Biological activity of cloned mammary tumor virus DNA fragments that bind purified glucocorticoid receptor protein in vitro

International Nuclear Information System (INIS)

Yamamoto, K.R.; Payvar, F.; Firestone, G.L.; Maler, B.A.; Wrange, O.; Carlstedt-Duke, J.; Gustafsson, J.A.; Chandler, V.L.; Karolinska Institutet, Stockholm, Sweden)

1983-01-01

To test whether high-affinity receptor:DNA interactions can be correlated with receptor effects on promoter function in vivo, we have mapped in greater detail the receptor-binding regions on murine mammary tumor virus DNA, using both nitrocellulose-filter binding and electron microscopy. Recombinant plasmids bearing these receptor-binding domains have been transfected into cultured cells, and the expression of the plasmid sequences has been monitored for hormonal regulation. The results are considered in terms of a speculative proposal that the glucocorticoid receptor may effect changes in promoter activity via specific alteration of chromatin and/or DNA structure. 37 references, 6 figures, 2 tables

Cloning of the cDNA and gene for a human D2 dopamine receptor

International Nuclear Information System (INIS)

Grady, D.K.; Makam, H.; Stofko, R.E.; Bunzow, J.R.; Civelli, O.; Marchionni, M.A.; Alfano, M.; Frothingham, L.; Fischer, J.B.; Burke-Howie, K.J.; Server, A.C.

1989-01-01

A clone encoding a human D 2 dopamine receptor was isolated from a pituitary cDNA library and sequenced. The deduced protein sequence is 96% identical with that of the cloned rat receptor with one major difference: the human receptor contains an additional 29 amino acids in its putative third cytoplasmic loop. Southern blotting demonstrated the presence of only one human D 2 receptor gene. Two overlapping phage containing the gene were isolated and characterized. DNA sequence analysis of these clones showed that the coding sequence is interrupted by six introns and that the additional amino acids present in the human pituitary receptor are encoded by a single exon of 87 base pairs. The involvement of this sequence in alternative splicing and its biological significance are discussed

Oxygen dependency of epidermal growth factor receptor binding and DNA synthesis of rat hepatocytes

International Nuclear Information System (INIS)

Hirose, Tetsuro; Terajima, Hiroaki; Yamauchi, Akira

1997-01-01

Background/Aims: Changes in oxygen availability modulate replicative responses in several cell types, but the effects on hepatocyte replication remain unclear. We have studied the effects of transient nonlethal hypoxia on epidermal growth factor receptor binding and epidermal growth factor-induced DNA synthesis of rat hepatocytes. Methods: Lactate dehydrogenase activity in culture supernatant, intracellular adenosine triphosphate content, 125 I-epidermal growth factor specific binding, epidermal growth factor receptor protein expression, and 3 H-thymidine incorporation were compared between hepatocytes cultured in hypoxia and normoxia. Results: Hypoxia up to 3 h caused no significant increase in lactate dehydrogenase activity in the culture supernatant, while intracellular adenosine triphosphate content decreased time-dependently and was restored to normoxic levels by reoxygenation (nonlethal hypoxia). Concomitantly, 125 I-epidermal growth factor specific binding to hepatocytes decreased time-dependently (to 54.1% of normoxia) and was restored to control levels by reoxygenation, although 125 I-insulin specific binding was not affected. The decrease in 125 I-epidermal growth factor specific binding was explained by the decrease in the number or available epidermal growth factor receptors (21.37±3.08 to 12.16±1.42 fmol/10 5 cells), while the dissociation constant of the receptor was not affected. The change in the number of available receptors was not considered to be due to receptor degradation-resynthesis, since immuno-detection of the epidermal growth factor receptor revealed that the receptor protein expression did not change during hypoxia and reoxygenation, and since neither actinomycin D nor cycloheximide affected the recovery of 125 I-epidermal growth factor binding by reoxygenation. Inhibition of epidermal growth factor-induced DNA synthesis after hypoxia (to 75.4% of normoxia by 3 h hypoxia) paralleled the decrease in 125 I-epidermal growth factor binding

Cloning of the γ-aminobutyric acid (GABA) ρ1 cDNA: A GABA receptor subunit highly expressed in the retina

International Nuclear Information System (INIS)

Cutting, G.R.; Lu, Luo; Kasch, L.M.; Montrose-Rafizadeh, C.; Antonarakis, S.E.; Guggino, W.B.; Kazazian, H.H. Jr.; O'Hara, B.F.; Donovan, D.M.; Shimada, Shoichi; Uhl, G.R.

1991-01-01

Type A γ-aminobutyric acid (GABA A ) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. The authors have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence is 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABA A subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA ρ 1 , with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family

Purine receptor P2Y_6 mediates cellular response to γ-ray-induced DNA damage

International Nuclear Information System (INIS)

Ide, Shunta; Nishimaki, Naoko; Tsukimoto, Mitsutoshi; Kojima, Shuji

2014-01-01

We previously showed that nucleotide P2 receptor agonists such as ATP and UTP amplify γ-ray-induced focus formation of phosphorylated histone H2A variant H2AX (γH2AX), which is considered to be an indicator of DNA damage so far, by activating purine P2Y_6 and P2Y_1_2 receptors. Therefore, we hypothesized that these P2 receptors play a role in inducing the repair response to γ-ray-induced DNA damage. In the present study, we tested this idea by using human lung cancer A549 cells. First, reverse-transcription polymerase chain reaction (RT-PCR) showed that P2Y_6 receptor is highly expressed in A549 cells, but P2Y_1_2 receptor is only weakly expressed. Next, colony formation assay revealed that P2Y_6 receptor antagonist MRS2578 markedly reduced the survival rate of γ-ray-exposed A549 cells. The survival rate was also significantly reduced in P2Y_6-knock-down cells, compared with scramble siRNA-transfected cells. Since it has reported that phosphorylation of ERK1/2 after activation of EGFR via P2Y_6 and P2Y_1_2 receptors is involved in the repair response to γ-ray-induced DNA damage, we next examined whether γ-ray-induced phosphorylation of ERK1/2 was also inhibited by MRS2578 in A549 cells. We found that it was. Taken together, these findings indicate that purinergic signaling through P2Y_6 receptor, followed by ERK1/2 activation, promotes the cellular repair response to γ-ray-induced DNA damage. (author)

Increased NMDA receptor inhibition at an increased Sevoflurane MAC

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Brosnan Robert J

2012-06-01

Full Text Available Abstract Background Sevoflurane potently enhances glycine receptor currents and more modestly decreases NMDA receptor currents, each of which may contribute to immobility. This modest NMDA receptor antagonism by sevoflurane at a minimum alveolar concentration (MAC could be reciprocally related to large potentiation of other inhibitory ion channels. If so, then reduced glycine receptor potency should increase NMDA receptor antagonism by sevoflurane at MAC. Methods Indwelling lumbar subarachnoid catheters were surgically placed in 14 anesthetized rats. Rats were anesthetized with sevoflurane the next day, and a pre-infusion sevoflurane MAC was measured in duplicate using a tail clamp method. Artificial CSF (aCSF containing either 0 or 4 mg/mL strychnine was then infused intrathecally at 4 μL/min, and the post-infusion baseline sevoflurane MAC was measured. Finally, aCSF containing strychnine (either 0 or 4 mg/mL plus 0.4 mg/mL dizocilpine (MK-801 was administered intrathecally at 4 μL/min, and the post-dizocilpine sevoflurane MAC was measured. Results Pre-infusion sevoflurane MAC was 2.26%. Intrathecal aCSF alone did not affect MAC, but intrathecal strychnine significantly increased sevoflurane requirement. Addition of dizocilpine significantly decreased MAC in all rats, but this decrease was two times larger in rats without intrathecal strychnine compared to rats with intrathecal strychnine, a statistically significant (P  Conclusions Glycine receptor antagonism increases NMDA receptor antagonism by sevoflurane at MAC. The magnitude of anesthetic effects on a given ion channel may therefore depend on the magnitude of its effects on other receptors that modulate neuronal excitability.

Overexpression of insulin-like growth factor (IGF)-I receptor enhances inhibition of DNA replication in mouse cells exposed to x-rays

International Nuclear Information System (INIS)

Wang, Y.; Cheong, N.; Miura, M.; Iliakis, G.

1997-01-01

Previous studies from our laboratory provided evidence for the operation of signal transduction pathways involving ras, myc, and staurosporine-sensitive protein kinases in the regulation of DNA replication in irradiated cells. Because ras and myc are also involved in the signal transduction elicited in response to ligand activation of growth factor receptors, we wondered whether growth factor receptors are upstream elements in the regulation of DNA replication in irradiated cells. Here, we report on the role of insulin-like growth factor I receptor (IGF-IR) in the regulation of DNA replication in irradiated cells. We compare radiation-induced inhibition of DNA replication in BALB/c 3T3 cells with that in P6 cells. P6 cells are derived from BALB/c 3T3 cells by transfection with a vector expressing IGF-IR, leading to 30-fold overexpression. We observe a significantly stronger inhibition of DNA replication after irradiation in P6 as compared with BALB/c 3T3 cells at all doses examined. Sedimentation in alkaline sucrose gradients shows that the increased inhibition in P6 cells is due to an increased inhibition of replicon initiation, the main controlling event in DNA replication. Staurosporine at 20 nM reduces radiation-induced inhibition of DNA replication in BALB/c 3T3 cells, but has only a small effect in P6 cells. Caffeine at a concentration of 1 mM, on the other hand, removes over 60% of the inhibition in both cell lines. The results implicate IGF-IR in the regulation of DNA replication in irradiated cells, but also suggest differences between cells of different origins in the proteins involved in the regulating signal transduction pathway. (orig.). With 5 figs

Transferrin-polycation-mediated introduction of DNA into human leukemic cells: Stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels

International Nuclear Information System (INIS)

Cotten, M.; Laengle-Rouault, F.; Kirlappos, H.; Wagner, E.; Mechtler, K.; Zenke, M.; Beug, H.; Birnstiel, M.L.

1990-01-01

The authors have subverted a receptor-mediated endocytosis event to transport genes into human leukemic cells. By coupling the natural iron-delivery protein transferrin to the DNA-binding polycations polylysine or protamine, they have created protein conjugates that bind nucleic acids and carry them into the cell during the normal transferrin cycle. They demonstrate here that this procedure is useful for a human leukemic cell line. They enhanced the rate of gene delivery by (i) increasing the transferrin receptor density through treatment of the cells with the cell permeable iron chelator desferrioxamine, (ii) interfering with the synthesis of heme with succinyl acetone treatment, or (iii) stimulating the degradation of heme with cobalt chloride treatment. Consistent with gene delivery as an endocytosis event, they show that the subsequent expression in K-562 cells of a gene included in the transported DNA depends upon the cellular presence of the lysosomotropic agent chloroquine. By contrast, monensin blocks transferrinfection, as does incubation of the cells at 18 degree C

Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein

International Nuclear Information System (INIS)

Gray, P.W.; Barrett, K.; Chantry, D.; Turner, M.; Feldmann, M.

1990-01-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extracellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10 -9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ)

Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

Science.gov (United States)

Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

1990-10-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

cDNA cloning and expression of a human platelet-derived growth factor (PDGF) receptor specific for B-chain-containing PDGF molecules

International Nuclear Information System (INIS)

Claesson-Welsh, L.; Eriksson, A.; Moren, A.; Severinsson, L.; Ek, B.; Ostman, A.; Betsholtz, C.; Heldin, C.H.

1988-01-01

The structure of the human receptor for platelet-derived growth factor (PDGF) has been deduced through cDNA cloning. A 5.45-kilobase-pair cDNA clone predicts a 1,106-amino-acid polypeptide, including the cleavable signal sequence. The overall amino acid sequence similarity with the murine PDGFR receptor is 85%. After transcription of the cDNA and translation in vitro, a PDGR receptor antiserum was used to immunoprecipitate a product of predicted size, which also could be phosphorylated in vitro. Stable introduction of the cDNA into Chinese hamster ovary (CHO) cells led to the expression of a 190-kilodalton component, which was immunoprecipitated by the PDGF receptor antiserum; this most probably represents the mature PDGF receptor. Binding assays with different /sup 125/I-labeled dimeric forms of PDGF A and B chains showed that the PDGFR receptor expressed in CHO cells bound PDGF-BB and, to a lesser extent, PDGF-AB, but not PDGF-AA

High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells

International Nuclear Information System (INIS)

Whittaker, J.; Okamoto, A.K.; Thys, R.; Bell, G.I.; Steiner, D.F.; Hofmann, C.A.

1987-01-01

In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10 6 receptors per cell. The cell line with the highest 125 I-insulin binding (NIH 3T3 HIR3.5) had 6 x 10 6 receptors with a K/sub d/ of 10 -9 M. This level was not dependent on exposure to metals but could be increased further to 2 x 10 7 receptors per cell by addition of sodium butyrate to the culture medium. The α and β subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the α and β subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis

Importance of the Sequence-Directed DNA Shape for Specific Binding Site Recognition by the Estrogen-Related Receptor

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Kareem Mohideen-Abdul

2017-06-01

Full Text Available Most nuclear receptors (NRs bind DNA as dimers, either as hetero- or as homodimers on DNA sequences organized as two half-sites with specific orientation and spacing. The dimerization of NRs on their cognate response elements (REs involves specific protein–DNA and protein–protein interactions. The estrogen-related receptor (ERR belongs to the steroid hormone nuclear receptor (SHR family and shares strong similarity in its DNA-binding domain (DBD with that of the estrogen receptor (ER. In vitro, ERR binds with high affinity inverted repeat REs with a 3-bps spacing (IR3, but in vivo, it preferentially binds to single half-site REs extended at the 5′-end by 3 bp [estrogen-related response element (ERREs], thus explaining why ERR was often inferred as a purely monomeric receptor. Since its C-terminal ligand-binding domain is known to homodimerize with a strong dimer interface, we investigated the binding behavior of the isolated DBDs to different REs using electrophoretic migration, multi-angle static laser light scattering (MALLS, non-denaturing mass spectrometry, and nuclear magnetic resonance. In contrast to ER DBD, ERR DBD binds as a monomer to EREs (IR3, such as the tff1 ERE-IR3, but we identified a DNA sequence composed of an extended half-site embedded within an IR3 element (embedded ERRE/IR3, where stable dimer binding is observed. Using a series of chimera and mutant DNA sequences of ERREs and IR3 REs, we have found the key determinants for the binding of ERR DBD as a dimer. Our results suggest that the sequence-directed DNA shape is more important than the exact nucleotide sequence for the binding of ERR DBD to DNA as a dimer. Our work underlines the importance of the shape-driven DNA readout mechanisms based on minor groove recognition and electrostatic potential. These conclusions may apply not only to ERR but also to other members of the SHR family, such as androgen or glucocorticoid, for which a strong well-conserved half

ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

Science.gov (United States)

Karayazi Atici, Ödül; Urbanska, Anna; Gopinathan, Sesha Gopal; Boutillon, Florence; Goffin, Vincent; Shemanko, Carrie S

2018-02-01

Prolactin (PRL) acts as a survival factor for breast cancer cells, but the PRL signaling pathway and the mechanism are unknown. Previously, we identified the master chaperone, heat shock protein 90 (HSP90) α, as a prolactin-Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) target gene involved in survival, and here we investigated the role of HSP90 in the mechanism of PRL-induced viability in response to DNA damage. The ataxia-telangiectasia mutated kinase (ATM) protein plays a critical role in the cellular response to double-strand DNA damage. We observed that PRL increased viability of breast cancer cells treated with doxorubicin or etoposide. The increase in cellular resistance is specific to the PRL receptor, because the PRL receptor antagonist, Δ1-9-G129R-hPRL, prevented the increase in viability. Two different HSP90 inhibitors, 17-allylamino-17-demethoxygeldanamycin and BIIB021, reduced the PRL-mediated increase in cell viability of doxorubicin-treated cells and led to a decrease in JAK2, ATM, and phosphorylated ATM protein levels. Inhibitors of JAK2 (G6) and ATM (KU55933) abolished the PRL-mediated increase in cell viability of DNA-damaged cells, supporting the involvement of each, as well as the crosstalk of ATM with the PRL pathway in the context of DNA damage. Drug synergism was detected between the ATM inhibitor (KU55933) and doxorubicin and between the HSP90 inhibitor (BIIB021) and doxorubicin. Short interfering RNA directed against ATM prevented the PRL-mediated increase in cell survival in two-dimensional cell culture, three-dimensional collagen gel cultures, and clonogenic cell survival, after doxorubicin treatment. Our results indicate that ATM contributes to the PRL-JAK2-STAT5-HSP90 pathway in mediating cellular resistance to DNA-damaging agents. Copyright © 2018 Endocrine Society.

Encoded novel forms of HSP70 or a cytolytic protein increase DNA vaccine potency.

Science.gov (United States)

Garrod, Tamsin; Grubor-Bauk, Branka; Yu, Stanley; Gargett, Tessa; Gowans, Eric J

2014-01-01

In humans, DNA vaccines have failed to demonstrate the equivalent levels of immunogenicity that were shown in smaller animals. Previous studies have encoded adjuvants, predominantly cytokines, within these vaccines in an attempt to increase antigen-specific immune responses. However, these strategies have lacked breadth of innate immune activation and have led to disappointing results in clinical trials. Damage associated molecular patterns (DAMPs) have been identified as pattern recognition receptor (PRR) agonists. DAMPs can bind to a wide range of PRRs on dendritic cells (DCs) and thus our studies have aimed to utilize this characteristic to act as an adjuvant in a DNA vaccine approach. Specifically, HSP70 has been identified as a DAMP, but has been limited by its lack of accessibility to PRRs in and on DCs. Here, we discuss the promising results achieved with the inclusion of membrane-bound or secreted HSP70 into a DNA vaccine encoding HIV gag as the model immunogen.

Hypoxia increases pulmonary arterial thromboxane receptor internalization independent of receptor sensitization.

Science.gov (United States)

Fediuk, J; Sikarwar, A S; Lizotte, P P; Hinton, M; Nolette, N; Dakshinamurti, S

2015-02-01

Persistent Pulmonary Hypertension of the Newborn (PPHN) is characterized by sustained vasospasm and an increased thromboxane:prostacyclin ratio. Thromboxane (TP) receptors signal via Gαq to mobilize IP3 and Ca(2+), causing pulmonary arterial constriction. We have previously reported increased TP internalization in hypoxic pulmonary arterial (PA) myocytes. Serum-deprived PA myocytes were grown in normoxia (NM) or hypoxia (HM) for 72 h. TP localization was visualized in agonist-naïve and -challenged NM and HM by immunocytochemistry. Pathways for agonist-induced TP receptor internalization were determined by inhibiting caveolin- or clathrin-mediated endocytosis, and caveolar fractionation. Roles of actin and tubulin in TP receptor internalization were assessed using inhibitors of tubulin, actin-stabilizing or -destabilizing agents. PKA, PKC or GRK activation and inhibition were used to determine the kinase responsible for post-agonist receptor internalization. Agonist-naïve HM had decreased cell surface TP, and greater TP internalization after agonist challenge. TP protein did not sort with caveolin-rich fractions. Inhibition of clathrin prevented TP internalization. Both actin-stabilizing and -destabilizing agents prevented TP endocytosis in NM, while normalizing TP internalization in HM. Velocity of TP internalization was unaffected by PKA activity, but PKC activation normalized TP receptor internalization in HM. GRK inhibition had no effect. We conclude that in hypoxic myocytes, TP is internalized faster and to a greater extent than in normoxic controls. Internalization of the agonist-challenged TP requires clathrin, dynamic actin and is sensitive to PKC activity. TP receptor trafficking and signaling in hypoxia are pivotal to understanding increased vasoconstrictor sensitivity. Copyright © 2014 Elsevier Ltd. All rights reserved.

DNA methylation of specific CpG sites in the promoter region regulates the transcription of the mouse oxytocin receptor.

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Shimrat Mamrut

Full Text Available Oxytocin is a peptide hormone, well known for its role in labor and suckling, and most recently for its involvement in mammalian social behavior. All central and peripheral actions of oxytocin are mediated through the oxytocin receptor, which is the product of a single gene. Transcription of the oxytocin receptor is subject to regulation by gonadal steroid hormones, and is profoundly elevated in the uterus and mammary glands during parturition. DNA methylation is a major epigenetic mechanism that regulates gene transcription, and has been linked to reduced expression of the oxytocin receptor in individuals with autism. Here, we hypothesized that transcription of the mouse oxytocin receptor is regulated by DNA methylation of specific sites in its promoter, in a tissue-specific manner. Hypothalamus-derived GT1-7, and mammary-derived 4T1 murine cell lines displayed negative correlations between oxytocin receptor transcription and methylation of the gene promoter, and demethylation caused a significant enhancement of oxytocin receptor transcription in 4T1 cells. Using a reporter gene assay, we showed that methylation of specific sites in the gene promoter, including an estrogen response element, significantly inhibits transcription. Furthermore, methylation of the oxytocin receptor promoter was found to be differentially correlated with oxytocin receptor expression in mammary glands and the uterus of virgin and post-partum mice, suggesting that it plays a distinct role in oxytocin receptor transcription among tissues and under different physiological conditions. Together, these results support the hypothesis that the expression of the mouse oxytocin receptor gene is epigenetically regulated by DNA methylation of its promoter.

Cloning of a cDNA encoding the human cation-dependent mannose 6-phosphate-specific receptor

International Nuclear Information System (INIS)

Pohlmann, R.; Nagel, G.; Schmidt, B.

1987-01-01

Complementary DNA clones for the human cation-dependent mannose 6-phosphate-specific receptor have been isolated from a human placenta library in λgt11. The nucleotide sequence of the 2463-base-pair cDNA insert includes a 145-base-pair 5' untranslated region, an open reading frame of 831 base pairs corresponding to 277 amino acids, and a 1487-base-pair 3' untranslated region. The deduced amino acid sequence is colinear with that determined by amino acid sequencing of the N-terminus peptide (41 residues) and nine tryptic peptides (93 additional residues). The receptor is synthesized as a precursor with a signal peptide of 20 amino acids. The hydrophobicity profile of the receptor indicates a single membrane-spanning domain, which separates an N-terminal region containing five potential N-glycosylation sites from a C-terminal region lacking N-glycosylation sites. Thus the N-terminal (M/sub r/ = 18,299) and C-terminal (M/sub r/ ≤ 7648) segments of the mature receptor are assumed to be exposed to the extracytosolic and cytosolic sides of the membrane, respectively. Analysis of a panel of somatic cell (mouse-human) hybrids shows that the gene for the receptor is located on human chromosome 12

Cells Lacking mtDNA Display Increased dNTP Pools upon DNA Damage

DEFF Research Database (Denmark)

Skovgaard, Tine; Rasmussen, Lene Juel; Munch-Petersen, Birgitte

Imbalanced dNTP pools are highly mutagenic due to a deleterious effect on DNA polymerase fidelity. Mitochondrial DNA defects, including mutations and deletions, are commonly found in a wide variety of different cancer types. In order to further study the interconnection between dNTP pools...... and mitochondrial function we have examined the effect of DNA damage on dNTP pools in cells deficient of mtDNA. We show that DNA damage induced by UV irradiation, in a dose corresponding to LD50, induces an S phase delay in different human osteosarcoma cell lines. The UV pulse also has a destabilizing effect...... shows that normal mitochondrial function is prerequisite for retaining stable dNTP pools upon DNA damage. Therefore it is likely that mitochondrial deficiency defects may cause an increase in DNA mutations by disrupting dNTP pool balance....

Enhancement of the priming efficacy of DNA vaccines encoding dendritic cell-targeted antigens by synergistic toll-like receptor ligands

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Kornbluth Richard S

2009-08-01

Full Text Available Abstract Background Targeting of protein antigens to dendritic cells (DC via the DEC205 receptor enhances presentation of antigen-derived peptides on MHC-I and MHC-II molecules and, in the presence of costimulatory signals, antigen-specific immune responses. The immunogenicity and efficacy of DNA vaccination can also be enhanced by fusing the encoded antigen to single chain antibodies directed against DEC205. To further improve this strategy, we evaluated different toll-like receptor ligands (TLR and CD40 ligands (CD40L as adjuvants for DNA vaccines encoding a DEC205-single-chain antibody fused to the ovalbumin model antigen or HIV-1 Gag and assessed the priming efficacy of DNA in a DNA prime adenoviral vector boost immunization regimen. Results Mice were primed with the adjuvanted DEC-205 targeted DNA vaccines and boosted with adenoviral vectors encoding the same antigens. CD8+ T cell responses were determined after the adenoviral booster immunization, to determine how well the different DNA immunization regimens prime for the adenoviral boost. In the absence of adjuvants, targeting of DNA-encoded ovalbumin to DCs suppressed CD8+ T-cell responses after the adenoviral booster immunization. CD8+ T-cell responses to the DEC205 targeted DNA vaccines increased only slightly by adding either the TLR-9 ligand CpG, the TLR-3 ligand Poly I:C, or CD40 ligand expression plasmids. However, the combination of both TLR-ligands led to a strong enhancement of CD8+ T-cell responses compared to a non-targeted DNA vaccine. This finding was confirmed using HIV Gag as antigen. Conclusion Although DNA prime adenoviral vector boost immunizations belong to the strongest inducers of cytotoxic T cell responses in different animal models and humans, the CD8+ T cell responses can be further improved by targeting the DNA encoded antigen to DEC205 in the presence of synergistic TLR ligands CpG and Poly I:C.

Effects of gamma irradiation on the DNA-protein complex between the estrogen response element and the estrogen receptor

Energy Technology Data Exchange (ETDEWEB)

Stisova, Viktorie [Department of Radiation Dosimetry, Nuclear Physics Institute AS CR, Na Truhlarce 39/64, 18086 Praha 8 (Czech Republic); Goffinont, Stephane; Spotheim-Maurizot, Melanie [Centre de Biophysique Moleculaire CNRS, rue Charles Sadron, 45071 Orleans Cedex 2 (France); Davidkova, Marie, E-mail: davidkova@ujf.cas.c [Department of Radiation Dosimetry, Nuclear Physics Institute AS CR, Na Truhlarce 39/64, 18086 Praha 8 (Czech Republic)

2010-08-15

Signaling by estrogens, risk factors in breast cancer, is mediated through their binding to the estrogen receptor protein (ER), followed by the formation of a complex between ER and a DNA sequence, called estrogen response element (ERE). Anti-estrogens act as competitive inhibitors by blocking the signal transduction. We have studied in vitro the radiosensitivity of the complex between ERalpha, a subtype of this receptor, and a DNA fragment bearing ERE, as well as the influence of an estrogen (estradiol) or an anti-estrogen (tamoxifen) on this radiosensitivity. We observe that the complex is destabilized upon irradiation with gamma rays in aerated aqueous solution. The analysis of the decrease of binding abilities of the two partners shows that destabilization is mainly due to the damage to the protein. The destabilization is reduced when irradiating in presence of tamoxifen and is increased in presence of estradiol. These effects are due to opposite influences of the ligands on the loss of binding ability of ER. The mechanism that can account for our results is: binding of estradiol or tamoxifen induces distinct structural changes of the ER ligand-binding domain that can trigger (by allostery) distinct structural changes of the ER DNA-binding domains and thus, can differently affect ER-ERE interaction.

DNA Methylation Modulates Nociceptive Sensitization after Incision.

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Yuan Sun

Full Text Available DNA methylation is a key epigenetic mechanism controlling DNA accessibility and gene expression. Blockade of DNA methylation can significantly affect pain behaviors implicated in neuropathic and inflammatory pain. However, the role of DNA methylation with regard to postoperative pain has not yet been explored. In this study we sought to investigate the role of DNA methylation in modulating incisional pain and identify possible targets under DNA methylation and contributing to incisional pain. DNA methyltranferase (DNMT inhibitor 5-Aza-2'-deoxycytidine significantly reduced incision-induced mechanical allodynia and thermal sensitivity. Aza-2'-deoxycytidine also reduced hindpaw swelling after incision, suggesting an anti-inflammatory effect. Global DNA methylation and DNMT3b expression were increased in skin after incision, but none of DNMT1, DNMT3a or DNMT3b was altered in spinal cord or DRG. The expression of proopiomelanocortin Pomc encoding β-endorphin and Oprm1 encoding the mu-opioid receptor were upregulated peripherally after incision; moreover, Oprm1 expression was further increased under DNMT inhibitor treatment. Finally, local peripheral injection of the opioid receptor antagonist naloxone significantly exacerbated incision-induced mechanical hypersensitivity. These results suggest that DNA methylation is functionally relevant to incisional nociceptive sensitization, and that mu-opioid receptor signaling might be one methylation regulated pathway controlling sensitization after incision.

AIM2-Like Receptors Positively and Negatively Regulate the Interferon Response Induced by Cytosolic DNA

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Yuki Nakaya

2017-07-01

Full Text Available Cytosolic DNAs derived from retrotransposons serve as pathogen-associated molecular patterns for pattern recognition receptors (PRRs that stimulate the induction of interferons (IFNs and other cytokines, leading to autoimmune disease. Cyclic GMP-AMP synthase is one PRR that senses retrotransposon DNA, activating type I IFN responses through the stimulator of IFN genes (STING. Absent in melanoma 2 (AIM2-like receptors (ALRs have also been implicated in these pathways. Here we show that the mouse ALR IFI205 senses cytosolic retrotransposon DNA independently of cyclic GMP-AMP production. AIM2 antagonizes IFI205-mediated IFN induction activity by sequestering it from STING. We also found that the complement of genes located in the ALR locus in C57BL/6 and AIM2 knockout mice are different and unique, which has implications for interpretation of the sensing of pathogens in different mouse strains. Our data suggest that members of the ALR family are critical to the host IFN response to endogenous DNA.

Pneumococcal DNA-binding proteins released through autolysis induce the production of proinflammatory cytokines via toll-like receptor 4.

Science.gov (United States)

Nagai, Kosuke; Domon, Hisanori; Maekawa, Tomoki; Oda, Masataka; Hiyoshi, Takumi; Tamura, Hikaru; Yonezawa, Daisuke; Arai, Yoshiaki; Yokoji, Mai; Tabeta, Koichi; Habuka, Rie; Saitoh, Akihiko; Yamaguchi, Masaya; Kawabata, Shigetada; Terao, Yutaka

2018-03-01

Streptococcus pneumoniae is a leading cause of bacterial pneumonia. Our previous study suggested that S. pneumoniae autolysis-dependently releases intracellular pneumolysin, which subsequently leads to lung injury. In this study, we hypothesized that pneumococcal autolysis induces the leakage of additional intracellular molecules that could increase the pathogenicity of S. pneumoniae. Liquid chromatography tandem-mass spectrometry analysis identified that chaperone protein DnaK, elongation factor Tu (EF-Tu), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were released with pneumococcal DNA by autolysis. We demonstrated that recombinant (r) DnaK, rEF-Tu, and rGAPDH induced significantly higher levels of interleukin-6 and tumor necrosis factor production in peritoneal macrophages and THP-1-derived macrophage-like cells via toll-like receptor 4. Furthermore, the DNA-binding activity of these proteins was confirmed by surface plasmon resonance assay. We demonstrated that pneumococcal DnaK, EF-Tu, and GAPDH induced the production of proinflammatory cytokines in macrophages, and might cause host tissue damage and affect the development of pneumococcal diseases. Copyright © 2018 Elsevier Inc. All rights reserved.

Endothelin-1 activation of ETB receptors leads to a reduced cellular proliferative rate and an increased cellular footprint

International Nuclear Information System (INIS)

Wilson, Jamie L.; Taylor, Linda; Polgar, Peter

2012-01-01

Endothelin-1 (ET-1) is a vasoactive peptide which signals through two G-protein coupled receptors, endothelin receptor A (ETA) and B (ETB). We determined that ET-1 activation of its ETB receptor in stably cDNA transfected CHO cells leads to a 55% reduction in cell number by end-point cell counting and a 35% decrease in cell growth by a real-time cell-substrate impedance-based assay after 24 h of cell growth. When CHO ETB cells were synchronized in the late G1 cell cycle phase, ET-1 delayed their S phase progression compared to control by 30% as determined by [ 3 H]-thymidine incorporation. On the other hand, no such delay was observed during late G2/M to G1 transit when cells were treated with ET-1 after release from mitotic arrest. Using the cell-substrate impedance-based assay, we observed that ET-1 induces opposing morphological changes in CHO ETA and CHO ETB cells with ETB causing an increase in the cell footprint and ETA a decrease. Likewise, in pulmonary artery smooth muscle cells, which express both ETA and ETB receptors, ET-1 induces an ETA-dependent contraction and an ETB dependent dilation. These results are shedding light on a possible beneficial role for ETB in diseases involving ET-1 dysfunction such as pulmonary hypertension. -- Highlights: ► ET- hinders cell proliferation in CHO cells transfected with ETB. ► ET-1 also decreases the rate of DNA synthesis in CHO ETB cells. ► JNK and PI3K appear to be involved in this reduction of DNA synthesis. ► ETB activation in CHO ETB cells and hSMCs leads to dilatory morphological changes. ► In CHO ETA and hSMCs, ETA activation leads to constrictive morphological changes.

Endothelin-1 activation of ETB receptors leads to a reduced cellular proliferative rate and an increased cellular footprint

Energy Technology Data Exchange (ETDEWEB)

Wilson, Jamie L.; Taylor, Linda; Polgar, Peter, E-mail: peterp@bu.edu

2012-06-10

Endothelin-1 (ET-1) is a vasoactive peptide which signals through two G-protein coupled receptors, endothelin receptor A (ETA) and B (ETB). We determined that ET-1 activation of its ETB receptor in stably cDNA transfected CHO cells leads to a 55% reduction in cell number by end-point cell counting and a 35% decrease in cell growth by a real-time cell-substrate impedance-based assay after 24 h of cell growth. When CHO ETB cells were synchronized in the late G1 cell cycle phase, ET-1 delayed their S phase progression compared to control by 30% as determined by [{sup 3}H]-thymidine incorporation. On the other hand, no such delay was observed during late G2/M to G1 transit when cells were treated with ET-1 after release from mitotic arrest. Using the cell-substrate impedance-based assay, we observed that ET-1 induces opposing morphological changes in CHO ETA and CHO ETB cells with ETB causing an increase in the cell footprint and ETA a decrease. Likewise, in pulmonary artery smooth muscle cells, which express both ETA and ETB receptors, ET-1 induces an ETA-dependent contraction and an ETB dependent dilation. These results are shedding light on a possible beneficial role for ETB in diseases involving ET-1 dysfunction such as pulmonary hypertension. -- Highlights: Black-Right-Pointing-Pointer ET- hinders cell proliferation in CHO cells transfected with ETB. Black-Right-Pointing-Pointer ET-1 also decreases the rate of DNA synthesis in CHO ETB cells. Black-Right-Pointing-Pointer JNK and PI3K appear to be involved in this reduction of DNA synthesis. Black-Right-Pointing-Pointer ETB activation in CHO ETB cells and hSMCs leads to dilatory morphological changes. Black-Right-Pointing-Pointer In CHO ETA and hSMCs, ETA activation leads to constrictive morphological changes.

Elevated Adenosine Induces Placental DNA Hypomethylation Independent of A2B Receptor Signaling in Preeclampsia.

Science.gov (United States)

Huang, Aji; Wu, Hongyu; Iriyama, Takayuki; Zhang, Yujin; Sun, Kaiqi; Song, Anren; Liu, Hong; Peng, Zhangzhe; Tang, Lili; Lee, Minjung; Huang, Yun; Ni, Xin; Kellems, Rodney E; Xia, Yang

2017-07-01

Preeclampsia is a prevalent pregnancy hypertensive disease with both maternal and fetal morbidity and mortality. Emerging evidence indicates that global placental DNA hypomethylation is observed in patients with preeclampsia and is linked to altered gene expression and disease development. However, the molecular basis underlying placental epigenetic changes in preeclampsia remains unclear. Using 2 independent experimental models of preeclampsia, adenosine deaminase-deficient mice and a pathogenic autoantibody-induced mouse model of preeclampsia, we demonstrate that elevated placental adenosine not only induces hallmark features of preeclampsia but also causes placental DNA hypomethylation. The use of genetic approaches to express an adenosine deaminase minigene specifically in placentas, or adenosine deaminase enzyme replacement therapy, restored placental adenosine to normal levels, attenuated preeclampsia features, and abolished placental DNA hypomethylation in adenosine deaminase-deficient mice. Genetic deletion of CD73 (an ectonucleotidase that converts AMP to adenosine) prevented the elevation of placental adenosine in the autoantibody-induced preeclampsia mouse model and ameliorated preeclampsia features and placental DNA hypomethylation. Immunohistochemical studies revealed that elevated placental adenosine-mediated DNA hypomethylation predominantly occurs in spongiotrophoblasts and labyrinthine trophoblasts and that this effect is independent of A2B adenosine receptor activation in both preeclampsia models. Extending our mouse findings to humans, we used cultured human trophoblasts to demonstrate that adenosine functions intracellularly and induces DNA hypomethylation without A2B adenosine receptor activation. Altogether, both mouse and human studies reveal novel mechanisms underlying placental DNA hypomethylation and potential therapeutic approaches for preeclampsia. © 2017 American Heart Association, Inc.

Expansion of microsatellite in the thyroid hormone receptor-alpha1 gene linked to increased receptor expression and less aggressive thyroid cancer

DEFF Research Database (Denmark)

Onda, Masamitsu; Li, Daisy; Suzuki, Shinichi

2002-01-01

PURPOSE: The purpose of this study was to determine whether the length of the THRA1 microsatellite, which resides in a noncoding portion of the thyroid hormone receptor-alpha1 gene, affects receptor expression and is linked to clinicopathological parameters in thyroid cancer. EXPERIMENTAL DESIGN......: In 30 cases of surgically resected sporadic thyroid cancer, the length of the THRA1 microsatellite was determined by DNA sequence analysis, and expression of thyroid hormone receptor-alpha1 was assessed immunohistochemically in thin sections cut from tumor blocks. The length of THRA1 and expression...... of thyroid hormone receptor-alpha1 were also assessed in seven cancer cell lines. Regression analysis was used to gauge the correlation between the size of THRA1 and receptor expression. Multivariate analysis was used to test for links to the clinical parameters of gender, age, histology, stage, nodal...

Signal transduction by the platelet-derived growth factor receptor

International Nuclear Information System (INIS)

Williams, L.T.; Escobedo, J.A.; Keating, M.T.; Coughlin, S.R.

1988-01-01

The mitogenic effects of platelet-derived growth factor (PDGF) are mediated by the PDGF receptor. The mouse PDGF receptor was recently purified on the basis of its ability to become tyrosine phosphorylated in response to the A-B human platelet form of PDGF, and the receptor amino acid sequence was determined from a full-length cDNA clone. Both the human and mouse receptor cDNA sequences have been expressed in Chinese hamster ovary fibroblast (CHO) cells that normally lack PDGF receptors. This paper summarizes recent results using this system to study signal transduction by the PDGF receptor. Some of the findings show that the KI domain of the PDGF receptor plays an important role in the stimulation of DNA synthesis by PDGF. Surprisingly, the kinase insert region is not essential for PDGF stimulation of PtdIns turnover, pH change, increase in cellular calcium, and receptor autophosphorylation. In addition, PDGF stimulates a conformational change in the receptor

Increased brain dopamine and dopamine receptors in schizophrenia

International Nuclear Information System (INIS)

Mackay, A.V.; Iversen, L.L.; Rossor, M.; Spokes, E.; Bird, E.; Arregui, A.; Creese, I.; Synder, S.H.

1982-01-01

In postmortem samples of caudate nucleus and nucleus accumbens from 48 schizophrenic patients, there were significant increases in both the maximum number of binding sites (Bmax) and the apparent dissociation constant (KD) for tritiated spiperone. The increase in apparent KD probably reflects the presence of residual neuroleptic drugs, but changes in Bmax for tritiated spiperone reflect genuine changes in receptor numbers. The increases in receptors were seen only in patients in whom neuroleptic medication had been maintained until the time of death, indicating that they may be entirely iatrogenic. Dopamine measurements for a larger series of schizophrenic and control cases (n greater than 60) show significantly increased concentrations in both the nucleus accumbens and caudate nucleus. The changes in dopamine were not obviously related to neuroleptic medication and, unlike the receptor changes, were most severe in younger patients

Gene polymorphisms and increased DNA damage in morbidly obese women.

Science.gov (United States)

Luperini, B C O; Almeida, D C; Porto, M P; Marcondes, J P C; Prado, R P; Rasera, I; Oliveira, M R M; Salvadori, D M F

2015-06-01

Obesity is characterized by increased adipose tissue mass resulting from a chronic imbalance between energy intake and expenditure. Furthermore, there is a clearly defined relationship among fat mass expansion, chronic low-grade systemic inflammation and reactive oxygen species (ROS) generation; leading to ROS-related pathological events. In the past years, genome-wide association studies have generated convincing evidence associating genetic variation at multiple regions of the genome with traits that reflect obesity. Therefore, the present study aimed to evaluate the relationships among the gene polymorphisms ghrelin (GHRL-rs26802), ghrelin receptor (GHSR-rs572169), leptin (LEP-rs7799039), leptin receptor (LEPR-rs1137101) and fat mass and obesity-associated (FTO-rs9939609) and obesity. The relationships among these gene variants and the amount of DNA damage were also investigated. Three hundred Caucasian morbidly obese and 300 eutrophic (controls) women were recruited. In summary, the results demonstrated that the frequencies of the GHRL, GHSR, LEP and LEPR polymorphisms were not different between Brazilian white morbidly obese and eutrophic women. Exceptions were the AA-FTO genotype and allele A, which were significantly more frequent in obese women than in the controls (0.23% vs. 0.10%; 0.46 vs. 0.36, respectively), and the TT-FTO genotype and the T allele, which were less frequent in morbidly obese women (p<0.01). Furthermore, significant differences in the amount of genetic lesions associated with FTO variants were observed only in obese women. In conclusion, this study demonstrated that the analyzed SNPs were not closely associated with morbid obesity, suggesting they are not the major contributors to obesity. Therefore, our data indicated that these gene variants are not good biomarkers for predicting risk susceptibility for obesity, whereas ROS generated by the inflammatory status might be one of the causes of DNA damage in obese women, favoring

Increased oxidative DNA damage in mononuclear leukocytes in vitiligo

Energy Technology Data Exchange (ETDEWEB)

Giovannelli, Lisa [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy)]. E-mail: lisag@pharm.unifi.it; Bellandi, Serena [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Pitozzi, Vanessa [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Fabbri, Paolo [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Dolara, Piero [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Moretti, Silvia [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy)

2004-11-22

Vitiligo is an acquired pigmentary disorder of the skin of unknown aetiology. The autocytotoxic hypothesis suggests that melanocyte impairment could be related to increased oxidative stress. Evidences have been reported that in vitiligo oxidative stress might also be present systemically. We used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, in peripheral blood cells from patients with active vitiligo and healthy controls. The basal level of oxidative DNA damage in mononuclear leukocytes was increased in vitiligo compared to normal subjects, whereas DNA strand breaks (SBs) were not changed. This alteration was not accompanied by a different capability to respond to in vitro oxidative challenge. No differences in the basal levels of DNA damage in polymorphonuclear leukocytes were found between patients and healthy subjects. Thus, this study supports the hypothesis that in vitiligo a systemic oxidative stress exists, and demonstrates for the first time the presence of oxidative alterations at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear component of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo.

Increased oxidative DNA damage in mononuclear leukocytes in vitiligo

International Nuclear Information System (INIS)

Giovannelli, Lisa; Bellandi, Serena; Pitozzi, Vanessa; Fabbri, Paolo; Dolara, Piero; Moretti, Silvia

2004-01-01

Vitiligo is an acquired pigmentary disorder of the skin of unknown aetiology. The autocytotoxic hypothesis suggests that melanocyte impairment could be related to increased oxidative stress. Evidences have been reported that in vitiligo oxidative stress might also be present systemically. We used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, in peripheral blood cells from patients with active vitiligo and healthy controls. The basal level of oxidative DNA damage in mononuclear leukocytes was increased in vitiligo compared to normal subjects, whereas DNA strand breaks (SBs) were not changed. This alteration was not accompanied by a different capability to respond to in vitro oxidative challenge. No differences in the basal levels of DNA damage in polymorphonuclear leukocytes were found between patients and healthy subjects. Thus, this study supports the hypothesis that in vitiligo a systemic oxidative stress exists, and demonstrates for the first time the presence of oxidative alterations at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear component of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo

Abnormal XPD-induced nuclear receptor transactivation in DNA repair disorders: trichothiodystrophy and xeroderma pigmentosum.

Science.gov (United States)

Zhou, Xiaolong; Khan, Sikandar G; Tamura, Deborah; Ueda, Takahiro; Boyle, Jennifer; Compe, Emmanuel; Egly, Jean-Marc; DiGiovanna, John J; Kraemer, Kenneth H

2013-08-01

XPD (ERCC2) is a DNA helicase involved in nucleotide excision repair and in transcription as a structural bridge tying the transcription factor IIH (TFIIH) core with the cdk-activating kinase complex, which phosphorylates nuclear receptors. Mutations in XPD are associated with several different phenotypes, including trichothiodystrophy (TTD), with sulfur-deficient brittle hair, bone defects, and developmental abnormalities without skin cancer, xeroderma pigmentosum (XP), with pigmentary abnormalities and increased skin cancer, or XP/TTD with combined features, including skin cancer. We describe the varied clinical features and mutations in nine patients examined at the National Institutes of Health who were compound heterozygotes for XPD mutations but had different clinical phenotypes: four TTD, three XP, and two combined XP/TTD. We studied TFIIH-dependent transactivation by nuclear receptor for vitamin D (VDR) and thyroid in cells from these patients. The vitamin D stimulation ratio of CYP24 and osteopontin was associated with specific pairs of mutations (reduced in 5, elevated in 1) but not correlated with distinct clinical phenotypes. Thyroid receptor stimulation ratio for KLF9 was not significantly different from normal. XPD mutations frequently were associated with abnormal VDR stimulation in compound heterozygote patients with TTD, XP, or XP/TTD.

Gefitinib radiosensitizes stem-like glioma cells: inhibition of epidermal growth factor receptor-Akt-DNA-PK signaling, accompanied by inhibition of DNA double-strand break repair.

Science.gov (United States)

Kang, Khong Bee; Zhu, Congju; Wong, Yin Ling; Gao, Qiuhan; Ty, Albert; Wong, Meng Cheong

2012-05-01

We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)-Akt-DNA-dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, γ-H(2)AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival, γ-H(2)AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G(2)/M arrest and increased γ-H(2)AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased γ-H(2)AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Stem-like gliomaspheres are resistant to irradiation-induced cytotoxicity, G(2)/M arrest, and DNA DSBs, compared with nonstem

Gefitinib Radiosensitizes Stem-Like Glioma Cells: Inhibition of Epidermal Growth Factor Receptor-Akt-DNA-PK Signaling, Accompanied by Inhibition of DNA Double-Strand Break Repair

Energy Technology Data Exchange (ETDEWEB)

Kang, Khong Bee, E-mail: dmskkb@nccs.com.sg [Brain Tumour Research Laboratory, Division of Medical Sciences, National Cancer Centre Singapore (Singapore); Zhu Congju; Wong Yinling; Gao Qiuhan; Ty, Albert; Wong, Meng Cheong [Brain Tumour Research Laboratory, Division of Medical Sciences, National Cancer Centre Singapore (Singapore)

2012-05-01

Purpose: We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)-Akt-DNA-dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Methods and Materials: Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, {gamma}-H{sub 2}AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival, {gamma}-H{sub 2}AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Results: Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G{sub 2}/M arrest and increased {gamma}-H{sub 2}AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased {gamma}-H{sub 2}AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Conclusions: Stem-like gliomaspheres are

Comparison of cDNA-derived protein sequences of the human fibronectin and vitronectin receptor α-subunits and platelet glycoprotein IIb

International Nuclear Information System (INIS)

Fitzgerald, L.A.; Poncz, M.; Steiner, B.; Rall, S.C. Jr.; Bennett, J.S.; Phillips, D.R.

1987-01-01

The fibronectin receptor (FnR), the vitronectin receptor (VnR), and the platelet membrane glycoprotein (GP) IIb-IIIa complex are members of a family of cell adhesion receptors, which consist of noncovalently associated α- and β-subunits. The present study was designed to compare the cDNA-derived protein sequences of the α-subunits of human FnR, VnR, and platelet GP IIb. cDNA clones for the α-subunit of the FnR (FnR/sub α/) were obtained from a human umbilical vein endothelial (HUVE) cell library by using an oligonucleotide probe designed from a peptide sequence of platelet GP IIb. cDNA clones for platelet GP IIb were isolated from a cDNA expression library of human erythroleukemia cells by using antibodies. cDNA clones of the VnR α-subunit (VnR/sub α/) were obtained from the HUVE cell library by using an oligonucleotide probe from the partial cDNA sequence for the VnR/sub α/. Translation of these sequences showed that the FNR/sub α/, the VnR/sub α/, and GP IIb are composed of disulfide-linked large (858-871 amino acids) and small (137-158 amino acids) chains that are posttranslationally processed from a single mRNA. A single hydrophobic segment located near the carboxyl terminus of each small chain appears to be a transmembrane domain. The large chains appear to be entirely extracellular, and each contains four repeated putative Ca 2+ -binding domains of about 30 amino acids that have sequence similarities to other Ca 2+ -binding proteins. The identity among the protein sequences of the three receptor α-subunits ranges from 36.1% to 44.5%, with the Ca 2+ -binding domains having the greatest homology. These proteins apparently evolved by a process of gene duplication

Increased DNA-repair in spleen cells of M. Hodgkin

International Nuclear Information System (INIS)

Frischauf, H.; Neumann, E.; Howanietz, L.; Dolejs, I.; Tuschl, H.; Altmann, H.

1974-11-01

In spleen cells of control patients and cells of Morbus Hodgkin, DNA-repair after gamma- and UV-irradiation was determined measuring the incorporated 3H-thymidine activity in the DNA. Additionally, the ratio of labeled cells compared to non-labeled cells and the grains per cell were evaluated by autoradiographic investigations. DNA-content per cell was measured using pulsecytophotometry. A significant increase of DNA-repair capacity after gamma-irradiation was found by density gradient centrifugation in alkaline sucrose. The same trend could be shown by investigations of unscheduled DNA-synthesis using autoradiographic method. (author)

Gefitinib Radiosensitizes Stem-Like Glioma Cells: Inhibition of Epidermal Growth Factor Receptor-Akt-DNA-PK Signaling, Accompanied by Inhibition of DNA Double-Strand Break Repair

International Nuclear Information System (INIS)

Kang, Khong Bee; Zhu Congju; Wong Yinling; Gao Qiuhan; Ty, Albert; Wong, Meng Cheong

2012-01-01

Purpose: We compared radiosensitivity of brain tumor stem cells (BTSCs) with matched nonstem glioma cells, and determined whether gefitinib enhanced BTSC radiosensitivity by inhibiting epidermal growth factor receptor (EGFR)–Akt-DNA–dependent protein kinase (DNA-PK) signaling, followed by enhanced DNA double-stand breaks (DSBs) and inhibition of DSB repair. Methods and Materials: Radiosensitivity of stem-like gliomaspheres and nonstem glioma cells (obtained at patient neurosurgical resection) were evaluated by clonogenic assays, γ-H 2 AX immunostaining and cell cycle distribution. Survival of irradiated and nonirradiated NOD-SCID mice intracranially implanted with stem-like gliomaspheres were monitored. Glioma cells treated with gefitinib, irradiation, or both were assayed for clonogenic survival, γ-H 2 AX immunostaining, DNA-PKcs expression, and phosphorylation of EGFR and Akt. Results: Stem-like gliomaspheres displayed BTSC characteristics of self-renewal; differentiation into lineages of neurons, oligodendrocytes, and astrocytes; and initiation of glioma growth in NOD-SCID mice. Irradiation dose-dependently reduced clonogenic survival, induced G 2 /M arrest and increased γ-H 2 AX immunostaining of nonstem glioma cells, but not stem-like gliomaspheres. There was no difference in survival of irradiated and nonirradiated mice implanted with stem-like gliomaspheres. The addition of gefitinib significantly inhibited clonogenic survival, increased γ-H 2 AX immunostaining, and reduced DNA-PKcs expression of irradiated stem-like gliomaspheres, without affecting irradiated-nonstem glioma cells. Gefitinib alone, and when combined with irradiation, inhibited phosphorylation of EGFR (Y1068 and Y1045) and Akt (S473) in stem-like gliomaspheres. In nonstem glioma cells, gefitinib alone inhibited EGFR Y1068 phosphorylation, with further inhibition by combined gefitinib and irradiation. Conclusions: Stem-like gliomaspheres are resistant to irradiation

Relation between serum xenobiotic induced receptor activities and sperm DNA damage and sperm apoptotic markers in European and Inuit populations

DEFF Research Database (Denmark)

Long, Manhai; Stronati, Alessanda; Bizzaro, Davide

2007-01-01

Persistent organic pollutants (POPs) can interfere with hormone activities and are suspected as endocrine disrupters involved in disorders, e.g. reproductive disorders. We investigated the possible relation between the actual integrated serum xenoestrogenic, xenoandrogenic and aryl hydrocarbon......, but higher xenoandrogenic activity. In contrast, in the European groups, xenobiotic-induced receptor activities were found to be positively correlated with the DNA damage. Further research must elucidate whether altered receptor activities in concerted action with genetic and/or nutrient factors may have...... protecting effect on sperm DNA damage of the Inuit population....

Dietary restriction but not angiotensin II type 1 receptor blockade improves DNA damage-related vasodilator dysfunction in rapidly aging Ercc1Δ/- mice.

Science.gov (United States)

Wu, Haiyan; van Thiel, Bibi S; Bautista-Niño, Paula K; Reiling, Erwin; Durik, Matej; Leijten, Frank P J; Ridwan, Yanto; Brandt, Renata M C; van Steeg, Harry; Dollé, Martijn E T; Vermeij, Wilbert P; Hoeijmakers, Jan H J; Essers, Jeroen; van der Pluijm, Ingrid; Danser, A H Jan; Roks, Anton J M

2017-08-01

DNA damage is an important contributor to endothelial dysfunction and age-related vascular disease. Recently, we demonstrated in a DNA repair-deficient, prematurely aging mouse model ( Ercc1 Δ/- mice) that dietary restriction (DR) strongly increases life- and health span, including ameliorating endothelial dysfunction, by preserving genomic integrity. In this mouse mutant displaying prominent accelerated, age-dependent endothelial dysfunction we investigated the signaling pathways involved in improved endothelium-mediated vasodilation by DR, and explore the potential role of the renin-angiotensin system (RAS). Ercc1 Δ/- mice showed increased blood pressure and decreased aortic relaxations to acetylcholine (ACh) in organ bath experiments. Nitric oxide (NO) signaling and phospho-Ser 1177 -eNOS were compromised in Ercc1 Δ / - DR improved relaxations by increasing prostaglandin-mediated responses. Increase of cyclo-oxygenase 2 and decrease of phosphodiesterase 4B were identified as potential mechanisms. DR also prevented loss of NO signaling in vascular smooth muscle cells and normalized angiotensin II (Ang II) vasoconstrictions, which were increased in Ercc1 Δ/- mice. Ercc1 Δ/ - mutants showed a loss of Ang II type 2 receptor-mediated counter-regulation of Ang II type 1 receptor-induced vasoconstrictions. Chronic losartan treatment effectively decreased blood pressure, but did not improve endothelium-dependent relaxations. This result might relate to the aging-associated loss of treatment efficacy of RAS blockade with respect to endothelial function improvement. In summary, DR effectively prevents endothelium-dependent vasodilator dysfunction by augmenting prostaglandin-mediated responses, whereas chronic Ang II type 1 receptor blockade is ineffective. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

The radioprotector O-phospho-tyrosine stimulates DNA-repair via epidermal growth factor receptor- and DNA-dependent kinase phosphorylation

International Nuclear Information System (INIS)

Dittmann, Klaus; Mayer, Claus; Wanner, Gabriele; Kehlbach, Rainer; Rodemann, H. Peter

2007-01-01

Background and purpose: Purpose of the study was to elucidate the underlying molecular mechanism of the radioprotector O-phospho-tyrosine (P-Tyr). Methods: Molecular effects of P-Tyr at the level of EGFR responses were investigated in vitro with bronchial carcinoma cell line A549. Nuclear EGFR transport and DNA-PK activation were quantified after Western blotting. Residual DNA-damages were quantified by help of γH 2 AX focus assay. Results: As determined by dose-response curves, treatment of cells with P-Tyr for 16 h before irradiation results in radioprotection. Simultaneous treatment with EGFR blocking antibody Cetuximab abolished P-Tyr associated radioprotection. At the molecular level P-Tyr mediated a general phosphorylation of EGFR and a pronounced phosphorylation of nuclear EGFR at residue Thr No. 654, also observed after treatment with ionizing radiation. This phosphorylation was associated with nuclear EGFR accumulation. Moreover, P-Tyr-triggered EGFR nuclear accumulation was associated with phosphorylation of DNA-PK at Thr 2609. This activated form of DNA-PK was not DNA associated, but after radiation, DNA binding increased, particularly after P-Tyr pre-treatment. These molecular effects of P-Tyr resulted in a reduction of residual DNA-damage after irradiation. Conclusions: Radioprotection by P-Tyr is mediated through its stimulation of nuclear EGFR transport and concurrent, but DNA-damage independent, activation of DNA-PK. Thus, subsequent irradiation results in increased binding of DNA-PK to DNA, improved DNA-repair and increased cell survival

Structural analysis of complementary DNA and amino acid sequences of human and rat androgen receptors

International Nuclear Information System (INIS)

Chang, C.; Kokontis, J.; Liao, S.

1988-01-01

Structural analysis of cDNAs for human and rat androgen receptors (ARs) indicates that the amino-terminal regions of ARs are rich in oligo- and poly(amino acid) motifs as in some homeotic genes. The human AR has a long stretch of repeated glycines, whereas rat AR has a long stretch of glutamines. There is a considerable sequence similarity among ARs and the receptors for glucocorticoids, progestins, and mineralocorticoids within the steroid-binding domains. The cysteine-rich DNA-binding domains are well conserved. Translation of mRNA transcribed from AR cDNAs yielded 94- and 76-kDa proteins and smaller forms that bind to DNA and have high affinity toward androgens. These rat or human ARs were recognized by human autoantibodies to natural Ars. Molecular hybridization studies, using AR cDNAs as probes, indicated that the ventral prostate and other male accessory organs are rich in AR mRNA and that the production of AR mRNA in the target organs may be autoregulated by androgens

Increasing Nucleosome Occupancy Is Correlated with an Increasing Mutation Rate so Long as DNA Repair Machinery Is Intact

Science.gov (United States)

Taylor, Jared F.; Khattab, Omar S.; Chen, Yu-Han; Chen, Yumay; Jacobsen, Steven E.; Wang, Ping H.

2015-01-01

Deciphering the multitude of epigenomic and genomic factors that influence the mutation rate is an area of great interest in modern biology. Recently, chromatin has been shown to play a part in this process. To elucidate this relationship further, we integrated our own ultra-deep sequenced human nucleosomal DNA data set with a host of published human genomic and cancer genomic data sets. Our results revealed, that differences in nucleosome occupancy are associated with changes in base-specific mutation rates. Increasing nucleosome occupancy is associated with an increasing transition to transversion ratio and an increased germline mutation rate within the human genome. Additionally, cancer single nucleotide variants and microindels are enriched within nucleosomes and both the coding and non-coding cancer mutation rate increases with increasing nucleosome occupancy. There is an enrichment of cancer indels at the theoretical start (74 bp) and end (115 bp) of linker DNA between two nucleosomes. We then hypothesized that increasing nucleosome occupancy decreases access to DNA by DNA repair machinery and could account for the increasing mutation rate. Such a relationship should not exist in DNA repair knockouts, and we thus repeated our analysis in DNA repair machinery knockouts to test our hypothesis. Indeed, our results revealed no correlation between increasing nucleosome occupancy and increasing mutation rate in DNA repair knockouts. Our findings emphasize the linkage of the genome and epigenome through the nucleosome whose properties can affect genome evolution and genetic aberrations such as cancer. PMID:26308346

Increasing Nucleosome Occupancy Is Correlated with an Increasing Mutation Rate so Long as DNA Repair Machinery Is Intact.

Directory of Open Access Journals (Sweden)

Puya G Yazdi

Full Text Available Deciphering the multitude of epigenomic and genomic factors that influence the mutation rate is an area of great interest in modern biology. Recently, chromatin has been shown to play a part in this process. To elucidate this relationship further, we integrated our own ultra-deep sequenced human nucleosomal DNA data set with a host of published human genomic and cancer genomic data sets. Our results revealed, that differences in nucleosome occupancy are associated with changes in base-specific mutation rates. Increasing nucleosome occupancy is associated with an increasing transition to transversion ratio and an increased germline mutation rate within the human genome. Additionally, cancer single nucleotide variants and microindels are enriched within nucleosomes and both the coding and non-coding cancer mutation rate increases with increasing nucleosome occupancy. There is an enrichment of cancer indels at the theoretical start (74 bp and end (115 bp of linker DNA between two nucleosomes. We then hypothesized that increasing nucleosome occupancy decreases access to DNA by DNA repair machinery and could account for the increasing mutation rate. Such a relationship should not exist in DNA repair knockouts, and we thus repeated our analysis in DNA repair machinery knockouts to test our hypothesis. Indeed, our results revealed no correlation between increasing nucleosome occupancy and increasing mutation rate in DNA repair knockouts. Our findings emphasize the linkage of the genome and epigenome through the nucleosome whose properties can affect genome evolution and genetic aberrations such as cancer.

Monoclonal antibody to the type I insulin-like growth factor (IGF-I) receptor blocks IGF-I receptor-mediated DNA synthesis: clarification of the mitogenic mechanisms of IGF-I and insulin in human skin fibroblasts

International Nuclear Information System (INIS)

Flier, J.S.; Usher, P.; Moses, A.C.

1986-01-01

Insulin and insulin-like growth factor type I (IGF-I) stimulate an overlapping spectrum of biological responses in human skin fibroblasts. Although insulin and IGF-I are known to stimulate the incorporation of [ 3 H]thymidine into DNA in these cells, the identify of the receptor(s) that mediates this effect has not been fully clarified. The mouse anti-human IGF-I receptor antibody αIR-3 binds with specificity to IGF-I but not to insulin receptors in human placental membranes; it also specifically inhibits the binding of 125 I-labeled IGF-I but not 125 I-labeled insulin to suspensions of human skin fibroblasts in a dose-dependent manner. αIR-3 competitively inhibits IGF-I-mediated stimulation of [ 3 H]thymidine incorporation into DNA. This inhibition is dependent on the concentration of αIR-3 and in the presence of a fixed antibody concentration can be partially overcome by high concentrations of IGF-I. In contrast, at concentrations of 3 H]thymidine incorporation is not inhibited by αIR-3. However, the incremental effects of higher concentrations (> 1 μg/ml) of insulin on [ 3 H]thymidine incorporation are inhibited by αIR-3. αIR-3 is a highly specific antagonist of IGF-I receptor-mediated mitogenesis in human skin fibroblasts. By using this antibody, it is shown directly that insulin can act through the IGF-I receptor to stimulate DNA synthesis but can also activate this effect through the insulin receptor itself

Identification of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a novel target of bisphenol A.

Science.gov (United States)

Ito, Yuki; Ito, Takumi; Karasawa, Satoki; Enomoto, Teruya; Nashimoto, Akihiro; Hase, Yasuyoshi; Sakamoto, Satoshi; Mimori, Tsuneyo; Matsumoto, Yoshihisa; Yamaguchi, Yuki; Handa, Hiroshi

2012-01-01

Bisphenol A (BPA) forms the backbone of plastics and epoxy resins used to produce packaging for various foods and beverages. BPA is also an estrogenic disruptor, interacting with human estrogen receptors (ER) and other related nuclear receptors. Nevertheless, the effects of BPA on human health remain unclear. The present study identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a novel BPA-binding protein. DNA-PKcs, in association with the Ku heterodimer (Ku70/80), is a critical enzyme involved in the repair of DNA double-strand breaks. Low levels of DNA-PK activity are previously reported to be associated with an increased risk of certain types of cancer. Although the Kd for the interaction between BPA and a drug-binding mutant of DNA-PKcs was comparatively low (137 nM), high doses of BPA were required before cellular effects were observed (100-300 μM). The results of an in vitro kinase assay showed that BPA inhibited DNA-PK kinase activity in a concentration-dependent manner. In M059K cells, BPA inhibited the phosphorylation of DNA-PKcs at Ser2056 and H2AX at Ser139 in response to ionizing radiation (IR)-irradiation. BPA also disrupted DNA-PKcs binding to Ku70/80 and increased the radiosensitivity of M059K cells, but not M059J cells (which are DNA-PKcs-deficient). Taken together, these results provide new evidence of the effects of BPA on DNA repair in mammalian cells, which are mediated via inhibition of DNA-PK activity. This study may warrant the consideration of the possible carcinogenic effects of high doses of BPA, which are mediated through its action on DNA-PK.

Cross-talk between an activator of nuclear receptors-mediated transcription and the D1 dopamine receptor signaling pathway.

Science.gov (United States)

Schmidt, Azriel; Vogel, Robert; Rutledge, Su Jane; Opas, Evan E; Rodan, Gideon A; Friedman, Eitan

2005-03-01

Nuclear receptors are transcription factors that usually interact, in a ligand-dependent manner, with specific DNA sequences located within promoters of target genes. The nuclear receptors can also be controlled in a ligand-independent manner via the action of membrane receptors and cellular signaling pathways. 5-Tetradecyloxy-2-furancarboxylic acid (TOFA) was shown to stimulate transcription from the MMTV promoter via chimeric receptors that consist of the DNA binding domain of GR and the ligand binding regions of the PPARbeta or LXRbeta nuclear receptors (GR/PPARbeta and GR/LXRbeta). TOFA and hydroxycholesterols also modulate transcription from NF-kappaB- and AP-1-controlled reporter genes and induce neurite differentiation in PC12 cells. In CV-1 cells that express D(1) dopamine receptors, D(1) dopamine receptor stimulation was found to inhibit TOFA-stimulated transcription from the MMTV promoter that is under the control of chimeric GR/PPARbeta and GR/LXRbeta receptors. Treatment with the D(1) dopamine receptor antagonist, SCH23390, prevented dopamine-mediated suppression of transcription, and by itself increased transcription controlled by GR/LXRbeta. Furthermore, combined treatment of CV-1 cells with TOFA and SCH23390 increased transcription controlled by the GR/LXRbeta chimeric receptor synergistically. The significance of this in vitro synergy was demonstrated in vivo, by the observation that SCH23390 (but not haloperidol)-mediated catalepsy in rats was potentiated by TOFA, thus showing that an agent that mimics the in vitro activities of compounds that activate members of the LXR and PPAR receptor families can influence D1 dopamine receptor elicited responses.

Disruption of Maternal DNA Repair Increases Sperm-DerivedChromosomal Aberrations

Energy Technology Data Exchange (ETDEWEB)

Marchetti, Francesco; Essers, Jeroun; Kanaar, Roland; Wyrobek,Andrew J.

2007-02-07

The final weeks of male germ cell differentiation occur in aDNA repair-deficient environment and normal development depends on theability of the egg to repair DNA damage in the fertilizing sperm. Geneticdisruption of maternal DNA double-strand break repair pathways in micesignificantly increased the frequency of zygotes with chromosomalstructural aberrations after paternal exposure to ionizing radiation.These findings demonstrate that radiation-induced DNA sperm lesions arerepaired after fertilization by maternal factors and suggest that geneticvariation in maternal DNA repair can modulate the risk of early pregnancylosses and of children with chromosomal aberrations of paternalorigin.

Inhibition of the epidermal growth factor receptor in bladder cancer cells treated with the DNA-damaging drug etoposide markedly increases apoptosis

DEFF Research Database (Denmark)

Munk, Mathias; Memon, Ashfaque Ahmed; Nexo, Ebba

2007-01-01

: The bladder cancer cell lines RT4 and T24, representing low- and high-malignancy grades respectively, were treated with VP16 (10 or 50 microM) and the level of apoptosis determined using a commercial kit. EGFR receptor activity was determined by western blotting using antibodies against phosphorylated EGFR....... The EGFR was either activated by heparin-binding (HB)-EGF (1 nM) or inhibited with the specific EGFR inhibitor gefitinib (1 or 5 microM). The pan-caspase inhibitor Z-VAD (30 microM) was used to test the involvement of caspase activity. RESULTS: Treatment of T24 bladder cancer cells with VP16 (50 micro...... suggest that activation of the EGFR induced a cell-survival function when bladder cancer cells were treated with the DNA-damaging drug VP16, and that combined treatment with VP16 and the EGFR inhibitor gefitinib might improve the efficacy of treatment. Udgivelsesdato: 2007-Jan...

β-Receptor-mediated increase in cerebral blood flow during hypoglycemia

International Nuclear Information System (INIS)

Hollinger, B.R.; Bryan, R.M.

1987-01-01

The authors tested the hypothesis that β-adrenergic receptor stimulation is involved with the increase in regional cerebral blood flow (rCBF) during hypoglycemia. Rats were surgically prepared with the use of halothane-nitrous oxide anesthesia. A plaster restraining cast was placed around the hindquarters, and anesthesia was discontinued. Hypoglycemia was produced by an intravenous injection of insulin; normoglycemic control rates were given saline. Propranolol was administered to some control and some hypoglycemic rats to block the β-adrenergic receptors. Regional CBF was measured using 4-[N-methyl- 14 C]iodoantipyrine. Regional CBF increased during hypoglycemia in rats that were not treated with propranolol. The increase varied from ∼60 to 200% depending on the brain region. During hypoglycemia, propranolol abolished the increase in rCBF in the hypothalamus, cerebellum, and pyramidal tract. In other regions the increase in rCBF was only 33-65% of the increase in hypoglycemic rats that were not treated with propranolol. They conclude that β-receptor stimulation plays a major role in the increase in rCBF during hypoglycemia

MMS exposure promotes increased MtDNA mutagenesis in the presence of replication-defective disease-associated DNA polymerase γ variants.

Science.gov (United States)

Stumpf, Jeffrey D; Copeland, William C

2014-10-01

Mitochondrial DNA (mtDNA) encodes proteins essential for ATP production. Mutant variants of the mtDNA polymerase cause mutagenesis that contributes to aging, genetic diseases, and sensitivity to environmental agents. We interrogated mtDNA replication in Saccharomyces cerevisiae strains with disease-associated mutations affecting conserved regions of the mtDNA polymerase, Mip1, in the presence of the wild type Mip1. Mutant frequency arising from mtDNA base substitutions that confer erythromycin resistance and deletions between 21-nucleotide direct repeats was determined. Previously, increased mutagenesis was observed in strains encoding mutant variants that were insufficient to maintain mtDNA and that were not expected to reduce polymerase fidelity or exonuclease proofreading. Increased mutagenesis could be explained by mutant variants stalling the replication fork, thereby predisposing the template DNA to irreparable damage that is bypassed with poor fidelity. This hypothesis suggests that the exogenous base-alkylating agent, methyl methanesulfonate (MMS), would further increase mtDNA mutagenesis. Mitochondrial mutagenesis associated with MMS exposure was increased up to 30-fold in mip1 mutants containing disease-associated alterations that affect polymerase activity. Disrupting exonuclease activity of mutant variants was not associated with increased spontaneous mutagenesis compared with exonuclease-proficient alleles, suggesting that most or all of the mtDNA was replicated by wild type Mip1. A novel subset of C to G transversions was responsible for about half of the mutants arising after MMS exposure implicating error-prone bypass of methylated cytosines as the predominant mutational mechanism. Exposure to MMS does not disrupt exonuclease activity that suppresses deletions between 21-nucleotide direct repeats, suggesting the MMS-induce mutagenesis is not explained by inactivated exonuclease activity. Further, trace amounts of CdCl2 inhibit mtDNA replication but

Prenatal stress, fearfulness, and the epigenome: Exploratory analysis of sex differences in DNA methylation of the glucocorticoid receptor gene.

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Brendan Dale Ostlund

2016-07-01

Full Text Available Exposure to stress in utero is a risk factor for the development of problem behavior in the offspring, though precise pathways are unknown. We examined whether DNA methylation of the glucocorticoid receptor gene, NR3C1, was associated with experiences of stress by an expectant mother and fearfulness in her infant. Mothers reported on prenatal stress and infant temperament when infants were 5 months old (n = 68. Buccal cells for methylation analysis were collected from each infant. Prenatal stress was not related to infant fearfulness or NR3C1 methylation in the sample as a whole. Exploratory sex-specific analysis revealed a trend-level association between prenatal stress and increased methylation of NR3C1 exon 1F for female, but not male, infants. In addition, increased methylation was significantly associated with greater fearfulness for females. Results suggest an experience-dependent pathway to fearfulness for female infants via epigenetic modification of the glucocorticoid receptor gene. Future studies should examine prenatal stress in a comprehensive fashion while considering sex differences in epigenetic processes underlying infant temperament.

Chitosan-based DNA delivery vector targeted to gonadotropin-releasing hormone (GnRH) receptor.

Science.gov (United States)

Boonthum, Chatwalee; Namdee, Katawut; Boonrungsiman, Suwimon; Chatdarong, Kaywalee; Saengkrit, Nattika; Sajomsang, Warayuth; Ponglowhapan, Suppawiwat; Yata, Teerapong

2017-02-10

The main purpose of this study was to investigate the application of modified chitosan as a potential vector for gene delivery to gonadotropin-releasing hormone receptor (GnRHR)-expressing cells. Such design of gene carrier could be useful in particular for gene therapy for cancers related to the reproductive system, gene disorders of sexual development, and contraception and fertility control. In this study, a decapeptide GnRH was successfully conjugated to chitosan (CS) as confirmed by proton nuclear magnetic resonance spectroscopy ( 1 H NMR) and Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The synthesized GnRH-conjugated chitosan (GnRH-CS) was able to condense DNA to form positively charged nanoparticles and specifically deliver plasmid DNA to targeted cells in both two-dimensional (2D) and three-dimensional (3D) cell cultures systems. Importantly, GnRH-CS exhibited higher transfection activity compared to unmodified CS. In conclusion, GnRH-conjugated chitosan can be a promising carrier for targeted DNA delivery to GnRHR-expressing cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

DNA methylation of the oxytocin receptor gene predicts neural response to ambiguous social stimuli

Directory of Open Access Journals (Sweden)

Allison eJack

2012-10-01

Full Text Available Oxytocin and its receptor (OXTR play an important role in a variety of social perceptual and affiliative processes. Individual variability in social information processing likely has a strong heritable component, and as such, many investigations have established an association between common genetic variants of OXTR and variability in the social phenotype. However, to date, these investigations have primarily focused only on changes in the sequence of DNA without considering the role of epigenetic factors. DNA methylation is an epigenetic mechanism by which cells control transcription through modification of chromatin structure. DNA methylation of OXTR decreases expression of the gene and high levels of methylation have been associated with autism spectrum disorders. This link between epigenetic variability and social phenotype allows for the possibility that social processes are under epigenetic control. We hypothesized that the level of DNA methylation of OXTR would predict individual variability in social perception. Using the brain’s sensitivity to displays of animacy as a neural endophenotype of social perception, we found significant associations between the degree of OXTR methylation and brain activity evoked by the perception of animacy. Our results suggest that consideration of DNA methylation may substantially improve our ability to explain individual differences in imaging genetic association studies.

Free energy calculations offer insights into the influence of receptor flexibility on ligand-receptor binding affinities.

Science.gov (United States)

Dolenc, Jožica; Riniker, Sereina; Gaspari, Roberto; Daura, Xavier; van Gunsteren, Wilfred F

2011-08-01

Docking algorithms for computer-aided drug discovery and design often ignore or restrain the flexibility of the receptor, which may lead to a loss of accuracy of the relative free enthalpies of binding. In order to evaluate the contribution of receptor flexibility to relative binding free enthalpies, two host-guest systems have been examined: inclusion complexes of α-cyclodextrin (αCD) with 1-chlorobenzene (ClBn), 1-bromobenzene (BrBn) and toluene (MeBn), and complexes of DNA with the minor-groove binding ligands netropsin (Net) and distamycin (Dist). Molecular dynamics simulations and free energy calculations reveal that restraining of the flexibility of the receptor can have a significant influence on the estimated relative ligand-receptor binding affinities as well as on the predicted structures of the biomolecular complexes. The influence is particularly pronounced in the case of flexible receptors such as DNA, where a 50% contribution of DNA flexibility towards the relative ligand-DNA binding affinities is observed. The differences in the free enthalpy of binding do not arise only from the changes in ligand-DNA interactions but also from changes in ligand-solvent interactions as well as from the loss of DNA configurational entropy upon restraining.

Presynaptic Glycine Receptors Increase GABAergic Neurotransmission in Rat Periaqueductal Gray Neurons

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Kwi-Hyung Choi

2013-01-01

Full Text Available The periaqueductal gray (PAG is involved in the central regulation of nociceptive transmission by affecting the descending inhibitory pathway. In the present study, we have addressed the functional role of presynaptic glycine receptors in spontaneous glutamatergic transmission. Spontaneous EPSCs (sEPSCs were recorded in mechanically dissociated rat PAG neurons using a conventional whole-cell patch recording technique under voltage-clamp conditions. The application of glycine (100 µM significantly increased the frequency of sEPSCs, without affecting the amplitude of sEPSCs. The glycine-induced increase in sEPSC frequency was blocked by 1 µM strychnine, a specific glycine receptor antagonist. The results suggest that glycine acts on presynaptic glycine receptors to increase the probability of glutamate release from excitatory nerve terminals. The glycine-induced increase in sEPSC frequency completely disappeared either in the presence of tetrodotoxin or Cd2+, voltage-gated Na+, or Ca2+ channel blockers, suggesting that the activation of presynaptic glycine receptors might depolarize excitatory nerve terminals. The present results suggest that presynaptic glycine receptors can regulate the excitability of PAG neurons by enhancing glutamatergic transmission and therefore play an important role in the regulation of various physiological functions mediated by the PAG.

Enterolactone: A novel radiosensitizer for human breast cancer cell lines through impaired DNA repair and increased apoptosis

International Nuclear Information System (INIS)

Bigdeli, Bahareh; Goliaei, Bahram; Masoudi-Khoram, Nastaran; Jooyan, Najmeh; Nikoofar, Alireza; Rouhani, Maryam; Haghparast, Abbas; Mamashli, Fatemeh

2016-01-01

Introduction: Radiotherapy is a potent treatment against breast cancer, which is the most commonly diagnosed cancer among women. However, the emergence of radioresistance due to increased DNA repair leads to radiotherapeutic failure. Applying polyphenols combined with radiation is a more promising method leading to better survival. Enterolactone, a phytoestrogenic polyphenol, has been reported to inhibit an important radioresistance signaling pathway, therefore we conjectured that enterolactone could enhance radiosensitivity in breast cancer. To assess this hypothesis, radiation response of enterolactone treated MDA-MB-231 and T47D cell lines and corresponding cellular mechanisms were investigated. Methods: Cytotoxicity of enterolactone was measured via MTT assay. Cells were treated with enterolactone before X-irradiation, and clonogenic assay was used to evaluate radiosensitivity. Cell cycle distribution and apoptosis were measured by flow cytometric analysis. In addition, DNA damages and corresponding repair, chromosomal damages, and aberrations were assessed by comet, micronucleus, and cytogenetic assays, respectively. Results: Enterolactone decreased the viability of cells in a concentration- and time dependent manner. Enterolactone significantly enhanced radiosensitivity of cells by abrogating G2/M arrest, impairing DNA repair, and increasing radiation-induced apoptosis. Furthermore, increased chromosomal damages and aberrations were detected in cells treated with enterolactone combined with X-rays than X-ray alone. These effects were more prominent in T47D than MDA-MB-231 cells. Discussion: To our knowledge, this is the first report that enterolactone is a novel radiosensitizer for breast cancer irrespective of estrogen receptor status. Authors propose enterolactone as a candidate for combined therapy to decrease the radiation dose delivered to patients and subsequent side effects. - Highlights: • Enterolactone is proposed to be a novel radiosensitizer for

Enterolactone: A novel radiosensitizer for human breast cancer cell lines through impaired DNA repair and increased apoptosis

Energy Technology Data Exchange (ETDEWEB)

Bigdeli, Bahareh, E-mail: bhr.bigdeli@ut.ac.ir [Department of Biophysics, Institute of Biochemistry and Biophysics, University of Tehran, 16th Azar St., Enghelab Sq., Tehran (Iran, Islamic Republic of); Goliaei, Bahram, E-mail: goliaei@ut.ac.ir [Department of Biophysics, Institute of Biochemistry and Biophysics, University of Tehran, 16th Azar St., Enghelab Sq., Tehran (Iran, Islamic Republic of); Masoudi-Khoram, Nastaran, E-mail: n.masoudi@alumni.ut.ac.ir [Department of Biophysics, Institute of Biochemistry and Biophysics, University of Tehran, 16th Azar St., Enghelab Sq., Tehran (Iran, Islamic Republic of); Jooyan, Najmeh, E-mail: n.jooyan@ut.ac.ir [Department of Biophysics, Institute of Biochemistry and Biophysics, University of Tehran, 16th Azar St., Enghelab Sq., Tehran (Iran, Islamic Republic of); Nikoofar, Alireza, E-mail: nikoofar@iums.ac.ir [Department of Radiotherapy, Iran University of Medical Sciences (IUMS), Shahid Hemmat Highway, Tehran (Iran, Islamic Republic of); Rouhani, Maryam, E-mail: rouhani@iasbs.ac.ir [Department of Biological Sciences, Institute for Advanced Studies in Basic Sciences (IASBS), Prof. Yousef Sobouti Blvd., Gava Zang, Zanjan (Iran, Islamic Republic of); Haghparast, Abbas, E-mail: Haghparast@sbmu.ac.ir [Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Daneshjo St., Evin, Tehran (Iran, Islamic Republic of); Mamashli, Fatemeh, E-mail: mamashli@ut.ac.ir [Department of Biophysics, Institute of Biochemistry and Biophysics, University of Tehran, 16th Azar St., Enghelab Sq., Tehran (Iran, Islamic Republic of)

2016-12-15

Introduction: Radiotherapy is a potent treatment against breast cancer, which is the most commonly diagnosed cancer among women. However, the emergence of radioresistance due to increased DNA repair leads to radiotherapeutic failure. Applying polyphenols combined with radiation is a more promising method leading to better survival. Enterolactone, a phytoestrogenic polyphenol, has been reported to inhibit an important radioresistance signaling pathway, therefore we conjectured that enterolactone could enhance radiosensitivity in breast cancer. To assess this hypothesis, radiation response of enterolactone treated MDA-MB-231 and T47D cell lines and corresponding cellular mechanisms were investigated. Methods: Cytotoxicity of enterolactone was measured via MTT assay. Cells were treated with enterolactone before X-irradiation, and clonogenic assay was used to evaluate radiosensitivity. Cell cycle distribution and apoptosis were measured by flow cytometric analysis. In addition, DNA damages and corresponding repair, chromosomal damages, and aberrations were assessed by comet, micronucleus, and cytogenetic assays, respectively. Results: Enterolactone decreased the viability of cells in a concentration- and time dependent manner. Enterolactone significantly enhanced radiosensitivity of cells by abrogating G2/M arrest, impairing DNA repair, and increasing radiation-induced apoptosis. Furthermore, increased chromosomal damages and aberrations were detected in cells treated with enterolactone combined with X-rays than X-ray alone. These effects were more prominent in T47D than MDA-MB-231 cells. Discussion: To our knowledge, this is the first report that enterolactone is a novel radiosensitizer for breast cancer irrespective of estrogen receptor status. Authors propose enterolactone as a candidate for combined therapy to decrease the radiation dose delivered to patients and subsequent side effects. - Highlights: • Enterolactone is proposed to be a novel radiosensitizer for

Rapid surface accumulation of NMDA receptors increases glutamatergic excitation during status epilepticus.

Science.gov (United States)

Naylor, David E; Liu, Hantao; Niquet, Jerome; Wasterlain, Claude G

2013-06-01

After 1h of lithium-pilocarpine status epilepticus (SE), immunocytochemical labeling of NMDA receptor NR1 subunits reveals relocation of subunits from the interior to the cell surface of dentate gyrus granule cells and CA3 pyramidal cells. Simultaneously, an increase in NMDA-miniature excitatory postsynaptic currents (mEPSC) as well as an increase in NMDA receptor-mediated tonic currents is observed in hippocampal slices after SE. Mean-variance analysis of NMDA-mEPSCs estimates that the number of functional postsynaptic NMDA receptors per synapse increases 38% during SE, and antagonism by ifenprodil suggests that an increase in the surface representation of NR2B-containing NMDA receptors is responsible for the augmentation of both the phasic and tonic excitatory currents with SE. These results provide a potential mechanism for an enhancement of glutamatergic excitation that maintains SE and may contribute to excitotoxic injury during SE. Therapies that directly antagonize NMDA receptors may be a useful therapeutic strategy during refractory SE. Copyright © 2013 Elsevier Inc. All rights reserved.

Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

Energy Technology Data Exchange (ETDEWEB)

Wang, Deng-Liang [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Yang, Hai-Tao; Wang, Jiang-Jie [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yao, Pei-Sen [Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Pan, Ru-Jun [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yang, Chaoyong James, E-mail: cyyang@xmu.edu.cn [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Kang, De-Zhi, E-mail: kdzy99988@163.com [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China)

2014-10-31

Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

International Nuclear Information System (INIS)

Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

2014-01-01

Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K d 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K d 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy

AIM2-Like Receptors Positively and Negatively Regulate the Interferon Response Induced by Cytosolic DNA.

Science.gov (United States)

Nakaya, Yuki; Lilue, Jingtao; Stavrou, Spyridon; Moran, Eileen A; Ross, Susan R

2017-07-05

Cytosolic DNAs derived from retrotransposons serve as pathogen-associated molecular patterns for pattern recognition receptors (PRRs) that stimulate the induction of interferons (IFNs) and other cytokines, leading to autoimmune disease. Cyclic GMP-AMP synthase is one PRR that senses retrotransposon DNA, activating type I IFN responses through the stimulator of IFN genes (STING). Absent in melanoma 2 (AIM2)-like receptors (ALRs) have also been implicated in these pathways. Here we show that the mouse ALR IFI205 senses cytosolic retrotransposon DNA independently of cyclic GMP-AMP production. AIM2 antagonizes IFI205-mediated IFN induction activity by sequestering it from STING. We also found that the complement of genes located in the ALR locus in C57BL/6 and AIM2 knockout mice are different and unique, which has implications for interpretation of the sensing of pathogens in different mouse strains. Our data suggest that members of the ALR family are critical to the host IFN response to endogenous DNA. IMPORTANCE Autoimmune diseases like Aicardi-Goutières syndrome and lupus erythematosus arise when cells of the immune system become activated and attack host cells and tissues. We found that DNA generated by endogenous retroviruses and retroelements in inbred mice and mouse cells is recognized by several host proteins found in macrophages that are members of the ALR family and that these proteins both suppress and activate the pathways leading to the generation of cytokines and IFNs. We also show that there is great genetic diversity between different inbred mouse strains in the ALR genes, which might contribute to differential susceptibility to autoimmunity. Understanding how immune cells become activated is important to the control of disease. Copyright © 2017 Nakaya et al.

Increasing global participation in genetics research through DNA barcoding.

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Adamowicz, Sarah J; Steinke, Dirk

2015-12-01

DNA barcoding--the sequencing of short, standardized DNA regions for specimen identification and species discovery--has promised to facilitate rapid access to biodiversity knowledge by diverse users. Here, we advance our opinion that increased global participation in genetics research is beneficial, both to scientists and for science, and explore the premise that DNA barcoding can help to democratize participation in genetics research. We examine publication patterns (2003-2014) in the DNA barcoding literature and compare trends with those in the broader, related domain of genomics. While genomics is the older and much larger field, the number of nations contributing to the published literature is similar between disciplines. Meanwhile, DNA barcoding exhibits a higher pace of growth in the number of publications as well as greater evenness among nations in their proportional contribution to total authorships. This exploration revealed DNA barcoding to be a highly international discipline, with growing participation by researchers in especially biodiverse nations. We briefly consider several of the challenges that may hinder further participation in genetics research, including access to training and molecular facilities as well as policy relating to the movement of genetic resources.

DMPD: Signal transduction pathways mediated by the interaction of CpG DNA withToll-like receptor 9. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 14751759 Signal transduction pathways mediated by the interaction of CpG DNA withTo...;16(1):17-22. (.png) (.svg) (.html) (.csml) Show Signal transduction pathways mediated by the interaction of... CpG DNA withToll-like receptor 9. PubmedID 14751759 Title Signal transduction pathways media

Risperidone treatment increases CB1 receptor binding in rat brain

DEFF Research Database (Denmark)

Secher, Anna; Husum, Henriette; Holst, Birgitte

2010-01-01

, the ghrelin receptor, neuropeptide Y, adiponectin and proopiomelanocortin. We investigated whether the expression of these factors was affected in rats chronically treated with the antipsychotic risperidone. METHODS: Male Sprague-Dawley rats were treated with risperidone (1.0 mg/kg/day) or vehicle (20...... showed that risperidone treatment altered CB(1) receptor binding in the rat brain. Risperidone-induced adiposity and metabolic dysfunction in the clinic may be explained by increased CB(1) receptor density in brain regions involved in appetite and regulation of metabolic function....

Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity.

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Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

2014-10-31

Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher's attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with Kd 56±7.3nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

Activation of glucocorticoid receptors increases 5-HT2A receptor levels

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Trajkovska, Viktorija; Kirkegaard, Lisbeth; Krey, Gesa

2009-01-01

an effect of GR activation on 5-HT2A levels, mature organotypic hippocampal cultures were exposed to corticosterone with or without GR antagonist mifepristone and mineralocorticoid receptor (MR) antagonist spironolactone. In GR under-expressing mice, hippocampal 5-HT2A receptor protein levels were decreased......Major depression is associated with both dysregulation of the hypothalamic pituitary adrenal axis and serotonergic deficiency, not the least of the 5-HT2A receptor. However, how these phenomena are linked to each other, and whether a low 5-HT2A receptor level is a state or a trait marker...... of depression is unknown. In mice with altered glucocorticoid receptor (GR) expression we investigated 5-HT2A receptor levels by Western blot and 3H-MDL100907 receptor binding. Serotonin fibre density was analyzed by stereological quantification of serotonin transporter immunopositive fibers. To establish...

LEDGF/p75 Deficiency Increases Deletions at the HIV-1 cDNA Ends.

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Bueno, Murilo T D; Reyes, Daniel; Llano, Manuel

2017-09-15

Processing of unintegrated linear HIV-1 cDNA by the host DNA repair system results in its degradation and/or circularization. As a consequence, deficient viral cDNA integration generally leads to an increase in the levels of HIV-1 cDNA circles containing one or two long terminal repeats (LTRs). Intriguingly, impaired HIV-1 integration in LEDGF/p75-deficient cells does not result in a correspondent increase in viral cDNA circles. We postulate that increased degradation of unintegrated linear viral cDNA in cells lacking the lens epithelium-derived growth factor (LEDGF/p75) account for this inconsistency. To evaluate this hypothesis, we characterized the nucleotide sequence spanning 2-LTR junctions isolated from LEDGF/p75-deficient and control cells. LEDGF/p75 deficiency resulted in a significant increase in the frequency of 2-LTRs harboring large deletions. Of note, these deletions were dependent on the 3' processing activity of integrase and were not originated by aberrant reverse transcription. Our findings suggest a novel role of LEDGF/p75 in protecting the unintegrated 3' processed linear HIV-1 cDNA from exonucleolytic degradation.

Concanavalin a increases beta-adrenergic and glucocorticoid receptors in porcine splenocytes

International Nuclear Information System (INIS)

Kelley, K.N.; Westly, H.J.

1986-01-01

We identified specific glucocorticoid and beta-adrenergic receptors on porcine splenocytes. There are 2000 to 4000 glucocorticoid receptors per cell with a K /SUB D/ of 2 to 4 nM and 1000 beta-adrenergic receptors with a K /SUB D/ of 0.3 to 0.6 nM. When splenocytes were incubated with concanavalin A (Con A), there was an approximate 2-fold increase in both gluococorticoid and beta-adrenergic receptors with no change in binding affinity. Incubation of splenocytes with cortisol as low as 40 nM (13 ng/ml) inhibited proliferation in response to Con A. This inhibitory effect of cortisol was not due to cytotoxic effects of glucocorticoids. At maximal physiologic concentrations (400 nM; 135 ng/ml), cortisol caused reductions in Con A activation of thymocytes and peripheral blood mononuclear cells. When eight wk old pigs were restrained, there was an increase in plasma cortisol, atrophy of thymus and reduction in skin test responses to phytohemagglutinin. On the basis of the data, we suggest that physiologic concentrations of stress asociated hormones affect functional activities of porcine lymphoid cells. Since activated splenocytes display increased numbers of receptors for these hormones, perhaps glucocorticoids or catecholamines normally function in vivo to suppress clonal expansion of antigen activated and autoreactive T lymphocytes

Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system

International Nuclear Information System (INIS)

Kajikawa, Mizuho; Sasaki, Kaori; Wakimoto, Yoshitaro; Toyooka, Masaru; Motohashi, Tomoko; Shimojima, Tsukasa; Takeda, Shigeki; Park, Enoch Y.; Maenaka, Katsumi

2009-01-01

Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (G i α) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [ 35 S]GTPγS-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.

Quantification of transcription factor-DNA binding affinity in a living cell.

Science.gov (United States)

Belikov, Sergey; Berg, Otto G; Wrange, Örjan

2016-04-20

The apparent dissociation constant (Kd) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [(3)H]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis. The fraction of DNA sites occupied by the expressed receptor was determined by dimethylsulphate in vivo footprinting and used for calculation of the receptor-DNA binding affinity. The forkhead transcription factor FoxA1 enhanced the DNA binding by GR with an apparent Kd of ∼1 μM and dramatically stimulated DNA binding by AR with an apparent Kd of ∼0.13 μM at a composite androgen responsive DNA element containing one FoxA1 binding site and one palindromic hormone receptor binding site known to bind one receptor homodimer. FoxA1 exerted a weak constitutive- and strongly cooperative DNA binding together with AR but had a less prominent effect with GR, the difference reflecting the licensing function of FoxA1 at this androgen responsive DNA element. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Expression and function of the human estrogen receptor in yeast

International Nuclear Information System (INIS)

White, J.H.; Metzger, D.; Chambon, P.

1988-01-01

Gene expression in eukaryotes is regulated at many levels. Moreover, there is increasing evidence that the basic control mechanisms of transcription initiation have been conserved across the range of eukaryotes from yeast to man. In vertebrates, the nuclear receptors, whose activity is dependent on the binding of specific ligands, stimulate transcription by interacting with specific cis-acting sequences and display all of the hallmarks of inducible enhancer factors. Alignment of their amino acid sequences indicates that they are composed of a series of conserved domains. The domain structure of the human estrogen receptor (hER) is typical of receptor proteins. Region C, containing two putative zinc fingers, comprises the DNA-binding domain responsible for specific recognition of estrogen response elements (ERE). Region E contains the hormone-binding domain and domain(s) responsible for transcription activation. A mutant of the hER, called HE15, which lacks the hormone-binding domain, binds DNA in vivo and in vitro but activates transcription only poorly in a constitutive manner in vivo in HeLa cells. A series of studies have demonstrated that the hormone- and DNA-binding domains of the nuclear receptors function independently. Chimeric proteins consisting of the DNA-binding domain of yeast GAL4 coupled to the hormone-binding domains of either the hER or glucocorticoid receptor element (GRE) will stimulate transcription in HeLa cells when bound to a UAS. Taken together, these results demonstrate that the hER and other nuclear receptors, as well as GAL4 and GCN4 proteins of yeast, consist of discrete and separable DNA-binding and transcription-activation functions. To investigate these striking parallels further, the authors have expressed the hER in the yeast Saccharomyces cerevisiae and have analyzed its hormone- and DNA-binding properties in vitro and its ability to stimulate transcription in vivo

Specificity of DNA-binding by the FAX-1 and NHR-67 nuclear receptors of Caenorhabditis elegans is partially mediated via a subclass-specific P-box residue

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Smith Eric L

2008-01-01

Full Text Available Abstract Background The nuclear receptors of the NR2E class play important roles in pattern formation and nervous system development. Based on a phylogenetic analysis of DNA-binding domains, we define two conserved groups of orthologous NR2E genes: the NR2E1 subclass, which includes C. elegans nhr-67, Drosophila tailless and dissatisfaction, and vertebrate Tlx (NR2E2, NR2E4, NR2E1, and the NR2E3 subclass, which includes C. elegans fax-1 and vertebrate PNR (NR2E5, NR2E3. PNR and Tll nuclear receptors have been shown to bind the hexamer half-site AAGTCA, instead of the hexamer AGGTCA recognized by most other nuclear receptors, suggesting unique DNA-binding properties for NR2E class members. Results We show that NR2E3 subclass member FAX-1, unlike NHR-67 and other NR2E1 subclass members, binds to hexamer half-sites with relaxed specificity: it will bind hexamers with the sequence ANGTCA, although it prefers a purine to a pyrimidine at the second position. We use site-directed mutagenesis to demonstrate that the difference between FAX-1 and NHR-67 binding preference is partially mediated by a conserved subclass-specific asparagine or aspartate residue at position 19 of the DNA-binding domain. This amino acid position is part of the "P box" that plays a critical role in defining binding site specificity and has been shown to make hydrogen-bond contacts to the second position of the hexamer in co-crystal structures for other nuclear receptors. The relaxed specificity allows FAX-1 to bind a much larger repertoire of half-sites than NHR-67. While NR2E1 class proteins bind both monomeric and dimeric sites, the NR2E3 class proteins bind only dimeric sites. The presence of a single strong site adjacent to a very weak site allows dimeric FAX-1 binding, further increasing the number of dimeric binding sites to which FAX-1 may bind in vivo. Conclusion These findings identify subclass-specific DNA-binding specificities and dimerization properties for the NR2E1

Vitamin D receptor displays DNA binding and transactivation as a heterodimer with the retinoid X receptor, but not with the thyroid hormone receptor.

Science.gov (United States)

Thompson, P D; Hsieh, J C; Whitfield, G K; Haussler, C A; Jurutka, P W; Galligan, M A; Tillman, J B; Spindler, S R; Haussler, M R

1999-12-01

The vitamin D receptor (VDR) is a transcription factor believed to function as a heterodimer with the retinoid X receptor (RXR). However, it was reported [Schräder et al., 1994] that, on putative vitamin D response elements (VDREs) within the rat 9k and mouse 28k calcium binding protein genes (rCaBP 9k and mCaBP 28k), VDR and thyroid hormone receptor (TR) form heterodimers that transactivate in response to both 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and triiodothyronine (T(3)). We, therefore, examined associations of these receptors on the putative rCaBP 9k and mCaBP 28k VDREs, as well as on established VDREs from the rat osteocalcin (rOC) and mouse osteopontin (mOP) genes, plus the thyroid hormone response element (TRE) from the rat myosin heavy chain (rMHC) gene. In gel mobility shift assays, we found no evidence for VDR-TR heterodimer interaction with any tested element. Further, employing these hormone response elements linked to reporter genes in transfected cells, VDR and TR mediated responses to their cognate ligands only from the rOC/mOP and rMHC elements, respectively, while the CaBP elements were unresponsive to any combination of ligand(s). Utilizing the rOC and mOP VDREs, two distinct repressive actions of TR on VDR-mediated signaling were demonstrated: a T(3)-independent action, presumably via direct TR-RXR competition for DNA binding, and a T(3)-dependent repression, likely by diversion of limiting RXR from VDR-RXR toward the formation of TR-RXR heterodimers. The relative importance of these two mechanisms differed in a response element-specific manner. These results may provide a partial explanation for the observed association between hyperthyroidism and bone demineralization/osteoporosis. Copyright 1999 Wiley-Liss, Inc.

Increased mitochondrial DNA deletions and copy number in transfusion-dependent thalassemia

Science.gov (United States)

Calloway, Cassandra

2016-01-01

BACKGROUND. Iron overload is the primary cause of morbidity in transfusion-dependent thalassemia. Increase in iron causes mitochondrial dysfunction under experimental conditions, but the occurrence and significance of mitochondrial damage is not understood in patients with thalassemia. METHODS. Mitochondrial DNA (mtDNA) to nuclear DNA copy number (Mt/N) and frequency of the common 4977-bp mitochondrial deletion (ΔmtDNA4977) were quantified using a quantitative PCR assay on whole blood samples from 38 subjects with thalassemia who were receiving regular transfusions. RESULTS. Compared with healthy controls, Mt/N and ΔmtDNA4977 frequency were elevated in thalassemia (P = 0.038 and P 15 mg/g dry-weight or splenectomy, with the highest levels observed in subjects who had both risk factors (P = 0.003). Myocardial iron (MRI T2* 40/1 × 107 mtDNA, respectively (P = 0.025). Subjects with Mt/N values below the group median had significantly lower Matsuda insulin sensitivity index (5.76 ± 0.53) compared with the high Mt/N group (9.11 ± 0.95, P = 0.008). CONCLUSION. Individuals with transfusion-dependent thalassemia demonstrate age-related increase in mtDNA damage in leukocytes. These changes are markedly amplified by splenectomy and are associated with extrahepatic iron deposition. Elevated mtDNA damage in blood cells may predict the risk of iron-associated organ damage in thalassemia. FUNDING. This project was supported by Children’s Hospital & Research Center Oakland Institutional Research Award and by the National Center for Advancing Translational Sciences, NIH, through UCSF-CTSI grant UL1 TR000004. PMID:27583305

Involvement of aberrant DNA methylation on reduced expression of lysophosphatidic acid receptor-1 gene in rat tumor cell lines

International Nuclear Information System (INIS)

Tsujiuchi, Toshifumi; Shimizu, Kyoko; Onishi, Mariko; Sugata, Eriko; Fujii, Hiromasa; Mori, Toshio; Honoki, Kanya; Fukushima, Nobuyuki

2006-01-01

Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, it has been reported that alterations of LPA receptor expression might be important in the malignant transformation of tumor cells. Therefore, to assess an involvement of DNA methylation in reduced expression of the LPA receptor-1 (lpa1) gene, we investigated the expression of the lpa1 gene and its DNA methylation patterns in rat tumor cell lines. Both rat brain-derived neuroblastoma B103 and liver-derived hepatoma RH7777 cells used in this study indicated no expression of lpa1. For the analysis of methylation status, bisulfite sequencing was performed with B103 and RH7777 cells, comparing with other lpa1 expressed cells and normal tissues of brain and liver. The lpa1 expressed cells and tissues were all unmethylated in this region of lpa1. In contrast, both B103 and RH7777 cells were highly methylated, correlating with reduced expression of the lpa1. Treatment with 5-aza 2'-deoxycytidine induced expression of lpa1 gene in B103 and RH7777 cells after 24 h. In RH7777 cells treated with 5-aza 2'-deoxycytidine, stress fiber formation was also observed in response to LPA in RH7777 cells, but not in untreated RH7777 cells. These results suggest that aberrant DNA methylation of the lpa1 gene may be involved in its reduced expression in rat tumor cells

LSD Increases Primary Process Thinking via Serotonin 2A Receptor Activation

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Rainer Kraehenmann

2017-11-01

Full Text Available Rationale: Stimulation of serotonin 2A (5-HT2A receptors by lysergic acid diethylamide (LSD and related compounds such as psilocybin has previously been shown to increase primary process thinking – an ontologically and evolutionary early, implicit, associative, and automatic mode of thinking which is typically occurring during altered states of consciousness such as dreaming. However, it is still largely unknown whether LSD induces primary process thinking under placebo-controlled, standardized experimental conditions and whether these effects are related to subjective experience and 5-HT2A receptor activation. Therefore, this study aimed to test the hypotheses that LSD increases primary process thinking and that primary process thinking depends on 5-HT2A receptor activation and is related to subjective drug effects.Methods: Twenty-five healthy subjects performed an audio-recorded mental imagery task 7 h after drug administration during three drug conditions: placebo, LSD (100 mcg orally and LSD together with the 5-HT2A receptor antagonist ketanserin (40 mg orally. The main outcome variable in this study was primary index (PI, a formal measure of primary process thinking in the imagery reports. State of consciousness was evaluated using the Altered State of Consciousness (5D-ASC rating scale.Results: LSD, compared with placebo, significantly increased primary index (p < 0.001, Bonferroni-corrected. The LSD-induced increase in primary index was positively correlated with LSD-induced disembodiment (p < 0.05, Bonferroni-corrected, and blissful state (p < 0.05, Bonferroni-corrected on the 5D-ASC. Both LSD-induced increases in primary index and changes in state of consciousness were fully blocked by ketanserin.Conclusion: LSD induces primary process thinking via activation of 5-HT2A receptors and in relation to disembodiment and blissful state. Primary process thinking appears to crucially organize inner experiences during both dreams and

LSD Increases Primary Process Thinking via Serotonin 2A Receptor Activation

Science.gov (United States)

Kraehenmann, Rainer; Pokorny, Dan; Aicher, Helena; Preller, Katrin H.; Pokorny, Thomas; Bosch, Oliver G.; Seifritz, Erich; Vollenweider, Franz X.

2017-01-01

Rationale: Stimulation of serotonin 2A (5-HT2A) receptors by lysergic acid diethylamide (LSD) and related compounds such as psilocybin has previously been shown to increase primary process thinking – an ontologically and evolutionary early, implicit, associative, and automatic mode of thinking which is typically occurring during altered states of consciousness such as dreaming. However, it is still largely unknown whether LSD induces primary process thinking under placebo-controlled, standardized experimental conditions and whether these effects are related to subjective experience and 5-HT2A receptor activation. Therefore, this study aimed to test the hypotheses that LSD increases primary process thinking and that primary process thinking depends on 5-HT2A receptor activation and is related to subjective drug effects. Methods: Twenty-five healthy subjects performed an audio-recorded mental imagery task 7 h after drug administration during three drug conditions: placebo, LSD (100 mcg orally) and LSD together with the 5-HT2A receptor antagonist ketanserin (40 mg orally). The main outcome variable in this study was primary index (PI), a formal measure of primary process thinking in the imagery reports. State of consciousness was evaluated using the Altered State of Consciousness (5D-ASC) rating scale. Results: LSD, compared with placebo, significantly increased primary index (p LSD-induced increase in primary index was positively correlated with LSD-induced disembodiment (p LSD-induced increases in primary index and changes in state of consciousness were fully blocked by ketanserin. Conclusion: LSD induces primary process thinking via activation of 5-HT2A receptors and in relation to disembodiment and blissful state. Primary process thinking appears to crucially organize inner experiences during both dreams and psychedelic states of consciousness. PMID:29167644

Allosteric analysis of glucocorticoid receptor-DNA interface induced by cyclic Py-Im polyamide: a molecular dynamics simulation study.

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Yaru Wang

Full Text Available BACKGROUND: It has been extensively developed in recent years that cell-permeable small molecules, such as polyamide, can be programmed to disrupt transcription factor-DNA interfaces and can silence aberrant gene expression. For example, cyclic pyrrole-imidazole polyamide that competes with glucocorticoid receptor (GR for binding to glucocorticoid response elements could be expected to affect the DNA dependent binding by interfering with the protein-DNA interface. However, how such small molecules affect the transcription factor-DNA interfaces and gene regulatory pathways through DNA structure distortion is not fully understood so far. METHODOLOGY/PRINCIPAL FINDINGS: In the present work, we have constructed some models, especially the ternary model of polyamides+DNA+GR DNA-binding domain (GRDBD dimer, and carried out molecular dynamics simulations and free energy calculations for them to address how polyamide molecules disrupt the GRDBD and DNA interface when polyamide and protein bind at the same sites on opposite grooves of DNA. CONCLUSIONS/SIGNIFICANCE: We found that the cyclic polyamide binding in minor groove of DNA can induce a large structural perturbation of DNA, i.e. a >4 Å widening of the DNA minor groove and a compression of the major groove by more than 4 Å as compared with the DNA molecule in the GRDBD dimer+DNA complex. Further investigations for the ternary system of polyamides+DNA+GRDBD dimer and the binary system of allosteric DNA+GRDBD dimer revealed that the compression of DNA major groove surface causes GRDBD to move away from the DNA major groove with the initial average distance of ∼4 Å to the final average distance of ∼10 Å during 40 ns simulation course. Therefore, this study straightforward explores how small molecule targeting specific sites in the DNA minor groove disrupts the transcription factor-DNA interface in DNA major groove, and consequently modulates gene expression.

HindIII identifies a two allele DNA polymorphism of the human cannabinoid receptor gene (CNR)

Energy Technology Data Exchange (ETDEWEB)

Caenazzo, L.; Hoehe, M.R.; Hsieh, W.T.; Berrettini, W.H.; Bonner, T.I.; Gershon, E.S. (National Inst. of Health, Bethesda, MD (United States))

1991-09-11

HCNR p5, a 0.9 kb BamHI/EcoRI fragment from the human cannabinoid receptor gene inserted into pUC19, was used as probe. The fragment is located in an intron approximately 14 kb 5{prime} of the initiation codon. This fragment is a clean single copy sequence by genomic blotting. Hybridization of human genomic DNA digested with HindIII identified a two allele RFLP with bands at 5.5 (A1) and 3.3 kb (A2). The human cannabinoid receptor gene has been genetically mapped in CEPH reference pedigrees to the centromeric/q region of chromosome 6. In situ hybridization localizes it to 6q14-q15. Codominant segregation has been observed in 26 informative two- and three-generation CEPH pedigrees and in 14 medium-sized disease families.

Disruption of mitochondrial DNA replication in Drosophila increases mitochondrial fast axonal transport in vivo.

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Rehan M Baqri

Full Text Available Mutations in mitochondrial DNA polymerase (pol gamma cause several progressive human diseases including Parkinson's disease, Alper's syndrome, and progressive external ophthalmoplegia. At the cellular level, disruption of pol gamma leads to depletion of mtDNA, disrupts the mitochondrial respiratory chain, and increases susceptibility to oxidative stress. Although recent studies have intensified focus on the role of mtDNA in neuronal diseases, the changes that take place in mitochondrial biogenesis and mitochondrial axonal transport when mtDNA replication is disrupted are unknown. Using high-speed confocal microscopy, electron microscopy and biochemical approaches, we report that mutations in pol gamma deplete mtDNA levels and lead to an increase in mitochondrial density in Drosophila proximal nerves and muscles, without a noticeable increase in mitochondrial fragmentation. Furthermore, there is a rise in flux of bidirectional mitochondrial axonal transport, albeit with slower kinesin-based anterograde transport. In contrast, flux of synaptic vesicle precursors was modestly decreased in pol gamma-alpha mutants. Our data indicate that disruption of mtDNA replication does not hinder mitochondrial biogenesis, increases mitochondrial axonal transport, and raises the question of whether high levels of circulating mtDNA-deficient mitochondria are beneficial or deleterious in mtDNA diseases.

Cytosolic DNA Sensor Upregulation Accompanies DNA Electrotransfer in B16.F10 Melanoma Cells

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Katarina Znidar

2016-01-01

Full Text Available In several preclinical tumor models, antitumor effects occur after intratumoral electroporation, also known as electrotransfer, of plasmid DNA devoid of a therapeutic gene. In mouse melanomas, these effects are preceded by significant elevation of several proinflammatory cytokines. These observations implicate the binding and activation of intracellular DNA-specific pattern recognition receptors or DNA sensors in response to DNA electrotransfer. In tumors, IFNβ mRNA and protein levels significantly increased. The mRNAs of several DNA sensors were detected, and DAI, DDX60, and p204 tended to be upregulated. These effects were accompanied with reduced tumor growth and increased tumor necrosis. In B16.F10 cells in culture, IFNβ mRNA and protein levels were significantly upregulated. The mRNAs for several DNA sensors were present in these cells; DNA-dependent activator of interferon regulatory factor (DAI, DEAD (Asp-Glu-Ala-Asp box polypeptide 60 (DDX60, and p204 were significantly upregulated while DDX60 protein levels were coordinately upregulated. Upregulation of DNA sensors in tumors could be masked by the lower transfection efficiency compared to in vitro or to dilution by other tumor cell types. Mirroring the observation of tumor necrosis, cells underwent a significant DNA concentration-dependent decrease in proliferation and survival. Taken together, these results indicate that DNA electrotransfer may cause the upregulation of several intracellular DNA sensors in B16.F10 cells, inducing effects in vitro and potentially in vivo.

Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system

Energy Technology Data Exchange (ETDEWEB)

Kajikawa, Mizuho; Sasaki, Kaori [Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Wakimoto, Yoshitaro; Toyooka, Masaru [Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); Motohashi, Tomoko; Shimojima, Tsukasa [National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540 (Japan); Takeda, Shigeki [Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma 376-8515 (Japan); Park, Enoch Y. [Laboratory of Biotechnology, Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 836 Oya, Suruga-ku, Shizuoka, Shizuoka 422-8529 (Japan); Maenaka, Katsumi, E-mail: kmaenaka-umin@umin.net [Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)

2009-07-31

Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (G{sub i}{alpha}) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [{sup 35}S]GTP{gamma}S-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.

Mu-opiate receptors measured by positron emission tomography are increased in temporal lobe epilepsy.

Science.gov (United States)

Frost, J J; Mayberg, H S; Fisher, R S; Douglass, K H; Dannals, R F; Links, J M; Wilson, A A; Ravert, H T; Rosenbaum, A E; Snyder, S H

1988-03-01

Neurochemical studies in animal models of epilepsy have demonstrated the importance of multiple neurotransmitters and their receptors in mediating seizures. The role of opiate receptors and endogenous opioid peptides in seizure mechanisms is well developed and is the basis for measuring opiate receptors in patients with epilepsy. Patients with complex partial seizures due to unilateral temporal seizure foci were studied by positron emission tomography using 11C-carfentanil to measure mu-opiate receptors and 18F-fluoro-deoxy-D-glucose to measure glucose utilization. Opiate receptor binding is greater in the temporal neocortex on the side of the electrical focus than on the opposite side. Modeling studies indicate that the increase in binding is due to an increase in affinity or the number of unoccupied receptors. No significant asymmetry of 11C-carfentanil binding was detected in the amygdala or hippocampus. Glucose utilization correlated inversely with 11C-carfentanil binding in the temporal neocortex. Increased opiate receptors in the temporal neocortex may represent a tonic anticonvulsant system that limits the spread of electrical activity from other temporal lobe structures.

cDNA cloning of chicken orexin receptor and tissue distribution: sexually dimorphic expression in chicken gonads.

Science.gov (United States)

Ohkubo, T; Tsukada, A; Shamoto, K

2003-12-01

Orexin-A and -B are known to stimulate food intake in mammals. However, the critical roles of orexins in birds are not fully understood, since orexins have no stimulatory effect on food intake in the chicken. To understand the physiological role(s) of orexins in birds, we have cloned chicken orexin receptor (cOXR) cDNA by RT-PCR, and analysed the tIssue distribution of OXR mRNA in the chicken. The cOXR cDNA is 1869 bp long and encodes 501 amino acids. The cloned cDNA for cOXR corresponds to the type 2 OXR in mammals, and shows approximately 80% similarity to those of mammals at the amino acid level. Expression analysis by RNase protection assay revealed OXR mRNA was distributed widely in brain regions, and expression in the cerebrum, hypothalamus and optic tectum were abundant. In peripheral tIssues, OXR mRNA was expressed in the pituitary gland, adrenal gland and testis, but no mRNA expression was observed in other tIssues examined. Furthermore, we found that the amount of cOXR mRNA was different between testis and ovary, while prepro-orexin mRNA is equally expressed in the gonads of both sexes in the chicken. These data indicate that the orexins have neuroendocrine actions in chickens, which are mediated through hypothalamic receptors as has been observed in mammals. In addition, orexin may have specific role(s) in the regulation of gonadal function in which sex-dependent mechanisms could be involved.

Increased hypothalamic 5-HT2A receptor gene expression and effects of pharmacologic 5-HT2A receptor inactivation in obese Ay mice

International Nuclear Information System (INIS)

Nonogaki, Katsunori; Nozue, Kana; Oka, Yoshitomo

2006-01-01

Serotonin (5-hydroxytryptamine; 5-HT) 2A receptors contribute to the effects of 5-HT on platelet aggregation and vascular smooth muscle cell proliferation, and are reportedly involved in decreases in plasma levels of adiponectin, an adipokine, in diabetic subjects. Here, we report that systemic administration of sarpogrelate, a 5-HT2A receptor antagonist, suppressed appetite and increased hypothalamic pro-opiomelanocortin and cocaine- and amphetamine-regulated transcript, corticotropin releasing hormone, 5-HT2C, and 5-HT1B receptor gene expression. A y mice, which have ectopic expression of the agouti protein, significantly increased hypothalamic 5-HT2A receptor gene expression in association with obesity compared with wild-type mice matched for age. Systemic administration of sarpogrelate suppressed overfeeding, body weight gain, and hyperglycemia in obese A y mice, whereas it did not increase plasma adiponectin levels. These results suggest that obesity increases hypothalamic 5-HT2A receptor gene expression, and pharmacologic inactivation of 5-HT2A receptors inhibits overfeeding and obesity in A y mice, but did not increase plasma adiponectin levels

ESR1 gene promoter region methylation in free circulating DNA and its correlation with estrogen receptor protein expression in tumor tissue in breast cancer patients

International Nuclear Information System (INIS)

Martínez-Galán, Joaquina; Ríos, Sandra; Delgado, Juan Ramón; Torres-Torres, Blanca; Núñez, María Isabel; López-Peñalver, Jesús; Del Moral, Rosario; Ruiz De Almodóvar, José Mariano; Menjón, Salomón; Concha, Ángel; Chamorro, Clara

2014-01-01

Tumor expression of estrogen receptor (ER) is an important marker of prognosis, and is predictive of response to endocrine therapy in breast cancer. Several studies have observed that epigenetic events, such methylation of cytosines and deacetylation of histones, are involved in the complex mechanisms that regulate promoter transcription. However, the exact interplay of these factors in transcription activity is not well understood. In this study, we explored the relationship between ER expression status in tumor tissue samples and the methylation of the 5′ CpG promoter region of the estrogen receptor gene (ESR1) isolated from free circulating DNA (fcDNA) in plasma samples from breast cancer patients. Patients (n = 110) with non-metastatic breast cancer had analyses performed of ER expression (luminal phenotype in tumor tissue, by immunohistochemistry method), and the ESR1-DNA methylation status (fcDNA in plasma, by quantitative methylation specific PCR technique). Our results showed a significant association between presence of methylated ESR1 in patients with breast cancer and ER negative status in the tumor tissue (p = 0.0179). There was a trend towards a higher probability of ESR1-methylation in those phenotypes with poor prognosis i.e. 80% of triple negative patients, 60% of HER2 patients, compared to 28% and 5.9% of patients with better prognosis such as luminal A and luminal B, respectively. Silencing, by methylation, of the promoter region of the ESR1 affects the expression of the estrogen receptor protein in tumors of breast cancer patients; high methylation of ESR1-DNA is associated with estrogen receptor negative status which, in turn, may be implicated in the patient’s resistance to hormonal treatment in breast cancer. As such, epigenetic markers in plasma may be of interest as new targets for anticancer therapy, especially with respect to endocrine treatment

Activation of protein kinase A and clustering of cell surface receptors by N-methyl-N'-nitro-N-nitrosoguanidine are independent of genomic DNA damage

Energy Technology Data Exchange (ETDEWEB)

Wang Zheng; Wang Guliang; Yang Jun; Guo Lei; Yu Yingnian

2003-07-25

Alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces cellular stress leading to chromosomal aberrations, mutations and cell death. Previous reports from our laboratory have shown that low concentration of MNNG induces untargeted mutation (UTM), which occurs on intact DNA in mammalian cells through changes in gene expression profile. It also causes the activation of cAMP-protein kinase A (PKA) and up-regulation of POL-{beta}, which is demonstrated to play a role in DNA repair system. In order to find out the possible initial signal involved in UTM, we try to investigate whether the activation of PKA-CREB signal pathway is closely related to DNA damage. Our data shows that the treatment of low concentration MNNG (0.2 {mu}M) activates PKA-CREB pathway in a comparable level both in a nuclear and enucleated cell system. And similar to the cell response caused by UV, the clustering of cell surface receptors of epidermal growth factor (EGF) and tumor necrosis factor {alpha} (TNF{alpha}) was also observed in cells exposed to MNNG. It was further demonstrated that the clustering of the surface receptors is independent of the genomic DNA damage, as this phenomenon was also observed in enucleated cells. These observations indicate that the initiation of signal cascades induced by low concentration of MNNG might be associated with its interaction with cell surface receptors and/or direct activation of related signal proteins but not its DNA damaging property.

Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage

DEFF Research Database (Denmark)

Syljuåsen, Randi G; Sørensen, Claus Storgaard; Hansen, Lasse Tengbjerg

2005-01-01

by increased amounts of nonextractable RPA protein, formation of single-stranded DNA, and induction of DNA strand breaks. Moreover, these responses were prevented by siRNA-mediated downregulation of Cdk2 or the replication initiation protein Cdc45, or by addition of the CDK inhibitor roscovitine. We propose...

Dopamine Receptor Activation Increases HIV Entry into Primary Human Macrophages

Science.gov (United States)

Gaskill, Peter J.; Yano, Hideaki H.; Kalpana, Ganjam V.; Javitch, Jonathan A.; Berman, Joan W.

2014-01-01

Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers. PMID:25268786

Cellular uptake of fluorophore-labeled glyco-DNA-gold nanoparticles

Energy Technology Data Exchange (ETDEWEB)

Witten, Katrin G.; Ruff, Julie [RWTH Aachen University, Institute of Inorganic Chemistry and JARA - Fundamentals of Future Information Technology (Germany); Mohr, Anne; Goertz, Dieter; Recker, Tobias; Rinis, Natalie [RWTH Aachen University, Institute of Biochemistry and Molecular Biology, University Hospital Aachen (Germany); Rech, Claudia; Elling, Lothar [RWTH Aachen University, Laboratory for Biomaterials, Institute of Biotechnology and Helmholtz-Institute for Biomedical Engineering (Germany); Mueller-Newen, Gerhard [RWTH Aachen University, Institute of Biochemistry and Molecular Biology, University Hospital Aachen (Germany); Simon, Ulrich, E-mail: ulrich.simon@ac.rwth-aachen.de [RWTH Aachen University, Institute of Inorganic Chemistry and JARA - Fundamentals of Future Information Technology (Germany)

2013-10-15

DNA-functionalized gold nanoparticles (AuNP-DNA) were hybridized with complementary di-N-acetyllactosamine-(di-LacNAc, [3Gal({beta}1-4)GlcNAc({beta}1-]2)-modified oligonucleotides to form glycol-functionalized particles, AuNP-DNA-di-LacNAc. While AuNP-DNA are known to be taken up by cells via scavenger receptors, glycol-functionalized particles have shown to be taken up via asialoglycoprotein receptors (ASGP-R). In this work, the interaction of these new particles with HepG2 cells was analyzed, which express scavenger receptors class B type I (SR-BI) and ASGP-R. To study the contribution of these receptors as potential mediators for cellular uptake, receptor-blocking experiments were performed with d-lactose, a ligand for ASGP-R, Fucoidan, a putative ligand for SR-BI, and a SR-BI blocking antibody. Labeling with Cy5-modified DNA ligands enabled us to monitor the particle uptake by confocal fluorescence microscopy and flow cytometry, in order to discriminate the two putative pathways by competitive binding studies. While SR-BI-antibody and d-lactose had no inhibiting effects on particle uptake Fucoidan led to a complete inhibition. Thus, a receptor-mediated uptake by the two receptors studied could not be proven and therefore other uptake mechanisms have to be considered.

Dihydrocoumarin, an HDAC Inhibitor, Increases DNA Damage Sensitivity by Inhibiting Rad52

Directory of Open Access Journals (Sweden)

Chin-Chuan Chen

2017-12-01

Full Text Available Effective DNA repair enables cancer cells to survive DNA damage induced by chemotherapeutic or radiotherapeutic treatments. Therefore, inhibiting DNA repair pathways is a promising therapeutic strategy for increasing the efficacy of such treatments. In this study, we found that dihydrocoumarin (DHC, a flavoring agent, causes deficiencies in double-stand break (DSB repair and prolonged DNA damage checkpoint recovery in yeast. Following DNA damage, Rad52 recombinase was revealed to be inhibited by DHC, which results in deficiencies in DSB repair and prolonged DNA damage checkpoint recovery. The deletion of RPD3, a class I histone deacetylase (HDAC, was found to mimic DHC-induced suppression of Rad52 expression, suggesting that the HDAC inhibitor activity of DHC is critical to DSB repair and DNA damage sensitivity. Overall, our findings delineate the regulatory mechanisms of DHC in DSB repair and suggest that it might potentially be used as an inhibitor of the DNA repair pathway in human cells.

Effects of gamma irradiation on the DNA-protein complex between the estrogen response element and the estrogen receptor

Czech Academy of Sciences Publication Activity Database

Štísová, Viktorie; Goffinont, S.; Maurizot, M. S.; Davídková, Marie

2010-01-01

Roč. 79, č. 8 (2010), s. 880-889 ISSN 0969-806X R&D Projects: GA MŠk 1P05OC085; GA MŠk OC09012 Institutional research plan: CEZ:AV0Z10480505 Keywords : DNA-protein complex * estrogen response element * estrogen receptor * ionizing radiation Subject RIV: BO - Biophysics Impact factor: 1.132, year: 2010

Dissecting direct and indirect readout of cAMP receptor protein DNA binding using an inosine and 2,6-diaminopurine in vitro selection system

DEFF Research Database (Denmark)

Lindemose, Søren; Nielsen, Peter E.; Møllegaard, Niels Erik

2008-01-01

The DNA interaction of the Escherichia coli cyclic AMP receptor protein (CRP) represents a typical example of a dual recognition mechanism exhibiting both direct and indirect readout. We have dissected the direct and indirect components of DNA recognition by CRP employing in vitro selection...... is functionally intact. The majority of the selected sites contain the natural consensus sequence TGTGAN(6)TCACA (i.e. TITIDN(6)TCDCD). Thus, direct readout of the consensus sequence is independent of minor groove conformation. Consequently, the indirect readout known to occur in the TG/CA base pair step (primary...... kink site) in the consensus sequence is not affected by I-D substitutions. In contrast, the flanking regions are selected as I/C rich sequences (mostly I-tracts) instead of A/T rich sequences which are known to strongly increase CRP binding, thereby demonstrating almost exclusive indirect readout...

Human circulating plasma DNA significantly decreases while lymphocyte DNA damage increases under chronic occupational exposure to low-dose gamma-neutron and tritium β-radiation

Energy Technology Data Exchange (ETDEWEB)

Korzeneva, Inna B., E-mail: inna.korzeneva@molgen.vniief.ru [Russian Federal Nuclear Center – All-Russian Research Institute of Experimental Physics (RFNC-VNIIEF) 607190, Sarov, 37 Mira ave., Nizhniy Novgorod Region (Russian Federation); Kostuyk, Svetlana V.; Ershova, Liza S. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, 115478 Moscow, 1 Moskvorechye str. (Russian Federation); Osipov, Andrian N. [Federal Medial and Biological Center named after Burnazyan of the Federal Medical and Biological Agency (FMBTz named after Burnazyan of FMBA), Moscow (Russian Federation); State Research Center - Burnasyan Federal Medical Biophysical Center of Federal Medical Biological Agency, Zhivopisnaya, 46, Moscow, 123098 (Russian Federation); Zhuravleva, Veronika F.; Pankratova, Galina V. [Russian Federal Nuclear Center – All-Russian Research Institute of Experimental Physics (RFNC-VNIIEF) 607190, Sarov, 37 Mira ave., Nizhniy Novgorod Region (Russian Federation); Porokhovnik, Lev N.; Veiko, Natalia N. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, 115478 Moscow, 1 Moskvorechye str. (Russian Federation)

2015-09-15

Highlights: • The chronic exposure to low-dose IR induces DSBs in human lymphocytes (TM index). • Exposure to IR decreases the level of human circulating DNA (cfDNA index). • IR induces an increase of DNase1 activity (DNase1 index) in plasma. • IR induces an increase of the level of antibodies to DNA (Ab DNA index) in plasma. • The ratio cfDNA/(DNase 1 × Ab DNA × TM) is a potential marker of human exposure to IR. - Abstract: The blood plasma of healthy people contains cell-fee (circulating) DNA (cfDNA). Apoptotic cells are the main source of the cfDNA. The cfDNA concentration increases in case of the organism’s cell death rate increase, for example in case of exposure to high-dose ionizing radiation (IR). The objects of the present research are the blood plasma and blood lymphocytes of people, who contacted occupationally with the sources of external gamma/neutron radiation or internal β-radiation of tritium N = 176). As the controls (references), blood samples of people, who had never been occupationally subjected to the IR sources, were used (N = 109). With respect to the plasma samples of each donor there were defined: the cfDNA concentration (the cfDNA index), DNase1 activity (the DNase1 index) and titre of antibodies to DNA (the Ab DNA index). The general DNA damage in the cells was defined (using the Comet assay, the tail moment (TM) index). A chronic effect of the low-dose ionizing radiation on a human being is accompanied by the enhancement of the DNA damage in lymphocytes along with a considerable cfDNA content reduction, while the DNase1 content and concentration of antibodies to DNA (Ab DNA) increase. All the aforementioned changes were also observed in people, who had not worked with the IR sources for more than a year. The ratio cfDNA/(DNase1 × Ab DNA × TM) is proposed to be used as a marker of the chronic exposure of a person to the external low-dose IR. It was formulated the assumption that the joint analysis of the cfDNA, DNase1, Ab

Association of Amine-Receptor DNA Sequence Variants with Associative Learning in the Honeybee.

Science.gov (United States)

Lagisz, Malgorzata; Mercer, Alison R; de Mouzon, Charlotte; Santos, Luana L S; Nakagawa, Shinichi

2016-03-01

Octopamine- and dopamine-based neuromodulatory systems play a critical role in learning and learning-related behaviour in insects. To further our understanding of these systems and resulting phenotypes, we quantified DNA sequence variations at six loci coding octopamine-and dopamine-receptors and their association with aversive and appetitive learning traits in a population of honeybees. We identified 79 polymorphic sequence markers (mostly SNPs and a few insertions/deletions) located within or close to six candidate genes. Intriguingly, we found that levels of sequence variation in the protein-coding regions studied were low, indicating that sequence variation in the coding regions of receptor genes critical to learning and memory is strongly selected against. Non-coding and upstream regions of the same genes, however, were less conserved and sequence variations in these regions were weakly associated with between-individual differences in learning-related traits. While these associations do not directly imply a specific molecular mechanism, they suggest that the cross-talk between dopamine and octopamine signalling pathways may influence olfactory learning and memory in the honeybee.

Cloning the interleukin 1 receptor from human T cells

International Nuclear Information System (INIS)

Sims, J.E.; Acres, R.B.; Grubin, C.E.; McMahan, C.J.; Wignall, J.M.; March, C.J.; Dower, S.K.

1989-01-01

cDNA clones of the interleukin 1 (IL-1) receptor expressed in a human T-cell clone have been isolated by using a murine IL-1 receptor cDNA as a probe. The human and mouse receptors show a high degree of sequence conservation. Both are integral membrane proteins possessing a single membrane-spanning segment. Similar to the mouse receptor, the human IL-1 receptor contains a large cytoplasmic region and an extracellular, IL-1 binding portion composed of three immunoglobulin-like domains. When transfected into COS cells, the human IL-1 receptor cDNA clone leads to expression of two different affinity classes of receptors, with K a values indistinguishable from those determined for IL-1 receptors in the original T-cell clone. An IL-1 receptor expressed in human dermal fibroblasts has also been cloned and sequenced and found to be identical to the IL-1 receptor expressed in T cells

GABAA receptors, but not dopamine, serotonin or NMDA receptors, are increased in the frontal cortex from schizophrenic subjects

International Nuclear Information System (INIS)

Daen, B.; Hussain, T.; Scarr, E.; Tomaskovic, E.; Kitsoulis, S.; Pavey, G.; Hill, C.; Keks, N.; Opeskin, K.; Copolov, D.L.

1998-01-01

Full text: Having shown changed 5HT 2A receptor density in the frontal cortex (FC) from schizophrenic subjects (1) we now report on further studies of the molecular neuroanatomy of the FC in schizophrenia. We used in situ radioligand binding and autoradiography to measure the density of [ 3 H]8OH-DPAT (1 nM) binding (5HT 1A receptors) and [ 3 H]GR113808 (2.4nM) binding (5HT 4 receptors) in Brodmann's areas (BA) 8, 9 and 10 from 10 schizophrenic and 10 controls subjects. In addition, [ 3 H]muscimol (100 nM) binding (GABA A receptors), [ 3 H]TCP (20nM) binding (NMDA receptors), [ 3 H]SCH 23390 (3nM) binding (DA D 1 like receptors) and [ 3 H]YM-09151-2 (4nM) binding (DA D 2 -like receptors) was measured in BA 9 from 17 schizophrenic and 17 control subjects. Subjects were matched for age and sex and the post-mortem interval for tissue collection did not differ. There was a significant increase (18%) in the density of GABA A receptors in BA 9 from subjects with schizophrenia (p<0.05) with no change in NMDA, dopamine or serotonin receptors. These data support the hypothesis that there are selective changes in neurotransmitter receptors in the FC of subjects with schizophrenia. It is not yet clear if such changes contribute to the pathology of the illness. Copyright (1998) Australian Neuroscience Society

A nonsense mutation causing decreased levels of insulin receptor mRNA: Detection by a simplified technique for direct sequencing of genomic DNA amplified by the polymerase chain reaction

International Nuclear Information System (INIS)

Kadowaki, T.; Kadowaki, H.; Taylor, S.I.

1990-01-01

Mutations in the insulin receptor gene can render the cell resistant to the biological action of insulin. The authors have studied a patient with leprechaunism (leprechaun/Minn-1), a genetic syndrome associated with intrauterine growth retardation and extreme insulin resistance. Genomic DNA from the patient was amplified by the polymerase chain reaction catalyzed by Thermus aquaticus (Taq) DNA polymerase, and the amplified DNA was directly sequenced. A nonsense mutations was identified at codon 897 in exon 14 in the paternal allele of the patient's insulin receptor gene. Levels of insulin receptor mRNA are decreased to <10% of normal in Epstein-Barr virus-transformed lymphoblasts and cultured skin fibroblasts from this patient. Thus, this nonsense mutation appears to cause a decrease in the levels of insulin receptor mRNA. In addition, they have obtained indirect evidence that the patient's maternal allele of the insulin receptor gene contains a cis-acting dominant mutation that also decreases the level of mRNA, but by a different mechanism. The nucleotide sequence of the entire protein-coding domain and the sequences of the intron-exon boundaries for all 22 exons of the maternal allele were normal. Presumably, the mutation in the maternal allele maps elsewhere in the insulin receptor gene. Thus, they conclude that the patient is a compound heterozygote for two cis-acting dominant mutations in the insulin receptor gene: (i) a nonsense mutation in the paternal allel that reduces the level of insulin receptor mRNA and (ii) an as yet unidentified mutation in the maternal allele that either decreases the rate of transcription or decreases the stability of the mRNA

DNA damage in blood lymphocytes in patients after {sup 177}Lu peptide receptor radionuclide therapy

Energy Technology Data Exchange (ETDEWEB)

Eberlein, Uta; Bluemel, Christina; Buck, Andreas Konrad; Werner, Rudolf Alexander; Lassmann, Michael [University of Wuerzburg, Department of Nuclear Medicine, Wuerzburg (Germany); Nowak, Carina; Scherthan, Harry [Bundeswehr Institute of Radiobiology affiliated to the University of Ulm, Munich (Germany)

2015-10-15

The aim of the study was to investigate DNA double strand break (DSB) formation and its correlation with the absorbed dose to the blood lymphocytes of patients undergoing their first peptide receptor radionuclide therapy (PRRT) with {sup 177}Lu-labelled DOTATATE/DOTATOC. The study group comprised 16 patients receiving their first PRRT. At least six peripheral blood samples were obtained before, and between 0.5 h and 48 h after radionuclide administration. From the time-activity curves of the blood and the whole body, residence times for blood self-irradiation and whole-body irradiation were determined. Peripheral blood lymphocytes were isolated, fixed with ethanol and subjected to immunofluorescence staining for colocalizing γ-H2AX/53BP1 DSB-marking foci. The average number of DSB foci per cell per patient sample was determined as a function of the absorbed dose to the blood and compared with an in vitro calibration curve established in our laboratory with {sup 131}I and {sup 177}Lu. The average number of radiation-induced foci (RIF) per cell increased over the first 5 h after radionuclide administration and decreased thereafter. A linear fit from 0 to 5 h as a function of the absorbed dose to the blood agreed with our in vitro calibration curve. At later time-points the number of RIF decreased, indicating progression of DNA repair. Measurements of RIF and the absorbed dose to the blood after systemic administration of {sup 177}Lu may be used to obtain data on the individual dose-response relationships in vivo. Individual patient data were characterized by a linear dose-dependent increase and an exponential decay function describing repair. (orig.)

Retinoid X Receptor α-Dependent HBV Minichromosome Remodeling and Viral Replication.

Science.gov (United States)

Zhang, Yan; He, Song; Guo, Jin-Jun; Peng, Hong; Fan, Jia-Hao; Li, Qing-Ling

2017-01-01

The HBV covalently closed circular DNA (cccDNA) is organized into a minichromosome in the nuclei of infected hepatocytes through interactions with histone and nonhistone proteins. Retinoid X receptor α (RXRα), a liver-enriched nuclear receptor, participates in regulation of HBV replication and transcription through modulation of HBV enhancer 1 and core promoter activity. This study investigated RXRα involvement in HBV cccDNA epigenetic modifications. Quantitative cccDNA chromatin immunoprecipitation (ChIP) was applied to study the recruitment of RXRα, histones, and chromatin-modifying enzymes to HBV minichromosome in HepG2 cells after transfection of the linear HBV genome. RXRα Was found to directly bind to HBV cccDNA; recruitment of RXRα to HBV mini-chromosome paralleled HBV replication, histone recruitment, and histone acetylation in HBVcccDNA. Moreover, RXRα overexpression or knock-down significantly increased or impaired the recruitment of the p300 acetyltransferase to cccDNAminichromosome. Our results confirmed the regulation of RXRα on HBV replication in vitro and demonstrated the modulation of RXRα on HBV cccDNA epigenetics. These findings provide a profound theoretical and experimental basis for late-model antiviral treatment acting on the HBV cccDNA and minichromosome.

Dopamine receptor activation increases HIV entry into primary human macrophages.

Directory of Open Access Journals (Sweden)

Peter J Gaskill

Full Text Available Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers.

Cisplatin-resistant cells express increased levels of a factor that recognizes damaged DNA

International Nuclear Information System (INIS)

Chu, G.; Chang, E.

1990-01-01

Cancer treatment with the drug cisplatin is often thwarted by the emergence of drug-resistant cells. To study this phenomenon, the authors identified two independent cellular factors that recognize cisplatin-damaged DNA. One of the two factors, designated XPE binding factor, is deficient in complementation group E of xeroderma pigmentosum, an inherited disease characterized by defective repair of DNA damaged by ultraviolet radiation, cisplatin, and other agents. Human tumor cell lines selected for resistance to cisplatin showed more efficient DNA repair and increased expression of XPE binding factor. These results suggest that XPE binding factor may be responsible, at least in part, for the development of cisplatin resistance in human tumors and that the mechanism may be increased DNA repair

Increased levels of mitochondrial DNA copy number in patients with vitiligo.

Science.gov (United States)

Vaseghi, H; Houshmand, M; Jadali, Z

2017-10-01

Oxidative stress is known to be involved in the pathogenesis of autoimmune diseases such as vitiligo. Evidence suggests that the human mitochondrial DNA copy number (mtDNAcn) is vulnerable to damage mediated by oxidative stress. The purpose of this study was to examine and compare peripheral blood mtDNAcn and oxidative DNA damage byproducts (8-hydroxy-2-deoxyguanosine; 8-OHdG) in patients with vitiligo and healthy controls (HCs). The relative mtDNAcn and the oxidative damage (formation of 8-OHdG in mtDNA) of each sample were determined by real-time quantitative PCR. Blood samples were obtained from 56 patients with vitiligo and 46 HCs. The mean mtDNAcn and the degree of mtDNA damage were higher in patients with vitiligo than in HCs. These data suggest that increase in mtDNAcn and oxidative DNA damage may be involved in the pathogenesis of vitiligo. © 2017 British Association of Dermatologists.

Acutely increasing δGABAA receptor activity impairs memory and inhibits synaptic plasticity in the hippocampus

Directory of Open Access Journals (Sweden)

Paul David Whissell

2013-09-01

Full Text Available Extrasynaptic γ-aminobutyric acid type A (GABAA receptors that contain the δ subunit (δGABAA receptors are expressed in several brain regions including the dentate gyrus (DG and CA1 subfields of the hippocampus. Drugs that increase δGABAA receptor activity have been proposed as treatments for a variety of disorders including insomnia, epilepsy and chronic pain. Also, long-term pretreatment with the δGABAA receptor–preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP enhances discrimination memory and increases neurogenesis in the DG. Despite the potential therapeutic benefits of such treatments, the effects of acutely increasing δGABAA receptor activity on memory behaviors remain unknown. Here, we studied the effects of THIP (4 mg/kg, i.p. on memory performance in wild-type (WT and δGABAA receptor null mutant (Gabrd–/– mice. Additionally, the effects of THIP on long-term potentiation (LTP, a molecular correlate of memory, were studied within the DG and CA1 subfields of the hippocampus using electrophysiological recordings of field potentials in hippocampal slices. The results showed that THIP impaired performance in the Morris water maze, contextual fear conditioning and object recognition tasks in WT mice but not Gabrd–/– mice. Furthermore, THIP inhibited LTP in hippocampal slices from WT but not Gabrd–/– mice, an effect that was blocked by GABAA receptor antagonist bicuculline. Thus, acutely increasing δGABAA receptor activity impairs memory behaviors and inhibits synaptic plasticity. These results have important implications for the development of therapies aimed at increasing δGABAA receptor activity.

Acutely increasing δGABAA receptor activity impairs memory and inhibits synaptic plasticity in the hippocampus

Science.gov (United States)

Whissell, Paul D.; Eng, Dave; Lecker, Irene; Martin, Loren J.; Wang, Dian-Shi; Orser, Beverley A.

2013-01-01

Extrasynaptic γ-aminobutyric acid type A (GABAA) receptors that contain the δ subunit (δGABAA receptors) are expressed in several brain regions including the dentate gyrus (DG) and CA1 subfields of the hippocampus. Drugs that increase δGABAA receptor activity have been proposed as treatments for a variety of disorders including insomnia, epilepsy and chronic pain. Also, long-term pretreatment with the δGABAA receptor–preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) enhances discrimination memory and increases neurogenesis in the DG. Despite the potential therapeutic benefits of such treatments, the effects of acutely increasing δGABAA receptor activity on memory behaviors remain unknown. Here, we studied the effects of THIP (4 mg/kg, i.p.) on memory performance in wild-type (WT) and δGABAA receptor null mutant (Gabrd−/−) mice. Additionally, the effects of THIP on long-term potentiation (LTP), a molecular correlate of memory, were studied within the DG and CA1 subfields of the hippocampus using electrophysiological recordings of field potentials in hippocampal slices. The results showed that THIP impaired performance in the Morris water maze, contextual fear conditioning and object recognition tasks in WT mice but not Gabrd−/− mice. Furthermore, THIP inhibited LTP in hippocampal slices from WT but not Gabrd−/− mice, an effect that was blocked by GABAA receptor antagonist bicuculline. Thus, acutely increasing δGABAA receptor activity impairs memory behaviors and inhibits synaptic plasticity. These results have important implications for the development of therapies aimed at increasing δGABAA receptor activity. PMID:24062648

Chronic Administration of Benzo(apyrene Induces Memory Impairment and Anxiety-Like Behavior and Increases of NR2B DNA Methylation.

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Wenping Zhang

Full Text Available Recently, an increasing number of human and animal studies have reported that exposure to benzo(apyrene (BaP induces neurological abnormalities and is also associated with adverse effects, such as tumor formation, immunosuppression, teratogenicity, and hormonal disorders. However, the exact mechanisms underlying BaP-induced impairment of neurological function remain unclear. The aim of this study was to examine the regulating mechanisms underlying the impact of chronic BaP exposure on neurobehavioral performance.C57BL mice received either BaP in different doses (1.0, 2.5, 6.25 mg/kg or olive oil twice a week for 90 days. Memory and emotional behaviors were evaluated using Y-maze and open-field tests, respectively. Furthermore, levels of mRNA expression were measured by using qPCR, and DNA methylation of NMDA receptor 2B subunit (NR2B was examined using bisulfate pyrosequencing in the prefrontal cortex and hippocampus.Compared to controls, mice that received BaP (2.5, 6.25 mg/kg showed deficits in short-term memory and an anxiety-like behavior. These behavioral alterations were associated with a down-regulation of the NR2B gene and a concomitant increase in the level of DNA methylation in the NR2B promoter in the two brain regions.Chronic BaP exposure induces an increase in DNA methylation in the NR2B gene promoter and down-regulates NR2B expression, which may contribute to its neurotoxic effects on behavioral performance. The results suggest that NR2B vulnerability represents a target for environmental toxicants in the brain.

miRNA-148a regulates the expression of the estrogen receptor through DNMT1-mediated DNA methylation in breast cancer cells

Science.gov (United States)

Xu, Yurui; Chao, Lin; Wang, Jianyu; Sun, Yonghong

2017-01-01

Breast cancer remains the most prevalent cancer among women worldwide. The expression of estrogen receptor-α (ER-α) is an important marker for prognosis. ER-α status may be positive or negative in breast cancer cells, although the cause of negative or positive status is not yet fully characterized. In the present study, the expression of ER-α and miRNA-148a was assessed in two breast cancer cell lines, HCC1937 and MCF7. An association between ER-α and miRNA-148a expression was identified. It was then demonstrated that DNA methyltransferase 1 (DNMT1) is a target of miRNA-148a, which may suppress the expression of ER-α via DNA methylation. Finally, an miRNA-148a mimic or inhibitor was transfected into MCF7 cells; the miRNA-148a mimic increased ER-α expression whereas the miRNA-148a inhibitor decreased ER-α expression. In conclusion, it was identified that miRNA-148a regulates ER-α expression through DNMT1-mediated DNA methylation in breast cancer cells. This may represent a potential miRNA-based strategy to modulate the expression of ER-α and provide a novel perspective for investigating the role of miRNAs in treating breast cancer. PMID:29085474

Intracellular Delivery of a Planar DNA Origami Structure by the Transferrin-Receptor Internalization Pathway

DEFF Research Database (Denmark)

Schaffert, David Henning; Okholm, Anders Hauge; Sørensen, Rasmus Schøler

2016-01-01

DNA origami provides rapid access to easily functionalized, nanometer-sized structures making it an intriguing platform for the development of defined drug delivery and sensor systems. Low cellular uptake of DNA nanostructures is a major obstacle in the development of DNA-based delivery platforms....... Herein, significant strong increase in cellular uptake in an established cancer cell line by modifying a planar DNA origami structure with the iron transport protein transferrin (Tf) is demonstrated. A variable number of Tf molecules are coupled to the origami structure using a DNA-directed, site...... on the origami surface....

Toll-like receptor 2 or toll-like receptor 4 deficiency does not modify lupus in MRLlpr mice.

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Simon J Freeley

Full Text Available Systemic lupus erythematosus is an autoimmune disease with a high morbidity and nephritis is a common manifestation. Previous studies in murine lupus models have suggest a role for Toll-like receptor 2 and 4. We examined the role of these molecules in MRL lpr mice which is one of the most established and robust murine models. We compared disease parameters in Toll-like receptor 2 or Toll-like receptor 4 deficient mice with their littermate controls. We found no difference in the severity of glomerulonephritis as assessed by histology, serum creatinine and albuminuria when Toll-like receptor 2 or Toll-like receptor 4 deficient MRLlpr mice were compared with Toll-like receptor sufficient controls. We also found similar levels of anti-dsDNA and anti-ssDNA antibodies. These results show that Toll-like receptor 2 and Toll-like receptor 4 do not play a significant role in MRLlpr mice, and therefore they may not be important in human lupus.

Intracellular Delivery of a Planar DNA Origami Structure by the Transferrin-Receptor Internalization Pathway.

Science.gov (United States)

Schaffert, David H; Okholm, Anders H; Sørensen, Rasmus S; Nielsen, Jesper S; Tørring, Thomas; Rosen, Christian B; Kodal, Anne Louise B; Mortensen, Michael R; Gothelf, Kurt V; Kjems, Jørgen

2016-05-01

DNA origami provides rapid access to easily functionalized, nanometer-sized structures making it an intriguing platform for the development of defined drug delivery and sensor systems. Low cellular uptake of DNA nanostructures is a major obstacle in the development of DNA-based delivery platforms. Herein, significant strong increase in cellular uptake in an established cancer cell line by modifying a planar DNA origami structure with the iron transport protein transferrin (Tf) is demonstrated. A variable number of Tf molecules are coupled to the origami structure using a DNA-directed, site-selective labeling technique to retain ligand functionality. A combination of confocal fluorescence microscopy and quantitative (qPCR) techniques shows up to 22-fold increased cytoplasmic uptake compared to unmodified structures and with an efficiency that correlates to the number of transferrin molecules on the origami surface. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Acutely increasing δGABA(A) receptor activity impairs memory and inhibits synaptic plasticity in the hippocampus.

Science.gov (United States)

Whissell, Paul D; Eng, Dave; Lecker, Irene; Martin, Loren J; Wang, Dian-Shi; Orser, Beverley A

2013-01-01

Extrasynaptic γ-aminobutyric acid type A (GABA(A)) receptors that contain the δ subunit (δGABA(A) receptors) are expressed in several brain regions including the dentate gyrus (DG) and CA1 subfields of the hippocampus. Drugs that increase δGABA(A) receptor activity have been proposed as treatments for a variety of disorders including insomnia, epilepsy and chronic pain. Also, long-term pretreatment with the δGABA(A) receptor-preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) enhances discrimination memory and increases neurogenesis in the DG. Despite the potential therapeutic benefits of such treatments, the effects of acutely increasing δGABA(A) receptor activity on memory behaviors remain unknown. Here, we studied the effects of THIP (4 mg/kg, i.p.) on memory performance in wild-type (WT) and δGABA(A) receptor null mutant (Gabrd(-/-)) mice. Additionally, the effects of THIP on long-term potentiation (LTP), a molecular correlate of memory, were studied within the DG and CA1 subfields of the hippocampus using electrophysiological recordings of field potentials in hippocampal slices. The results showed that THIP impaired performance in the Morris water maze, contextual fear conditioning and object recognition tasks in WT mice but not Gabrd(-/-) mice. Furthermore, THIP inhibited LTP in hippocampal slices from WT but not Gabrd(-/-) mice, an effect that was blocked by GABA(A) receptor antagonist bicuculline. Thus, acutely increasing δGABA(A) receptor activity impairs memory behaviors and inhibits synaptic plasticity. These results have important implications for the development of therapies aimed at increasing δGABA(A) receptor activity.

Hyperinsulinemia is associated with increased soluble insulin receptors release from hepatocytes

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Marcia eHiriart

2014-06-01

Full Text Available It has been generally assumed that insulin circulates freely in blood. However it can also interact with plasma proteins. Insulin receptors are located in the membrane of target cells and consist of an alpha and beta subunits with a tyrosine kinase cytoplasmic domain. The ectodomain, called soluble insulin receptor (SIR has been found elevated in patients with diabetes mellitus. We explored if insulin binds to SIRs in circulation under physiological conditions and hypothesize that this SIR may be released by hepatocytes in response to high insulin concentrations. The presence of SIR in rat and human plasmas and the culture medium of hepatocytes was explored using Western blot analysis. A purification protocol was performed to isolated SIR using affinity, gel filtration and ion exchange chromatographies. A modified reverse hemolytic plaque assay was used to measure SIR release from cultured hepatocytes. Incubation with 1 nmol l-1 insulin induces the release of the insulin receptor ectodomains from normal rat hepatocytes. This effect can be partially prevented by blocking protease activity. Furthermore, plasma levels of SIR were higher in a model of metabolic syndrome, where rats are hyperinsulinemic. We also found increased SIR levels in hyperinsulinemic humans. SIR may be an important regulator of the amount of free insulin in circulation. In hyperinsulinemia the amount of this soluble receptor increases, this could lead to higher amounts of insulin bound to this receptor, rather than free insulin, which is the biologically active form of the hormone. This observation could enlighten the mechanisms of insulin resistance.

Increased rate of repair of ultraviolet-induced DNA strand breaks in mitogen stimulated lymphocytes

Energy Technology Data Exchange (ETDEWEB)

Hamlet, S.M.; Lavin, M.F.; Jennings, P.A. (Queensland Univ., St. Lucia (Australia). Dept. of Biochemistry; Queensland Univ., St. Lucia (Australia). Dept. of Veterinary Pathology; Queensland Univ. St. Lucia (Australia). Dept. of Public Health)

1982-05-01

Previous results have shown that phytohaemagglutinin-stimulated bovine lymphocytes exhibit a peak of ultraviolet-induced DNA repair synthesis 3 to 4 days after addition of mitogen. The level of repair synthesis was approximately tenfold higher than that in unstimulated lymphocytes. These studies have been extended to examine the rate of repair of strand breaks in U.V.-irradiated bovine lymphocytes. The extent of breakage of DNA was shown to be the same in mitogen-stimulated and unstimulated lymphocytes from two breeds of cattle, when determined by sedimentation of nucleoids on sucrose gradients. However, in mitogen-stimulated cells the time taken to repair DNA strand breaks was 6 hours compared with 12 hours in stationary phase lymphocytes after a U.V. dose of 5 J/m/sup 2/. These results suggest that the increased rate of repair of strand breaks is due to the induction of enzymes involved at the post-incision stage of DNA repair. Thus the increased level of repair synthesis observed in earlier work correlates with an increased rate of repair of DNA strand breaks in phytohaemagglutinin-stimulated bovine lymphocytes.

Increased rate of repair of ultraviolet-induced DNA strand breaks in mitogen stimulated lymphocytes

International Nuclear Information System (INIS)

Hamlet, S.M.; Lavin, M.F.; Jennings, P.A.; Queensland Univ., St. Lucia; Queensland Univ. St. Lucia

1982-01-01

Previous results have shown that phytohaemagglutinin-stimulated bovine lymphocytes exhibit a peak of ultraviolet-induced DNA repair synthesis 3 to 4 days after addition of mitogen. The level of repair synthesis was approximately tenfold higher than that in unstimulated lymphocytes. These studies have been extended to examine the rate of repair of strand breaks in U.V.-irradiated bovine lymphocytes. The extent of breakage of DNA was shown to be the same in mitogen-stimulated and unstimulated lymphocytes from two breeds of cattle, when determined by sedimentation of nucleoids on sucrose gradients. However, in mitogen-stimulated cells the time taken to repair DNA strand breaks was 6 hours compared with 12 hours in stationary phase lymphocytes after a U.V. dose of 5 J/m 2 . These results suggest that the increased rate of repair of strand breaks is due to the induction of enzymes involved at the post-incision stage of DNA repair. Thus the increased level of repair synthesis observed in earlier work correlates with an increased rate of repair of DNA strand breaks in phytohaemagglutinin-stimulated bovine lymphocytes. (author)

Molecular cloning of a second subunit of the receptor for human granulocyte - macrophage colony-stimulating factor (GM-CSF): Reconstitution of a high-affinity GM-CSF receptor

International Nuclear Information System (INIS)

Hayashida, Kazuhiro; Kitamura, Toshio; Gorman, D.M.; Miyajima, Atsushi; Arai, Kenichi; Yokota, Takashi

1990-01-01

Using the mouse interleukin 3 (IL-3) receptor cDNA as a probe, the authors obtained a monologous cDNA (KH97) from a cDNA library of a human hemopoietic cell line, TF-1. The protein encoded by the KH97 cDNA has 56% amino acid sequence identity with the mouse IL-3 receptor and retains features common to the family of cytokine receptors. Fibroblasts transfected with the KH97 cDNA expressed a protein of 120 kDa but did not bind any human cytokines, including IL-3 and granulocyte - macrophage colony-stimulating factor (GM-CSF). Interestingly, cotransfection of cDNAs for KH97 and the low-affinity human GM-CSF receptor in fibroblasts resulted in formation of a high-affinity receptor for GM-CSF. The dissociation rate of GM-CSF from the reconstituted high-affinity receptor was slower than that from the low-affinity site, whereas the association rate was unchanged. Cross-linking of 125 I-labeled GM-CSF to fibroblasts cotransfected with both cDNAs revealed the same cross-linking patterns as in TF-1 cells - i.e., two major proteins of 80 and 120 kDa which correspond to the low-affinity GM-CSF receptor and the KH97 protein, respectively. These results indicate that the high-affinity GM-CSF receptor is composed of at least two components in a manner analogous to the IL-2 receptor. They therefore propose to designate the low-affinity GM-CSF receptor and the KH97 protein as the α and β subunits of the GM-CSF receptor, respectively

CRF1 receptor-deficiency increases cocaine reward.

Science.gov (United States)

Contarino, Angelo; Kitchener, Pierre; Vallée, Monique; Papaleo, Francesco; Piazza, Pier-Vincenzo

2017-05-01

Stimulant drugs produce reward but also activate stress-responsive systems. The corticotropin-releasing factor (CRF) and the related hypothalamus-pituitary-adrenal (HPA) axis stress-responsive systems are activated by stimulant drugs. However, their role in stimulant drug-induced reward remains poorly understood. Herein, we report that CRF 1 receptor-deficient (CRF 1 -/-), but not wild-type, mice show conditioned place preference (CPP) responses to a relatively low cocaine dose (5 mg/kg, i.p.). Conversely, wild-type, but not CRF 1 -/-, mice display CPP responses to a relatively high cocaine dose (20 mg/kg, i.p.), indicating that CRF 1 receptor-deficiency alters the rewarding effects of cocaine. Acute pharmacological antagonism of the CRF 1 receptor by antalarmin also eliminates cocaine reward. Nevertheless, CRF 1 -/- mice display higher stereotypy responses to cocaine than wild-type mice. Despite the very low plasma corticosterone concentration, CRF 1 -/- mice show higher nuclear glucocorticoid receptor (GR) levels in the brain region of the hippocampus than wild-type mice. Full rescue of wild-type-like corticosterone and GR circadian rhythm and level in CRF 1 -/- mice by exogenous corticosterone does not affect CRF 1 receptor-dependent cocaine reward but induces stereotypy responses to cocaine. These results indicate a critical role for the CRF 1 receptor in cocaine reward, independently of the closely related HPA axis activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mood stabilizer treatment increases serotonin type 1A receptor binding in bipolar depression

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Nugent, Allison C; Carlson, Paul J; Bain, Earle E; Eckelman, William; Herscovitch, Peter; Manji, Husseini; Zarate, Carlos A; Drevets, Wayne C

2013-01-01

Abnormal serotonin type 1A (5-HT1A) receptor function and binding have been implicated in the pathophysiology of mood disorders. Preclinical studies have consistently shown that stress decreases the gene expression of 5-HT1A receptors in experimental animals, and that the associated increase in hormone secretion plays a crucial role in mediating this effect. Chronic administration of the mood stabilizers lithium and divalproex (valproate semisodium) reduces glucocorticoid signaling and function in the hippocampus. Lithium has further been shown to enhance 5-HT1A receptor function. To assess whether these effects translate to human subject with bipolar disorder (BD), positron emission tomography (PET) and [18F]trans-4-fluoro-N-(2-[4-(2-methoxyphenyl) piperazino]-ethyl)-N-(2-pyridyl) cyclohexanecarboxamide ([18F]FCWAY) were used to acquire PET images of 5-HT1A receptor binding in 10 subjects with BD, before and after treatment with lithium or divalproex. Mean 5-HT1A binding potential (BPP) significantly increased following mood stabilizer treatment, most prominently in the mesiotemporal cortex (hippocampus plus amygdala). When mood state was also controlled for, treatment was associated with increases in BPP in widespread cortical areas. These preliminary findings are consistent with the hypothesis that these mood stabilizers enhance 5-HT1A receptor expression in BD, which may underscore an important component of these agents' mechanism of action. PMID:23926239

Unsaturated free fatty acids increase benzodiazepine receptor agonist binding depending on the subunit composition of the GABAA receptor complex.

Science.gov (United States)

Witt, M R; Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nielsen, M

1996-11-01

It has been shown previously that unsaturated free fatty acids (FFAs) strongly enhance the binding of agonist benzodiazepine receptor ligands and GABAA receptor ligands in the CNS in vitro. To investigate the selectivity of this effect, recombinant human GABAA/benzodiazepine receptor complexes formed by different subunit compositions (alpha x beta y gamma 2, x = 1, 2, 3, and 5; y = 1, 2, and 3) were expressed using the baculovirus-transfected Sf9 insect cell system. At 10(-4) M, unsaturated FFAs, particularly arachidonic (20:4) and docosahexaenoic (22:6) acids, strongly stimulated (> 200% of control values) the binding of [3H]flunitrazepam ([3H]FNM) to the alpha 3 beta 2 gamma 2 receptor combination in whole cell preparations. No effect or small increases in levels of unsaturated FFAs on [3H]FNM binding to alpha 1 beta x gamma 2 and alpha 2 beta x gamma 2 receptor combinations were observed, and weak effects (130% of control values) were detected using the alpha 5 beta 2 gamma 2 receptor combination. The saturated FFAs, stearic and palmitic acids, were without effect on [3H]FNM binding to any combination of receptor complexes. The hydroxylated unsaturated FFAs, ricinoleic and ricinelaidic acids, were shown to decrease the binding of [3H]FNM only if an alpha 1 beta 2 gamma 2 receptor combination was used. Given the heterogeneity of the GABAA/ benzodiazepine receptor subunit distribution in the CNS, the effects of FFAs on the benzodiazepine receptor can be assumed to vary at both cellular and regional levels.

Goatpoxvirus ATPase activity is increased by dsDNA and decreased by zinc ion.

Science.gov (United States)

Lee, Ming-Liang; Hsu, Wei-Li; Wang, Chi-Young; Chen, Hui-Yu; Lin, Fong-Yuan; Chang, Ming-Huang; Chang, Hong-You; Wong, Min-Liang; Chan, Kun-Wei

2016-10-01

Viral-encoded ATPase can act as a part of molecular motor in genome packaging of DNA viruses, such as vaccinia virus and adenovirus, by ATP hydrolysis and interaction with DNA. Poxviral ATPase (also called A32) is involved in genomic double-stranded DNA (dsDNA) encapsidation, and inhibition of the expression of A32 causes formation of immature virions lacking viral DNA. However, the role of A32 in goatpoxvirus genome packaging and its dsDNA binding property are not known. In this study, purified recombinant goatpoxvirus A32 protein (rA32) was examined for its dsDNA binding property as well as the effect of dsDNA on ATP hydrolysis. We found that rA32 could bind dsDNA, and its ATPase activity was significant increased with dsDNA binding. Effects of magnesium and calcium ions on ATP hydrolysis were investigated also. The ATPase activity was dramatically enhanced by dsDNA in the presence of Mg(2+); in contrast, ATPase function was not altered by Ca(2+). Furthermore, the enzyme activity of rA32 was completely blocked by Zn(2+). Regarding DNA-protein interaction, the rA32-ATP-Mg(2+) showed lower dsDNA binding affinity than that of rA32-ATP-Ca(2+). The DNA-protein binding was stronger in the presence of zinc ion. Our results implied that A32 may play a role in viral genome encapsidation and DNA condensation.

Human circulating plasma DNA significantly decreases while lymphocyte DNA damage increases under chronic occupational exposure to low-dose gamma-neutron and tritium β-radiation.

Science.gov (United States)

Korzeneva, Inna B; Kostuyk, Svetlana V; Ershova, Liza S; Osipov, Andrian N; Zhuravleva, Veronika F; Pankratova, Galina V; Porokhovnik, Lev N; Veiko, Natalia N

2015-09-01

The blood plasma of healthy people contains cell-fee (circulating) DNA (cfDNA). Apoptotic cells are the main source of the cfDNA. The cfDNA concentration increases in case of the organism's cell death rate increase, for example in case of exposure to high-dose ionizing radiation (IR). The objects of the present research are the blood plasma and blood lymphocytes of people, who contacted occupationally with the sources of external gamma/neutron radiation or internal β-radiation of tritium N = 176). As the controls (references), blood samples of people, who had never been occupationally subjected to the IR sources, were used (N = 109). With respect to the plasma samples of each donor there were defined: the cfDNA concentration (the cfDNA index), DNase1 activity (the DNase1 index) and titre of antibodies to DNA (the Ab DNA index). The general DNA damage in the cells was defined (using the Comet assay, the tail moment (TM) index). A chronic effect of the low-dose ionizing radiation on a human being is accompanied by the enhancement of the DNA damage in lymphocytes along with a considerable cfDNA content reduction, while the DNase1 content and concentration of antibodies to DNA (Ab DNA) increase. All the aforementioned changes were also observed in people, who had not worked with the IR sources for more than a year. The ratio cfDNA/(DNase1×Ab DNA × TM) is proposed to be used as a marker of the chronic exposure of a person to the external low-dose IR. It was formulated the assumption that the joint analysis of the cfDNA, DNase1, Ab DNA and TM values may provide the information about the human organism's cell resistivity to chronic exposure to the low-dose IR and about the development of the adaptive response in the organism that is aimed, firstly, at the effective cfDNA elimination from the blood circulation, and, secondly - at survival of the cells, including the cells with the damaged DNA. Copyright © 2015. Published by Elsevier B.V.

Increased neutrophil expression of pattern recognition receptors during COPD exacerbations

NARCIS (Netherlands)

Pouwels, Simon D.; Van Geffen, Wouter H.; Jonker, Marnix R.; Kerstjens, Huib A. M.; Nawijn, Martijn C.; Heijink, Irene H.

Previously, we observed increased serum levels of damage-associated molecular patterns (DAMPs) during COPD exacerbations. Here, gene expression of DAMP receptors was measured in peripheral blood neutrophils of COPD patients during stable disease and severe acute exacerbation. The expression of

Food restriction prevents an age-associated increase in rat liver beta-adrenergic receptors

Energy Technology Data Exchange (ETDEWEB)

Dax, E.M.; Ingram, D.K.; Partilla, J.S.; Gregerman, R.I.

1989-05-01

In male Wistar rats fed ad libitum (24% protein, 4.5 Kcal/gm), the (/sup 125/I)iodopindolol binding capacity of the beta-adrenergic receptors in liver of 24-month-old animals is 3-4 times greater than that of 6-month-old counterparts. In rats fed the same diet, on alternate days from weaning, the receptor capacity did not increase significantly between 6 and 24 months (10.20 +/- 0.55 vs 9.20 +/- 0.72 fmol/mg) or between 24 and 30 months. This was not due to acute dietary deprivation, as rats food-restricted for only 2 weeks, at 23.5 months of age, also showed elevated receptor capacities compared to 6-month-old ad libitum fed animals. Moreover, intermittent feeding produced no significant effects among 6-month-old animals, whether restricted since weaning or for two weeks prior to sacrifice. Many biochemical parameters that decrease with aging in rats fed ad libitum are prevented by dietary restriction. Our results demonstrate that a reproducible biochemical process that increases with aging is also prevented with dietary restriction. The age-related, liver beta-receptor increase may be a potentially reliable marker for studying biochemical perturbations that modify life span.

Food restriction prevents an age-associated increase in rat liver beta-adrenergic receptors

International Nuclear Information System (INIS)

Dax, E.M.; Ingram, D.K.; Partilla, J.S.; Gregerman, R.I.

1989-01-01

In male Wistar rats fed ad libitum (24% protein, 4.5 Kcal/gm), the [ 125 I]iodopindolol binding capacity of the beta-adrenergic receptors in liver of 24-month-old animals is 3-4 times greater than that of 6-month-old counterparts. In rats fed the same diet, on alternate days from weaning, the receptor capacity did not increase significantly between 6 and 24 months (10.20 +/- 0.55 vs 9.20 +/- 0.72 fmol/mg) or between 24 and 30 months. This was not due to acute dietary deprivation, as rats food-restricted for only 2 weeks, at 23.5 months of age, also showed elevated receptor capacities compared to 6-month-old ad libitum fed animals. Moreover, intermittent feeding produced no significant effects among 6-month-old animals, whether restricted since weaning or for two weeks prior to sacrifice. Many biochemical parameters that decrease with aging in rats fed ad libitum are prevented by dietary restriction. Our results demonstrate that a reproducible biochemical process that increases with aging is also prevented with dietary restriction. The age-related, liver beta-receptor increase may be a potentially reliable marker for studying biochemical perturbations that modify life span

No increased sperm DNA fragmentation index in semen containing human papillomavirus or herpesvirus

DEFF Research Database (Denmark)

Kaspersen, Maja Døvling; Bungum, Mona; Fedder, Jens

2013-01-01

It remains unknown whether human papillomaviruses (HPVs) or human herpesviruses (HHVs) in semen affect sperm DNA integrity. We investigated whether the presence of these viruses in semen was associated with an elevated sperm DNA fragmentation index. Semen from 76 sperm donors was examined by a PCR......-based hybridization array that identifies all HHVs and 35 of the most common HPVs. Sperm DNA integrity was determined by the sperm chromatin structure assay. HPVs or HHVs, or both, were found in 57% of semen samples; however, sperm DNA fragmentation index was not increased in semen containing these viruses....

Delayed repair of DNA single-strand breaks does not increase cytogenetic damage

International Nuclear Information System (INIS)

Morgan, W.F.; Djordjevic, M.C.; Jostes, R.F.; Pantelias, G.E.

1985-01-01

DNA damage and cytogenetic effects of ionizing radiation were investigated in Chinese hamster ovary (CHO) cells and unstimulated human peripheral blood lymphocytes. DNA damage and repair were analysed by alkaline elution under conditions that predominantly measured DNA single-strand breaks (ssb). X-radiation (2.5 Gy) induced ssb in both CHO cells and unstimulated lymphocytes, and the breaks were repaired within 30 and 90 min, respectively. This rapid repair was delayed by the poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide (3AB). The cytogenetic effects of the 3AB-induced delay in DNA repair were examined by analysing sister chromatid exchange (SCE) frequency in CHO cells and fragmentation of prematurely condensed chromosomes (PCC) in unstimulated human lymphocytes after 2.5 Gy of X-rays. Although 3AB delayed the rejoining of DNA ssb, this delay did not result in increased cytogenetic damage manifested as either SCE or fragmentation of PCC. These results indicate that the rapidly rejoining DNA ssb are not important in the production of chromosome damage. (author)

Pretreatment with mixed-function oxidase inducers increases the sensitivity of the hepatocyte/DNA repair assay

International Nuclear Information System (INIS)

Shaddock, J.G.; Heflich, R.H.; McMillan, D.C.; Hinson, J.A.; Casciano, D.A.

1989-01-01

A recent National Toxicology Program evaluation indicates that the rat hepatocyte/DNA repair assay has a high false-negative rate and that it is insensitive to some genotoxic hepatocarcinogens as well as other species and organ-specific carcinogens. In this study, the authors examined whether the sensitivity of the hepatocyte/DNA repair assay might be increased through animal pretreatment with various hepatic mixed-function oxidase inducers, i.e., Aroclor 1254, phenobarbital, and 3,3',4,4'-tetrachloroazobenzene (TCAB). The effects on unscheduled DNA synthesis (UDS), a measured of DNA damage and repair, were studied in cultures exposed to known and/or potential carcinogens that had been evaluated as negative or questionable or that produced conflicting results with hepatocytes isolated from uninduced animals. 4,4'-Oxydianiline, 1-nitropy-rene, and TCAB produced concentration-dependent increases in UDS in hepatocytes from rats pretreated with Aroclor 1254. 4,4'-Oxydianiline and TCAB also induced a dose-dependent increase in DNA repair in hepatocytes from rats pretreated with phenobarbital, whereas 1-nitropyrene was negative. These data indicate that the limited sensitivity to chemical carcinogens displayed by the hepatocyte/DNA repair assay may be increased by using hepatocytes isolated from animals exposed to hepatic mixed-function oxidase inducers

Pretreatment with mixed-function oxidase inducers increases the sensitivity of the hepatocyte/DNA repair assay

Energy Technology Data Exchange (ETDEWEB)

Shaddock, J.G.; Heflich, R.H.; McMillan, D.C.; Hinson, J.A.; Casciano, D.A. (National Center for Toxicological Research, Jefferson, AK (USA) Univ. of Arkansas for Medical Sciences, Little Rock (USA))

1989-01-01

A recent National Toxicology Program evaluation indicates that the rat hepatocyte/DNA repair assay has a high false-negative rate and that it is insensitive to some genotoxic hepatocarcinogens as well as other species and organ-specific carcinogens. In this study, the authors examined whether the sensitivity of the hepatocyte/DNA repair assay might be increased through animal pretreatment with various hepatic mixed-function oxidase inducers, i.e., Aroclor 1254, phenobarbital, and 3,3{prime},4,4{prime}-tetrachloroazobenzene (TCAB). The effects on unscheduled DNA synthesis (UDS), a measured of DNA damage and repair, were studied in cultures exposed to known and/or potential carcinogens that had been evaluated as negative or questionable or that produced conflicting results with hepatocytes isolated from uninduced animals. 4,4{prime}-Oxydianiline, 1-nitropy-rene, and TCAB produced concentration-dependent increases in UDS in hepatocytes from rats pretreated with Aroclor 1254. 4,4{prime}-Oxydianiline and TCAB also induced a dose-dependent increase in DNA repair in hepatocytes from rats pretreated with phenobarbital, whereas 1-nitropyrene was negative. These data indicate that the limited sensitivity to chemical carcinogens displayed by the hepatocyte/DNA repair assay may be increased by using hepatocytes isolated from animals exposed to hepatic mixed-function oxidase inducers.

Increased NMDA and AMPA receptor densities in the anterior cingulate cortex in schizophrenia

International Nuclear Information System (INIS)

Zavitsanou, K.; Huang, X.-F.

2002-01-01

Full text: The anterior cingulate cortex (ACC) is a brain area of potential importance to our understanding of the pathophysiology of schizophrenia. Since a disturbed balance between excitatory and inhibitory activity is suggested to occur in the ACC in schizophrenia, the present study has focused on the analysis of binding of [ 3 H]MK801, [ 3 H]AMPA and [ 3 H]kainate, radioligands which respectively label the NMDA, AMPA and kainate receptors of the ionotropic glutamate receptor family in the ACC of 10 schizophrenia patients and 10 matched controls, using quantitative autoradiography. AMPA receptor densities were higher in cortical layer II whereas NMDA receptor densities were higher in cortical layers II-III in the ACC of both control and schizophrenia group. In contrast, kainate receptors displayed the highest density in cortical layer V. [ 3 H]AMPA binding was significantly increased by 25% in layer II in the schizophrenia group as compared to the control group. Similarly, a significant 17% increase of [ 3 H]MK801 binding was observed in layers II-III in the schizophrenia group. No statistically significant differences were observed for [ 3 H] kainate binding between the two groups. These results suggest that ionotropic glutamate receptors are differentially altered in the ACC of schizophrenia. The increase in [ 3 H]AMPA and [ 3 H]MK801 binding points to a postsynaptic compensation for impaired glutamatergic neurotransmission in the ACC in schizophrenia. Such abnormality could lead to an imbalance between the excitatory and inhibitory neurotransmission in this brain area that may contribute to the emergence of some schizophrenia symptoms. Copyright (2002) Australian Neuroscience Society

Nanocarriers for DNA Vaccines: Co-Delivery of TLR-9 and NLR-2 Ligands Leads to Synergistic Enhancement of Proinflammatory Cytokine Release

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Johanna Poecheim

2015-12-01

Full Text Available Adjuvants enhance immunogenicity of vaccines through either targeted antigen delivery or stimulation of immune receptors. Three cationic nanoparticle formulations were evaluated for their potential as carriers for a DNA vaccine, and muramyl dipeptide (MDP as immunostimulatory agent, to induce and increase immunogenicity of Mycobacterium tuberculosis antigen encoding plasmid DNA (pDNA. The formulations included (1 trimethyl chitosan (TMC nanoparticles, (2 a squalene-in-water nanoemulsion, and (3 a mineral oil-in-water nanoemulsion. The adjuvant effect of the pDNA-nanocomplexes was evaluated by serum antibody analysis in immunized mice. All three carriers display a strong adjuvant effect, however, only TMC nanoparticles were capable to bias immune responses towards Th1. pDNA naturally contains immunostimulatory unmethylated CpG motifs that are recognized by Toll-like receptor 9 (TLR-9. In mechanistic in vitro studies, activation of TLR-9 and the ability to enhance immunogenicity by simultaneously targeting TLR-9 and NOD-like receptor 2 (NLR-2 was determined by proinflammatory cytokine release in RAW264.7 macrophages. pDNA in combination with MDP was shown to significantly increase proinflammatory cytokine release in a synergistic manner, dependent on NLR-2 activation. In summary, novel pDNA-Ag85A loaded nanoparticle formulations, which induce antigen specific immune responses in mice were developed, taking advantage of the synergistic combinations of TLR and NLR agonists to increase the adjuvanticity of the carriers used.

Increased migration of monocytes in essential hypertension is associated with increased transient receptor potential channel canonical type 3 channels

DEFF Research Database (Denmark)

Zhao, Zhigang; Ni, Yinxing; Chen, Jing

2012-01-01

Increased transient receptor potential canonical type 3 (TRPC3) channels have been observed in patients with essential hypertension. In the present study we tested the hypothesis that increased monocyte migration is associated with increased TRPC3 expression. Monocyte migration assay was performe...

Activation-induced proteolysis of cytoplasmic domain of zeta in T cell receptors and Fc receptors.

Science.gov (United States)

Taupin, J L; Anderson, P

1994-12-01

The CD3-T cell receptor (TCR) complex on T cells and the Fc gamma receptor type III (Fc gamma RIII)-zeta-gamma complex on natural killer cells are functionally analogous activation receptors that associate with a family of disulfide-linked dimers composed of the related subunits zeta and gamma. Immunochemical analysis of receptor complexes separated on two-dimensional diagonal gels allowed the identification of a previously uncharacterized zeta-p14 heterodimer. zeta-p14 is a component of both CD3-TCR and Fc gamma RIII-zeta-gamma. Peptide mapping analysis shows that p14 is structurally related to zeta, suggesting that it is either: (i) derived from zeta proteolytically or (ii) the product of an alternatively spliced mRNA. The observation that COS cells transformed with a cDNA encoding zeta express zeta-p14 supports the former possibility. The expression of CD3-TCR complexes including zeta-p14 increases following activation with phorbol 12-myristate 13-acetate or concanavalin A, suggesting that proteolysis of zeta may contribute to receptor modulation or desensitization.

Hormone Receptor Expression Analyses in Neoplastic and Non-Neoplastic Canine Mammary Tissue by a Bead Based Multiplex Branched DNA Assay: A Gene Expression Study in Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Samples.

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Annika Mohr

Full Text Available Immunohistochemistry (IHC is currently considered the method of choice for steroid hormone receptor status evaluation in human breast cancer and, therefore, it is commonly utilized for assessing canine mammary tumors. In case of low hormone receptor expression, IHC is limited and thus is complemented by molecular analyses. In the present study, a multiplex bDNA assay was evaluated as a method for hormone receptor gene expression detection in canine mammary tissues. Estrogen receptor (ESR1, progesterone receptor (PGR, prolactin receptor (PRLR and growth hormone receptor (GHR gene expressions were evaluated in neoplastic and non-neoplastic canine mammary tissues. A set of 119 fresh frozen and 180 formalin-fixed, paraffin-embedded (FFPE was comparatively analyzed and used for assay evaluation. Furthermore, a possible association between the hormone receptor expression in different histological subtypes of canine malignant mammary tumors and the castration status, breed and invasive growth of the tumor were analyzed. The multiplex bDNA assay proved to be more sensitive for fresh frozen specimens. Hormone receptor expression found was significantly decreased in malignant mammary tumors in comparison to non-neoplastic tissue and benign mammary tumors. Among the histological subtypes the lowest gene expression levels of ESR1, PGR and PRLR were found in solid, anaplastic and ductal carcinomas. In summary, the evaluation showed that the measurement of hormone receptors with the multiplex bDNA assay represents a practicable method for obtaining detailed quantitative information about gene expression in canine mammary tissue for future studies. Still, comparison with IHC or quantitative real-time PCR is needed for further validation of the present method.

A retinoic acid receptor cDNA probe (RAR2) identifies a moderately frequent RFLP on chromosome 17

Energy Technology Data Exchange (ETDEWEB)

Bale, A E; Weinberger, C; McBride, O W [National Cancer Institute, Bethesda, MD (USA)

1988-08-11

RAR2, a 0.72 kb EcoRI, PvuII fragment from the 5{prime} end of the retinoic acid receptor cDNA probe was isolated. PstI identifies a constant band at 0.87 kb and a simple two allele polymorphism with a band at either 3.3 kb (A1) or 2.9 kb (A2). In 38 random blood donors, the frequency of the 3.3 kb allele (A1) was 0.29 and of the 2.9 kb allele (A2) was 0.71. The polymorphic bands and the 0.87 kb constant band segregated with chromosome 17 in 88 human-rodent somatic cell hybrids. Co-dominant inheritance was shown in 35 individuals from 5 informative families. Weak constant bands at 6.4 kb, 4.0 kb and 1.4 kb did not cosegregate with the polymorphic bands in somatic cell hybrids and could be eliminated by increasing the wash stringency.

High Glucose-Induced Oxidative Stress Increases the Copy Number of Mitochondrial DNA in Human Mesangial Cells

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Ghada Al-Kafaji

2013-01-01

Full Text Available Oxidative damage to mitochondrial DNA (mtDNA has been linked to the pathogenicity of diabetic nephropathy. We tested the hypothesis that mtDNA copy number may be increased in human mesangial cells in response to high glucose-induced reactive oxygen species (ROS to compensate for damaged mtDNA. The effect of manganese superoxide dismutase mimetic (MnTBAP on glucose-induced mtDNA copy number was also examined. The copy number of mtDNA was determined by real-time PCR in human mesangial cells cultured in 5 mM glucose, 25 mM glucose, and mannitol (osmotic control, as well as in cells cultured in 25 mM glucose in the presence and absence of 200 μM MnTBAP. Intracellular ROS was assessed by confocal microscopy and flow cytometry in human mesangial cells. The copy number of mtDNA was significantly increased when human mesangial cells were incubated with 25 mM glucose compared to 5 mM glucose and mannitol. In addition, 25 mM glucose rapidly generated ROS in the cells, which was not detected in 5 mM glucose. Furthermore, mtDNA copy number was significantly decreased and maintained to normal following treatment of cells with 25 mM glucose and MnTBAP compared to 25 mM glucose alone. Inclusion of MnTBAP during 25 mM glucose incubation inhibited mitochondrial superoxide in human mesangial cells. Increased mtDNA copy number in human mesangial cells by high glucose could contribute to increased mitochondrial superoxide, and prevention of mtDNA copy number could have potential in retarding the development of diabetic nephropathy.

The role of oxytocin receptor gene (OXTR) DNA methylation (DNAm) in human social and emotional functioning: a systematic narrative review.

Science.gov (United States)

Maud, Catherine; Ryan, Joanne; McIntosh, Jennifer E; Olsson, Craig A

2018-05-29

The neuropeptide Oxytocin (OXT) plays a central role in birthing, mother-infant bonding and a broad range of related social behaviours in mammals. More recently, interest has extended to epigenetic programming of genes involved in oxytocinergic neurotransmission. This review brings together early findings in a rapidly developing field of research, examining relationships between DNA methylation (DNAm) of the Oxytocin Receptor Gene (OXTR) and social and emotional behaviour in human populations. A systematic search across Web of Knowledge/Science, Scopus, Medline and EMBASE captured all published studies prior to June 2017 examining the association between OXTR DNAm and human social and emotional outcomes. Search terms included 'oxytocin gene' or 'oxytocin receptor gene' and 'epigenetics' or 'DNA methylation'. Any article with a focus on social and emotional functioning was then identified from this set by manual review. Nineteen studies met eligibility criteria. There was considerable heterogeneity of study populations, tissue samples, instrumentation, measurement, and OXTR site foci. Only three studies examined functional consequences of OXTR DNAm on gene expression and protein synthesis. Increases in OXTR DNAm were associated with callous-unemotional traits in youth, social cognitive deficits in Autistic Spectrum Disorder (ASD), rigid thinking in anorexia nervosa, affect regulation problems, and problems with facial and emotional recognition. In contrast, reductions in DNAm were associated with perinatal stress, postnatal depression, social anxiety and autism in children. Consistent with an emerging field of inquiry, there is not yet sufficient evidence to draw conclusions about the role of OXTR DNAm in human social and emotional behaviour. However, taken together, findings point to increased OXTR DNAm in general impairments in social, cognitive and emotional functioning, and decreased OXTR DNAm in specific patterns of impairment related to mood and anxiety

Peroxisome proliferator-activated receptor delta activation leads to increased transintestinal cholesterol efflux

NARCIS (Netherlands)

Vrins, Carlos L. J.; van der Velde, Astrid E.; van den Oever, Karin; Levels, Johannes H. M.; Huet, Stephane; Oude Elferink, Ronald P. J.; Kuipers, Folkert; Groen, Albert K.

2009-01-01

Peroxisome proliferator-activated receptor delta (PPARdelta) is involved in regulation of energy homeostasis. Activation of PPARdelta markedly increases fecal neutral sterol secretion, the last step in reverse cholesterol transport. This phenomenon can neither be explained by increased hepatobiliary

Peroxisome proliferator-activated receptor delta activation leads to increased transintestinal cholesterol efflux

NARCIS (Netherlands)

Vrins, Carlos L. J.; van der Velde, Astrid E.; van den Oever, Karin; Levels, Johannes H. M.; Huet, Stephane; Elferink, Ronald P. J. Oude; Kuipers, Folkert; Groen, Albert K.

2009-01-01

Peroxisome proliferator-activated receptor delta (PPAR delta) is involved in regulation of energy homeostasis. Activation of PPAR delta markedly increases fecal neutral sterol secretion, the last step in reverse cholesterol transport. This phenomenon can neither be explained by increased

Relationship of Structure and Function of DNA-Binding Domain in Vitamin D Receptor

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Lin-Yan Wan

2015-07-01

Full Text Available While the structure of the DNA-binding domain (DBD of the vitamin D receptor (VDR has been determined in great detail, the roles of its domains and how to bind the motif of its target genes are still under debate. The VDR DBD consists of two zinc finger modules and a C-terminal extension (CTE, at the end of the C-terminal of each structure presenting α-helix. For the first zinc finger structure, N37 and S-box take part in forming a dimer with 9-cis retinoid X receptor (RXR, while V26, R50, P-box and S-box participate in binding with VDR response elements (VDRE. For the second zinc finger structure, P61, F62 and H75 are essential in the structure of the VDR homodimer with the residues N37, E92 and F93 of the downstream of partner VDR, which form the inter-DBD interface. T-box of the CTE, especially the F93 and I94, plays a critical role in heterodimerization and heterodimers–VDRE binding. Six essential residues (R102, K103, M106, I107, K109, and R110 of the CTE α-helix of VDR construct one interaction face, which packs against the DBD core of the adjacent symmetry mate. In 1,25(OH2D3-activated signaling, the VDR-RXR heterodimer may bind to DR3-type VDRE and ER9-type VDREs of its target gene directly resulting in transactivation and also bind to DR3-liked nVDRE of its target gene directly resulting in transrepression. Except for this, 1α,25(OH2D3 ligand VDR-RXR may bind to 1αnVDRE indirectly through VDIR, resulting in transrepression of the target gene. Upon binding of 1α,25(OH2D3, VDR can transactivate and transrepress its target genes depending on the DNA motif that DBD binds.

Experimental diabetes increases insulin-like growth factor I and II receptor concentration and gene expression in kidney

International Nuclear Information System (INIS)

Werner, H.; Shen-Orr, Z.; Stannard, B.; Burguera, B.; Roberts, C.T. Jr.; LeRoith, D.

1990-01-01

Insulinlike growth factor I (IGF-I) is a mitogenic hormone with important regulatory roles in growth and development. One of the target organs for IGF-I action is the kidney, which synthesizes abundant IGF-I receptors and IGF-I itself. To study the involvement of IGF-I and the IGF-I receptor in the development of nephropathy, one of the major complications of diabetes mellitus, we measured the expression of these genes in the kidney and in other tissues of the streptozocin-induced diabetic rat. The binding of 125I-labeled IGF-I to crude membranes was measured in the same tissues. We observed a 2.5-fold increase in the steady-state level of IGF-I-receptor mRNA in the diabetic kidney, which was accompanied by a 2.3-fold increase in IGF-I binding. In addition to this increase in IGF-I binding to the IGF-I receptor, there was also binding to a lower-molecular-weight material that may represent an IGF-binding protein. No change was detected in the level of IGF-I-peptide mRNA. Similarly, IGF-II-receptor mRNA levels and IGF-II binding were significantly increased in the diabetic kidney. IGF-I- and IGF-II-receptor mRNA levels and IGF-I and IGF-II binding returned to control values after insulin treatment. Because the IGF-I receptor is able to transduce mitogenic signals on activation of its tyrosine kinase domain, we hypothesize that, among other factors, high levels of receptor in the diabetic kidney may also be involved in the development of diabetic nephropathy. Increased IGF-II-receptor expression in the diabetic kidney may be important for the intracellular transport and packaging of lysosomal enzymes, although a role for this receptor in signal transduction cannot be excluded. Finally, the possible role of IGF-binding proteins requires further study

Increased sensitivity of DNA damage response-deficient cells to stimulated microgravity-induced DNA lesions.

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Nan Li

Full Text Available Microgravity is a major stress factor that astronauts have to face in space. In the past, the effects of microgravity on genomic DNA damage were studied, and it seems that the effect on genomic DNA depends on cell types and the length of exposure time to microgravity or simulated microgravity (SMG. In this study we used mouse embryonic stem (MES and mouse embryonic fibroblast (MEF cells to assess the effects of SMG on DNA lesions. To acquire the insight into potential mechanisms by which cells resist and/or adapt to SMG, we also included Rad9-deleted MES and Mdc1-deleted MEF cells in addition to wild type cells in this study. We observed significant SMG-induced DNA double strand breaks (DSBs in Rad9-/- MES and Mdc1-/- MEF cells but not in their corresponding wild type cells. A similar pattern of DNA single strand break or modifications was also observed in Rad9-/- MES. As the exposure to SMG was prolonged, Rad9-/- MES cells adapted to the SMG disturbance by reducing the induced DNA lesions. The induced DNA lesions in Rad9-/- MES were due to SMG-induced reactive oxygen species (ROS. Interestingly, Mdc1-/- MEF cells were only partially adapted to the SMG disturbance. That is, the induced DNA lesions were reduced over time, but did not return to the control level while ROS returned to a control level. In addition, ROS was only partially responsible for the induced DNA lesions in Mdc1-/- MEF cells. Taken together, these data suggest that SMG is a weak genomic DNA stress and can aggravate genomic instability in cells with DNA damage response (DDR defects.

Hormone-dependent nuclear export of estradiol receptor and DNA synthesis in breast cancer cells

Science.gov (United States)

Lombardi, Maria; Castoria, Gabriella; Migliaccio, Antimo; Barone, Maria Vittoria; Di Stasio, Rosina; Ciociola, Alessandra; Bottero, Daniela; Yamaguchi, Hiroshi; Appella, Ettore; Auricchio, Ferdinando

2008-01-01

In breast cancer cells, cytoplasmic localization of the estradiol receptor α (ERα) regulates estradiol-dependent S phase entry. We identified a nuclear export sequence (NES) in ERα and show that its export is dependent on both estradiol-mediated phosphatidylinositol-3-kinase (PI3K)/AKT activation and chromosome region maintenance 1 (CRM1). A Tat peptide containing the ERα NES disrupts ERα–CRM1 interaction and prevents nuclear export of ERα- and estradiol-induced DNA synthesis. NES-ERα mutants do not exit the nucleus and inhibit estradiol-induced S phase entry; ERα-dependent transcription is normal. ERα is associated with Forkhead proteins in the nucleus, and estradiol stimulates nuclear exit of both proteins. ERα knockdown or ERα NES mutations prevent ERα and Forkhead nuclear export. A mutant of forkhead in rhabdomyosarcoma (FKHR), which cannot be phosphorylated by estradiol-activated AKT, does not associate with ERα and is trapped in the nucleus, blocking S phase entry. In conclusion, estradiol-induced AKT-dependent phosphorylation of FKHR drives its association with ERα, thereby triggering complex export from the nucleus necessary for initiation of DNA synthesis and S phase entry. PMID:18644889

Exploring optimization parameters to increase ssDNA recombineering in Lactococcus lactis and Lactobacillus reuteri.

Science.gov (United States)

Van Pijkeren, Jan-Peter; Neoh, Kar Mun; Sirias, Denise; Findley, Anthony S; Britton, Robert A

2012-01-01

Single-stranded DNA (ssDNA) recombineering is a technology which is used to make subtle changes in the chromosome of several bacterial genera. Cells which express a single-stranded DNA binding protein (RecT or Bet) are transformed with an oligonucleotide which is incorporated via an annealing and replication-dependent mechanism. By in silico analysis we identified ssDNA binding protein homologs in the genus Lactobacillus and Lactococcus lactis. To assess whether we could further improve the recombineering efficiency in Lactobacillus reuteri ATCC PTA 6475 we expressed several RecT homologs in this strain. RecT derived from Enterococcus faecalis CRMEN 19 yielded comparable efficiencies compared with a native RecT protein, but none of the other proteins further increased the recombineering efficiency. We successfully improved recombineering efficiency 10-fold in L. lactis by increasing oligonucleotide concentration combined with the use of oligonucleotides containing phosphorothioate-linkages (PTOs). Surprisingly, neither increased oligonucleotide concentration nor PTO linkages enhanced recombineering in L. reuteri 6475. To emphasize the utility of this technology in improving probiotic features we modified six bases in a transcriptional regulatory element region of the pdu-operon of L. reuteri 6475, yielding a 3-fold increase in the production of the antimicrobial compound reuterin. Directed genetic modification of lactic acid bacteria through ssDNA recombineering will simplify strain improvement in a way that, when mutating a single base, is genetically indistinguishable from strains obtained through directed evolution.

Increased immunogenicity of avian influenza DNA vaccine delivered to the skin using a microneedle patch

Science.gov (United States)

Kim, Yeu-Chun; Song, Jae-Min; Lipatov, Aleksandr S.; Choi, Seong-O; Lee, Jeong Woo; Donis, Ruben O.; Compans, Richard W.; Kang, Sang-Moo; Prausnitz, Mark R.

2012-01-01

Effective public health responses to an influenza pandemic require an effective vaccine that can be manufactured and administered to large populations in the shortest possible time. In this study, we evaluated a method for vaccination against avian influenza virus that uses a DNA vaccine for rapid manufacturing and delivered by a microneedle skin patch for simplified administration and increased immunogenicity. We prepared patches containing 700 µm-long microneedles coated with an avian H5 influenza hemagglutinin DNA vaccine from A/Viet Nam/1203/04 influenza virus. The coating DNA dose increased with DNA concentration in the coating solution and the number of dip coating cycles. Coated DNA was released into the skin tissue by dissolution within minutes. Vaccination of mice using microneedles induced higher levels of antibody responses and hemagglutination inhibition titers, and improved protection against lethal infection with avian influenza as compared to conventional intramuscular delivery of the same dose of the DNA vaccine. Additional analysis showed that the microneedle coating solution containing carboxymethylcellulose and a surfactant may have negatively affected the immunogenicity of the DNA vaccine. Overall, this study shows that DNA vaccine delivery by microneedles can be a promising approach for improved vaccination to mitigate an influenza pandemic. PMID:22504442

Receptor-like protein-tyrosine phosphatase alpha specifically inhibits insulin-increased prolactin gene expression

DEFF Research Database (Denmark)

Jacob, K K; Sap, J; Stanley, F M

1998-01-01

A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin...... is specific by two criteria. A number of potential RPTPalpha targets were ruled out by finding (a) that they are not affected or (b) that they are not on the pathway to insulin-increased prolactin-CAT activity. The negative effect of RPTPalpha on insulin activation of the prolactin promoter is not due...... to reduced phosphorylation or kinase activity of the insulin receptor or to reduced phosphorylation of insulin receptor substrate-1 or Shc. Inhibitor studies suggest that insulin-increased prolactin gene expression is mediated by a Ras-like GTPase but is not mitogen-activated protein kinase dependent...

Loss of vitamin D receptor produces polyuria by increasing thirst.

Science.gov (United States)

Kong, Juan; Zhang, Zhongyi; Li, Dongdong; Wong, Kari E; Zhang, Yan; Szeto, Frances L; Musch, Mark W; Li, Yan Chun

2008-12-01

Vitamin D receptor (VDR)-null mice develop polyuria, but the underlying mechanism remains unknown. In this study, we investigated the relationship between vitamin D and homeostasis of water and electrolytes. VDR-null mice had polyuria, but the urine osmolarity was normal as a result of high salt excretion. The urinary responses to water restriction and to vasopressin were similar between wild-type and VDR-null mice, suggesting intact fluid-handling capacity in VDR-null mice. Compared with wild-type mice, however, renin and angiotensin II were dramatically upregulated in the kidney and brain of VDR-null mice, leading to a marked increase in water intake and salt appetite. Angiotensin II-mediated upregulation of intestinal NHE3 expression partially explained the increased salt absorption and excretion in VDR-null mice. In the brain of VDR-null mice, expression of c-Fos, which is known to associate with increased water intake, was increased in the hypothalamic paraventricular nucleus and the subfornical organ. Treatment with an angiotensin II type 1 receptor antagonist normalized water intake, urinary volume, and c-Fos expression in VDR-null mice. Furthermore, despite a salt-deficient diet to reduce intestinal salt absorption, VDR-null mice still maintained the increased water intake and urinary output. Together, these data indicate that the polyuria observed in VDR-null mice is not caused by impaired renal fluid handling or increased intestinal salt absorption but rather is the result of increased water intake induced by the increase in systemic and brain angiotensin II.

CB1 receptor antagonism increases hippocampal acetylcholine release: site and mechanism of action.

Science.gov (United States)

Degroot, Aldemar; Köfalvi, Attila; Wade, Mark R; Davis, Richard J; Rodrigues, Ricardo J; Rebola, Nelson; Cunha, Rodrigo A; Nomikos, George G

2006-10-01

Evidence indicates that blockade of cannabinoid receptors increases acetylcholine (ACh) release in brain cortical regions. Although it is assumed that this type of effect is mediated through CB1 receptor (CB1R) antagonism, several in vitro functional studies recently have suggested non-CB1R involvement. In addition, neither the precise neuroanatomical site nor the exact mechanisms underlying this effect are known. We thoroughly examined these issues using a combination of systemic and local administration of CB1R antagonists, different methods of in vivo microdialysis, CB1R knockout (KO) mice, tissue measurements of ACh, and immunochemistry. First, we showed that systemic injections of the CB1R antagonists N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR-141716A) and N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) dose-dependently increased hippocampal ACh efflux. Likewise, local hippocampal, but not septal, infusions of SR141716A or AM251 increased hippocampal ACh release. It is noteworthy that the stimulatory effects of systemically administered CB1R antagonists on hippocampal ACh release were completely abolished in CB1R KO mice. CB1R KO mice had similar basal but higher stress-enhanced hippocampal ACh levels compared with wild-type controls. It is interesting that dopamine D1 receptor antagonism counteracted the stimulatory effect of CB1R blockade on hippocampal ACh levels. Finally, immunohistochemical methods revealed that a high proportion of CB1R-positive nerve terminals were found in hippocampus and confirmed the colocalization of CB1 receptors with cholinergic and dopaminergic nerve terminals. In conclusion, hippocampal ACh release may specifically be controlled through CB1Rs located on both cholinergic and dopaminergic neuronal projections, and CB1R antagonism increases hippocampal ACh release, probably through both a direct

A hotspot in the glucocorticoid receptor DNA-binding domain susceptible to loss of function mutation

Science.gov (United States)

Banuelos, Jesus; Shin, Soon Cheon; Lu, Nick Z.

2015-01-01

Glucocorticoids (GCs) are used to treat a variety of inflammatory disorders and certain cancers. However, GC resistance occurs in subsets of patients. We found that EL4 cells, a GC-resistant mouse thymoma cell line, harbored a point mutation in their GC receptor (GR) gene, resulting in the substitution of arginine 493 by a cysteine in the second zinc finger of the DNA-binding domain. Allelic discrimination analyses revealed that the R493C mutation occurred on both alleles. In the absence of GCs, the GR in EL4 cells localized predominantly in the cytoplasm and upon dexamethasone treatment underwent nuclear translocation, suggesting the ligand binding ability of the GR in EL4 cells was intact. In transient transfection assays, the R493C mutant could not transactivate the MMTV-luciferase reporter. Site-directed mutagenesis to revert the R493C mutation restored the transactivation activity. Cotransfection experiments showed that the R493C mutant did not inhibit the transcriptional activities of the wild-type GR. In addition, the R493C mutant did not repress either the AP-1 or NF-κB reporters as effectively as WT GR. Furthermore, stable expression of the WT GR in the EL4 cells enabled GC-mediated gene regulation, specifically upregulation of IκBα and downregulation of interferon γ and interleukin 17A. Arginine 493 is conserved among multiple species and all human nuclear receptors and its mutation has also been found in the human GR, androgen receptor, and mineralocorticoid receptor. Thus, R493 is necessary for the transcriptional activity of the GR and a hotspot for mutations that result in GC resistance. PMID:25676786

Role of adrenergic receptors in the caffeine-induced increase in ...

African Journals Online (AJOL)

The present study was designed to investigate the effects of alpha and beta adrenergic receptor blockers on caffeine-induced increase in canine hindlimb glucose uptake. The study was carried out on fasted male anaesthetized dogs divided into five groups (5dogs per group). Each dog was given a bolus injection of normal ...

Radioresistant DNA synthesis in cells of patients showing increased chromosomal sensitivity to ionizing radiation

International Nuclear Information System (INIS)

Barenfeld, L.S.; Pleskach, N.M.; Bildin, V.N.; Prokofjeva, V.V.; Mikhelson, V.M.

1986-01-01

The rate of DNA synthesis after γ-irradiation was studied either by analysis of the steady-state distribution of daughter [ 3 H]DNA in alkaline sucrose gradients or by direct assay of the amount of [ 3 H]thymidine incorporated into DNA of fibroblasts derived from a normal donor (LCH882) and from Down's syndrome (LCH944), Werner's syndrome (WS1LE) and xeroderma pigmentosum (XP2LE) patients with chromosomal sensitivity to ionizing radiation. Doses of γ-irradiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition of DNA synthesis in the cells from the affected individuals. The radioresistant DNA synthesis in Down's syndrome cells was mainly due to a much lower inhibition of replicon initiation than that in normal cells; these cells were also more resistant to damage that inhibited replicon elongation. Our data suggest that radioresistant DNA synthesis may be an intrinsic feature of all genetic disorders showing increased radiosensitivity in terms of chromosome aberrations. (orig.)

ssDNA Pairing Accuracy Increases When Abasic Sites Divide Nucleotides into Small Groups.

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Alexandra Peacock-Villada

Full Text Available Accurate sequence dependent pairing of single-stranded DNA (ssDNA molecules plays an important role in gene chips, DNA origami, and polymerase chain reactions. In many assays accurate pairing depends on mismatched sequences melting at lower temperatures than matched sequences; however, for sequences longer than ~10 nucleotides, single mismatches and correct matches have melting temperature differences of less than 3°C. We demonstrate that appropriately grouping of 35 bases in ssDNA using abasic sites increases the difference between the melting temperature of correct bases and the melting temperature of mismatched base pairings. Importantly, in the presence of appropriately spaced abasic sites mismatches near one end of a long dsDNA destabilize the annealing at the other end much more effectively than in systems without the abasic sites, suggesting that the dsDNA melts more uniformly in the presence of appropriately spaced abasic sites. In sum, the presence of appropriately spaced abasic sites allows temperature to more accurately discriminate correct base pairings from incorrect ones.

cDNA for the human β2-adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor

International Nuclear Information System (INIS)

Kobilka, B.K.; Dixon, R.A.F.; Frielle, T.

1987-01-01

The authors have isolated and sequenced a cDNA encoding the human β 2 -adrenergic receptor. The deduced amino acid sequence (413 residues) is that of a protein containing seven clusters of hydrophobic amino acids suggestive of membrane-spanning domains. While the protein is 87% identical overall with the previously cloned hamster β 2 -adrenergic receptor, the most highly conserved regions are the putative transmembrane helices (95% identical) and cytoplasmic loops (93% identical), suggesting that these regions of the molecule harbor important functional domains. Several of the transmembrane helices also share lesser degrees of identity with comparable regions of select members of the opsin family of visual pigments. They have localized the gene for the β 2 -adrenergic receptor to q31-q32 on chromosome 5. This is the same position recently determined for the gene encoding the receptor for platelet-derived growth factor and is adjacent to that for the FMS protooncogene, which encodes the receptor for the macrophage colony-stimulating factor

Hypoxia increases exercise heart rate despite combined inhibition of β-adrenergic and muscarinic receptors

DEFF Research Database (Denmark)

Siebenmann, Christoph; Rasmussen, Peter; Sørensen, Henrik

2015-01-01

Hypoxia increases the heart rate (HR) response to exercise but the mechanism(s) remain unclear. We tested the hypothesis that the tachycardic effect of hypoxia persists during separate but not combined inhibition of β-adrenergic and muscarinic receptors. Nine subjects performed incremental exercise...... combined β-adrenergic and muscarinic receptor inhibition....

Positive transcriptional regulation of the human micro opioid receptor gene by poly(ADP-ribose) polymerase-1 and increase of its DNA binding affinity based on polymorphism of G-172 -> T.

Science.gov (United States)

Ono, Takeshi; Kaneda, Toshio; Muto, Akihiro; Yoshida, Tadashi

2009-07-24

Micro opioid receptor (MOR) agonists such as morphine are applied widely in clinical practice as pain therapy. The effects of morphine through MOR, such as analgesia and development of tolerance and dependence, are influenced by individual specificity. Recently, we analyzed single nucleotide polymorphisms on the human MOR gene to investigate the factors that contribute to individual specificity. In process of single nucleotide polymorphisms analysis, we found that specific nuclear proteins bound to G(-172) --> T region in exon 1 in MOR gene, and its affinity to DNA was increased by base substitution from G(-172) to T(-172). The isolated protein was identified by mass spectrometry and was confirmed by Western blotting to be poly(ADP-ribose) polymerase-1 (PARP-1). The overexpressed PARP-1 bound to G(-172) --> T and enhanced the transcription of reporter vectors containing G(-172) and T(-172). Furthermore, PARP-1 inhibitor (benzamide) decreased PARP-1 binding to G(-172) --> T without affecting mRNA or protein expression level of PARP-1 and down-regulated the subsequent MOR gene expression in SH-SY5Y cells. Moreover, we found that tumor necrosis factor-alpha enhanced MOR gene expression as well as increased PARP-1 binding to the G(-172) --> T region and G(-172) --> T-dependent transcription in SH-SY5Y cells. These effects were also inhibited by benzamide. In this study, our data suggest that PARP-1 positively regulates MOR gene transcription via G(-172) --> T, which might influence individual specificity in therapeutic opioid effects.

DNA methylation and histone deacetylation regulating insulin sensitivity due to chronic cold exposure.

Science.gov (United States)

Wang, Xiaoqing; Wang, Lai; Sun, Yizheng; Li, Ruiping; Deng, Jinbo; Deng, Jiexin

2017-02-01

In this study, we investigated the causal relationship between chronic cold exposure and insulin resistance and the mechanisms of how DNA methylation and histone deacetylation regulate cold-reduced insulin resistance. 46 adult male mice from postnatal day 90-180 were randomly assigned to control group and cold-exposure group. Mice in cold-exposure group were placed at temperature from -1 to 4 °C for 30 days to mimic chronic cold environment. Then, fasting blood glucose, blood insulin level and insulin resistance index were measured with enzymatic methods. Immunofluorescent labeling was carried out to visualize the insulin receptor substrate 2 (IRS2), Obese receptor (Ob-R, a leptin receptor), voltage-dependent anion channel protein 1 (VDAC1), cytochrome C (cytC), 5-methylcytosine (5-mC) positive cells in hippocampal CA1 area. Furthermore, the expressions of some proteins mentioned above were detected with Western blot. The results showed: ① Chronic cold exposure could reduce the insulin resistance index (P cold-exposure group than in control group with both immunohistochemical staining and Western blot (P cold exposure increased DNA methylation and histone deacetylation in the pyramidal cells of CA1 area and led to an increase in the expression of histone deacetylase 1 (HDAC1) and DNA methylation relative enzymes (P cold exposure can improve insulin sensitivity, with the involvement of DNA methylation, histone deacetylation and the regulation of mitochondrial energy metabolism. These epigenetic modifications probably form the basic mechanism of cold-reduced insulin resistance. Copyright © 2016 Elsevier Inc. All rights reserved.

HMGB1-mediated DNA bending: Distinct roles in increasing p53 binding to DNA and the transactivation of p53-responsive gene promoters.

Science.gov (United States)

Štros, Michal; Kučírek, Martin; Sani, Soodabeh Abbasi; Polanská, Eva

2018-03-01

HMGB1 is a chromatin-associated protein that has been implicated in many important biological processes such as transcription, recombination, DNA repair, and genome stability. These functions include the enhancement of binding of a number of transcription factors, including the tumor suppressor protein p53, to their specific DNA-binding sites. HMGB1 is composed of two highly conserved HMG boxes, linked to an intrinsically disordered acidic C-terminal tail. Previous reports have suggested that the ability of HMGB1 to bend DNA may explain the in vitro HMGB1-mediated increase in sequence-specific DNA binding by p53. The aim of this study was to reinvestigate the importance of HMGB1-induced DNA bending in relationship to the ability of the protein to promote the specific binding of p53 to short DNA duplexes in vitro, and to transactivate two major p53-regulated human genes: Mdm2 and p21/WAF1. Using a number of HMGB1 mutants, we report that the HMGB1-mediated increase in sequence-specific p53 binding to DNA duplexes in vitro depends very little on HMGB1-mediated DNA bending. The presence of the acidic C-terminal tail of HMGB1 and/or the oxidation of the protein can reduce the HMGB1-mediated p53 binding. Interestingly, the induction of transactivation of p53-responsive gene promoters by HMGB1 requires both the ability of the protein to bend DNA and the acidic C-terminal tail, and is promoter-specific. We propose that the efficient transactivation of p53-responsive gene promoters by HMGB1 depends on complex events, rather than solely on the promotion of p53 binding to its DNA cognate sites. Copyright © 2018 Elsevier B.V. All rights reserved.

Cow's milk increases the activities of human nuclear receptors peroxisome proliferator-activated receptors alpha and delta and retinoid X receptor alpha involved in the regulation of energy homeostasis, obesity, and inflammation.

Science.gov (United States)

Suhara, W; Koide, H; Okuzawa, T; Hayashi, D; Hashimoto, T; Kojo, H

2009-09-01

The nuclear peroxisome proliferator-activated receptors (PPAR) have been shown to play crucial roles in regulating energy homeostasis including lipid and carbohydrate metabolism, inflammatory responses, and cell proliferation, differentiation, and survival. Because PPAR agonists have the potential to prevent or ameliorate diseases such as hyperlipidemia, diabetes, atherosclerosis, and obesity, we have explored new natural agonists for PPAR. For this purpose, cow's milk was tested for agonistic activity toward human PPAR subtypes using a reporter gene assay. Milk increased human PPARalpha activity in a dose-dependent manner with a 3.2-fold increase at 0.5% (vol/vol). It also enhanced human PPARdelta activity in a dose-dependent manner with an 11.5-fold increase at 0.5%. However, it only slightly affected human PPARgamma activity. Ice cream, butter, and yogurt also increased the activities of PPARalpha and PPARdelta, whereas vegetable cream affected activity of PPARdelta but not PPARalpha. Skim milk enhanced the activity of PPAR to a lesser degree than regular milk. Milk and fresh cream increased the activity of human retinoid X receptor (RXR)alpha as well as PPARalpha and PPARdelta, whereas neither affected vitamin D3 receptor, estrogen receptors alpha and beta, or thyroid receptors alpha and beta. Both milk and fresh cream were shown by quantitative real-time PCR to increase the quantity of mRNA for uncoupling protein 2 (UCP2), an energy expenditure gene, in a dose-dependent manner. The increase in UCP2 mRNA was found to be reduced by treatment with PPARdelta-short interfering (si)RNA. This study unambiguously clarified at the cellular level that cow's milk increased the activities of human PPARalpha, PPARdelta, and RXRalpha. The possible role in enhancing the activities of PPARalpha, PPARdelta, and RXRalpha, and the health benefits of cow's milk were discussed.

DNA homologous recombination factor SFR1 physically and functionally interacts with estrogen receptor alpha.

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Yuxin Feng

Full Text Available Estrogen receptor alpha (ERα, a ligand-dependent transcription factor, mediates the expression of its target genes by interacting with corepressors and coactivators. Since the first cloning of SRC1, more than 280 nuclear receptor cofactors have been identified, which orchestrate target gene transcription. Aberrant activity of ER or its accessory proteins results in a number of diseases including breast cancer. Here we identified SFR1, a protein involved in DNA homologous recombination, as a novel binding partner of ERα. Initially isolated in a yeast two-hybrid screen, the interaction of SFR1 and ERα was confirmed in vivo by immunoprecipitation and mammalian one-hybrid assays. SFR1 co-localized with ERα in the nucleus, potentiated ER's ligand-dependent and ligand-independent transcriptional activity, and occupied the ER binding sites of its target gene promoters. Knockdown of SFR1 diminished ER's transcriptional activity. Manipulating SFR1 expression by knockdown and overexpression revealed a role for SFR1 in ER-dependent and -independent cancer cell proliferation. SFR1 differs from SRC1 by the lack of an intrinsic activation function. Taken together, we propose that SFR1 is a novel transcriptional modulator for ERα and a potential target in breast cancer therapy.

Role of fetal DNA in preeclampsia (review).

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Konečná, Barbora; Vlková, Barbora; Celec, Peter

2015-02-01

Preeclampsia is an autoimmune disorder characterized by hypertension. It begins with abnormal cytotrophoblast apoptosis, which leads to inflammation and an increase in the levels of anti-angiogenic factors followed by the disruption of the angiogenic status. Increased levels of fetal DNA and RNA coming from the placenta, one of the most commonly affected organs in pregnancies complicated by preeclampsia, have been found in pregnant women with the condition. However, it remains unknown as to whether this is a cause or a consequence of preeclampsia. Few studies have been carried out on preeclampsia in which an animal model of preeclampsia was induced by an injection of different types of DNA that are mimic fetal DNA and provoke inflammation through Toll-like receptor 9 (TLR9) or cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). The specific mechanisms involved in the development of preeclampsia are not yet fully understood. It is hypothesized that the presence of different fragments of fetal DNA in maternal plasma may cause for the development of preeclampsia. The function of DNase during preeclampsia also remains unresolved. Studies have suggested that its activity is decreased or the DNA is protected against its effects. Further research is required to uncover the pathogenesis of preeclampsia and focus more on the condition of patients with the condition.

An examination of the characteristics, concentration, and distribution of androgen receptor in rat testis during sexual maturation

International Nuclear Information System (INIS)

Buzek, S.W.

1989-01-01

In these studies a nuclear exchange assay was established in rat testis in which exchange after 86 hours at 4 degree C was greater than 85% complete and receptor was stable. Receptor concentration per DNA measured by exchange declined between 15 and 25 days of age in the rat testis, then increased 4-fold during sexual maturation. Proliferation of germ cells which had low receptor concentration appeared to account for the early decline in testicular receptor concentration, whereas increase in receptor number per Sertoli cell between 25 and 35 days of age contributed to the later increase. Detailed studies showed that other possible explanations for changes in receptor number were not likely. Androgen receptor dynamics in testicular cells showed rapid, specific uptake of [ 3 H]-testosterone that was easily blocked by unlabeled testosterone, and medroxyprogesterone acetate, but not as well as by the anti-androgens cyproterone acetate and hydroxyflutamide

Single-molecule photobleaching reveals increased MET receptor dimerization upon ligand binding in intact cells

International Nuclear Information System (INIS)

Dietz, Marina S; Haße, Daniel; Ferraris, Davide M; Göhler, Antonia; Niemann, Hartmut H; Heilemann, Mike

2013-01-01

The human receptor tyrosine kinase MET and its ligand hepatocyte growth factor/scatter factor are essential during embryonic development and play an important role during cancer metastasis and tissue regeneration. In addition, it was found that MET is also relevant for infectious diseases and is the target of different bacteria, amongst them Listeria monocytogenes that induces bacterial uptake through the surface protein internalin B. Binding of ligand to the MET receptor is proposed to lead to receptor dimerization. However, it is also discussed whether preformed MET dimers exist on the cell membrane. To address these issues we used single-molecule fluorescence microscopy techniques. Our photobleaching experiments show that MET exists in dimers on the membrane of cells in the absence of ligand and that the proportion of MET dimers increases significantly upon ligand binding. Our results indicate that partially preformed MET dimers may play a role in ligand binding or MET signaling. The addition of the bacterial ligand internalin B leads to an increase of MET dimers which is in agreement with the model of ligand-induced dimerization of receptor tyrosine kinases.

The C'-terminal interaction domain of the thyroid hormone receptor confers the ability of the DNA site to dictate positive or negative transcriptional activity

International Nuclear Information System (INIS)

Holloway, J.M.; Glass, C.K.; Adler, S.; Nelson, C.A.; Rosenfeld, M.G.

1990-01-01

To investigate mechanisms responsible for positive and negative transcriptional control, the authors have utilized two types of promoters that are diffferentially regulated by thyroid hormone (T 3 ) receptors. Promoters containing the palindromic T 3 response element TCAGGTCA TGACCTGA are positively regulated by the T 3 receptor after the administration of T 3 , whereas otherwise identical promoters containing the estrogen response element TCAGGTCA CTG TGACCTGA can be regulated negatively; converse effects are observed with the estrogen receptor. They describe evidence that the transcriptional inhibitory effects of the T 3 or estrogen receptors on the estrogen or T 3 response elements, respectively, are imposed by amino acid sequences in the C'-terminal region that colocalize with dimerization and hormone-binding domains and that these sequences can transfer inhibitory functions to other classes of transcription factors. Removal of the C'-terminal dimerization and hormone-binding domains of either the αT 3 or estrogen receptors permits each receptor to act constitutively to enhance transcription on both T 3 and estrogen response elements. It is, therefore, suggested that protein-protein interactions between receptor C' termini limit the subset of DNA binding sites on which transcriptional activation occurs

Increased dopamine D1 receptor binding in the human mesocortical system following central cholinergic activation

International Nuclear Information System (INIS)

Fedi, M.; Berkovic, S.F.; Tochon-Danguy, H.J.; Reutens, D.C.

2002-01-01

Full text: The interaction between the cholinergic and dopaminergic system has been implicated in many pathological processes including, Alzheimer's disease, schizophrenia and drug addiction. Little is known about the control of dopamine (DA) release following central cholinergic activation in humans, but experimental studies suggest that endogenously released Acetylcholine (ACh) achieved by the administration of cholinesterase inhibitors, can increase dopamine efflux in different regions of the brain. This leads to the activation of different types of post-synaptic dopaminergic receptors which belong to the family of G-protein coupled receptors (GPCRs). A common paradigm of the GPCRs desensitization is that agonist-induced receptor signaling is rapidly attenuated by receptor internalisation. Several experiments have shown that the activation of Dl receptors in acute conditions leads, within minutes, to translocation of the receptor from the surface of the neurons to the endosomal compartment in the cytoplasm and increased receptor turnover. To assess changes in Dl receptor density following an intravenous infusion of the selective cholinesterase inhibitor physostigmine salicylate (PHY), we studied eleven normal subjects (10 male and 1 female, mean age 36.1 and 61617; 9.9) using [11C]-SCH23390 and PET The binding potential (BP) for SCH23390 was significantly (p 0.05). There was no statistically significant difference between baseline and physostigmine Kl ratio (p>0.05) suggesting that BP changes observed were not secondary to regional blood flow changes or to an order effect of the scans. Copyright (2002) The Australian and New Zealand Society of Nuclear Medicine Inc

The immune receptor Trem1 cooperates with diminished DNA damage response to induce preleukemic stem cell expansion.

Science.gov (United States)

Du, W; Amarachintha, S; Wilson, A; Pang, Q

2017-02-01

Fanconi anemia (FA) is an inherited bone marrow failure syndrome with extremely high risk of leukemic transformation. Here we investigate the relationship between DNA damage response (DDR) and leukemogenesis using the Fanca knockout mouse model. We found that chronic exposure of the Fanca -/- hematopoietic stem cells to DNA crosslinking agent mitomycin C in vivo leads to diminished DDR, and the emergence/expansion of pre-leukemia stem cells (pre-LSCs). Surprisingly, although genetic correction of Fanca deficiency in the pre-LSCs restores DDR and reduces genomic instability, but fails to prevent pre-LSC expansion or delay leukemia development in irradiated recipients. Furthermore, we identified transcription program underlying dysregulated DDR and cell migration, myeloid proliferation, and immune response in the Fanca -/- pre-LSCs. Forced expression of the downregulated DNA repair genes, Rad51c or Trp53i13, in the Fanca -/- pre-LSCs partially rescues DDR but has no effect on leukemia, whereas shRNA knockdown of the upregulated immune receptor genes Trem1 or Pilrb improves leukemia-related survival, but not DDR or genomic instability. Furthermore, Trem1 cooperates with diminished DDR in vivo to promote Fanca -/- pre-LSC expansion and leukemia development. Our study implicates diminishing DDR as a root cause of FA leukemogenesis, which subsequently collaborates with other signaling pathways for leukemogenic transformation.

Estradiol-induced increase in the magnitude of long-term potentiation is prevented by blocking NR2B-containing receptors.

Science.gov (United States)

Smith, Caroline C; McMahon, Lori L

2006-08-16

Estradiol, through activation of genomic estrogen receptors, induces changes in synaptic morphology and function in hippocampus, a brain region important for memory acquisition. Specifically, this hormone increases CA1 pyramidal cell dendritic spine density, NMDA receptor (NMDAR)-mediated transmission, and the magnitude of long-term potentiation (LTP) at CA3-CA1 synapses. We recently reported that the estradiol-induced increase in LTP magnitude occurs only when there is a simultaneous increase in the fractional contribution of NMDAR-mediated transmission relative to AMPA receptor transmission, suggesting a direct role for the increase in NMDAR transmission to the heightened LTP magnitude. Estradiol has been shown to increase expression of the NMDAR subunit NR2B, but whether this translates into an increase in function of NR2B-containing receptors remains to be determined. Here we show that not only is the estradiol-induced increase in NMDAR transmission mediated by NR2B-containing receptors, but blocking these receptors using RO25-6981 [R-(R,S)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidine propranol] (0.5 microM), an NR2B selective antagonist, prevents the estradiol-induced increase in LTP magnitude. Thus, our data show a causal link between the estradiol-induced increase in transmission mediated by NR2B-containing NMDARs and the increase in LTP magnitude.

Mechanism for iron control of the Vibrio fischeri luminescence system: involvement of cyclic AMP and cyclic AMP receptor protein and modulation of DNA level.

Science.gov (United States)

Dunlap, P V

1992-07-01

Iron controls luminescence in Vibrio fischeri by an indirect but undefined mechanism. To gain insight into that mechanism, the involvement of cyclic AMP (cAMP) and cAMP receptor protein (CRP) and of modulation of DNA levels in iron control of luminescence were examined in V. fischeri and in Escherichia coli containing the cloned V. fischeri lux genes on plasmids. For V. fischeri and E. coli adenylate cyclase (cya) and CRP (crp) mutants containing intact lux genes (luxR luxICDABEG), presence of the iron chelator ethylenediamine-di(o-hydroxyphenyl acetic acid) (EDDHA) increased expression of the luminescence system like in the parent strains only in the cya mutants in the presence of added cAMP. In the E. coli strains containing a plasmid with a Mu dl(lacZ) fusion in luxR, levels of beta-galactosidase activity (expression from the luxR promoter) and luciferase activity (expression from the lux operon promoter) were both 2-3-fold higher in the presence of EDDHA in the parent strain, and for the mutants this response to EDDHA was observed only in the cya mutant in the presence of added cAMP. Therefore, cAMP and CRP are required for the iron restriction effect on luminescence, and their involvement in iron control apparently is distinct from the known differential control of transcription from the luxR and luxICDABEG promoters by cAMP-CRP. Furthermore, plasmid and chromosomal DNA levels were higher in E. coli and V. fischeri in the presence of EDDHA. The higher DNA levels correlated with an increase in expression of chromosomally encoded beta-galactosidase in E. coli and with a higher level of autoinducer in cultures of V. fischeri. These results implicate cAMP-CRP and modulation of DNA levels in the mechanism of iron control of the V. fischeri luminescence system.

Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA

Energy Technology Data Exchange (ETDEWEB)

Chowdhury, E.H., E-mail: md.ezharul.hoque@med.monash.edu.my [Jeffrey Cheah School of Medicine and Health Sciences, Monash University Sunway Campus, Jalan Lagoon Selatan, Bandar Sunway, Selangor Darul Ehsan (Malaysia)

2011-06-17

Highlights: {yields} Cytoplasmic stability of plasmid DNA is enhanced by fluoride incorporation into carbonate apatite carrier. {yields} Fluoridated carbonate apatite promotes a robust increase in transgene expression. {yields} Controlled dissolution of fluoridated carbonate apatite in endosomal acidic environment might buffer the endosomes and prevent degradation of the released DNA. -- Abstract: Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particle-forming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA.

Fluoride enhances transfection activity of carbonate apatite by increasing cytoplasmic stability of plasmid DNA

International Nuclear Information System (INIS)

Chowdhury, E.H.

2011-01-01

Highlights: → Cytoplasmic stability of plasmid DNA is enhanced by fluoride incorporation into carbonate apatite carrier. → Fluoridated carbonate apatite promotes a robust increase in transgene expression. → Controlled dissolution of fluoridated carbonate apatite in endosomal acidic environment might buffer the endosomes and prevent degradation of the released DNA. -- Abstract: Intracellular delivery of a functional gene or a nucleic acid sequence to specifically knockdown a harmful gene is a potential approach to precisely treat a critical human disease. The intensive efforts in the last few decades led to the development of a number of viral and non-viral synthetic vectors. However, an ideal delivery tool in terms of the safety and efficacy has yet to be established. Recently, we have developed pH-sensing inorganic nanocrystals of carbonate apatite for efficient and cell-targeted delivery of gene and gene-silencing RNA. Here we show that addition of very low level of fluoride to the particle-forming medium facilitates a robust increase in transgene expression following post-incubation of the particles with HeLa cells. Confocal microscopic observation and Southern blotting prove the cytoplasmic existence of plasmid DNA delivered by likely formed fluoridated carbonate apatite particles while degradation of plasmid DNA presumably by cytoplasmic nucleases was noticed following delivery with apatite particles alone. The beneficial role of fluoride in enhancing carbonate apatite-mediated gene expression might be due to the buffering potential of generated fluoridated apatite in endosomal acidic environment, thereby increasing the half-life of delivered plasmid DNA.

Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA

DEFF Research Database (Denmark)

Barnathan, E S; Kuo, A; Karikó, K

1990-01-01

Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC...... an endothelial cell cDNA library using the polymerase chain reaction (PCR) and oligonucleotide primers corresponding to the DNA sequence of the receptor cloned from transformed human fibroblasts (Roldan et al, EMBO J 9:467, 1990). The size of the cDNA (approximately 1,054 base pairs, bp) and the presence...

Variants of sequence family B Thermococcus kodakaraensis DNA polymerase with increased mismatch extension selectivity.

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Claudia Huber

Full Text Available Fidelity and selectivity of DNA polymerases are critical determinants for the biology of life, as well as important tools for biotechnological applications. DNA polymerases catalyze the formation of DNA strands by adding deoxynucleotides to a primer, which is complementarily bound to a template. To ensure the integrity of the genome, DNA polymerases select the correct nucleotide and further extend the nascent DNA strand. Thus, DNA polymerase fidelity is pivotal for ensuring that cells can replicate their genome with minimal error. DNA polymerases are, however, further optimized for more specific biotechnological or diagnostic applications. Here we report on the semi-rational design of mutant libraries derived by saturation mutagenesis at single sites of a 3'-5'-exonuclease deficient variant of Thermococcus kodakaraensis DNA polymerase (KOD pol and the discovery for variants with enhanced mismatch extension selectivity by screening. Sites of potential interest for saturation mutagenesis were selected by their proximity to primer or template strands. The resulting libraries were screened via quantitative real-time PCR. We identified three variants with single amino acid exchanges-R501C, R606Q, and R606W-which exhibited increased mismatch extension selectivity. These variants were further characterized towards their potential in mismatch discrimination. Additionally, the identified enzymes were also able to differentiate between cytosine and 5-methylcytosine. Our results demonstrate the potential in characterizing and developing DNA polymerases for specific PCR based applications in DNA biotechnology and diagnostics.

Molecular characterization of a rat α2B-adrenergic receptor

International Nuclear Information System (INIS)

Zeng, D.; Harrison, J.K.; D'Angelo, D.D.; Barber, C.M.; Tucker, A.L.; Lu, Z.; Lynch, K.R.

1990-01-01

α 2 -Adrenergic receptors comprise a heterogeneous population based on pharmacologic and molecular evidence. The authors have isolated a cDNA clone (pRNGα 2 ) encoding a rat α 2 -adrenergic receptor. A rat kidney cDNA library was screened with an oligonucleotide complementary to a highly conserved region found in all biogenic amine receptors described to date. The deduced amino acid sequence displays many features of guanyl nucleotide-binding protein-coupled receptors except it does not have a consensus N-linked glycosylation site near the amino terminus. Membranes prepared from COS cells transfected with pRNGα 2 DNA display high affinity an saturable binding to [ 3 H]rauwolscine. Competition curve data analysis shows that RNGα 2 protein binds to a variety of adrenergic drugs with the following rank order of potency: yohimbine ≥ chlorpromazine > prazosin ≥ clonidine > norepinephrine ≥ oxymetazoline. RNGα 2 RNA accumulates in both rat kidney and neonatal rat lung. When a cysteine residue (Cys-169) that is conserved among all members of the seven-transmembrane-region superfamily is changed to phenylalanine, the RNGα 2 protein fails to bind [ 3 H]rauwolscine after expression in COS cells. They conclude that pRNGα 2 likely represents a cDNA for a rat α 2B -adrenergic receptor

Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA

DEFF Research Database (Denmark)

Stangegaard, Michael; Dufva, I.H.; Dufva, Hans Martin

2006-01-01

oligonucleotides (pentadecamers) consistently, yielded at least 2 fold as much cDNA as did random hexamers using either-poly(A) RNA or an amplified version of messenger RNA (aRNA) as a template. The cDNA generated using pentadecamers did not differ in size distribution or the amount of incorporated label compared...... with cDNA generated with random hexamers. The increased efficiency of priming using random pentadecamers resulted in reverse transcription of > 80% of the template aRNA, while random hexamers induced reverse transcription of only 40% of the template aRNA. This suggests a better coverage...... that random pentadecamers can replace random hexamers in reverse transcription reactions on both poly(A) RNA and amplified RNA, resulting in higher cDNA yields and quality....

Generation of Oxtr cDNA(HA)-Ires-Cre Mice for Gene Expression in an Oxytocin Receptor Specific Manner.

Science.gov (United States)

Hidema, Shizu; Fukuda, Tomokazu; Hiraoka, Yuichi; Mizukami, Hiroaki; Hayashi, Ryotaro; Otsuka, Ayano; Suzuki, Shingo; Miyazaki, Shinji; Nishimori, Katsuhiko

2016-05-01

The neurohypophysial hormone oxytocin (OXT) and its receptor (OXTR) have critical roles in the regulation of pro-social behaviors, including social recognition, pair bonding, parental behavior, and stress-related responses. Supporting this hypothesis, a portion of patients suffering from autism spectrum disorder have mutations, such as single nucleotide polymorphisms, or epigenetic modifications in their OXTR gene. We previously reported that OXTR-deficient mice exhibit pervasive social deficits, indicating the critical role of OXTR in social behaviors. In the present study, we generated Oxtr cDNA(HA)-Ires-Cre knock-in mice, expressing both OXTR and Cre recombinase under the control of the endogenous Oxtr promoter. Knock-in cassette of Oxtr cDNA(HA)-Ires-Cre consisted of Oxtr cDNA tagged with the hemagglutinin epitope at the 3' end (Oxtr cDNA(HA)), internal ribosomal entry site (Ires), and Cre. Cre was expressed in the uterus, mammary gland, kidney, and brain of Oxtr cDNA(HA)-Ires-Cre knock-in mice. Furthermore, the distribution of Cre in the brain was similar to that observed in Oxtr-Venus fluorescent protein expressing mice (Oxtr-Venus), another animal model previously generated by our group. Social behavior of Oxtr cDNA(HA)-Ires-Cre knock-in mice was similar to that of wild-type animals. We demonstrated that this construct is expressed in OXTR-expressing neurons specifically after an infection with the recombinant adeno-associated virus carrying the flip-excision switch vector. Using this system, we showed the transport of the wheat-germ agglutinin tracing molecule from the OXTR-expressing neurons to the innervated neurons in knock-in mice. This study might contribute to the monosynaptic analysis of neuronal circuits and to the optogenetic analysis of neurons expressing OXTR. © 2015 Wiley Periodicals, Inc.

Mitochondrial DNA: An Endogenous Trigger for Immune Paralysis.

Science.gov (United States)

Schäfer, Simon T; Franken, Lars; Adamzik, Michael; Schumak, Beatrix; Scherag, André; Engler, Andrea; Schönborn, Niels; Walden, Jennifer; Koch, Susanne; Baba, Hideo A; Steinmann, Jörg; Westendorf, Astrid M; Fandrey, Joachim; Bieber, Thomas; Kurts, Christian; Frede, Stilla; Peters, Jürgen; Limmer, Andreas

2016-04-01

Critically ill patients are at high risk to suffer from sepsis, even in the absence of an initial infectious source, but the molecular mechanisms for their increased sepsis susceptibility, including a suppressed immune system, remain unclear. Although microbes and pathogen-associated molecular pattern are accepted inducers of sepsis and septic immunosuppression, the role of endogenous Toll-like receptor (TLR) ligands, such as mitochondrial DNA (mtDNA), in altering the immune response is unknown. Mitochondrial DNA serum concentrations of the mitochondrial genes D-Loop and adenosine triphosphatase 6 were determined (quantitative polymerase chain reaction) in 165 septic patients and 50 healthy volunteers. Furthermore, cytotoxic T-cell activity was analyzed in wild-type and TLR9 knockout mice, with/without previous mtDNA administration, followed by injection of an ovalbumin-expressing adenoviral vector. Mitochondrial DNA serum concentrations were increased in septic patients (adenosine triphosphatase 6, 123-fold; D-Loop, 76-fold, P < 0.0001) compared with volunteers. Furthermore, a single mtDNA injection caused profound, TLR9-dependent immunosuppression of adaptive T-cell cytotoxicity in wild-type but not in TLR9 knockout mice and evoked various immunosuppressive mechanisms including the destruction of the splenic microstructure, deletion of cross-presenting dendritic cells, and up-regulation of programmed cell death ligand 1 and indoleamine 2,3-dioxygenase. Several of these findings in mice were mirrored in septic patients, and mtDNA concentrations were associated with an increased 30-day mortality. The findings of this study imply that mtDNA, an endogenous danger associated molecular pattern, is a hitherto unknown inducer of septic immunoparalysis and one possible link between initial inflammation and subsequent immunosuppression in critically ill patients.

Galactosylated DNA lipid nanocapsules for efficient hepatocyte targeting.

Science.gov (United States)

Morille, M; Passirani, C; Letrou-Bonneval, E; Benoit, J-P; Pitard, B

2009-09-11

The main objective of gene therapy via a systemic pathway is the development of a stable and non-toxic gene vector that can encapsulate and deliver foreign genetic materials into specific cell types with the transfection efficiency of viral vectors. With this objective, DNA complexed with cationic lipids of DOTAP/DOPE was encapsulated into lipid nanocapsules (LNCs) forming nanocarriers (DNA LNCs) with a size suitable for systemic injection (109+/-6 nm). With the goal of increasing systemic delivery, LNCs were stabilised with long chains of poly(ethylene glycol) (PEG), either from a PEG lipid derivative (DSPE-mPEG(2000)) or from an amphiphilic block copolymer (F108). In order to overcome internalisation difficulties encountered with PEG shield, a specific ligand (galactose) was covalently added at the distal end of the PEG chains, in order to provide active targeting of the asialoglycoprotein-receptor present on hepatocytes. This study showed that DNA LNCs were as efficient as positively charged DOTAP/DOPE lipoplexes for transfection. In primary hepatocytes, when non-galactosylated, the two polymers significantly decreased the transfection, probably by creating a barrier around the DNA LNCs. Interestingly, galactosylated F108 coated DNA LNCs led to a 18-fold increase in luciferase expression compared to non-galactosylated ones.

Effects of temperature and relative humidity on DNA methylation.

Science.gov (United States)

Bind, Marie-Abele; Zanobetti, Antonella; Gasparrini, Antonio; Peters, Annette; Coull, Brent; Baccarelli, Andrea; Tarantini, Letizia; Koutrakis, Petros; Vokonas, Pantel; Schwartz, Joel

2014-07-01

Previous studies have found relationships between DNA methylation and various environmental contaminant exposures. Associations with weather have not been examined. Because temperature and humidity are related to mortality even on non-extreme days, we hypothesized that temperature and relative humidity may affect methylation. We repeatedly measured methylation on long interspersed nuclear elements (LINE-1), Alu, and 9 candidate genes in blood samples from 777 elderly men participating in the Normative Aging Study (1999-2009). We assessed whether ambient temperature and relative humidity are related to methylation on LINE-1 and Alu, as well as on genes controlling coagulation, inflammation, cortisol, DNA repair, and metabolic pathway. We examined intermediate-term associations of temperature, relative humidity, and their interaction with methylation, using distributed lag models. Temperature or relative humidity levels were associated with methylation on tissue factor (F3), intercellular adhesion molecule 1 (ICAM-1), toll-like receptor 2 (TRL-2), carnitine O-acetyltransferase (CRAT), interferon gamma (IFN-γ), inducible nitric oxide synthase (iNOS), and glucocorticoid receptor, LINE-1, and Alu. For instance, a 5°C increase in 3-week average temperature in ICAM-1 methylation was associated with a 9% increase (95% confidence interval: 3% to 15%), whereas a 10% increase in 3-week average relative humidity was associated with a 5% decrease (-8% to -1%). The relative humidity association with ICAM-1 methylation was stronger on hot days than mild days. DNA methylation in blood cells may reflect biological effects of temperature and relative humidity. Temperature and relative humidity may also interact to produce stronger effects.

Functional expression of the 5-HT1c receptor in neuronal and nonneuronal cells

International Nuclear Information System (INIS)

Julius, D.; MacDermott, A.B.; Jessel, T.M.; Huang, K.; Molineaux, S.; Schieren, I.; Axel, R.

1988-01-01

The isolation of the genes encoding the multiple serotonin receptor subtypes and the ability to express these receptors in new cellular environments will help to elucidate the molecular mechanisms of action of serotonin in the mammalian brain. The cloning of most neurotransmitter receptors has required the purification of receptor, the determination of partial protein sequence, and the synthesis of oligonucleotide probes with which to obtain cDNA or genomic clones. However, the serotonin receptors have not been purified and antibodies have not been generated. The authors therefore designed a cDNA expression system that permits the identification of functional cDNA clones encoding serotonin receptors in the absence of protein sequence information. They have combined cloning in RNA expression vectors with an electrophysiological assay in oocytes to isolate a functional cDNA clone encoding the entire 5-HT 1c receptor. The sequence of this clone reveals that the 5-HT 1c receptor belongs to a family of G-protein-coupled receptors that are thought to traverse the membrane seven times. Mouse fibroblasts transformed with this clone bind serotonergic ligands and respond to serotonin with an elevation in intracellular calcium. Moreover, in situ hybridization and Northern blot analysis indicate that the 5-HT 1c receptor mRNA is expressed in a wide variety of neurons in the rat central nervous system, suggesting that this receptor plays a prominent role in neuronal function

Simultaneous Expression from Both the Sense and Antisense Strand of the Erythropoietin Receptor Gene Mitigates Acute Lung Injury

Science.gov (United States)

2017-09-01

concept efficacy that increasing EpoR or RopE expression by cDNA delivery to lung cells in vitro enhances cytoprotection against hyperoxia-induced injury...oxidative damage, cell culture, rodent model, inhalation cDNA delivery, sense and antisense erythropoietin receptor transcripts 16. SECURITY...prevention of acute lung injury. 1-6 50% Subtask 1: Prepare plasmid cDNA of EpoR and RopE in nanoparticle formulation. 1 Completed 06.2017 Subtask 2

Colonic vascular conductance increased by Daikenchuto via calcitonin gene-related peptide and receptor-activity modifying protein 1.

Science.gov (United States)

Kono, Toru; Koseki, Takashi; Chiba, Shinichi; Ebisawa, Yoshiaki; Chisato, Naoyuki; Iwamoto, Jun; Kasai, Shinichi

2008-11-01

Daikencyuto (DKT) is a traditional Japanese medicine (Kampo) and is a mixture of extract powders from dried Japanese pepper, processed ginger, ginseng radix, and maltose powder and has been used as the treatment of paralytic ileus. DKT may increase gastrointestinal motility by an up-regulation of the calcitonin gene-related peptide (CGRP). CGRP is also the most powerful vasoactive substance. In the present study, we investigated whether DKT has any effect on the colonic blood flow in rats. Experiments were performed on fasted anesthetized and artificially ventilated Wistar rats. Systemic mean arterial blood pressure and heart rate were recorded. Red blood cell flux in colonic blood flow was measured using noncontact laser tissue blood flowmetry, and colonic vascular conductance (CVC) was calculated as the ratio of flux to mean arterial blood pressure. We examined four key physiological mechanisms underlying the response using blocker drugs: CGRP1 receptor blocker (CGRP(8-37)), nitric oxide synthase inhibitor, vasoactive intestinal polypeptide (VIP) receptor blocker ([4-Cl-DPhe6, Leu17]-VIP), and substance P receptor blocker (spantide). Reverse transcription-polymerase chain reaction was used for the detection of mRNA of calcitonin receptor-like receptor, receptor-activity modifying protein 1, the component of CGRP 1 receptor and CGRP. After laparotomy, a cannula was inserted into the proximal colon to administer the DKT and to measure CVC at the distal colon. Intracolonal administration of DKT (10, 100, and 300 mg/kg) increased CVC (basal CVC, 0.10 mL/mmHg) from the first 15-min observation period (0.14, 0.17, and 0.17 mL/mmHg, respectively) and with peak response at either 45 min (0.17 mL/mmHg by 10 mg/kg), or 75 and 60 min (0.23 and 0.21 mL/mmHg by 100 and 300 mg/kg, respectively). CGRP(8-37) completely abolished the DKT-induced hyperemia, whereas nitric oxide synthase inhibitor partially attenuated the DKT-induced hyperemia. [4-Cl-DPhe6, Leu17]-VIP and spantide

Functional expression of Squalus acanthias melanocortin-5 receptor in CHO cells: ligand selectivity and interaction with MRAP.

Science.gov (United States)

Reinick, Christina L; Liang, Liang; Angleson, Josepha K; Dores, Robert M

2012-04-05

The melanocortin-5 receptor (MC(5)) of the dogfish Squalus acanthias (SacMC(5) receptor) can be functionally expressed in CHO cells in the absence of the co-expression of an exogenous MRAP cDNA. Both human ACTH(1-24) and dogfish ACTH(1-25) were much better stimulators of the SacMC(5) receptor than any of the mammalian or dogfish MSH ligands that were tested. The order of ligand selectivity for the dogfish melanocortins was ACTH(1-25)>αMSH>γ-MSH=δ-MSH>β-MSH. Unlike mammalian MC(5) receptors, the functional expression of the SacMC(5) receptor was not negatively impacted when the receptor was co-expressed with a cartilaginous fish (Callorhinchus milii) MRAP2 cDNA. However, co-expression with either mouse mMRAP1 or zebrafish zfMRAP1 increased the sensitivity of SacMC(5) receptor for hACTH(1-24) by at least one order of magnitude. Hence, SacMC(5) receptor has the potential to interact with MRAP1 orthologs and in this regard behaved more like a melanocortin MC(2) receptor ortholog than a melanocortin MC(5) receptor ortholog. These observations are discussed in light of the evolution of the melanocortin receptor gene family in cartilaginous fish, and the physiological implications of these observations are considered. Copyright © 2012 Elsevier B.V. All rights reserved.

The multiligand α₂-macroglobulin receptor/low density lipoprotein receptor-related protein

DEFF Research Database (Denmark)

Gliemann, Jørgen; Nykjær, Anders; Petersen, Claus Munck

1994-01-01

The fusion of separate lines of research has greatly helped in elucidating the function of the giant members of the low density lipoprotein (LDL) receptor (LDLR) supergene family. The cDNA encoding a large protein structurally closely related to LDLR, and hence named LDLR-related protein (LRP......), was cloned by Herz et al. in 1988.'Evidence was provided demonstrating that LRP can function as a receptor for chylomicron remnants@-migrating very low density lipoproteins (P-VLDL) rich in apolipoprotein E (apoE)?' The a2-macroglobulin (a2M) receptor (a2MR) was purified from rat livep and human p l a~e n t...... from the observation that affinity-purified a2MR/LRP contains a 40-kDa5.8 or 39-kDa6.' protein, designated a2MRAP, in addition to the a2MFULRP a- and P-chains. cDNA cloning" disclosed the 323-residue protein as both the human homologue of mouse heparin binding protein 44 (see reference 11) and...

Computational modeling of a carbon nanotube-based DNA nanosensor

Energy Technology Data Exchange (ETDEWEB)

Kalantari-Nejad, R; Bahrami, M [Mechanical Engineering Department, Amirkabir University of Technology, Tehran (Iran, Islamic Republic of); Rafii-Tabar, H [Department of Medical Physics and Biomedical Engineering and Research Centre for Medical Nanotechnology and Tissue Engineering, Shahid Beheshti University of Medical Sciences, Evin, Tehran (Iran, Islamic Republic of); Rungger, I; Sanvito, S, E-mail: mbahrami@aut.ac.ir [School of Physics and CRANN, Trinity College, Dublin 2 (Ireland)

2010-11-05

During the last decade the design of biosensors, based on quantum transport in one-dimensional nanostructures, has developed as an active area of research. Here we investigate the sensing capabilities of a DNA nanosensor, designed as a semiconductor single walled carbon nanotube (SWCNT) connected to two gold electrodes and functionalized with a DNA strand acting as a bio-receptor probe. In particular, we have considered both covalent and non-covalent bonding between the DNA probe and the SWCNT. The optimized atomic structure of the sensor is computed both before and after the receptor attaches itself to the target, which consists of another DNA strand. The sensor's electrical conductance and transmission coefficients are calculated at the equilibrium geometries via the non-equilibrium Green's function scheme combined with the density functional theory in the linear response limit. We demonstrate a sensing efficiency of 70% for the covalently bonded bio-receptor probe, which drops to about 19% for the non-covalently bonded one. These results suggest that a SWCNT may be a promising candidate for a bio-molecular FET sensor.

Computational modeling of a carbon nanotube-based DNA nanosensor

International Nuclear Information System (INIS)

Kalantari-Nejad, R; Bahrami, M; Rafii-Tabar, H; Rungger, I; Sanvito, S

2010-01-01

During the last decade the design of biosensors, based on quantum transport in one-dimensional nanostructures, has developed as an active area of research. Here we investigate the sensing capabilities of a DNA nanosensor, designed as a semiconductor single walled carbon nanotube (SWCNT) connected to two gold electrodes and functionalized with a DNA strand acting as a bio-receptor probe. In particular, we have considered both covalent and non-covalent bonding between the DNA probe and the SWCNT. The optimized atomic structure of the sensor is computed both before and after the receptor attaches itself to the target, which consists of another DNA strand. The sensor's electrical conductance and transmission coefficients are calculated at the equilibrium geometries via the non-equilibrium Green's function scheme combined with the density functional theory in the linear response limit. We demonstrate a sensing efficiency of 70% for the covalently bonded bio-receptor probe, which drops to about 19% for the non-covalently bonded one. These results suggest that a SWCNT may be a promising candidate for a bio-molecular FET sensor.

Cloning and functional characterization of the DA2 receptor gene in Chinese mitten crab (Eriocheir sinensis)

Science.gov (United States)

Xu, Min-jie; Zhang, Cong; Yang, Zhigang

2018-01-01

Dopamine (DA) plays a modulatory role in numerous physiological processes such as light adaptation and food intake, and exerts these functions through DA receptors (DARs). This study presents, for the first time, isolation and characterization of the dopamine receptor 2(DA2 receptor) cDNA from the intestinal tissue of Eriocheir sinensis, an economically important freshwater aquaculture species in China. The DA2 receptor cDNA sequence, which was obtained by rapid amplification of cDNA ends, is 2369bp long, encode peptide of 589 amino acid, and is highly homologous to related sequences in crustaceans. Analysis of the deduced amino acid sequence and the structure of the DA2 indicated that this receptor is a member of the family of G protein-coupled receptors (GPCRs), as it contains seven transmembrane domains and other common signatures of GPCRs. RT-PCR showed that the expression of the DA2 receptor gene was distributed in various tissues, and high expression levels were observed in the cranial ganglia and the thoracic ganglia. Further study of the effect of photoperiod on DA2 expression showed that constant darkness induced a significant increase in DA2 expression in the cranial ganglia. Finally, analysis of DA2 receptor expression under different feeding statuses showed that there was significantly greater expression in the hepatopancreas and intestines after feeding than before feeding, but there were no differences in expression between the before feeding and during feeding periods in either tissue. Our results indicate that the DA2 receptor structurally belongs to the family of G protein-coupled receptors, and that the cranial ganglia are the main tissues in which the DA2 receptor participates in light adaptation during dark hours. In addition, the DA2 receptor in E. sinensis may be involved in the physiological regulation of the hepatopancreas and digestive tract after the ingestion of food. This study provides a foundation for further exploration of the light

Gly184 of the Escherichia coli cAMP receptor protein provides optimal context for both DNA binding and RNA polymerase interaction.

Science.gov (United States)

Hicks, Matt N; Gunasekara, Sanjiva; Serate, Jose; Park, Jin; Mosharaf, Pegah; Zhou, Yue; Lee, Jin-Won; Youn, Hwan

2017-10-01

The Escherichia coli cAMP receptor protein (CRP) utilizes the helix-turn-helix motif for DNA binding. The CRP's recognition helix, termed F-helix, includes a stretch of six amino acids (Arg180, Glu181, Thr182, Val183, Gly184, and Arg185) for direct DNA contacts. Arg180, Glu181 and Arg185 are known as important residues for DNA binding and specificity, but little has been studied for the other residues. Here we show that Gly184 is another F-helix residue critical for the transcriptional activation function of CRP. First, glycine was repeatedly selected at CRP position 184 for its unique ability to provide wild type-level transcriptional activation activity. To dissect the glycine requirement, wild type CRP and mutants G184A, G184F, G184S, and G184Y were purified and their in vitro DNA-binding activity was measured. G184A and G184F displayed reduced DNA binding, which may explain their low transcriptional activation activity. However, G184S and G184Y displayed apparently normal DNA affinity. Therefore, an additional factor is needed to account for the diminished transcriptional activation function in G184S and G184Y, and the best explanation is perturbations in their interaction with RNA polymerase. The fact that glycine is the smallest amino acid could not fully warrant its suitability, as shown in this study. We hypothesize that Gly184 fulfills the dual functions of DNA binding and RNA polymerase interaction by conferring conformational flexibility to the F-helix.

Assignment of the human gene for the glucocorticoid receptor to chromosome 5.

OpenAIRE

Gehring, U; Segnitz, B; Foellmer, B; Francke, U

1985-01-01

Human lymphoblastic leukemia cells of line CEM-C7 are glucocroticoid-sensitive and contain glucocorticoid receptors of wild-type characteristics. EL4 mouse lymphoma cells are resistant to lysis by glucocorticoids due to mutant receptors that exhibit abnormal DNA binding. Hybrids between the two cell lines were prepared and analyzed with respect to glucocorticoid responsiveness and to receptor types by DNA-cellulose chromatrography. Sensitive hybrid cell clones contained the CEM-C7-specific re...

The role of GH receptor tyrosine phosphorylation in Stat5 activation

DEFF Research Database (Denmark)

Hansen, J A; Hansen, L H; Wang, X

1997-01-01

Stimulation of GH receptors leads to rapid activation of Jak2 kinase and subsequent tyrosine phosphorylation of the GH receptor. Three specific tyrosines located in the C-terminal domain of the GH receptor have been identified as being involved in GH-stimulated transcription of the Spi 2.1 promoter....... Mutated GH receptors lacking all but one of these three tyrosines are able to mediate a transcriptional response when transiently transfected into CHO cells together with a Spi 2.1 promoter/luciferase construct. Similarly, these GH receptors were found to be able to mediate activation of Stat5 DNA......-binding activity, whereas the GH receptor mutant lacking all intracellular tyrosines was not. Synthetic tyrosine phosphorylated peptides corresponding to the GH receptor sequence around the three tyrosines inhibited Stat5 DNA-binding activity while their non-phosphorylated counterparts were ineffective. Tyrosine...

MDMA Increases Excitability in the Dentate Gyrus: Role of 5HT2A Receptor Induced PGE2 Signaling

Science.gov (United States)

Collins, Stuart A.; Huff, Courtney; Chiaia, Nicolas; Gudelsky, Gary A.; Yamamoto, Bryan K.

2015-01-01

MDMA is a widely abused psychostimulant which causes release of serotonin in various forebrain regions. Recently, we reported that MDMA increases extracellular glutamate concentrations in the dentate gyrus, via activation of 5HT2A receptors. We examined the role of prostaglandin signaling in mediating the effects of 5HT2A receptor activation on the increases in extracellular glutamate and the subsequent long-term loss of parvalbumin interneurons in the dentate gyrus caused by MDMA. Administration of MDMA into the dentate gyrus of rats increased PGE2 concentrations which was prevented by coadministration of MDL100907, a 5HT2A receptor antagonist. MDMA-induced increases in extracellular glutamate were inhibited by local administration of SC-51089, an inhibitor of the EP1 prostaglandin receptor. Systemic administration of SC-51089 during injections of MDMA prevented the decreases in parvalbumin interneurons observed 10 days later. The loss of parvalbumin immunoreactivity after MDMA exposure coincided with a decrease in paired-pulse inhibition and afterdischarge threshold in the dentate gyrus. These changes were prevented by inhibition of EP1 and 5HT2A receptors during MDMA. Additional experiments revealed an increased susceptibility to kainic acid-induced seizures in MDMA treated rats which could be prevented with SC51089 treatments during MDMA exposure. Overall, these findings suggest that 5HT2A receptors mediate MDMA-induced PGE2 signaling and subsequent increases in glutamate. This signaling mediates parvalbumin cell losses as well as physiologic changes in the dentate gyrus, suggesting that the lack of the inhibition provided by these neurons increases the excitability within the dentate gyrus of MDMA treated rats. PMID:26670377

Significance of the Melanocortin 1 and Endothelin B Receptors in Melanocyte Homeostasis and Prevention of Sun-Induced Genotoxicity

Directory of Open Access Journals (Sweden)

Zalfa A. Abdel-Malek

2016-08-01

Full Text Available The membrane bound melanocortin 1 receptor (MC1R, and the endothelin B receptor (ENDBR are two G-protein coupled receptors that play important roles in constitutive regulation of melanocytes and their response to ultraviolet radiation (UVR, the main etiological factor for melanoma. The human MC1R is a Gs protein-coupled receptor, which is activated by its agonists α-melanocyte stimulating hormone (α-melanocortin; α-MSH and adrenocorticotropic hormone (ACTH. The ENDBR is a Gq coupled-receptor, which is activated by Endothelin (ET-3 during embryonic development, and ET-1 postnatally. Pigmentation and the DNA repair capacity are two major factors that determine the risk for melanoma. Activation of the MC1R by its agonists stimulates the synthesis of eumelanin, the dark brown photoprotective pigment. In vitro studies showed that α-MSH and ET-1 interact synergistically in the presence of basic fibroblast growth factor (bFGF to stimulate human melanocyte proliferation and melanogenesis, and to inhibit UVR-induced apoptosis. An important function of the MC1R is reduction of oxidative stress and activation of DNA repair pathways. The human MC1R is highly polymorphic, and MC1R variants, particularly those that cause loss of function of the expressed receptor, are associated with increased melanoma risk independently of pigmentation. These variants compromise the DNA repair and antioxidant capacities of human melanocytes. Recently, activation of ENDBR by ET-1 was reported to reduce the induction and enhance the repair of UVR-induced DNA photoproducts. We conclude that α-MSH and ET-1 and their cognate receptors MC1R and ENDBR reduce the risk for melanoma by maintaining genomic stability of melanocytes via modulating the DNA damage response to solar UVR. Elucidating the response of melanocytes to UVR should improve our understanding of the process of melanomagenesis, and lead to effective melanoma chemoprevention, as well as therapeutic strategies.

Hepatocellular hypertrophy and cell proliferation in Sprague–Dawley rats from dietary exposure to potassium perfluorooctanesulfonate results from increased expression of xenosensor nuclear receptors PPARα and CAR/PXR

International Nuclear Information System (INIS)

Elcombe, Clifford R.; Elcombe, Barbara M.; Foster, John R.; Chang, Shu-Ching; Ehresman, David J.; Butenhoff, John L.

2012-01-01

Highlights: ► The mechanism of K + PFOS-induced hepatic hypertrophy and tumors was studied in rats. ► Plasma ALT and AST values and histology indicated a lack of overt hepatotoxicity. ► Plasma cholesterol and triglycerides were lowered by K + PFOS treatment. ► K + PFOS increased proliferation index and decreased apoptotic index in liver. ► Liver effects of K + PFOS result from increased expression of PPARα and CAR/PXR. -- Abstract: The present study investigated the potential role for activation of PPARα and CAR/PXR by potassium PFOS (K + PFOS) with respect to the etiology of hepatic hypertrophy and hepatocellular adenoma in rats. Male Sprague–Dawley rats were fed K + PFOS (20 or 100 ppm) for either 1, 7, or 28 days. Wyeth 14,643 (Wy 14,643, 50 ppm) and phenobarbital (PB, 500 ppm) were the controls for PPARα and CAR/PXR activation, respectively. Measurements included: plasma ALT, AST, cholesterol, triglycerides, and glucose; liver protein and DNA content; liver activities of palmitoyl CoA oxidase (ACOX), Cyp4A, CYP2B, and CYP3A; induction of liver CYP4A1, CYP2E1, CYP2B1/2, and CYP3A1 proteins (SDS-PAGE and Western blots); liver and thyroid microscopic histopathology, apoptotic index, and cell proliferation index. Terminal body weight was decreased by K + PFOS (100 ppm) and Wy 14,643. All test-compound treatments increased liver weight. Plasma lipids were decreased by both PFOS and Wy 14,643. After treatment for 1 day, K + PFOS (100 ppm), PB, and Wy 14,643 increased mean hepatic DNA concentration and total hepatic DNA, and total DNA remained elevated after treatment for 7 days and 28 days (PB and Wy 14,643 only). Hepatic P450 concentration was elevated after 7 and 28 days by K + PFOS and by PB. K + PFOS and Wy 14,643 increased liver activities of ACOX and CYP4A as well as increased liver CYP4A1 protein. By 28 days of treatment, K + PFOS and PB increased liver activities of CYP2B and CYP3A as well as increased liver CYP2B1/2 and CYP3A1 proteins, and Wy

The phytochemical 3,3'-diindolylmethane decreases expression of AR-controlled DNA damage repair genes through repressive chromatin modifications and is associated with DNA damage in prostate cancer cells.

Science.gov (United States)

Palomera-Sanchez, Zoraya; Watson, Gregory W; Wong, Carmen P; Beaver, Laura M; Williams, David E; Dashwood, Roderick H; Ho, Emily

2017-09-01

Androgen receptor (AR) is a transcription factor involved in normal prostate physiology and prostate cancer (PCa) development. 3,3'-Diindolylmethane (DIM) is a promising phytochemical agent against PCa that affects AR activity and epigenetic regulators in PCa cells. However, whether DIM suppresses PCa via epigenetic regulation of AR target genes is unknown. We assessed epigenetic regulation of AR target genes in LNCaP PCa cells and showed that DIM treatment led to epigenetic suppression of AR target genes involved in DNA repair (PARP1, MRE11, DNA-PK). Decreased expression of these genes was accompanied by an increase in repressive chromatin marks, loss of AR occupancy and EZH2 recruitment to their regulatory regions. Decreased DNA repair gene expression was associated with an increase in DNA damage (γH2Ax) and up-regulation of genomic repeat elements LINE1 and α-satellite. Our results suggest that DIM suppresses AR-dependent gene transcription through epigenetic modulation, leading to DNA damage and genome instability in PCa cells. Published by Elsevier Inc.

Increased anticoagulant activity of thrombin-binding DNA aptamers by nanoscale organization on DNA nanostructures

DEFF Research Database (Denmark)

Rangnekar, Abhijit; Zhang, Alex M.; Shiyuan Li, Susan

2012-01-01

Control over thrombin activity is much desired to regulate blood clotting in surgical and therapeutic situations. Thrombin-binding RNA and DNA aptamers have been used to inhibit thrombin activity and thus the coagulation cascade. Soluble DNA aptamers, as well as two different aptamers tethered by...

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

Energy Technology Data Exchange (ETDEWEB)

Feng, Shan-Li [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China); Sun, Ming-Rui [Department of Pharmacology, Qiqihaer Medical College, Qiqihaer 160001 (China); Li, Ting-Ting; Yin, Xin [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China); Xu, Chang-Qing [Department of Pathophysiology, Harbin Medical University, Harbin 150086 (China); Sun, Yi-Hua, E-mail: syh200415@126.com [Department of Clinical Laboratory, The Second Affiliated Hospital of Harbin Medical University, Harbin 150086 (China)

2011-03-11

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

Progesterone receptor antagonist CDB-4124 increases depression-like behavior in mice without affecting locomotor ability

Science.gov (United States)

Beckley, Ethan H.; Scibelli, Angela C.; Finn, Deborah A.

2010-01-01

Progesterone withdrawal has been proposed as an underlying factor in premenstrual syndrome and postpartum depression. Progesterone withdrawal induces forced swim test (FST) immobility in mice, a depression-like behavior, but the contribution of specific receptors to this effect is unclear. The role of progesterone’s GABAA receptor-modulating metabolite allopregnanolone in depression- and anxiety-related behaviors has been extensively documented, but little attention has been paid to the role of progesterone receptors. We administered the classic progesterone receptor antagonist mifepristone (RU-38486) and the specific progesterone receptor antagonist CDB-4124 to mice that had been primed with progesterone for five days, and found that both compounds induced FST immobility reliably, robustly, and in a dose-dependent fashion. Although CDB-4124 increased FST immobility, it did not suppress initial activity in a locomotor test. These findings suggest that decreased progesterone receptor activity contributes to depression-like behavior in mice, consistent with the hypothesis that progesterone withdrawal may contribute to the symptoms of premenstrual syndrome or postpartum depression. PMID:21163582

Progesterone receptor antagonist CDB-4124 increases depression-like behavior in mice without affecting locomotor ability.

Science.gov (United States)

Beckley, Ethan H; Scibelli, Angela C; Finn, Deborah A

2011-07-01

Progesterone withdrawal has been proposed as an underlying factor in premenstrual syndrome and postpartum depression. Progesterone withdrawal induces forced swim test (FST) immobility in mice, a depression-like behavior, but the contribution of specific receptors to this effect is unclear. The role of progesterone's GABA(A) receptor-modulating metabolite allopregnanolone in depression- and anxiety-related behaviors has been extensively documented, but little attention has been paid to the role of progesterone receptors. We administered the classic progesterone receptor antagonist mifepristone (RU-38486) and the specific progesterone receptor antagonist CDB-4124 to mice that had been primed with progesterone for five days, and found that both compounds induced FST immobility reliably, robustly, and in a dose-dependent fashion. Although CDB-4124 increased FST immobility, it did not suppress initial activity in a locomotor test. These findings suggest that decreased progesterone receptor activity contributes to depression-like behavior in mice, consistent with the hypothesis that progesterone withdrawal may contribute to the symptoms of premenstrual syndrome or postpartum depression. Copyright © 2010 Elsevier Ltd. All rights reserved.

Running on empty: does mitochondrial DNA mutation limit replicative lifespan in yeast?: Mutations that increase the division rate of cells lacking mitochondrial DNA also extend replicative lifespan in Saccharomyces cerevisiae.

Science.gov (United States)

Dunn, Cory D

2011-10-01

Mitochondrial DNA (mtDNA) mutations escalate with increasing age in higher organisms. However, it has so far been difficult to experimentally determine whether mtDNA mutation merely correlates with age or directly limits lifespan. A recent study shows that budding yeast can also lose functional mtDNA late in life. Interestingly, independent studies of replicative lifespan (RLS) and of mtDNA-deficient cells show that the same mutations can increase both RLS and the division rate of yeast lacking the mitochondrial genome. These exciting, parallel findings imply a potential causal relationship between mtDNA mutation and replicative senescence. Furthermore, these results suggest more efficient methods for discovering genes that determine lifespan. Copyright © 2011 WILEY Periodicals, Inc.

Molecular verification on male sterile mutant after injected exogenous λDNA into wheat

International Nuclear Information System (INIS)

Yang Jingcheng; Yu Yuanjie; Liu Fengzhen; Qi Yanfang; Shen Fafu

2000-01-01

A cytoplasmic male sterile mutant and then a stable CMS line named D-type sterile line were obtained after injected exogenous λDNA into wheat line 814527, and line 814527 could be its maintainer line. By using λDNA labelled with 32 P as probe, unlabelled λDNA as positive check, dot blotting of nuclear DNA and chloroplast DNA of receptor 814527, D-type sterile line and its hybrid F 1 with Lumai 14 were carried out. Positive dots appeared in nuclear DNA and chloroplast DNA of D-type sterile line and its hybrid F 1 , but did not appear in the receptor. It showed that fragments of exogenous λDNA existed in nuclear genome and chloroplast genome of D-type sterile line, and could be inherited stably. All these results, on a molecular level, proved the reliability of exogenous DNA injection

Iron uptake and increased intracellular enzyme activity follow host lactoferrin binding by Trichomonas vaginalis receptors

Energy Technology Data Exchange (ETDEWEB)

Peterson, K.M.; Alderete, J.F.

1984-08-01

Lactoferrin acquisition and iron uptake by pathogenic Trichomonas vaginalis was examined. Saturation binding kinetics were obtained for trichomonads using increasing amounts of radioiodinated lactoferrin, while no significant binding by transferrin under similar conditions was achieved. Only unlabeled lactoferrin successfully and stoichiometrically competed with 125I-labeled lactoferrin binding. Time course studies showed maximal lactoferrin binding by 30 min at 37 degrees C. Data suggest no internalization of bound lactoferrin. The accumulation of radioactivity in supernatants after incubation of T. vaginalis with 125I-labeled lactoferrin and washing in PBS suggested the presence of low affinity sites for this host macromolecule. Scatchard analysis indicated the presence of 90,000 receptors per trichomonad with an apparent Kd of 1.0 microM. Two trichomonad lactoferrin binding proteins were identified by affinity chromatography and immunoprecipitation of receptor-ligand complexes. A 30-fold accumulation of iron was achieved using 59Fe-lactoferrin when compared to the steady state concentration of bound lactoferrin. The activity of pyruvate/ferrodoxin oxidoreductase, an enzyme involved in trichomonal energy metabolism, increased more than sixfold following exposure of the parasites to lactoferrin, demonstrating a biologic response to the receptor-mediated binding of lactoferrin. These data suggest that T. vaginalis possesses specific receptors for biologically relevant host proteins and that these receptors contribute to the metabolic processes of the parasites.

Iron uptake and increased intracellular enzyme activity follow host lactoferrin binding by Trichomonas vaginalis receptors

International Nuclear Information System (INIS)

Peterson, K.M.; Alderete, J.F.

1984-01-01

Lactoferrin acquisition and iron uptake by pathogenic Trichomonas vaginalis was examined. Saturation binding kinetics were obtained for trichomonads using increasing amounts of radioiodinated lactoferrin, while no significant binding by transferrin under similar conditions was achieved. Only unlabeled lactoferrin successfully and stoichiometrically competed with 125I-labeled lactoferrin binding. Time course studies showed maximal lactoferrin binding by 30 min at 37 degrees C. Data suggest no internalization of bound lactoferrin. The accumulation of radioactivity in supernatants after incubation of T. vaginalis with 125I-labeled lactoferrin and washing in PBS suggested the presence of low affinity sites for this host macromolecule. Scatchard analysis indicated the presence of 90,000 receptors per trichomonad with an apparent Kd of 1.0 microM. Two trichomonad lactoferrin binding proteins were identified by affinity chromatography and immunoprecipitation of receptor-ligand complexes. A 30-fold accumulation of iron was achieved using 59Fe-lactoferrin when compared to the steady state concentration of bound lactoferrin. The activity of pyruvate/ferrodoxin oxidoreductase, an enzyme involved in trichomonal energy metabolism, increased more than sixfold following exposure of the parasites to lactoferrin, demonstrating a biologic response to the receptor-mediated binding of lactoferrin. These data suggest that T. vaginalis possesses specific receptors for biologically relevant host proteins and that these receptors contribute to the metabolic processes of the parasites

A fractal analysis of protein to DNA binding kinetics using biosensors.

Science.gov (United States)

Sadana, Ajit

2003-08-01

A fractal analysis of a confirmative nature only is presented for the binding of estrogen receptor (ER) in solution to its corresponding DNA (estrogen response element, ERE) immobilized on a sensor chip surface [J. Biol. Chem. 272 (1997) 11384], and for the cooperative binding of human 1,25-dihydroxyvitamin D(3) receptor (VDR) to DNA with the 9-cis-retinoic acid receptor (RXR) [Biochemistry 35 (1996) 3309]. Ligands were also used to modulate the first reaction. Data taken from the literature may be modeled by using a single- or a dual-fractal analysis. Relationships are presented for the binding rate coefficient as a function of either the analyte concentration in solution or the fractal dimension that exists on the biosensor surface. The binding rate expressions developed exhibit a wide range of dependence on the degree of heterogeneity that exists on the surface, ranging from sensitive (order of dependence equal to 1.202) to very sensitive (order of dependence equal to 12.239). In general, the binding rate coefficient increases as the degree of heterogeneity or the fractal dimension of the surface increases. The predictive relationships presented provide further physical insights into the reactions occurring on the biosensor surface. Even though these reactions are occurring on the biosensor surface, the relationships presented should assist in understanding and in possibly manipulating the reactions occurring on cellular surfaces.

The Toll-like receptor gene family is integrated into human DNA damage and p53 networks.

Directory of Open Access Journals (Sweden)

Daniel Menendez

2011-03-01

Full Text Available In recent years the functions that the p53 tumor suppressor plays in human biology have been greatly extended beyond "guardian of the genome." Our studies of promoter response element sequences targeted by the p53 master regulatory transcription factor suggest a general role for this DNA damage and stress-responsive regulator in the control of human Toll-like receptor (TLR gene expression. The TLR gene family mediates innate immunity to a wide variety of pathogenic threats through recognition of conserved pathogen-associated molecular motifs. Using primary human immune cells, we have examined expression of the entire TLR gene family following exposure to anti-cancer agents that induce the p53 network. Expression of all TLR genes, TLR1 to TLR10, in blood lymphocytes and alveolar macrophages from healthy volunteers can be induced by DNA metabolic stressors. However, there is considerable inter-individual variability. Most of the TLR genes respond to p53 via canonical as well as noncanonical promoter binding sites. Importantly, the integration of the TLR gene family into the p53 network is unique to primates, a recurrent theme raised for other gene families in our previous studies. Furthermore, a polymorphism in a TLR8 response element provides the first human example of a p53 target sequence specifically responsible for endogenous gene induction. These findings-demonstrating that the human innate immune system, including downstream induction of cytokines, can be modulated by DNA metabolic stress-have many implications for health and disease, as well as for understanding the evolution of damage and p53 responsive networks.

DNA fragmentation and cell death mediated by T cell antigen receptor/CD3 complex on a leukemia T cell line.

Science.gov (United States)

Takahashi, S; Maecker, H T; Levy, R

1989-10-01

An anti-T cell receptor (TcR) monoclonal antibody (mAb), LC4, directed against a human leukemic T cell line, SUP-T13, caused DNA fragmentation ("apoptosis") and cell death upon binding to this cell line. Cross-linking of receptor molecules was necessary for this effect since F(ab')2, but not Fab', fragments of LC4 could induce cell death. Five anti-CD3 mAb tested also caused apoptosis, but only when they were presented on a solid phase. Interestingly, soluble anti-CD3 mAb induced calcium flux and had an additive effect on the calcium flux and interleukin 2 receptor expression induced by LC4, but these anti-CD3 mAb reversed the growth inhibition and apoptosis caused by LC4. The calcium ionophore A23187, but not the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), also induced apoptosis, suggesting that protein kinase C activation alone does not cause apoptosis, although PMA is growth inhibitory. These results suggest that two distinct biological phenomena can accompany stimulation of the TcR/CD3 complex. In both cases, calcium flux and interleukin 2 receptor expression is induced, but only in one case is apoptosis and cell death seen. The signal initiating apoptosis can be selectively prevented by binding CD3 portion of the receptor in this cell line. This difference in signals mediated by the TcR/CD3 complex may be important in explaining the process of thymic selection, as well as in choosing anti-TcR mAb for therapeutic use.

Protective effects of folic acid on DNA damage and DNA methylation levels induced by N-methyl- N'-nitro- N-nitrosoguanidine in Kazakh esophageal epithelial cells.

Science.gov (United States)

Chen, Y; Feng, H; Chen, D; Abuduwaili, K; Li, X; Zhang, H

2018-01-01

The protective effects of folic acid on DNA damage and DNA methylation induced by N-methyl- N'-nitro- N-nitrosoguanidine (MNNG) in Kazakh esophageal epithelial cells were investigated using a 3 × 3 factorial design trial. The cells were cultured in vitro and exposed to media containing different concentrations of folic acid and MNNG, after which growth indices were detected. DNA damage levels were measured using comet assays, and genome-wide DNA methylation levels (MLs) were measured using high-performance liquid chromatography. The DNA methylation of methylenetetrahydrofolate reductase (MTHFR) and folate receptor- α (FR α) genes was detected by bisulfite sequencing polymerase chain reaction (PCR). The results showed significant increases in tail DNA concentration, tail length, and Olive tail moment ( p methylation frequencies of MTHFR and FR α genes. In particular, significant differences were observed in the promoter regions of both genes ( p methylation in Kazakh esophageal epithelial cells upon MNNG exposure. Thus, sufficient folic acid levels could play a protective role against the damage induced by this compound.

Tyrosine 370 phosphorylation of ATM positively regulates DNA damage response

Science.gov (United States)

Lee, Hong-Jen; Lan, Li; Peng, Guang; Chang, Wei-Chao; Hsu, Ming-Chuan; Wang, Ying-Nai; Cheng, Chien-Chia; Wei, Leizhen; Nakajima, Satoshi; Chang, Shih-Shin; Liao, Hsin-Wei; Chen, Chung-Hsuan; Lavin, Martin; Ang, K Kian; Lin, Shiaw-Yih; Hung, Mien-Chie

2015-01-01

Ataxia telangiectasia mutated (ATM) mediates DNA damage response by controling irradiation-induced foci formation, cell cycle checkpoint, and apoptosis. However, how upstream signaling regulates ATM is not completely understood. Here, we show that upon irradiation stimulation, ATM associates with and is phosphorylated by epidermal growth factor receptor (EGFR) at Tyr370 (Y370) at the site of DNA double-strand breaks. Depletion of endogenous EGFR impairs ATM-mediated foci formation, homologous recombination, and DNA repair. Moreover, pretreatment with an EGFR kinase inhibitor, gefitinib, blocks EGFR and ATM association, hinders CHK2 activation and subsequent foci formation, and increases radiosensitivity. Thus, we reveal a critical mechanism by which EGFR directly regulates ATM activation in DNA damage response, and our results suggest that the status of ATM Y370 phosphorylation has the potential to serve as a biomarker to stratify patients for either radiotherapy alone or in combination with EGFR inhibition. PMID:25601159

ZRBA1, a Mixed EGFR/DNA Targeting Molecule, Potentiates Radiation Response Through Delayed DNA Damage Repair Process in a Triple Negative Breast Cancer Model

Energy Technology Data Exchange (ETDEWEB)

Heravi, Mitra [Department of Human Genetics, McGill University, Montreal (Canada); Department of Radiation Oncology, McGill University, Montreal (Canada); Segal Cancer Center, Jewish General Hospital, Montreal (Canada); Kumala, Slawomir [Department of Radiation Oncology, McGill University, Montreal (Canada); Segal Cancer Center, Jewish General Hospital, Montreal (Canada); Rachid, Zakaria; Jean-Claude, Bertrand J. [Cancer Drug Research Laboratory, McGill University Health Center, Montreal (Canada); Radzioch, Danuta [Department of Human Genetics, McGill University, Montreal (Canada); Muanza, Thierry M., E-mail: tmuanza@yahoo.com [Department of Radiation Oncology, McGill University, Montreal (Canada); Segal Cancer Center, Jewish General Hospital, Montreal (Canada)

2015-06-01

Purpose: ZRBA1 is a combi-molecule designed to induce DNA alkylating lesions and to block epidermal growth factor receptor (EGFR) TK domain. Inasmuch as ZRBA1 downregulates the EGFR TK-mediated antisurvival signaling and induces DNA damage, we postulated that it might be a radiosensitizer. The aim of this study was to further investigate the potentiating effect of ZRBA1 in combination with radiation and to elucidate the possible mechanisms of interaction between these 2 treatment modalities. Methods and Materials: The triple negative human breast MDA-MB-468 cancer cell line and mouse mammary cancer 4T1 cell line were used in this study. Clonogenic assay, Western blot analysis, and DNA damage analysis were performed at multiple time points after treatment. To confirm our in vitro findings, in vivo tumor growth delay assay was performed. Results: Our results show that a combination of ZRBA1 and radiation increases the radiation sensitivity of both cell lines significantly with a dose enhancement factor of 1.56, induces significant numbers of DNA strand breaks, prolongs higher DNA damage up to 24 hours after treatment, and significantly increases tumor growth delay in a syngeneic mouse model. Conclusions: Our data suggest that the higher efficacy of this combination could be partially due to increased DNA damage and delayed DNA repair process and to the inhibition of EGFR. The encouraging results of this combination demonstrated a significant improvement in treatment efficiency and therefore could be applicable in early clinical trial settings.

Increase in DNA vaccine efficacy by virosome delivery and co-expression of a cytolytic protein.

Science.gov (United States)

Gargett, Tessa; Grubor-Bauk, Branka; Miller, Darren; Garrod, Tamsin; Yu, Stanley; Wesselingh, Steve; Suhrbier, Andreas; Gowans, Eric J

2014-06-01

The potential of DNA vaccines has not been realised due to suboptimal delivery, poor antigen expression and the lack of localised inflammation, essential for antigen presentation and an effective immune response to the immunogen. Initially, we examined the delivery of a DNA vaccine encoding a model antigen, luciferase (LUC), to the respiratory tract of mice by encapsulation in a virosome. Virosomes that incorporated influenza virus haemagglutinin effectively delivered DNA to cells in the mouse respiratory tract and resulted in antigen expression and systemic and mucosal immune responses to the immunogen after an intranasal (IN) prime/intradermal (ID) boost regimen, whereas a multidose ID regimen only generated systemic immunity. We also examined systemic immune responses to LUC after ID vaccination with a DNA vaccine, which also encoded one of the several cytolytic or toxic proteins. Although the herpes simplex virus thymidine kinase, in the presence of the prodrug, ganciclovir, resulted in cell death, this failed to increase the humoral or cell-mediated immune responses. In contrast, the co-expression of LUC with the rotavirus non-structural protein 4 (NSP4) protein or a mutant form of mouse perforin, proteins which are directly cytolytic, resulted in increased LUC-specific humoral and cell-mediated immunity. On the other hand, co-expression of LUC with diphtheria toxin subunit A or overexpression of perforin or NSP4 resulted in a lower level of immunity. In summary, the efficacy of DNA vaccines can be improved by targeted IN delivery of DNA or by the induction of cell death in vaccine-targeted cells after ID delivery.

Frequency of distribution of leptin receptor gene polymorphism in obstructive sleep apnea patients.

Science.gov (United States)

Popko, K; Gorska, E; Wasik, M; Stoklosa, A; Pływaczewski, R; Winiarska, M; Gorecka, D; Sliwinski, P; Demkow, U

2007-11-01

Leptin is an adipocyte-derived hormone regulating energy homeostasis and body weight. Leptin concentration is increased in patients with the obstructive sleep apnea syndrome (OSAS). Leptin receptor (LEPR) is a single transmembrane protein belonging to the superfamily of cytokine receptors related by a structure to the hemopoietin receptor family. The aim of the present study was to evaluate the frequency of distribution of leptin receptor gene polymorphism GLN223ARG in OSAS patients compared with healthy controls. The examined group included 179 subjects: 102 OSAS patients (74 men and 28 women) and 77 non-apneic controls (39 men and 38 women). Genomic DNA was isolated with the use of a column method and genotyping of DNA sequence variation was carried out by restriction enzyme analysis of PCR-amplified DNA. The results revealed a significant correlation between the polymorphism of LEPR and OSAS. Carriers of Arg allele in homozygotic genotype Arg/Arg and heterozygotic genotype Gln/Arg were more often obese and developed OSAS than the group of carriers of homozygotic Gln/Gln genotype. This tendency was observed in the whole examined population and in the group of obese women. We also found the highest levels of total cholesterol, LDL, HDL, and triglycerides in the group of homozygotic Arg/Arg genotype carriers, lower in heterozygotic Gln/Arg genotype carriers, and the lowest in the group of persons carring homozygotic Gln/Gln genotype. The presence of Arg allel seems linked to a higher risk of obesity and higher lipid levels in OSAS patients. OSAS may have a strong genetic basis due to the effects from a variety of genes including those for leptin receptor.

Increased thermolability of benzodiazepine receptors in cerebral cortex of a baboon with spontaneous seizures: a case report.

Science.gov (United States)

Squires, R; Naquet, R; Riche, D; Braestrup, C

1979-06-01

The benzodiazepine receptor in the cortex of 1 spontaneously epileptic baboon exhibited an increased rate of thermal inactivation at 65 degrees C when compared with those from 3 other baboons. In other respects (receptor concentration, affinities for flunitrazepam and diazepam, and response to changing pH), the benzodiazepine receptor from this animal was very similar to the receptors in the cortex of 3 other baboons. The 3H-QNB (muscarinic) and 3H-naloxone (opiate) binding sites in the brain of all 4 baboons appeared very similar with respect to all parameters studied (thermal stability, concentration, regional distribution, and affinities for respective ligands). An endogenous factor stabilizing the benzodiazepine receptor could be lacking in the spontaneously epileptic baboon.

Impact of the Tamsulosin in Alpha Adrenergic Receptor of Airways at Patients with Increased Bronchial Reactibility.

Science.gov (United States)

Mustafa, Lirim; Ilazi, Ali; Dauti, Arta; Islami, Pellumb; Kastrati, Bashkim; Islami, Hilmi

2015-08-01

In this work, effect of tamsulosin as antagonist of alpha1A and alpha1B adrenergic receptor and effect of agonists of beta2 adrenergic receptor-salbutamol in patients with increased bronchial reactibility was studied. Parameters of the lung function are determined with Body plethysmography six (6) hours after administration of tamsulosin. Raw and ITGV were registered and specific resistance (SRaw) was calculated as well. Tamsulosin was administered in per os manner as a preparation in the shape of the capsules with a brand name of "Prolosin", produced by Niche Generics Limited, Hitchin, Herts. After six (6) hours of administration of tamsulosin, results gained indicate that blockage of alpha1A and alpha1B-adrenergic receptor (0.8 mg per os) has not changed significantly (p > 0.1) the bronchomotor tonus of tracheobronchial tree in comparison to the check-up that has inhaled salbutamol agonist of adrenergic beta2 receptor (2 inh. x 0.2 mg), (p tamsulosin. This suggests that even after six hours of administration of tamsulosin, and determining of lung function parameters, the activity of alpha1A and alpha1B-adrenergic receptor in the smooth bronchial musculature has not changed in patients with increased bronchial reactibility.

The O-β-linked N-acetylglucosaminylation of the Lamin B receptor and its impact on DNA binding and phosphorylation.

Science.gov (United States)

Smet-Nocca, Caroline; Page, Adeline; Cantrelle, François-Xavier; Nikolakaki, Eleni; Landrieu, Isabelle; Giannakouros, Thomas

2018-04-01

Lamin B Receptor (LBR) is an integral protein of the interphase inner nuclear membrane that is implicated in chromatin anchorage to the nuclear envelope. Phosphorylation of a stretch of arginine-serine (RS) dipeptides in the amino-terminal nucleoplasmic domain of LBR regulates the interactions of the receptor with other nuclear proteins, DNA and RNA and thus modulates tethering of heterochromatin to the nuclear envelope. While phosphorylation has been extensively studied, very little is known about other post-translational modifications of the protein. There is only one report on the O-β-linked N-acetyl-glucosaminylation (O-GlcNAcylation) of a serine residue downstream of the RS domain of rat LBR. In the present study we identify additional O-GlcNAcylation sites by using as substrates of O-β-N-acetylglucosaminyltransferase (OGT) a set of peptides containing the entire LBR RS domain or parts of it as well as flanking sequences. The in vitro activity of OGT was assessed by tandem mass spectrometry and NMR spectroscopy. Furthermore, we provide evidence that O-GlcNAcylation hampers DNA binding while it marginally affects RS domain phosphorylation mediated by SRPK1, Akt2 and cdk1 kinases. Our methodology providing a quantitative description of O-GlcNAc patterns based on a combination of mass spectrometry and high resolution NMR spectroscopy on short peptide substrates allows subsequent functional analyses. Hence, our approach is of general interest to a wide audience of biologists aiming at deciphering the functional role of O-GlcNAc glycosylation and its crosstalk with phosphorylation. Copyright © 2018 Elsevier B.V. All rights reserved.

Oxidative DNA damage and mammary cell proliferation by alcohol-derived salsolinol.

Science.gov (United States)

Murata, Mariko; Midorikawa, Kaoru; Kawanishi, Shosuke

2013-10-21

Drinking alcohol is a risk factor for breast cancer. Salsolinol (SAL) is endogenously formed by a condensation reaction of dopamine with acetaldehyde, a major ethanol metabolite, and SAL is detected in blood and urine after alcohol intake. We investigated the possibility that SAL can participate in tumor initiation and promotion by causing DNA damage and cell proliferation, leading to alcohol-associated mammary carcinogenesis. SAL caused oxidative DNA damage including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), in the presence of transition metal ions, such as Cu(II) and Fe(III)EDTA. Inhibitory effects of scavengers on SAL-induced DNA damage and the electron spin resonance study indicated the involvement of H₂O₂, which is generated via the SAL radical. Experiments on scavengers and site specificity of DNA damage suggested ·OH generation via a Fenton reaction and copper-peroxide complexes in the presence of Fe(III)EDTA and Cu(II), respectively. SAL significantly increased 8-oxodG formation in normal mammary epithelial MCF-10A cells. In addition, SAL induced cell proliferation in estrogen receptor (ER)-negative MCF-10A cells, and the proliferation was inhibited by an antioxidant N-acetylcysteine and an epidermal growth factor receptor (EGFR) inhibitor AG1478, suggesting that reactive oxygen species may participate in the proliferation of MCF-10A cells via EGFR activation. Furthermore, SAL induced proliferation in estrogen-sensitive breast cancer MCF-7 cells, and a surface plasmon resonance sensor revealed that SAL significantly increased the binding activity of ERα to the estrogen response element but not ERβ. In conclusion, SAL-induced DNA damage and cell proliferation may play a role in tumor initiation and promotion of multistage mammary carcinogenesis in relation to drinking alcohol.

Interaction of the phosphorylated DNA-binding domain in nuclear receptor CAR with its ligand-binding domain regulates CAR activation.

Science.gov (United States)

Shizu, Ryota; Min, Jungki; Sobhany, Mack; Pedersen, Lars C; Mutoh, Shingo; Negishi, Masahiko

2018-01-05

The nuclear protein constitutive active/androstane receptor (CAR or NR1I3) regulates several liver functions such as drug and energy metabolism and cell growth or death, which are often involved in the development of diseases such as diabetes and hepatocellular carcinoma. CAR undergoes a conversion from inactive homodimers to active heterodimers with retinoid X receptor α (RXRα), and phosphorylation of the DNA-binding domain (DBD) at Thr-38 in CAR regulates this conversion. Here, we uncovered the molecular mechanism by which this phosphorylation regulates the intramolecular interaction between CAR's DBD and ligand-binding domain (LBD), enabling the homodimer-heterodimer conversion. Phosphomimetic substitution of Thr-38 with Asp increased co-immunoprecipitation of the CAR DBD with CAR LBD in Huh-7 cells. Isothermal titration calorimetry assays also revealed that recombinant CAR DBD-T38D, but not nonphosphorylated CAR DBD, bound the CAR LBD peptide. This DBD-LBD interaction masked CAR's dimer interface, preventing CAR homodimer formation. Of note, EGF signaling weakened the interaction of CAR DBD T38D with CAR LBD, converting CAR to the homodimer form. The DBD-T38D-LBD interaction also prevented CAR from forming a heterodimer with RXRα. However, this interaction opened up a CAR surface, allowing interaction with protein phosphatase 2A. Thr-38 dephosphorylation then dissociated the DBD-LBD interaction, allowing CAR heterodimer formation with RXRα. We conclude that the intramolecular interaction of phosphorylated DBD with the LBD enables CAR to adapt a transient monomer configuration that can be converted to either the inactive homodimer or the active heterodimer.

Genetic transformation of watermelon with pumpkin DNA by low energy ion beam-mediated introduction

International Nuclear Information System (INIS)

Wang Haobo; Guo Jinhua; Huang Qunce; Yu Zengliang

2002-01-01

The No.601 watermelon (citrullus lanatus) seeds were treated with 25 keV N + implantation at the dosage of 7.8 x 10 16 ions/cm 2 . After treatment, watermelon seeds were incubated with 380 μg/μl pumpkin (Cucubita, maxima Duch) DNA solution at 35 degree C for 5 hours. By two-generations of selection and resistance screening at seedling stage, one transformed material was selected out, whose rind color is similar to that of the donor pumpkin and whose size of seeds is between that of the donor and the receptor. Using AFLP (amplified fragment length polymorphism) technique, two polymorphic DNA fragments were amplified. This primarily testified that the donor DNA fragments/gene were introduced into the receptor cell and integrated into the genomic DNA of the receptor

Genetic Transformation of Watermelon with Pumpkin DNA by Low Energy Ion Beam-Mediated Introduction

Science.gov (United States)

Wang, Hao-bo; Gao, Xiu-wu; Guo, Jin-hua; Huang, Qun-ce; Yu, Zeng-liang

2002-12-01

The No.601 watermelon (citrullus lanatus) seeds were treated with 25 keV N+ implantation at the dosage of 7.8 × 1016 ions/cm2. After treatment, watermelon seeds were incubated with 380 μg/μl pumpkin (Cucubita, maxima Duch) DNA solution at 35 °C for 5 hours. By two-generations of selection and resistance screening at seedling stage, one transformed material was selected out, whose rind color is similar to that of the donor pumpkin and whose size of seeds is between that of the donor and the receptor. Using AFLP (amplified fragment length polymorphism) technique, two polymorphic DNA fragments were amplified. This primarily testified that the donor DNA fragments/gene were introduced into the receptor cell and integrated into the genomic DNA of the receptor.

Transcriptional targets shared by estrogen receptor- related receptors (ERRs) and estrogen receptor (ER) alpha, but not by ERbeta.

Science.gov (United States)

Vanacker, J M; Pettersson, K; Gustafsson, J A; Laudet, V

1999-01-01

The physiological activities of estrogens are thought to be mediated by specific nuclear receptors, ERalpha and ERbeta. However, certain tissues, such as the bone, that are highly responsive to estrogens only express a low level of these receptors. Starting from this apparent contradiction, we have evaluated the potentials of two related receptors ERRalpha and ERRbeta to intervene in estrogen signaling. ERalpha, ERRalpha and ERRbeta bind to and activate transcription through both the classical estrogen response element (ERE) and the SF-1 response element (SFRE). In contrast, ERbeta DNA-binding and transcriptional activity is restricted to the ERE. Accordingly, the osteopontin gene promoter is stimulated through SFRE sequences, by ERRalpha as well as by ERalpha, but not by ERbeta. Analysis of the cross-talk within the ER/ERR subgroup of nuclear receptors thus revealed common targets but also functional differences between the two ERs. PMID:10428965

Effect of the caffeine on treated and non-treated plasmid DNA with stannic chloride; Efeito da cafeina em DNA plasmidial tratado e nao tratado com cloreto estanoso

Energy Technology Data Exchange (ETDEWEB)

Moreno, Silvana Ramos F. [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Inst. de Biologia. Dept. de Biofisica e Biometria]|[Universidade Federal Fluminense, Niteroi, RJ (Brazil). Faculdade de Ciencias Medicas. Curso de Pos-graduacao em Patologia Experimental; Mattos, Jose C.P. de; Dantas, Flavio; Araujo, Adriano Caldeira de; Bernardo-Filho, Mario [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Inst. de Biologia. Dept. de Biofisica e Biometria]. E-mail: bernardo@uerj.br

2000-07-01

Caffeine, a methilxantine drug is a component of coffee, tea, stimulants and other drinks. Caffeine inhibits phosphodiesterase leading to intracellular accumulation of cyclic AMP, blocks adenosine receptors, and increases the release of Ca{sup 2+}. We have studied the possible effect of caffeine in DNA plasmid treated or not with stannous chloride (SnCl{sub 2}). Previous evaluations of the effect of caffeine on the labeling of red blood cells and plasma proteins with technetium-99m have showed a decrease of % ATI in the insoluble fraction of plasma proteins. Samples of DNA were treated with SnCl{sub 2} (0 and 200{mu}g/ml) in 0.8% agarose. SnCl{sub 2} has induced break on DNA and caffeine has not showed effect on the DNA. This indicates that caffeine does not eliminate the oxidant action of SnCl{sub 2} and does not promote break in isolated DNA plasmid. (author)

Increased transient receptor potential canonical type 3 channels in vasculature from hypertensive rats

DEFF Research Database (Denmark)

Liu, Daoyan; Yang, Dachun; He, Hongbo

2009-01-01

We tested the hypothesis that transient receptor potential canonical type 3 (TRPC3) channels are increased in vascular smooth muscle cells and aortic tissue from spontaneously hypertensive rats (SHR) compared with normotensive Wistar Kyoto rats. Expression of TRPC3 was analyzed by immunohistochem...

Sensing of Blood Pressure Increase by Transient Receptor Potential Vanilloid 1 Receptors on Baroreceptors

OpenAIRE

Sun, Hao; Li, De-Pei; Chen, Shao-Rui; Hittelman, Walter N.; Pan, Hui-Lin

2009-01-01

The arterial baroreceptor is critically involved in the autonomic regulation of homoeostasis. The transient receptor potential vanilloid 1 (TRPV1) receptor is expressed on both somatic and visceral sensory neurons. Here, we examined the TRPV1 innervation of baroreceptive pathways and its functional significance in the baroreflex. Resiniferatoxin (RTX), an ultrapotent analog of capsaicin, was used to ablate TRPV1-expressing afferent neurons and fibers in adult rats. Immunofluorescence labeling...

Classification of viral zoonosis through receptor pattern analysis.

Science.gov (United States)

Bae, Se-Eun; Son, Hyeon Seok

2011-04-13

Viral zoonosis, the transmission of a virus from its primary vertebrate reservoir species to humans, requires ubiquitous cellular proteins known as receptor proteins. Zoonosis can occur not only through direct transmission from vertebrates to humans, but also through intermediate reservoirs or other environmental factors. Viruses can be categorized according to genotype (ssDNA, dsDNA, ssRNA and dsRNA viruses). Among them, the RNA viruses exhibit particularly high mutation rates and are especially problematic for this reason. Most zoonotic viruses are RNA viruses that change their envelope proteins to facilitate binding to various receptors of host species. In this study, we sought to predict zoonotic propensity through the analysis of receptor characteristics. We hypothesized that the major barrier to interspecies virus transmission is that receptor sequences vary among species--in other words, that the specific amino acid sequence of the receptor determines the ability of the viral envelope protein to attach to the cell. We analysed host-cell receptor sequences for their hydrophobicity/hydrophilicity characteristics. We then analysed these properties for similarities among receptors of different species and used a statistical discriminant analysis to predict the likelihood of transmission among species. This study is an attempt to predict zoonosis through simple computational analysis of receptor sequence differences. Our method may be useful in predicting the zoonotic potential of newly discovered viral strains.

Initiation of lambda DNA replication. The Escherichia coli small heat shock proteins, DnaJ and GrpE, increase DnaK's affinity for the lambda P protein.

Science.gov (United States)

Osipiuk, J; Georgopoulos, C; Zylicz, M

1993-03-05

It is known that the initiation of bacteriophage lambda replication requires the orderly assembly of the lambda O.lambda P.DnaB helicase protein preprimosomal complex at the ori lambda DNA site. The DnaK, DnaJ, and GrpE heat shock proteins act together to destabilize the lambda P.DnaB complex, thus freeing DnaB and allowing it to unwind lambda DNA near the ori lambda site. The first step of this disassembly reaction is the binding of DnaK to the lambda P protein. In this report, we examined the influence of the DnaJ and GrpE proteins on the stability of the lambda P.DnaK complex. We present evidence for the existence of the following protein-protein complexes: lambda P.DnaK, lambda P.DnaJ, DnaJ.DnaK, DnaK.GrpE, and lambda P.DnaK.GrpE. Our results suggest that the presence of GrpE alone destabilizes the lambda P.DnaK complex, whereas the presence of DnaJ alone stabilizes the lambda P.DnaK complex. Using immunoprecipitation, we show that in the presence of GrpE, DnaK exhibits a higher affinity for the lambda P.DnaJ complex than it does alone. Using cross-linking with glutaraldehyde, we show that oligomeric forms of DnaK exhibit a higher affinity for lambda P than monomeric DnaK. However, in the presence of GrpE, monomeric DnaK can efficiently bind lambda P protein. These findings help explain our previous results, namely that in the GrpE-dependent lambda DNA replication system, the DnaK protein requirement can be reduced up to 10-fold.

TLR9 and NF-κB are partially involved in activation of human neutrophils by Helicobacter pylori and its purified DNA.

Directory of Open Access Journals (Sweden)

Lourdes Alvarez-Arellano

Full Text Available Helicobacter pylori infection represents one of the most common bacterial infections worldwide. The inflammatory response to this bacterium involves a large influx of neutrophils to the lamina propria of the gastric mucosa. However, little is known about the receptors and molecular mechanisms involved in activation of these neutrophils. In this study, we aimed to determine the role of toll-like receptor 9 (TLR9 in the response of human neutrophils to H. pylori and purified H. pylori DNA (Hp-DNA. Neutrophils were isolated from the blood of adult volunteers and challenged with either H. pylori or Hp-DNA. We found that both, H. pylori and Hp-DNA induced increased expression and release of IL-8. Furthermore, we showed that TLR9 is involved in the induction of IL-8 production by H. pylori and Hp-DNA. IL-8 production induced by H. pylori but not by Hp-DNA was partially mediated by NF-κB. In conclusion, this study showed for first time that both, H. pylori and Hp-DNA activate TLR9 and induce a different inflammatory response that leads to activation of neutrophils.

Lung function discordance in monozygotic twins and associated differences in blood DNA methylation

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Bolund, Anneli C S; Starnawska, Anna; Miller, Martin R

2017-01-01

Background: Lung function is an important predictor of morbidity and mortality, with accelerated lung function decline reported to have immense consequences for the world's healthcare systems. The lung function decline across individual's lifetime is a consequence of age-related changes in lung...... as TGF-β-receptor-related genes, may be involved in the cross-sectional level and longitudinal change in lung function in middle-aged monozygotic twins....... and genetic factors. DNA methylation plays a crucial role in regulation of gene expression, with increasing evidence linking aberrant DNA methylation levels with a number of common human diseases. In this study, we investigated possible associations between genome-wide DNA methylation levels and lung function...

PPARGC1A DNA methylation in subcutaneous adipose tissue in low birth weight subjects

DEFF Research Database (Denmark)

Gillberg, Linn; Jacobsen, Stine; Rönn, Tina

2014-01-01

OBJECTIVE: Increased DNA methylation of the metabolic regulator peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) has been reported in skeletal muscle from type 2 diabetes (T2D) subjects and from low birth weight (LBW) subjects with an increased risk of T2D. High...... and insulin-stimulated SAT from LBW and matched normal birth weight (NBW) subjects during control and high-fat overfeeding. MATERIALS/METHODS: Nineteen young healthy men with LBW and 26 NBW controls were studied after both a 5-day high-fat overfeeding and a control diet in a randomized crossover setting. DNA...... methylation was assessed with bisulfite sequencing and mRNA expression with quantitative real-time PCR. RESULTS: Following high-fat overfeeding, increased SAT PPARGC1A DNA methylation was observed in LBW subjects but not in NBW controls. Basal SAT PPARGC1A mRNA expression was unaffected by diet and similar...

Melanocortin 1 receptor genotype: an important determinant of the damage response of melanocytes to ultraviolet radiation

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Kadekaro, Ana Luisa; Leachman, Sancy; Kavanagh, Renny J.; Swope, Viki; Cassidy, Pamela; Supp, Dorothy; Sartor, Maureen; Schwemberger, Sandy; Babcock, George; Wakamatsu, Kazumasa; Ito, Shosuke; Koshoffer, Amy; Boissy, Raymond E.; Manga, Prashiela; Sturm, Richard A.; Abdel-Malek, Zalfa A.

2010-01-01

The melanocortin 1 receptor gene is a main determinant of human pigmentation, and a melanoma susceptibility gene, because its variants that are strongly associated with red hair color increase melanoma risk. To test experimentally the association between melanocortin 1 receptor genotype and melanoma susceptibility, we compared the responses of primary human melanocyte cultures naturally expressing different melanocortin 1 receptor variants to α-melanocortin and ultraviolet radiation. We found that expression of 2 red hair variants abolished the response to α-melanocortin and its photoprotective effects, evidenced by lack of functional coupling of the receptor, and absence of reduction in ultraviolet radiation-induced hydrogen peroxide generation or enhancement of repair of DNA photoproducts, respectively. These variants had different heterozygous effects on receptor function. Microarray data confirmed the observed differences in responses of melanocytes with functional vs. nonfunctional receptor to α-melanocortin and ultraviolet radiation, and identified DNA repair and antioxidant genes that are modulated by α-melanocortin. Our findings highlight the molecular mechanisms by which the melanocortin 1 receptor genotype controls genomic stability of and the mutagenic effect of ultraviolet radiation on human melanocytes.—Kadekaro, A. L., Leachman, S., Kavanagh, R. J., Swope, V., Cassidy, P., Supp, D., Sartor, M., Schwemberger, S., Babcock, G., Wakamatsu, K., Ito, S., Koshoffer, A., Boissy, R. E., Manga, P., Sturm, R. A., Abdel-Malek, Z. A. Melanocortin 1 receptor genotype: an important determinant of the damage response of melanocytes to ultraviolet radiation. PMID:20519635

Receptor-Mediated Entry of Pristine Octahedral DNA Nanocages in Mammalian Cells

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Vindigni, Giulia; Raniolo, Sofia; Ottaviani, Alessio

2016-01-01

, more recently, identified as a tumor marker. For this purpose a truncated octahedral DNA nanocage functionalized with a single biotin molecule, which allows DNA cage detection through the biotin–streptavidin assays, was constructed. The results indicate that DNA nanocages are stable in biological...

Analysis of the hormone-binding domain of steroid receptors using chimeras generated by homologous recombination

International Nuclear Information System (INIS)

Martinez, Elisabeth D.; Pattabiraman, Nagarajan; Danielsen, Mark

2005-01-01

The glucocorticoid receptor and the mineralocorticoid receptor are members of the steroid receptor family that exhibit ligand cross-reactivity. Specificity of steroid receptor action is investigated in the present work by the construction and characterization of chimeras between the glucocorticoid receptor and the mineralocorticoid receptor. We used an innovative approach to make novel steroid receptor proteins in vivo that in general, contrary to our expectations, show increased ligand specificity compared to the parental receptors. We describe a receptor that is specific for the potent synthetic glucocorticoid triamcinolone acetonide and does not bind aldosterone. A further set of chimeras has an increased ability to discriminate between ligands, responding potently to mineralocorticoids and only very weakly to synthetic glucocorticoids. A chimera with the fusion site in the hinge highlights the importance of the region between the DNA-binding and the hormone-binding domains since, unlike both the glucocorticoid and mineralocorticoid receptors, it only responds to mineralocorticoids. One chimera has reduced specificity in that it acts as a general corticoid receptor, responding to glucocorticoids and mineralocorticoids with similar potency and efficacy. Our data suggest that regions of the glucocorticoid and mineralocorticoid receptor hormone-binding domains are functionally non-reciprocal. We present transcriptional, hormone-binding, and structure-modeling evidence that suggests that receptor-specific interactions within and across domains mediate aspects of specificity in transcriptional responses to steroids

Superior induction of T cell responses to conserved HIV-1 regions by electroporated alphavirus replicon DNA compared to that with conventional plasmid DNA vaccine.

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Knudsen, Maria L; Mbewe-Mvula, Alice; Rosario, Maximillian; Johansson, Daniel X; Kakoulidou, Maria; Bridgeman, Anne; Reyes-Sandoval, Arturo; Nicosia, Alfredo; Ljungberg, Karl; Hanke, Tomás; Liljeström, Peter

2012-04-01

Vaccination using "naked" DNA is a highly attractive strategy for induction of pathogen-specific immune responses; however, it has been only weakly immunogenic in humans. Previously, we constructed DNA-launched Semliki Forest virus replicons (DREP), which stimulate pattern recognition receptors and induce augmented immune responses. Also, in vivo electroporation was shown to enhance immune responses induced by conventional DNA vaccines. Here, we combine these two approaches and show that in vivo electroporation increases CD8(+) T cell responses induced by DREP and consequently decreases the DNA dose required to induce a response. The vaccines used in this study encode the multiclade HIV-1 T cell immunogen HIVconsv, which is currently being evaluated in clinical trials. Using intradermal delivery followed by electroporation, the DREP.HIVconsv DNA dose could be reduced to as low as 3.2 ng to elicit frequencies of HIV-1-specific CD8(+) T cells comparable to those induced by 1 μg of a conventional pTH.HIVconsv DNA vaccine, representing a 625-fold molar reduction in dose. Responses induced by both DREP.HIVconsv and pTH.HIVconsv were further increased by heterologous vaccine boosts employing modified vaccinia virus Ankara MVA.HIVconsv and attenuated chimpanzee adenovirus ChAdV63.HIVconsv. Using the same HIVconsv vaccines, the mouse observations were supported by an at least 20-fold-lower dose of DNA vaccine in rhesus macaques. These data point toward a strategy for overcoming the low immunogenicity of DNA vaccines in humans and strongly support further development of the DREP vaccine platform for clinical evaluation.

Generation of Gene-Engineered Chimeric DNA Molecules for Specific Therapy of Autoimmune Diseases

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Gesheva, Vera; Szekeres, Zsuzsanna; Mihaylova, Nikolina; Dimitrova, Iliyana; Nikolova, Maria; Erdei, Anna; Prechl, Jozsef

2012-01-01

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the development of self-reactive B and T cells and autoantibody production. In particular, double-stranded DNA-specific B cells play an important role in lupus progression, and their selective elimination is a reasonable approach for effective therapy of SLE. DNA-based vaccines aim at the induction of immune response against the vector-encoded antigen. Here, we are exploring, as a new DNA-based therapy of SLE, a chimeric DNA molecule encoding a DNA-mimotope peptide, and the Fv but not the immunogenic Fc fragment of an FcγRIIb-specific monoclonal antibody. This DNA construct was inserted in the expression vector pNut and used as a naked DNA vaccine in a mouse model of lupus. The chimeric DNA molecule can be expressed in eukaryotic cells and cross-links cell surface receptors on DNA-specific B cells, delivering an inhibitory intracellular signal. Intramuscular administration of the recombinant DNA molecule to lupus-prone MRL/lpr mice prevented increase in IgG anti-DNA antibodies and was associated with a low degree of proteinuria, modulation of cytokine profile, and suppression of lupus nephritis. PMID:23075110

Insulin receptor substrates 1 and 2 but not Shc can activate the insulin receptor independent of insulin and induce proliferation in CHO-IR cells

International Nuclear Information System (INIS)

Niessen, Markus; Jaschinski, Frank; Item, Flurin; McNamara, Morgan P.; Spinas, Giatgen A.; Trueb, Thomas

2007-01-01

Ligand-activated insulin receptor (IR) attracts and phosphorylates various substrates such as insulin receptor substrates 1-4 (IRS) and Shc. To investigate how binding affinity for substrate affects signalling we generated chimeric receptors with the β-chain of the insulin receptor containing NPXY motives with different affinities for receptor substrates. We found that the extent of receptor tyrosine phosphorylation positively correlates with binding affinity towards IRS1/2 but not towards Shc. Moreover, overexpression of IRS1 or IRS2 but not of Shc increased IR tyrosine phosphorylation in a dose-dependent manner, also independent of insulin. Molecular truncations of IRS1 revealed that neither the isolated PH and PTB domains nor the C-terminus with the tyrosine phosphorylation sites alone are sufficient for substrate-dependent receptor activation. Overexpression of IRS1 and IRS2 impaired insulin-induced internalization of the IR in a dose-dependent manner suggesting that IRS proteins prevent endosome-associated receptor dephosphorylation/inactivation. IRS1 and IRS2 could therefore target the activated IR to different cellular compartments. Overexpression of IRS1 and IRS2 inhibited insulin-stimulated activation of the MAP kinases Erk1/2 while it increased/induced activation of Akt/PKB. Finally, overexpression of IRS1 and IRS2 but not of Shc induced DNA synthesis in starved CHO-IR cells independent of exogenous growth factors. Our results demonstrate that variations in cellular IRS1 and IRS2 concentration affect insulin signalling both upstream and downstream and that IRS proteins could play instructive rather than just permissive roles in signal transmission

Synaptic glutamate spillover increases NMDA receptor reliability at the cerebellar glomerulus

OpenAIRE

Mitchell, Cassie S.; Lee, Robert H.

2011-01-01

Glutamate spillover in the mossy fiber to granule cell cerebellar glomeruli has been hypothesized to increase neurotransmission reliability. In this study, we evaluate this hypothesis using an experimentally-based quantitative model of glutamate spillover on the N-methyl-d-aspartate receptors (NMDA-Rs) at the cerebellar glomerulus. The transient and steady-state responses of NMDA-Rs were examined over a physiological range of firing rates. Examined cases included direct glutamate release acti...

Ethanol-induced increase in portal blood flow: Role of acetate and A1- and A2-adenosine receptors

International Nuclear Information System (INIS)

Carmichael, F.J.; Saldivia, V.; Varghese, G.A.; Israel, Y.; Orrego, H.

1988-01-01

The increase in portal blood flow induced by ethanol appears to be adenosine mediated. Acetate, which is released by the liver during ethanol metabolism, is known to increase adenosine levels in tissues and in blood. The effects of acetate on portal blood flow were investigated in rats using the microsphere technique. The intravenous infusion of acetate resulted in vasodilation of the preportal vasculature and in a dose-dependent increase in portal blood flow. This acetate-induced increase in portal blood flow was suppressed by the adenosine receptor blocker, 8-phenyltheophylline. Using the A 1 -adenosine receptor agonist N-6-cyclohexyl adenosine and the A 2 -agonist 5'-N-ethylcarboxamido adenosine, we demonstrate that the effect of adenosine on the preportal vasculature is mediated by the A 2 -subtype of adenosine receptors. In conclusion, these data support the hypothesis that the increase in portal blood flow after ethanol administration results from a preportal vasodilatory effect of adenosine formed from acetate metabolism in extrahepatic tissues

Increased natriuretic peptide receptor A and C gene expression in rats with pressure-overload cardiac hypertrophy

DEFF Research Database (Denmark)

Christoffersen, Tue E.H.; Aplin, Mark; Strom, Claes C.

2006-01-01

also affects cardiac hypertrophy and fibrosis. In this study we examined the expression of genes for the NPRs in rats with pressure-overload cardiac hypertrophy. The ANG II type 1 receptor was blocked with losartan (10 mg.kg(-1).day(-1)) to investigate a possible role of the renin-angiotensin system......RNAs for the natriuretic peptides or their receptors. Although increased gene expression does not necessarily convey a higher concentration of the protein, the data suggest that pressure overload is accompanied by upregulation of not only ANP and BNP but also their receptors NPR-A and NPR-C in the left ventricle....

Altered DNA Methylation Patterns Associated With Clinically Relevant Increases in PTSD Symptoms and PTSD Symptom Profiles in Military Personnel.

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Martin, Christiana; Cho, Young-Eun; Kim, Hyungsuk; Yun, Sijung; Kanefsky, Rebekah; Lee, Hyunhwa; Mysliwiec, Vincent; Cashion, Ann; Gill, Jessica

2018-05-01

Military personnel experience posttraumatic stress disorder (PTSD), which is associated with differential DNA methylation across the whole genome. However, the relationship between these DNA methylation patterns and clinically relevant increases in PTSD severity is not yet clearly understood. The purpose of this study was to identify differences in DNA methylation associated with PTSD symptoms and investigate DNA methylation changes related to increases in the severity of PTSD in military personnel. In this pilot study, a cross-sectional comparison was made between military personnel with PTSD (n = 8) and combat-matched controls without PTSD (n = 6). Symptom measures were obtained, and genome-wide DNA methylation was measured using methylated DNA immunoprecipitation (MeDIP-seq) from whole blood samples at baseline and 3 months later. A longitudinal comparison measured DNA methylation changes in military personnel with clinically relevant increases in PTSD symptoms between time points (PTSD onset) and compared methylation patterns to controls with no clinical changes in PTSD. In military personnel with elevated PTSD symptoms 3 months following baseline, 119 genes exhibited reduced methylation and 8 genes exhibited increased methylation. Genes with reduced methylation in the PTSD-onset group relate to the canonical pathways of netrin signaling, Wnt/Ca + pathway, and axonal guidance signaling. These gene pathways relate to neurological disorders, and the current findings suggest that these epigenetic changes potentially relate to PTSD symptomology. This study provides some novel insights into the role of epigenetic changes in PTSD symptoms and the progression of PTSD symptoms in military personnel.

Thioredoxin and thioredoxin reductase influence estrogen receptor α-mediated gene expression in human breast cancer cells

OpenAIRE

Rao, Abhi K; Ziegler, Yvonne S; McLeod, Ian X; Yates, John R; Nardulli, Ann M

2009-01-01

Accumulation of reactive oxygen species (ROS) in cells damages resident proteins, lipids, and DNA. In order to overcome the oxidative stress that occurs with ROS accumulation, cells must balance free radical production with an increase in the level of antioxidant enzymes that convert free radicals to less harmful species. We identified two antioxidant enzymes, thioredoxin (Trx) and Trx reductase (TrxR), in a complex associated with the DNA-bound estrogen receptor α (ERα). Western analysis and...

Mung bean nuclease treatment increases capture specificity of microdroplet-PCR based targeted DNA enrichment.

Directory of Open Access Journals (Sweden)

Zhenming Yu

Full Text Available Targeted DNA enrichment coupled with next generation sequencing has been increasingly used for interrogation of select sub-genomic regions at high depth of coverage in a cost effective manner. Specificity measured by on-target efficiency is a key performance metric for target enrichment. Non-specific capture leads to off-target reads, resulting in waste of sequencing throughput on irrelevant regions. Microdroplet-PCR allows simultaneous amplification of up to thousands of regions in the genome and is among the most commonly used strategies for target enrichment. Here we show that carryover of single-stranded template genomic DNA from microdroplet-PCR constitutes a major contributing factor for off-target reads in the resultant libraries. Moreover, treatment of microdroplet-PCR enrichment products with a nuclease specific to single-stranded DNA alleviates off-target load and improves enrichment specificity. We propose that nuclease treatment of enrichment products should be incorporated in the workflow of targeted sequencing using microdroplet-PCR for target capture. These findings may have a broad impact on other PCR based applications for which removal of template DNA is beneficial.

Increased DNA damage in blood cells of rat treated with lead as assessed by comet assay

Directory of Open Access Journals (Sweden)

Mohammad Arif

2008-06-01

Full Text Available A growing body of evidence suggests that oxidative stress is the key player in the pathogenesis of lead-induced toxicity. The present study investigated lead induced oxidative DNA damage, if any in rat blood cells by alkaline comet assay. Lead was administered intraperitoneally to rats at doses of 25, 50 and 100 mg/kg body weight for 5 days consecutively. Blood collected on day six from sacrificed lead-treated rats was used to assess the extent of DNA damage by comet assay which entailed measurement of comet length, olive tail moment, tail DNA (% and tail length. The results showed that treatment with lead significantly increased DNA damage in a dose-dependent manner. Therefore, our data suggests that lead treatment is associated with oxidative stress-induced DNA damage in rat blood cells which could be used as an early bio-marker of lead-toxicity.

Increased levels of unscheduled DNA synthesis in UV-irradiated human fibroblasts pretreated with sodium butyrate

International Nuclear Information System (INIS)

Williams, J.I.; Friedberg, E.C.

1982-01-01

Pretreatment of growing normal and xeroderma pigmentosum (XP) human fibroblasts with sodium butyrate at concentrations of 5-20 mM results in increased levels of DNA repair synthesis measured by autoradiography after exposure of the cells to 254 nm UV radiation in the fluence range 0-25 J/m 2 . The phenomenon manifests as an increased extent and an increased initial rate of unscheduled DNA synthesis (UDS). This experimental result is not due to an artifact of autoradiography related to cell size. Xeroderma pigmentosum cells from complementation groups A, C, D and E and XP variant cells all exhibit increases in the levels of UV-induced UDS in response to sodium butyrate proportional to those observed with normal cells. These UDS increases associated with butyrate pretreatment correlate with demonstrable changes in intracellular thymidine pool size and suggest that sodium butyrate enhances uptake of exogenous radiolabeled thymidine during UV-induced repair synthesis by reducing endogenous levels of thymidine. (author)

Ribosomal DNA transcription in the dorsal raphe nucleus is increased in residual but not in paranoid schizophrenia.

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Krzyżanowska, Marta; Steiner, Johann; Brisch, Ralf; Mawrin, Christian; Busse, Stefan; Braun, Katharina; Jankowski, Zbigniew; Bernstein, Hans-Gert; Bogerts, Bernhard; Gos, Tomasz

2015-03-01

The central serotonergic system is implicated in the pathogenesis of schizophrenia, where the imbalance between dopamine, serotonin and glutamate plays a key pathophysiological role. The dorsal raphe nucleus (DRN) is the main source of serotonergic innervation of forebrain limbic structures disturbed in schizophrenia patients. The study was carried out on paraffin-embedded brains from 17 (8 paranoid and 9 residual) schizophrenia patients and 28 matched controls without mental disorders. The transcriptional activity of ribosomal DNA (rDNA) in DRN neurons was evaluated by the AgNOR silver-staining method. An increased rDNA transcriptional activity was found in schizophrenia patients in the cumulative analysis of all DRN subnuclei (t test, P = 0.02). Further subgroup analysis revealed that it was an effect specific for residual schizophrenia versus paranoid schizophrenia or control groups (ANOVA, P = 0.002). This effect was confounded neither by suicide nor by antipsychotic medication. Our findings suggest that increased activity of rDNA in DRN neurons is a distinct phenomenon in schizophrenia, particularly in residual patients. An activation of the rDNA transcription in DRN neurons may represent a compensatory mechanism to overcome the previously described prefrontal serotonergic hypofunction in this diagnostic subgroup.

Stimulation of LDL receptor activity in Hep-G2 cells by a serum factor(s)

International Nuclear Information System (INIS)

Ellsworth, J.L.; Brown, C.; Cooper, A.D.

1988-01-01

The regulation of low-density lipoprotein (LDL) receptor activity in the human hepatoma cell line Hep-G2 by serum components was examined. Incubation of dense monolayers of Hep-G2 cells with fresh medium containing 10% fetal calf serum (FM) produced a time-dependent increase in LDL receptor activity. Uptake and degradation of 125I-LDL was stimulated two- to four-fold, as compared with that of Hep-G2 cells cultured in the same media in which they had been grown to confluence (CM); the maximal 125I-LDL uptake plus degradation increased from 0.2 microgram/mg cell protein/4 h to 0.8 microgram/mg cell protein/4 h. In addition, a two-fold increase in cell surface binding of 125I-LDL to Hep-G2 cells was observed when binding was measured at 4 degrees C. There was no change in the apparent Kd. The stimulation of LDL receptor activity was suppressed in a concentration-dependent manner by the addition of cholesterol, as LDL, to the cell medium. In contrast to the stimulation of LDL receptor activity, FM did not affect the uptake or degradation of 125I-asialoorosomucoid. Addition of FM increased the protein content per dish, and DNA synthesis was stimulated approximately five-fold, as measured by [3H]thymidine incorporation into DNA; however, the cell number did not change. Cellular cholesterol biosynthesis was also stimulated by FM; [14C]acetate incorporation into unesterified and esterified cholesterol was increased approximately five-fold. Incubation of Hep-G2 cells with high-density lipoproteins (200 micrograms protein/ml) or albumin (8.0 mg/ml) in the absence of the serum factor did not significantly increase the total processed 125I-LDL. Stimulation of LDL receptor activity was dependent on a heat-stable, nondialyzable serum component that eluted in the inclusion volume of a Sephadex G-75 column

Cloning and expression of a rat brain α2B-adrenergic receptor

International Nuclear Information System (INIS)

Flordellis, C.S.; Handy, D.E.; Bresnahan, M.R.; Zannis, V.I.; Gavras, H.

1991-01-01

The authors isolated a cDNA clone (RBα 2B ) and its homologous gene (GRα 2B ) encoding an α 2B -adrenergic receptor subtype by screening a rat brain cDNA and a rat genomic library. Nucleotide sequence analysis showed that both clones code for a protein of 458 amino acids, which is 87% homologous to the human kidney glycosylated adrenergic receptor (α 2 -C4) and divergent from the rat kidney nonglycosylated α 2B subtype (RNGα 2 ). Transient expression of RBα 2B in COS-7 cells resulted in high-affinity saturable binding for [ 3 H]rauwolscine and a high receptor number in the membranes of transfected COS-7 cells. Pharmacological analysis demonstrated that the expressed receptor bound adrenergic ligands with the following order of potency: rauwolscine > yohimbine > prazosin > oxymetazoline, with a prazosin-to-oxymetazoline K i ratio of 0.34. This profile is characteristic of the α 2B -adrenergic receptor subtype. Blotting analysis of rat brain mRNA gave one major and two minor mRNA species, and hybridization with strand-specific probes showed that both DNA strands of GRα 2B may be transcriptionally active. These findings show that rat brain expresses an α 2B -adrenergic receptor subtype that is structurally different from the rat kidney nonglycosylated α 2B subtype. Thus the rat expresses at least two divergent α 2B -adrenergic receptors

Activation of GLP-1 receptors on vascular smooth muscle cells reduces the autoregulatory response in afferent arterioles and increases renal blood flow

DEFF Research Database (Denmark)

Jensen, Elisa Pouline; Poulsen, Steen Seier; Kissow, Hannelouise

2015-01-01

was to localize renal GLP-1 receptors and describe GLP-1 mediated effects on the renal vasculature. We hypothesized that renal GLP-1 receptors are located in the renal microcirculation and activation of these affects renal autoregulation and increases renal blood flow. In vivo autoradiography using 125I-GLP-1......, 125I-exendin-4 (GLP-1 analog) and 125I-exendin 9-39 (GLP-1 receptor antagonist) was performed in rodents to localize specific GLP-1 receptor binding. GLP-1 mediated effects on blood pressure (BP), renal blood flow (RBF), heart rate (HR), renin secretion, urinary flow rate and Na+ and K+ excretion were...... conclude that GLP-1 receptors are located in the renal vasculature including afferent arterioles. Activation of these receptors reduces the autoregulatory response of afferent arterioles to acute pressure increases and increases renal blood flow in normotensive rats....

Mitochondrial DNA as an inflammatory mediator in cardiovascular diseases.

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Nakayama, Hiroyuki; Otsu, Kinya

2018-03-06

Mitochondria play a central role in multiple cellular functions, including energy production, calcium homeostasis, and cell death. Currently, growing evidence indicates the vital roles of mitochondria in triggering and maintaining inflammation. Chronic inflammation without microbial infection - termed sterile inflammation - is strongly involved in the development of heart failure. Sterile inflammation is triggered by the activation of pattern recognition receptors (PRRs) that sense endogenous ligands called damage-associated molecular patterns (DAMPs). Mitochondria release multiple DAMPs including mitochondrial DNA, peptides, and lipids, which induce inflammation via the stimulation of multiple PRRs. Among the mitochondrial DAMPs, mitochondrial DNA (mtDNA) is currently highlighted as the DAMP that mediates the activation of multiple PRRs, including Toll-like receptor 9, Nod-like receptors, and cyclic GMP-AMP synthetase/stimulator of interferon gene pathways. These PRR signalling pathways, in turn, lead to the activation of nuclear factor-κB and interferon regulatory factor, which enhances the transcriptional activity of inflammatory cytokines and interferons, and induces the recruitment of inflammatory cells. As the heart is an organ comprising abundant mitochondria for its ATP consumption (needed to maintain constant cyclic contraction and relaxation), the generation of massive amounts of mitochondrial radical oxygen species and mitochondrial DAMPs are predicted to occur and promote cardiac inflammation. Here, we will focus on the role of mtDNA in cardiac inflammation and review the mechanism and pathological significance of mtDNA-induced inflammatory responses in cardiac diseases. © 2018 The Author(s).

Improved Yield of High Molecular Weight DNA Coincides with Increased Microbial Diversity Access from Iron Oxide Cemented Sub-Surface Clay Environments

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Hurt, Richard A.; Robeson, Michael S.; Shakya, Migun; Moberly, James G.; Vishnivetskaya, Tatiana A.; Gu, Baohua; Elias, Dwayne A.

2014-01-01

Despite over three decades of progress, extraction of high molecular weight (HMW) DNA from high clay soils or iron oxide cemented clay has remained challenging. HMW DNA is desirable for next generation sequencing as it yields the most comprehensive coverage. Several DNA extraction procedures were compared from samples that exhibit strong nucleic acid adsorption. pH manipulation or use of alternative ion solutions offered no improvement in nucleic acid recovery. Lysis by liquid N2 grinding in concentrated guanidine followed by concentrated sodium phosphate extraction supported HMW DNA recovery from clays high in iron oxides. DNA recovered using 1 M sodium phosphate buffer (PB) as a competitive desorptive wash was 15.22±2.33 µg DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25 µg DNA/g clay with the Powerlyzer system (MoBio). Increasing PB concentration in the lysis reagent coincided with increasing DNA fragment length during initial extraction. Rarefaction plots of 16S rRNA (V1–V3 region) pyrosequencing from A-horizon and clay soils showed an ∼80% and ∼400% larger accessed diversity compared to the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more operational taxonomic units (OTU) recovered. PMID:25033199

Estradiol increases the expression of TNF-α and TNF receptor 1 in lactotropes.

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Zaldivar, Verónica; Magri, María Laura; Zárate, Sandra; Jaita, Gabriela; Eijo, Guadalupe; Radl, Daniela; Ferraris, Jimena; Pisera, Daniel; Seilicovich, Adriana

2011-01-01

Estrogens are recognized modulators of pituitary cell renewal, sensitizing cells to mitogenic and apoptotic signals. Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine that plays an important role in tissue homeostasis modulating cell proliferation, differentiation and death. We previously demonstrated that TNF-α-induced apoptosis of anterior pituitary cells from female rats is estrogen-dependent and predominant in cells from rats at proestrus when estradiol levels are the highest. Considering that one of the mechanisms involved in the apoptotic action of estrogens can result from increased expression of cytokines and/or their receptors, the aim of the present study was to evaluate the effect of estrogens on the expression of TNF-α and its receptor, TNF receptor 1 (TNFR1), in anterior pituitary cells. TNFR1 expression, determined by Western blot, was higher in anterior pituitary glands from rats at proestrus than at diestrus. Incubation of anterior pituitary cells from ovariectomized rats with 17β-estradiol enhanced TNFR1 protein expression. As determined by double immunocytochemistry, the expression of TNF-α and TNFR1 was detected in prolactin-, GH-, LH- and ACTH-bearing cells. 17β-estradiol increased the percentage of TNF-α and TNFR1-immunoreactive lactotropes but did not modify the number of GH-bearing cells expressing TNF-α or TNFR1. Our results demonstrate that estradiol increases the expression of TNF-α and TNFR1 in anterior pituitary cells, especially in lactotropes. The sensitizing action of estrogens to proapoptotic stimuli at proestrus in the anterior pituitary gland may involve changes in the expression of the TNF-α/TNFR1 system. Copyright © 2011 S. Karger AG, Basel.

Dietary selenomethionine increases exon-specific DNA methylation of the p53 gene in rat liver and colon mucosa.

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Zeng, Huawei; Yan, Lin; Cheng, Wen-Hsing; Uthus, Eric O

2011-08-01

The regulation of site-specific DNA methylation of tumor suppressor genes has been considered as a leading mechanism by which certain nutrients exert their anticancer property. This study was to investigate whether selenium (Se) affects the methylation of globe genomic DNA and the exon-specific p53 gene. Three groups of rats (n = 6-7/group) were fed the AIN-93G basal diet supplemented with 0 [Se deficient (D)], 0.15 [Se adequate (A)], or 4 mg [Se supranutritional (S)] (Se as l-selenomethionine)/kg diet for 104 d, respectively. Rats fed the A or S diet had greater plasma and liver glutathione peroxidase activity, liver thioredoxin reductase activity, and plasma homocysteine concentration than those fed the D diet. However, compared with the A diet, rats fed the S diet did not further increase these Se-dependent enzyme activities or homocysteine concentration. In contrast, Se concentrations in kidney, liver, gastrocnemius muscle, and plasma were increased in a Se-dose-dependent manner. Interestingly, rats fed the S diet had significantly less global liver genomic DNA methylation than those fed the D diet. However, the S diet significantly increased the methylation of the p53 gene (exons 5-8) but not the β-actin gene (exons 2-3) DNA in liver and colon mucosa compared with those fed the D diet. Taken together, long-term Se consumption not only affects selenoprotein enzyme activities, homocysteine, tissue Se concentrations, and global genomic DNA methylation but also increases exon-specific DNA methylation of the p53 gene in a Se-dose-dependent manner in rat liver and colon mucosa.

Structural organization of the human glucocorticoid receptor determined by one- and two-dimensional gel electrophoresis of proteolytic receptor fragments

International Nuclear Information System (INIS)

Smith, A.C.; Harmon, J.M.

1987-01-01

The structural organization of the steroid-binding protein of the IM-9 cell glucocorticoid receptor was investigated by using one- and two-dimensional gel electrophoresis of proteolytic receptor fragments. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of receptor fragments isolated after trypsin digestion of immunopurified [ 3 H]dexamethasone 21-mesylate ([ 3 H]DM-) labeled receptor revealed the presence of a stable 26.5-kilodalton (kDa) steroid-containing non-DNA-binding fragment, derived from a larger, less stable, 29-kDa fragment. The 26.5-kDa tryptic fragment appeared to be completely contained within a 41-kDa, steroid-containing, DNA-binding species isolated after chymotrypsin digestion of the intact protein. Two-dimensional electrophoretic analysis of the [ 3 H]DM-labeled tryptic fragments resolved two 26.5-kDa and two 29-kDa components. This was the same number of isoforms seen in the intact protein, indicating that the charge heterogeneity of the steroid-binding protein is the result of modification within the steroid-containing, non-DNA-binding, 26.5-kDa tryptic fragment. Two-dimensional analysis of the 41-kDa [ 3 H]DM-labeled chymotryptic species revealed a pattern of isoforms more complex than that seen either in the intact protein or in the steroid-containing tryptic fragments. These results suggest that the 41-kDa [ 3 H]DM-labeled species resolved by one-dimensional SDS-PAGE after chymotrypsin digestion may be composed of several distinct proteolytic fragments

Opposing roles of Toll-like receptor and cytosolic DNA-STING signaling pathways for Staphylococcus aureus cutaneous host defense.

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Philip O Scumpia

2017-07-01

Full Text Available Successful host defense against pathogens requires innate immune recognition of the correct pathogen associated molecular patterns (PAMPs by pathogen recognition receptors (PRRs to trigger the appropriate gene program tailored to the pathogen. While many PRR pathways contribute to the innate immune response to specific pathogens, the relative importance of each pathway for the complete transcriptional program elicited has not been examined in detail. Herein, we used RNA-sequencing with wildtype and mutant macrophages to delineate the innate immune pathways contributing to the early transcriptional response to Staphylococcus aureus, a ubiquitous microorganism that can activate a wide variety of PRRs. Unexpectedly, two PRR pathways-the Toll-like receptor (TLR and Stimulator of Interferon Gene (STING pathways-were identified as dominant regulators of approximately 95% of the genes that were potently induced within the first four hours of macrophage infection with live S. aureus. TLR signaling predominantly activated a pro-inflammatory program while STING signaling activated an antiviral/type I interferon response with live but not killed S. aureus. This STING response was largely dependent on the cytosolic DNA sensor cyclic guanosine-adenosine synthase (cGAS. Using a cutaneous infection model, we found that the TLR and STING pathways played opposite roles in host defense to S. aureus. TLR signaling was required for host defense, with its absence reducing interleukin (IL-1β production and neutrophil recruitment, resulting in increased bacterial growth. In contrast, absence of STING signaling had the opposite effect, enhancing the ability to restrict the infection. These results provide novel insights into the complex interplay of innate immune signaling pathways triggered by S. aureus and uncover opposing roles of TLR and STING in cutaneous host defense to S. aureus.

Virus-encoded chemokine receptors--putative novel antiviral drug targets

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Rosenkilde, Mette M

2005-01-01

Large DNA viruses, in particular herpes- and poxviruses, have evolved proteins that serve as mimics or decoys for endogenous proteins in the host. The chemokines and their receptors serve key functions in both innate and adaptive immunity through control of leukocyte trafficking, and have...... receptors belong to the superfamily of G-protein coupled 7TM receptors that per se are excellent drug targets. At present, non-peptide antagonists have been developed against many chemokine receptors. The potentials of the virus-encoded chemokine receptors as drug targets--ie. as novel antiviral strategies...

PET-scan shows peripherally increased neurokinin 1 receptor availability in chronic tennis elbow: visualizing neurogenic inflammation?

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Magnus Peterson

Full Text Available In response to pain, neurokinin 1 (NK1 receptor availability is altered in the central nervous system. The NK1 receptor and its primary agonist, substance P, also play a crucial role in peripheral tissue in response to pain, as part of neurogenic inflammation. However, little is known about alterations in NK1 receptor availability in peripheral tissue in chronic pain conditions and very few studies have been performed on human beings. Ten subjects with chronic tennis elbow were therefore examined by positron emission tomography (PET with the NK1 specific radioligand [(11C]GR205171 before and after treatment with graded exercise. The radioligand signal intensity was higher in the affected arm as compared with the unaffected arm, measured as differences between the arms in volume of voxels and signal intensity of this volume above a reference threshold set as 2.5 SD above mean signal intensity of the unaffected arm before treatment. In the eight subjects examined after treatment, pain ratings decreased in all subjects but signal intensity decreased in five and increased in three. In conclusion, NK1 receptors may be activated, or up-regulated in the peripheral, painful tissue of a chronic pain condition. This up-regulation does, however, have moderate correlation to pain ratings. The increased NK1 receptor availability is interpreted as part of ongoing neurogenic inflammation and may have correlation to the pathogenesis of chronic tennis elbow.ClinicalTrials.gov NCT00888225 http://clinicaltrials.gov/

Increased Circulating and Urinary Levels of Soluble TAM Receptors in Diabetic Nephropathy.

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Ochodnicky, Peter; Lattenist, Lionel; Ahdi, Mohamed; Kers, Jesper; Uil, Melissa; Claessen, Nike; Leemans, Jaklien C; Florquin, Sandrine; Meijers, Joost C M; Gerdes, Victor E A; Roelofs, Joris J T H

2017-09-01

TAM receptors (Tyro3, Axl, and Mer) have been implicated in innate immunity. Circulating TAM receptor soluble forms (sTyro3, sAxl, sMer) are related to autoimmune disorders. We investigated TAM and their ligand protein S in patients with diabetes. Urinary and plasma levels of protein S, sTyro3, sAxl, and sMer were determined in 126 patients with diabetes assigned to a normoalbuminuric or macroalbuminuric (urinary albumin excretion 300 mg/24 hours, respectively) study group and 18 healthy volunteers. TAM and protein S immunostaining was performed on kidney biopsy specimens from patients with diabetic nephropathy (n = 9) and controls (n = 6). TAM expression and shedding by tubular epithelial cells were investigated by PCR and enzyme-linked immunosorbent assay in an in vitro diabetes model. Patients with macroalbuminuria diabetes had higher circulating levels of sMer and more urinary sTyro3 and sMer than normoalbuminuric diabetics. Increased clearance of sTyro3 and sMer was associated with loss of tubular Tyro3 and Mer expression in diabetic nephropathy tissue and glomerular depositions of protein S. During in vitro diabetes, human kidney cells had down-regulation of Tyro3 and Mer mRNA and increased shedding of sTyro3 and sMer. Renal injury in diabetes is associated with elevated systemic and urine levels of sMer and sTyro3. This is the first study reporting excretion of sTAM receptors in urine, identifying the kidney as a source of sTAM. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Increased cell-free DNA concentrations in patients with obstructive sleep apnea.

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Shin, Chol; Kim, Jin K; Kim, Je H; Jung, Ki H; Cho, Kyung J; Lee, Chang K; Lee, Seung G

2008-12-01

Blood concentrations of cell-free DNA, which is considered to be released during apoptosis, are elevated under some pathological conditions such as cardiovascular disease and cancer. The association between obstructive sleep apnea (OSA) and cell-free DNA concentrations has not been reported so far. The purpose of the present study was to examine the association between OSA and plasma DNA concentrations. A case-control study was conducted using a total of 164 men aged 39-67 years, who were free of coronary heart disease and cancer. Laboratory-based overnight polysomnography was performed for all participants. On the basis of polysomnography, patients with an apnea-hypopnea index (AHI) = 5-30 events/h were defined as having mild-moderate OSA (n = 33) and those with >30 events/h were defined as having severe OSA (n = 49). All 82 controls had AHI DNA concentrations from all participants were analyzed for the beta-globin gene using fluorescence-based real-time polymerase chain reaction. Patients with severe OSA had significantly higher plasma DNA concentrations than persons with mild-moderate OSA and those without OSA (P DNA concentration (P DNA concentrations (>8 microg/L) had approximately fourfold higher odds of OSA than those with low DNA levels. Further data are warranted to confirm the association for men and to evaluate the association for women.

Anxiolytic-Like Effects of Increased Ghrelin Receptor Signaling in the Amygdala

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Jensen, Morten; Ratner, Cecilia; Rudenko, Olga

2016-01-01

BACKGROUND: Besides the well-known effects of ghrelin on adiposity and food intake regulation, the ghrelin system has been shown to regulate aspects of behavior including anxiety and stress. However, the effect of virus-mediated overexpression of the ghrelin receptor in the amygdala has...... not previously been addressed directly. METHOD: First, we examined the acute effect of peripheral ghrelin administration on anxiety- and depression-like behavior using the open field, elevated plus maze, forced swim and tail suspension tests. Next, we examined the effect of peripheral ghrelin administration...... and ghrelin receptor deficiency on stress in a familiar and social environment using the Intellicage system. Importantly, we also used a novel approach to study ghrelin receptor signaling in the brain by overexpressing the ghrelin receptor in the amygdala. We examined the effect of ghrelin receptor...

Deep Sequencing of T-cell Receptor DNA as a Biomarker of Clonally Expanded TILs in Breast Cancer after Immunotherapy.

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Page, David B; Yuan, Jianda; Redmond, David; Wen, Y Hanna; Durack, Jeremy C; Emerson, Ryan; Solomon, Stephen; Dong, Zhiwan; Wong, Phillip; Comstock, Christopher; Diab, Adi; Sung, Janice; Maybody, Majid; Morris, Elizabeth; Brogi, Edi; Morrow, Monica; Sacchini, Virgilio; Elemento, Olivier; Robins, Harlan; Patil, Sujata; Allison, James P; Wolchok, Jedd D; Hudis, Clifford; Norton, Larry; McArthur, Heather L

2016-10-01

In early-stage breast cancer, the degree of tumor-infiltrating lymphocytes (TIL) predicts response to chemotherapy and overall survival. Combination immunotherapy with immune checkpoint antibody plus tumor cryoablation can induce lymphocytic infiltrates and improve survival in mice. We used T-cell receptor (TCR) DNA sequencing to evaluate both the effect of cryoimmunotherapy in humans and the feasibility of TCR sequencing in early-stage breast cancer. In a pilot clinical trial, 18 women with early-stage breast cancer were treated preoperatively with cryoablation, single-dose anti-CTLA-4 (ipilimumab), or cryoablation + ipilimumab. TCRs within serially collected peripheral blood and tumor tissue were sequenced. In baseline tumor tissues, T-cell density as measured by TCR sequencing correlated with TIL scores obtained by hematoxylin and eosin (H&E) staining. However, tumors with little or no lymphocytes by H&E contained up to 3.6 × 10 6 TCR DNA sequences, highlighting the sensitivity of the ImmunoSEQ platform. In this dataset, ipilimumab increased intratumoral T-cell density over time, whereas cryoablation ± ipilimumab diversified and remodeled the intratumoral T-cell clonal repertoire. Compared with monotherapy, cryoablation plus ipilimumab was associated with numerically greater numbers of peripheral blood and intratumoral T-cell clones expanding robustly following therapy. In conclusion, TCR sequencing correlates with H&E lymphocyte scoring and provides additional information on clonal diversity. These findings support further study of the use of TCR sequencing as a biomarker for T-cell responses to therapy and for the study of cryoimmunotherapy in early-stage breast cancer. Cancer Immunol Res; 4(10); 835-44. ©2016 AACR. ©2016 American Association for Cancer Research.

Pharmacologic implications of peroxisome proliferator activated receptors (PPAR Implicaciones farmacológicas de los receptores activados por los proliferadores de peroxisomas (PPAR

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Luis Carlos Mejía Rivera

2001-01-01

Full Text Available PPAR are a group of proteins, members of the receptors located within the nucleus. These receptors modulate DNA transcriptional activity by binding to specific response elements on target genes. To date, three main types of PPAR have been identified designed α, δand γthese receptors are involved in the regulation of diferent metabolic processes, being the group of receptors more intensely studied. PPARαare greatly involved in both catabolism of fatty acids and transport of extracellular lipids; fibrates, their agonists, are of proved usefulness in some dyslipidemias. Thiazolidinediones used as antihyperglicemiant agents are PPARγagonists, but their relationship with carbohydrate metabolism is not yet clear; nevertheless, their use in the management of type 2 diabetes mellitus is of increasing importance. On the other hand, nonsteroidal anti-inflammatory agents are somehow related with PPARδfunctions; up to date a molecular and epidemiologic relationship of these drugs and receptors with colon cancer has been established. Los receptores activados por los proliferadores de peroxisomas (PPAR son un grupo de proteínas pertenecientes a la familia de receptores de ubicación nuclear que se comportan como factores que modulan la transcripción del DNA al unirse a elementos de respuesta específicos de ciertos genes blanco. Hasta el momento se han descrito tres tipos principales de PPAR designados como α, δ, y γ; estos receptores se encuentran involucrados en la regulación de diferentes procesos metabólicos; por esto se han convertido en uno de los grupos de receptores más intensamente estudiados. Los PPARα participan tanto en el catabolismo de los ácidos grasos como en el transporte extracelular de lípidos; los fibratos, sus agonistas, tienen utilidad ampliamente demostrada en el manejo de algunas dislipidemias. Las tiazolidindionas utilizadas como fármacos antihiperglicemiantes son agonistas de los PPARγ; todav

Deficiency of the DNA repair protein nibrin increases the basal but not the radiation induced mutation frequency in vivo

International Nuclear Information System (INIS)

Wessendorf, Petra; Vijg, Jan; Nussenzweig, André; Digweed, Martin

2014-01-01

Highlights: • lacZ mutant frequencies measured in vivo in mouse models of radiosensitive Nijmegen Breakage Syndrome. • Spontaneous mutation frequencies are increased in lymphatic tissue due to Nbn mutation. • Single base transitions, not deletions, dominate the mutation spectrum. • Radiation induced mutation frequencies are not increased due to Nbn mutation. - Abstract: Nibrin (NBN) is a member of a DNA repair complex together with MRE11 and RAD50. The complex is associated particularly with the repair of DNA double strand breaks and with the regulation of cell cycle check points. Hypomorphic mutation of components of the complex leads to human disorders characterised by radiosensitivity and increased tumour occurrence, particularly of the lymphatic system. We have examined here the relationship between DNA damage, mutation frequency and mutation spectrum in vitro and in vivo in mouse models carrying NBN mutations and a lacZ reporter plasmid. We find that NBN mutation leads to increased spontaneous DNA damage in fibroblasts in vitro and high basal mutation rates in lymphatic tissue of mice in vivo. The characteristic mutation spectrum is dominated by single base transitions rather than the deletions and complex rearrangements expected after abortive repair of DNA double strand breaks. We conclude that in the absence of wild type nibrin, the repair of spontaneous errors, presumably arising during DNA replication, makes a major contribution to the basal mutation rate. This applies also to cells heterozygous for an NBN null mutation. Mutation frequencies after irradiation in vivo were not increased in mice with nibrin mutations as might have been expected considering the radiosensitivity of NBS patient cells in vitro. Evidently apoptosis is efficient, even in the absence of wild type nibrin

Deficiency of the DNA repair protein nibrin increases the basal but not the radiation induced mutation frequency in vivo

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Wessendorf, Petra [Institute of Medical and Human Genetics, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin (Germany); Vijg, Jan [Albert Einstein College of Medicine, Michael F. Price Center, 1301 Morris Park Avenue, Bronx, NY 10461 (United States); Nussenzweig, André [Laboratory of Genome Integrity, National Cancer Institute, National Institute of Health, 37 Convent Drive, Room 1106, Bethesda, MD 20892 (United States); Digweed, Martin, E-mail: martin.digweed@charite.de [Institute of Medical and Human Genetics, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin (Germany)

2014-11-15

Highlights: • lacZ mutant frequencies measured in vivo in mouse models of radiosensitive Nijmegen Breakage Syndrome. • Spontaneous mutation frequencies are increased in lymphatic tissue due to Nbn mutation. • Single base transitions, not deletions, dominate the mutation spectrum. • Radiation induced mutation frequencies are not increased due to Nbn mutation. - Abstract: Nibrin (NBN) is a member of a DNA repair complex together with MRE11 and RAD50. The complex is associated particularly with the repair of DNA double strand breaks and with the regulation of cell cycle check points. Hypomorphic mutation of components of the complex leads to human disorders characterised by radiosensitivity and increased tumour occurrence, particularly of the lymphatic system. We have examined here the relationship between DNA damage, mutation frequency and mutation spectrum in vitro and in vivo in mouse models carrying NBN mutations and a lacZ reporter plasmid. We find that NBN mutation leads to increased spontaneous DNA damage in fibroblasts in vitro and high basal mutation rates in lymphatic tissue of mice in vivo. The characteristic mutation spectrum is dominated by single base transitions rather than the deletions and complex rearrangements expected after abortive repair of DNA double strand breaks. We conclude that in the absence of wild type nibrin, the repair of spontaneous errors, presumably arising during DNA replication, makes a major contribution to the basal mutation rate. This applies also to cells heterozygous for an NBN null mutation. Mutation frequencies after irradiation in vivo were not increased in mice with nibrin mutations as might have been expected considering the radiosensitivity of NBS patient cells in vitro. Evidently apoptosis is efficient, even in the absence of wild type nibrin.

Typical and Atypical Antipsychotic Drugs Increase Extracellular Histamine Levels in the Rat Medial Prefrontal Cortex: Contribution of Histamine H1 Receptor Blockade

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Kjell A Svensson

2012-05-01

Full Text Available Atypical antipsychotics such as clozapine and olanzapine have been shown to enhance histamine turnover and this effect has been hypothesized to contribute to their improved therapeutic profile compared to typical antipsychotics. In the present study, we examined the effects of antipsychotic drugs on histamine (HA efflux in the mPFC of the rat by means of in vivo microdialysis and sought to differentiate the receptor mechanisms which underlie such effects. Olanzapine and clozapine increased mPFC HA efflux in a dose related manner. Increased HA efflux was also observed after quetiapine, chlorpromazine and perphenazine treatment. We found no effect of the selective 5-HT2A antagonist MDL100907, 5-HT2c antagonist SB242084 or the 5-HT6 antagonist Ro 04-6790 on mPFC HA efflux. HA efflux was increased following treatment with selective H1 receptor antagonists pyrilamine, diphenhydramine and triprolidine, the H3 receptor antagonist ciproxifan and the mixed 5HT2A/H1 receptor antagonist ketanserin. The potential novel antipsychotic drug FMPD, which has a lower affinity at H1 receptors than olanzapine, did not affect HA efflux. Similarly, other antipsychotics with lower H1 receptor affinity (risperidone, aripiprazole and haloperidol were also without effect on HA efflux. Perfusion of clozapine and pyrilamine into the TMN, but not the mPFC, increased local HA efflux. Finally, HA efflux after antipsychotic treatment was significantly correlated with affinity at H1 receptors whereas 9 other receptors, including 5-HT2A, were not. These results demonstrate that both typical and atypical antipsychotics increase mPFC histamine efflux and this effect may be mediated via antagonism of histamine H1 receptors.

FXR silencing in human colon cancer by DNA methylation and KRAS signaling.

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Bailey, Ann M; Zhan, Le; Maru, Dipen; Shureiqi, Imad; Pickering, Curtis R; Kiriakova, Galina; Izzo, Julie; He, Nan; Wei, Caimiao; Baladandayuthapani, Veerabhadran; Liang, Han; Kopetz, Scott; Powis, Garth; Guo, Grace L

2014-01-01

Farnesoid X receptor (FXR) is a bile acid nuclear receptor described through mouse knockout studies as a tumor suppressor for the development of colon adenocarcinomas. This study investigates the regulation of FXR in the development of human colon cancer. We used immunohistochemistry of FXR in normal tissue (n = 238), polyps (n = 32), and adenocarcinomas, staged I-IV (n = 43, 39, 68, and 9), of the colon; RT-quantitative PCR, reverse-phase protein array, and Western blot analysis in 15 colon cancer cell lines; NR1H4 promoter methylation and mRNA expression in colon cancer samples from The Cancer Genome Atlas; DNA methyltransferase inhibition; methyl-DNA immunoprecipitation (MeDIP); bisulfite sequencing; and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) knockdown assessment to investigate FXR regulation in colon cancer development. Immunohistochemistry and quantitative RT-PCR revealed that expression and function of FXR was reduced in precancerous lesions and silenced in a majority of stage I-IV tumors. FXR expression negatively correlated with phosphatidylinositol-4, 5-bisphosphate 3 kinase signaling and the epithelial-to-mesenchymal transition. The NR1H4 promoter is methylated in ~12% colon cancer The Cancer Genome Atlas samples, and methylation patterns segregate with tumor subtypes. Inhibition of DNA methylation and KRAS silencing both increased FXR expression. FXR expression is decreased early in human colon cancer progression, and both DNA methylation and KRAS signaling may be contributing factors to FXR silencing. FXR potentially suppresses epithelial-to-mesenchymal transition and other oncogenic signaling cascades, and restoration of FXR activity, by blocking silencing mechanisms or increasing residual FXR activity, represents promising therapeutic options for the treatment of colon cancer.

Chewing ameliorates stress-induced suppression of spatial memory by increasing glucocorticoid receptor expression in the hippocampus.

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Miyake, Shinjiro; Yoshikawa, Gota; Yamada, Kentaro; Sasaguri, Ken-Ichi; Yamamoto, Toshiharu; Onozuka, Minoru; Sato, Sadao

2012-03-29

Chewing alters hypothalamic-pituitary-adrenal axis function and improves the ability to cope with stress in rodents. Given that stress negatively influences hippocampus-dependent learning and memory, we aimed to elucidate whether masticatory movements, namely chewing, improve the stress-induced impairment of spatial memory in conjunction with increased hippocampal glucocorticoid receptor expression. Male Sprague-Dawley rats were subjected to restraint stress by immobilization for 2h: the stress with chewing (SC) group were allowed to chew on a wooden stick during the latter half of the immobilization period, whereas the stress without chewing (ST) group were not allowed to do so. Performance in the Morris water maze test was significantly impaired in the ST group compared with the SC group. Further, the numbers of glucocorticoid receptor immunopositive neurons in the hippocampal cornu ammonis 1 region were significantly lower in the ST group than in the control and SC groups. The control and SC rats showed no significant differences in both the water maze performance and the numbers of glucocorticoid receptor-immunopositive neurons. The immunohistochemical finding correlated with the performance in the water maze test. These results suggest that chewing is a behavioral mechanism to cope with stress by increasing hippocampal glucocorticoid receptor expression. Copyright © 2012 Elsevier B.V. All rights reserved.

Increased leptin/leptin receptor pathway affects systemic and airway inflammation in COPD former smokers

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Bruno A

2011-05-01

Full Text Available Andreina Bruno1, Marinella Alessi2, Simona Soresi2, Anna Bonanno1, Loredana Riccobono1, Angela Marina Montalbano1, Giusy Daniela Albano1, Mark Gjomarkaj1, Mirella Profita11Institute of Biomedicine and Molecular Immunology, Italian National Research Council, Palermo, Italy; 2Dipartimento Biomedico di Biomedicina Interna e Specialistica, University Palermo, ItalyBackground: Leptin, a hormone produced mainly by adipose tissue, regulates food intake and energy expenditure. It is involved in inflammatory diseases such as chronic obstructive pulmonary disease (COPD and its deficiency is associated with increased susceptibility to the infection. The leptin receptor is expressed in the lung and in the neutrophils.Methods: We measured the levels of leptin, tumor necrosis factor alpha (TNF-a and soluble form of intercellular adhesion molecule-1 (sICAM-1 in sputum and plasma from 27 smoker and former smoker patients with stable COPD using ELISA methods. Further we analyzed leptin and its receptor expression in sputum cells from 16 COPD patients using immunocytochemistry.Results: In plasma of COPD patients, leptin was inversely correlated with TNF-a and positively correlated with the patient weight, whereas the levels of sICAM-1 were positively correlated with TNF-a. In sputum of COPD patients leptin levels were correlated with forced expiratory volume in 1 second/forced vitality capacity. Additionally, increased levels of sputum leptin and TNF-a were observed in COPD former smokers rather than smokers. Further the expression of leptin receptor in sputum neutrophils was significantly higher in COPD former smokers than in smokers, and the expression of leptin and its receptor was positively correlated in neutrophils of COPD former smokers.Conclusion: Our findings suggest a role of leptin in the local and systemic inflammation of COPD and, taking into account the involvement of neutrophils in this inflammatory disease, describe a novel aspect of the leptin

Effect of the caffeine on treated and non-treated plasmid DNA with stannic chloride

International Nuclear Information System (INIS)

Moreno, Silvana Ramos F.; Universidade Federal Fluminense, Niteroi, RJ; Mattos, Jose C.P. de; Dantas, Flavio; Araujo, Adriano Caldeira de; Bernardo-Filho, Mario

2000-01-01

Caffeine, a methilxantine drug is a component of coffee, tea, stimulants and other drinks. Caffeine inhibits phosphodiesterase leading to intracellular accumulation of cyclic AMP, blocks adenosine receptors, and increases the release of Ca 2+ . We have studied the possible effect of caffeine in DNA plasmid treated or not with stannous chloride (SnCl 2 ). Previous evaluations of the effect of caffeine on the labeling of red blood cells and plasma proteins with technetium-99m have showed a decrease of % ATI in the insoluble fraction of plasma proteins. Samples of DNA were treated with SnCl 2 (0 and 200μg/ml) in 0.8% agarose. SnCl 2 has induced break on DNA and caffeine has not showed effect on the DNA. This indicates that caffeine does not eliminate the oxidant action of SnCl 2 and does not promote break in isolated DNA plasmid. (author)

Genomic and epigenetic evidence for oxytocin receptor deficiency in autism

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Worley Gordon

2009-10-01

Full Text Available Abstract Background Autism comprises a spectrum of behavioral and cognitive disturbances of childhood development and is known to be highly heritable. Although numerous approaches have been used to identify genes implicated in the development of autism, less than 10% of autism cases have been attributed to single gene disorders. Methods We describe the use of high-resolution genome-wide tilepath microarrays and comparative genomic hybridization to identify copy number variants within 119 probands from multiplex autism families. We next carried out DNA methylation analysis by bisulfite sequencing in a proband and his family, expanding this analysis to methylation analysis of peripheral blood and temporal cortex DNA of autism cases and matched controls from independent datasets. We also assessed oxytocin receptor (OXTR gene expression within the temporal cortex tissue by quantitative real-time polymerase chain reaction (PCR. Results Our analysis revealed a genomic deletion containing the oxytocin receptor gene, OXTR (MIM accession no.: 167055, previously implicated in autism, was present in an autism proband and his mother who exhibits symptoms of obsessive-compulsive disorder. The proband's affected sibling did not harbor this deletion but instead may exhibit epigenetic misregulation of this gene through aberrant gene silencing by DNA methylation. Further DNA methylation analysis of the CpG island known to regulate OXTR expression identified several CpG dinucleotides that show independent statistically significant increases in the DNA methylation status in the peripheral blood cells and temporal cortex in independent datasets of individuals with autism as compared to control samples. Associated with the increase in methylation of these CpG dinucleotides is our finding that OXTR mRNA showed decreased expression in the temporal cortex tissue of autism cases matched for age and sex compared to controls. Conclusion Together, these data provide

Increased DNA damage in progression of COPD: a response by poly(ADP-ribose polymerase-1.

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Ingrid Oit-Wiscombe

Full Text Available Chronic oxidative stress (OS, a major mechanism of chronic obstructive pulmonary disease (COPD, may cause significant damage to DNA. Poly(ADP-ribose polymerase (PARP-1 is rapidly activated by OS-induced DNA lesions. However, the degree of DNA damage along with the evolution of COPD is unclear. In peripheral blood mononuclear cells of non-smoking individuals, non-obstructive smokers, patients with COPD of all stages and those with COPD exacerbation, we evaluated DNA damage, PARP activity and PARP-1 mRNA expression using Comet Assay IV, biotinylated-NAD incorporation assay and qRT-PCR, respectively and subjected results to ordinal logistic regression modelling. Adjusted for demographics, smoking-related parameters and lung function, novel comet parameters, tail length/cell length ratio and tail migration/cell length ratio, showed the greatest increase along the study groups corresponding to the evolution of COPD [odds ratio (OR 7.88, 95% CI 4.26-14.57; p<0.001 and OR 3.91, 95% CI 2.69-5.66; p<0.001, respectively]. Analogously, PARP activity increased significantly over the groups (OR = 1.01; 95%; p<0.001. An antioxidant tetrapeptide UPF17 significantly reduced the PARP-1 mRNA expression in COPD, compared to that in non-obstructive individuals (p = 0.040. Tail length/cell length and tail migration/cell length ratios provide novel progression-sensitive tools for assessment of DNA damage. However, it remains to be elucidated whether inhibition of an elevated PARP-1 activity has a safe enough potential to break the vicious cycle of the development and progression of COPD.

Expression of somatotropin receptor messenger ribonucleic acid in bovine tissues

International Nuclear Information System (INIS)

Lucy, M.C.; Boyd, C.K.; Koenigsfeld, A.T.; Okamura, C.S.

1998-01-01

The somatotropin receptor mRNA is controlled by at least two different gene promoters that generate 2 two variants with different exon 1 sequences (1A and 1B). The location of 1A and 1B somatotropin receptor mRNA within cattle tissues and, hence, the tissue specificity of the 1A and 1B promoters are unknown. In addition, the cDNA sequence of the 1B somatotropin receptor has not been determined. Our objective, therefore, was to sequence a cDNA for the 1B somatotropin receptor and to analyze bovine tissues for expression of 1A and 1B somatotropin receptor mRNA. Twenty adult tissues and six fetal tissues were collected at slaughter from each of four cows and two fetuses. Messenger RNA was analyzed using ribonuclease protection assays. The adult liver expressed both 1A and 1B mRNA. All other adult tissues expressed 1B mRNA but not 1A mRNA. The greatest amount of 1B mRNA was detected in liver and adipose (abdominal and subcutaneous) tissues. Other tissues had approximately one-half to one-tenth of the amount of 1B mRNA in the liver or adipose tissue. Fetal tissues (including fetal liver) expressed 1B mRNA and not 1A mRNA. Based on cDNA sequencing, the protein encoded by the 1A and 1B mRNA was nearly identical. We concluded that 1A somatotropin receptor mRNA is specific to adult bovine liver. Other adult and fetal bovine tissues expressed 1B somatotropin receptor mRNA with a predicted protein sequence that was similar to the 1A somatotropin receptor

CDNA CLONING OF FATHEAD MINNOW (PIMEPHALES PROMELAS) ESTROGEN AND ANDROGEN RECEPTORS FOR USE IN STEROID RECEPTOR EXTRAPOLATION STUDIES FOR ENDOCRINE DISRUPTING CHEMICALS

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cDNA Cloning of Fathead minnow (Pimephales promelas) Estrogen and Androgen Receptors for Use in Steroid Receptor Extrapolation Studies for Endocrine Disrupting Chemicals. Wilson, V.S.1,, Korte, J.2, Hartig P. 1, Ankley, G.T.2, Gray, L.E., Jr 1, , and Welch, J.E.1. 1U.S...

Electromagnetic noise inhibits radiofrequency radiation-induced DNA damage and reactive oxygen species increase in human lens epithelial cells

Science.gov (United States)

Wu, Wei; Wang, KaiJun; Ni, Shuang; Ye, PanPan; Yu, YiBo; Ye, Juan; Sun, LiXia

2008-01-01

Purpose The goal of this study was to investigate whether superposing of electromagnetic noise could block or attenuate DNA damage and intracellular reactive oxygen species (ROS) increase of cultured human lens epithelial cells (HLECs) induced by acute exposure to 1.8 GHz radiofrequency field (RF) of the Global System for Mobile Communications (GSM). Methods An sXc-1800 RF exposure system was used to produce a GSM signal at 1.8 GHz (217 Hz amplitude-modulated) with the specific absorption rate (SAR) of 1, 2, 3, and 4 W/kg. After 2 h of intermittent exposure, the ROS level was assessed by the fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). DNA damage to HLECs was examined by alkaline comet assay and the phosphorylated form of histone variant H2AX (γH2AX) foci formation assay. Results After exposure to 1.8 GHz RF for 2 h, HLECs exhibited significant intracellular ROS increase in the 2, 3, and 4 W/kg groups. RF radiation at the SAR of 3 W/kg and 4 W/kg could induce significant DNA damage, examined by alkaline comet assay, which was used to detect mainly single strand breaks (SSBs), while no statistical difference in double strand breaks (DSBs), evaluated by γH2AX foci, was found between RF exposure (SAR: 3 and 4 W/kg) and sham exposure groups. When RF was superposed with 2 μT electromagnetic noise could block RF-induced ROS increase and DNA damage. Conclusions DNA damage induced by 1.8 GHz radiofrequency field for 2 h, which was mainly SSBs, may be associated with the increased ROS production. Electromagnetic noise could block RF-induced ROS formation and DNA damage. PMID:18509546

Suicide Gene Therapy to Increase the Safety of Chimeric Antigen Receptor-Redirected T Lymphocytes

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Monica Casucci, Attilio Bondanza

2011-01-01

Full Text Available Chimeric antigen receptors (CARs are generated by fusing the antigen-binding motif of a monoclonal antibody (mAb with the signal transduction machinery of the T-cell receptor (TCR. The genetic modification of T lymphocytes with chimeric receptors specific for tumor-associated antigens (TAAs allows for the redirection towards tumor cells. Clinical experience with CAR-redirected T cells suggests that antitumor efficacy associates with some degree of toxicity, especially when TAA expression is shared with healthy tissues. This situation closely resembles the case of allogeneic hematopoietic stem cell transplantation (HSCT, wherein allorecognition causes both the graft-versus-leukemia (GVL effect and graft-versus-host disease (GVHD. Suicide gene therapy, i.e. the genetic induction of a conditional suicide phenotype into donor T cells, enables dissociating the GVL effect from GVHD. Applying suicide gene modification to CAR-redirected T cells may therefore greatly increase their safety profile and facilitate their clinical development.

RTA, a candidate G protein-coupled receptor: Cloning, sequencing, and tissue distribution

International Nuclear Information System (INIS)

Ross, P.C.; Figler, R.A.; Corjay, M.H.; Barber, C.M.; Adam, N.; Harcus, D.R.; Lynch, K.R.

1990-01-01

Genomic and cDNA clones, encoding a protein that is a member of the guanine nucleotide-binding regulatory protein (G protein)-coupled receptor superfamily, were isolated by screening rat genomic and thoracic aorta cDNA libraries with an oligonucleotide encoding a highly conserved region of the M 1 muscarinic acetylcholine receptor. Sequence analyses of these clones showed that they encode a 343-amino acid protein (named RTA). The RTA gene is single copy, as demonstrated by restriction mapping and Southern blotting of genomic clones and rat genomic DNA. RTA RNA sequences are relatively abundant throughout the gut, vas deferens, uterus, and aorta but are only barely detectable (on Northern blots) in liver, kidney, lung, and salivary gland. In the rat brain, RTA sequences are markedly abundant in the cerebellum. TRA is most closely related to the mas oncogene (34% identity), which has been suggested to be a forebrain angiotensin receptor. They conclude that RTA is not an angiotensin receptor; to date, they have been unable to identify its ligand

Cerebral 5-HT2A receptor binding is increased in patients with Tourette's syndrome

DEFF Research Database (Denmark)

Haugbøl, Steven; Pinborg, Lars H.; Regeur, Lisbeth

2007-01-01

Experimental and clinical data have suggested that abnormalities in the serotonergic neurotransmissions in frontal-subcortical circuits are involved in Tourette's syndrome. To test the hypothesis that the brain's 5-HT2A receptor binding is increased in patients with Tourette's syndrome, PET imagi...

A ketogenic diet accelerates neurodegeneration in mice with induced mitochondrial DNA toxicity in the forebrain.

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Lauritzen, Knut H; Hasan-Olive, Md Mahdi; Regnell, Christine E; Kleppa, Liv; Scheibye-Knudsen, Morten; Gjedde, Albert; Klungland, Arne; Bohr, Vilhelm A; Storm-Mathisen, Jon; Bergersen, Linda H

2016-12-01

Mitochondrial genome maintenance plays a central role in preserving brain health. We previously demonstrated accumulation of mitochondrial DNA damage and severe neurodegeneration in transgenic mice inducibly expressing a mutated mitochondrial DNA repair enzyme (mutUNG1) selectively in forebrain neurons. Here, we examine whether severe neurodegeneration in mutUNG1-expressing mice could be rescued by feeding the mice a ketogenic diet, which is known to have beneficial effects in several neurological disorders. The diet increased the levels of superoxide dismutase 2, and mitochondrial mass, enzymes, and regulators such as SIRT1 and FIS1, and appeared to downregulate N-methyl-D-aspartic acid (NMDA) receptor subunits NR2A/B and upregulate γ-aminobutyric acid A (GABA A ) receptor subunits α 1 . However, unexpectedly, the ketogenic diet aggravated neurodegeneration and mitochondrial deterioration. Electron microscopy showed structurally impaired mitochondria accumulating in neuronal perikarya. We propose that aggravation is caused by increased mitochondrial biogenesis of generally dysfunctional mitochondria. This study thereby questions the dogma that a ketogenic diet is unambiguously beneficial in mitochondrial disorders. Copyright © 2016 Elsevier Inc. All rights reserved.

Increased levels of etheno-DNA adducts and genotoxicity biomarkers of long-term exposure to pure diesel engine exhaust.

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Shen, Meili; Bin, Ping; Li, Haibin; Zhang, Xiao; Sun, Xin; Duan, Huawei; Niu, Yong; Meng, Tao; Dai, Yufei; Gao, Weimin; Yu, Shanfa; Gu, Guizhen; Zheng, Yuxin

2016-02-01

Etheno-DNA adducts are biomarkers for assessing oxidative stress. In this study, the aim was to detect the level of etheno-DNA adducts and explore the relationship between the etheno-DNA adducts and genotoxicity biomarkers of the diesel engine exhaust (DEE)-exposed workers. We recruited 86 diesel engine testing workers with long-term exposure to DEE and 99 non-DEE-exposed workers. The urinary mono-hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) and etheno-DNA adducts (εdA and εdC) were detected by HPLC-MS/MS and UPLC-MS/MS, respectively. Genotoxicity biomarkers were also evaluated by comet assay and cytokinesis-block micronucleus assay. The results showed that urinary εdA was significantly higher in the DEE-exposed workers (p<0.001), exhibited 2.1-fold increase compared with the non-DEE-exposed workers. The levels of urinary OH-PAHs were positively correlated with the level of εdA among all the study subjects (p<0.001). Moreover, we found that the increasing level of εdA was significantly associated with the increased olive tail moment, percentage of tail DNA, or frequency of micronucleus in the study subjects (p<0.01). No significant association was observed between the εdC level and any measured genotoxicity biomarkers. In summary, εdA could serve as an indicator for DEE exposure in the human population. Copyright © 2015 Elsevier B.V. All rights reserved.

Changing the insulin receptor to possess insulin-like growth factor I ligand specificity

International Nuclear Information System (INIS)

Andersen, A.S.; Kjeldsen, T.; Wiberg, F.C.; Christensen, P.M.; Rasmussen, J.S.; Norris, K.; Moeller, K.B.; Moeller, N.P.H.

1990-01-01

To examine the role of the N-terminal part of the insulin-like growth factor I (IGF-I) receptor and insulin receptor in determining ligand specificity, the authors prepared an expression vector encoding a hybrid receptor where exon 1 (encoding the signal peptide and seven amino acids of the α-subunit), exon 2, and exon 3 of the insulin receptor were replaced with the corresponding IGF-I receptor cDNA (938 nucleotides). To allow direct quantitative comparison of the binding capabilities of this hybrid receptor with those of the human IGF-I receptor and the insulin receptor, all three receptors were expressed in baby hamster kidney (BHK) cells as soluble molecules and partially purified before characterization. The hybrid IGF-I/insulin receptor bound IGF-I with an affinity comparable to that of the wild-type IGF-I receptor. In contrast, the hybrid receptor no longer displayed high-affinity binding of insulin. These results directly demonstrate that it is possible to change the specificity of the insulin receptor to that of the IGF-I receptor and, furthermore, that the binding specificity for IGF-I is encoded within the nucleotide sequence from 135 to 938 of the IGF-I receptor cDNA. Since the hybrid receptor only bound insulin with low affinity, the insulin binding region is likely to be located within exons 2 and 3 of the insulin receptor

Cloning and expression of a widely expressed receptor tyrosine phosphatase

DEFF Research Database (Denmark)

Sap, J; D'Eustachio, P; Givol, D

1990-01-01

We describe the identification of a widely expressed receptor-type (transmembrane) protein tyrosine phosphatase (PTPase; EC 3.1.3.48). Screening of a mouse brain cDNA library under low-stringency conditions with a probe encompassing the intracellular (phosphatase) domain of the CD45 lymphocyte...... antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid...

Location analysis for the estrogen receptor-α reveals binding to diverse ERE sequences and widespread binding within repetitive DNA elements

Science.gov (United States)

Mason, Christopher E.; Shu, Feng-Jue; Wang, Cheng; Session, Ryan M.; Kallen, Roland G.; Sidell, Neil; Yu, Tianwei; Liu, Mei Hui; Cheung, Edwin; Kallen, Caleb B.

2010-01-01

Location analysis for estrogen receptor-α (ERα)-bound cis-regulatory elements was determined in MCF7 cells using chromatin immunoprecipitation (ChIP)-on-chip. Here, we present the estrogen response element (ERE) sequences that were identified at ERα-bound loci and quantify the incidence of ERE sequences under two stringencies of detection: ERE sequence. We demonstrate that ∼50% of all ERα-bound loci do not have a discernable ERE and show that most ERα-bound EREs are not perfect consensus EREs. Approximately one-third of all ERα-bound ERE sequences reside within repetitive DNA sequences, most commonly of the AluS family. In addition, the 3-bp spacer between the inverted ERE half-sites, rather than being random nucleotides, is C(A/T)G-enriched at bona fide receptor targets. Diverse ERα-bound loci were validated using electrophoretic mobility shift assay and ChIP-polymerase chain reaction (PCR). The functional significance of receptor-bound loci was demonstrated using luciferase reporter assays which proved that repetitive element ERE sequences contribute to enhancer function. ChIP-PCR demonstrated estrogen-dependent recruitment of the coactivator SRC3 to these loci in vivo. Our data demonstrate that ERα binds to widely variant EREs with less sequence specificity than had previously been suspected and that binding at repetitive and nonrepetitive genomic targets is favored by specific trinucleotide spacers. PMID:20047966

Location analysis for the estrogen receptor-alpha reveals binding to diverse ERE sequences and widespread binding within repetitive DNA elements.

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Mason, Christopher E; Shu, Feng-Jue; Wang, Cheng; Session, Ryan M; Kallen, Roland G; Sidell, Neil; Yu, Tianwei; Liu, Mei Hui; Cheung, Edwin; Kallen, Caleb B

2010-04-01

Location analysis for estrogen receptor-alpha (ERalpha)-bound cis-regulatory elements was determined in MCF7 cells using chromatin immunoprecipitation (ChIP)-on-chip. Here, we present the estrogen response element (ERE) sequences that were identified at ERalpha-bound loci and quantify the incidence of ERE sequences under two stringencies of detection: ERE sequence. We demonstrate that approximately 50% of all ERalpha-bound loci do not have a discernable ERE and show that most ERalpha-bound EREs are not perfect consensus EREs. Approximately one-third of all ERalpha-bound ERE sequences reside within repetitive DNA sequences, most commonly of the AluS family. In addition, the 3-bp spacer between the inverted ERE half-sites, rather than being random nucleotides, is C(A/T)G-enriched at bona fide receptor targets. Diverse ERalpha-bound loci were validated using electrophoretic mobility shift assay and ChIP-polymerase chain reaction (PCR). The functional significance of receptor-bound loci was demonstrated using luciferase reporter assays which proved that repetitive element ERE sequences contribute to enhancer function. ChIP-PCR demonstrated estrogen-dependent recruitment of the coactivator SRC3 to these loci in vivo. Our data demonstrate that ERalpha binds to widely variant EREs with less sequence specificity than had previously been suspected and that binding at repetitive and nonrepetitive genomic targets is favored by specific trinucleotide spacers.

Functional analysis of the interdependence between DNA uptake sequence and its cognate ComP receptor during natural transformation in Neisseria species.

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Jamie-Lee Berry

Full Text Available Natural transformation is the widespread biological process by which "competent" bacteria take up free DNA, incorporate it into their genomes, and become genetically altered or "transformed". To curb often deleterious transformation by foreign DNA, several competent species preferentially take up their own DNA that contains specific DUS (DNA uptake sequence watermarks. Our recent finding that ComP is the long sought DUS receptor in Neisseria species paves the way for the functional analysis of the DUS-ComP interdependence which is reported here. By abolishing/modulating ComP levels in Neisseria meningitidis, we show that the enhancement of transformation seen in the presence of DUS is entirely dependent on ComP, which also controls transformation in the absence of DUS. While peripheral bases in the DUS were found to be less important, inner bases are essential since single base mutations led to dramatically impaired interaction with ComP and transformation. Strikingly, naturally occurring DUS variants in the genomes of human Neisseria commensals differing from DUS by only one or two bases were found to be similarly impaired for transformation of N. meningitidis. By showing that ComPsub from the N. subflava commensal specifically binds its cognate DUS variant and mediates DUS-enhanced transformation when expressed in a comP mutant of N. meningitidis, we confirm that a similar mechanism is used by all Neisseria species to promote transformation by their own, or closely related DNA. Together, these findings shed new light on the molecular events involved in the earliest step in natural transformation, and reveal an elegant mechanism for modulating horizontal gene transfer between competent species sharing the same niche.

Estrogen signalling and the DNA damage response in hormone dependent breast cancers

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C Elizabeth Caldon

2014-05-01

Full Text Available Estrogen is necessary for the normal growth and development of breast tissue, but high levels of estrogen are a major risk factor for breast cancer. One mechanism by which estrogen could contribute to breast cancer is via the induction of DNA damage. This perspective discusses the mechanisms by which estrogen alters the DNA damage response (DDR and DNA repair through the regulation of key effector proteins including ATM, ATR, CHK1, BRCA1 and p53 and the feedback on estrogen receptor signalling from these proteins. We put forward the hypothesis that estrogen receptor signalling converges to suppress effective DNA repair and apoptosis in favour of proliferation. This is important in hormone-dependent breast cancer as it will affect processing of estrogen-induced DNA damage, as well as other genotoxic insults. DDR and DNA repair proteins are frequently mutated or altered in estrogen responsive breast cancer which will further change the processing of DNA damage. Finally the action of estrogen signalling on DNA damage is also relevant to the therapeutic setting as the suppression of a DNA damage response by estrogen has the potential to alter the response of cancers to anti-hormone treatment or chemotherapy that induces DNA damage.

G protein-coupled estrogen receptor 1 (GPER1)/GPR30 increases ERK1/2 activity through PDZ motif-dependent and -independent mechanisms.

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Gonzalez de Valdivia, Ernesto; Broselid, Stefan; Kahn, Robin; Olde, Björn; Leeb-Lundberg, L M Fredrik

2017-06-16

G protein-coupled receptor 30 (GPR30), also called G protein-coupled estrogen receptor 1 (GPER1), is thought to play important roles in breast cancer and cardiometabolic regulation, but many questions remain about ligand activation, effector coupling, and subcellular localization. We showed recently that GPR30 interacts through the C-terminal type I PDZ motif with SAP97 and protein kinase A (PKA)-anchoring protein (AKAP) 5, which anchor the receptor in the plasma membrane and mediate an apparently constitutive decrease in cAMP production independently of G i/o Here, we show that GPR30 also constitutively increases ERK1/2 activity. Removing the receptor PDZ motif or knocking down specifically AKAP5 inhibited the increase, showing that this increase also requires the PDZ interaction. However, the increase was inhibited by pertussis toxin as well as by wortmannin but not by AG1478, indicating that G i/o and phosphoinositide 3-kinase (PI3K) mediate the increase independently of epidermal growth factor receptor transactivation. FK506 and okadaic acid also inhibited the increase, implying that a protein phosphatase is involved. The proposed GPR30 agonist G-1 also increased ERK1/2 activity, but this increase was only observed at a level of receptor expression below that required for the constitutive increase. Furthermore, deleting the PDZ motif did not inhibit the G-1-stimulated increase. Based on these results, we propose that GPR30 increases ERK1/2 activity via two G i/o -mediated mechanisms, a PDZ-dependent, apparently constitutive mechanism and a PDZ-independent G-1-stimulated mechanism. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular characterization of a novel human hybrid-type receptor that binds the alpha2-macroglobulin receptor-associated protein

DEFF Research Database (Denmark)

Jacobsen, Linda; Madsen, P; Moestrup, S K

1996-01-01

the corresponding cDNA. The gene, designated SORL1, maps to chromosome 11q 23/24 and encodes a 2214-residue type 1 receptor containing a furin cleavage site immediately preceding the N terminus determined in the purified protein. The receptor, designated sorLA-1, has a short cytoplasmic tail containing a tyrosine...... density lipoprotein receptor gene family receptors, and 3) six tandemly arranged fibronectin type III repeats also found in certain neural adhesion proteins. sorLA-1 may therefore be classified as a hybrid receptor. Northern blotting revealed specific mRNA transcripts in brain, spinal cord, and testis......The 39-40-kDa receptor-associated protein (RAP) binds to the members of the low density lipoprotein receptor gene family and functions as a specialized endoplasmic reticulum/Golgi chaperone. Using RAP affinity chromatography, we have purified a novel approximately 250-kDa brain protein and isolated...

Association between iron level, glucose impairment and increased DNA damage during pregnancy.

Science.gov (United States)

Zein, Salam; Rachidi, Samar; Shami, Nadine; Sharara, Iman; Cheikh-Ali, Khawla; Gauchez, Anne-Sophie; Moulis, Jean-Marc; Ayoubi, Jean-Marc; Salameh, Pascale; Hininger-Favier, Isabelle

2017-09-01

Elevated circulating ferritin has been reported to increase the risk of gestational diabetes mellitus (GDM). When high ferritin translates into high iron stores, iron excess is also a condition leading to free radical damage. We aimed to evaluate the relationship between oxidative stress (OS) induced by iron status and GDM risk in non iron-supplemented pregnant women. This was a pilot observational study conducted on 93 non-anemic pregnant women. Iron status was assessed at the first trimester of gestation. Blood sampling was done at 24-28 weeks' gestation for oral glucose tolerance test (OGTT), insulin and biological markers of oxidative damage tests. A significant increase in DNA damage was found in patients who developed GDM. Women with elevated DNA damage had a six-fold increased risk of developing GDM (Exp (B)=6.851, P=0.038; 95% CI [1.108-42.375]). The serum ferritin levels at first trimester were significantly correlated to lipid peroxidation (rho=0.24, p=0.012). The stratified analysis suggests that ferritin is a modifying factor for the correlation of oxidative stress (OS) and glucose intolerance. Moderate ferritin levels due to iron intake without iron-supplement, at early pregnancy is a modifying factor for the correlation of oxidative damage and glucose intolerance in pregnant women. Larger studies to evaluate the risk of food iron intake induced increased oxidative damage in offspring are warranted to propose nutrition advice regarding iron intake in women with a high risk of GDM. Copyright © 2016 Elsevier GmbH. All rights reserved.

Short-term exposure to oleandrin enhances responses to IL-8 by increasing cell surface IL-8 receptors

Science.gov (United States)

Raviprakash, Nune; Manna, Sunil Kumar

2014-01-01

BACKGROUND AND PURPOSE One of the first steps in host defence is the migration of leukocytes. IL-8 and its receptors are a chemokine system essential to such migration. Up-regulation of these receptors would be a viable strategy to treat dysfunctional host defence. Here, we studied the effects of the plant glycoside oleandrin on responses to IL-8 in a human monocytic cell line. EXPERIMENTAL APPROACH U937 cells were incubated with oleandrin (1-200 ng mL−1) for either 1 h (pulse) or for 24 h (non-pulse). Apoptosis; activation of NF-κB, AP-1 and NFAT; calcineurin activity and IL-8 receptors (CXCR1 and CXCR2) were measured using Western blotting, RT-PCR and reporter gene assays. KEY RESULTS Pulse exposure to oleandrin did not induce apoptosis or cytoxicity as observed after non-pulse exposure. Pulse exposure enhanced activation of NF-κB induced by IL-8 but not that induced by TNF-α, IL-1, EGF or LPS. Exposure to other apoptosis-inducing compounds (azadirachtin, resveratrol, thiadiazolidine, or benzofuran) did not enhance activation of NF-κB. Pulse exposure to oleandrin increased expression of IL-8 receptors and chemotaxis, release of enzymes and activation of NF-κB, NFAT and AP-1 along with increased IL-8-mediated calcineurin activation, and wound healing. Pulse exposure increased numbers of cell surface IL-8 receptors. CONCLUSIONS AND IMPLICATIONS Short-term (1 h; pulse) exposure to a toxic glycoside oleandrin, enhanced biological responses to IL-8 in monocytic cells, without cytoxicity. Pulse exposure to oleandrin could provide a viable therapy for those conditions where leukocyte migration is defective. PMID:24172227

Activation of GLP-1 receptors on vascular smooth muscle cells reduces the autoregulatory response in afferent arterioles and increases renal blood flow.

Science.gov (United States)

Jensen, Elisa P; Poulsen, Steen S; Kissow, Hannelouise; Holstein-Rathlou, Niels-Henrik; Deacon, Carolyn F; Jensen, Boye L; Holst, Jens J; Sorensen, Charlotte M

2015-04-15

Glucagon-like peptide (GLP)-1 has a range of extrapancreatic effects, including renal effects. The mechanisms are poorly understood, but GLP-1 receptors have been identified in the kidney. However, the exact cellular localization of the renal receptors is poorly described. The aim of the present study was to localize renal GLP-1 receptors and describe GLP-1-mediated effects on the renal vasculature. We hypothesized that renal GLP-1 receptors are located in the renal microcirculation and that activation of these affects renal autoregulation and increases renal blood flow. In vivo autoradiography using (125)I-labeled GLP-1, (125)I-labeled exendin-4 (GLP-1 analog), and (125)I-labeled exendin 9-39 (GLP-1 receptor antagonist) was performed in rodents to localize specific GLP-1 receptor binding. GLP-1-mediated effects on blood pressure, renal blood flow (RBF), heart rate, renin secretion, urinary flow rate, and Na(+) and K(+) excretion were investigated in anesthetized rats. Effects of GLP-1 on afferent arterioles were investigated in isolated mouse kidneys. Specific binding of (125)I-labeled GLP-1, (125)I-labeled exendin-4, and (125)I-labeled exendin 9-39 was observed in the renal vasculature, including afferent arterioles. Infusion of GLP-1 increased blood pressure, RBF, and urinary flow rate significantly in rats. Heart rate and plasma renin concentrations were unchanged. Exendin 9-39 inhibited the increase in RBF. In isolated murine kidneys, GLP-1 and exendin-4 significantly reduced the autoregulatory response of afferent arterioles in response to stepwise increases in pressure. We conclude that GLP-1 receptors are located in the renal vasculature, including afferent arterioles. Activation of these receptors reduces the autoregulatory response of afferent arterioles to acute pressure increases and increases RBF in normotensive rats. Copyright © 2015 the American Physiological Society.

Prenatal Alcohol Exposure Increases Histamine H3 Receptor-Mediated Inhibition of Glutamatergic Neurotransmission in Rat Dentate Gyrus.

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Varaschin, Rafael K; Allen, Nyika A; Rosenberg, Martina J; Valenzuela, C Fernando; Savage, Daniel D

2018-02-01

We have reported that prenatal alcohol exposure (PAE)-induced deficits in dentate gyrus, long-term potentiation (LTP), and memory are ameliorated by the histamine H 3 receptor inverse agonist ABT-239. Curiously, ABT-239 did not enhance LTP or memory in control offspring. Here, we initiated an investigation of how PAE alters histaminergic neurotransmission in the dentate gyrus and other brain regions employing combined radiohistochemical and electrophysiological approaches in vitro to examine histamine H 3 receptor number and function. Long-Evans rat dams voluntarily consumed either a 0% or 5% ethanol solution 4 hours each day throughout gestation. This pattern of drinking, which produces a mean peak maternal serum ethanol concentration of 60.8 ± 5.8 mg/dl, did not affect maternal weight gain, litter size, or offspring birthweight. Radiohistochemical studies in adult offspring revealed that specific [ 3 H]-A349821 binding to histamine H 3 receptors was not different in PAE rats compared to controls. However, H 3 receptor-mediated G i /G o protein-effector coupling, as measured by methimepip-stimulated [ 35 S]-GTPγS binding, was significantly increased in cerebral cortex, cerebellum, and dentate gyrus of PAE rats compared to control. A LIGAND analysis of detailed methimepip concentration-response curves in dentate gyrus indicated that PAE significantly elevates receptor-effector coupling by a lower affinity H 3 receptor population without significantly altering the affinities of H 3 receptor subpopulations. In agreement with the [ 35 S]-GTPγS studies, a similar range of methimepip concentrations also inhibited electrically evoked field excitatory postsynaptic potential responses and increased paired-pulse ratio, a measure of decreased glutamate release, to a significantly greater extent in dentate gyrus slices from PAE rats than in controls. These results suggest that a PAE-induced elevation in H 3 receptor-mediated inhibition of glutamate release from

Direct uptake and degradation of DNA by lysosomes

Science.gov (United States)

Fujiwara, Yuuki; Kikuchi, Hisae; Aizawa, Shu; Furuta, Akiko; Hatanaka, Yusuke; Konya, Chiho; Uchida, Kenko; Wada, Keiji; Kabuta, Tomohiro

2013-01-01

Lysosomes contain various hydrolases that can degrade proteins, lipids, nucleic acids and carbohydrates. We recently discovered “RNautophagy,” an autophagic pathway in which RNA is directly taken up by lysosomes and degraded. A lysosomal membrane protein, LAMP2C, a splice variant of LAMP2, binds to RNA and acts as a receptor for this pathway. In the present study, we show that DNA is also directly taken up by lysosomes and degraded. Like RNautophagy, this autophagic pathway, which we term “DNautophagy,” is dependent on ATP. The cytosolic sequence of LAMP2C also directly interacts with DNA, and LAMP2C functions as a receptor for DNautophagy, in addition to RNautophagy. Similarly to RNA, DNA binds to the cytosolic sequences of fly and nematode LAMP orthologs. Together with the findings of our previous study, our present findings suggest that RNautophagy and DNautophagy are evolutionarily conserved systems in Metazoa. PMID:23839276

Adaptive increase in D3 dopamine receptors in the brain reward circuits of human cocaine fatalities.

Science.gov (United States)

Staley, J K; Mash, D C

1996-10-01

The mesolimbic dopaminergic system plays a primary role in mediating the euphoric and rewarding effects of most abused drugs. Chronic cocaine use is associated with an increase in dopamine neurotransmission resulting from the blockade of dopamine uptake and is mediated by the activation of dopamine receptors. Recent studies have suggested that the D3 receptor subtype plays a pivotal role in the reinforcing effects of cocaine. The D3 receptor-preferring agonist 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT) is a reinforcer in rhesus monkeys trained to self-administer cocaine, but not in cocainenaive monkeys. In vitro autoradiographic localization of [3H]-(+)-7-OH-DPAT binding in the human brain demonstrated that D3 receptors were prevalent and highly localized over the ventromedial sectors of the striatum. Pharmacological characterization of [3H]-(+)-7-OH-DPAT binding to the human nucleus accumbens demonstrated a rank order of potency similar to that observed for binding to the cloned D3 receptor expressed in transfected cell lines. Region-of-interest analysis of [3H]-(+)-7-OH-DPAT binding to the D3 receptor demonstrated a one- to threefold elevation in the number of binding sites over particular sectors of the striatum and substantia nigra in cocaine overdose victims as compared with age-matched and drug-free control subjects. The elevated number of [3H]-(+)-7-OH-DPAT binding sites demonstrates that adaptive changes in the D3 receptor in the reward circuitry of the brain are associated with chronic cocaine abuse. These results suggest that the D3 receptor may be a useful target for drug development of anticocaine medications.

Angiotensin II type 2 receptor stimulation increases the rate of NG108-15 cell migration via actin depolymerization.

Science.gov (United States)

Kilian, Peter; Campbell, Shirley; Bilodeau, Lyne; Guimond, Marie-Odile; Roberge, Claude; Gallo-Payet, Nicole; Payet, Marcel Daniel

2008-06-01

Angiotensin II (Ang II) has been reported to induce migration in neuronal cell types. Using time-lapse microscopy, we show here that Ang II induces acceleration in NG108-15 cell migration. This effect was antagonized by PD123319, a selective AT2 receptor antagonist, but not by DUP753, a selective AT1 receptor antagonist, and was mimicked by the specific AT2 receptor agonist CGP42112. This Ang II-induced acceleration was not sensitive to the inhibition of previously described signaling pathways of the AT2 receptor, guanylyl cyclase/cyclic GMP or p42/p44 mapk cascades, but was abolished by pertussis toxin treatment and involved PP2A activation. Immunofluorescence studies indicate that Ang II or CGP42112 decreased the amount of filamentous actin at the leading edge of the cells. This decrease was accompanied by a concomitant increase in globular actin levels. Regulation of actin turnover in actin-based motile systems is known to be mainly under the control of the actin depolymerizing factor and cofilin. Basal migration speed decreased by 77.2% in cofilin-1 small interfering RNA-transfected NG108-15 cells, along with suppression of the effect of Ang II. In addition, the Ang II-induced increase in cell velocity was abrogated in serum-free medium as well as by genistein or okadaic acid treatment in a serum-containing medium. Such results indicate that the AT2 receptor increases the migration speed of NG108-15 cells and involves a tyrosine kinase activity, followed by phosphatase activation, which may be of the PP2A type. Therefore, the present study identifies actin depolymerization and cofilin as new targets of AT2 receptor action, in the context of cellular migration.

Cloning of zebrafish activin type IIB receptor (ActRIIB) cDNA and mRNA expression of ActRIIB in embryos and adult tissues.

Science.gov (United States)

Garg, R R; Bally-Cuif, L; Lee, S E; Gong, Z; Ni, X; Hew, C L; Peng, C

1999-07-20

A full-length cDNA encoding for activin type IIB receptor (ActRIIB) was cloned from zebrafish embryos. It encodes a protein with 509 amino acids consisting of a signal peptide, an extracellular ligand binding domain, a single transmembrane region, and an intracellular kinase domain with predicted serine/threonine specificity. The extracellular domain shows 74-91% sequence identity to human, bovine, mouse, rat, chicken, Xenopus and goldfish activin type IIB receptors, while the transmembrane region and the kinase domain show 67-78% and 82-88% identity to these known activin IIB receptors, respectively. In adult zebrafish, ActRIIB mRNA was detected by RT-PCR in the gonads, as well as in non-reproductive tissues, including the brain, heart and muscle. In situ hybridization on ovarian sections further localized ActRIIB mRNA to cytoplasm of oocytes at different stages of development. Using whole-mount in situ hybridization, ActRIIB mRNA was found to be expressed at all stages of embryogenesis examined, including the sphere, shield, tail bud, and 6-7 somite. These results provide the first evidence that ActRIIB mRNA is widely distributed in fish embryonic and adult tissues. Cloning of zebrafish ActRIIB demonstrates that this receptor is highly conserved during vertebrate evolution and provides a basis for further studies on the role of activin in reproduction and development in lower vertebrates.

Structure and function of DNA polymerase μ

International Nuclear Information System (INIS)

Matsumoto, Takuro; Maezawa, So

2013-01-01

DNA polymerases are enzymes playing the central role in DNA metabolism, including DNA replication, DNA repair and recombination. DNA polymerase μ (pol μ DNA polymerase λ (pol λ) and terminal deoxynucleotidyltransferase (TdT) in X family DNA polymerases function in non-homologous end-joining (NHEJ), which is the predonmiant repair pathway for DNA double-strand breaks (DSBs). NHEJ involves enzymes that capture both ends of the broken DNA strand, bring them together in a synaptic DNA-protein complex, and repair the DSB. Pol μ and pol λ fill in the gaps at the junction to maintain the genomic integrity. TdT synthesizes N region at the junction during V(D)J recombination and promotes diversity of immunoglobulin or T-cell receptor gene. Among these three polymerases, the regulatory mechanisms of pol μ remain rather unclear. We have approached the mechanism of pol μ from both sides of structure and cellular dynamics. Here, we propose some new insights into pol μ and the probable NHEJ model including our findings. (author)

High and increasing Oxa-51 DNA load predict mortality in Acinetobacter baumannii bacteremia: implication for pathogenesis and evaluation of therapy.

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Yu-Chung Chuang

Full Text Available BACKGROUND: While quantification of viral loads has been successfully employed in clinical medicine and has provided valuable insights and useful markers for several viral diseases, the potential of measuring bacterial DNA load to predict outcome or monitor therapeutic responses remains largely unexplored. We tested this possibility by investigating bacterial loads in Acinetobacter baumannii bacteremia, a rapidly increasing nosocomial infection characterized by high mortality, drug resistance, multiple and complicated risk factors, all of which urged the need of good markers to evaluate therapeutics. METHODS AND FINDINGS: We established a quantitative real-time PCR assay based on an A. baumannii-specific gene, Oxa-51, and conducted a prospective study to examine A. baumannii loads in 318 sequential blood samples from 51 adults patients (17 survivors, 34 nonsurvivors with culture-proven A. baumannii bacteremia in the intensive care units. Oxa-51 DNA loads were significantly higher in the nonsurvivors than survivors on day 1, 2 and 3 (P=0.03, 0.001 and 0.006, respectively. Compared with survivors, nonsurvivors had higher maximum Oxa-51 DNA load and a trend of increase from day 0 to day 3 (P<0.001, which together with Pitt bacteremia score were independent predictors for mortality by multivariate analysis (P=0.014 and 0.016, for maximum Oxa-51 DNA and change of Oxa-51 DNA, respectively. Kaplan-Meier analysis revealed significantly different survival curves in patients with different maximum Oxa-51 DNA and change of Oxa-51 DNA from day 0 to day 3. CONCLUSIONS: High Oxa-51 DNA load and its initial increase could predict mortality. Moreover, monitoring Oxa-51 DNA load in blood may provide direct parameters for evaluating new regimens against A. baumannii in future clinical studies.

Vitamin D receptor FokI genotype may modify the susceptibility to schizophrenia and bipolar mood disorder by regulation of dopamine D1 receptor gene expression.

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Ahmadi, S; Mirzaei, K; Hossein-Nezhad, A; Shariati, G

2012-10-01

This study is designed to test association of FOKI polymorphism in Vitamin D receptor (VDR) gene and its potential effect on expression of dopamine D1 receptor in schizophrenia and bipolar mood disorder as well as in healthy individuals. In this case-control study 196 patient with schizophrenia, 119 patients with bipolar mood disorder and 192 healthy individuals as the control group were recruited. All psychiatric disorders were diagnosed according to DSM IV criteria. Healthy control group denied any family history of such disorders. FOKI was genotyped by means of PCR-RFLP method. The mRNA was extracted from the peripheral blood mononuclear cells (PBMC) and the cDNA was synthesized. Frequency of ff genotype was more common in patients with bipolar disorders compared to the healthy control group (Odds ratio=1.84, 95% CI; 0.81 to 4.17) with increased relative risk (Relative risk=1.31, CI 95%; 0.86 to 1.99). There were significant differences between relative expressions of dopamine D1 receptor gene in various genotypes. Our results indicated that the ff genotype was associated with lower expression of dopamine D1 receptor gene. VDR as a nuclear receptor may contribute to bipolar disorders via modification of the expression of the neurotransmitters receptor such as dopamine.

Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage.

Science.gov (United States)

Waraky, Ahmed; Lin, Yingbo; Warsito, Dudi; Haglund, Felix; Aleem, Eiman; Larsson, Olle

2017-11-03

We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases ( e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G 1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Evolution of the Immune Repertoire with and without Somatic DNA Recombination

NARCIS (Netherlands)

Takumi, K.; Hogeweg, P.

1998-01-01

Repertoire of an immune system is a set of antigen receptors each having a unique specificity to bind an antigen. In many vertebrate species, antigen receptors are produced via combinatorial arrangements of DNA segments in specialized immune cells. Due to this molecular mechanism, repertoire

Parkinson's disease brain mitochondria have impaired respirasome assembly, age-related increases in distribution of oxidative damage to mtDNA and no differences in heteroplasmic mtDNA mutation abundance

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Keeney Paula M

2009-09-01

Full Text Available Abstract Background Sporadic Parkinson's disease (sPD is a nervous system-wide disease that presents with a bradykinetic movement disorder and is frequently complicated by depression and cognitive impairment. sPD likely has multiple interacting causes that include increased oxidative stress damage to mitochondrial components and reduced mitochondrial bioenergetic capacity. We analyzed mitochondria from postmortem sPD and CTL brains for evidence of oxidative damage to mitochondrial DNA (mtDNA, heteroplasmic mtDNA point mutations and levels of electron transport chain proteins. We sought to determine if sPD brains possess any mtDNA genotype-respiratory phenotype relationships. Results Treatment of sPD brain mtDNA with the mitochondrial base-excision repair enzyme 8-oxyguanosine glycosylase-1 (hOGG1 inhibited, in an age-dependent manner, qPCR amplification of overlapping ~2 kbase products; amplification of CTL brain mtDNA showed moderate sensitivity to hOGG1 not dependent on donor age. hOGG1 mRNA expression was not different between sPD and CTL brains. Heteroplasmy analysis of brain mtDNA using Surveyor nuclease® showed asymmetric distributions and levels of heteroplasmic mutations across mtDNA but no patterns that statistically distinguished sPD from CTL. sPD brain mitochondria displayed reductions of nine respirasome proteins (respiratory complexes I-V. Reduced levels of sPD brain mitochondrial complex II, III and V, but not complex I or IV proteins, correlated closely with rates of NADH-driven electron flow. mtDNA levels and PGC-1α expression did not differ between sPD and CTL brains. Conclusion PD brain mitochondria have reduced mitochondrial respiratory protein levels in complexes I-V, implying a generalized defect in respirasome assembly. These deficiencies do not appear to arise from altered point mutational burden in mtDNA or reduction of nuclear signaling for mitochondrial biogenesis, implying downstream etiologies. The origin of age

A candidate pheromone receptor and two odorant receptors of the hawkmoth Manduca sexta.

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Patch, Harland M; Velarde, Rodrigo A; Walden, Kimberly K O; Robertson, Hugh M

2009-05-01

In this study, we cloned and characterized three Manduca sexta odorant receptors (ORs). One receptor is a putative pheromone receptor expressed exclusively in a cell associated with male-specific type-I trichoid sensilla. We describe the results of real-time PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) experiments that show MsextaOR1 is expressed only in male antennae. In situ hybridization labels a single cell associated with type-1 trichoid sensilla, which houses two neurons that have been previously determined to respond to the major components of the pheromone blend. The second receptor, MsextaOR2, was discovered using degenerate primers designed to conserved motifs of a unique group ORs that share as much as 88% identity. Comparison of RT-PCR, qRT-PCR, and in situ hybridization results with those of ORs in the Drosophila melanogaster Or83b subfamily shows a strong sequence and expression pattern similarity. The third receptor, MsextaOR3, was found by 5'-end sequencing of a normalized and subtracted cDNA library from male M. sexta antennae. RT-PCR and qRT-PCR show that this receptor is expressed only in male and female antennae. These are the first ORs, including a putative pheromone receptor, to be described from M. sexta.

Increased yield of PCR products by addition of T4 gene 32 protein to the SMART PCR cDNA synthesis system.

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Villalva, C; Touriol, C; Seurat, P; Trempat, P; Delsol, G; Brousset, P

2001-07-01

Under certain conditions, T4 gene 32 protein is known to increase the efficiency of different enzymes, such as Taq DNA polymerase, reverse transcriptase, and telomerase. In this study, we compared the efficiency of the SMART PCR cDNA synthesis kit with and without the T4 gene 32 protein. The use of this cDNA synthesis procedure, in combination with T4 gene 32 protein, increases the yield of RT-PCR products from approximately 90% to 150%. This effect is even observed for long mRNA templates and low concentrations of total RNA (25 ng). Therefore, we suggest the addition of T4 gene 32 protein in the RT-PCR mixture to increase the efficiency of cDNA synthesis, particularly in cases when low amounts of tissue are used.

Increased rhythmicity in hypertensive arterial smooth muscle is linked to transient receptor potential canonical channels

DEFF Research Database (Denmark)

Chen, Xiaoping; Yang, Dachun; Ma, Shuangtao

2010-01-01

Vasomotion describes oscillations of arterial vascular tone due to synchronized changes of intracellular calcium concentrations. Since increased calcium influx into vascular smooth muscle cells from spontaneously hypertensive rats (SHR) has been associated with variances of transient receptor pot...

Genetic basis of olfactory cognition: extremely high level of DNA sequence polymorphism in promoter regions of the human olfactory receptor genes revealed using the 1000 Genomes Project dataset.

Science.gov (United States)

Ignatieva, Elena V; Levitsky, Victor G; Yudin, Nikolay S; Moshkin, Mikhail P; Kolchanov, Nikolay A

2014-01-01

The molecular mechanism of olfactory cognition is very complicated. Olfactory cognition is initiated by olfactory receptor proteins (odorant receptors), which are activated by olfactory stimuli (ligands). Olfactory receptors are the initial player in the signal transduction cascade producing a nerve impulse, which is transmitted to the brain. The sensitivity to a particular ligand depends on the expression level of multiple proteins involved in the process of olfactory cognition: olfactory receptor proteins, proteins that participate in signal transduction cascade, etc. The expression level of each gene is controlled by its regulatory regions, and especially, by the promoter [a region of DNA about 100-1000 base pairs long located upstream of the transcription start site (TSS)]. We analyzed single nucleotide polymorphisms using human whole-genome data from the 1000 Genomes Project and revealed an extremely high level of single nucleotide polymorphisms in promoter regions of olfactory receptor genes and HLA genes. We hypothesized that the high level of polymorphisms in olfactory receptor promoters was responsible for the diversity in regulatory mechanisms controlling the expression levels of olfactory receptor proteins. Such diversity of regulatory mechanisms may cause the great variability of olfactory cognition of numerous environmental olfactory stimuli perceived by human beings (air pollutants, human body odors, odors in culinary etc.). In turn, this variability may provide a wide range of emotional and behavioral reactions related to the vast variety of olfactory stimuli.

Changes in amount and intracellular distribution of androgen receptor in human foreskin as a function of age.

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Roehrborn, C G; Lange, J L; George, F W; Wilson, J D

1987-01-01

To provide insight into the factors that control growth of the penis we measured the amount and intracellular distribution of specific high affinity androgen receptor in foreskins obtained at circumcision from 49 males varying in age from newborn to 59 yr. Total (cytosolic plus nuclear extract) androgen receptor decreased from approximately 40 fmol/g tissue weight in newborn foreskins to approximately 25 fmol/g by 1 yr of age. The amount of receptor rose in childhood to approximately 180 fmol/g in the late teenage years and fell thereafter to approximately 20-40 fmol/g in men older than 40 yr. The amount of receptor in the nuclear fraction increased at the time of puberty and subsequently decreased in parallel with the decline in total receptor level. These changes in androgen-receptor amount are similar when expressed per milligram DNA or per milligram protein. Images PMID:3491838

Arsenic exposure from drinking water is associated with decreased gene expression and increased DNA methylation in peripheral blood

Energy Technology Data Exchange (ETDEWEB)

Ameer, Syeda Shegufta [Department of Laboratory Medicine, Division of Occupational and Environmental Medicine, Lund University, Lund (Sweden); Engström, Karin [Department of Laboratory Medicine, Division of Occupational and Environmental Medicine, Lund University, Lund (Sweden); Institute of Environmental Medicine, Unit of Metals & Health, Karolinska Institutet, Stockholm (Sweden); Hossain, Mohammad Bakhtiar [Department of Laboratory Medicine, Division of Occupational and Environmental Medicine, Lund University, Lund (Sweden); Concha, Gabriela [Science Department, Risk Benefit Assessment Unit, National Food Agency, Uppsala (Sweden); Vahter, Marie [Institute of Environmental Medicine, Unit of Metals & Health, Karolinska Institutet, Stockholm (Sweden); Broberg, Karin, E-mail: Karin.broberg@ki.se [Institute of Environmental Medicine, Unit of Metals & Health, Karolinska Institutet, Stockholm (Sweden)

2017-04-15

Background: Exposure to inorganic arsenic increases the risk of cancer and non-malignant diseases. Inefficient arsenic metabolism is a marker for susceptibility to arsenic toxicity. Arsenic may alter gene expression, possibly by altering DNA methylation. Objectives: To elucidate the associations between arsenic exposure, gene expression, and DNA methylation in peripheral blood, and the modifying effects of arsenic metabolism. Methods: The study participants, women from the Andes, Argentina, were exposed to arsenic via drinking water. Arsenic exposure was assessed as the sum of arsenic metabolites in urine (U-As), using high performance liquid-chromatography hydride-generation inductively-coupled-plasma-mass-spectrometry, and arsenic metabolism efficiency was assessed by the urinary fractions (%) of the individual metabolites. Genome-wide gene expression (N = 80 women) and DNA methylation (N = 93; 80 overlapping with gene expression) in peripheral blood were measured using Illumina DirectHyb HumanHT-12 v4.0 and Infinium Human-Methylation 450K BeadChip, respectively. Results: U-As concentrations, ranging 10–1251 μg/L, was associated with decreased gene expression: 64% of the top 1000 differentially expressed genes were down-regulated with increasing U-As. U-As was also associated with hypermethylation: 87% of the top 1000 CpGs were hypermethylated with increasing U-As. The expression of six genes and six individual CpG sites were significantly associated with increased U-As concentration. Pathway analyses revealed enrichment of genes related to cell death and cancer. The pathways differed somewhat depending on arsenic metabolism efficiency. We found no overlap between arsenic-related gene expression and DNA methylation for individual genes. Conclusions: Increased arsenic exposure was associated with lower gene expression and hypermethylation in peripheral blood, but with no evident overlap. - Highlights: • Women exposed to inorganic arsenic were studied for

Reduced DNA methylation of FKBP5 in Cushing's syndrome.

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Resmini, Eugenia; Santos, Alicia; Aulinas, Anna; Webb, Susan M; Vives-Gilabert, Yolanda; Cox, Olivia; Wand, Gary; Lee, Richard S

2016-12-01

FKBP5 encodes a co-chaperone of HSP90 protein that regulates intracellular glucocorticoid receptor sensitivity. When it is bound to the glucocorticoid receptor complex, cortisol binds with lower affinity to glucocorticoid receptor. Cushing's syndrome is associated with memory deficits, smaller hippocampal volumes, and wide range of cognitive impairments. We aimed at evaluating blood DNA methylation of FKBP5 and its relationship with memory and hippocampal volumes in Cushing's syndrome patients. Polymorphism rs1360780 in FKBP5 has also been assessed to determine whether genetic variations can also govern CpG methylation. Thirty-two Cushing's syndrome patients and 32 matched controls underwent memory tests, 3-Tesla MRI of the brain, and DNA extraction from total leukocytes. DNA samples were bisulfite treated, PCR amplified, and pyrosequenced to assess a total of 41CpG-dinucleotides in the introns 1, 2, 5, and 7 of FKBP5. Significantly lower intronic FKBP5 DNA methylation in CS patients compared to controls was observed in ten CpG-dinucleotides. DNA methylation at these CpGs correlated with left and right HV (Intron-2-Region-2-CpG-3: LHV, r = 0.73, p = 0.02; RHV, r = 0.58, p = 0.03). Cured and active CS patients showed both lower methylation of intron 2 (92.37, 91.8, and 93.34 %, respectively, p = 0.03 for both) and of intron 7 (77.08, 73.74, and 79.71 %, respectively, p = 0.02 and p < 0.01) than controls. Twenty-two subjects had the CC genotype, 34 had the TC genotype, and eight had the TT genotype. Lower average DNA methylation in intron 7 was observed in the TT subjects compared to CC (72.5vs. 79.5 %, p = 0.02) and to TC (72.5 vs. 79.0 %, p = 0.03). Our data demonstrate, for the first time, a reduction of intronic DNA methylation of FKBP5 in CS patients.

A monoclonal antibody against PDGF B-chain inhibits PDGF-induced DNA synthesis in C3H fibroblasts and prevents binding of PDGF to its receptor.

Science.gov (United States)

Vassbotn, F S; Langeland, N; Hagen, I; Holmsen, H

1990-09-01

A monoclonal antibody (MAb 6D11) against platelet-derived growth factor (PDGF) was studied. We found that the MAb 6D11 in concentrations equimolar to PDGF blocked the [3H]thymidine incorporation in C3H/10T1/2 C18 fibroblasts stimulated by PDGF B-B and PDGF A-B. This inhibition was overcome by high doses of PDGF. The [3H]thymidine incorporation stimulated by other growth factors (aFGF, bFGF and bombesin) was not inhibited by the antibody. The MAb 6D11 blocked receptor binding of PDGF B-B, but not PDGF A-A. These findings suggest that the MAb 6D11 abolishes PDGF-induced DNA synthesis by blocking PDGF receptor binding. In this communication we demonstrate an isoform-specific monoclonal antibody against PDGF.

Expression of glucocorticoid and progesterone nuclear receptor genes in archival breast cancer tissue

International Nuclear Information System (INIS)

Smith, Robert A; Lea, Rod A; Curran, Joanne E; Weinstein, Stephen R; Griffiths, Lyn R

2003-01-01

Previous studies in our laboratory have shown associations of specific nuclear receptor gene variants with sporadic breast cancer. In order to investigate these findings further, we conducted the present study to determine whether expression levels of the progesterone and glucocorticoid nuclear receptor genes vary in different breast cancer grades. RNA was extracted from paraffin-embedded archival breast tumour tissue and converted into cDNA. Sample cDNA underwent PCR using labelled primers to enable quantitation of mRNA expression. Expression data were normalized against the 18S ribosomal gene multiplex and analyzed using analysis of variance. Analysis of variance indicated a variable level of expression of both genes with regard to breast cancer grade (P = 0.00033 for glucocorticoid receptor and P = 0.023 for progesterone receptor). Statistical analysis indicated that expression of the progesterone nuclear receptor is elevated in late grade breast cancer tissue

Irbesartan increased PPARγ activity in vivo in white adipose tissue of atherosclerotic mice and improved adipose tissue dysfunction

International Nuclear Information System (INIS)

Iwai, Masaru; Kanno, Harumi; Senba, Izumi; Nakaoka, Hirotomo; Moritani, Tomozo; Horiuchi, Masatsugu

2011-01-01

Research highlights: → Atherosclerotic apolipoprotein E-deficient (ApoEKO) mice were treated with irbesartan. → Irbesartan decreased white adipose tissue weight without affecting body weight. → DNA-binding for PPARγ was increased in white adipose tissue in vivo by irbesartan. → Irbesartan increased adipocyte number in white adipose tissue. → Irbesatan increased the expression of adiponectin and leptin in white adipose tissue. -- Abstract: The effect of the PPARγ agonistic action of an AT 1 receptor blocker, irbesartan, on adipose tissue dysfunction was explored using atherosclerotic model mice. Adult male apolipoprotein E-deficient (ApoEKO) mice at 9 weeks of age were treated with a high-cholesterol diet (HCD) with or without irbesartan at a dose of 50 mg/kg/day for 4 weeks. The weight of epididymal and retroperitoneal adipose tissue was decreased by irbesartan without changing food intake or body weight. Treatment with irbesartan increased the expression of PPARγ in white adipose tissue and the DNA-binding activity of PPARγ in nuclear extract prepared from adipose tissue. The expression of adiponectin, leptin and insulin receptor was also increased by irbesartan. These results suggest that irbesartan induced activation of PPARγ and improved adipose tissue dysfunction including insulin resistance.

A Model of Post-Infection Fatigue Is Associated with Increased TNF and 5-HT2A Receptor Expression in Mice.

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Yvonne Couch

Full Text Available It is well documented that serotonin (5-HT plays an important role in psychiatric illness. For example, myalgic encephalomyelitis (ME/CFS, which is often provoked by infection, is a disabling illness with an unknown aetiology and diagnosis is based on symptom-specific criteria. However, 5-HT2A receptor expression and peripheral cytokines are known to be upregulated in ME. We sought to examine the relationship between the 5-HT system and cytokine expression following systemic bacterial endotoxin challenge (LPS, 0.5 mg/kg i.p., at a time when the acute sickness behaviours have largely resolved. At 24 hours post-injection mice exhibit no overt changes in locomotor behaviour, but do show increased immobility in a forced swim test, as well as decreased sucrose preference and reduced marble burying activity, indicating a depressive-like state. While peripheral IDO activity was increased after LPS challenge, central activity levels remained stable and there was no change in total brain 5-HT levels or 5-HIAA/5-HT. However, within the brain, levels of TNF and 5-HT2A receptor mRNA within various regions increased significantly. This increase in receptor expression is reflected by an increase in the functional response of the 5-HT2A receptor to agonist, DOI. These data suggest that regulation of fatigue and depressive-like moods after episodes of systemic inflammation may be regulated by changes in 5-HT receptor expression, rather than by levels of enzyme activity or cytokine expression in the CNS.

Cetuximab Induces Eme1-Mediated DNA Repair: a Novel Mechanism for Cetuximab Resistance

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Agnieszka Weinandy

2014-03-01

Full Text Available Overexpression of the epidermal growth factor receptor (EGFR is observed in a large number of neoplasms. The monoclonal antibody cetuximab/Erbitux is frequently applied to treat EGFR-expressing tumors. However, the application of cetuximab alone or in combination with radio- and/or chemotherapy often yields only little benefit for patients. In the present study, we describe a mechanism that explains resistance of both tumor cell lines and cultured primary human glioma cells to cetuximab. Treatment of these cells with cetuximab promoted DNA synthesis in the absence of increased proliferation, suggesting that DNA repair pathways were activated. Indeed, we observed that cetuximab promoted the activation of the DNA damage response pathway and prevented the degradation of essential meiotic endonuclease 1 homolog 1 (Eme1, a heterodimeric endonuclease involved in DNA repair. The increased levels of Eme1 were necessary for enhanced DNA repair, and the knockdown of Eme1 was sufficient to prevent efficient DNA repair in response to ultraviolet-C light or megavoltage irradiation. These treatments reduced the survival of tumor cells, an effect that was reversed by cetuximab application. Again, this protection was dependent on Eme1. Taken together, these results suggest that cetuximab initiates pathways that result in the stabilization of Eme1, thereby resulting in enhanced DNA repair. Accordingly, cetuximab enhances DNA repair, reducing the effectiveness of DNA-damaging therapies. This aspect should be considered when using cetuximab as an antitumor agent and suggests that Eme1 is a negative predictive marker.

Gene Transfer and Molecular Cloning of the Human NGF Receptor

Science.gov (United States)

Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

1986-04-01

Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

Molecular analysis of the nerve growth factor receptor

International Nuclear Information System (INIS)

Hempstead, B.; Patil, N.; Olson, K.; Chao, M.

1988-01-01

An essential molecule in the translocation of information by nerve growth factor (NGF) to responsive cells is the cell-surface receptor for NGF. This paper presents information on the genomic structure of the NGF receptor gene, NGF receptor models, and transfection of NGF receptors. Equilibrium binding of [ 125 I]NGF to cells reveals two distinct affinity states for the NGF receptor. The human NGF receptor gene is a single-copy gene, consisting of six exons that span 23 kb. The receptor gene is capable of being transferred to fibroblast cells from human genomic DNA and expressed at high levels. The constitutive nature of the receptor promoter sequence is a partial explanation of why this tissue-specific gene is expressed efficiently in a variety of nonneuronal cells after genomic gene transfer. The two kinetic forms of the NGF receptor appear to be encoded by the same protein, which is the product of a single gene

Obligatory Role of EP1 Receptors in the Increase in Cerebral Blood Flow Produced by Hypercapnia in the Mice.

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Ken Uekawa

Full Text Available Hypercapnia induces potent vasodilation in the cerebral circulation. Although it has long been known that prostanoids participate in the cerebrovascular effects of hypercapnia, the role of prostaglandin E2 (PGE2 and PGE2 receptors have not been fully investigated. In this study, we sought to determine whether cyclooxygenase-1 (COX-1-derived PGE2 and EP1 receptors are involved in the cerebrovascular response induced by hypercapnia. Cerebral blood flow (CBF was recorded by laser-Doppler flowmetry in the somatosenasory cortex of anesthetized male EP1-/- mice and wild type (WT littermates. In WT mice, neocortical application of the EP1 receptor antagonist SC-51089 attenuated the increase in CBF elicited by systemic hypercapnia (pCO2 = 50-60 mmHg. SC-51089 also attenuated the increase in CBF produced by neocortical treatment of arachidonic acid or PGE2. These CBF responses were also attenuated in EP1-/- mice. In WT mice, the COX-1 inhibitor SC-560, but not the COX-2 inhibitor NS-398, attenuated the hypercapnic CBF increase. Neocortical application of exogenous PGE2 restored the attenuation in resting CBF and the hypercapnic response induced by SC-560. In contrast, exogenous PGE2 failed to rescue the attenuation both in WT mice induced by SC-51089 and EP1-/- mice, attesting to the obligatory role of EP1 receptors in the response. These findings indicate that the hypercapnic vasodilatation depends on COX-1-derived PGE2 acting on EP1 receptors and highlight the critical role that COX-1-derived PGE2 and EP1 receptors play in the hypercapnic regulation of the cerebral circulation.

Increased binding of LDL and VLDL to apo B,E receptors of hepatic plasma membrane of rats treated with Fibernat.

Science.gov (United States)

Venkatesan, Nandini; Devaraj, S Niranjali; Devaraj, H

2003-10-01

Research has focussed on the hypocholesterolemic effects of certain types of dietary fiber such as enhancing conversion of hepatic cholesterol to bile acids or increase in catabolism of low density lipoprotein (LDL) via the apo B,E receptor. The effect of oral administration of a unique fibre cocktail of fenugreek seed powder, guar gum and wheat bran (Fibernat) and its varied effects on some aspects of lipid metabolism and cholesterol homeostasis in rats were examined. Rats were administered Fibernat along with the atherogenic diet containing 1.5 % cholesterol and 0.1 % cholic acid. Amounts of hepatic lipids, hepatic and fecal bile acids and activity of hepatic triglyceride lipase (HTGL) were determined. Transmission electron microscopic examination of the liver tissue and extent of uptake of (125)I-LDL and (125)I-VLDL by the hepatic apo B,E receptor was carried out. Food intake and body weight gain were similar between the 3 different dietary groups. Fibernat intake significantly increased apo B,E receptor expression in rat liver as reflected by an increase in the maximum binding capacity (B(max)) of the apo B,E receptor to (125)I-LDL and (125)I-VLDL. The activity of HTGL was increased by approximately 1.5-fold in Fibernat-fed rats as compared to those fed the atherogenic diet alone. A marked hypocholesterolemic effect was observed. Cholesterol homeostasis was achieved in Fibernat-fed rats. Two possible mechanisms are postulated to be responsible for the observed hypocholesterolemic effect a) an increase in conversion of cholesterol to bile acids and b) possibly by intra-luminal binding which resulted in increased fecal excretion of bile acids and neutral sterols. The resulting reduction in cholesterol content of liver cells coupled with upregulation of hepatic apo B,E receptors and increased clearance of circulating atherogenic lipoproteins-LDL and very low density lipoprotein (LDL and VLDL)-is the main mechanism involved in the hypocholesterolemic effect of

Transient Receptor Potential Vanilloid 4 Activation-Induced Increase in Glycine-Activated Current in Mouse Hippocampal Pyramidal Neurons

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Mengwen Qi

2018-02-01

Full Text Available Background/Aims: Glycine plays an important role in regulating hippocampal inhibitory/ excitatory neurotransmission through activating glycine receptors (GlyRs and acting as a co-agonist of N-methyl-d-aspartate-type glutamate receptors. Activation of transient receptor potential vanilloid 4 (TRPV4 is reported to inhibit hippocampal A-type γ-aminobutyric acid receptor, a ligand-gated chloride ion channel. GlyRs are also ligand-gated chloride ion channels and this paper aimed to explore whether activation of TRPV4 could modulate GlyRs. Methods: Whole-cell patch clamp recording was employed to record glycine-activated current (IGly and Western blot was conducted to assess GlyRs subunits protein expression. Results: Application of TRPV4 agonist (GSK1016790A or 5,6-EET increased IGly in mouse hippocampal CA1 pyramidal neurons. This action was blocked by specific antagonists of TRPV4 (RN-1734 or HC-067047 and GlyR (strychnine, indicating that activation of TRPV4 increases strychnine-sensitive GlyR function in mouse hippocampal pyramidal neurons. GSK1016790A-induced increase in IGly was significantly attenuated by protein kinase C (PKC (BIM II or D-sphingosine or calcium/calmodulin-dependent protein kinase II (CaMKII (KN-62 or KN-93 antagonists but was unaffected by protein kinase A or protein tyrosine kinase antagonists. Finally, hippocampal protein levels of GlyR α1 α2, α3 and β subunits were not changed by treatment with GSK1016790A for 30 min or 1 h, but GlyR α2, α3 and β subunits protein levels increased in mice that were intracerebroventricularly (icv. injected with GSK1016790A for 5 d. Conclusion: Activation of TRPV4 increases GlyR function and expression, and PKC and CaMKII signaling pathways are involved in TRPV4 activation-induced increase in IGly. This study indicates that GlyRs may be effective targets for TRPV4-induced modulation of hippocampal inhibitory neurotransmission.

Inhibition of transcription of abscisic acid in relation to the binding with DNA

International Nuclear Information System (INIS)

Basak, Sukla; Basu, P.S.; Biswas, B.B.

1976-01-01

Abscisic acid (ABA), a plant substance inhibits RNA synthesis in vivo and vitro. In vitro inhibition by ABA has been demonstrated in isolated RNA polymerase system from coconut endosperm chromatin. This inhibition can be partly reversible with indole acetic acid-receptor protein complex if added in the system. To find the mechanism of inhibition of transcription by ABA, it has been found that ABA (10 -4 -10 -5 M) can bind with DNA and can prevent strand separation. This binding increases the Tm value. ABA binds with DNA but not with RNA. Moreover, ABA can equally bind and prevent denaturation of calfthymus DNA and E. coli DNA. pH optimum for this binding is 8.0. The bound complex is resistant to alkali and alcohol but susceptible to acid below pH 5.0. It has further been demonstrated that free aBA at this pH is changed to another component which has tentatively been identified as lactone form of ABA. (author)

Ku proteins function as corepressors to regulate farnesoid X receptor-mediated gene expression

International Nuclear Information System (INIS)

Ohno, Masae; Kunimoto, Masaaki; Nishizuka, Makoto; Osada, Shigehiro; Imagawa, Masayoshi

2009-01-01

The farnesoid X receptor (FXR; NR1H4) is a member of the nuclear receptor superfamily and regulates the expression of genes involved in enterohepatic circulation and the metabolism of bile acids. Based on functional analyses, nuclear receptors are divided into regions A-F. To explore the cofactors interacting with FXR, we performed a pull-down assay using GST-fused to the N-terminal A/B region and the C region, which are required for the ligand-independent transactivation and DNA-binding, respectively, of FXR, and nuclear extracts from HeLa cells. We identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku80, and Ku70 as FXR associated factors. These proteins are known to have an important role in DNA repair, recombination, and transcription. DNA-PKcs mainly interacted with the A/B region of FXR, whereas the Ku proteins interacted with the C region and with the D region (hinge region). Chromatin immunoprecipitation assays revealed that the Ku proteins associated with FXR on the bile salt export pump (BSEP) promoter. Furthermore, we demonstrated that ectopic expression of the Ku proteins decreased the promoter activity and expression of BSEP gene mediated by FXR. These results suggest that the Ku proteins function as corepressors for FXR.

Functional cloning using pFB retroviral cDNA expression libraries.

Science.gov (United States)

Felts, Katherine A; Chen, Keith; Zaharee, Kim; Sundar, Latha; Limjoco, Jamie; Miller, Anna; Vaillancourt, Peter

2002-09-01

Retroviral cDNA expression libraries allow the efficient introduction of complex cDNA libraries into virtually any mitotic cell type for screening based on gene function. The cDNA copy number per cell can be easily controlled by adjusting the multiplicity of infection, thus cell populations may be generated in which >90% of infected cells contain one to three cDNAs. We describe the isolation of two known oncogenes and one cell-surface receptor from a human Burkitt's lymphoma (Daudi) cDNA library inserted into the high-titer retroviral vector pFB.

RETINOID RECEPTORS IN BONE AND THEIR ROLE IN BONE REMODELING

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Petra eHenning

2015-03-01

Full Text Available Vitamin A (retinol is a necessary and important constituent of the body which is provided by food intake of retinyl esters and carotenoids. Vitamin A is known best for being important for vision, but in addition to the eye, vitamin A is necessary in numerous other organs in the body, including the skeleton. Vitamin A is converted to an active compound, all-trans-retinoic acid (ATRA, which is responsible for most of its biological actions. ATRA binds to intracellular nuclear receptors called retinoic acid receptors (RAR, RAR, RAR. RARs and closely related retinoid X receptors (RXR, RXR, RXR form heterodimers which bind to DNA and function as ligand activated transcription factors. It has been known for many years that hypervitaminosis A promotes skeleton fragility by increasing osteoclast formation and decreasing cortical bone mass. Some epidemiological studies have suggested that increased intake of vitamin A and increased serum levels of retinoids may decrease bone mineral density and increase fracture rate, but the literature on this is not conclusive. The current review summarizes how vitamin A is taken up by the intestine, metabolized, stored in the liver and processed to ATRA. ATRA’s effects on formation and activity of osteoclasts and osteoblasts are outlined, and a summary of clinical data pertaining to vitamin A and bone is presented.

Extracellular matrix components influence DNA synthesis of rat hepatocytes in primary culture

International Nuclear Information System (INIS)

Sawada, N.; Tomomura, A.; Sattler, C.A.; Sattler, G.L.; Kleinman, H.K.; Pitot, H.C.

1986-01-01

The effects of several extracellular matrix components (EMCs) - fibronectin (Fn), laminin (Ln), type I (C-I) and type IV (C-IV) collagen - on DNA synthesis in rat hepatocytes in primary culture were examined by both quantitative scintillation spectrometry and autoradiography of [ 3 H]thymidine incorporation. Hepatocytes cultured on Fn showed the most active DNA synthesis initiated by epidermal growth factor (EGF) with decreasing levels of [ 3 H]thymidine uptake exhibited in the cell cultured on C-IV, C-I, and Ln, respectively. The decreasing level of DNA synthesis in hepatocytes cultured on Fn, C-IV, C-I, and Ln respectively was not influenced by cell density. The number of EGF receptors of hepatocytes was also not influenced by EMCs. These data suggest that EMCs modify hepatocyte DNA synthesis by means of post-EGF-receptor mechanisms which are regulated by both growth factors and cell density

Isolation of the human anionic glutathione S-transferase cDNA and the relation of its gene expression to estrogen-receptor content in primary breast cancer

International Nuclear Information System (INIS)

Moscow, J.A.; Townsend, A.J.; Goldsmith, M.E.; Whang-Peng, J.; Vickers, P.J.; Poisson, R.; Legault-Poisson, S.; Myers, C.E.; Cowan, K.H.

1988-01-01

The development of multidrug resistance in MCF7 human breast cancer cells is associated with overexpression of P-glycoprotein, changes in activities of several detoxication enzymes, and loss of hormone sensitivity and estrogen receptors (ERs). The authors have cloned the cDNA for one of the drug-detoxifying enzymes overexpressed in multidrug-resistant MCF7 cells (Adr R MCF7), the anionic isozyme of glutathione S-transferase (GSTπ). Hybridization with this GSTπ cDNA, GSTπ-1, demonstrated that increased GSTπ activity in Adr R MCF7 cells is associated with overexpression but not with amplification of the gene. They mapped the GSTπ gene to human chromosome 11q13 by in situ hybridization. Since multidrug resistance and GSTπ overexpression are associated with the loss of ERs in Adr R MCF7 cells, they examined several other breast cancer cell lines that were not selected for drug resistance. In each of these cell lines they found an inverse association between GSTπ expression and ER content. They also examined RNA from 21 primary breast cancers and found a similar association between GSTπ expression and ER content in vivo. The finding of similar patterns of expression of a drug-detoxifying enzyme and of ERs in vitro as well as in vivo suggests that ER-negative breast cancer cells may have greater protection against antineoplastic agents conferred by GSTπ than ER-positive tumors

Detection of melatonin receptor mRNA in human muscle

International Nuclear Information System (INIS)

Li Lei

2004-01-01

To verify the expression of melatonin receptor mRNA in human, muscle, muscle beside vertebrae was collected to obtain total RNA and the mRNA of melatonin receptor was detected by RT-PCR method. The electrophoretic results of RT-PCR products by mt 1 and MT 2 primer were all positive and the sequence is corresponding with human melatonin receptor cDNA. It suggests that melatonin may act on the muscle beside vertebrae directly and regulate its growth and development. (authors)

Dopamine D4 receptor activation increases hippocampal gamma oscillations by enhancing synchronization of fast-spiking interneurons.

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Richard Andersson

Full Text Available BACKGROUND: Gamma oscillations are electric activity patterns of the mammalian brain hypothesized to serve attention, sensory perception, working memory and memory encoding. They are disrupted or altered in schizophrenic patients with associated cognitive deficits, which persist in spite of treatment with antipsychotics. Because cognitive symptoms are a core feature of schizophrenia it is relevant to explore signaling pathways that potentially regulate gamma oscillations. Dopamine has been reported to decrease gamma oscillation power via D1-like receptors. Based on the expression pattern of D4 receptors (D4R in hippocampus, and pharmacological effects of D4R ligands in animals, we hypothesize that they are in a position to regulate gamma oscillations as well. METHODOLOGY/PRINCIPAL FINDINGS: To address this hypothesis we use rat hippocampal slices and kainate-induced gamma oscillations. Local field potential recordings as well as intracellular recordings of pyramidal cells, fast-spiking and non-fast-spiking interneurons were carried out. We show that D4R activation with the selective ligand PD168077 increases gamma oscillation power, which can be blocked by the D4R-specific antagonist L745,870 as well as by the antipsychotic drug Clozapine. Pyramidal cells did not exhibit changes in excitatory or inhibitory synaptic current amplitudes, but inhibitory currents became more coherent with the oscillations after application of PD168077. Fast-spiking, but not non-fast spiking, interneurons, increase their action potential phase-coupling and coherence with regard to ongoing gamma oscillations in response to D4R activation. Among several possible mechanisms we found that the NMDA receptor antagonist AP5 also blocks the D4R mediated increase in gamma oscillation power. CONCLUSIONS/SIGNIFICANCE: We conclude that D4R activation affects fast-spiking interneuron synchronization and thereby increases gamma power by an NMDA receptor-dependent mechanism. This

Effect of insulin analogues on insulin/IGF1 hybrid receptors: increased activation by glargine but not by its metabolites M1 and M2.

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Cécile Pierre-Eugene

Full Text Available BACKGROUND: In diabetic patients, the pharmacokinetics of injected human insulin does not permit optimal control of glycemia. Fast and slow acting insulin analogues have been developed, but they may have adverse properties, such as increased mitogenic or anti-apoptotic signaling. Insulin/IGF1 hybrid receptors (IR/IGF1R, present in most tissues, have been proposed to transmit biological effects close to those of IGF1R. However, the study of hybrid receptors is difficult because of the presence of IR and IGF1R homodimers. Our objective was to perform the first study on the pharmacological properties of the five marketed insulin analogues towards IR/IGF1R hybrids. METHODOLOGY: To study the effect of insulin analogues on IR/IGF1R hybrids, we used our previously developed Bioluminescence Resonance Energy Transfer (BRET assay that permits specific analysis of the pharmacological properties of hybrid receptors. Moreover, we have developed a new, highly sensitive BRET-based assay to monitor phophatidylinositol-3 phosphate (PIP(3 production in living cells. Using this assay, we performed a detailed pharmacological analysis of PIP(3 production induced by IGF1, insulin and insulin analogues in living breast cancer-derived MCF-7 and MDA-MB231 cells. RESULTS: Among the five insulin analogues tested, only glargine stimulated IR/IGF1R hybrids with an EC50 that was significantly lower than insulin and close to that of IGF1. Glargine more efficiently stimulated PIP(3 production in MCF-7 cells but not in MDA-MB231 cells as compared to insulin. In contrast, glargine metabolites M1 and M2 showed lower potency for hybrid receptors stimulation, PIP(3 production, Akt and Erk1/2 phosphorylation and DNA synthesis in MCF-7 cells, compared to insulin. CONCLUSION: Glargine, possibly acting through IR/IGF1R hybrids, displays higher potency, whereas its metabolites M1 and M2 display lower potency than insulin for the stimulation of proliferative/anti-apoptotic pathways in

Next-generation sequencing-based detection of circulating tumour DNA After allogeneic stem cell transplantation for lymphoma.

Science.gov (United States)

Herrera, Alex F; Kim, Haesook T; Kong, Katherine A; Faham, Malek; Sun, Heather; Sohani, Aliyah R; Alyea, Edwin P; Carlton, Victoria E; Chen, Yi-Bin; Cutler, Corey S; Ho, Vincent T; Koreth, John; Kotwaliwale, Chitra; Nikiforow, Sarah; Ritz, Jerome; Rodig, Scott J; Soiffer, Robert J; Antin, Joseph H; Armand, Philippe

2016-12-01

Next-generation sequencing (NGS)-based circulating tumour DNA (ctDNA) detection is a promising monitoring tool for lymphoid malignancies. We evaluated whether the presence of ctDNA was associated with outcome after allogeneic haematopoietic stem cell transplantation (HSCT) in lymphoma patients. We studied 88 patients drawn from a phase 3 clinical trial of reduced-intensity conditioning HSCT in lymphoma. Conventional restaging and collection of peripheral blood samples occurred at pre-specified time points before and after HSCT and were assayed for ctDNA by sequencing of the immunoglobulin or T-cell receptor genes. Tumour clonotypes were identified in 87% of patients with adequate tumour samples. Sixteen of 19 (84%) patients with disease progression after HSCT had detectable ctDNA prior to progression at a median of 3·7 months prior to relapse/progression. Patients with detectable ctDNA 3 months after HSCT had inferior progression-free survival (PFS) (2-year PFS 58% vs. 84% in ctDNA-negative patients, P = 0·033). In multivariate models, detectable ctDNA was associated with increased risk of progression/death (Hazard ratio 3·9, P = 0·003) and increased risk of relapse/progression (Hazard ratio 10·8, P = 0·0006). Detectable ctDNA is associated with an increased risk of relapse/progression, but further validation studies are necessary to confirm these findings and determine the clinical utility of NGS-based minimal residual disease monitoring in lymphoma patients after HSCT. © 2016 John Wiley & Sons Ltd.

A feed-forward regulation of endothelin receptors by c-Jun in human non-pigmented ciliary epithelial cells and retinal ganglion cells.

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Junming Wang

Full Text Available c-Jun, c-Jun N-terminal kinase(JNK and endothelin B (ETB receptor have been shown to contribute to the pathogenesis of glaucoma. Previously, we reported that an increase of c-Jun and CCAAT/enhancer binding protein β (C/EBPβ immunohistostaining is associated with upregulation of the ETB receptor within the ganglion cell layer of rats with elevated intraocular pressure (IOP. In addition, both transcription factors regulate the expression of the ETB receptor in human non-pigmented ciliary epithelial cells (HNPE. The current study addressed the mechanisms by which ET-1 produced upregulation of ET receptors in primary rat retinal ganglion cells (RGCs and HNPE cells. Treatment of ET-1 and ET-3 increased the immunocytochemical staining of c-Jun and C/EBPβ in primary rat RGCs and co-localization of both transcription factors was observed. A marked increase in DNA binding activity of AP-1 and C/EBPβ as well as elevated protein levels of c-Jun and c-Jun-N-terminal kinase (JNK were detected following ET-1 treatment in HNPE cells. Overexpression of ETA or ETB receptor promoted the upregulation of c-Jun and also elevated its promoter activity. In addition, upregulation of C/EBPβ augmented DNA binding and mRNA expression of c-Jun, and furthermore, the interaction of c-Jun and C/EBPβ was confirmed using co-immunoprecipitation. Apoptosis of HNPE cells was identified following ET-1 treatment, and overexpression of the ETA or ETB receptor produced enhanced apoptosis. ET-1 mediated upregulation of c-Jun and C/EBPβ and their interaction may represent a novel mechanism contributing to the regulation of endothelin receptor expression. Reciprocally, c-Jun was also found to regulate the ET receptors and C/EBPβ appeared to play a regulatory role in promoting expression of c-Jun. Taken together, the data suggests that ET-1 triggers the upregulation of c-Jun through both ETA and ETB receptors, and conversely c-Jun also upregulates endothelin receptor expression

Systemic blockade of dopamine D2-like receptors increases high-voltage spindles in the globus pallidus and motor cortex of freely moving rats.

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Chen Yang

Full Text Available High-voltage spindles (HVSs have been reported to appear spontaneously and widely in the cortical-basal ganglia networks of rats. Our previous study showed that dopamine depletion can significantly increase the power and coherence of HVSs in the globus pallidus (GP and motor cortex of freely moving rats. However, it is unclear whether dopamine regulates HVS activity by acting on dopamine D₁-like receptors or D₂-like receptors. We employed local-field potential and electrocorticogram methods to simultaneously record the oscillatory activities in the GP and primary motor cortex (M1 in freely moving rats following systemic administration of dopamine receptor antagonists or saline. The results showed that the dopamine D₂-like receptor antagonists, raclopride and haloperidol, significantly increased the number and duration of HVSs, and the relative power associated with HVS activity in the GP and M1 cortex. Coherence values for HVS activity between the GP and M1 cortex area were also significantly increased by dopamine D₂-like receptor antagonists. On the contrary, the selective dopamine D₁-like receptor antagonist, SCH23390, had no significant effect on the number, duration, or relative power of HVSs, or HVS-related coherence between M1 and GP. In conclusion, dopamine D₂-like receptors, but not D₁-like receptors, were involved in HVS regulation. This supports the important role of dopamine D₂-like receptors in the regulation of HVSs. An siRNA knock-down experiment on the striatum confirmed our conclusion.

Systemic blockade of dopamine D2-like receptors increases high-voltage spindles in the globus pallidus and motor cortex of freely moving rats.

Science.gov (United States)

Yang, Chen; Ge, Shun-Nan; Zhang, Jia-Rui; Chen, Lei; Yan, Zhi-Qiang; Heng, Li-Jun; Zhao, Tian-Zhi; Li, Wei-Xin; Jia, Dong; Zhu, Jun-Ling; Gao, Guo-Dong

2013-01-01

High-voltage spindles (HVSs) have been reported to appear spontaneously and widely in the cortical-basal ganglia networks of rats. Our previous study showed that dopamine depletion can significantly increase the power and coherence of HVSs in the globus pallidus (GP) and motor cortex of freely moving rats. However, it is unclear whether dopamine regulates HVS activity by acting on dopamine D₁-like receptors or D₂-like receptors. We employed local-field potential and electrocorticogram methods to simultaneously record the oscillatory activities in the GP and primary motor cortex (M1) in freely moving rats following systemic administration of dopamine receptor antagonists or saline. The results showed that the dopamine D₂-like receptor antagonists, raclopride and haloperidol, significantly increased the number and duration of HVSs, and the relative power associated with HVS activity in the GP and M1 cortex. Coherence values for HVS activity between the GP and M1 cortex area were also significantly increased by dopamine D₂-like receptor antagonists. On the contrary, the selective dopamine D₁-like receptor antagonist, SCH23390, had no significant effect on the number, duration, or relative power of HVSs, or HVS-related coherence between M1 and GP. In conclusion, dopamine D₂-like receptors, but not D₁-like receptors, were involved in HVS regulation. This supports the important role of dopamine D₂-like receptors in the regulation of HVSs. An siRNA knock-down experiment on the striatum confirmed our conclusion.

DP97, a DEAD box DNA/RNA helicase, is a target gene-selective co-regulator of the constitutive androstane receptor

International Nuclear Information System (INIS)

Kanno, Yuichiro; Serikawa, Takafumi; Inajima, Jun; Inouye, Yoshio

2012-01-01

Highlights: ► DP97 interacts with nuclear receptor CAR. ► DP97 enhances CAR-mediated transcriptional activation. ► DP97 synergistically enhances transactivity of CAR by the co-expression of SRC-1 or PGC1α. ► DP97 is a gene-selective co-activator for hCAR. -- Abstract: The constitutive androstane receptor (CAR) plays a key role in the expression of xenobiotic/steroid and drug metabolizing enzymes and their transporters. In this study, we demonstrated that DP97, a member of the DEAD box DNA/RNA helicase protein family, is a novel CAR-interacting protein. Using HepG2 cells expressing human CAR in the presence of tetracycline, we showed that knockdown of DP97 with small interfering RNAs suppressed tetracycline-inducible mRNA expression of CYP2B6 and UGT1A1 but not CYP3A4. Thus, DP97 was found to be a gene (or promoter)-selective co-activator for hCAR. DP97-mediated CAR transactivation was synergistically enhanced by the co-expression of SRC-1 or PGC1α, therefore it might act as mediator between hCAR and appropriate co-activators.

Receptor-receptor interactions within receptor mosaics. Impact on neuropsychopharmacology.

Science.gov (United States)

Fuxe, K; Marcellino, D; Rivera, A; Diaz-Cabiale, Z; Filip, M; Gago, B; Roberts, D C S; Langel, U; Genedani, S; Ferraro, L; de la Calle, A; Narvaez, J; Tanganelli, S; Woods, A; Agnati, L F

2008-08-01

Future therapies for diseases associated with altered dopaminergic signaling, including Parkinson's disease, schizophrenia and drug addiction or drug dependence may substantially build on the existence of intramembrane receptor-receptor interactions within dopamine receptor containing receptor mosaics (RM; dimeric or high-order receptor oligomers) where it is believed that the dopamine D(2) receptor may operate as the 'hub receptor' within these complexes. The constitutive adenosine A(2A)/dopamine D(2) RM, located in the dorsal striato-pallidal GABA neurons, are of particular interest in view of the demonstrated antagonistic A(2A)/D(2) interaction within these heteromers; an interaction that led to the suggestion and later demonstration that A(2A) antagonists could be used as novel anti-Parkinsonian drugs. Based on the likely existence of A(2A)/D(2)/mGluR5 RM located both extrasynaptically on striato-pallidal GABA neurons and on cortico-striatal glutamate terminals, multiple receptor-receptor interactions within this RM involving synergism between A(2A)/mGluR5 to counteract D(2) signaling, has led to the proposal of using combined mGluR5 and A(2A) antagonists as a future anti-Parkinsonian treatment. Based on the same RM in the ventral striato-pallidal GABA pathways, novel strategies for the treatment of schizophrenia, building on the idea that A(2A) agonists and/or mGluR5 agonists will help reduce the increased dopaminergic signaling associated with this disease, have been suggested. Such treatment may ensure the proper glutamatergic drive from the mediodorsal thalamic nucleus to the prefrontal cortex, one which is believed to be reduced in schizophrenia due to a dominance of D(2)-like signaling in the ventral striatum. Recently, A(2A) receptors also have been shown to counteract the locomotor and sensitizing actions of cocaine and increases in A(2A) receptors have also been observed in the nucleus accumbens after extended cocaine self-administration, probably

Long-term activation of group I metabotropic glutamate receptors increases functional TRPV1-expressing neurons in mouse dorsal root ganglia

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Takayoshi eMasuoka

2016-03-01

Full Text Available Damaged tissues release glutamate and other chemical mediators for several hours. These chemical mediators contribute to modulation of pruritus and pain. Herein, we investigated the effects of long-term activation of excitatory glutamate receptors on functional expression of transient receptor potential vaniloid type 1 (TRPV1 in dorsal root ganglion (DRG neurons and then on thermal pain behavior. In order to detect the TRPV1-mediated responses in cultured DRG neurons, we monitored intracellular calcium responses to capsaicin, a TRPV1 agonist, with Fura-2. Long-term (4 h treatment with glutamate receptor agonists (glutamate, quisqualate or DHPG increased the proportion of neurons responding to capsaicin through activation of metabotropic glutamate receptor mGluR1, and only partially through the activation of mGluR5; engagement of these receptors was evident in neurons responding to allylisothiocyanate (AITC, a transient receptor potential ankyrin type 1 (TRPA1 agonist. Increase in the proportion was suppressed by phospholipase C, protein kinase C, mitogen/extracellular signal-regulated kinase, p38 mitogen-activated protein kinase or transcription inhibitors. Whole-cell recording was performed to record TRPV1-mediated membrane current; TRPV1 current density significantly increased in the AITC-sensitive neurons after the quisqualate treatment. To elucidate the physiological significance of this phenomenon, a hot plate test was performed. Intraplantar injection of quisqualate or DHPG induced heat hyperalgesia that lasted for 4 h post injection. This chronic hyperalgesia was attenuated by treatment with either mGluR1 or mGluR5 antagonists. These results suggest that long-term activation of mGluR1/5 by peripherally released glutamate may increase the number of neurons expressing functional TRPV1 in DRG, which may be strongly associated with chronic hyperalgesia.

DNA clasping by mycobacterial HU: the C-terminal region of HupB mediates increased specificity of DNA binding.

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Sandeep Kumar

Full Text Available BACKGROUND: HU a small, basic, histone like protein is a major component of the bacterial nucleoid. E. coli has two subunits of HU coded by hupA and hupB genes whereas Mycobacterium tuberculosis (Mtb has only one subunit of HU coded by ORF Rv2986c (hupB gene. One noticeable feature regarding Mtb HupB, based on sequence alignment of HU orthologs from different bacteria, was that HupB(Mtb bears at its C-terminal end, a highly basic extension and this prompted an examination of its role in Mtb HupB function. METHODOLOGY/PRINCIPAL FINDINGS: With this objective two clones of Mtb HupB were generated; one expressing full length HupB protein (HupB(Mtb and another which expresses only the N terminal region (first 95 amino acid of hupB (HupB(MtbN. Gel retardation assays revealed that HupB(MtbN is almost like E. coli HU (heat stable nucleoid protein in terms of its DNA binding, with a binding constant (K(d for linear dsDNA greater than 1000 nM, a value comparable to that obtained for the HUalphaalpha and HUalphabeta forms. However CTR (C-terminal Region of HupB(Mtb imparts greater specificity in DNA binding. HupB(Mtb protein binds more strongly to supercoiled plasmid DNA than to linear DNA, also this binding is very stable as it provides DNase I protection even up to 5 minutes. Similar results were obtained when the abilities of both proteins to mediate protection against DNA strand cleavage by hydroxyl radicals generated by the Fenton's reaction, were compared. It was also observed that both the proteins have DNA binding preference for A:T rich DNA which may occur at the regulatory regions of ORFs and the oriC region of Mtb. CONCLUSIONS/SIGNIFICANCE: These data thus point that HupB(Mtb may participate in chromosome organization in-vivo, it may also play a passive, possibly an architectural role.

Dexamethasone Enhances 1α,25-Dihydroxyvitamin D3 Effects by Increasing Vitamin D Receptor Transcription*

Science.gov (United States)

Hidalgo, Alejandro A.; Deeb, Kristin K.; Pike, J. Wesley; Johnson, Candace S.; Trump, Donald L.

2011-01-01

Calcitriol, the active form of vitamin D, in combination with the glucocorticoid dexamethasone (Dex) has been shown to increase the antitumor effects of calcitriol in squamous cell carcinoma. In this study we found that pretreatment with Dex potentiates calcitriol effects by inhibiting cell growth and increasing vitamin D receptor (VDR) and VDR-mediated transcription. Treatment with actinomycin D inhibits Vdr mRNA synthesis, indicating that Dex regulates VDR expression at transcriptional level. Real time PCR shows that treatment with Dex increases Vdr transcripts in a time- and a dose-dependent manner, indicating that Dex directly regulates expression of Vdr. RU486, an inhibitor of glucocorticoids, inhibits Dex-induced Vdr expression. In addition, the silencing of glucocorticoid receptor (GR) abolishes the induction of Vdr by Dex, indicating that Dex increases Vdr transcripts in a GR-dependent manner. A fragment located 5.2 kb upstream of Vdr transcription start site containing two putative glucocorticoid response elements (GREs) was evaluated using a luciferase-based reporter assay. Treatment with 100 nm Dex induces transcription of luciferase driven by the fragment. Deletion of the GRE distal to transcription start site was sufficient to abolish Dex induction of luciferase. Also, chromatin immunoprecipitation reveals recruitment of GR to distal GRE with Dex treatment. We conclude that Dex increases VDR and vitamin D effects by increasing Vdr de novo transcription in a GR-dependent manner. PMID:21868377

Genetic basis of olfactory cognition: extremely high level of DNA sequence polymorphism in promoter regions of the human olfactory receptor genes revealed using the 1000 Genomes Project dataset

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Elena V. Ignatieva

2014-03-01

Full Text Available The molecular mechanism of olfactory cognition is very complicated. Olfactory cognition is initiated by olfactory receptor proteins (odorant receptors, which are activated by olfactory stimuli (ligands. Olfactory receptors are the initial player in the signal transduction cascade producing a nerve impulse, which is transmitted to the brain. The sensitivity to a particular ligand depends on the expression level of multiple proteins involved in the process of olfactory cognition: olfactory receptor proteins, proteins that participate in signal transduction cascade, etc. The expression level of each gene is controlled by its regulatory regions, and especially, by the promoter (a region of DNA about 100–1000 base pairs long located upstream of the transcription start site. We analyzed single nucleotide polymorphisms using human whole-genome data from the 1000 Genomes Project and revealed an extremely high level of single nucleotide polymorphisms in promoter regions of olfactory receptor genes and HLA genes. We hypothesized that the high level of polymorphisms in olfactory receptor promoters was responsible for the diversity in regulatory mechanisms controlling the expression levels of olfactory receptor proteins. Such diversity of regulatory mechanisms may cause the great variability of olfactory cognition of numerous environmental olfactory stimuli perceived by human beings (air pollutants, human body odors, odors in culinary etc.. In turn, this variability may provide a wide range of emotional and behavioral reactions related to the vast variety of olfactory stimuli.

Pentadecapeptide BPC 157 Enhances the Growth Hormone Receptor Expression in Tendon Fibroblasts

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Chung-Hsun Chang

2014-11-01

Full Text Available BPC 157, a pentadecapeptide derived from human gastric juice, has been demonstrated to promote the healing of different tissues, including skin, muscle, bone, ligament and tendon in many animal studies. However, the underlying mechanism has not been fully clarified. The present study aimed to explore the effect of BPC 157 on tendon fibroblasts isolated from Achilles tendon of male Sprague-Dawley rat. From the result of cDNA microarray analysis, growth hormone receptor was revealed as one of the most abundantly up-regulated genes in tendon fibroblasts by BPC 157. BPC 157 dose- and time-dependently increased the expression of growth hormone receptor in tendon fibroblasts at both the mRNA and protein levels as measured by RT/real-time PCR and Western blot, respectively. The addition of growth hormone to BPC 157-treated tendon fibroblasts dose- and time-dependently increased the cell proliferation as determined by MTT assay and PCNA expression by RT/real-time PCR. Janus kinase 2, the downstream signal pathway of growth hormone receptor, was activated time-dependently by stimulating the BPC 157-treated tendon fibroblasts with growth hormone. In conclusion, the BPC 157-induced increase of growth hormone receptor in tendon fibroblasts may potentiate the proliferation-promoting effect of growth hormone and contribute to the healing of tendon.

Ultraviolet irradiation produces cytotoxic synergy and increased DNA interstrand crosslinking with cis- and trans-diamminedichloroplatinum(II)

International Nuclear Information System (INIS)

Swinnen, L.J.; Erickson, L.C.

1989-01-01

The excision-repair mechanism responsible for the removal of UV-induced thymine dimers may also play a role in the repair of cis-diamminedichloroplatinum(II) (cis-DDP)-induced DNA adducts in both bacteria and mammalian cells. It was hypothesized that UV dimers and cis-DDP adducts, when present simultaneously, might compete for a common repair system. Colony survival assays were performed in HT-29 human colon carcinoma cells exposed either to cis-DDP alone or to cis-DDP immediately followed by UV exposure. Progressively greater cytotoxic synergy with both increasing UV dose and cis-DDP dose was observed, to a point of saturation beyond which further toxicity was purely additive. An approximate doubling in DNA crosslink frequency, relative to cis-DDP alone, was found in cells exposed to cis-DDP plus UV. Since cis-DDP produces both inter- and intrastrand DNA crosslinks similar studies were performed with trans-DDP, which is incapable of producing intrastrand crosslinks, but does produce interstrand crosslinks. Cytotoxic synergy and increased interstrand crosslinking again resulted from the addition of UV exposure, but not to the same extent as seen with cis-DDP. (author)

Androgen insensitivity syndrome: gonadal androgen receptor activity

International Nuclear Information System (INIS)

Coulam, C.B.; Graham, M.L.; Spelsberg, T.C.

1984-01-01

To determine whether abnormalities of the androgen receptor previously observed in skin fibroblasts from patients with androgen insensitivity syndrome also occur in the gonads of affected individuals, androgen receptor activity in the gonads of a patient with testicular feminization syndrome was investigated. Using conditions for optimal recovery of androgen receptor from human testes established by previous studies, we detected the presence of a high-affinity (dissociation constant . 3.2 X 10(-10) mol/L), low-capacity (4.2 X 10(-12) mol/mg DNA), androgen-binding protein when tritium-labeled R1881 was incubated at 4 degrees C with nuclear extracts from the gonads of control patients or from a patient with testicular feminization syndrome but not when incubated at 37 degrees C. Thus this patient has an androgen receptor with a temperature lability similar to that of receptors from normal persons

Conformational destabilization of Immunoglobulin G increases the low pH-binding affinity with the Neonatal Fc Receptor

DEFF Research Database (Denmark)

Walters, Benjamin T; Jensen, Pernille Foged; Larraillet, Vincent

2016-01-01

Crystallographic evidence suggests that the pH-dependent affinity of IgG molecules for the neonatal Fc receptor (FcRn) receptor primarily arises from salt bridges involving IgG histidine residues, resulting in moderate affinity at mildly acidic conditions. However, this view does not explain the ......H-dependent affinity in IgG-FcRn interactions and exemplify the important and often ignored role of intrinsic conformational dynamics in a protein ligand, to dictate affinity for biologically important receptors.......Crystallographic evidence suggests that the pH-dependent affinity of IgG molecules for the neonatal Fc receptor (FcRn) receptor primarily arises from salt bridges involving IgG histidine residues, resulting in moderate affinity at mildly acidic conditions. However, this view does not explain...... the diversity in affinity found in IgG variants, such as the YTE mutant (M252Y,S254T,T256E), which increases affinity to FcRn by up to 10×. Here we compare hydrogen exchange measurements at pH 7.0 and pH 5.5 with and without FcRn bound with surface plasmon resonance estimates of dissociation constants and Fc...

DNA Methyltransferase 3B Gene Promoter and Interleukin-1 Receptor Antagonist Polymorphisms in Childhood Immune Thrombocytopenia

Directory of Open Access Journals (Sweden)

Margarita Pesmatzoglou

2012-01-01

Full Text Available Primary immune thrombocytopenia (ITP is one of the most common blood diseases as well as the commonest acquired bleeding disorder in childhood. Although the etiology of ITP is unclear, in the pathogenesis of the disease, both environmental and genetic factors including polymorphisms of TNF-a, IL-10, and IL-4 genes have been suggested to be involved. In this study, we investigated the rs2424913 single-nucleotide polymorphism (SNP (C46359T in DNA methyltransferase 3B (DNMT3B gene promoter and the VNTR polymorphism of IL-1 receptor antagonist (IL-1 Ra intron-2 in 32 children (17 boys with the diagnosis of ITP and 64 healthy individuals. No significant differences were found in the genotype distribution of DNMT3B polymorphism between the children with ITP and the control group, whereas the frequency of allele T appeared significantly increased in children with ITP (P = 0.03, OR = 2, 95% CI: 1.06–3.94. In case of IL-1 Ra polymorphism, children with ITP had a significantly higher frequency of genotype I/II, compared to control group (P = 0.043, OR = 2.60, 95% CI: 1.02–6.50. Moreover, genotype I/I as well as allele I was overrepresented in the control group, suggesting that allele I may have a decreased risk for development of ITP. Our findings suggest that rs2424913 DNMT3B SNP as well as IL-1 Ra VNTR polymorphism may contribute to the susceptibility to ITP.

Chromatin mobility is increased at sites of DNA double-strand breaks

NARCIS (Netherlands)

Krawczyk, P. M.; Borovski, T.; Stap, J.; Cijsouw, T.; ten Cate, R.; Medema, J. P.; Kanaar, R.; Franken, N. A. P.; Aten, J. A.

2012-01-01

DNA double-strand breaks (DSBs) can efficiently kill cancer cells, but they can also produce unwanted chromosome rearrangements when DNA ends from different DSBs are erroneously joined. Movement of DSB-containing chromatin domains might facilitate these DSB interactions and promote the formation of

Quantification of Cooperativity in Heterodimer-DNA Binding Improves the Accuracy of Binding Specificity Models*

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Isakova, Alina; Berset, Yves; Hatzimanikatis, Vassily; Deplancke, Bart

2016-01-01

Many transcription factors (TFs) have the ability to cooperate on DNA elements as heterodimers. Despite the significance of TF heterodimerization for gene regulation, a quantitative understanding of cooperativity between various TF dimer partners and its impact on heterodimer DNA binding specificity models is still lacking. Here, we used a novel integrative approach, combining microfluidics-steered measurements of dimer-DNA assembly with mechanistic modeling of the implicated protein-protein-DNA interactions to quantitatively interrogate the cooperative DNA binding behavior of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ):retinoid X receptor α (RXRα) heterodimer. Using the high throughput MITOMI (mechanically induced trapping of molecular interactions) platform, we derived equilibrium DNA binding data for PPARγ, RXRα, as well as the PPARγ:RXRα heterodimer to more than 300 target DNA sites and variants thereof. We then quantified cooperativity underlying heterodimer-DNA binding and derived an integrative heterodimer DNA binding constant. Using this cooperativity-inclusive constant, we were able to build a heterodimer-DNA binding specificity model that has superior predictive power than the one based on a regular one-site equilibrium. Our data further revealed that individual nucleotide substitutions within the target site affect the extent of cooperativity in PPARγ:RXRα-DNA binding. Our study therefore emphasizes the importance of assessing cooperativity when generating DNA binding specificity models for heterodimers. PMID:26912662

Quantification of Cooperativity in Heterodimer-DNA Binding Improves the Accuracy of Binding Specificity Models.

Science.gov (United States)

Isakova, Alina; Berset, Yves; Hatzimanikatis, Vassily; Deplancke, Bart

2016-05-06

Many transcription factors (TFs) have the ability to cooperate on DNA elements as heterodimers. Despite the significance of TF heterodimerization for gene regulation, a quantitative understanding of cooperativity between various TF dimer partners and its impact on heterodimer DNA binding specificity models is still lacking. Here, we used a novel integrative approach, combining microfluidics-steered measurements of dimer-DNA assembly with mechanistic modeling of the implicated protein-protein-DNA interactions to quantitatively interrogate the cooperative DNA binding behavior of the adipogenic peroxisome proliferator-activated receptor γ (PPARγ):retinoid X receptor α (RXRα) heterodimer. Using the high throughput MITOMI (mechanically induced trapping of molecular interactions) platform, we derived equilibrium DNA binding data for PPARγ, RXRα, as well as the PPARγ:RXRα heterodimer to more than 300 target DNA sites and variants thereof. We then quantified cooperativity underlying heterodimer-DNA binding and derived an integrative heterodimer DNA binding constant. Using this cooperativity-inclusive constant, we were able to build a heterodimer-DNA binding specificity model that has superior predictive power than the one based on a regular one-site equilibrium. Our data further revealed that individual nucleotide substitutions within the target site affect the extent of cooperativity in PPARγ:RXRα-DNA binding. Our study therefore emphasizes the importance of assessing cooperativity when generating DNA binding specificity models for heterodimers. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

KSHV strategies for host dsDNA sensing machinery.

Science.gov (United States)

Gao, Hang; Song, Yanyan; Liu, Chengrong; Liang, Qiming

2016-12-01

The innate immune system utilizes pattern recognition receptors cyclic GMP-AMP synthase (cGAS) to sense cytosolic double-stranded (ds) DNA and initiate type 1 interferon signaling and autophagy pathway, which collaborate to limit pathogen infections as well as alarm the adaptive immune response. The genomes of herpesviruses are large dsDNA, which represent a major class of pathogen signatures recognized by cellular DNA sensor cGAS. However, to successfully establish the persistent infection, herpesviruses have evolved their viral genes to modulate different aspects of host immune signaling. This review summarizes the evasion strategies of host cGAS DNA sensing pathway by Kaposi's Sarcoma-associated Herpesvirus (KSHV) and their contributions to KSHV life cycles.

IFI16 and cGAS cooperate in the activation of STING during DNA sensing in human keratinocytes.

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Almine, Jessica F; O'Hare, Craig A J; Dunphy, Gillian; Haga, Ismar R; Naik, Rangeetha J; Atrih, Abdelmadjid; Connolly, Dympna J; Taylor, Jordan; Kelsall, Ian R; Bowie, Andrew G; Beard, Philippa M; Unterholzner, Leonie

2017-02-13

Many human cells can sense the presence of exogenous DNA during infection though the cytosolic DNA receptor cyclic GMP-AMP synthase (cGAS), which produces the second messenger cyclic GMP-AMP (cGAMP). Other putative DNA receptors have been described, but whether their functions are redundant, tissue-specific or integrated in the cGAS-cGAMP pathway is unclear. Here we show that interferon-γ inducible protein 16 (IFI16) cooperates with cGAS during DNA sensing in human keratinocytes, as both cGAS and IFI16 are required for the full activation of an innate immune response to exogenous DNA and DNA viruses. IFI16 is also required for the cGAMP-induced activation of STING, and interacts with STING to promote STING phosphorylation and translocation. We propose that the two DNA sensors IFI16 and cGAS cooperate to prevent the spurious activation of the type I interferon response.

Increased urinary excretion of 8-oxo-2'-deoxyguanosine, a biomarker of oxidative DNA damage, in urban bus drivers

DEFF Research Database (Denmark)

Loft, S; Poulsen, H E; Vistisen, K

1999-01-01

Oxidative damage to DNA could be involved in the increased risk of cancer associated with exposure to polluted urban air, which contains a number of oxidants. CYP1A2 is induced by and metabolizes polyaromatic hydrocarbons (PAH) and aromatic amines and could modify effects of exposure to ambient air...... pollution. Similarly, DNA repair may be influenced by occupational and other exposures as well as modify the effect of DNA damaging agents. As part of a large investigation of the genotoxic burden to diesel exposed workers in transport sectors we studied oxidative DNA damage in 57 non-smoking bus drivers...... from the greater Copenhagen area. The drivers were studied on a workday and on a day off work. Comparisons were made between drivers from the central (n=30) and rural/suburban (n=27) areas of Copenhagen. The rate of oxidative DNA damage was estimated from 24 h urinary excretion of 8-oxo-2...

Identifying a Mechanism for Crosstalk Between the Estrogen and Glucocorticoid Receptors | Center for Cancer Research

Science.gov (United States)

Estrogen has long been known to play important roles in the development and progression of breast cancer. Its receptor (ER), a member of the steroid receptor family, binds to estrogen response elements (EREs) in DNA and regulates gene transcription. More recently, another steroid receptor family member, the glucocorticoid receptor (GR), has been implicated in breast cancer

Acetylation Increases EWS-FLI1 DNA Binding and Transcriptional Activity

International Nuclear Information System (INIS)

Schlottmann, Silke; Erkizan, Hayriye V.; Barber-Rotenberg, Julie S.; Knights, Chad; Cheema, Amrita; Üren, Aykut; Avantaggiati, Maria L.; Toretsky, Jeffrey A.

2012-01-01

Ewing Sarcoma (ES) is associated with a balanced chromosomal translocation that in most cases leads to the expression of the oncogenic fusion protein and transcription factor EWS-FLI1. EWS-FLI1 has been shown to be crucial for ES cell survival and tumor growth. However, its regulation is still enigmatic. To date, no functionally significant post-translational modifications of EWS-FLI1 have been shown. Since ES are sensitive to histone deacetylase inhibitors (HDI), and these inhibitors are advancing in clinical trials, we sought to identify if EWS-FLI1 is directly acetylated. We convincingly show acetylation of the C-terminal FLI1 (FLI1-CTD) domain, which is the DNA binding domain of EWS-FLI1. In vitro acetylation studies showed that acetylated FLI1-CTD has higher DNA binding activity than the non-acetylated protein. Over-expression of PCAF or treatment with HDI increased the transcriptional activity of EWS-FLI1, when co-expressed in Cos7 cells. However, our data that evaluates the acetylation of full-length EWS-FLI1 in ES cells remains unclear, despite creating acetylation specific antibodies to four potential acetylation sites. We conclude that EWS-FLI1 may either gain access to chromatin as a result of histone acetylation or undergo regulation by direct acetylation. These data should be considered when patients are treated with HDAC inhibitors. Further investigation of this phenomenon will reveal if this potential acetylation has an impact on tumor response.

The assay of thyrotropin receptor antibodies with human TSH/LH-CG chimeric receptor expressed on chinese hamster ovary cells

International Nuclear Information System (INIS)

Yi, Ka Hee; Kim, Chang Min

1996-12-01

TSH/LH-CG chimera cDNA is transfected to CHO-K1 cell to obtain the chimeric receptor expressed on the cell surface. The optimal conditions for TSAb and TSBAb measurements are determined using chimeric receptors and under these conditions activity of TSAb and TSBAb in the sera of the Graves' patients. The results obtained are compared to those of TSAb assays using FRTL5 cells CHO-TSHR cells which have wild type human TSH receptor. The transfection procedure of chimeric receptor gene to CHO-K1 cells are on going. The optimal conditions for TSAb and TSBAb measurement using chimeric receptor will be determined after success of transfection procedure. If this study is successfully completed, not only the heterogeneity of Graves. IgG but also pathogenesis of Graves' disease will be elucidated. (author). 25 refs

The assay of thyrotropin receptor antibodies with human TSH/LH-CG chimeric receptor expressed on chinese hamster ovary cells

Energy Technology Data Exchange (ETDEWEB)

Yi, Ka Hee; Kim, Chang Min [Korea Cancer Center Hospital, Seoul (Korea, Republic of)

1996-12-01

TSH/LH-CG chimera cDNA is transfected to CHO-K1 cell to obtain the chimeric receptor expressed on the cell surface. The optimal conditions for TSAb and TSBAb measurements are determined using chimeric receptors and under these conditions activity of TSAb and TSBAb in the sera of the Graves` patients. The results obtained are compared to those of TSAb assays using FRTL5 cells CHO-TSHR cells which have wild type human TSH receptor. The transfection procedure of chimeric receptor gene to CHO-K1 cells are on going. The optimal conditions for TSAb and TSBAb measurement using chimeric receptor will be determined after success of transfection procedure. If this study is successfully completed, not only the heterogeneity of Graves. IgG but also pathogenesis of Graves` disease will be elucidated. (author). 25 refs.

Endocannabinoid receptor 1 gene variations increase risk for obesity and modulate body mass index in European populations

DEFF Research Database (Denmark)

Benzinou, Michael; Chèvre, Jean-Claude; Ward, Kirsten J

2008-01-01

The therapeutic effects of cannabinoid receptor blockade on obesity-associated phenotypes underline the importance of the endocannabinoid pathway on the energy balance. Using a staged-approach, we examined the contribution of the endocannabinoid receptor 1 gene (CNR1) on obesity and body mass ind...... variations increase the risk for obesity and modulate BMI in our European population. As CB1 is a drug target for obesity, a pharmacogenetic analysis of the endocannabinoid blockade obesity treatment may be of interest to identify best responders....

Hippocampal low-frequency stimulation inhibits afterdischarge and increases GABA (A) receptor expression in amygdala-kindled pharmacoresistant epileptic rats.

Science.gov (United States)

Wu, Guofeng; Wang, Likun; Hong, Zhen; Ren, Siying; Zhou, Feng

2017-08-01

The purpose of the present study was to observe the effects of hippocampal low-frequency stimulation (Hip-LFS) on amygdala afterdischarge and GABA (A) receptor expression in pharmacoresistant epileptic (PRE) rats. A total of 110 healthy adult male Wistar rats were used to generate a model of epilepsy by chronic stimulation of the amygdala. Sixteen PRE rats were selected from 70 amygdala-kindled rats by testing their response to Phenytoin and Phenobarbital, and they were randomly assigned to a pharmacoresistant stimulation group (PRS group, 8 rats) or a pharmacoresistant control group (PRC group, 8 rats). A stimulation electrode was implanted into the hippocampus of all of the rats. Hip-LFS was administered twice per day in the PRS group for two weeks. Simultaneously, amygdala stimulus-induced seizures and afterdischarge were recorded. After the hippocampal stimulation was terminated, the brain tissues were obtained to determine the GABA (A) receptors by a method of immumohistochemistry and a real-time polymerase chain reaction. The stages and duration of the amygdala stimulus-induced epileptic seizures were decreased in the PRS group. The afterdischarge threshold was increased and the duration as well as the afterdischarge frequency was decreased. Simultaneously, the GABA (A) expression was significantly increased in the PRS group. Hip-LFS may inhibit amygdala stimulus-induced epileptic seizures and up-regulate GABA (A) receptor expression in PRE rats. The antiepileptic effects of hippocampal stimulation may be partly achieved by increasing the GABA (A) receptor.

Study on effect of ionizing radiation on costimulatory molecular receptor and its relationship with T cell apoptosis

International Nuclear Information System (INIS)

Zhou Jianhua; Su Liaoyuan; Tong Jian; Wang Chunlei; Wang Aiqing

2001-01-01

Effects of different dose γ-ray irradiation on T lymphocyte co-stimulative receptor and apoptosis were investigated to explore the mechanisms of ionizing radiation leading to lymphocyte apoptosis and relationship between CD28 and Fas in T lymphocytes. The effects of irradiation with 0.5, 2, 4 and 8 Gy γ-rays on CD28 and Fas (CD95) molecule expression of human T lymphocytes were measured by flow cytometry (FCM) with direct immunofluorescence technique. Before and after CD28 McAb and IL-18 dealing with T cells the T cells DNA fragmentation and apoptosis were determined by JAM test and FCM technique. With increasing radiation dosage, the CD28 molecule expression decreased and CD95 molecule expression increased in CD3 + T cells, which could affect signal transduction and increase cell DNA fragmentation and apoptosis. However, CD28 McAb and IL-18 could resist DNA fragmentation and the apoptosis

Enhancement of immune response induced by DNA vaccine cocktail expressing complete LACK and TSA genes against Leishmania major.

Science.gov (United States)

Ghaffarifar, Fatemeh; Jorjani, Ogholniaz; Sharifi, Zohreh; Dalimi, Abdolhossein; Hassan, Zuhair M; Tabatabaie, Fatemeh; Khoshzaban, Fariba; Hezarjaribi, Hajar Ziaei

2013-04-01

Leishmaniasis is an important disease in humans. Leishmania homologue of receptor for Activated C Kinase (LACK) and thiol specific antioxidant (TSA) as immuno-dominant antigens of Leishmania major are considered the most promising molecules for a DNA vaccine. We constructed a DNA cocktail, containing plasmids encoding LACK and TSA genes of Leishmania major and evaluated the immune response and survival rate in BALB/c mice. IgG and Interferon gamma values were noticeably increased in the immunized group with DNA cocktail vaccine, which were significantly higher than those in the single-gene vaccinated and control groups (p 0.05). The immunized mice with the cocktail DNA vaccine presented a considerable reduction in diameter of lesion compared to other groups and a significant difference was observed (p < 0.05) in this regard. The survival time of the immunized mice with the cocktail DNA vaccine was significantly higher than that in the other groups (p < 0.05) after their being challenged with Leishmania major. The findings of this study indicated that the cocktail DNA vaccine increased the cellular response and survival rate and induced protection against infection with Leishmania in the mice. © 2012 The Authors © 2012 APMIS.

A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors.

Science.gov (United States)

Sarris, Panagiotis F; Duxbury, Zane; Huh, Sung Un; Ma, Yan; Segonzac, Cécile; Sklenar, Jan; Derbyshire, Paul; Cevik, Volkan; Rallapalli, Ghanasyam; Saucet, Simon B; Wirthmueller, Lennart; Menke, Frank L H; Sohn, Kee Hoon; Jones, Jonathan D G

2015-05-21

Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a "decoy" domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain. Copyright © 2015 Elsevier Inc. All rights reserved.

Meiosis observation of the sterile mutant after injection of exogenous DNA into wheat

International Nuclear Information System (INIS)

Yang Jingcheng; Yu Yuanjie; Qi Yanfang; Shen Fafu; Liu Fengzhen

2001-01-01

A male sterile mutant was obtained after injection of exogenous λ DNA into wheat line 814527. Meiosis of pollen mother cells (PMC) of the mutant and its receptor (line 814527) were observed. The results showed that the frequency of chromosomal variation of the sterile line was 18%, and that of the receptor was 0.8%. The main types of variation included univalent, chromosome lagging, chromosome fragment, chromosome bridge, micronucleus, abnormal ditrad and tetrad. The fragment of DNA injected into the receptor may influence the normal genetic process of chromosomes in pollen mother cells, and this may cause variations of chromosomes. The chromosome variation in meiosis may cause a part of pollen mother cells to abort, but it is not the main cause of abortion

Function and distribution of 5-HT2 receptors in the honeybee (Apis mellifera.

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Markus Thamm

Full Text Available BACKGROUND: Serotonin plays a pivotal role in regulating and modulating physiological and behavioral processes in both vertebrates and invertebrates. In the honeybee (Apis mellifera, serotonin has been implicated in division of labor, visual processing, and learning processes. Here, we present the cloning, heterologous expression, and detailed functional and pharmacological characterization of two honeybee 5-HT2 receptors. METHODS: Honeybee 5-HT2 receptor cDNAs were amplified from brain cDNA. Recombinant cell lines were established constitutively expressing receptor variants. Pharmacological properties of the receptors were investigated by Ca(2+ imaging experiments. Quantitative PCR was applied to explore the expression patterns of receptor mRNAs. RESULTS: The honeybee 5-HT2 receptor class consists of two subtypes, Am5-HT2α and Am5-HT2β. Each receptor gene also gives rise to alternatively spliced mRNAs that possibly code for truncated receptors. Only activation of the full-length receptors with serotonin caused an increase in the intracellular Ca(2+ concentration. The effect was mimicked by the agonists 5-methoxytryptamine and 8-OH-DPAT at low micromolar concentrations. Receptor activities were blocked by established 5-HT receptor antagonists such as clozapine, methiothepin, or mianserin. High transcript numbers were detected in exocrine glands suggesting that 5-HT2 receptors participate in secretory processes in the honeybee. CONCLUSIONS: This study marks the first molecular and pharmacological characterization of two 5-HT2 receptor subtypes in the same insect species. The results presented should facilitate further attempts to unravel central and peripheral effects of serotonin mediated by these receptors.

Function and distribution of 5-HT2 receptors in the honeybee (Apis mellifera).

Science.gov (United States)

Thamm, Markus; Rolke, Daniel; Jordan, Nadine; Balfanz, Sabine; Schiffer, Christian; Baumann, Arnd; Blenau, Wolfgang

2013-01-01

Serotonin plays a pivotal role in regulating and modulating physiological and behavioral processes in both vertebrates and invertebrates. In the honeybee (Apis mellifera), serotonin has been implicated in division of labor, visual processing, and learning processes. Here, we present the cloning, heterologous expression, and detailed functional and pharmacological characterization of two honeybee 5-HT2 receptors. Honeybee 5-HT2 receptor cDNAs were amplified from brain cDNA. Recombinant cell lines were established constitutively expressing receptor variants. Pharmacological properties of the receptors were investigated by Ca(2+) imaging experiments. Quantitative PCR was applied to explore the expression patterns of receptor mRNAs. The honeybee 5-HT2 receptor class consists of two subtypes, Am5-HT2α and Am5-HT2β. Each receptor gene also gives rise to alternatively spliced mRNAs that possibly code for truncated receptors. Only activation of the full-length receptors with serotonin caused an increase in the intracellular Ca(2+) concentration. The effect was mimicked by the agonists 5-methoxytryptamine and 8-OH-DPAT at low micromolar concentrations. Receptor activities were blocked by established 5-HT receptor antagonists such as clozapine, methiothepin, or mianserin. High transcript numbers were detected in exocrine glands suggesting that 5-HT2 receptors participate in secretory processes in the honeybee. This study marks the first molecular and pharmacological characterization of two 5-HT2 receptor subtypes in the same insect species. The results presented should facilitate further attempts to unravel central and peripheral effects of serotonin mediated by these receptors.

Placental expression of asialoglycoprotein receptor associated with Hepatitis B virus transmission from mother to child.

Science.gov (United States)

Vyas, Ashish Kumar; Ramakrishna, Usha; Sen, Bijoya; Islam, Mojahidul; Ramakrishna, Gayatri; Patra, Sharda; Rastogi, Archana; Sarin, Shiv Kumar; Trehanpati, Nirupma

2018-04-30

Asialoglycoprotein receptor expression on hepatocytes has been associated with endocytosis, binding and uptake of hepatitis B virus. The role of asialoglycoprotein receptor in hepatitis B virus vertical transmission and its expression on placenta has not yet been studied. Thirty-four HBsAg+ve and 13 healthy pregnant mothers along with their newborns were enrolled. The former were categorized into transmitting and non-transmitting mothers based on their newborns being hepatitis B surface antigen and hepatitis B virus DNA positive. Expression of asialoglycoprotein receptor and hepatitis B surface antigen in placenta and isoform of asialoglycoprotein receptor on dendritic cell in peripheral and cord blood dendritic cells were analysed using flowcytometry, immune histochemistry, immune florescence and qRT-PCR. Twelve HBsAg+ve mothers transmitted hepatitis B virus to their newborns whereas the rest (n = 22) did not. Hepatitis B virus-transmitting mothers showed increased expression of asialoglycoprotein receptor in trophoblasts of placenta. Immunofluorescence microscopy revealed colocalization of hepatitis B surface antigen and asialoglycoprotein receptor in placenta as well as in DCs of transmitting mothers. There was no significant difference in the expression of asialoglycoprotein receptor on peripheral blood mononuclear cells or chord blood mononuclear cells between the 2 groups. However, hepatitis B virus-transmitting mothers and their HBsAg+ve newborns showed increased mRNA levels of isoform of asialoglycoprotein receptor on dendritic cell in peripheral blood mononuclear cells. Hepatitis B virus-transmitting mothers and their HBsAg+ve newborns showed an increased expression of isoform of asialoglycoprotein receptor on dendritic cell on circulating dendritic cells compared to hepatitis B virus non-transmitting mothers and their negative newborns. This study revealed that increased expression of asialoglycoprotein receptor in placenta and colocalization with

Role of laminin receptor in tumor cell migration

DEFF Research Database (Denmark)

Wewer, U M; Taraboletti, G; Sobel, M E

1987-01-01

Polyclonal antisera were made against biochemically purified laminin receptor protein as well as against synthetic peptides deduced from a complementary DNA clone corresponding to the COOH-terminal end of the laminin receptor (U.M. Wewer et al., Proc. Natl. Acad. Sci. USA, 83: 7137-7141, 1986......). These antisera were used to study the potential role of laminin receptor in laminin-mediated attachment and haptotactic migration of human A2058 melanoma cells. The anti-laminin receptor antisera reacted with the surface of suspended, nonpermeabilized melanoma and carcinoma cells. The anti-laminin receptor...... antisera blocked the surface interaction of A2058 cells with endogenous laminin, resulting in the inhibition of laminin-mediated cell attachment. The A2058 melanoma cells migrated toward a gradient of solid phase laminin or fibronectin (haptotaxis). Anti-laminin antiserum abolished haptotaxis on laminin...

The cellular receptors of exogenous RNA

Directory of Open Access Journals (Sweden)

Patryk Reniewicz

2016-04-01

Full Text Available One of the key determinants of survival for organisms is proper recognition of exogenous and endogenous nucleic acids. Therefore, high eukaryotes developed a number of receptors that allow for discrimination between friend or foe DNA and RNA. Appearance of exogenous RNA in cytoplasm provides a signal of danger and triggers cellular responses that facilitate eradication of a pathogen. Recognition of exogenous RNA is additionally complicated by fact that large amount of endogenous RNA is present in cytoplasm Thus, number of different receptors, found in eukaryotic cells, is able to recognize that nucleic acid. First group of those receptors consist endosomal Toll like receptors, namely TLR3, TLR7, TLR8 and TLR13. Those receptors recognize RNA released from pathogens that enter the cell by endocytosis. The second group includes cytoplasmic sensors like PKR and the family of RLRs comprised of RIG-I, MDA5 and LGP2. Cytoplasmic receptors recognize RNA from pathogens invading the cell by non-endocytic pathway. In both cases binding of RNA by its receptors results in activation of the signalling cascades that lead to the production of interferon and other cytokines.

Increased expression of (pro)renin receptor does not cause hypertension or cardiac and renal fibrosis in mice

NARCIS (Netherlands)

Rosendahl, Alva; Niemann, Gianina; Lange, Sascha; Ahadzadeh, Erfan; Krebs, Christian; Contrepas, Aurelie; van Goor, Harry; Wiech, Thorsten; Bader, Michael; Schwake, Michael; Peters, Judith; Stahl, Rolf; Nguyen, Genevieve; Wenzel, Ulrich

Binding of renin and prorenin to the (pro)renin receptor (PRR) increases their enzymatic activity and upregulates the expression of pro-fibrotic genes in vitro. Expression of PRR is increased in the heart and kidney of hypertensive and diabetic animals, but its causative role in organ damage is