A weak band at 26 kDa corresponding to the human 5{alpha}R1 protein was detected by Western blot in both DPCs, but not in fibroblasts. Immuonofluorescence analysis confirmed that 5{alpha}R1 was localized to the cytoplasm rather than in the nuclei in both DPCs Furthermore, a 5{alpha}R assay using [{sup 14}C]testosterone labeling in intact cells revealed that testosterone was transformed primarily into androstenedione, and in small amounts, into DHT. Our results demonstrate that the 5{alpha}R activities of either bDPCs or sDPCs are stronger than that of dermal fibroblasts, despite the fact that the major steroidogenic activity is attributed to 17{beta}-HSD rather than 5{alpha}R among the three cell types. The 5{alpha}R1 inhibitor MK386 exhibited a more potent inhibitory effect on 5{alpha}R activity than finasteride (5{alpha}R2 inhibitor) in bDPCs.

  19. Differential global and extra-cellular matrix focused gene expression patterns between normal and glaucomatous human lamina cribrosa cells

    PubMed Central

    Wordinger, Robert J.; Clark, Abbot F.; O'Brien, Colm J.

    2009-01-01

    Purpose Marked extracellular matrix (ECM) remodeling occurs in the human optic nerve head in primary open angle glaucoma (POAG). The glial fibrillary acid protein (GFAP) negative lamina cribrosa cell may play an important role in this remodeling process. We report the first study of global and ECM-focused gene transcription differentials between GFAP-negative lamina cribrosa (LC) cells from normal and POAG human donors. Methods GFAP-negative LC cell lines were generated from the optic nerve tissue of four normal (n=4) and four POAG (n=4) human donors. Using Affymetrix U133A arrays the transcriptional profile between the normal and diseased groups were compared. Bioinformatic analysis was performed using robust multichip average (RMA Express) and EASE/David. Real time TaqMan PCR and immunohistochemistry analyses were performed to validate the microarray data. Results 183 genes were upregulated by greater than 1.5 fold and 220 were down regulated by greater than 1.5 fold in the POAG LC cells versus normal controls. Upregulated genes in POAG LC cells included, transforming growth factor beta 1 (TGFβ1), secreted acid protein cysteine rich (SPARC), periostin (POSTN), thrombospondin-1 (THBS1), cartilage linking protein-1 (CRTL-1), and collagen type I (COL1A1), collagen type V (COL5A1), and collagen type XI (COL11A1). Downregulated ECM genes in POAG included fibulin 1 (FBLN1), decorin (DCN), and collagen type XVIII (COL18A1). All TaqMan PCR validation assays were significant (*p<0.05) and consistent with the array data. Immunohistochemistry of one target (periostin) confirmed its differential expression at the protein level in POAG optic nerve head tissue compared with non-glaucomatous controls. Functional annotation and over-representation analysis identified ECM genes as a statistically over-represented class of genes in POAG LC cells compared with normal LC cells. Conclusions This study reports for the first time that POAG LC cells in-vitro demonstrate upregulated ECM

  20. Exercise Decreases Lipogenic Gene Expression in Adipose Tissue and Alters Adipocyte Cellularity during Weight Regain After Weight Loss

    PubMed Central

    Giles, Erin D.; Steig, Amy J.; Jackman, Matthew R.; Higgins, Janine A.; Johnson, Ginger C.; Lindstrom, Rachel C.; MacLean, Paul S.

    2016-01-01

    Exercise is a potent strategy to facilitate long-term weight maintenance. In addition to increasing energy expenditure and reducing appetite, exercise also favors the oxidation of dietary fat, which likely helps prevent weight re-gain. It is unclear whether this exercise-induced metabolic shift is due to changes in energy balance, or whether exercise imparts additional adaptations in the periphery that limit the storage and favor the oxidation of dietary fat. To answer this question, adipose tissue lipid metabolism and related gene expression were studied in obese rats following weight loss and during the first day of relapse to obesity. Mature, obese rats were weight-reduced for 2 weeks with or without daily treadmill exercise (EX). Rats were weight maintained for 6 weeks, followed by relapse on: (a) ad libitum low fat diet (LFD), (b) ad libitum LFD plus EX, or (c) a provision of LFD to match the positive energy imbalance of exercised, relapsing animals. 24 h retention of dietary- and de novo-derived fat were assessed directly using 14C palmitate/oleate and 3H20, respectively. Exercise decreased the size, but increased the number of adipocytes in both retroperitoneal (RP) and subcutaneous (SC) adipose depots, and prevented the relapse-induced increase in adipocyte size. Further, exercise decreased the expression of genes involved in lipid uptake (CD36 and LPL), de novo lipogenesis (FAS, ACC1), and triacylglycerol synthesis (MGAT and DGAT) in RP adipose during relapse following weight loss. This was consistent with the metabolic data, whereby exercise reduced retention of de novo-derived fat even when controlling for the positive energy imbalance. The decreased trafficking of dietary fat to adipose tissue with exercise was explained by reduced energy intake which attenuated energy imbalance during refeeding. Despite having decreased expression of lipogenic genes, the net retention of de novo-derived lipid was higher in both the RP and SC adipose of exercising

  1. Exercise Decreases Lipogenic Gene Expression in Adipose Tissue and Alters Adipocyte Cellularity during Weight Regain After Weight Loss.

    PubMed

    Giles, Erin D; Steig, Amy J; Jackman, Matthew R; Higgins, Janine A; Johnson, Ginger C; Lindstrom, Rachel C; MacLean, Paul S

    2016-01-01

    Exercise is a potent strategy to facilitate long-term weight maintenance. In addition to increasing energy expenditure and reducing appetite, exercise also favors the oxidation of dietary fat, which likely helps prevent weight re-gain. It is unclear whether this exercise-induced metabolic shift is due to changes in energy balance, or whether exercise imparts additional adaptations in the periphery that limit the storage and favor the oxidation of dietary fat. To answer this question, adipose tissue lipid metabolism and related gene expression were studied in obese rats following weight loss and during the first day of relapse to obesity. Mature, obese rats were weight-reduced for 2 weeks with or without daily treadmill exercise (EX). Rats were weight maintained for 6 weeks, followed by relapse on: (a) ad libitum low fat diet (LFD), (b) ad libitum LFD plus EX, or (c) a provision of LFD to match the positive energy imbalance of exercised, relapsing animals. 24 h retention of dietary- and de novo-derived fat were assessed directly using (14)C palmitate/oleate and (3)H20, respectively. Exercise decreased the size, but increased the number of adipocytes in both retroperitoneal (RP) and subcutaneous (SC) adipose depots, and prevented the relapse-induced increase in adipocyte size. Further, exercise decreased the expression of genes involved in lipid uptake (CD36 and LPL), de novo lipogenesis (FAS, ACC1), and triacylglycerol synthesis (MGAT and DGAT) in RP adipose during relapse following weight loss. This was consistent with the metabolic data, whereby exercise reduced retention of de novo-derived fat even when controlling for the positive energy imbalance. The decreased trafficking of dietary fat to adipose tissue with exercise was explained by reduced energy intake which attenuated energy imbalance during refeeding. Despite having decreased expression of lipogenic genes, the net retention of de novo-derived lipid was higher in both the RP and SC adipose of exercising

  2. Exercise Decreases Lipogenic Gene Expression in Adipose Tissue and Alters Adipocyte Cellularity during Weight Regain After Weight Loss.

    PubMed

    Giles, Erin D; Steig, Amy J; Jackman, Matthew R; Higgins, Janine A; Johnson, Ginger C; Lindstrom, Rachel C; MacLean, Paul S

    2016-01-01

    Exercise is a potent strategy to facilitate long-term weight maintenance. In addition to increasing energy expenditure and reducing appetite, exercise also favors the oxidation of dietary fat, which likely helps prevent weight re-gain. It is unclear whether this exercise-induced metabolic shift is due to changes in energy balance, or whether exercise imparts additional adaptations in the periphery that limit the storage and favor the oxidation of dietary fat. To answer this question, adipose tissue lipid metabolism and related gene expression were studied in obese rats following weight loss and during the first day of relapse to obesity. Mature, obese rats were weight-reduced for 2 weeks with or without daily treadmill exercise (EX). Rats were weight maintained for 6 weeks, followed by relapse on: (a) ad libitum low fat diet (LFD), (b) ad libitum LFD plus EX, or (c) a provision of LFD to match the positive energy imbalance of exercised, relapsing animals. 24 h retention of dietary- and de novo-derived fat were assessed directly using (14)C palmitate/oleate and (3)H20, respectively. Exercise decreased the size, but increased the number of adipocytes in both retroperitoneal (RP) and subcutaneous (SC) adipose depots, and prevented the relapse-induced increase in adipocyte size. Further, exercise decreased the expression of genes involved in lipid uptake (CD36 and LPL), de novo lipogenesis (FAS, ACC1), and triacylglycerol synthesis (MGAT and DGAT) in RP adipose during relapse following weight loss. This was consistent with the metabolic data, whereby exercise reduced retention of de novo-derived fat even when controlling for the positive energy imbalance. The decreased trafficking of dietary fat to adipose tissue with exercise was explained by reduced energy intake which attenuated energy imbalance during refeeding. Despite having decreased expression of lipogenic genes, the net retention of de novo-derived lipid was higher in both the RP and SC adipose of exercising

  3. Cellular zinc is required for intestinal epithelial barrier maintenance via the regulation of claudin-3 and occludin expression.

    PubMed

    Miyoshi, Yuka; Tanabe, Soichi; Suzuki, Takuya

    2016-07-01

    Intracellular zinc is required for a variety of cell functions, but its precise roles in the maintenance of the intestinal tight junction (TJ) barrier remain unclear. The present study investigated the essential roles of intracellular zinc in the preservation of intestinal TJ integrity and the underlying molecular mechanisms. Depletion of intracellular zinc in both intestinal Caco-2 cells and mouse colons through the application of a cell-permeable zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) induced a disruption of the TJ barrier, as indicated by increased FITC-labeled dextran flux and decreased transepithelial electrical resistance. The TPEN-induced TJ disruption is associated with downregulation of two TJ proteins, occludin and claudin-3. Biotinylation of cell surface proteins revealed that the zinc depletion induced the proteolysis of occludin but not claudin-3. Occludin proteolysis was sensitive to the inhibition of calpain activity, and increased calpain activity was observed in the zinc-depleted cells. Although quantitative PCR analysis and promoter reporter assay have demonstrated that the zinc depletion-induced claudin-3 downregulation occurred at transcriptional levels, a site-directed mutation in the egr1 binding site in the claudin-3 promoter sequence induced loss of both the basal promoter activity and the TPEN-induced decreases. Reduced egr1 expression by a specific siRNA also inhibited claudin-3 expression and transepithelial electrical resistance maintenance in cells. This study shows that intracellular zinc has an essential role in the maintenance of the intestinal epithelial TJ barrier through regulation of occludin proteolysis and claudin-3 transcription. PMID:27151944

  4. Cellular and molecular determinants of all-trans retinoic acid sensitivity in breast cancer: Luminal phenotype and RARα expression

    PubMed Central

    Centritto, Floriana; Paroni, Gabriela; Bolis, Marco; Garattini, Silvio Ken; Kurosaki, Mami; Barzago, Maria Monica; Zanetti, Adriana; Fisher, James Neil; Scott, Mark Francis; Pattini, Linda; Lupi, Monica; Ubezio, Paolo; Piccotti, Francesca; Zambelli, Alberto; Rizzo, Paola; Gianni', Maurizio; Fratelli, Maddalena; Terao, Mineko; Garattini, Enrico

    2015-01-01

    Forty-two cell lines recapitulating mammary carcinoma heterogeneity were profiled for all-trans retinoic acid (ATRA) sensitivity. Luminal and ER+ (estrogen-receptor-positive) cell lines are generally sensitive to ATRA, while refractoriness/low sensitivity is associated with a Basal phenotype and HER2 positivity. Indeed, only 2 Basal cell lines (MDA-MB157 and HCC-1599) are highly sensitive to the retinoid. Sensitivity of HCC-1599 cells is confirmed in xenotransplanted mice. Short-term tissue-slice cultures of surgical samples validate the cell-line results and support the concept that a high proportion of Luminal/ER+ carcinomas are ATRA sensitive, while triple-negative (Basal) and HER2-positive tumors tend to be retinoid resistant. Pathway-oriented analysis of the constitutive gene-expression profiles in the cell lines identifies RARα as the member of the retinoid pathway directly associated with a Luminal phenotype, estrogen positivity and ATRA sensitivity. RARα3 is the major transcript in ATRA-sensitive cells and tumors. Studies in selected cell lines with agonists/antagonists confirm that RARα is the principal mediator of ATRA responsiveness. RARα over-expression sensitizes retinoid-resistant MDA-MB453 cells to ATRA anti-proliferative action. Conversely, silencing of RARα in retinoid-sensitive SKBR3 cells abrogates ATRA responsiveness. All this is paralleled by similar effects on ATRA-dependent inhibition of cell motility, indicating that RARα may mediate also ATRA anti-metastatic effects. We define gene sets of predictive potential which are associated with ATRA sensitivity in breast cancer cell lines and validate them in short-term tissue cultures of Luminal/ER+ and triple-negative tumors. In these last models, we determine the perturbations in the transcriptomic profiles afforded by ATRA. The study provides fundamental information for the development of retinoid-based therapeutic strategies aimed at the stratified treatment of breast cancer subtypes

  5. Calculating Formulas of Coefficient and Mean Neutron Exposure in the Exponential Expression of Neutron Exposure Distribution

    NASA Astrophysics Data System (ADS)

    Zhang, F. H.; Zhou, G. D.; Ma, K.; Ma, W. J.; Cui, W. Y.; Zhang, B.

    2015-11-01

    Present studies have shown that, in the main stages of the development and evolution of asymptotic giant branch (AGB) star s-process models, the distributions of neutron exposures in the nucleosynthesis regions can all be expressed by an exponential function ({ρ_{AGB}}(τ) = C/{τ_0}exp ( - τ/{τ_0})) in the effective range of values. However, the specific expressions of the proportional coefficient C and the mean neutron exposure ({τ_0}) in the formula for different models are not completely determined in the related literatures. Through dissecting the basic solving method of the exponential distribution of neutron exposures, and systematically combing the solution procedure of exposure distribution for different stellar models, the general calculating formulas as well as their auxiliary equations for calculating C and ({τ_0}) are reduced. Given the discrete distribution of neutron exposures ({P_k}), i.e. the mass ratio of the materials which have exposed to neutrons for (k) ((k = 0, 1, 2 \\cdots )) times when reaching the final distribution with respect to the materials of the He intershell, (C = - {P_1}/ln R), and ({τ_0} = - Δ τ /ln R) can be obtained. Here, (R) expresses the probability that the materials can successively experience neutron irradiation for two times in the He intershell. For the convective nucleosynthesis model (including the Ulrich model and the ({}^{13}{C})-pocket convective burning model), (R) is just the overlap factor r, namely the mass ratio of the materials which can undergo two successive thermal pulses in the He intershell. And for the (^{13}{C})-pocket radiative burning model, (R = sumlimits_{k = 1}^∞ {{P_k}} ). This set of formulas practically give the corresponding relationship between C or ({τ_0}) and the model parameters. The results of this study effectively solve the problem of analytically calculating the distribution of neutron exposures in the low-mass AGB star s-process nucleosynthesis model of (^{13}{C

  6. Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines.

    PubMed

    Zhu, Cui; Chen, Zhuang; Jiang, Zongyong

    2016-08-29

    Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs) represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1-11) have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes), goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines.

  7. Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines

    PubMed Central

    Zhu, Cui; Chen, Zhuang; Jiang, Zongyong

    2016-01-01

    Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs) represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1–11) have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes), goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines. PMID:27589719

  8. Expression, Distribution and Role of Aquaporin Water Channels in Human and Animal Stomach and Intestines.

    PubMed

    Zhu, Cui; Chen, Zhuang; Jiang, Zongyong

    2016-01-01

    Stomach and intestines are involved in the secretion of gastrointestinal fluids and the absorption of nutrients and fluids, which ensure normal gut functions. Aquaporin water channels (AQPs) represent a major transcellular route for water transport in the gastrointestinal tract. Until now, at least 11 AQPs (AQP1-11) have been found to be present in the stomach, small and large intestines. These AQPs are distributed in different cell types in the stomach and intestines, including gastric epithelial cells, gastric glands cells, absorptive epithelial cells (enterocytes), goblet cells and Paneth cells. AQP1 is abundantly distributed in the endothelial cells of the gastrointestinal tract. AQP3 and AQP4 are mainly distributed in the basolateral membrane of epithelial cells in the stomach and intestines. AQP7, AQP8, AQP10 and AQP11 are distributed in the apical of enterocytes in the small and large intestines. Although AQP-null mice displayed almost no phenotypes in gastrointestinal tracts, the alterations of the expression and localization of these AQPs have been shown to be associated with the pathology of gastrointestinal disorders, which suggests that AQPs play important roles serving as potential therapeutic targets. Therefore, this review provides an overview of the expression, localization and distribution of AQPs in the stomach, small and large intestine of human and animals. Furthermore, this review emphasizes the potential roles of AQPs in the physiology and pathophysiology of stomach and intestines. PMID:27589719

  9. Adolescence methylphenidate treatment in a rodent model of attention deficit/hyperactivity disorder: dopamine transporter function and cellular distribution in adulthood.

    PubMed

    Somkuwar, Sucharita S; Darna, Mahesh; Kantak, Kathleen M; Dwoskin, Linda P

    2013-07-15

    Attention deficit/hyperactivity disorder (ADHD) is attributed to dysfunction of the prefrontal cortex. Methylphenidate, an inhibitor of dopamine and norepinephrine transporters (DAT and NET, respectively), is a standard treatment for ADHD. The Spontaneously Hypertensive Rat (SHR) is a well-established animal model of ADHD. Our previous results showed that methylphenidate treatment in adolescent SHR enhanced cocaine self-administration during adulthood, and alterations in DAT function in prefrontal cortex play a role in this response. Importantly, prefrontal cortex subregions, orbitofrontal cortex (OFC) and medial prefrontal cortex (mPFC), have been shown to have distinct roles in ADHD and cocaine self-administration. In the current study, SHR, Wistar-Kyoto (WKY) and Wistar (WIS) rats received a therapeutically relevant dose of methylphenidate (1.5mg/kg, p.o.) or vehicle during adolescence and then OFC and mPFC DAT function and cellular expression were assessed during adulthood. In both OFC and mPFC, no strain differences in Vmax or Km for dopamine uptake into synaptosomes were found between vehicle-treated SHR, WKY and WIS. Methylphenidate increased DAT Vmax in SHR mPFC and decreased DAT Vmax in WKY OFC. Also, methylphenidate decreased DAT Km in WIS OFC. Further, methylphenidate did not alter DAT cellular localization, indicating that methylphenidate treatment during adolescence regulated DAT function in SHR mPFC in a trafficking-independent manner. Thus, the increase in mPFC DAT function was an SHR-specific long term consequence of methylphenidate treatment during adolescence, which may be responsible for the treatment-induced alterations in behavior including the observed increases in cocaine self-administration.

  10. Analysis of expression, cellular localization, and function of three inhibitors of apoptosis (IAPs) from Litopenaeus vannamei during WSSV infection and in regulation of antimicrobial peptide genes (AMPs).

    PubMed

    Wang, Pei-Hui; Wan, Ding-Hui; Gu, Zhi-Hua; Qiu, Wei; Chen, Yong-Gui; Weng, Shao-Ping; Yu, Xiao-Qiang; He, Jian-Guo

    2013-01-01

    Inhibitors of apoptosis (IAPs) play important roles in apoptosis and NF-κB activation. In this study, we cloned and characterized three IAPs (LvIAP1-3) from the Pacific white shrimp, Litopenaeusvannamei. LvIAP1-3 proteins shared signature domains and exhibited significant similarities with other IAP family proteins. The tissue distributions of LvIAP1-3 were studied. The expression of LvIAP1-3 was induced in the muscle after white spot syndrome virus (WSSV) infection. LvIAP1 expression in the gill, hemocytes, hepatopancreas, and intestine was responsive to WSSV and Vibrioalginolyticus infections. LvIAP2 expression in the gill, hemocytes, and hepatopancreas was also responsive to WSSV infection. The expression of LvIAP3 in the gill, hemocytes, and intestine was reduced after V. alginolyticus infection. When overexpressed in Drosophila S2 cells, GFP labeled-LvIAP2 was distributed in the cytoplasm and appeared as speck-like aggregates in the nucleus. Both LvIAP1 and LvIAP3 were widely distributed throughout the cytoplasm and nucleus. The expression of LvIAP1, LvIAP2, and LvIAP3 was significantly knocked down by dsRNA-mediated gene silencing. In the gill of LvIAP1- or LvIAP3-silenced shrimp, the expression of WSSV VP28 was significantly higher than that of the dsGFP control group, suggesting that LvIAP1 and LvIAP3 may play protective roles in host defense against WSSV infection. Intriguingly, the LvIAP2-silenced shrimp all died within 48 hours after dsLvIAP2 injection. In the hemocytes of LvIAP2-silenced shrimps, the expression of antimicrobial peptide genes (AMPs), including Penaeidins, lysozyme, crustins, Vibriopenaeicidae-induced cysteine and proline-rich peptides (VICPs), was significantly downregulated, while the expression of anti-lipopolysaccharide factors (ALFs) was upregulated. Moreover, LvIAP2 activated the promoters of the NF-κB pathway-controlled AMPs, such as shrimp Penaeidins and Drosophila drosomycin and attacin A, in Drosophila S2 cells. Taken together

  11. The cellular distribution of Na+/H+ exchanger regulatory factor 1 is determined by the PDZ-I domain and regulates the malignant progression of breast cancer

    PubMed Central

    Du, Guifang; Gu, Yanan; Hao, Chengcheng; Yuan, Zhu; He, Junqi; Jiang, Wen G.; Cheng, Shan

    2016-01-01

    The oncogenic role of ectopic expression of Na+/H+ exchanger regulatory factor 1 (NHERF1) was recently suggested. Here, we show that NHERF1 was upregulated in high grades compared with low grades. Increased NHERF1 expression was correlated with poor prognosis and poor survival. NHERF1 expression was higher in the nucleus of cancer cells than in contiguous non- mammary epithelial cells. A novel mutation, namely NHERF1 Y24S, was identified in human breast cancer tissues and shown to correspond to a conserved residue in the PDZ-I domain of NHERF1. Truncation and mutation of the PDZ-I domain of NHERF1 increased the nuclear distribution of the NHERF1 protein, and this redistribution was associated with the malignant phenotype of breast cancer cells, including growth, migration, and adhesion. The present results suggest a role for NHERF1 in the progression of breast cancer mediated by the nuclear distribution of the NHERF1 protein, as determined by the truncation or key site mutation of the PDZ-I domain. PMID:27097111

  12. No effect of sustained systemic growth retardation on the distribution of Reelin-expressing interneurons in the neuron-producing hippocampal dentate gyrus in rats.

    PubMed

    Ohishi, Takumi; Wang, Liyun; Ogawa, Bunichiro; Fujisawa, Kenichi; Taniai, Eriko; Hayashi, Hitomi; Mitsumori, Kunitoshi; Shibutani, Makoto

    2010-12-01

    Reelin signaling plays a role in neuronal migration and positioning during brain development. To clarify the effect of systemic growth retardation on the distribution of Reelin-expressing interneurons in the hilus of the hippocampal dentate gyrus, pregnant rats were fed a synthetic diet with either a normal (20% casein) or low (10% casein) protein concentration from gestational day 10 to postnatal day (PND) 21 at weaning. Male offspring were immunohistochemically examined at PND 21 and on PND 77. Protein-restricted offspring displayed systemic growth retardation through PND 77 and had decreased absolute brain weights and an increased number of external granular cells in the cerebellar cortex, suggestive of retarded brain growth at weaning. However, maternal protein restriction did not change the cellular distribution of immunoreactivity for Reelin, Calbindin-D-28K, or glutamic acid decarboxylase 67 or of NeuN-positive postmitotic neurons in the dentate hilus either at PND 21 or PND 77, which suggests that the population of γ-aminobutyric acid-ergic interneurons involving synthesis of Reelin was not affected. Furthermore, as well as the distribution of hilar neurons expressing neurogenesis-related FoxG1, cell proliferation and apoptosis in the subgranular zone were unaffected through PND 77. These results suggest that systemic growth retardation caused by maternal protein restriction does not affect neuronal migration and postnatal neurogenesis of the dentate gyrus resulting in unaltered distribution of Reelin-synthesizing interneurons.

  13. Pyrroloquinoline Quinone Induces Cancer Cell Apoptosis via Mitochondrial-Dependent Pathway and Down-Regulating Cellular Bcl-2 Protein Expression.

    PubMed

    Min, Zhihui; Wang, Lingyan; Jin, Jianjun; Wang, Xiangdong; Zhu, Bijun; Chen, Hao; Cheng, Yunfeng

    2014-01-01

    Pyrroloquinoline quinone (PQQ) has been reported as a promising agent that might contribute to tumor cell apoptosis and death, yet little is known on its mechanisms. In current study, the effect of PQQ on cell proliferation and mitochondrial-dependent apoptosis were examined in 3 solid tumor cell lines (A549, Neuro-2A and HCC-LM3). PQQ treatment at low to medium dosage exhibited potent anti-tumor activity on A549 and Neuro-2A cells, while had comparably minimal impact on the viabilities of 2 human normal cell lines (HRPTEpiC and HUVEC). The apoptosis of the 3 tumor cell lines induced by PQQ were increased in a concentration-dependent manner, which might be attributed to the accumulation of intracellular reactive oxygen species (ROS), decline in ATP levels and dissipation of mitochondrial membrane potential (MMP), in conjunction with down-regulation of Bcl-2 protein expression, up-regulation of activated caspase-3, and disturbed phosphorylated MAPK protein levels. PQQ induced tumor cells apoptosis was significantly alleviated by pan-caspase inhibitor Z-VAD-FMK. The present work highlights the potential capability of PQQ as an anti-tumor agent with low toxicity towards normal cells through activating mitochondrial-dependent apoptosis pathways, and warrants its development for cancer therapy.

  14. Cholesterol-induced changes of type VIII collagen expression and distribution in carotid arteries of rabbit.

    PubMed

    Plenz, G; Dorszewski, A; Völker, W; Ko, Y S; Severs, N J; Breithardt, G; Robenek, H

    1999-10-01

    Lipoproteins play a major role in cardiovascular disease and atherosclerosis. In the vascular wall, they strongly influence the organization of extracellular matrix. The present study set out to investigate the changes in the extracellular matrix of the vessel wall induced by atherogenic diet, focusing on type VIII collagen, a vascular collagen that has not previously been investigated in detail. The influence of cholesterol diet on the expression, distribution, and deposition of type VIII collagen was examined in carotid arteries of New Zealand White rabbits. Carotid arteries of rabbits receiving diet supplemented with 1% cholesterol for 6 weeks and those on the same regimen followed by normal chow for 1 day, 10 days, 5 weeks, and 12 weeks were studied and compared with controls not exposed to the cholesterol diet. Carotid arteries of normocholesterolemic rabbits contained type VIII collagen-expressing cells in all layers, with focal accumulations of expressing cells in the subendothelial areas, the outer medial zone, and the adventitia. In response to cholesterol diet, type VIII collagen synthesis was reduced in media and adventitia and the distribution patterns changed. Expressing cells were found predominantly in the endothelium, and type VIII collagen accumulated in the intimal space. Immunogold labeling for electron microscopy revealed that type VIII collagen in the intima is associated with microfibrils extending from the internal elastic lamina. Withdrawal of cholesterol resulted in reestablishment of the normal distribution pattern. Northern and Western blot analyses supported the immunoconfocal and in situ hybridization data, demonstrating decreased type VIII collagen expression in response to cholesterol diet and progressive recovery to normal levels with time after withdrawal of cholesterol. Our study demonstrates that type VIII collagen is modulated in the presence of cholesterol. The data indicate that type VIII collagen is specifically remodeled

  15. Regulation of gene expression by NFAT transcription factors in hibernating ground squirrels is dependent on the cellular environment.

    PubMed

    Zhang, Yichi; Storey, Kenneth B

    2016-09-01

    Calcineurin is a calmodulin-stimulated phosphatase that regulates the nuclear translocation of nuclear factor of activated T cell (NFAT) c1-4 through dephosphorylation. We believe that this mechanism plays various roles in the remodeling and maintenance of Ictidomys tridecemlineatus skeletal muscle. During hibernation, bouts of torpor and arousal take place, and squirrels do not lose muscle mass despite being inactive. Protein expression of Ca(2+) signaling proteins were studied using immunoblotting. A DNA-protein interaction ELISA technique was created to test the binding of NFATs in the nucleus to DNA probes containing the NFAT response element under environmental conditions reflective of those during hibernation. Calcineurin protein levels increased by 3.08-fold during torpor (compared to euthermic control), whereas calpain1 levels also rose by 3.66-fold during torpor. Calmodulin levels were elevated upon entering torpor. NFATc4 binding to DNA showed a 1.4-fold increase during torpor, and we found that this binding was further enhanced when 600 nM of Ca(2+) was supplemented. We also found that decreasing the temperature of ELISAs resulted in progressive decreases in the binding of NFATs c1, c3, and c4 to DNA. In summary, calmodulin and calpain1 appear to activate calcineurin and NFATc4 during torpor. NFAT binding to target promoters is affected by intranuclear [Ca(2+)] and environmental temperatures. Therefore, Ca(2+) signaling and temperature changes play key roles in regulation of the NFAT-calcineurin pathway in skeletal muscle of hibernating 13-lined ground squirrels over the torpor-arousal cycle, and they may contribute to the avoidance of disuse-induced muscle atrophy that occurs naturally in these animals.

  16. Regulation of gene expression by NFAT transcription factors in hibernating ground squirrels is dependent on the cellular environment.

    PubMed

    Zhang, Yichi; Storey, Kenneth B

    2016-09-01

    Calcineurin is a calmodulin-stimulated phosphatase that regulates the nuclear translocation of nuclear factor of activated T cell (NFAT) c1-4 through dephosphorylation. We believe that this mechanism plays various roles in the remodeling and maintenance of Ictidomys tridecemlineatus skeletal muscle. During hibernation, bouts of torpor and arousal take place, and squirrels do not lose muscle mass despite being inactive. Protein expression of Ca(2+) signaling proteins were studied using immunoblotting. A DNA-protein interaction ELISA technique was created to test the binding of NFATs in the nucleus to DNA probes containing the NFAT response element under environmental conditions reflective of those during hibernation. Calcineurin protein levels increased by 3.08-fold during torpor (compared to euthermic control), whereas calpain1 levels also rose by 3.66-fold during torpor. Calmodulin levels were elevated upon entering torpor. NFATc4 binding to DNA showed a 1.4-fold increase during torpor, and we found that this binding was further enhanced when 600 nM of Ca(2+) was supplemented. We also found that decreasing the temperature of ELISAs resulted in progressive decreases in the binding of NFATs c1, c3, and c4 to DNA. In summary, calmodulin and calpain1 appear to activate calcineurin and NFATc4 during torpor. NFAT binding to target promoters is affected by intranuclear [Ca(2+)] and environmental temperatures. Therefore, Ca(2+) signaling and temperature changes play key roles in regulation of the NFAT-calcineurin pathway in skeletal muscle of hibernating 13-lined ground squirrels over the torpor-arousal cycle, and they may contribute to the avoidance of disuse-induced muscle atrophy that occurs naturally in these animals. PMID:27344571

  17. Biochemical and cellular characterization of Helicobacter pylori RecA, a protein with high-level constitutive expression.

    PubMed

    Orillard, Emilie; Radicella, J Pablo; Marsin, Stéphanie

    2011-12-01

    Helicobacter pylori is a bacterial pathogen colonizing half of the world's human population. It has been implicated in a number of gastric diseases, from asymptomatic gastritis to cancer. It is characterized by an amazing genetic variability that results from high mutation rates and efficient DNA homologous recombination and transformation systems. Here, we report the characterization of H. pylori RecA (HpRecA), a protein shown to be involved in DNA repair, transformation, and mouse colonization. The biochemical characterization of the purified recombinase reveals activities similar to those of Escherichia coli RecA (EcRecA). We show that in H. pylori, HpRecA is present in about 80,000 copies per cell during exponential growth and decreases to about 50,000 copies in stationary phase. The amount of HpRecA remains unchanged after induction of DNA lesions, suggesting that HpRecA is always expressed at a high level in order to repair DNA damage or facilitate recombination. We performed HpRecA localization analysis by adding a Flag tag to the protein, revealing two different patterns of localization. During exponential growth, RecA-Flag presents a diffuse pattern, overlapping with the DAPI (4',6-diamidino-2-phenylindole) staining of DNA, whereas during stationary phase, the protein is present in more defined areas devoid of DAPI staining. These localizations are not affected by inactivation of competence or DNA recombination genes. Neither UV irradiation nor gamma irradiation modified HpRecA localization, suggesting the existence of a constitutive DNA damage adaptation system. PMID:21949074

  18. The expression and distribution of α-Gal gene in various species ocular surface tissue

    PubMed Central

    Shao, Yi; Yu, Yao; Pei, Chong-Gang; Qu, Yangluowa; Gao, Gui-Ping; Yang, Ji-Lin; Zhou, Qiong; Yang, Lu; Liu, Qiu-Ping

    2012-01-01

    AIM To examine the α-Gal gene expression and distribution in the different species/genus and developing phase animal ocular surface tissue. METHODS α-Gal binding assay were carried out on various animal eye sections. Photograph, slit-lamp observation on various eye showed normal corneal transparence. RESULTS A strong α-Gal expression in invertebrates and some vertebrates ocular tissue, but no α-Gal binding in birds, fish and mammal. α-Gal expression change in the development of mice ocular surface tissue (except sclera) and display genus dependency in the different murine ocular surface tissue. CONCLUSION This study identified specific α-Gal epitopes binding area in the ocular surface of several species and may solve the problem that naive ocular surface may be used as natural α-Gal gene knockout model/high risk immunologic rejection model or ocular surface scaffold material. PMID:23166862

  19. Plant simple sequence repeats: distribution, variation, and effects on gene expression.

    PubMed

    Sharopova, Natalya

    2008-02-01

    Genome-wide simple sequence repeat (SSR) information was analyzed together with functional annotations of Arabidopsis genes and public gene expression data for Arabidopsis and rice. Analysis of more than 15,000 Arabidopsis and more than 16,000 rice SSRs indicated that SSRs may affect the expression of hundreds of genes. Data from experiments on DNA methylation, histone acetylation, and transcript turnover suggest that SSRs may affect gene expression at transcriptional and posttranscriptional levels. Members of some functional groups were shown to be enriched with SSRs and often contained similar but non-homologous repeats within the same gene regions. In addition, the distribution of perfect and imperfect SSRs in some Arabidopsis, maize, and rice genes was used to demonstrate how two-level control of SSR variation may contribute to protein evolution.

  20. Differences in the Expression and Distribution of Flotillin-2 in Chick, Mice and Human Muscle Cells

    PubMed Central

    Possidonio, Ana Claudia Batista; Soares, Carolina Pontes; Portilho, Débora Morueco; Midlej, Victor; Benchimol, Marlene; Butler-Browne, Gillian; Costa, Manoel Luis; Mermelstein, Claudia

    2014-01-01

    Myoblasts undergo a series of changes in the composition and dynamics of their plasma membranes during the initial steps of skeletal muscle differentiation. These changes are crucial requirements for myoblast fusion and allow the formation of striated muscle fibers. Membrane microdomains, or lipid rafts, have been implicated in myoblast fusion. Flotillins are scaffold proteins that are essential for the formation and dynamics of lipid rafts. Flotillins have been widely studied over the last few years, but still little is known about their role during skeletal muscle differentiation. In the present study, we analyzed the expression and distribution of flotillin-2 in chick, mice and human muscle cells grown in vitro. Primary cultures of chick myogenic cells showed a decrease in the expression of flotillin-2 during the first 72 hours of muscle differentiation. Interestingly, flotillin-2 was found to be highly expressed in chick myogenic fibroblasts and weakly expressed in chick myoblasts and multinucleated myotubes. Flotillin-2 was distributed in vesicle-like structures within the cytoplasm of chick myogenic fibroblasts, in the mouse C2C12 myogenic cell line, and in neonatal human muscle cells. Cryo-immunogold labeling revealed the presence of flotillin-2 in vesicles and in Golgi stacks in chick myogenic fibroblasts. Further, brefeldin A induced a major reduction in the number of flotillin-2 containing vesicles which correlates to a decrease in myoblast fusion. These results suggest the involvement of flotillin-2 during the initial steps of skeletal myogenesis. PMID:25105415

  1. Differences in the expression and distribution of flotillin-2 in chick, mice and human muscle cells.

    PubMed

    Possidonio, Ana Claudia Batista; Soares, Carolina Pontes; Portilho, Débora Morueco; Midlej, Victor; Benchimol, Marlene; Butler-Browne, Gillian; Costa, Manoel Luis; Mermelstein, Claudia

    2014-01-01

    Myoblasts undergo a series of changes in the composition and dynamics of their plasma membranes during the initial steps of skeletal muscle differentiation. These changes are crucial requirements for myoblast fusion and allow the formation of striated muscle fibers. Membrane microdomains, or lipid rafts, have been implicated in myoblast fusion. Flotillins are scaffold proteins that are essential for the formation and dynamics of lipid rafts. Flotillins have been widely studied over the last few years, but still little is known about their role during skeletal muscle differentiation. In the present study, we analyzed the expression and distribution of flotillin-2 in chick, mice and human muscle cells grown in vitro. Primary cultures of chick myogenic cells showed a decrease in the expression of flotillin-2 during the first 72 hours of muscle differentiation. Interestingly, flotillin-2 was found to be highly expressed in chick myogenic fibroblasts and weakly expressed in chick myoblasts and multinucleated myotubes. Flotillin-2 was distributed in vesicle-like structures within the cytoplasm of chick myogenic fibroblasts, in the mouse C2C12 myogenic cell line, and in neonatal human muscle cells. Cryo-immunogold labeling revealed the presence of flotillin-2 in vesicles and in Golgi stacks in chick myogenic fibroblasts. Further, brefeldin A induced a major reduction in the number of flotillin-2 containing vesicles which correlates to a decrease in myoblast fusion. These results suggest the involvement of flotillin-2 during the initial steps of skeletal myogenesis.

  2. A genetically engineered live-attenuated simian-human immunodeficiency virus that co-expresses the RANTES gene improves the magnitude of cellular immunity in rhesus macaques

    SciTech Connect

    Shimizu, Yuya; Inaba, Katsuhisa; Kaneyasu, Kentaro; Ibuki, Kentaro; Himeno, Ai; Okoba, Masashi; Goto, Yoshitaka; Hayami, Masanori; Miura, Tomoyuki; Haga, Takeshi . E-mail: a0d518u@cc.miyazaki-u.ac.jp

    2007-04-25

    Regulated-on-activation-normal-T-cell-expressed-and-secreted (RANTES), a CC-chemokine, enhances antigen-specific T helper (Th) type-1 responses against HIV-1. To evaluate the adjuvant effects of RANTES against HIV vaccine candidate in SHIV-macaque models, we genetically engineered a live-attenuated SHIV to express the RANTES gene (SHIV-RANTES) and characterized the virus's properties in vivo. After the vaccination, the plasma viral loads were same in the SHIV-RANTES-inoculated monkeys and the parental nef-deleted SHIV (SHIV-NI)-inoculated monkeys. SHIV-RANTES provided some immunity in monkeys by remarkably increasing the antigen-specific CD4{sup +} Th cell-proliferative response and by inducing an antigen-specific IFN-{gamma} ELISpot response. The magnitude of the immunity in SHIV-RANTES-immunized animals, however, failed to afford greater protection against a heterologous pathogenic SHIV (SHIV-C2/1) challenge compared to control SHIV-NI-immunized animals. SHIV-RANTES immunized monkeys, elicited robust cellular CD4{sup +} Th responses and IFN-{gamma} ELISpot responses after SHIV-C2/1 challenge. These findings suggest that the chemokine RANTES can augment vaccine-elicited, HIV-specific CD4{sup +} T cell responses.

  3. La Autoantigen Induces Ribosome Binding Protein 1 (RRBP1) Expression through Internal Ribosome Entry Site (IRES)-Mediated Translation during Cellular Stress Condition

    PubMed Central

    Gao, Wenqing; Li, Qi; Zhu, Ruiyu; Jin, Jian

    2016-01-01

    The function of ribosome binding protein 1 (RRBP1) is regulating the transportation and secretion of some intracellular proteins in mammalian cells. Transcription of RRBP1 is induced by various cytokines. However, few studies focused on the process of RRPB1 mRNA translation. The RRBP1 mRNA has a long 5′ untranslated region that potentially formed a stable secondary structure. In this study, we show that the 5′ UTR of RRBP1 mRNA contains an internal ribosome entry site (IRES). Moreover, the RRBP1 expression is induced by chemotherapeutic drug paclitaxel or adriamycin in human hepatocellular carcinoma cells and accompanied with the increased expression of La autoantigen (La), which binds to RRBP1 IRES element and facilitates translation initiation. Interestingly, we found IRES-mediated RRBP1 translation is also activated during serum-starvation condition which can induce cytoplasmic localization of La. After mapping the entire RRBP1 5′ UTR, we determine the core IRES activity is located between nt-237 and -58. Furthermore, two apical GARR loops within the functional RRBP1 IRES elements may be important for La binding. These results strongly suggest an important role for IRES-dependent translation of RRBP1 mRNA in hepatocellular carcinoma cells during cellular stress conditions. PMID:27447629

  4. Inhibition of MDR1 gene expression and enhancing cellular uptake for effective colon cancer treatment using dual-surface–functionalized nanoparticles

    PubMed Central

    Xiao, Bo; Zhang, Mingzhen; Viennois, Emilie; Zhang, Yuchen; Wei, Na; Baker, Mark T.; Jung, Yunjin; Merlin, Didier

    2015-01-01

    Nanomedicine options for colon cancer therapy have been limited by the lack of suitable carriers capable of delivering sufficient drug into tumors to cause lethal toxicity. To circumvent this limitation, we fabricated a camptothecin (CPT)-loaded poly(lactic-co-glycolic acid) nanoparticle (NP) with dual-surface functionalization—Pluronic F127 and chitosan—for inhibiting multi-drug resistant gene 1 (MDR1) expression and enhancing tumor uptake. The resultant spherical NPs-P/C had a desirable particle size (~268 nm), slightly positive zeta-potential, and the ability to efficiently down-regulate the expression of MDR1. In vitro cytotoxicity tests revealed that the 24 and 48 h IC50 values of NPs-P/C1 were 2.03 and 0.67 µM, respectively, which were much lower than those for free CPT and other NPs. Interestingly, NPs-P/C1 showed the highest cellular uptake efficiency (approximately 85.5%) among the different drug formulations. Most importantly, treatment of colon tumor-bearing mice with various drug formulations confirmed that the introduction of Pluronic F127 and chitosan to the NP surface significantly enhanced the therapeutic efficacy of CPT, induced tumor cell apoptosis, and reduced systemic toxicity. Collectively, these findings suggest that our one-step–fabricated, dual-surface–functionalized NPs may hold promise as a readily scalable and effective drug carrier with clinical potential in colon cancer therapy. PMID:25701040

  5. Primary structure and developmental expression of Bufo arenarum cellular nucleic acid-binding protein: changes in subcellular localization during early embryogenesis.

    PubMed

    Armas, P; Cabada, M O; Calcaterra, N B

    2001-02-01

    A Bufo arenarum cellular nucleic acid-binding protein (bCNBP) full-length cDNA was cloned. bCNBP is a 19.4 kDa protein containing seven CCHC zinc finger motifs, an RGG box and a Ser-rich region. Amino acid comparisons showed high values of homology in vertebrates and smaller values in insects or inferior eukaryotes. Northern blot analysis during oogenesis and early development revealed two transcripts with different expressions of pattern behavior. One of them is present in all stages analyzed, whereas the other is only detected from the beginning of zygotic transcription. Immunocytochemistry assays carried out on sections of ovary and early embryos showed that there was no specific staining of previtellogenic oocytes. In early vitellogenic oocytes, in oocytes at stages V/VI and in embryos at early blastula stage, reaction was observed inside the cytoplasm. At mid-blastula stage, CNBP was mainly detected in the epiblast. At the late gastrula stage, two layers of cells were stained in the archenteron roof, in which the internal one presented as strong staining. Nuclei in this layer were stained even stronger than the cytoplasm. Changes in mRNA expression patterns, accompanied by changes in subcellular localization, suggest that CNBP might interact with both nuclear and cytoplasmic nucleic acids.

  6. La Autoantigen Induces Ribosome Binding Protein 1 (RRBP1) Expression through Internal Ribosome Entry Site (IRES)-Mediated Translation during Cellular Stress Condition.

    PubMed

    Gao, Wenqing; Li, Qi; Zhu, Ruiyu; Jin, Jian

    2016-01-01

    The function of ribosome binding protein 1 (RRBP1) is regulating the transportation and secretion of some intracellular proteins in mammalian cells. Transcription of RRBP1 is induced by various cytokines. However, few studies focused on the process of RRPB1 mRNA translation. The RRBP1 mRNA has a long 5' untranslated region that potentially formed a stable secondary structure. In this study, we show that the 5' UTR of RRBP1 mRNA contains an internal ribosome entry site (IRES). Moreover, the RRBP1 expression is induced by chemotherapeutic drug paclitaxel or adriamycin in human hepatocellular carcinoma cells and accompanied with the increased expression of La autoantigen (La), which binds to RRBP1 IRES element and facilitates translation initiation. Interestingly, we found IRES-mediated RRBP1 translation is also activated during serum-starvation condition which can induce cytoplasmic localization of La. After mapping the entire RRBP1 5' UTR, we determine the core IRES activity is located between nt-237 and -58. Furthermore, two apical GARR loops within the functional RRBP1 IRES elements may be important for La binding. These results strongly suggest an important role for IRES-dependent translation of RRBP1 mRNA in hepatocellular carcinoma cells during cellular stress conditions. PMID:27447629

  7. Direct combinatorial interaction between a herpes simplex virus regulatory protein and a cellular octamer-binding factor mediates specific induction of virus immediate-early gene expression.

    PubMed Central

    O'Hare, P; Goding, C R; Haigh, A

    1988-01-01

    We provide evidence for a novel mechanism of transcriptional regulation in which the immediate-early (IE) transactivating protein of herpes simplex virus, Vmw65, is assembled into a specific DNA-binding complex together with a cellular octamer-binding factor (TRF). The assembly of Vmw65/TRF complex requires not only the core TRF recognition site, but also flanking sequences which are dispensable for TRF binding alone. We show from functional analyses that TRF binding by a motif is required but not sufficient to confer induction on a heterologous promoter, and it is the ability of the motif to allow TRF/Vmw65 complex assembly which correlates with functional activity. Thus, for the induction of HSV IE expression, Vmw65 forms a complex with TRF by recognition of the specific subset of appropriately flanked TRF binding sites present in each of the IE genes. This mechanism may provide a paradigm for the selective utilization of the same transcription factor in differential gene expression. Images PMID:2854058

  8. Cellular Effect of High Doses of Silica-Coated Quantum Dot Profiled with High Throughput Gene Expression Analysis and High Content Cellomics Measurements

    PubMed Central

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

    2009-01-01

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

  9. Tissue distribution and developmental changes of ghrelin and GOAT expression in broiler chickens during embryogenesis.

    PubMed

    Ma, Jinyou; Yu, Yan; Xue, Jiajia; Ou, Changbo; Mo, Haizhen; Liu, Xingyou

    2015-03-01

    Ghrelin plays important roles, such as regulating growth hormone release and energy metabolism, but little is known about its developmental changes in the proventriculi of chicken embryos. This study was designed to elucidate the distributions and developmental changes of ghrelin and ghrelin-O-acyltransferase (GOAT) expression in broiler embryos using qRT-PCR and immunohistochemistry. Our results demonstrated the following: (1) on E18, ghrelin and GOAT are ubiquitously expressed in every tissue examined. The expression level of ghrelin mRNA was the highest in the proventriculus, reaching a level that was 50-fold higher than that in the hypothalamus, while GOAT mRNA expression was low in the proventriculus and it was only 67.6% as high as that of hypothalamus; (2) ghrelin and GOAT mRNA expression were detected in the proventriculus on E9, but only at 1.9% and 1.7% of the level expressed on E18, respectively, and their expression levels increased rapidly from E18 to E21. There was similar developmental pattern in the ghrelin and GOAT mRNA expression; and (3) ghrelin-immunopositive cells were first detected in the proventriculus on E15, were located only in the compound tubular glands of the proventriculus, and were of the closed-cell type. The density of ghrelin-immunopositive cells increased significantly from E15 to E21. These results suggest that ghrelin may be an important regulating factor that plays a vital role during the development of chicken embryos. PMID:25449181

  10. Effect of Carbon Distribution During the Microstructure Evolution of Dual-Phase Steels Studied Using Cellular Automata, Genetic Algorithms, and Experimental Strategies

    NASA Astrophysics Data System (ADS)

    Halder, Chandan; Karmakar, Anish; Hasan, Sk. Md.; Chakrabarti, Debalay; Pietrzyk, Maciej; Chakraborti, Nirupam

    2016-09-01

    The development of ferrite-martensite dual-phase microstructures by cold-rolling and intercritical annealing of 0.06 wt pct carbon steel was systematically studied using a dilatometer for two different heating rates (1 and 10 K/s). A step quenching treatment has been designed to develop dual-phase structures having a similar martensite fraction for two different heating rates. An increase in heating rate seemed to refine the ferrite grain size, but it increased the size and spacing of the martensitic regions. As a result, the strength of the steel increased with heating rate; however, the formability was affected. It has been concluded that the distribution of C during the annealing treatment of cold-rolled steel determines the size, distribution, and morphology of martensite, which ultimately influences the mechanical properties. Experimental detection of carbon distribution in austenite is difficult during annealing of the cold-rolled steel as the phase transformation occurs at a high temperature and C is an interstitial solute, which diffuses fast at that temperature. Therefore, a cellular automata (CA)-based phase transformation model is proposed in the present study for the prediction of C distribution in austenite during annealing of steel as the function of C content and heating rate. The CA model predicts that the carbon distribution in austenite becomes more inhomogeneous when the heating rate increases. In the CA model, the extent of carbon inhomogeneity is measured using a kernel averaging method for different orders of neighbors, which accounts for the different physical space during calculation. The obtained results reveal that the 10th order (covering 10-µm physical spaces around the cell of interest) is showing the maximum inhomogeneity of carbon and the same effect has been investigated and confirmed using auger electron spectroscopy (AES) for 0.06 wt pct carbon steel. Furthermore, the optimization of carbon homogeneity with respect to heating rate

  11. Mitigating effects of L-selenomethionine on low-dose iron ion radiation-induced changes in gene expression associated with cellular stress.

    PubMed

    Nuth, Manunya; Kennedy, Ann R

    2013-07-01

    Ionizing radiation associated with highly energetic and charged heavy (HZE) particles poses a danger to astronauts during space travel. The aim of the present study was to evaluate the patterns of gene expression associated with cellular exposure to low-dose iron ion irradiation, in the presence and absence of L-selenomethionine (SeM). Human thyroid epithelial cells (HTori-3) were exposed to low-dose iron ion (1 GeV/n) irradiation at 10 or 20 cGy with or without SeM pretreatment. The cells were harvested 6 and 16 h post-irradiation and analyzed by the Affymetrix U133Av2 gene chip arrays. Genes exhibiting a 1.5-fold expression cut-off and 5% false discovery rate (FDR) were considered statistically significant and subsequently analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) for pathway analysis. Representative genes were further validated by real-time RT-PCR. Even at low doses of radiation from iron ions, global genome profiling of the irradiated cells revealed the upregulation of genes associated with the activation of stress-related signaling pathways (ubiquitin-mediated proteolysis, p53 signaling, cell cycle and apoptosis), which occurred in a dose-dependent manner. A 24-h pretreatment with SeM was shown to reduce the radiation effects by mitigating stress-related signaling pathways and downregulating certain genes associated with cell adhesion. The mechanism by which SeM prevents radiation-induced transformation in vitro may involve the suppression of the expression of genes associated with stress-related signaling and certain cell adhesion events.

  12. Cellular expression and localization of DGKζ-interacting NAP1-like proteins in the brain and functional implications under hypoxic stress.

    PubMed

    Takahashi, Nobuya; Hozumi, Yasukazu; Tanaka, Toshiaki; Okada, Masashi; Iseki, Ken; Hayasaka, Kiyoshi; Goto, Kaoru

    2014-11-01

    Diacylglycerol kinase (DGK) catalyzes conversion of a lipid second messenger diacylglycerol to another messenger molecule phosphatidic acid. Consequently, DGK plays a pivotal role in cellular pathophysiology by regulating the levels of these two messengers. We reported previously that DGKζ translocates from the nucleus to cytoplasm in hippocampal neurons under ischemic/hypoxic stress. In addition, we also identified nucleosome assembly protein 1 (NAP1)-like proteins NAP1L1 and NAP1L4 as novel DGKζ-interacting partners using a proteomic approach and revealed that these NAP1-like proteins induce cytoplasmic translocation of DGKζ in overexpressed cells because NAP1-like proteins associate with the nuclear localization signal of DGKζ and block its nuclear import via importin α. In the present study, we examined whether NAP1-like proteins are expressed in the brain and whether the molecular interaction of DGKζ and NAP1-like proteins would be changed in the brain after hypoxic stress. Immunohistochemistry revealed that NAP1L1 and NAP1L4 are widely expressed in neurons and glial cells in the brain with some differences. After 3 days of transient whole-body hypoxic stress, DGKζ translocated from the nucleus to cytoplasm in hippocampal pyramidal neurons, whereas NAP1-like proteins remained in the cytoplasm. Contrary to our expectations, NAP1-like proteins showed no change in their expression levels. The molecular interaction between DGKζ and NAP1-like proteins was attenuated after hypoxic stress. These results suggest that DGKζ cytoplasmic translocation in neurons under hypoxic stress is regulated by some mechanism which differs from that mediated by NAP1-like proteins. PMID:24893663

  13. Bone morphogenetic protein 15 and growth differentiation factor 9 expression in the ovary of European sea bass (Dicentrarchus labrax): cellular localization, developmental profiles, and response to unilateral ovariectomy.

    PubMed

    García-López, Ángel; Sánchez-Amaya, María Isabel; Halm, Silke; Astola, Antonio; Prat, Francisco

    2011-12-01

    Vertebrate oocytes actively contribute to follicle development by secreting a variety of growth factors, among which bone morphogenetic protein 15 (BMP15/Bmp15) and growth differentiation factor 9 (GDF9/Gdf9) have been paid particular attention. In the present study, we describe the cellular localization, the developmental profiles, and the response to unilateral ovariectomy (a procedure implying the surgical removal of one of the ovaries) of protein and mRNA steady-state levels of Bmp15 and Gdf9 in the ovary of European sea bass, an important fish species for marine aquaculture industry. In situ hybridization and immunohistochemistry demonstrated that the oocyte is the main production site of Bmp15 and Gdf9 in European sea bass ovary. During oocyte development, Bmp15 protein expression started to be detected only from the lipid vesicle stage onwards but not in primary pre-vitellogenic (i.e. perinucleolar) oocytes as the bmp15 mRNA already did. Gdf9 protein and gdf9 mRNA expression were both detected in primary perinucleolar oocytes and followed similar decreasing patterns thereafter. Unilateral ovariectomy induced a full compensatory growth of the remaining ovary in the 2-month period following surgery (Á. García-López, M.I. Sánchez-Amaya, C.R. Tyler, F. Prat 2011). The compensatory growth elicited different changes in the expression levels of mRNA and protein of both factors, although the involvement of Bmp15 and Gdf9 in the regulatory network orchestrating such process remains unclear at present. Altogether, our results establish a solid base for further studies focused on elucidating the specific functions of Bmp15 and Gdf9 during primary and secondary oocyte growth in European sea bass.

  14. The Dopamine D2 Receptor Gene in Lamprey, Its Expression in the Striatum and Cellular Effects of D2 Receptor Activation

    PubMed Central

    Robertson, Brita; Huerta-Ocampo, Icnelia; Ericsson, Jesper; Stephenson-Jones, Marcus; Pérez-Fernández, Juan; Bolam, J. Paul; Diaz-Heijtz, Rochellys; Grillner, Sten

    2012-01-01

    All basal ganglia subnuclei have recently been identified in lampreys, the phylogenetically oldest group of vertebrates. Furthermore, the interconnectivity of these nuclei is similar to mammals and tyrosine hydroxylase-positive (dopaminergic) fibers have been detected within the input layer, the striatum. Striatal processing is critically dependent on the interplay with the dopamine system, and we explore here whether D2 receptors are expressed in the lamprey striatum and their potential role. We have identified a cDNA encoding the dopamine D2 receptor from the lamprey brain and the deduced protein sequence showed close phylogenetic relationship with other vertebrate D2 receptors, and an almost 100% identity within the transmembrane domains containing the amino acids essential for dopamine binding. There was a strong and distinct expression of D2 receptor mRNA in a subpopulation of striatal neurons, and in the same region tyrosine hydroxylase-immunoreactive synaptic terminals were identified at the ultrastructural level. The synaptic incidence of tyrosine hydroxylase-immunoreactive boutons was highest in a region ventrolateral to the compact layer of striatal neurons, a region where most striatal dendrites arborise. Application of a D2 receptor agonist modulates striatal neurons by causing a reduced spike discharge and a diminished post-inhibitory rebound. We conclude that the D2 receptor gene had already evolved in the earliest group of vertebrates, cyclostomes, when they diverged from the main vertebrate line of evolution (560 mya), and that it is expressed in striatum where it exerts similar cellular effects to that in other vertebrates. These results together with our previous published data (Stephenson-Jones et al. 2011, 2012) further emphasize the high degree of conservation of the basal ganglia, also with regard to the indirect loop, and its role as a basic mechanism for action selection in all vertebrates. PMID:22563388

  15. Nuclear factor-kappa B directs carcinoembryonic antigen-related cellular adhesion molecule 1 receptor expression in Neisseria gonorrhoeae-infected epithelial cells.

    PubMed

    Muenzner, Petra; Billker, Oliver; Meyer, Thomas F; Naumann, Michael

    2002-03-01

    The human-specific pathogen Neisseria gonorrhoeae expresses opacity-associated (Opa) protein adhesins that bind to various members of the carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) family. In this study, we have analyzed the mechanism underlying N. gonorrhoeae-induced CEACAM up-regulation in epithelial cells. Epithelial cells represent the first barrier for the microbial pathogen. We therefore characterized CEACAM expression in primary human ovarian surface epithelial (HOSE) cells and found that CEACAM1-3 (L, S) and CEACAM1-4 (L, S) splice variants mediate an increased Opa(52)-dependent gonoccocal binding to HOSE cells. Up-regulation of these CEACAM molecules in HOSE cells is a direct process that takes place within 2 h postinfection and depends on close contact between microbial pathogen and HOSE cells. N. gonorrhoeae-triggered CEACAM1 up-regulation involves activation of the transcription factor nuclear factor kappaB (NF-kappaB), which translocates as a p50/p65 heterodimer into the nucleus, and an NF-kappaB-specific inhibitory peptide inhibited CEACAM1-receptor up-regulation in N. gonorrhoeae-infected HOSE cells. Bacterial lipopolysaccharides did not induce NF-kappaB and CEACAM up-regulation, which corresponds to our findings that HOSE cells do not express toll-like receptor 4. The ability of N. gonorrhoeae to up-regulate its epithelial receptor CEACAM1 through NF-kappaB suggests an important mechanism allowing efficient bacterial colonization during the initial infection process. PMID:11751883

  16. Mitigating effects of L-selenomethionine on low-dose iron ion radiation-induced changes in gene expression associated with cellular stress.

    PubMed

    Nuth, Manunya; Kennedy, Ann R

    2013-07-01

    Ionizing radiation associated with highly energetic and charged heavy (HZE) particles poses a danger to astronauts during space travel. The aim of the present study was to evaluate the patterns of gene expression associated with cellular exposure to low-dose iron ion irradiation, in the presence and absence of L-selenomethionine (SeM). Human thyroid epithelial cells (HTori-3) were exposed to low-dose iron ion (1 GeV/n) irradiation at 10 or 20 cGy with or without SeM pretreatment. The cells were harvested 6 and 16 h post-irradiation and analyzed by the Affymetrix U133Av2 gene chip arrays. Genes exhibiting a 1.5-fold expression cut-off and 5% false discovery rate (FDR) were considered statistically significant and subsequently analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) for pathway analysis. Representative genes were further validated by real-time RT-PCR. Even at low doses of radiation from iron ions, global genome profiling of the irradiated cells revealed the upregulation of genes associated with the activation of stress-related signaling pathways (ubiquitin-mediated proteolysis, p53 signaling, cell cycle and apoptosis), which occurred in a dose-dependent manner. A 24-h pretreatment with SeM was shown to reduce the radiation effects by mitigating stress-related signaling pathways and downregulating certain genes associated with cell adhesion. The mechanism by which SeM prevents radiation-induced transformation in vitro may involve the suppression of the expression of genes associated with stress-related signaling and certain cell adhesion events. PMID:23946774

  17. Interferon-gamma increases cellular calcium ion concentration and inositol 1,4,5-trisphosphate formation in human renal carcinoma cells: relation to ICAM-1 antigen expression.

    PubMed Central

    Hansen, A. B.; Bouchelouche, P. N.; Lillevang, S. T.; Andersen, C. B.

    1994-01-01

    In the present study, we investigated the effect of interferon-gamma (IFN-gamma) on cellular calcium ion concentration [Ca2+]i and inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) formation in the human renal carcinoma cell line CaKi-1. We also examined the possible role of a Ca(2+)-dependent mechanism during IFN-gamma-induced intercellular adhesion molecule 1 (ICAM-1) antigen expression. IFN-gamma caused a rapid concentration-dependent rise in [Ca2+]i, which was partly inhibited by diltiazem, a calcium channel blocker, TMB-8, an inhibitor of intracellular calcium redistribution, and in calcium-free medium. IFN-gamma caused a fourfold increase in Ins 1,4,5-P3 formation. The induction of ICAM-1 antigen expression was synergistically enhanced by 4-bromocalcium ionophore A23187. Finally, the calcium antagonists diltiazem. TMB-8 and EGTA, as well as two potent inhibitors of Ca(2+)-dependent kinases, calmidazolium (R24571) and W7, had no or only a minor inhibitory effect on IFN-gamma induction. Our data suggest that IFN-gamma increases [Ca2+]i in CaKi-1 cells by stimulating influx of Ca2+ and release of Ca2+ from intracellular stores, probably via Ins 1,4,5-P3 formation. IFN-gamma signal transduction in our model may not be limited to an increase in [Ca2+]i and Ins 1,4,5-P3, since IFN-gamma-induced ICAM-1 antigen expression was abrogated to a minor degree by calcium antagonists and not coupled to Ins 1,4,5-P3 formation. PMID:7905278

  18. Increased tumor distribution and expression of histidine-rich plasmid polyplexes

    PubMed Central

    Leng, Qixin; Chou, Szu-Ting; Scaria, Puthupparampil V; Woodle, Martin C; Mixson, A James

    2014-01-01

    Background Selecting nonviral carriers for in vivo gene delivery is often dependent on determining the optimal carriers from transfection assays in vitro. The rationale behind this in vitro strategy is to cast a net sufficiently wide to identify the few effective carriers of plasmids for in vivo studies. Nevertheless, many effective in vivo carriers may be overlooked by this strategy because of the marked differences between in vitro and in vivo assays. Methods After solid-phase synthesis of linear and branched histidine/lysine (HK) peptides, the two peptide carriers were compared for their ability to transfect MDA-MB-435 tumor cells in vitro and then in vivo. Results By contrast to their transfection activity in vitro, the linear H2K carrier of plasmids was far more effective in vivo compared to the branch H2K4b. Surprisingly, negatively-charged polyplexes formed by the linear H2K peptide gave higher transfection in vivo than did those with a positive surface charge. To examine the distribution of plasmid expression within the tumor from H2K polyplexes, we found widespread expression by immunohistochemical staining. With a fluorescent tdTomato expressing-plasmid, we confirmed a pervasive distribution and gene expression within the tumor mediated by the H2K polyplex. Conclusions Although mechanisms underlying the efficiency of gene expression are probably multifactorial, unpacking of the H2K polyplex within the tumor appears to have a significant role. Further development of these H2K polyplexes represents an attractive approach for plasmid-based therapies of cancer. © 2014 The Authors. The Journal of Gene Medicine published by John Wiley & Sons, Ltd. PMID:25303767

  19. Nitric Oxide Synthase in the Central Nervous System and Peripheral Organs of Stramonita haemastoma: Protein Distribution and Gene Expression in Response to Thermal Stress

    PubMed Central

    Toni, Mattia; De Angelis, Federica; Bonaccorsi di Patti, Maria Carmela; Cioni, Carla

    2015-01-01

    Nitric oxide (NO) is generated via the oxidation of l-arginine by the enzyme NO synthase (NOS) both in vertebrates and invertebrates. Three NOS isoforms, nNOS, iNOS and eNOS, are known in vertebrates, whereas a single NOS isoform is usually expressed in invertebrates, sharing structural and functional characteristics with nNOS or iNOS depending on the species. The present paper is focused on the constitutive Ca2+/calmodulin-dependent nNOS recently sequenced by our group in the neogastropod Stramonita haemastoma (ShNOS). In this paper we provide new data on cellular distribution of ShNOS in the CNS (pedal ganglion) and peripheral organs (osphradium, tentacle, eye and foot) obtained by WB, IF, CM and NADPHd. Results demonstrated that NOS-like proteins are widely expressed in sensory receptor elements, neurons and epithelial cells. The detailed study of NOS distribution in peripheral and central neurons suggested that NOS is both intracellular and presynaptically located. Present findings confirm that NO may have a key role in the central neuronal circuits of gastropods and in sensory perception. The physiological relevance of NOS enzymes in the same organs was suggested by thermal stress experiments demonstrating that the constitutive expression of ShNOS is modulated in a time- and organ-dependent manner in response to environmental stressors. PMID:26528988

  20. Nitric Oxide Synthase in the Central Nervous System and Peripheral Organs of Stramonita haemastoma: Protein Distribution and Gene Expression in Response to Thermal Stress.

    PubMed

    Toni, Mattia; De Angelis, Federica; di Patti, Maria Carmela Bonaccorsi; Cioni, Carla

    2015-11-01

    Nitric oxide (NO) is generated via the oxidation of l-arginine by the enzyme NO synthase (NOS) both in vertebrates and invertebrates. Three NOS isoforms, nNOS, iNOS and eNOS, are known in vertebrates, whereas a single NOS isoform is usually expressed in invertebrates, sharing structural and functional characteristics with nNOS or iNOS depending on the species. The present paper is focused on the constitutive Ca(2+)/calmodulin-dependent nNOS recently sequenced by our group in the neogastropod Stramonita haemastoma (ShNOS). In this paper we provide new data on cellular distribution of ShNOS in the CNS (pedal ganglion) and peripheral organs (osphradium, tentacle, eye and foot) obtained by WB, IF, CM and NADPHd. Results demonstrated that NOS-like proteins are widely expressed in sensory receptor elements, neurons and epithelial cells. The detailed study of NOS distribution in peripheral and central neurons suggested that NOS is both intracellular and presynaptically located. Present findings confirm that NO may have a key role in the central neuronal circuits of gastropods and in sensory perception. The physiological relevance of NOS enzymes in the same organs was suggested by thermal stress experiments demonstrating that the constitutive expression of ShNOS is modulated in a time- and organ-dependent manner in response to environmental stressors.

  1. The immunohistochemical expression profile of osteopontin in normal human tissues using two site-specific antibodies reveals a wide distribution of positive cells and extensive expression in the central and peripheral nervous systems.

    PubMed

    Kunii, Yasuto; Niwa, Shin-ichi; Hagiwara, Yoshiaki; Maeda, Masahiro; Seitoh, Tsutomu; Suzuki, Toshimitsu

    2009-09-01

    To elucidate the cellular distribution of osteopontin (OPN) in normal human tissues, we undertook immunohistochemistry using two site-specific OPN antibodies. The 10A16 monoclonal antibody was raised against the amino acid sequence just downstream of the thrombin cleavage site, while the O-17 polyclonal antibody was raised against the N-terminal peptide. Each antibody has been confirmed previously to react with both whole OPN and its relevant fragments. The expression pattern for these two antibodies was similar in distribution. In addition, we also identified expression in Ebner's gland, type II pneumocytes, Kupffer cells, cells of the endocrine organs, anterior lens capsule and ciliary body, synovial type A cells, mesothelia, adipocytes, and mast cells. Neurons and glia in the central nervous system and spinal cord, cranial and peripheral nerve sheaths, ganglion cells in the sympathetic ganglion, intestinal plexuses, retina, and choroid plexus also regularly exhibited OPN positivity. Testicular germ cells, pancreatic exocrine cells, and follicular dendritic cells reacted with 10A16 only, whereas lutein cells and taste bud cells exhibited O-17 reactivity alone. These minor differences were hypothesized to reflect the state of OPN in the cells; that is, whether OPN was in its whole molecule or fragmented form. In conclusion, we demonstrate that OPN is widely distributed in normal human cells, particularly those comprising the central and peripheral nervous systems.

  2. High-Content Imaging and Gene Expression Approaches To Unravel the Effect of Surface Functionality on Cellular Interactions of Silver Nanoparticles.

    PubMed

    Manshian, Bella B; Pfeiffer, Christian; Pelaz, Beatriz; Heimerl, Thomas; Gallego, Marta; Möller, Marco; del Pino, Pablo; Himmelreich, Uwe; Parak, Wolfgang J; Soenen, Stefaan J

    2015-10-27

    The toxic effects of Ag nanoparticles (NPs) remain an issue of debate, where the respective contribution of the NPs themselves and of free Ag(+) ions present in the NP stock suspensions and after intracellular NP corrosion are not fully understood. Here, we employ a recently set up methodology based on high-content (HC) imaging combined with high-content gene expression studies to examine the interaction of three types of Ag NPs with identical core sizes, but coated with either mercaptoundecanoic acid (MUA), dodecylamine-modified poly(isobutylene-alt-maleic anhydride) (PMA), or poly(ethylene glycol) (PEG)-conjugated PMA with two types of cultured cells (primary human umbilical vein endothelial cells (HUVEC) and murine C17.2 neural progenitor cells). As a control, cells were also exposed to free Ag(+) ions at the same concentration as present in the respective Ag NP stock suspensions. The data reveal clear effects of the NP surface properties on cellular interactions. PEGylation of the NPs significantly reduces their cellular uptake efficiency, whereas MUA-NPs are more prone to agglomeration in complex tissue culture media. PEG-NPs display the lowest levels of toxicity, which is in line with their reduced cell uptake. MUA-NPs display the highest levels of toxicity, caused by autophagy, cell membrane damage, mitochondrial damage, and cytoskeletal deformations. At similar intracellular NP levels, PEG-NPs induce the highest levels of reactive oxygen species (ROS), but do not affect the cell cytoskeleton, in contrast to MUA- and PMA-NPs. Gene expression studies support the findings above, defining autophagy and cell membrane damage-related necrosis as main toxicity pathways. Additionally, immunotoxicity, DNA damage responses, and hypoxia-like toxicity were observed for PMA- and especially MUA-NPs. Together, these data reveal that Ag(+) ions do contribute to Ag NP-associated toxicity, particularly upon intracellular degradation. The different surface properties of the

  3. Cellular distribution of the renal bumetanide-sensitive Na-K-2Cl cotransporter BSC-1 in the inner stripe of the outer medulla during the development of hypertension in the spontaneously hypertensive rat.

    PubMed

    Sonalker, Prajakta A; Tofovic, Stevan P; Jackson, Edwin K

    2007-12-01

    1. The renal bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1) is expressed only in the thick ascending limb and selectively traffics from intracellular vesicles (IVs) to apical plasma membranes (PMs), where BSC-1 regulates sodium reabsorption. We showed previously that in kidneys from adult spontaneously hypertensive rats (SHR; model of essential hypertension) total protein expression of BSC-1 was higher compared with kidneys from normotensive Wistar-Kyoto (WKY) rats. However, whether this change is associated with an increased trafficking of BSC-1 from IVs to PMs is unknown. The goal of the present study was to test the hypothesis that the increase in total renal BSC-1 protein expression in SHR is accompanied by an augmented distribution of BSC-1 from IVs to PMs. 2. To test the hypothesis, we obtained renal tissue from the inner stripe of the outer medulla (ISOM; enriched in thick ascending limbs) and isolated IVs and PMs from this tissue by differential centrifugation. Total BSC-1 protein expression in ISOM and BSC-1 protein expression in ISOM IVs and PMs were measured by semiquantitative western blotting in SHR and aged-matched WKY rats at different ages and stages of hypertension. 3. At 5 weeks of age, SHR were prehypertensive (mean arterial blood pressure (MABP) 97 mmHg). At this age, both the total abundance and cellular distribution of BSC-1 were similar in ISOM from SHR and WKY rats. 4. As SHR aged, their hypertension progressed (MABP 137 and 195 mmHg at 8 and 14 weeks of age, respectively). Associated with the increase in MABP was an increase in both steady state protein levels of ISOM BSC-1 and the distribution of ISOM BSC-1 to PMs (four- and sixfold increases at 8 and 14 weeks of age, respectively, compared with age-matched WKY rats; P < 0.001). 5. Using semiquantitative reverse transcription-polymerase chain reaction, BSC-1 mRNA was measured and was found not to differ between SHR and WKY rat ISOM at any age or level of MABP. 6. We conclude that as

  4. Distributed Function Mining for Gene Expression Programming Based on Fast Reduction.

    PubMed

    Deng, Song; Yue, Dong; Yang, Le-chan; Fu, Xiong; Feng, Ya-zhou

    2016-01-01

    For high-dimensional and massive data sets, traditional centralized gene expression programming (GEP) or improved algorithms lead to increased run-time and decreased prediction accuracy. To solve this problem, this paper proposes a new improved algorithm called distributed function mining for gene expression programming based on fast reduction (DFMGEP-FR). In DFMGEP-FR, fast attribution reduction in binary search algorithms (FAR-BSA) is proposed to quickly find the optimal attribution set, and the function consistency replacement algorithm is given to solve integration of the local function model. Thorough comparative experiments for DFMGEP-FR, centralized GEP and the parallel gene expression programming algorithm based on simulated annealing (parallel GEPSA) are included in this paper. For the waveform, mushroom, connect-4 and musk datasets, the comparative results show that the average time-consumption of DFMGEP-FR drops by 89.09%%, 88.85%, 85.79% and 93.06%, respectively, in contrast to centralized GEP and by 12.5%, 8.42%, 9.62% and 13.75%, respectively, compared with parallel GEPSA. Six well-studied UCI test data sets demonstrate the efficiency and capability of our proposed DFMGEP-FR algorithm for distributed function mining. PMID:26751200

  5. Distributed Function Mining for Gene Expression Programming Based on Fast Reduction.

    PubMed

    Deng, Song; Yue, Dong; Yang, Le-chan; Fu, Xiong; Feng, Ya-zhou

    2016-01-01

    For high-dimensional and massive data sets, traditional centralized gene expression programming (GEP) or improved algorithms lead to increased run-time and decreased prediction accuracy. To solve this problem, this paper proposes a new improved algorithm called distributed function mining for gene expression programming based on fast reduction (DFMGEP-FR). In DFMGEP-FR, fast attribution reduction in binary search algorithms (FAR-BSA) is proposed to quickly find the optimal attribution set, and the function consistency replacement algorithm is given to solve integration of the local function model. Thorough comparative experiments for DFMGEP-FR, centralized GEP and the parallel gene expression programming algorithm based on simulated annealing (parallel GEPSA) are included in this paper. For the waveform, mushroom, connect-4 and musk datasets, the comparative results show that the average time-consumption of DFMGEP-FR drops by 89.09%%, 88.85%, 85.79% and 93.06%, respectively, in contrast to centralized GEP and by 12.5%, 8.42%, 9.62% and 13.75%, respectively, compared with parallel GEPSA. Six well-studied UCI test data sets demonstrate the efficiency and capability of our proposed DFMGEP-FR algorithm for distributed function mining.

  6. Distributed Function Mining for Gene Expression Programming Based on Fast Reduction

    PubMed Central

    Deng, Song; Yue, Dong; Yang, Le-chan; Fu, Xiong; Feng, Ya-zhou

    2016-01-01

    For high-dimensional and massive data sets, traditional centralized gene expression programming (GEP) or improved algorithms lead to increased run-time and decreased prediction accuracy. To solve this problem, this paper proposes a new improved algorithm called distributed function mining for gene expression programming based on fast reduction (DFMGEP-FR). In DFMGEP-FR, fast attribution reduction in binary search algorithms (FAR-BSA) is proposed to quickly find the optimal attribution set, and the function consistency replacement algorithm is given to solve integration of the local function model. Thorough comparative experiments for DFMGEP-FR, centralized GEP and the parallel gene expression programming algorithm based on simulated annealing (parallel GEPSA) are included in this paper. For the waveform, mushroom, connect-4 and musk datasets, the comparative results show that the average time-consumption of DFMGEP-FR drops by 89.09%%, 88.85%, 85.79% and 93.06%, respectively, in contrast to centralized GEP and by 12.5%, 8.42%, 9.62% and 13.75%, respectively, compared with parallel GEPSA. Six well-studied UCI test data sets demonstrate the efficiency and capability of our proposed DFMGEP-FR algorithm for distributed function mining. PMID:26751200

  7. Collectivity, Distributivity, and the Interpretation of Plural Numerical Expressions in Child and Adult Language

    PubMed Central

    Musolino, Julien

    2013-01-01

    Sentences containing plural numerical expressions (e.g., two boys) can give rise to two interpretations (collective and distributive), arising from the fact that their representation admits of a part-whole structure. We present the results of a series of experiments designed to explore children’s understanding of this distinction and its implications for the acquisition of linguistic expressions with number words. We show that preschoolers access both interpretations, indicating that they have the requisite linguistic and conceptual machinery to generate the corresponding representations. Furthermore, they can shift their interpretation in response to structural and lexical manipulations. However, they are not fully adult-like: unlike adults, they are drawn to the distributive interpretation, and are not yet aware of the lexical semantics of each and together, which should favor one or another interpretation. This research bridges a gap between a well-established body of work in cognitive psychology on the acquisition of number words and more recent work investigating children’s knowledge of the syntactic and semantic properties of sentences featuring numerical expressions. PMID:24223477

  8. Evaluation of humoral, mucosal, and cellular immune responses following co-immunization of HIV-1 Gag and Env proteins expressed by Newcastle disease virus.

    PubMed

    Khattar, Sunil K; Palaniyandi, Senthilkumar; Samal, Sweety; LaBranche, Celia C; Montefiori, David C; Zhu, Xiaoping; Samal, Siba K

    2015-01-01

    The combination of multiple HIV antigens in a vaccine can broaden antiviral immune responses. In this study, we used NDV vaccine strain LaSota to generate rNDV (rLaSota/optGag) expressing human codon optimized p55 Gag protein of HIV-1. We examined the effect of co-immunization of rLaSota/optGag with rNDVs expressing different forms of Env protein gp160, gp120, gp140L [a version of gp140 that lacked cytoplasmic tail and contained complete membrane-proximal external region (MPER)] and gp140S (a version of gp140 that lacked cytoplasmic tail and distal half of MPER) on magnitude and breadth of humoral, mucosal and cellular immune responses in guinea pigs and mice. Our results showed that inclusion of rLaSota/optGag with rNDVs expressing different forms of Env HIV Gag did not affect the Env-specific humoral and mucosal immune responses in guinea pigs and that the potent immune responses generated against Env persisted for at least 13 weeks post immunization. The highest Env-specific humoral and mucosal immune responses were observed with gp140S+optGag group. The neutralizing antibody responses against HIV strains BaL.26 and MN.3 induced by gp140S+optGag and gp160+optGag were higher than those elicited by other groups. Inclusion of Gag with gp160, gp140S and gp140L enhanced the level of Env-specific IFN-γ-producing CD8(+) T cells in mice. Inclusion of Gag with gp160 and gp140L also resulted in increased Env-specific CD4(+) T cells. The level of Gag-specific CD8(+) and CD4(+) T cells was also enhanced in mice immunized with Gag along with gp140S and gp120. These results indicate lack of antigen interference in a vaccine containing rNDVs expressing Env and Gag proteins.

  9. Fluoxetine up-regulates expression of cellular FLICE-inhibitory protein and inhibits LPS-induced apoptosis in hippocampus-derived neural stem cell

    SciTech Connect

    Chiou, S.-H. . E-mail: shchiou@vghtpe.gov.tw; Chen, S.-J. . E-mail: sjchen@vghtpe.gov.tw; Peng, C-H.; Chang, Y.-L.; Ku, H.-H.; Hsu, W.-M.; Ho, Larry L.-T.; Lee, C.-H.

    2006-05-05

    Fluoxetine is a widely used antidepressant compound which inhibits the reuptake of serotonin in the central nervous system. Recent studies have shown that fluoxetine can promote neurogenesis and improve the survival rate of neurons. However, whether fluoxetine modulates the proliferation or neuroprotection effects of neural stem cells (NSCs) needs to be elucidated. In this study, we demonstrated that 20 {mu}M fluoxetine can increase the cell proliferation of NSCs derived from the hippocampus of adult rats by MTT test. The up-regulated expression of Bcl-2, Bcl-xL and the cellular FLICE-inhibitory protein (c-FLIP) in fluoxetine-treated NSCs was detected by real-time RT-PCR. Our results further showed that fluoxetine protects the lipopolysaccharide-induced apoptosis in NSCs, in part, by activating the expression of c-FLIP. Moreover, c-FLIP induction by fluoxetine requires the activation of the c-FLIP promoter region spanning nucleotides -414 to -133, including CREB and SP1 sites. This effect appeared to involve the phosphatidylinositol-3-kinase-dependent pathway. Furthermore, fluoxetine treatment significantly inhibited the induction of proinflammatory factor IL-1{beta}, IL-6, and TNF-{alpha} in the culture medium of LPS-treated NSCs (p < 0.01). The results of high performance liquid chromatography coupled to electrochemical detection further confirmed that fluoxentine increased the functional production of serotonin in NSCs. Together, these data demonstrate the specific activation of c-FLIP by fluoxetine and indicate the novel role of fluoxetine for neuroprotection in the treatment of depression.

  10. Changes in gene expression and cellular localization of insulin-like growth factors 1 and 2 in the ovaries during ovary development of the yellowtail, Seriola quinqueradiata.

    PubMed

    Higuchi, Kentaro; Gen, Koichiro; Izumida, Daisuke; Kazeto, Yukinori; Hotta, Takuro; Takashi, Toshinori; Aono, Hideaki; Soyano, Kiyoshi

    2016-06-01

    A method of controlling the somatic growth and reproduction of yellowtail fish (Seriola quinqueradiata) is needed in order to establish methods for the efficient aquaculture production of the species. However, little information about the hormonal interactions between somatic growth and reproduction is available for marine teleosts. There is accumulating evidence that insulin-like growth factor (IGF), a major hormone related somatic growth, plays an important role in fish reproduction. As the first step toward understanding the physiological role of IGF in the development of yellowtail ovaries, we characterized the expression and cellular localization of IGF-1 and IGF-2 in the ovary during development. We histologically classified the maturity of two-year-old females with ovaries at various developmental stages into the perinucleolar (Pn), yolk vesicle (Yv), primary yolk (Py), secondary yolk and tertiary yolk (Ty) stages, according to the most advanced type of oocyte present. The IGF-1 gene expression showed constitutively high levels at the different developmental stages, although IGF-1 mRNA levels tended to increase from the Py to the Ty stage with vitellogenesis, reaching maximum levels during the Ty stage. The IGF-2 mRNA levels increased as ovarian development advanced. Using immunohistochemistry methods, immunoreactive IGF-1 was mainly detected in the theca cells of ovarian follicles during late secondary oocyte growth, and in part of the granulosa cells of Ty stage oocytes. IGF-2 immunoreactivity was observed in all granulosa cells in layer in Ty stage oocytes. These results indicate that follicular IGFs may be involved in yellowtail reproduction via autocrine/paracrine mechanisms. PMID:26764214

  11. Co-expression of four baculovirus proteins, IE1, LEF3, P143, and PP31, elicits a cellular chromatin-containing reticulate structure in the nuclei of uninfected cells

    SciTech Connect

    Nagamine, Toshihiro; Abe, Atsushi; Suzuki, Takehiro; Dohmae, Naoshi; Matsumoto, Shogo

    2011-08-15

    Baculovirus DNA replication, transcription, and nucleocapsid assembly occur within a subnuclear structure called the virogenic stroma (VS) that consists of two subcompartments. Specific components of the VS sub-compartments have not been identified except for PP31, a DNA-binding protein that localizes specifically to the electron-dense region of VS. Here, we investigate the dynamic structure of VS using a GFP-tagged PP31 molecule (GFP-PP31). GFP-PP31 localizes to the VS throughout the course of infection. At later times post-infection, a PP31 reticulum distributed within VS was also apparent, indicating that VS sub-compartments compose a reticulate structure. Transient expression of PP31 with the viral proteins, IE1, LEF3, and P143, in uninfected cells resulted in the formation of a reticulate structure containing cellular chromatin and the spatial arrangements of the four proteins within the induced reticulum were the same as those within VS reticulum, suggesting that the two reticula are formed by a similar mechanism.

  12. Multiple Regions of Kaposi’s Sarcoma-Associated Herpesvirus ORF59 RNA are Required for Its Expression Mediated by Viral ORF57 and Cellular RBM15

    PubMed Central

    Massimelli, Maria Julia; Majerciak, Vladimir; Kang, Jeong-Gu; Liewehr, David J.; Steinberg, Seth M.; Zheng, Zhi-Ming

    2015-01-01

    KSHV ORF57 (MTA) promotes RNA stability of ORF59, a viral DNA polymerase processivity factor. Here, we show that the integrity of both ORF59 RNA ends is necessary for ORF57-mediated ORF59 expression and deletion of both 5’ and 3’ regions, or one end region with a central region, of ORF59 RNA prevents ORF57-mediated translation of ORF59. The ORF59 sequence between nt 96633 and 96559 resembles other known MTA-responsive elements (MREs). ORF57 specifically binds to a stem-loop region from nt 96596–96572 of the MRE, which also binds cellular RBM15. Internal deletion of the MRE from ORF59 led to poor export, but accumulation of nuclear ORF59 RNA in the presence of ORF57 or RBM15. Despite of being translatable in the presence of ORF57, this deletion mutant exhibits translational defect in the presence of RBM15. Together, our results provide novel insight into the roles of ORF57 and RBM15 in ORF59 RNA accumulation and protein translation. PMID:25690794

  13. Polyamine depletion down-regulates expression of the Trichomonas vaginalis cytotoxic CP65, a 65-kDa cysteine proteinase involved in cellular damage.

    PubMed

    Alvarez-Sánchez, María Elizbeth; Carvajal-Gamez, Bertha Isabel; Solano-González, Eduardo; Martínez-Benitez, Máximo; Garcia, Ana F; Alderete, John F; Arroyo, Rossana

    2008-01-01

    Recently, we found that inhibition of putrescine synthesis by ornithine decarboxylase (ODC) significantly increased Trichomonas vaginalis adherence mediated by protein adhesins. Surprisingly and unexpectedly, trichomonal contact-dependent cytotoxicity was absent. Therefore, a role for polyamine depletion on regulation of T. vaginalis cytotoxicity mediated by the cysteine proteinase (CP) of 65-kDa, CP65, was investigated. We performed cytotoxicity and cell-binding assays followed by zymograms, as well as Western blot and indirect immunofluorescence assays using specific anti-CP65 antibodies to detect CP65. Trichomonads grown in the presence of the ODC inhibitor, 1-4-diamino-2-butanone (DAB) had lower levels of cytotoxicity that corresponded with diminished CP65 proteolytic activity when compared to untreated organisms handled identically. Likewise, semiquantitative and qRT-PCR as well as Western blot and immunofluorescence assays showed decreased amounts of tvcp65 mRNA and CP65 protein in DAB-treated parasites. These effects were reversed by addition of exogenous putrescine. These data show a direct link between polyamine metabolism and expression of the cytotoxic CP65 proteinase involved in trichomonal host cellular damage.

  14. Expression and distribution of the duck enteritis virus UL51 protein in experimentally infected ducks.

    PubMed

    Shen, Chanjuan; Cheng, Anchun; Wang, Mingshu; Xu, Chao; Jia, Renyong; Chen, Xiaoyue; Zhu, Dekang; Luo, Qihui; Cui, Hengmin; Zhou, Yi; Wang, Yin; Xu, Zhiwen; Chen, Zhengli; Wang, Xiaoyu

    2010-06-01

    To determine the expression and distribution of tegument proteins encoded by duck enteritis virus (DEV) UL51 gene in tissues of experimentally infected ducks, for the first time, an immunoperoxidase staining method to detect UL51 protein (UL51p) in paraffin-embedded tissues is reported. A rabbit anti-UL51 polyclonal serum, raised against a recombinant 6-His-UL51 fusion protein expressed in Escherichia coli, was prepared, purified, and used as primary antibodies. Fifty-eight 30-day-old DEV-free ducks were intramuscularly inoculated with the pathogenic DEV CHv strain as infection group, and two ducks were selected as preinfection group. The tissues were collected at sequential time points between 2 and 480 hr postinoculation (PI) and prepared for immunoperoxidase staining. DEV UL51p was first found in the spleen and liver at 8 hr PI; in the bursa of Fabricius and thymus at 12 hr PI; in the Harders glands, esophagus, small intestine (including the duodenum, jejunum, and ileum), and large intestine (including the caecum and rectum) at 24 hr PI; in the glandularis ventriculus at 48 hr PI; and in the pancreas, cerebrum, kidney, lung, and myocardium at 72 hr PI. Throughout the infection process, the UL51p was not seen in the muscle. Furthermore, the intensity of positive staining of DEV UL51p antigen in various tissues increased sharply from 8 to 96 hr PI, peaked during 120-144 hr PI, and then decreased steadily from 216 to 480 hr PI, suggesting that the expressional levels of DEV UL51p in systemic organs have a close correlation with the progression of duck virus enteritis (DVE) disease. A number of DEV UL51p was distributed in the bursa of Fabricius, thymus, spleen, liver, esophagus, small intestine, and large intestine of DEV-infected ducks, whereas less DEV UL51p was distributed in the Harders glands, glandularis ventriculus, cerebrum, kidney, lung, pancreas, and myocardium of DEV-infected ducks. Moreover, DEV UL51p can be expressed in the cytoplasm of various types

  15. Fibrous wound repair associated with biodegradable poly-L/D-lactide copolymer implants: study of the expression of tenascin and cellular fibronectin.

    PubMed

    Kontio, R; Salo, A; Suuronen, R; Lindqvist, C; Meurman, J H; Virtanen, I

    1998-10-01

    Extracellular matrix (ECM) proteins are known to play a role in inflammatory and hyperplastic processes. Our aim in the present study was to study the distribution of tenascin (Tn), cellular fibronectins (cFn) and myofibroblasts around biodegradable poly-L/D-lactide (PLA) implants with monoclonal antibodies (MAb). Ethylene-oxide and gamma-irradiation sterilized PLA plate-type implants were inserted into the dorsal subcutaneous tissue of ten adult rabbits. Follow-up times were 4, 12, 16, 36 and 48 wk. Only some inflammatory cells were observed. In electron microscopy, a close coherence between the implant and the stromal tissue was seen. Immunoreactivity for Tn, cFn and alpha-actin was detected as a distinct layer bordering the implant, regardless of the sterilization method for the first 36 wk. From week 36 onwards, Tn immunoreactivity was downregulated while cFn immunoreactivity still persisted. A moderate upregulation for myofibroblasts was seen on the week 48 specimens, when hydrolysation of PLA implant had started. The persistent content of myofibroblasts, Tn and cFn suggests a prolonged wound healing produced by PLA implants. The absence of Tn at the week 48 specimens suggests that cFn, rather than Tn may be needed for alpha-actin-mediated contraction by myofibroblasts. PMID:15348694

  16. Cellular and molecular characterization of multipolar Map5-expressing cells: a subset of newly generated, stage-specific parenchymal cells in the mammalian central nervous system.

    PubMed

    Crociara, Paola; Parolisi, Roberta; Conte, Daniele; Fumagalli, Marta; Bonfanti, Luca

    2013-01-01

    Although extremely interesting in adult neuro-glio-genesis and promising as an endogenous source for repair, parenchymal progenitors remain largely obscure in their identity and physiology, due to a scarce availability of stage-specific markers. What appears difficult is the distinction between real cell populations and various differentiation stages of the same population. Here we focused on a subset of multipolar, polydendrocyte-like cells (mMap5 cells) expressing the microtubule associated protein 5 (Map5), which is known to be present in most neurons. We characterized the morphology, phenotype, regional distribution, proliferative dynamics, and stage-specific marker expression of these cells in the rabbit and mouse CNS, also assessing their existence in other mammalian species. mMap5 cells were never found to co-express the Ng2 antigen. They appear to be a population of glial cells sharing features but also differences with Ng2+progenitor cells. We show that mMap5 cells are newly generated, postmitotic parenchymal elements of the oligodendroglial lineage, thus being a stage-specific population of polydendrocytes. Finally, we report that the number of mMap5 cells, although reduced within the brain of adult/old animals, can increase in neurodegenerative and traumatic conditions.

  17. BDNF Expression in Larval and Adult Zebrafish Brain: Distribution and Cell Identification

    PubMed Central

    Cacialli, Pietro; Gueguen, Marie-Madeleine; Coumailleau, Pascal; D’Angelo, Livia; Kah, Olivier; Lucini, Carla; Pellegrini, Elisabeth

    2016-01-01

    Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, has emerged as an active mediator in many essential functions in the central nervous system of mammals. BDNF plays significant roles in neurogenesis, neuronal maturation and/or synaptic plasticity and is involved in cognitive functions such as learning and memory. Despite the vast literature present in mammals, studies devoted to BDNF in the brain of other animal models are scarse. Zebrafish is a teleost fish widely known for developmental genetic studies and is emerging as model for translational neuroscience research. In addition, its brain shows many sites of adult neurogenesis allowing higher regenerative properties after traumatic injuries. To add further knowledge on neurotrophic factors in vertebrate brain models, we decided to determine the distribution of bdnf mRNAs in the larval and adult zebrafish brain and to characterize the phenotype of cells expressing bdnf mRNAs by means of double staining studies. Our results showed that bdnf mRNAs were widely expressed in the brain of 7 days old larvae and throughout the whole brain of mature female and male zebrafish. In adults, bdnf mRNAs were mainly observed in the dorsal telencephalon, preoptic area, dorsal thalamus, posterior tuberculum, hypothalamus, synencephalon, optic tectum and medulla oblongata. By combining immunohistochemistry with in situ hybridization, we showed that bdnf mRNAs were never expressed by radial glial cells or proliferating cells. By contrast, bdnf transcripts were expressed in cells with neuronal phenotype in all brain regions investigated. Our results provide the first demonstration that the brain of zebrafish expresses bdnf mRNAs in neurons and open new fields of research on the role of the BDNF factor in brain mechanisms in normal and brain repairs situations. PMID:27336917

  18. BDNF Expression in Larval and Adult Zebrafish Brain: Distribution and Cell Identification.

    PubMed

    Cacialli, Pietro; Gueguen, Marie-Madeleine; Coumailleau, Pascal; D'Angelo, Livia; Kah, Olivier; Lucini, Carla; Pellegrini, Elisabeth

    2016-01-01

    Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, has emerged as an active mediator in many essential functions in the central nervous system of mammals. BDNF plays significant roles in neurogenesis, neuronal maturation and/or synaptic plasticity and is involved in cognitive functions such as learning and memory. Despite the vast literature present in mammals, studies devoted to BDNF in the brain of other animal models are scarse. Zebrafish is a teleost fish widely known for developmental genetic studies and is emerging as model for translational neuroscience research. In addition, its brain shows many sites of adult neurogenesis allowing higher regenerative properties after traumatic injuries. To add further knowledge on neurotrophic factors in vertebrate brain models, we decided to determine the distribution of bdnf mRNAs in the larval and adult zebrafish brain and to characterize the phenotype of cells expressing bdnf mRNAs by means of double staining studies. Our results showed that bdnf mRNAs were widely expressed in the brain of 7 days old larvae and throughout the whole brain of mature female and male zebrafish. In adults, bdnf mRNAs were mainly observed in the dorsal telencephalon, preoptic area, dorsal thalamus, posterior tuberculum, hypothalamus, synencephalon, optic tectum and medulla oblongata. By combining immunohistochemistry with in situ hybridization, we showed that bdnf mRNAs were never expressed by radial glial cells or proliferating cells. By contrast, bdnf transcripts were expressed in cells with neuronal phenotype in all brain regions investigated. Our results provide the first demonstration that the brain of zebrafish expresses bdnf mRNAs in neurons and open new fields of research on the role of the BDNF factor in brain mechanisms in normal and brain repairs situations. PMID:27336917

  19. Prenatal ethanol consumption alters the expression of cellular retinol binding protein and retinoic acid receptor mRNA in fetal rat embryo and brain.

    PubMed

    Grummer, M A; Zachman, R D

    1995-12-01

    The mechanism by which prenatal ethanol ingestion causes fetal alcohol syndrome (FAS) is unknown. We hypothesize that ethanol disrupts the normal function of retinoids in embryogenesis and differentiation, resulting in FAS. The present work was designed to determine if prenatal ethanol ingestion affects the expression of cellular retinol binding protein (CRBP) and nuclear retinoic acid receptors (RARs). Paired timed pregnant rats were fed a liquid diet, one group treated with 36% of carbohydrate calories replaced with ethanol. Maternal serum retinol concentrations during pregnancy peaked on the 6th day of pregnancy, but no difference was noted between the ethanol and control group. At the 12th and 20th day of gestation, embryos or fetal brain were removed, and RNA was isolated for Northern hybridization. The abundance of CRBP mRNA was significantly elevated by ethanol consumption. In both the 12-day embryo (relative density of control: 1.00 +/- 0.10; vs. ethanol: 1.87 +/- 0.30, p < 0.05) and 20-day fetal brain (relative density of control: 1.00 +/- 0.09; vs. ethanol: 1.46 +/- 0.09, p < 0.01). In the embryo, ethanol ingestion resulted in a decrease in the level of RAR-beta mRNA (control: 1.00 +/- 0.05; vs. ethanol: 0.71 +/- 0.07, p < 0.01), but had no effect on RAR-alpha or RAR-gamma mRNA. In contrast to the embryo, the expression of both the 3.7- and 2.7-kb RAR-alpha transcripts was significantly greater in day 20 fetal brain of ethanol-treated rats (3.7-kb RAR-alpha control: 1.00 +/- 0.11; vs. ethanol: 1.65 +/- 0.06; p < 0.001; 2.7-kb RAR-alpha control: 1.00 +/- 0.14; vs. ethanol: 1.74 +/- 0.27, p < 0.05), whereas RAR-beta and RAR-gamma expression were not altered. These observations suggest that altered vitamin A function is a potential factor in the embryopathy of prenatal ethanol exposure. PMID:8749798

  20. Localization of complement factor H gene expression and protein distribution in the mouse outer retina

    PubMed Central

    Smit-McBride, Zeljka; Oltjen, Sharon L.; Radu, Roxana A.; Estep, Jason; Nguyen, Anthony T.; Gong, Qizhi

    2015-01-01

    Purpose To determine the localization of complement factor H (Cfh) mRNA and its protein in the mouse outer retina. Methods Quantitative real-time PCR (qPCR) was used to determine the expression of Cfh and Cfh-related (Cfhr) transcripts in the RPE/choroid. In situ hybridization (ISH) was performed using the novel RNAscope 2.0 FFPE assay to localize the expression of Cfh mRNA in the mouse outer retina. Immunohistochemistry (IHC) was used to localize Cfh protein expression, and western blots were used to characterize CFH antibodies used for IHC. Results Cfh and Cfhr2 transcripts were detected in the mouse RPE/choroid using qPCR, while Cfhr1, Cfhr3, and Cfhrc (Gm4788) were not detected. ISH showed abundant Cfh mRNA in the RPE of all mouse strains (C57BL/6, BALB/c, 129/Sv) tested, with the exception of the Cfh−/− eye. Surprisingly, the Cfh protein was detected by immunohistochemistry in photoreceptors rather than in RPE cells. The specificity of the CFH antibodies was tested by western blotting. Our CFH antibodies recognized purified mouse Cfh protein, serum Cfh protein in wild-type C57BL/6, BALB/c, and 129/Sv, and showed an absence of the Cfh protein in the serum of Cfh−/− mice. Greatly reduced Cfh protein immunohistological signals in the Cfh−/− eyes also supported the specificity of the Cfh protein distribution results. Conclusions Only Cfh and Cfhr2 genes are expressed in the mouse outer retina. Only Cfh mRNA was detected in the RPE, but no protein. We hypothesize that the steady-state concentration of Cfh protein is low in the cells due to secretion, and therefore is below the detection level for IHC. PMID:25684976

  1. Distribution and Expression of the ZmpA Metalloprotease in the Burkholderia cepacia Complex

    PubMed Central

    Gingues, S.; Kooi, C.; Visser, M. B.; Subsin, B.; Sokol, P. A.

    2005-01-01

    The distribution of the metalloprotease gene zmpA was determined among strains of the Burkholderia cepacia complex (Bcc). The zmpA gene was present in B. cepacia, B. cenocepacia, B. stabilis, B. ambifaria and B. pyrrocinia but absent from B. multivorans, B. vietnamiensis, B. dolosa, and B. anthina. The presence of zmpA generally correlated with extracellular proteolytic activity with the exception of five strains, which had zmpA but had no detectable proteolytic activity when skim milk agar was used as a substrate (zmpA protease deficient). Western immunoblot experiments with anti-ZmpA antibodies suggest that the zmpA protease-deficient strains do not secrete or accumulate detectable ZmpA. Transcriptional zmpA::lacZ fusions were introduced in selected strains of the Bcc. zmpA::lacZ was expressed in all strains, but expression was generally lower in the zmpA protease-deficient strains than in the zmpA protease-proficient strains. Quantitative reverse transcriptase real-time PCR demonstrated that zmpA protease-deficient strains did express zmpA mRNA, although at various levels. ZmpA has previously been shown to be positively regulated by the CepIR quorum-sensing system. Addition of exogenous AHLs did not restore extracellular protease production to any of the zmpA protease-deficient strains; however, introduction of cepR in trans complemented protease activity in two of five strains. Extracellular proteolytic activity was restored by the presence of zmpA in trans in two of the five strains. These studies suggest that although some strains of the Bcc contain the zmpA gene, multiple factors may influence its expression. PMID:16321929

  2. Distribution of type 1 cannabinoid receptor-expressing neurons in the septal-hypothalamic region of the mouse: colocalization with GABAergic and glutamatergic markers.

    PubMed

    Hrabovszky, Erik; Wittmann, Gábor; Kalló, Imre; Füzesi, Tamás; Fekete, Csaba; Liposits, Zsolt

    2012-04-01

    Type 1 cannabinoid receptor (CB1) is the principal mediator of retrograde endocannabinoid signaling in the brain. In this study, we addressed the topographic distribution and amino acid neurotransmitter phenotype of endocannabinoid-sensitive hypothalamic neurons in mice. The in situ hybridization detection of CB1 mRNA revealed high levels of expression in the medial septum (MS) and the diagonal band of Broca (DBB), moderate levels in the preoptic area and the hypothalamic lateroanterior (LA), paraventricular (Pa), ventromedial (VMH), lateral mammillary (LM), and ventral premammillary (PMV) nuclei, and low levels in many other hypothalamic regions including the suprachiasmatic (SCh) and arcuate (Arc) nuclei. This regional distribution pattern was compared with location of γ-aminobutyric acid (GABA)ergic and glutamatergic cell groups, as identified by the expression of glutamic acid decarboxylase 65 (GAD65) and type 2 vesicular glutamate transporter (VGLUT2) mRNAs, respectively. The MS, DBB, and preoptic area showed overlaps between GABAergic and CB1-expressing neurons, whereas hypothalamic sites with moderate CB1 signals, including the LA, Pa, VMH, LM, and PMV, were dominated by glutamatergic neurons. Low CB1 mRNA levels were also present in other glutamatergic and GABAergic regions. Dual-label in situ hybridization experiments confirmed the cellular co-expression of CB1 with both glutamatergic and GABAergic markers. In this report we provide a detailed anatomical map of hypothalamic glutamatergic and GABAergic systems whose neurotransmitter release is controlled by retrograde endocannabinoid signaling from hypothalamic and extrahypothalamic target neurons. This neuroanatomical information contributes to an understanding of the role that the endocannabinoid system plays in the regulation of endocrine and metabolic functions.

  3. Regulation of N-methyl-D-aspartate receptor expression and N-methyl-D-aspartate-induced cellular response during chronic hypoxia in differentiated rat PC12 cells.

    PubMed

    Kobayashi, S; Millhorn, D E

    2000-01-01

    The purpose of the present study was to examine the effect of chronic hypoxia on N-methyl-D-aspartate-mediated cellular responses in differentiated PC12 cells. PC12 cells were differentiated by treatment with nerve growth factor. Patch-clamp analysis in differentiated PC12 cells showed that extracellularly applied N-methyl-D-aspartate induced an inward current that was abolished by the presence of the N-methyl-D-aspartate receptor antagonist MK-801. Results from Ca(2+) imaging experiments showed that N-methyl-D-aspartate induced an elevation in intracellular free Ca(2+) which was also abolished by MK-801. We also examined the effect of hypoxia on the N-methyl-D-aspartate-induced current in nerve growth factor-treated cells. We found that the N-methyl-D-aspartate-induced inward current and the N-methyl-D-aspartate-induced elevation in intracellular free Ca(2+) were markedly attenuated by chronic hypoxia. We next examined the possibility that the reduced N-methyl-D-aspartate responsiveness was due to down-regulation of N-methyl-D-aspartate receptor levels. Northern blot and immunoblot analyses showed that both messenger RNA and protein levels for N-methyl-D-aspartate receptor subunit 1 were markedly decreased during hypoxia. However, the messenger RNA for N-methyl-D-aspartate receptor subunit 2C was increased, whereas the protein level for subunit 2C did not change. Our results indicate that differentiated PC12 cells express functional N-methyl-D-aspartate receptors and that chronic exposure to hypoxia attenuates the N-methyl-D-aspartate-induced Ca(2+) accumulation in these cells via down-regulation of N-methyl-D-aspartate receptor subunit 1. This mechanism may play an important role in protecting PC12 cells against hypoxic stress. PMID:11113364

  4. EXPRESSION AND DISTRIBUTION OF THIOL-REGULATING ENZYME GLUTAREDOXIN 2 (GRX2) IN PORCINE OCULAR TISSUES*

    PubMed Central

    Upadhyaya, Bijaya; Tian, Xiaoli; Wu, Hongli; Lou, Marjorie F.

    2014-01-01

    Glutaredoxin2 (Grx2) is a mitochondrial isozyme of the cytosolic glutaredoxin1 (thioltransferase or TTase). Both belong to the large oxidoreductase family and play an important role in maintaining thiol/disulfide redox homeostasis in the cells. Grx2 is recently found in the lens where its activities of disulfide reductase and peroxidase, similar to TTase, can protect the lens against oxidative stress. Since other eye tissues are also highly sensitive to oxidative stress, and TTase’s distribution in the eye is known, we focused on this study by investigating the Grx2 distribution in the ocular tissues in comparison to the lens. Fresh porcine eyes were dissected into cornea, iris, ciliary body, the lens, vitreous humor, retina, and optic nerve. Each tissue (pooled from three eyes) was homogenized and processed for mitochondrial isolation. The mitochondrial fraction was analyzed for Grx2 protein using Western blotting with anti-Grx2 antibody, and Grx2 activity using the published procedure. The eye tissues were also measured for Grx2 mRNA expression by RT-PCR with GAPDH as the control. Grx2-rich mouse liver and purified recombinant mouse Grx2 were used as positive controls for the above analyses. It was found that Grx2 was present in all the tested ocular tissues, except vitreous humor. In comparison with the mouse liver, the protein levels of Grx2 in porcine ciliary body and the lens were 27-fold and 0.75-fold, respectively. Comparing to the lens, Grx2 protein was highest in the ciliary body (13.5-fold), followed by retina (9.2-fold), iris and optic nerve (2-fold), and cornea (1.2-fold). Enzyme activity assays showed that the retina had the highest Grx2 specific activity (3.9 mU/mg protein), followed by ciliary body (3.1 mU/mg), the lens (0.58 mU/mg), and optic nerve (0.32 mU/mg). Grx2 gene expression in these ocular tissues was further confirmed by RT-PCR analysis. Grx2 mRNA expression showed the highest in ciliary body, followed by retina, optic nerve, cornea

  5. The membrane-associated progesterone-binding protein 25-Dx: expression, cellular localization and up-regulation after brain and spinal cord injuries.

    PubMed

    Guennoun, R; Meffre, D; Labombarda, F; Gonzalez, S L; Gonzalez Deniselle, M C; Stein, D G; De Nicola, A F; Schumacher, M

    2008-03-01

    Progesterone has neuroprotective effects in the injured and diseased spinal cord and after traumatic brain injury (TBI). In addition to intracellular progesterone receptors (PR), membrane-binding sites of progesterone may be involved in neuroprotection. A first putative membrane receptor of progesterone, distinct from the classical intracellular PR isoforms, with a single membrane-spanning domain, has been cloned from porcine liver. Homologous proteins were cloned in rats (25-Dx), mice (PGRMC1) and humans (Hpr.6). We will refer to this progesterone-binding protein as 25-Dx. The distribution and regulation of 25-Dx in the nervous system may provide some clues to its functions. In spinal cord, 25-Dx is localized in cell membranes of dorsal horn neurons and ependymal cells lining the central canal. A role of 25-Dx in mediating the protective effects of progesterone in the spinal cord is supported by the observation that its mRNA and protein are up-regulated by progesterone in dorsal horn of the injured spinal cord. In contrast, the classical intracellular PRs were down-regulated under these conditions. In brain, 25-Dx is particularly abundant in the hypothalamic area, circumventricular organs, ependymal cells of the ventricular walls, and the meninges. Interestingly, it is co-expressed with vasopressin in neurons of the paraventricular, supraoptic and retrochiasmatic nuclei. In response to TBI, 25-Dx expression is up-regulated in neurons and induced in astrocytes. The expression of 25-Dx in structures involved in cerebrospinal fluid production and osmoregulation, and its up-regulation after brain damage, point to a potentially important role of this progesterone-binding protein in the maintenance of water homeostasis after TBI. Our observations suggest that progesterone's actions may involve different signaling mechanisms depending on the pathophysiological context, and that 25-Dx may be involved in the neuroprotective effect of progesterone in the injured brain and

  6. Molecular characterization, tissue distribution, and immune reaction expression of karyopherins in the domestic silkworm (Bombyx mori).

    PubMed

    Li, J; Wang, L; Qian, C; Zhang, C F; Dai, L S; Liu, Q N; Wei, G Q; Sun, Y; Liu, D R; Zhu, B J; Liu, C L

    2015-01-01

    Karyopherins, including alpha and beta types, are transport proteins in the eukaryotic cell that carry cargoes across nuclear pore complexes into or out of the nucleus. In this study, full open reading frames of one beta and three alpha types of karyopherin were cloned from cDNA of the domestic silkworm (Bombyx mori). The one beta and three alpha types' open reading frames were 2661, 1563, 1515, and 1551 base pairs long, respectively, and coded 886, 520, 504, and 516 amino acids, respectively. The alphas all had one importin-beta-binding (IBB) domain, and eight, four, or seven armadillo/beta-catenin-like repeats. The beta had 19 HEAT repeat domains, which constructed one importin-beta-N-terminal domain and one IBB domain. The recombinant proteins were expressed in Escherichia coli cells. The molecular weight of the beta type was approximately 100 kDa, and the alphas weighed approximately 60 kDa. Phylogenic tree construction revealed that the alphas could be classified into three known karyopherin-alpha subfamilies. We detected mRNA of the four karyopherins in normal 3rd day of 5th instar larvae, and in larvae injected with Gram-positive bacteria, Gram-negative bacteria, viruses, and fungi using real-time fluorescence quantitative reverse transcriptase-polymerase chain reaction, and found that the four karyopherins were widely distributed, but their expression levels were related to tissues type, the microbe injected, and the time point.

  7. Molecular characterization, tissue distribution, and immune reaction expression of karyopherins in the domestic silkworm (Bombyx mori).

    PubMed

    Li, J; Wang, L; Qian, C; Zhang, C F; Dai, L S; Liu, Q N; Wei, G Q; Sun, Y; Liu, D R; Zhu, B J; Liu, C L

    2015-01-01

    Karyopherins, including alpha and beta types, are transport proteins in the eukaryotic cell that carry cargoes across nuclear pore complexes into or out of the nucleus. In this study, full open reading frames of one beta and three alpha types of karyopherin were cloned from cDNA of the domestic silkworm (Bombyx mori). The one beta and three alpha types' open reading frames were 2661, 1563, 1515, and 1551 base pairs long, respectively, and coded 886, 520, 504, and 516 amino acids, respectively. The alphas all had one importin-beta-binding (IBB) domain, and eight, four, or seven armadillo/beta-catenin-like repeats. The beta had 19 HEAT repeat domains, which constructed one importin-beta-N-terminal domain and one IBB domain. The recombinant proteins were expressed in Escherichia coli cells. The molecular weight of the beta type was approximately 100 kDa, and the alphas weighed approximately 60 kDa. Phylogenic tree construction revealed that the alphas could be classified into three known karyopherin-alpha subfamilies. We detected mRNA of the four karyopherins in normal 3rd day of 5th instar larvae, and in larvae injected with Gram-positive bacteria, Gram-negative bacteria, viruses, and fungi using real-time fluorescence quantitative reverse transcriptase-polymerase chain reaction, and found that the four karyopherins were widely distributed, but their expression levels were related to tissues type, the microbe injected, and the time point. PMID:26535618

  8. Competition between clonal plasma cells and normal cells for potentially overlapping bone marrow niches is associated with a progressively altered cellular distribution in MGUS vs myeloma.

    PubMed

    Paiva, B; Pérez-Andrés, M; Vídriales, M-B; Almeida, J; de las Heras, N; Mateos, M-V; López-Corral, L; Gutiérrez, N C; Blanco, J; Oriol, A; Hernández, M T; de Arriba, F; de Coca, A G; Terol, M-J; de la Rubia, J; González, Y; Martín, A; Sureda, A; Schmidt-Hieber, M; Schmitz, A; Johnsen, H E; Lahuerta, J-J; Bladé, J; San-Miguel, J F; Orfao, A

    2011-04-01

    Disappearance of normal bone marrow (BM) plasma cells (PC) predicts malignant transformation of monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM) into symptomatic multiple myeloma (MM). The homing, behavior and survival of normal PC, but also CD34(+) hematopoietic stem cells (HSC), B-cell precursors, and clonal PC largely depends on their interaction with stromal cell-derived factor-1 (SDF-1) expressing, potentially overlapping BM stromal cell niches. Here, we investigate the distribution, phenotypic characteristics and competitive migration capacity of these cell populations in patients with MGUS, SMM and MM vs healthy adults (HA) aged >60 years. Our results show that BM and peripheral blood (PB) clonal PC progressively increase from MGUS to MM, the latter showing a slightly more immature immunophenotype. Of note, such increased number of clonal PC is associated with progressive depletion of normal PC, B-cell precursors and CD34(+) HSC in the BM, also with a parallel increase in PB. In an ex vivo model, normal PC, B-cell precursors and CD34(+) HSC from MGUS and SMM, but not MM patients, were able to abrogate the migration of clonal PC into serial concentrations of SDF-1. Overall, our results show that progressive competition and replacement of normal BM cells by clonal PC is associated with more advanced disease in patients with MGUS, SMM and MM. PMID:21252988

  9. APP expression, distribution and accumulation are altered by aluminum in a rodent model for Alzheimer's disease.

    PubMed

    Walton, J R; Wang, M-X

    2009-11-01

    Up-regulated expression of amyloid precursor protein (APP) occurs early in the cascade of events that leads to amyloid plaque formation in the human brain. APP gene up-regulation, mediated by activated NF-kappaB, is a response to stress from nM concentrations of aluminum ions, aluminum-disregulated iron ions, reactive-oxygen species, cytokines, and physical trauma. We examined in vivo effects of aluminum on APP in aged rats, obtained from previously-reported longitudinal studies, that chronically ingested aluminum in amounts equivalent to total dietary aluminum levels that Americans routinely ingest. These rats exhibited two outcomes: one group remained cognitively-intact, scoring as well on a memory-discrimination task in old age as in middle age. The other developed cognitive deterioration, obtaining significantly lower mean performance scores in old age than in middle age and exhibiting abnormal behaviors associated with dementia. We compared the expression, distribution and accumulation of APP in hippocampal and cortical tissue of these two rat groups. Compared to results from cognitively-intact rats, hippocampal and cortical tissue from the cognitively-deteriorated rats showed elevated APP gene expression, significantly more dense APP deposits in cytoplasm of neural cells, and APP-immunoreactive neurites that were swollen and varicose. This study shows aluminum routinely derived from chronic oral ingestion, that gradually accumulates in brain regions important for memory-processing, is sufficient to increase APP levels in neural cells of those regions. Aluminum may thus launch the cascade that results in the formation of amyloid plaques in human brain.

  10. Shaping protein distributions in stochastic self-regulated gene expression networks

    NASA Astrophysics Data System (ADS)

    Pájaro, Manuel; Alonso, Antonio A.; Vázquez, Carlos

    2015-09-01

    In this work, we study connections between dynamic behavior and network parameters, for self-regulatory networks. To that aim, a method to compute the regions in the space of parameters that sustain bimodal or binary protein distributions has been developed. Such regions are indicative of stochastic dynamics manifested either as transitions between absence and presence of protein or between two positive protein levels. The method is based on the continuous approximation of the chemical master equation, unlike other approaches that make use of a deterministic description, which as will be shown can be misleading. We find that bimodal behavior is a ubiquitous phenomenon in cooperative gene expression networks under positive feedback. It appears for any range of transcription and translation rate constants whenever leakage remains below a critical threshold. Above such a threshold, the region in the parameters space which sustains bimodality persists, although restricted to low transcription and high translation rate constants. Remarkably, such a threshold is independent of the transcription or translation rates or the proportion of an active or inactive promoter and depends only on the level of cooperativity. The proposed method can be employed to identify bimodal or binary distributions leading to stochastic dynamics with specific switching properties, by searching inside the parameter regions that sustain such behavior.

  11. Expression and distribution of carboxypeptidase B in the hippocampal subregions of normal and Alzheimer's disease brain.

    PubMed

    Papp, Henrietta; Török, I; Matsumoto, A; Enomoto, T; Matsuyama, S; Kása, P

    2003-01-01

    Earlier neurochemical studies suggested that human brain carboxypeptidase B may play a significant role in the degradation of amyloid-beta1-42 in the brain. Using an immimohistochemical technique we report here on the neuronal expression and distribution of this enzyme in the segments (CA1a, CA1b and CA1c) of the CA1 subfield and in area CA4 of the hippocampus in normal and Alzheimer's disease brain samples. Its distribution was compared with the appearance of neurofibrillary tangles in the same brain sample. For immunohistochemical localization of carboxypeptidase B, a specific C14-module antibody was applied, together with the Gallyas silver impregnation technique for the demonstration of neurofibrillary tangles. The results revealed that, in the control samples, most of the immunoreactivity appeared in segment CA1a in the pyramidal cells, less in segment CA1b and least in segment CA1c. In the Alzheimer's disease samples, there was no particular immunostaining in the neurons, but, a large number of silver-impregnated degenerated neurons appeared. The results support the suggestion that carboxypeptidase B may play a significant role in elimination of the intracellular accumulation and toxicity of amyloid-beta in the human brain and thereby protect the neurons from degeneration. PMID:12705322

  12. Effect of 6-O-α-maltosyl-β cyclodextrin and its cholesterol inclusion complex on cellular cholesterol levels and ABCA1 and ABCG1 expression in mouse mastocytoma P-815 cells.

    PubMed

    Okada, Yasuyo; Ueyama, Kiyomi; Nishikawa, Jyun-ichi; Semma, Masanori; Ichikawa, Atsushi

    2012-08-01

    We have previously described 6-O-α-maltosyl-β cyclodextrin (Mal-βCD), which forms soluble inclusion complex with cholesterol. Here we further investigated the effect of Mal-βCD and cholesterol/Mal-βCD inclusion complex (CLM) on cellular cholesterol levels in a mouse mast cell line, mastocytoma P-815 cells (P-815 cells). Mal-βCD removes cellular cholesterol forming inclusion complexes, while Mal-βCD-induced lack of cellular cholesterol was replenished by the addition of CLM without cytotoxicity. Reduction and replenishment of cellular cholesterol in Mal-βCD- and/or CLM-treated P-815 cells, respectively, were demonstrated by LC/MS and fluorescence microscopy with filipin III. CLM rather than free Mal-βCD and free cholesterol was efficiently incorporated into P-815 cells and its incorporation was inhibited by incubation at low temperature, or with sodium azide and cytochalasin D. P-815 cells have been confirmed to express ATP-binding cassette (ABC) transporters, ABCA1, ABCG1, and P-glycoprotein (P-gp), by Western blot and mRNA analysis. Cholesterol reduction by Mal-βCD abolishes the mRNA and protein expression of ABCA1 and ABCG1, but not of P-gp. Cholesterol loading by CLM restores the diminished ABCA1 and ABCG1 mRNA expression in Mal-βCD-treated P-815 cells. However, both Mal-βCD and CLM had no effect on P-gp activity measured by the rhodamine 123 efflux assay. These results indicate that alteration of cholesterol levels with Mal-βCD or CLM led to down- or up-regulation of ABCA1 and ABCG1 expression in P-815 cells.

  13. HISTONE DEACETYLASE6-Defective Mutants Show Increased Expression and Acetylation of ENHANCER OF TRIPTYCHON AND CAPRICE1 and GLABRA2 with Small But Significant Effects on Root Epidermis Cellular Pattern1

    PubMed Central

    Li, Dong-Xu; Chen, Wen-Qian; Xu, Zhi-Hong; Bai, Shu-Nong

    2015-01-01

    Cellular patterning in the Arabidopsis (Arabidopsis thaliana) root epidermis is dependent on positional information, the transmission of which involves histone acetylation. Here, we report that HISTONE DEACETYLASE6 (HDA6) has significant effects on this cellular patterning. Mutation of HDA6 led to ectopic hair cells in the nonhair positions of root epidermis in Arabidopsis, based on an analysis of paraffin sections stained with Toluidine Blue. While HDA6 was present throughout the root tip, epidermis-specific complementation with HDA6 could rescue the hda6 phenotype. Both transcript levels and expression patterns of ENHANCER OF TRIPTYCHON AND CAPRICE1 (ETC1) and GLABRA2 (GL2) in the root tip were affected in hda6. Consistent with these changes in expression, HDA6 directly bound to the promoter regions of ETC1 and GL2, and acetylation of histone H3 on these promoter regions and acetylation of histone H4 on the ETC1 promoter region was increased in the hda6 mutant. Taken together, these results indicate that HDA6 affects the cellular patterning of Arabidopsis root epidermis through altering the histone acetylation status of ETC1 and GL2 promoters and thereby affects the expression of these two components of the core transcription factor network determining epidermal cell fates. Our findings thus provide new insights into the role of histone acetylation in root epidermis cell patterning. PMID:26143251

  14. In vitro activation of T lymphocytes from human immunodeficiency virus (HIV)-seropositive blood donors. I. Soluble interleukin 2 receptor (IL2R) production parallels cellular IL2R expression and DNA synthesis.

    PubMed

    Prince, H E; Kleinman, S H; Maino, V C; Jackson, A L

    1988-03-01

    We investigated the relationship of soluble interleukin 2 receptor (sIL2R) production to cellular IL2R expression and DNA synthesis by mitogen-stimulated mononuclear cells from blood donors seropositive for human immunodeficiency virus (HIV). SIL2R was measured using an enzyme-linked immunosorbent assay which employed 2 anti-IL2R monoclonal antibodies recognizing distinct IL2R epitopes. Decreased phytohemagglutinin-induced DNA synthesis and cellular IL2R expression were accompanied by decreased levels of sIL2R in cell culture supernatants. Similar findings were observed for pokeweed mitogen-induced responses. There was no detectable spontaneous secretion of sIL2R into culture supernatants by unstimulated mononuclear cells from either HIV-seropositive or control seronegative donors. These findings indicate that the in vitro T-cell activation defects which characterize HIV infection include decreased sIL2R production, as well as decreased cellular IL2R expression and DNA synthesis. Further, they show that assessment of supernatant sIL2R levels can be used as a valid, reliable assay for T-cell activation.

  15. In situ hybridization reveals temporal and spatial changes in cellular expression of mRNA for a laminin receptor, laminin, and basement membrane (type IV) collagen in the developing kidney

    PubMed Central

    1989-01-01

    The appearance of extracellular matrix molecules and their receptors represent key events in the differentiation of cells of the kidney. Steady-state mRNA levels for a laminin receptor, the laminin B1, B2, and A chains, and the alpha 1-chain of collagen IV (alpha 1[IV]), were examined in mouse kidneys at 16 d gestation and birth, when cell differentiation is active, and 1-3 wk after birth when this activity has subsided. Northern analysis revealed that mRNA expression of laminin receptor precedes the alpha 1(IV) and laminin B chains whereas laminin A chain mRNA expression was very low. In situ hybridization reflected this pattern and revealed the cells responsible for expression. At 16 d gestation, laminin receptor mRNA was elevated in cells of newly forming glomeruli and proximal and distal tubules of the nephrogenic zone located in the kidney cortex. These cells also expressed mRNA for alpha 1(IV) and laminin chains. At birth, mRNA expression of receptor and all chains remained high in glomeruli but was reduced in proximal and distal tubules. At 1 wk after birth, expression was located in the medulla over collecting ducts and loops of Henle. Little expression was detectable by 3 wk. These results suggest that cellular expression of steady-state mRNA for laminin receptor, laminin, and collagen IV is temporally linked, with laminin receptor expression proceeding first and thereafter subsiding. PMID:2527859

  16. Cellular and Tumor Radiosensitivity is Correlated to Epidermal Growth Factor Receptor Protein Expression Level in Tumors Without EGFR Amplification;Epidermal growth factor receptor; Radiotherapy; Squamous cell carcinoma; Biomarker; Local tumor control

    SciTech Connect

    Kasten-Pisula, Ulla; Saker, Jarob; Eicheler, Wolfgang; Krause, Mechthild; Yaromina, Ala; Meyer-Staeckling, Soenke; Scherkl, Benjamin; Kriegs, Malte; Brandt, Burkhard; Grenman, Reidar; Petersen, Cordula; Baumann, Michael; Dikomey, Ekkehard

    2011-07-15

    Purpose: There is conflicting evidence for whether the expression of epidermal growth factor receptor in human tumors can be used as a marker of radioresponse. Therefore, this association was studied in a systematic manner using squamous cell carcinoma (SCC) cell lines grown as cell cultures and xenografts. Methods and Materials: The study was performed with 24 tumor cell lines of different tumor types, including 10 SCC lines, which were also investigated as xenografts on nude mice. Egfr gene dose and the length of CA-repeats in intron 1 were determined by polymerase chain reaction, protein expression in vitro by Western blot and in vivo by enzyme-linked immunosorbent assay, and radiosensitivity in vitro by colony formation. Data were correlated with previously published tumor control dose 50% data after fractionated irradiation of xenografts of the 10 SCC. Results: EGFR protein expression varies considerably, with most tumor cell lines showing moderate and only few showing pronounced upregulation. EGFR upregulation could only be attributed to massive gene amplification in the latter. In the case of little or no amplification, in vitro EGFR expression correlated with both cellular and tumor radioresponse. In vivo EGFR expression did not show this correlation. Conclusions: Local tumor control after the fractionated irradiation of tumors with little or no gene amplification seems to be dependent on in vitro EGFR via its effect on cellular radiosensitivity.

  17. Expression dynamics of the pea rbcS multigene family and organ distribution of the transcripts

    PubMed Central

    Fluhr, Robert; Moses, Phyllis; Morelli, Giorgio; Coruzzi, Gloria; Chua, Nam-Hai

    1986-01-01

    We have determined the nucleotide sequence of two members (rbcS-3A and -3C) of the pea nuclear gene family encoding the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase. Both rbcS-3A and -3C are interrupted by two introns located at the same positions as those of the other three pea rbcS genes. Compared with the other pea rbcS genes the rbcS-3C gene has the most divergent 5'- and 3'-flanking sequences while the rbcS-3A gene has a larger and highly divergent intron 1. All five pea rbcS genes are conserved in their coding regions but show considerable sequence differences in their 3'-untranslated portion. The 3' sequence divergence of the rbcS genes has allowed us to use S1 nuclease mapping procedures to compare their expression levels in different organs and during light induction. All the rbcS genes are differentially expressed in various organs of the pea plants; moreover, specific rbcS transcripts are under-represented in seeds and petals. In leaves there is a 10-fold difference between the highest and lowest specific rbcS transcript levels. By quantitating the distribution of rbcS transcripts during light, phytochrome and blue light induction of immature (etiolated), and mature (green), pea leaves, we show that the genes are differentially activated during leaf development. ImagesFig. 3.Fig. 4.Fig. 5.Fig. 6.Fig. 7.Fig. 8. PMID:16453702

  18. Buthionine sulfoximine, an inhibitor of glutathione biosynthesis, induces expression of soluble epoxide hydrolase and markers of cellular hypertrophy in a rat cardiomyoblast cell line: roles of the NF-κB and MAPK signaling pathways.

    PubMed

    Abdelhamid, Ghada; El-Kadi, Ayman O S

    2015-05-01

    Evidence suggests that upregulation of soluble epoxide hydrolase (sEH) is associated with the development of myocardial infarction, dilated cardiomyopathy, cardiac hypertrophy, and heart failure. However, the upregulation mechanism is still unknown. In this study, we treated H9C2 cells with buthionine sulfoximine (BSO) to explore whether oxidative stress upregulates sEH gene expression and to identify the molecular and cellular mechanisms behind this upregulatory response. Real-time PCR and Western blot analyses were used to measure mRNA and protein expression, respectively. We demonstrated that BSO significantly upregulated sEH at mRNA levels in a concentration- and time-dependent manner, leading to a significant increase in the cellular hypertrophic markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Furthermore, BSO significantly increased the cytosolic phosphorylated IκB-α and translocation of NF-κB p50 subunits, as measured by Western blot analysis. This level of translocation was paralleled by an increase in the DNA-binding activity of NF-κB P50 subunits. Moreover, our results demonstrated that pretreatment with the NF-κB inhibitor PDTC significantly inhibited BSO-mediated induction of sEH and cellular hypertrophic marker gene expression in a dose-dependent manner. Additionally, mitogen-activated protein kinases (MAPKs) were transiently phosphorylated by BSO treatment. To understand further the role of MAPKs pathway in BSO-mediated induction of sEH mRNA, we examined the role of extracellular signal-regulated kinase (ERK), c-JunN-terminal kinase (JNK), and p38 MAPK. Indeed, treatment with the MEK/ERK signal transduction inhibitor, PD98059, partially blocked the activation of IκB-α and translocation of NF-κB p50 subunits induced by BSO. Moreover, pretreatment with MEK/ERK signal transduction inhibitors, PD98059 and U0126, significantly inhibited BSO-mediated induction of sEH and cellular hypertrophic marker gene expression

  19. Buthionine sulfoximine, an inhibitor of glutathione biosynthesis, induces expression of soluble epoxide hydrolase and markers of cellular hypertrophy in a rat cardiomyoblast cell line: roles of the NF-κB and MAPK signaling pathways.

    PubMed

    Abdelhamid, Ghada; El-Kadi, Ayman O S

    2015-05-01

    Evidence suggests that upregulation of soluble epoxide hydrolase (sEH) is associated with the development of myocardial infarction, dilated cardiomyopathy, cardiac hypertrophy, and heart failure. However, the upregulation mechanism is still unknown. In this study, we treated H9C2 cells with buthionine sulfoximine (BSO) to explore whether oxidative stress upregulates sEH gene expression and to identify the molecular and cellular mechanisms behind this upregulatory response. Real-time PCR and Western blot analyses were used to measure mRNA and protein expression, respectively. We demonstrated that BSO significantly upregulated sEH at mRNA levels in a concentration- and time-dependent manner, leading to a significant increase in the cellular hypertrophic markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Furthermore, BSO significantly increased the cytosolic phosphorylated IκB-α and translocation of NF-κB p50 subunits, as measured by Western blot analysis. This level of translocation was paralleled by an increase in the DNA-binding activity of NF-κB P50 subunits. Moreover, our results demonstrated that pretreatment with the NF-κB inhibitor PDTC significantly inhibited BSO-mediated induction of sEH and cellular hypertrophic marker gene expression in a dose-dependent manner. Additionally, mitogen-activated protein kinases (MAPKs) were transiently phosphorylated by BSO treatment. To understand further the role of MAPKs pathway in BSO-mediated induction of sEH mRNA, we examined the role of extracellular signal-regulated kinase (ERK), c-JunN-terminal kinase (JNK), and p38 MAPK. Indeed, treatment with the MEK/ERK signal transduction inhibitor, PD98059, partially blocked the activation of IκB-α and translocation of NF-κB p50 subunits induced by BSO. Moreover, pretreatment with MEK/ERK signal transduction inhibitors, PD98059 and U0126, significantly inhibited BSO-mediated induction of sEH and cellular hypertrophic marker gene expression

  20. Probabilistic Cellular Automata

    PubMed Central

    Agapie, Alexandru; Giuclea, Marius

    2014-01-01

    Abstract Cellular automata are binary lattices used for modeling complex dynamical systems. The automaton evolves iteratively from one configuration to another, using some local transition rule based on the number of ones in the neighborhood of each cell. With respect to the number of cells allowed to change per iteration, we speak of either synchronous or asynchronous automata. If randomness is involved to some degree in the transition rule, we speak of probabilistic automata, otherwise they are called deterministic. With either type of cellular automaton we are dealing with, the main theoretical challenge stays the same: starting from an arbitrary initial configuration, predict (with highest accuracy) the end configuration. If the automaton is deterministic, the outcome simplifies to one of two configurations, all zeros or all ones. If the automaton is probabilistic, the whole process is modeled by a finite homogeneous Markov chain, and the outcome is the corresponding stationary distribution. Based on our previous results for the asynchronous case—connecting the probability of a configuration in the stationary distribution to its number of zero-one borders—the article offers both numerical and theoretical insight into the long-term behavior of synchronous cellular automata. PMID:24999557

  1. Extending breath analysis to the cellular level: current thoughts on the human microbiome and the expression of organic compounds in the human exposome

    EPA Science Inventory

    Human biomarkers are comprised of compounds from cellular metabolism, oxidative stress, and the microbiome of bacteria in the gut, genitourinary, and pulmonary tracts. When we examine patterns in human biomarkers to discern human health state or diagnose specific diseases, it is...

  2. Phylogenetic sequence analysis, recombinant expression, and tissue distribution of a channel catfish estrogen receptor beta

    USGS Publications Warehouse

    Xia, Zhenfang; Gale, William L.; Chang, Xiaotian; Langenau, David; Patino, Reynaldo; Maule, Alec G.; Densmore, Llewellyn D.

    2000-01-01

    An estrogen receptor β (ERβ) cDNA fragment was amplified by RT-PCR of total RNAextracted from liver and ovary of immature channel catfish. This cDNA fragment was used to screen an ovarian cDNA library made from an immature female fish. A clone was obtained that contained an open reading frame encoding a 575-amino-acid protein with a deduced molecular weight of 63.9 kDa. Maximum parsimony and Neighbor Joining analyses were used to generate a phylogenetic classification of channel catfish ERβ on the basis of 25 full-length teleost and tetrapod ER sequences. The consensus tree obtained indicated the existence of two major vertebrate ER subtypes, α and β. Within each subtype, and in accordance with established phylogenetic relationships, teleost and tetrapod ER were monophyletic confirming the results of a previous analysis (Z. Xiaet al., 1999, Gen. Comp. Endocrinol. 113, 360–368). Extracts of COS-7 cells transfectedwith channel catfish ERβ cDNA bound estrogen with high affinity (Kd = 0.21 nM) and specificity. The affinity of channel catfish ERβ for estrogen was higher than previously reported for channel catfish ERα. As determined by qualitative RT-PCR, the tissue distributions of ERα and ERβ were similar but not identical. Both ER subtypes were present in ovary and testis. ERα was found in all other tissues examined from juvenile and mature fish of both sexes. ERβ was also found in most tissues except, in most cases, whole blood and head kidney. Interestingly, the pattern of expression of ER subtypes in head kidney always corresponded to the pattern in whole blood. In conclusion, we isolated a channel catfish ERβ with ligand-binding affinity and tissue expression patterns different from ERα. Also, we confirmed the validity of our previously proposed general classification scheme for vertebrate ER into α and β subtypes and within each subtype, into teleost and tetrapod clades.

  3. Diverse Kir expression contributes to distinct bimodal distribution of resting potentials and vasotone responses of arterioles.

    PubMed

    Yang, Yuqin; Chen, Fangyi; Karasawa, Takatoshi; Ma, Ke-Tao; Guan, Bing-Cai; Shi, Xiao-Rui; Li, Hongzhe; Steyger, Peter S; Nuttall, Alfred L; Jiang, Zhi-Gen

    2015-01-01

    The resting membrane potential (RP) of vascular smooth muscle cells (VSMCs) is a major determinant of cytosolic calcium concentration and vascular tone. The heterogeneity of RPs and its underlying mechanism among different vascular beds remain poorly understood. We compared the RPs and vasomotion properties between the guinea pig spiral modiolar artery (SMA), brain arterioles (BA) and mesenteric arteries (MA). We found: 1) RPs showed a robust bimodal distribution peaked at -76 and -40 mV evenly in the SMA, unevenly at -77 and -51 mV in the BA and ~-71 and -52 mV in the MA. Ba(2+) 0.1 mM eliminated their high RP peaks ~-75 mV. 2) Cells with low RP (~-45 mV) hyperpolarized in response to 10 mM extracellular K(+), while cells with a high RP depolarized, and cells with intermediate RP (~-58 mV) displayed an initial hyperpolarization followed by prolonged depolarization. Moderate high K(+) typically induced dilation, constriction and a dilation followed by constriction in the SMA, MA and BA, respectively. 3) Boltzmann-fit analysis of the Ba(2+)-sensitive inward rectifier K(+) (Kir) whole-cell current showed that the maximum Kir conductance density significantly differed among the vessels, and the half-activation voltage was significantly more negative in the MA. 4) Corresponding to the whole-cell data, computational modeling simulated the three RP distribution patterns and the dynamics of RP changes obtained experimentally, including the regenerative swift shifts between the two RP levels after reaching a threshold. 5) Molecular works revealed strong Kir2.1 and Kir2.2 transcripts and Kir2.1 immunolabeling in all 3 vessels, while Kir2.3 and Kir2.4 transcript levels varied. We conclude that a dense expression of functional Kir2.X channels underlies the more negative RPs in endothelial cells and a subset of VSMC in these arterioles, and the heterogeneous Kir function is primarily responsible for the distinct bimodal RPs among these arterioles. The fast Kir

  4. Discriminating cellular heterogeneity using microwell-based RNA cytometry

    PubMed Central

    Dimov, Ivan K.; Lu, Rong; Lee, Eric P.; Seita, Jun; Sahoo, Debashis; Park, Seung-min; Weissman, Irving L.; Lee, Luke P.

    2014-01-01

    Discriminating cellular heterogeneity is important for understanding cellular physiology. However, it is limited by the technical difficulties of single-cell measurements. Here, we develop a two-stage system to determine cellular heterogeneity. In the first stage, we perform multiplex single-cell RNA-cytometry in a microwell array containing over 60,000 reaction chambers. In the second stage, we use the RNA-cytometry data to determine cellular heterogeneity by providing a heterogeneity likelihood score. Moreover, we use Monte-Carlo simulation and RNA-cytometry data to calculate the minimum number of cells required for detecting heterogeneity. We applied this system to characterize the RNA distributions of aging related genes in a highly purified mouse hematopoietic stem cell population. We identified genes that reveal novel heterogeneity of these cells. We also show that changes in expression of genes such as Birc6 during aging can be attributed to the shift of relative portions of cells in the high-expressing subgroup versus low-expressing subgroup. PMID:24667995

  5. Cellular Bases of Light-regulated Gravity Responses

    NASA Technical Reports Server (NTRS)

    Roux, Stanley J.

    2003-01-01

    This report summarizes the most significant research accomplished in our NAG2-1347 project on the cellular bases of light-regulated gravity responses, It elaborates mainly on our discovery of the role of calcium currents in gravity-directed polar development in single germinating spore cells of the fern Ceratopteris, our development of RNA silencing as a viable method of suppressing the expression of specific genes in Ceratopteris, and on the structure, expression and distribution of members of the annexin family in flowering plants, especially Arabidopsis.

  6. Fabrication of cellular materials

    NASA Astrophysics Data System (ADS)

    Prud'homme, Robert K.; Aksay, Ilhan A.; Garg, Rajeev

    1996-02-01

    Nature uses cellular materials in applications requiring strength while, simultaneously, minimizing raw materials requirements. Minimizing raw materials is efficient both in terms of the energy expended by the organism to synthesize the structure and in terms of the strength- to-weight ratio of the structure. Wood is the most obvious example of cellular bio-materials, and it is the focus of other presentations in this symposium. The lightweight bone structure of birds is another excellent example where weight is a key criterion. The anchoring foot of the common muscle [Mytilus edulis] whereby it attaches itself to objects is a further example of a biological system that uses a foam to fill space and yet conserve on raw materials. In the case of the muscle the foam is water filled and the foot structure distributes stress over a larger area so that the strength of the byssal thread from which it is suspended is matched to the strength of interfacial attachment of the foot to a substrate. In these examples the synthesis and fabrication of the cellular material is directed by intercellular, genetically coded, biochemical reactions. The resulting cell sizes are microns in scale. Cellular materials at the next larger scale are created by organisms at the next higher level of integration. For example an African tree frog lays her eggs in a gas/fluid foam sack she builds on a branch overhanging a pond. The outside of the foam sack hardens in the sun and prevents water evaporation. The foam structure minimizes the amount of fluid that needs to be incorporated into the sack and minimizes its weight. However, as far as the developing eggs are concerned, they are in an aqueous medium, i.e. the continuous fluid phase of the foam. After precisely six days the eggs hatch, and the solidified outer wall re-liquefies and dumps the emerging tadpoles into the pond below. The bee honeycomb is an example of a cellular material with exquisite periodicity at millimeter length scales. The

  7. Age-associated reduction of cellular spreading/mechanical force up-regulates matrix metalloproteinase-1 expression and collagen fibril fragmentation via c-Jun/AP-1 in human dermal fibroblasts.

    PubMed

    Qin, Zhaoping; Voorhees, John J; Fisher, Gary J; Quan, Taihao

    2014-12-01

    The dermal compartment of human skin is largely composed of dense collagen-rich fibrils, which provide structural and mechanical support. Skin dermal fibroblasts, the major collagen-producing cells, are interact with collagen fibrils to maintain cell spreading and mechanical force for function. A characteristic feature of aged human skin is fragmentation of collagen fibrils, which is initiated by matrix metalloproteinase 1 (MMP-1). Fragmentation impairs fibroblast attachment and thereby reduces spreading. Here, we investigated the relationship among fibroblast spreading, mechanical force, MMP-1 expression, and collagen fibril fragmentation. Reduced fibroblast spreading due to cytoskeletal disruption was associated with reduced cellular mechanical force, as determined by atomic force microscopy. These reductions substantially induced MMP-1 expression, which led to collagen fibril fragmentation and disorganization in three-dimensional collagen lattices. Constraining fibroblast size by culturing on slides coated with collagen micropatterns also significantly induced MMP-1 expression. Reduced spreading/mechanical force induced transcription factor c-Jun and its binding to a canonical AP-1 binding site in the MMP-1 proximal promoter. Blocking c-Jun function with dominant negative mutant c-Jun significantly reduced induction of MMP-1 expression in response to reduced spreading/mechanical force. Furthermore, restoration of fibroblast spreading/mechanical force led to decline of c-Jun and MMP-1 levels and eliminated collagen fibril fragmentation and disorganization. These data reveal a novel mechanism by which alteration of fibroblast shape/mechanical force regulates c-Jun/AP-1-dependent expression of MMP-1 and consequent collagen fibril fragmentation. This mechanism provides a foundation for understanding the cellular and molecular basis of age-related collagen fragmentation in human skin.

  8. Molecular Characterization, Tissue Distribution and Expression, and Potential Antiviral Effects of TRIM32 in the Common Carp (Cyprinus carpio)

    PubMed Central

    Wang, Yeda; Li, Zeming; Lu, Yuanan; Hu, Guangfu; Lin, Li; Zeng, Lingbing; Zhou, Yong; Liu, Xueqin

    2016-01-01

    Tripartite motif-containing protein 32 (TRIM32) belongs to the tripartite motif (TRIM) family, which consists of a large number of proteins containing a RING (Really Interesting New Gene) domain, one or two B-box domains, and coiled coil motif followed by different C-terminal domains. The TRIM family is known to be implicated in multiple cellular functions, including antiviral activity. However, it is presently unknown whether TRIM32 of common carp (Cyprinus carpio) has the antiviral effect. In this study, the sequence, expression, and antiviral function of TRIM32 homolog from common carp were analyzed. The full-length coding sequence region of trim32 was cloned from common carp. The results showed that the expression of TRIM32 (mRNA) was highest in the brain, remained stably expressed during embryonic development, and significantly increased following spring viraemia of carp virus (SVCV) infection. Transient overexpression of TRIM32 in affected Epithelioma papulosum cyprinid cells led to significant decrease of SVCV production as compared to the control group. These results suggested a potentially important role of common carp TRIM32 in enhancing host immune response during SVCV infection both in vivo and in vitro. PMID:27735853

  9. Therapeutic efficacy of PD-L1 blockade in a breast cancer model is enhanced by cellular vaccines expressing B7-1 and glycolipid-anchored IL-12

    PubMed Central

    Bozeman, Erica N; He, Sara; Shafizadeh, Yalda; Selvaraj, Periasamy

    2016-01-01

    Immunotherapeutic approaches have emerged as promising strategies to treat various cancers, including breast cancer. A single approach, however, is unlikely to effectively combat the complex, immune evasive strategies found within the tumor microenvironment, thus novel, effective combination treatments must be explored. In this study, we investigated the efficacy of a combination therapy consisting of PD-L1 immune checkpoint blockade and whole cell vaccination in a HER-2 positive mouse model of breast cancer. We demonstrate that tumorigenicity is completely abrogated when adjuvanted with immune stimulatory molecules (ISMs) B7-1 and a cell-surface anchored (GPI) form of IL-12 or GM-CSF. Irradiated cellular vaccines expressing the combination of adjuvants B7-1 and GPI-IL-12 completely inhibited tumor formation which was correlative with robust HER-2 specific CTL activity. However, in a therapeutic setting, both cellular vaccination and PD-L1 blockade induced only 10–20% tumor regression when administered alone but resulted in 50% tumor regression as a combination therapy. This protection was significantly hindered following CD4 or CD8 depletion indicating the essential role played by cellular immunity. Collectively, these pre-clinical studies provide a strong rationale for further investigation into the efficacy of combination therapy with tumor cell vaccines adjuvanted with membrane-anchored ISMs along with PD-L1 blockade for the treatment of breast cancer. PMID:26308597

  10. The regular distribution and expression pattern of immunosuppressive cytokine IL-35 in mouse uterus during early pregnancy.

    PubMed

    Jin, Erhui; Wang, Chenfang; Hu, Qianqian; Jin, Guangming; Li, Shenghe

    2014-01-01

    Cytokines within the uterus are critical in the maternal-fetal immune regulation. Immunosuppressive cytokine IL-35 was recently discovered inhibitory cytokine, which were pivotal in the establishment of immune tolerance against self-antigens and antigens encountered in foreign implantation. In order to analyze the role of IL-35 in maternal-fetal immune tolerance, the expression patterns of IL-35 in mouse endometrium were studied during early pregnancy by immunohistochemistry, ELISA and quantitative real-time PCR. As results, we found that IL-35 positive cells in the uterus showed significant distribution difference after fetal implantation, which mainly distributed in luminal epithelium and glandular epithelium of mouse uterus from gestational day 1 to 2, and glandular epithelium and stroma from gestational day 4 to 7. The number of positive cells, immunoreactive scores, protein and mRNA expression of IL-35 showed firstly increased and then decreased with the increase of pregnancy day. The largest contents of IL-35 in the uterus were detected on gestational day 4. Compared with non-pregnant mice, pregnant mice showed the significantly increased mRNA expression of Ebi3 (Epstein-Barr virus-induced gene 3, IL-35 subunit) in the endometrium on gestational day 2 and the highest level of expression on gestational day 4. The mRNA expression of p35 (IL-35 subunit) was significantly lower than that of Ebi3 gene and showed the inconsistent change from gestational day 5 to 7. However, the significant correlation existed between the immunohistochemical expression, contents and mRNA expression of IL-35. These results indicated that IL-35 contributed to the establishment and maintenance of maternal-fetal tolerance during early pregnancy.

  11. Multiscale Modeling of Antibody-Drug Conjugates: Connecting Tissue and Cellular Distribution to Whole Animal Pharmacokinetics and Potential Implications for Efficacy.

    PubMed

    Cilliers, Cornelius; Guo, Hans; Liao, Jianshan; Christodolu, Nikolas; Thurber, Greg M

    2016-09-01

    Antibody-drug conjugates exhibit complex pharmacokinetics due to their combination of macromolecular and small molecule properties. These issues range from systemic concerns, such as deconjugation of the small molecule drug during the long antibody circulation time or rapid clearance from nonspecific interactions, to local tumor tissue heterogeneity, cell bystander effects, and endosomal escape. Mathematical models can be used to study the impact of these processes on overall distribution in an efficient manner, and several types of models have been used to analyze varying aspects of antibody distribution including physiologically based pharmacokinetic (PBPK) models and tissue-level simulations. However, these processes are quantitative in nature and cannot be handled qualitatively in isolation. For example, free antibody from deconjugation of the small molecule will impact the distribution of conjugated antibodies within the tumor. To incorporate these effects into a unified framework, we have coupled the systemic and organ-level distribution of a PBPK model with the tissue-level detail of a distributed parameter tumor model. We used this mathematical model to analyze new experimental results on the distribution of the clinical antibody-drug conjugate Kadcyla in HER2-positive mouse xenografts. This model is able to capture the impact of the drug-antibody ratio (DAR) on tumor penetration, the net result of drug deconjugation, and the effect of using unconjugated antibody to drive ADC penetration deeper into the tumor tissue. This modeling approach will provide quantitative and mechanistic support to experimental studies trying to parse the impact of multiple mechanisms of action for these complex drugs. PMID:27287046

  12. Transcriptional Activity, Chromosomal Distribution and Expression Effects of Transposable Elements in Coffea Genomes

    PubMed Central

    da Silva, Carlos R. M.; Andrade, Alan C.; Marraccini, Pierre; Teixeira, João B.; Carazzolle, Marcelo F.; Pereira, Gonçalo A. G.; Pereira, Luiz Filipe P.; Vanzela, André L. L.; Wang, Lu; Jordan, I. King; Carareto, Claudia M. A.

    2013-01-01

    Plant genomes are massively invaded by transposable elements (TEs), many of which are located near host genes and can thus impact gene expression. In flowering plants, TE expression can be activated (de-repressed) under certain stressful conditions, both biotic and abiotic, as well as by genome stress caused by hybridization. In this study, we examined the effects of these stress agents on TE expression in two diploid species of coffee, Coffea canephora and C. eugenioides, and their allotetraploid hybrid C. arabica. We also explored the relationship of TE repression mechanisms to host gene regulation via the effects of exonized TE sequences. Similar to what has been seen for other plants, overall TE expression levels are low in Coffea plant cultivars, consistent with the existence of effective TE repression mechanisms. TE expression patterns are highly dynamic across the species and conditions assayed here are unrelated to their classification at the level of TE class or family. In contrast to previous results, cell culture conditions per se do not lead to the de-repression of TE expression in C. arabica. Results obtained here indicate that differing plant drought stress levels relate strongly to TE repression mechanisms. TEs tend to be expressed at significantly higher levels in non-irrigated samples for the drought tolerant cultivars but in drought sensitive cultivars the opposite pattern was shown with irrigated samples showing significantly higher TE expression. Thus, TE genome repression mechanisms may be finely tuned to the ideal growth and/or regulatory conditions of the specific plant cultivars in which they are active. Analysis of TE expression levels in cell culture conditions underscored the importance of nonsense-mediated mRNA decay (NMD) pathways in the repression of Coffea TEs. These same NMD mechanisms can also regulate plant host gene expression via the repression of genes that bear exonized TE sequences. PMID:24244387

  13. mRNA expression profile of serotonin receptor subtypes and distribution of serotonergic terminations in marmoset brain

    PubMed Central

    Shukla, Rammohan; Watakabe, Akiya; Yamamori, Tetsuo

    2014-01-01

    To better understand serotonin function in the primate brain, we examined the mRNA expression patterns of all the 13 members of the serotonin receptor (5HTR) family, by in situ hybridization (ISH) and the distribution of serotonergic terminations by serotonin transporter (SERT) protein immunohistochemical analysis. Ten of the 13 5HTRs showed significant mRNA expressions in the marmoset brain. Our study shows several new features of the organization of serotonergic systems in the marmoset brain. (1) The thalamus expressed only a limited number of receptor subtypes compared with the cortex, hippocampus, and other subcortical regions. (2) In the cortex, there are layer-selective and area-selective mRNA expressions of 5HTRs. (3) Highly localized mRNA expressions of 5HT1F and 5HT3A were observed. (4) There was a conspicuous overlap of the mRNA expressions of receptor subtypes known to have somatodendritic localization of receptor proteins with dense serotonergic terminations in the visual cortex, the central lateral (CL) nucleus of the thalamus, the presubiculum, and the medial mammillary nucleus of the hypothalamus. This suggests a high correlation between serotonin availability and receptor expression at these locations. (5) The 5HTRs show differences in mRNA expression pattern between the marmoset and mouse cortices whereas the patterns of both the species were much similar in the hippocampus. We discuss the possible roles of 5HTRs in the marmoset brain revealed by the analysis of their overall mRNA expression patterns. PMID:24904298

  14. Establishment of a luciferase assay-based screening system: Fumitremorgin C selectively inhibits cellular proliferation of immortalized astrocytes expressing an active form of AKT

    SciTech Connect

    Wang Lei; Sasai, Ken Akagi, Tsuyoshi; Tanaka, Shinya

    2008-08-29

    The AKT pathway is frequently activated in glioblastoma, and as such, inhibitors of this pathway could prove very useful as anti-glioblastoma therapies. Here we established immortalized astrocytes expressing Renilla luciferase as well as those expressing both an active form of AKT and firefly luciferase. Since both luciferase activities represent the numbers of corresponding cell lines, novel inhibitors of the AKT pathway can be identified by treating co-cultures containing the two types of luciferase-expressing cells with individual compounds. Indeed, such a screening system succeeded in identifying fumitremorgin C as an efficient inhibitor of the AKT pathway, which was further confirmed by the ability of fumitremorgin C to selectively inhibit the growth of immortalized astrocytes expressing an active form of AKT. The present study proposes a broadly applicable approach for identifying therapeutic agents that target the pathways and/or molecules responsible for cancer development.

  15. Immunohistochemical Expression of Estrogen and Progesterone Receptors in Human Colorectal Adenoma and Carcinoma Using Specified Automated Cellular Image Analysis System: A Clinicopathological Study

    PubMed Central

    Qasim, Ban Jumaa; Ali, Hussam Hasson; Hussein, Alaa Ghani

    2011-01-01

    Objectives To evaluate the immunohistochemical expression of estrogen receptors (ER) and progesterone receptors (PR) in colorectal adenoma and adenocarcinoma and to correlate this immunohistochemical expression with different clinicopathological parameters. Methods The study was retrospectively designed. A total of 86 tissue samples, including 33 paraffin blocks from patients with colorectal adenomas, 33 paraffin blocks from patients with colorectal adenocarcinomas and a control group of 20 samples of non-tumorous colonic tissue, were included in the study. Results The frequency of expression of ER and PR showed a gradual increase from control through adenoma to carcinoma. The frequencies of expression of ER in the control, adenoma and carcinoma were (10%, 15.15% and 42.42% respectively, p<0.001), while the frequency of expression for PR were (10%, 24.24% and 36.36% respectively, p<0.001). Strong ER and PR staining was mainly seen in carcinoma cases (42.42%, 36.36%, respectively) in comparison with adenoma (9.09%, 15.15%, respectively) and control (0%, 0%, respectively). The three digital parameters of ER and PR immunohistochemical expression (Area [A], Number of objects [N], and intensity [I]) were significantly increased in a sequence of normal mucosa-adenoma-carcinoma. There was a significant positive correlation between ER and PR in adenoma (r=0.312, p=0.034) and carcinoma (r=0.321, p=0.0398). Conclusion ER and PR expression increased in a sequence of; normal colonic mucosa-adenoma-carcinoma, and a positive correlation was observed between ER and PR expression in colonic adenoma and carcinoma specimen indicating that ER and PR may play a role in colorectal carcinogenesis. PMID:22125723

  16. Metal-induced stress in bivalves living along a gradient of Cd contamination: relating sub-cellular metal distribution to population-level responses.

    PubMed

    Perceval, Olivier; Couillard, Yves; Pinel-Alloul, Bernadette; Giguère, Anik; Campbell, Peter G C

    2004-09-20

    The use of biomarkers to assess the impacts of contaminants on aquatic ecosystems has noticeably increased over the past few years. Few of these studies, however, have contributed to the prediction of ecologically significant effects (i.e., at the population or community levels). The present field study was designed to evaluate the potential of metallothionein (MT) and sub-cellular metal partitioning measurements for predicting toxic effects at higher levels of the biological organization in freshwater bivalves (Pyganodon grandis) chronically exposed to Cd. For that purpose, we quantitatively sampled P. grandis populations in the littoral zone of nine lakes on the Precambrian Canadian Shield during two consecutive summers (1998 and 1999); lakes were characterized by contrasting Cd levels but similar trophic status. We tested relationships between the population status of P. grandis (i.e., growth parameters, density, biomass, secondary production, turnover ratio and cumulative fecundity) and (i) ambient Cd concentrations, (ii) sub-organismal responses (MT concentrations in the gill cytosol of individuals and Cd concentrations in three metal-ligand pools identified as M-HMW, the high molecular weight pool, M-MT, the metallothionein-like pool and M-LMW, the low molecular weight pool) and (iii) ecological confounding factors (food resources, presence of host fishes for the obligatory parasitic larval stage of P. grandis). Our results show that littoral density, live weight, dry viscera biomass, production and cumulative fecundity decreased with increasing concentrations of the free-cadmium ion in the environment (Pearson's r ranging from -0.63 to -0.78). On the other hand, theoretical maximum shell lengths (L( infinity )) in our populations were related to both the dissolved Ca concentration and food quality (sestonic C and N concentrations). Overall, Cd concentrations in the gill cytosolic HMW pool of the individual molluscs were the biomarker response that was most

  17. Metal-induced stress in bivalves living along a gradient of Cd contamination: relating sub-cellular metal distribution to population-level responses.

    PubMed

    Perceval, Olivier; Couillard, Yves; Pinel-Alloul, Bernadette; Giguère, Anik; Campbell, Peter G C

    2004-09-20

    The use of biomarkers to assess the impacts of contaminants on aquatic ecosystems has noticeably increased over the past few years. Few of these studies, however, have contributed to the prediction of ecologically significant effects (i.e., at the population or community levels). The present field study was designed to evaluate the potential of metallothionein (MT) and sub-cellular metal partitioning measurements for predicting toxic effects at higher levels of the biological organization in freshwater bivalves (Pyganodon grandis) chronically exposed to Cd. For that purpose, we quantitatively sampled P. grandis populations in the littoral zone of nine lakes on the Precambrian Canadian Shield during two consecutive summers (1998 and 1999); lakes were characterized by contrasting Cd levels but similar trophic status. We tested relationships between the population status of P. grandis (i.e., growth parameters, density, biomass, secondary production, turnover ratio and cumulative fecundity) and (i) ambient Cd concentrations, (ii) sub-organismal responses (MT concentrations in the gill cytosol of individuals and Cd concentrations in three metal-ligand pools identified as M-HMW, the high molecular weight pool, M-MT, the metallothionein-like pool and M-LMW, the low molecular weight pool) and (iii) ecological confounding factors (food resources, presence of host fishes for the obligatory parasitic larval stage of P. grandis). Our results show that littoral density, live weight, dry viscera biomass, production and cumulative fecundity decreased with increasing concentrations of the free-cadmium ion in the environment (Pearson's r ranging from -0.63 to -0.78). On the other hand, theoretical maximum shell lengths (L( infinity )) in our populations were related to both the dissolved Ca concentration and food quality (sestonic C and N concentrations). Overall, Cd concentrations in the gill cytosolic HMW pool of the individual molluscs were the biomarker response that was most

  18. Estrogen Receptor Alpha Distribution and Expression in the Social Neural Network of Monogamous and Polygynous Peromyscus.

    PubMed

    Cushing, Bruce S

    2016-01-01

    In microtine and dwarf hamsters low levels of estrogen receptor alpha (ERα) in the bed nucleus of the stria terminalis (BST) and medial amygdala (MeA) play a critical role in the expression of social monogamy in males, which is characterized by high levels of affiliation and low levels of aggression. In contrast, monogamous Peromyscus males display high levels of aggression and affiliative behavior with high levels of testosterone and aromatase activity. Suggesting the hypothesis that in Peromyscus ERα expression will be positively correlated with high levels of male prosocial behavior and aggression. ERα expression was compared within the social neural network, including the posterior medial BST, MeA posterodorsal, medial preoptic area (MPOA), ventromedial hypothalamus (VMH), and arcuate nucleus in two monogamous species, P. californicus and P. polionotus, and two polygynous species, P. leucopus and P. maniculatus. The results supported the prediction, with male P. polionotus and P. californicus expressing higher levels of ERα in the BST than their polygynous counter parts, and ERα expression was sexually dimorphic in the polygynous species, with females expressing significantly more than males in the BST in both polygynous species and in the MeA in P. leucopus. Peromyscus ERα expression also differed from rats, mice and microtines as in neither the MPOA nor the VMH was ERα sexually dimorphic. The results supported the hypothesis that higher levels of ERα are associated with monogamy in Peromyscus and that differential expression of ERα occurs in the same regions of the brains regardless of whether high or low expression is associated with social monogamy. Also discussed are possible mechanisms regulating this differential relationship. PMID:26959827

  19. Estrogen Receptor Alpha Distribution and Expression in the Social Neural Network of Monogamous and Polygynous Peromyscus.

    PubMed

    Cushing, Bruce S

    2016-01-01

    In microtine and dwarf hamsters low levels of estrogen receptor alpha (ERα) in the bed nucleus of the stria terminalis (BST) and medial amygdala (MeA) play a critical role in the expression of social monogamy in males, which is characterized by high levels of affiliation and low levels of aggression. In contrast, monogamous Peromyscus males display high levels of aggression and affiliative behavior with high levels of testosterone and aromatase activity. Suggesting the hypothesis that in Peromyscus ERα expression will be positively correlated with high levels of male prosocial behavior and aggression. ERα expression was compared within the social neural network, including the posterior medial BST, MeA posterodorsal, medial preoptic area (MPOA), ventromedial hypothalamus (VMH), and arcuate nucleus in two monogamous species, P. californicus and P. polionotus, and two polygynous species, P. leucopus and P. maniculatus. The results supported the prediction, with male P. polionotus and P. californicus expressing higher levels of ERα in the BST than their polygynous counter parts, and ERα expression was sexually dimorphic in the polygynous species, with females expressing significantly more than males in the BST in both polygynous species and in the MeA in P. leucopus. Peromyscus ERα expression also differed from rats, mice and microtines as in neither the MPOA nor the VMH was ERα sexually dimorphic. The results supported the hypothesis that higher levels of ERα are associated with monogamy in Peromyscus and that differential expression of ERα occurs in the same regions of the brains regardless of whether high or low expression is associ