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Cytosine methylation in DNA is a major epigenetic signal, and plays a central role in propagating chromatin status during cell division. However the mechanistic links between DNA methylation and histone methylation are poorly understood. A multi-domain protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is required for DNA CpG maintenance methylation at replication forks, and mouse UHRF1-null cells show enhanced susceptibility to DNA replication arrest and DNA damaging agents. Recent data demonstrated that the SET and RING associated (SRA) domain of UHRF1 binds hemimethylated CpG and flips 5-methylcytosine out of the DNA helix, whereas its tandom tudor domain and PHD domain bind the tail of histone H3 in a highly methylation sensitive manner. We hypothesize that UHRF1 brings the two components (histones and DNA) carrying appropriate markers (on the tails of H3 and hemimethylated CpG sites) ready to be assembled into a ...

UK PubMed Central (United Kingdom)

CAAX proteins are widely involved in global cellular functions such as proliferation, differentiation, and carcinogenesis. As an important modulator of biological activity, signal transduction via protein...Full Text Available

UK PubMed Central (United Kingdom)

In addition to inducing new transcriptional activities that lead within a few hours to the accumulation of heat shock proteins (Hsps), heat shock activates within minutes the major signaling transduction...Full Text Available

UK PubMed Central (United Kingdom)

AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism; its activity is regulated by a plethora of physiological conditions, exercises and many anti-diabetic drugs. Recent...Full Text Available

UK PubMed Central (United Kingdom)

Thioredoxin, a small, ubiquitous protein which participates in redox reactions through the reversible oxidation of its active center dithiol to a disulfide, is an essential protein in Bacillus...Full Text Available

UK PubMed Central (United Kingdom)

The activity of Src-related protein-tyrosine kinases is repressed by the phosphorylation of a conserved carboxyl-terminal tyrosine by another cytoplasmic protein-tyrosine kinase termed p50csk. In this...Full Text Available

UK PubMed Central (United Kingdom)

AMP-activated-protein-kinase (AMPK) is a key sensor and regulator of cellular and whole-body energy metabolism and plays a key role in regulation of lipid metabolism. Since lipid metabolism...Full Text Available

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Energy Technology Data Exchange (ETDEWEB)

In isolated rat adipocytes, insulin inhibits lipolysis to a greater extent than would be predicted by the decrease in (-/+)cAMP activity ratio of cAMP-dependent protein kinase (A-kinase), from which it was speculated that insulin promotes the dephosphorylation of hormone-sensitive lipase. They have examined the phosphorylation state of cellular proteins under conditions of varying A-kinase activities in the presence and absence of insulin. Protein phosphorylation was determined by SDS-PAGE electrophoresis of extracts from /sup 32/P-loaded cells; glycerol and A-kinase activity ratios were measured in the cytosolic extracts from control, non-radioactive cells. Increased protein phosphorylation in general occurred over the same range of A-kinase activity ratios, 0.1-0.3, associated with increased glycerol release. The ...

UK PubMed Central (United Kingdom)

Wiskott-Aldrich syndrome protein (WASp) is essential for optimal T cell activation. Patients with WAS exhibit both immunodeficiency and a marked susceptibility to systemic autoimmunity. We investigated...Full Text Available

UK PubMed Central (United Kingdom)

We had previously identified a macrophage surface protein whose expression is highly induced, transient, and specific, as it is restricted to actively fusing macrophages in vitro and in vivo. This protein...Full Text Available

UK PubMed Central (United Kingdom)

Mammals possess multiple insulin-like growth factor (IGF) binding proteins (IGFBPs), and related proteins, that modulate the activity of insulin/IGF signalling (IIS), a conserved neuroendocrine signalling...Full Text Available

UK PubMed Central (United Kingdom)

Sustained nigrostriatal dopamine depletion increases the serine/threonine phosphorylation of multiple striatal proteins that play a role in corticostriatal synaptic plasticity, including Thr²⁸⁶...Full Text Available

UK PubMed Central (United Kingdom)

CAP18 (an 18-kDa cationic antimicrobial protein) is a granulocyte-derived protein that can bind lipopolysaccharide (LPS) and inhibit various activities of LPS in vitro. The present study examined the...Full Text Available

British Library Electronic Table of Contents (United Kingdom)

This study was conducted to investigate the antibacterial effect of BSAP-254 on Bacillus cereus with the induced stress proteins. The BSAP-254 is an antimicrobial peptide isolated from soybean-fermenting bacteria, Bacillus subtilis SC-8. It had a narrow spectrum of activity against B. cereus group. The growth inhibitory effect of BSAP-254 (50??g/mL) reduced the population of B. cereus from >108 to 104 colony-forming units per milliliter within 30?min. In B. cereus exposed to BSAP-254, 14 intracellular proteins were differentially expressed as determined by 2-DE coupled with MS. Of the differentially expressed proteins identified, the stress protein GroEL, which is heat shock protein, was induced in B. cereus exposed to antibacterial peptide.

British Library Electronic Table of Contents (United Kingdom)

Amyloid precursor protein (APP), a key protein in pathogenesis of Alzheimer's disease (AD), is a type I transmembrane protein which can be cleaved by b- and g-secretase to release the amyloidogenic b-amyloid peptides (Ab) and the APP intracellular domain (AICD). While Ab has been widely believed to initiate pathogenic cascades culminating AD, the physiological functions and regulations of AICD remain elusive. In present study, endogenous AICD was demonstrated to be increased by canonical Wnt signal. Instead of due to g-secretase activity, enhanced AICD expression was found due to the increased protein stability by Wnt/b-catenin. b-Catenin was demonstrated to be an associating partner of AICD, capable of promoting AICD mediated transcriptional activity. Investigation by AICD mutants proved ...

UK PubMed Central (United Kingdom)

On rod disc membranes, single photoactivated rhodopsin (R*) molecules catalytically activate many copies of the G-protein (Gt), which in turn binds and activates the effector (phosphodiesterase). We...Full Text Available

UK PubMed Central (United Kingdom)

The soluble subcellular fraction of a chlB mutant contains an inactive precursor form of the molybdoenzyme nitrate reductase, which can be activated by the addition to the soluble fraction of protein...Full Text Available

UK PubMed Central (United Kingdom)

Large-conductance voltage- and calcium-activated potassium (BK) channels have been shown to play a role in detrusor overactivity (DO). The goal of this study was to determine whether bladder outlet...Full Text Available

UK PubMed Central (United Kingdom)

Summary5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICA riboside) has been extensively used in vitro and in vivo to...Full Text Available

UK PubMed Central (United Kingdom)

The products of trithorax group (trxG) genes maintain active transcription of many important developmental regulatory genes, including homeotic genes. Several trxG proteins have been shown to act in...Full Text Available

UK PubMed Central (United Kingdom)

The p90 ribosomal S6 kinases (RSKs) also known as MAPKAP-Ks are serine/threonine protein kinases that are activated by ERK or PDK1 and act as downstream effectors of mitogen-activated protein kinase...Full Text Available

UK PubMed Central (United Kingdom)

Inhibition of large conductance calcium-activated potassium (BK_Ca) channels mediates, in part, oxygen sensing by carotid body type I cells. However, BK_Ca channels remain active...Full Text Available

Science.gov (United States)

The Drosophila melanogaster tissue-specific transcription factor NTF-1 was originally identified in vitro as a protein that could bind to and activate transcription from the Dopa decarboxylase (Ddc) gene. A structure-function analysis of NTF-1 led to the identification of a discrete amino-terminal activation domain. Here, we report that an NTF-1 mutant lacking the activation domain acts as a trans-dominant inhibitor of NTF-1 activation in tissue culture cells by forming inactive heterodimers with the full-length protein. Ectopically expressing this dominant-negative protein or the full-length protein in developing Drosophila embryos leads to dire developmental consequences. Overexpressing the trans-dominant NTF-1 leads to lethality, while overexpressing full-length NTF-1 results in both lethality and morphogenetic ...

Science.gov (United States)

Exposure of the freshwater field crab Barytelphusa guerini to a sublethal concentration of NaF (30 mg litre(-1)) caused significant alterations in protein metabolism. After an exposure time of 15 days, the crab was found to have a marked depletion of total protein and free amino acid content. A significant elevation in transaminases, Aspartate aminotransferase (AAT) and Alanine aminotransferase (ALAT), and Glutamate dehydrogenase (GDH) activities was reflected in the free amino acid levels of the tissues. PMID:15092215

UK PubMed Central (United Kingdom)

Uteroglobin (UG) gene encodes a cytokine-like, multifunctional, antiinflammatory protein, with potent phospholipase A2-inhibitory activity. It has been suggested that during implantation this protein...Full Text Available

Science.gov (United States)

AIMS: To clarify the levels of protein oxidation markers such as protein carbonyl (PCO), protein hydroperoxides (P-OOH), advanced oxidation protein products (AOPP) and nitrotyrosine (NT), as well as antioxidative enzymes such as paraoxonase (PON-1) in women with and without gestational diabetes mellitus (GDM). METHODS: The study was conducted on 23 women with GDM and 22 women without GDM. The levels of the P-OOH, AOPP, and PON-1 were determined by colorimetric methods; whereas NT and PCO levels were measured by ELISA. RESULTS: The concentrations of protein oxidation markers were significantly increased and PON1 activity was significantly decreased in GDM group compared to those of normal pregnant women. The control group showed a significant negative correlation between PON-1 and PCO (r=-0.451, p=0.027); whereas in GDM group, there was a significant positive ...

UK PubMed Central (United Kingdom)

Engineered overexpression of protein kinase Cα (PKCα) was previously shown to endow nonmotile MCF-10A human breast cells with aggressive motility. A traceable mutant of PKCα...Full Text Available

UK PubMed Central (United Kingdom)

The positive control function of the bacterial enhancer-binding protein NtrC resides in its central domain, which is highly conserved among activators of ς⁵⁴ holoenzyme. Previous...Full Text Available

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The serpin ZPI is a protein Z (PZ)-dependent specific inhibitor of membrane-associated factor Xa (fXa) despite having an unfavorable P1 Tyr. PZ accelerates the inhibition reaction ∼2000-fold...Full Text Available

UK PubMed Central (United Kingdom)

Bcl-2 homologues (such as Bcl-x_L) promote survival in part through sequestration of “activator” BH3-only proteins (such as Puma), preventing them from directly activating...Full Text Available

UK PubMed Central (United Kingdom)

We use a novel normal mode analysis of an elastic network model drawn from configurations generated during microsecond all-atom molecular dynamics simulations to analyze the mechanism of auto-inhibition...Full Text Available

UK PubMed Central (United Kingdom)

Recognition of acetylated chromatin by the bromodomains and extra-terminal domain (BET) family proteins is a hallmark for transcriptional activation and anchoring viral genomes to mitotic chromosomes...Full Text Available

UK PubMed Central (United Kingdom)

BackgroundGlycochenodeoxycholate (GCDA) is one of the major human bile salts. Bile salts stimulate cell survival and proliferation through the mitogen-activated protein kinase, but...Full Text Available

UK PubMed Central (United Kingdom)

IntroductionAcute lung injury (ALI) may arise both after sepsis and non-septic inflammatory conditions and is often associated with the release of fatty acids, including oleic acid...Full Text Available

UK PubMed Central (United Kingdom)

The ubiquitin–proteasome pathway is the principal system for extralysosomal protein degradation in eukaryotic cells, and is essential for the regulation and maintenance of basic cellular processes,...Full Text Available

UK PubMed Central (United Kingdom)

Cancer cells constantly adapt to oxidative phosphorylation (OXPHOS) suppression resulting from hypoxia or mitochondria defects. Under the OXPHOS suppression, AMP-activated protein kinase (AMPK) regulates...Full Text Available

UK PubMed Central (United Kingdom)

Experimental metastases in the brain of mice are infiltrated by microglia, and parabiosis experiments of green fluorescent protein (GFP⁺) and GFP^- mice revealed that these microglia...Full Text Available

UK PubMed Central (United Kingdom)

BackgroundCalcium signaling plays an important role in B lymphocyte survival and activation, and is critically dependent on the inositol-1,4,5-tris-phosphate-induced...Full Text Available

UK PubMed Central (United Kingdom)

We describe the lethal, recessive accelerated-cell-death11 Arabidopsis mutant (acd11). Cell death in acd11 exhibits characteristics of animal apoptosis...Full Text Available

UK PubMed Central (United Kingdom)

The proprotein convertases (PCs) are calcium-dependent proteases responsible for processing precursor proteins into their active forms in eukariotes. The PC1/3 is a pivotal enzyme of this family that...Full Text Available

UK PubMed Central (United Kingdom)

Small RAC/ROP-family G proteins regulate development and stress responses in plants. Transient overexpression and RNA interference experiments suggested that the barley (Hordeum vulgare)...Full Text Available

UK PubMed Central (United Kingdom)

BackgroundMicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression by binding to the messenger RNA (mRNA) of protein coding genes. They control gene expression by either...Full Text Available

UK PubMed Central (United Kingdom)

The cellular properties of long-term potentiation (LTP) following pairing of pre- and postsynaptic activity were examined at a known glutamatergic synapse in the leech, specifically between the pressure...Full Text Available

UK PubMed Central (United Kingdom)

Studies on alcoholic liver injury mechanisms show a significant inhibition of the proteasome activity. To investigate this phenomenon, we isolated proteasome complexes from the liver of rats...Full Text Available

UK PubMed Central (United Kingdom)

IntroductionBRAF (v raf murine sarcoma viral oncogene homologue B1) is a serine-threonine kinase involved in the mitogen-activated protein kinase (MAPK) signalling pathway, known...Full Text Available

UK PubMed Central (United Kingdom)

The endoplasmic reticulum (ER) is an essential cellular compartment responsible for Ca²⁺ sequestration, signaling, protein translation, folding as well as transport. Several acute...Full Text Available

UK PubMed Central (United Kingdom)

BackgroundObesity is one of the principal causative factors involved in the development of metabolic syndrome. AMP-activated protein kinase (AMPK) is an energy sensor that regulates...Full Text Available

British Library Electronic Table of Contents (United Kingdom)

Aims A depressed cGMP-dependent protein kinase (PKG) activity is implicated in nitrate tolerance. The present study determines whether the leucine zipper-positive (LZ+) isoform of myosin phosphatase target subunit 1 (MYPT1), a key target protein for PKG actions, is involved in the development of nitrate tolerance. Methods and results Nitrate tolerance in in vitro preparations was obtained by a 24 h incubation with nitroglycerin (NTG). Nitrate tolerance in in vivo preparations was obtained by subcutaneous injection of mice with NTG, and the aortas were used. Protein levels of total MYPT1, MYPT1 (LZ+), PP1Cd, myosin light chain (MLC), and phosphorylated MLC were determined by Western blot analysis. Isometric vessel tension was determined by an organ chamber technique. Protein levels of MYPT1...

Science.gov (United States)

In view of the findings that several Kunitz-type protein inhibitors suppress tumor invasion and metastasis, the aim of the present study is to explore whether Bungarus multicinctus protease inhibitor-like protein-2 (PILP-2) and PILP-3 exhibit anti-tumor activity. Although approximately 28% of amino acid substitutions occurred between PILP-2 and PILP-3, molecular modeling suggested that PILP-2 and PILP-3 shared similar folded structures. Unlike PILP-2, PILP-3 showed a notable activity in abolishing migration and invasion of human neuroblastoma SK-N-SH cells. The ability of PILP-3 to inhibit matrix metalloprotease-2 (MMP-2) activity was higher than that of PILP-2. Pull-down assay revealed protein-protein interaction between PILP-3 and MMP-2. In contrast to mutation on N-terminal region, replacement of amino acids at C-terminus attenuated notably the ability of ...

Energy Technology Data Exchange (ETDEWEB)

Enzyme-linked Immunosorbent assay systems for the identification of irradiated egg, pork and chicken was developed. Eggs were irradiated in their shells to 0.5{approx}7kGy. Pork was irradiated to 0.5{approx}3kGy and chicken irradiated to 0.5kGy{approx}5kGy. The most sensitive proteins to irradiation were screened by SDS-PAGE and purified. Ovalbumin from egg, salt soluble protein(p) from pork, and salt soluble protein(c) from chicken showed the most sensitivity to irradiation. To investigate for a practical use in identifying of irradiated egg, pork and chicken, competitive ELISA was performed. The binding activity of ovalbumin to anti-ovalbumin IgG was reduced in a dose-dependent manner by irradiating up to 7kGy, and considerably lowered after irradiating at 7kGy. The concentration of 50% inhibition of ovalbumin to IgG was increased to 1.5(0.5kGy){approx}3.7(7kGy) times in an dose-dependent ...

International Nuclear Information System (INIS)

Active oxygens produced by low-dose irradiation can induce the synthesis of proteins involved in the active oxygen reduction path and can activate the chemical self-defense mechanisms in vivo, which can alleviate the injuries caused by active oxygens themselves. The following findings suggests that it is not only by low-dose irradiation that can induce the responses described above but also by any physical and/or chemical stresses which can produce small amount of active oxygens in vivo. 1. X-irradiation with 0.5 Gy and/or water immersion and restraint induced heat shock protein (HSP)70 in rat stomach cells. HSP70 was also induced in adrenal cells by paraquat administration. 2. The SOD activity in pancreas of rat significantly increased by #gamma#-irradiation with 0.5 Gy. 0.5 Gy irradiation preceeding the alloxan ...

UK PubMed Central (United Kingdom)

BackgroundWhen eukaryotic cells are deprived of amino acids, uncharged tRNAs accumulate and activate the conserved GCN2 protein kinase. Activated Gcn2p up-regulates the general amino...Full Text Available

UK PubMed Central (United Kingdom)

Motor proteins couple steps in ATP binding and hydrolysis to conformational switching both in and remote from the active site. In our kinesin·AMPPPNP crystal structure, closure of the active...Full Text Available

Energy Technology Data Exchange (ETDEWEB)

The elucidation on the metabolic products of the {sup 99}mTc-antibody conjugates may provide insights and approaches that would reduce the undesirable deposition of radioactive species in normal tissue. In this investigation, the radiolabeled species in blood, urine, bile and extracts of liver and kidney obtained at different times after the injection of a model antibody, {sup 99}mTc, into mice were analyzed with various chromatographic methods. Ninety-nine to 100% of the radioactivity in serum was associated with intact Mab 170. The radioactivity in liver homogenate extract was strictly protein-bound to either intact Mab or low molecular weight species (LMW). In kidney extracts, the majority of the radioactivity was protein bound {sup 99}mTc, with less than 8% of the activity being non- protein bound . Multiple {sup 99}mTc -containing protein and non-protein ...

Energy Technology Data Exchange (ETDEWEB)

Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthesized, purified and tested for their effect on preparations of membrane-bound, Lubrol solubilized and forskolin affinity-purified adenylate cyclase (AC). All analogs of forskolin significantly activated AC. However, in the presence of 0.1 to 0.3 microM forskolin, the less active forskolin photoaffinity probes at 100 microM caused inhibition. This inhibition was dose-dependent for PF, suggesting that PF may complete with F for the same binding site(s). After cross-linking (125I)PF-M to either membrane or Lubrol-solubilized AC preparations by photolysis, a radiolabeled 100-110 kDa protein band was observed after autoradiography following SDS-PAGE. F at 100 microM blocked the photoradiolabeling of this protein. Radioiodination of forskolin-affinity purified AC showed several ...

Energy Technology Data Exchange (ETDEWEB)

The effect of insulin on protein biosynthesis was examined in differentiated 3T3-L1 and 3T3-F442A adipocytes. Insulin altered the relative rate of synthesis of specific proteins independent of its ability to hasten conversion of the fibroblast (preadipocyte) phenotype to the adipocyte phenotype. Although more than one pattern of response to insulin was observed, the authors focused on the induction of a Mr 33,000 protein which was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Exposure of 3T3 adipocytes to insulin throughout differentiation specifically increased GAPDH activity and protein content by 2- to 3-fold as compared to 3T3 adipocytes differentiated in the absence of insulin. These changes in enzyme activity and content could be accounted for by a 4-fold increase in the relative rate of synthesis of GAPDH and a ...

Science.gov (United States)

Although the mechanism is unknown, Calculus Bovis and its active components, cholic acid analogs (CAAs), have been used in China to treat a wide range of diseases. Based on the previous finding that the potency of CAA is strongly dependent on the intrinsic surface activity, this paper aimed to investigate the role of the plasma membrane in the pharmacological activity of CAAs. First, CAAs (0.1 mM) caused a surface activity-dependent depression on ATPase activity in the cell membrane extract, but it had no effects on other cellular extracts, suggesting an indispensable role of the membrane environment for pharmacological activity. Second, CAAs lowered the membrane fluidity of cultured Caco-2 cells with the same rank-order of potency sequence. Third, the hypothesis that any functional protein located on the membrane is influenced by changes in ...

Energy Technology Data Exchange (ETDEWEB)

Homology analyses of the protein sequences of chicken liver and rat mammary gland fatty acid synthases were carried out. The amino acid sequences of the chicken and rat enzymes are 67% identical. If conservative substitutions are allowed, 78% of the amino acids are matched. A region of low homologies exists between the functional domains, in particular around amino acid residues 1059-1264 of the chicken enzyme. Homologies between the active sites of chicken and rat and of chicken and yeast enzymes have been analyzed by an alignment method. A high degree of homology exists between the active sites of the chicken and rat enzymes. However, the chicken and yeast enzymes show a lower degree of homology. The DADPH-binding dinucleotide folds of the {beta}-ketoacyl reductase and the enoyl reductase sites were identified by comparison with a known consensus sequence for the DADP- and FAD-binding dinucleotide folds. The ...

British Library Electronic Table of Contents (United Kingdom)

Abstract The Hippo pathway is an evolutionally conserved protein kinase cascade involved in regulating organ size in vivo and cell contact inhibition in vitro by governing cell proliferation and apoptosis. Deregulation of the Hippo pathway is linked to cancer development. Its first core kinase Warts was identified in Drosophila more than 15 years ago, but it gained much attention when other core components of the pathway were identified 8 years later. Major discoveries of the pathway were made during past several years. The core kinase components Hippo, Salvador, Warts, and Mats in the fly and Mst1/2, WW45, Lats1/2, and Mob1 in mammals phosphorylate and inactivate downstream transcriptional co-activators Yorkie in the fly, Yes-associated protein (YAP) and transcriptional co-activator with ...

British Library Electronic Table of Contents (United Kingdom)

Lung cancer is still difficult to treat by current chemotherapeutic procedures. We recently found that MVL, an anti-HIV lectin from blue-green algae Microcystis viridis, also has antitumor activity. The objective of this study was to investigate apoptosis-inducing activity of recombinant MVL (R-MVL) and proteomic changes in A549 cells, and to identify the molecular pathways responsible for the anti-cancer action of R-MVL. We found that R-MVL induces A549 cells apoptosis in a dose-dependent manner by using MTT assay, fluorescent microscope (FM) and flow cytometry (FCM), and the IC50 was calculated to be 24.12??g/ml. Subsequently, 7 altered proteins in R-MVL-treated A549 cells were identified, including upregulated aldehyde dehydrogenase 1 and ?-actin, and five downregulated proteins: heat s...

International Nuclear Information System (INIS)

Each of the three cysteine residues in the Escherichia coli RecA protein was replaced with a number of other amino acids. To do this, each cysteine codon was first converted to a chain-terminating amber codon by oligonucleotide-directed mutagenesis. These amber mutants were then either assayed for function in different suppressor strains or reverted by a second round of mutagenesis with oligonucleotides that had random sequences at the amber codon. Thirty-three different amino acid substitutions were obtained. Mutants were tested for three functions of RecA: survival following UV irradiation, homologous recombination, and induction of the SOS response. It was found that although none of the cysteines is essential for activity, mutations at each of these positions can affect one or more of the activities of RecA, depending on the particular amino acid substitution. In addition, the cysteine at position 116 appears to be ...

British Library Electronic Table of Contents (United Kingdom)

Synchronized cultures of the green alga Scenedesmus quadricauda were grown in the absence (untreated cultures) or in the presence (FdUrd-treated cultures) of 5-fluorodeoxyuridine, the specific inhibitor of nuclear DNA replication. The attainment of commitment points, at which the cells become committed to nuclear DNA replication, mitosis and cellular division, and the course of committed processes themselves were determined for cell cycle characterization. FdUrd-treated cultures showed nearly unaffected growth and attainment of the commitment points, while DNA replication(s), nuclear division(s) and protoplast fission(s) were blocked. Interestingly, the FdUrd-treated cells possessed a very high mitotic histone H1 kinase activity in the absence of any nuclear division(s). Compared with the ...

Energy Technology Data Exchange (ETDEWEB)

A hyaluronic acid binding fraction was purified from the supernatant media of both 3T3 and murine sarcoma virus (MSV) transformed 3T3 cultures by hyaluronate and immunoaffinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the hyaluronate affinity-purified fraction into three major protein bands of estimated molecular weight (M/sub r,e/) 70K, 66K, and 56K which contained hyaluronate binding activity and which were termed hyaluronate binding proteins (HABP). Hyaluronate affinity chromatography combined with immunoaffinity chromatography, using antibody directed against the larger HABP, allowed a 20-fold purification of HABP. Fractions isolated from 3T3 supernatant medium also contained additional binding molecules in the molecular weight range of 20K. This material was present in vanishingly small amounts and was not detected with a silver stain or with (/sup 35/S)methionine label. The ...

Energy Technology Data Exchange (ETDEWEB)

The human zinc finger protein genes (ZFX/Y) were identified as a result of a systematic search for the testis-determining factor gene on the human Y chromosome. Although they play no direct role in sex determination, they are of particular interest because they are highly conserved among mammals, birds, and amphibians and because, in eutherian mammals at least, they have active alleles on both the X and the Y chromosomes outside the pseudoautosomal region. We used in situ hybridization to localize the homologues of the zinc finger protein gene to chromosome 1 of the Australian echidna and to an equivalent position on chromosomes 1 and 2 of the playtpus. The localization to platypus chromosome 1 was confirmed by Southern analysis of a Chinese hamster [times] platypus cell hybrid retaining most of platypus chromosome 1. This localization is consistent with the cytological homology of chromosome 1 between the two species. The ...

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The uncoupling protein (UCP) is a proton/anion transporter found in the inner mitochondrial membrane of brown adipocyte. Although UCP has nor been detected in mitochondria from any other tissue, it shares structural and catalytic properties with several other mitochondrial carrier proteins. Although UCP was discovered only recently it is one of the most extensively studied mitochondrial carrier proteins.More recently, the mouse, rat, and human genes encoding for UCP have been isolated and sequenced. The availability of these various tools has led to several significant observations. UCP gene expression is strongly controlled at the level of transcription by signals that are activated after the stimulation of brown adipocytes by norepinephrine. The comparison of UCP gene with the genes encoding the adenine nucleotide translocator revealed the existence of structural and evolutionary homologies. Moreover, ...

Energy Technology Data Exchange (ETDEWEB)

In vivo effects of toxaphene on calcium pump activity in rat brain synaptosomes was studied. Male Sprague-Dawley rats were dosed with toxaphene at 0,25,50, and 100 mg/kg/day for 3 days and sacrificed 24 h after last dose. Ca/sup 2 +/-ATPase activity and /sup 45/Ca uptake were determined in brain P/sub 2/ fraction. Toxaphene inhibited both Ca/sup 2 +/-ATPase activity and /sup 45/Ca/sup 2 +/ uptake and the inhibition was dose dependent. Both substrate and Ca/sup 2 +/ activation kinetics of Ca/sup 2 +/-ATPase indicated non-competitive type of inhibition as evidenced by decreased catalytic velocity but not enzyme-substrate affinity. The inhibited Ca/sup 2 +/-ATPase activity and Ca/sup 2 +/ uptake were restored to normal level by exogenously added calmodulin which increased both velocity and affinity. The inhibition of Ca/sup 2 +/-ATPase activity and Ca/sup 2 +/ ...

British Library Electronic Table of Contents (United Kingdom)

This study describes the role of "inflammatory" oxidized (Ox) phospholipids in regulation of rahU (PA0122) expression and biofilm formation in Pseudomonas aeruginosa (383) wild type (rahU+) and rahU mutant (rahU-) strains. Functional analysis of RahU protein from P. aeruginosa in presence of Ox-phospholipids show: (a) LysoPC modulates RahU gene/and protein expression in rahU+ cells; (b) rahU promoter activity is increased by lysoPC and inhibited by PAPC, Ox-PAPC and arachidonic acid; the latter inhibitory effect can be reversed by lysoPC, which was enzymatically derived from PAPC; (c) biofilm formation increased in rahU- cells as compared to rahU+; and (d) inhibition of rahU promoter activity by PAPC and AA (but not lysoPC) showed significantly augmented biofilm formation in rahU+ but not ...

Energy Technology Data Exchange (ETDEWEB)

Two lines of mouse tumor cells were shown to be capable of aggregating mouse and rabbit platelets in vitro. This process required higher Mg/sup 2 +/ concentrations than were needed by other commonly used platelet-aggregating agents. Platelet-aggregating activity was also found in tumor cell membrane fragments. This membrane-bound platelet-aggregating material contained protein, lipid, and carbohydrate moieties. The presence of all three appeared to be essential for stimulating platelet aggregation. Destruction of any component abolished its activity. Platelet aggregation induced by tumor cell membrane fragments was associated with a secretory release reaction. In this process, growth-promoting activity for tumor cells was also released from platelets. These results underline the importance of platelets in establishing tumor metastases.

UK PubMed Central (United Kingdom)

BackgroundAdipokines, e.g. TNFα, IL-6 and leptin increase insulin resistance, and consequent hyperinsulinaemia influences breast cancer progression. Beside its mitogenic...Full Text Available

UK PubMed Central (United Kingdom)

BackgroundThe pre-fusion form of the herpes simplex virus (HSV) fusion protein gB undergoes pH-triggered conformational change in vitro and during viral entry (Dollery...Full Text Available

UK PubMed Central (United Kingdom)

In monocytes, sulfatide, a lipid from Mycobacterium tuberculosis, blocked priming for enhanced release of superoxide (O2-) by the macrophage activating factors lipopolysaccharide, gamma interferon,...Full Text Available

UK PubMed Central (United Kingdom)

BackgroundTristetraprolin (TTP/ZFP36) family proteins have anti-inflammatory activity by binding to and destabilizing pro-inflammatory mRNAs such as Tnf mRNA, and represent a potential...Full Text Available

UK PubMed Central (United Kingdom)

Human Fas/Apo-1 is a cell-surface protein that mediates apoptosis upon ligation with Fas ligand. The gene lies on the long arm of chromosome 10, consists of nine exons, and spans more than 26 kb of...Full Text Available

UK PubMed Central (United Kingdom)

Chaperones (stress proteins) are essential proteins to help the formation and maintenance of the proper conformation of other proteins and to promote cell survival after a large variety of environmental...Full Text Available

Energy Technology Data Exchange (ETDEWEB)

The modification of polymers by radiation grafting has been utilized for several decades. The penetrability of gamma rays allows to modify the internal surfaces of porous materials retaining its mechanical properties. In recent years applications of these materials to obtain chromatographic supports and biocatalysts have been reported. In this work, we described the grafting of glycidyl methacrylate (GMA) onto a macroporous polysulfone polymer. Reproducible amount of grafting, from 10% to 60% was obtained by choosing favourable monomer concentration and gamma radiation doses from 6 kGy up. Afterwards, iminodiacetic acid (IDA) and amino phenyl arsine oxide (PAO) were covalently attached to the grafted polyGMA, in correspondence with the grafting degree. Later on, a recombinant histidin-patch thioredoxin protein (HP-rTrx) was immobilized onto this surface by two different ways, involving specific protein orientations. The first one involves an ...

Science.gov (United States)

The Drosophila sequence-specific DNA binding protein, Adf-1, is capable of activating transcription of the alcohol dehydrogenase gene, Adh, and is implicated in the transcriptional control of other developmentally regulated genes. We have cloned the cDNA encoding Adf-1 by generating specific DNA probes deduced from partial amino acid sequence of the protein. Several cDNA clones encoding an extended open reading frame were isolated from a phage lambda library. The complete amino acid sequence of Adf-1 deduced from the longest cDNA reveals structural similarities to the putative helix-turn-helix DNA binding motif of Myb and Myb-related proteins. DNA sequence analysis of genomic clones and Northern blot analysis of mRNA suggest that Adf-1 is a single-copy gene encoding a 1.9-kb transcript. Purified recombinant Adf-1 expressed in Escherichia coli binds specifically to Adf-1 recognition sites and ...

British Library Electronic Table of Contents (United Kingdom)

The immune system modulates many key biological processes in humans. However, the exact role of the immune system in particle-associated periprosthetic osteolysis is controversial. Human tissue retrieval studies, in vivo and in vitro experiments suggest that the immune response to polymer particles is non-specific and macrophage-mediated. Lymphocytes may modulate this response. However direct lymphocyte activation by polymer particle-protein complexes seems unlikely. However, metallic byproducts may complex with serum proteins and lead to a Type IV, lymphocyte-mediated immune reaction. In predisposed individuals, this reaction may rarely lead to persistent painful joint effusions, necessitating debridement and excision of the bearing surfaces of the prosthesis. In these patients, retrieved...

British Library Electronic Table of Contents (United Kingdom)

DFNA5 was first identified as a gene causing autosomal dominant hearing loss (HL). Different mutations have been found, all exerting a highly specific gain-of-function effect, in which skipping of exon 8 causes the HL. Later reports revealed the involvement of the gene in different types of cancer. Epigenetic silencing of DFNA5 in a large percentage of gastric, colorectal and breast tumors and p53-dependent transcriptional activity have been reported, concluding that DFNA5 acts as a tumor suppressor gene in different frequent types of cancer. Despite these data, the molecular function of DFNA5 has not been investigated properly. Previous transfection studies with mutant DFNA5 in yeast and in mammalian cells showed a toxic effect of the mutant protein, which was not seen after transfection ...

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Heme peroxidases are a class of multifunctional redox-active proteins found in all organisms. We recently cloned, expressed, and characterized an ascorbate peroxidase from Leishmania major (LmAPX) that was capable of detoxifying hydrogen peroxide. Localization studies using green fluorescent protein fusions revealed that LmAPX was localized within the mitochondria by its N-terminal signal sequence. Subcellular fractionation analysis of the cell homogenate by the Percoll density-gradient method and subsequent Western blot analysis with anti-LmAPX antibody further confirmed the mitochondrial localization of mature LmAPX. Submitochondrial fractionation analysis showed that the mature enzyme (?3.6?kDa shorter than the theoretical value of the whole gene) was present in the intermembrane space ...

Science.gov (United States)

Plerocercoids of the tapeworm Spirometra mansonoides produce a substance that stimulates growth of experimental hosts. We report purification of plerocercoid growth factor (PGF) to homogeneity by a process involving isolation and solubilization of plerocercoid membranes, isoelectric point selection by chromatofocusing chromatography or preparative isoelectric focusing, and anion-exchange chromatography. A radioreceptor assay (RRA) for human growth hormone (hGH) was used to detect PGF and purity of the 27.5-kDa protein was judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteolytic activity was detected in the 27.5-kDa protein by gelatin substrate PAGE. Characterization of PGF as a neutral cysteine proteinase was based on substrate and inhibitor specificities and dependence on pH and thiol-containing reagents. The association of hGH agonist and proteinase activities was ...

Science.gov (United States)

The glass gene is required for proper photo-receptor differentiation during development of the Drosophila eye glass codes for a DNA-binding protein containing five zinc fingers that we show is a transcriptional activator. A comparison of the sequences of the glass genes from two species of Drosophila and a detailed functional domain analysis of the Drosophila melanogaster glass gene reveal that both the DNA-binding domain and the transcriptional-activation domain are highly conserved between the two species. Analysis of the DNA-binding domain of glass indicates that the three carboxyl-terminal zinc fingers alone are necessary and sufficient for DNA binding. We also show that a deletion mutant of glass containing only the DNA-binding domain can behave in a dominant-negative manner both in vivo and in a cell culture assay that measures transcriptional activation. PMID:7604032

DEFF Research Database (Denmark)

Udgivelsesdato: 2008/5

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A multifunctional protein kinase, purified from rat liver as ATP-citrate lyase kinase, has been identified as a glycogen synthase kinase. This kinase catalyzed incorporation of up to 1.5 mol of and)2numberSPO4/mol of synthase subunit associated with a decrease in the glycogen synthase activity ratio from 0.85 to a value of 0.15. Approximately 65-70% of the TUPO4 was incorporated into site 3 and 30-35% into site 2 as determined by reverse phase high performance liquid chromatography. This multifunctional kinase was distinguished from glycogen synthase kinase-3 on the basis of nucleotide and protein substrate specificities. Since the phosphate contents in glycogen synthase of sites 3 and 2 are altered in diabetes and by insulin administration, the possible involvement of the multifunctional kinase was explored. Glycogen synthase purified from diabetic rabbits was phosphorylated in vitro by this multifunctional kinase at only ...

UK PubMed Central (United Kingdom)

The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical...Full Text Available

International Nuclear Information System (INIS)

Genes encoding mutants of the thymine photodimer repair enzyme from bacteriophage T4 (T4 endonuclease V) having an amino acid substitution (T127M, W128A, W128S, Y129A, K130L, Y131A, Y132A) were constructed by use of a previously obtained synthetic gene and expressed in Escherichia coli under the control of the E. coli tryptophan promoter. An in vitro assay of partially fractionated mutant proteins for glycosylase activity was performed with chemically synthesized substrates containing a thymine photodimer. T127M and K130L showed almost the same activity as the wild-type protein. Although W128S, Y131A, and Y132A were slightly active, W128A and Y129A lost activity. The results indicated that the aromatic amino acids around position 130 may be important for the glycosylase activity. Mutant T127M was purified, and the Km value was found to be of ...

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The authors have demonstrated that induction of mitogenesis in human T lymphocytes is associated with the tyrosine phosphorylation of a 66KD soluble substrate-TPP 66. Since DMSO has been shown to be a non-specific stimulator of tyrosine protein kinases they have examined the effect of DMSO on both activation and tyrosine phosphorylation in human T cells. Human peripheral blood T lymphocytes were isolated by dextran sedimentation, Ficol/Paque centrifugation and nylon wool filtration. Phosphorylation was performed in cells incubated with ["3"2P] orthophosphate followed by DMSO for 30 min. TPP 66 was identified by 2-D PAGE, autoradiography, and HV electrophoresis of the hydrolyzed protein. Concentrations of DMSO from 1% to 50% induced the tyrosine phosphorylation of TPP 66 with maximal stimulation seen at 20%. DMSO alone did not activate the T cells (measured by ["3H] thymidine incorporation) when tested ...

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We report on the effect of potato maltodextrins with variable dextrose equivalent (Paselli SA-2, SA-6 and SA-10) on the surface behavior at the air-water interface of the mixture: legumin+small-molecule surfactant. Distinct in nature small-molecule surfactants (model: sodium salt of capric acid, Na-caprate; and commercially important: a citric acid ester of monoglyceride, CITREM) have been under our consideration. The role of the structure of both of the maltodextrins and the small-molecule surfactants in the effect studied has been elucidated by measurements in a bulk aqueous medium of the enthalpy of their interaction from mixing calorimetry, value of weight average molecular weight of the maltodextrins and the thermodynamics of the pair maltodextrin-solvent and maltodextrin-protein interactions from laser static light scattering. The combined data of mixing calorimetry and light scattering suggest some complex formation between the small-molecule surfactants and ...

Science.gov (United States)

The insulin signaling pathway, involving protein kinase B (PKB) and mitogen-activated protein kinase (MAPK), mediates the biological response to insulin and several growth factors and cytokines. To investigate the correlation between glucose transporter (Glut) biosynthesis and the insulin signaling pathway activated by novel compounds of Liriope platyphylla (LP9M80-H), alterations in Glut and key protein expression in the insulin signaling pathway were analyzed in the liver and brain of ICR mice treated with LP9M80-H. An in vitro assay showed that the highest level of insulin concentration was observed in the LP9M80-H-treated group, followed by the LP-H, LP-M, LP-E, and LP9M80-C-treated groups. Therefore, LP9M80-H was selected for use in studying the detailed mechanism of the insulin signaling pathway in animal systems. In an in vivo experiment, LP9M80-H induced a significant ...

International Nuclear Information System (INIS)

Ultraviolet light can be used to immobilize biomolecules onto thiol reactive surfaces in order to, e.g., make biosensors. The mechanism involves light-induced formation of free, reactive thiol groups in disulphide containing molecules. This technology allows for the creation of arrays of biomolecules with a high degree of reproducibility, circumventing the need for often expensive nano/micro-dispensing technologies. The ultimate size of the immobilized spots is defined by the focal area of the UV beam. Light-induced immobilization has the added benefit that the immobilized molecules will be spatially oriented and covalently bound to the surface. In this paper, we demonstrate the utility of a sensor array created with the new sensor technology when integrated into a microfluidic system. Protein arrays made using light-induced immobilization showed successful antigen/antibody binding in a flow cell allowing the visualisation of real time binding and enzyme ...

International Nuclear Information System (INIS)

The inducible transcription factor NF-#kappa#B regulates divergent signaling pathways including inflammatory response and cancer development. Selective inhibitors for NF-#kappa#B signaling are potentially useful for treatment of inflammation and cancer. NF-#kappa#B is canonically activated by preferential disposal of its inhibitory protein; I#kappa#B, which suppresses the nuclear translocation of NF-#kappa#B. I#kappa#B#alpha# (a major member of I#kappa#B family proteins) is phosphorylated with an I#kappa#B kinase (IKK) and subsequently polyubiquitylated by SCF"#beta#"T"r"C"P"1 ubiquitin-ligase in the presence of E1 and E2 prior to proteasomal degradation. Here, we describe a novel inhibitor termed GS143, which suppressed I#kappa#B#alpha# ubiquitylation, but not I#kappa#B#alpha# phosphorylation, MDM2-directed p53 ubiquitylation, and proteasome activity in vitro. GS143 markedly suppressed the destruction ...

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Various thiol reactive compounds, including phenylarsine oxide (PheAsO) and mersalyl, activate K/sup +/ transport into respiring mitochondria. Dicyclohexylcarbodiimide (DCCD) inhibits K/sup +/ influx. In the present studies, unidirectional fluxes of K/sup +/ or Mg/sup + +/ into respiring rat liver mitochondria were measured by means of /sup 42/K or /sup 28/Mg, with samples separated from incubation media by centrifugation through silicon. Experiments show that the % stimulation of K/sup +/ influx by PheAsO (8.5 nmol/mg protein) or by mersalyl (150 ..mu..M) was the same (53-55%) for control mitochondria as for samples pretreated with DCCD (30 nmol/mg protein). This means that DCCD caused the same % inhibition of K/sup +/ flux into control mitochondria and mitochondria in the presence of PheAsO or mersalyl (36-37%). Thus, the DCCD reactive site associated with the K/sup +/ influx mechanism appears to be independent of the ...

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The authors made peritoneal dialisis with 5% of glucose (7 ml/kg of body weight) in 11 dogs under the conditions of an acute experiments. They examined cortiosl and ADH activity, hematocrite and plasma protein in the samples of blood, obtained on the 20th and 50th minute. ADH was titrated biologicaly by a new method, proposed by Nacev. The results were compared with the changes in the circulatory and renal indices, obtained at the same procedure in the preceding investigations. There was an increase in the cortisol and ADH activity, which could be explained by the total hypovolemia, induced by peritoneal dialisis. The increase of the cortisol level is described as a separate link in a more complex mechanism, assuring metabolic homeostasis. PMID:1222705

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When labeled cAMP is incubated with isolated mitochondria from rat liver, it penetrates not only through the outer membrane of the mitochondria but also into the mitoplasts, where it is accumulated chiefly in the matrix. Damage to the mitochondrial membrane by freezing-thawing promotes not an influx but an efflux of cAMP out of the mitoplasts. A substantial part of the labeled nucleotide in all the investigated submitochondrial fractions was identified as intact cAMP by the method of thin-layer chromatography. The transport of cAMP into the mitochondria can explain the activation of their functions by the hormones that activate cytoplasmic adenylate cyclase and by extramitochondrial (cytosol) cAMP, as well as the presence of cAMP-dependent protein kinase and cAMP-phosphodiesterase in the mitochondria.

British Library Electronic Table of Contents (United Kingdom)

Cyclooxygenase-2 (COX-2) content is increased in many types of tumor cells. We have investigated the mechanism by which resveratrol, a stilbene that is pro-apoptotic in many tumor cell lines, causes apoptosis in human head and neck squamous cell carcinoma UMSCC-22B cells by a mechanism involving cellular COX-2. UMSCC-22B cells treated with resveratrol for 24 h, with or without selected inhibitors, were examined: (1) for the presence of nuclear activated ERK1/2, p53 and COX-2, (2) for evidence of apoptosis, and (3) by chromatin immunoprecipitation to demonstrate p53 binding to the p21 promoter. Stilbene-induced apoptosis was concentration-dependent, and associated with ERK1/2 activation, serine-15 p53 phosphorylation and nuclear accumulation of these proteins. These effects were blocked by ...

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This document consist of brief reports prepared by postdoctoral students supported by the project, each describing his accomplishments under the grant. Topics include (1) Malignant Transformation of MSU-1. 1 Cells by Gamma Radiation, (2) Correlation between Levels of ras Expression and Presence of Transformed Phenotypes Including Tumorigenicity, Using a Modulatable Promoter, (3) Relation between Specific rad Oncogene Expression, (4) Correlation of Genetic Changes in Fibroblastic Tumors with Malignancies, (5)Transformation of MSU-1.1 Cells by sis Oncogene, (6) Malignant Transformation of MSU-1.0 Cells, (7) Correlation of Urokinase Plasminogen Activation (mu-PA) with Malignant Phenotype, (8)Two Dimensional Gel Electrophoresis Studies of the Proteins of the Major Cell Strains of the MSU-1 Family of Cells, and (9) Correlation between Proteinase Activity Levels and Malignancy.

British Library Electronic Table of Contents (United Kingdom)

Mutations in sclerostin function or expression cause sclerosing bone dysplasias, involving decreased antagonism of Wnt/Lrp5 signaling. Conversely, deletion of the VHL tumor suppressor in osteoblasts, which stabilize HIF-a isoforms and thereby enables HIF-a/b-driven gene transcription, increases bone mineral content and cross-sectional area compared to wild-type controls. We examined the influence of cellular hypoxia (1% oxygen) upon sclerostin expression and canonical Wnt signaling. Osteoblasts and osteocytes cultured under hypoxia revealed decreased sclerostin transcript and protein, and increased expression and nuclear localization of activated b-catenin. Similarly, both hypoxia and the hypoxia mimetic DFO increased b-catenin gene reporter activity. Hypoxia and its mimetics increased exp...

Science.gov (United States)

Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is

UK PubMed Central (United Kingdom)

Many mutations associated with retinal degeneration lead to the production of misfolded proteins by cells of the retina. Emerging evidence suggests that these abnormal proteins cause cell death...Full Text Available

International Nuclear Information System (INIS)

Three cDNAs, designated IIA3, IIA3v, and IIA4, coding for P450s in the CYP2A gene subfamily were isolated from a #lambda#gt11 library prepared from human hepatic mRNA. Only three nucleotide differences and a single amino acid difference, Leu"1"6"0#->#His, were found between IIA3 and IIA3v, indicating that they are probably allelic variants. IIA4 displayed 94% amino acid similarity with IIA3 and IIA3v. The three cDNAs were inserted into vaccinia virus, and recombinant viruses were used to infect human hepatoma Hep G2 cells. Only IIA3 was able to produce an enzyme that had a reduced CO-bound spectrum with a #lambda#_m_a_x at 450 nm. This expressed enzyme was able to carry out coumarin 7-hydroxylation and ethoxycoumarin O-deethylation. cDNA-expressed IIA3v and IIA4 failed to incorporate heme and were enzymatically inactive. Analysis of IIA proteins in human liver microsomes, using antibody against rat IIA2, revealed two proteins of 49 and 50 ...

Science.gov (United States)

Receptor interacting protein 140 (RIP140) is a coregulator for numerous nuclear receptors and transcription factors and primarily exerts gene-repressive activities on various target genes. We previously identified a spectrum of posttranslational modifications on RIP140 that augment its property and biological activity. In T(3)-triggered biphasic regulation of cellular retinoic acid binding protein 1 (Crabp1) gene along the course of fibroblast-adipocyte differentiation, we found TRAP220(MED1) critical for T(3)-activated chromatin remodeling whereas RIP140 essential for T(3)-repressive chromatin remodeling of this gene promoter. In this current study, we aim to examine whether and how RIP140 replaces TRAP220(MED1) on the CrabpI promoter in differentiating adipocyte cultures. We find increasing recruitment of RIP140 to this promoter, with corresponding reduction in TRAP220(MED1) ...

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Constitutive activation of the oncogenic transcription factor STAT3 frequently occurs in various human malignancies. STAT3 activation involves dimerization via intermolecular pTyr-SH2 interaction. Thus, antagonizing this interaction is a feasible approach to inhibit STAT3 activation for cancer therapy. In order to identify selective STAT3 inhibitors, we developed a biochemical HTS system based on AlphaScreen technology, which measures the abilities of test compounds to antagonize pTyr-SH2 interactions. We screened our chemical libraries using this system and identified 5,15-diphenylporphyrin (5,15-DPP) as a selective STAT3-SH2 antagonist. Selective inhibition of STAT3 nuclear translocation and DNA biding activity was observed in cells treated with 5,15-DPP. IL-6-dependent dimerization of STAT3, c-myc promoter binding and c-myc protein expression were all suppressed by 5,15-DPP, ...

Science.gov (United States)

S100A4, a member of the Ca(2+)-activated S100 protein family, regulates the motility and invasiveness of cancer cells. Moreover, high S100A4 expression levels correlate with poor patient survival in several cancers. Although biochemical, biophysical, and structural data indicate that S100A4 is a noncovalent dimer, it is unknown if two functional S100A4 monomers are required for the productive recognition of protein targets and the promotion of cell invasion. To address this question, we created covalently linked S100A4 dimers using a glycine rich flexible linker. The single-chain S100A4 (sc-S100A4) proteins exhibited wild-type affinities for calcium and nonmuscle myosin-IIA, retained the ability to regulate nonmuscle myosin-IIA assembly, and promoted tumor cell invasion when expressed in S100A4-deficient colon carcinoma cells. Mutation of the two calcium-binding EF-hands in one monomer, while leaving ...

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Peroxisome proliferating agents (PPAs) are a structurally diverse group of chemicals that include environmental chemical contaminants such as certain chlorinated herbicides, solvents and plasticizers. PPAs have previously been shown to induce anti-trout laruci acid hydroxylase immunoreactive proteins in bluegill and catfish. In this investigation, induction of lauric acid hydroxylase activity and immunoreactive proteins was confirmed, and the mass spectral analysis of specific hydroxylation products was performed in order to identify possible species-specific differences in fatty acid metabolism. Male bluegill (Lepomis macrochirus) and channel catfish (Ictalurus punctatus) were administered clofibrate or ciprofibrate 48 hr prior to hepatic or trunk kidney (catfish only) microsome preparation. While no significant differences were observed in male catfish, male bluegill had significant decreases in hematocrit and plasma ...

International Nuclear Information System (INIS)

We have previously shown that budded viruses of Bombyx mori nucleopolyhedrovirus (BmNPV) enter the cell cytoplasm but do not migrate into the nuclei of non-permissive Sf9 cells that support a high titer of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) multiplication. Here we show, using the syncytium formation assay, that low-pH-triggered membrane fusion of BmNPV GP64 protein (Bm-GP64) is significantly lower than that of AcMNPV GP64 protein (Ac-GP64). Mutational analyses of GP64 proteins revealed that a single amino acid substitution between Ac-GP64 H155 and Bm-GP64 Y153 can have significant positive or negative effects on membrane fusion activity. Studies using bacmid-based GP64 recombinant AcMNPV harboring point-mutated ac-gp64 and bm-gp64 genes showed that Ac-GP64 H155Y and Bm-GP64 Y153H substitutions decreased and increased, respectively, the multiplication and cell-to-cell spread ...

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PMID:20880255

KoreaScience (Korea)

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Sulfate activation occurs in Euglena mitochondria the authors now find that the sulfate activating enzymes are absent from Euglena chloroplasts. Cells of mutant W/sub 10/BSmL lacking plastids also lack detectable sulfolipid (SL) when grown on /sup 35/SO/sub 4//sup 2 -/ indicating that SL is absent from the mitochondria and is exclusively in the plastids. Plastids alone will convert /sup 35/S-cysteine to /sup 35/SL in the presence of ATP and Mg/sup 2 +/; light is stimulatory. Under similar conditions, chloroplasts and mitochondria incubated together convert /sup 35/SO/sub 4//sup 2 -/ to plastid-localized /sup 35/SL but either organelle incubated alone fails to effect this conversion. Unlabeled cysteine blocks SL labeling from sulfate in the mixed incubation; since cysteine is formed from sulfate by Euglena mitochrondria, cysteine (and other compounds) may move from the mitochondrion to the chloroplast to provide the sulfo group for SL formation. ...

International Nuclear Information System (INIS)

Purpose: To analyze p53, bcl-2, c-myc, and cyclooxygenase-2 protein expression changes and examine their relationship with various antioxidant enzymes in esophageal carcinoma patients. Methods and Materials: Patients in Group 1 underwent transhiatal esophagectomy and those in Group 2 were administered chemoradiotherapy followed by surgery after 4 weeks of neoadjuvant therapy. Results: The relationship analysis among the various protein markers and antioxidant enzymes showed an inverse correlation between bcl-2 and superoxide dismutase/catalase in tumor tissues, irrespective of the treatment arm followed. An important positive association was observed between bcl-2 and reduced glutathione levels in the tumor tissue of patients receiving neoadjuvant therapy. Another apoptosis-modulating marker, c-myc, in the tumor tissue of Group 2 patients showed similar pattern levels (high and low) as that of superoxide dismutase/catalase. The association of ...

International Nuclear Information System (INIS)

We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to ...

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The in situ assembly states of the glucose transport carrier protein in the plasma membrane and in the intracellular (microsomal) storage pool of rat adipocytes were assessed by studying radiation-induced inactivation of the D-glucose-sensitive cytochalasin B binding activities. High energy radiation inactivated the glucose-sensitive cytochalasin B binding of each of these membrane preparations by reducing the total number of the binding sites without affecting the dissociation constant. The reduction in total number of binding sites was analyzed as a function of radiation dose based on target theory, from which a radiation-sensitive mass (target size) was calculated. When the plasma membranes of insulin-treated adipocytes were used, a target size of approximately 58,000 daltons was obtained. For adipocyte microsomal membranes, we obtained target sizes of approximately 112,000 and 109,000 daltons prior to and after insulin treatment, ...

Science.gov (United States)

The acute effects of injections of the human growth hormone-like factor purified from plerocercoids of the tapeworm Spirometra mansonoides on carbohydrate, lipid, and protein metabolisms were determined in intact rats. Male rats were injected ip with saline, insulin, or various doses of partially purified PGF. The rats injected with insulin had significantly reduced serum glucose concentrations but no dose of PGF caused a change in serum glucose levels. Insulin and PGF stimulated [14C]glucose and [14C]leucine oxidation to 14CO2 in adipose tissue and muscle and increased incorporation of both [14C]glucose carbons into lipids and [14C]leucine into protein in fat and muscle. The responses to PGF were dose-dependent and persisted after 3 hr of incubation in vitro. Injections of naloxone prior to injecting PGF to block the stress response did not prevent the stimulation of insulin-like responses by PGF. Therefore, PGF has intrinsic insulin-like ...

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Functionalization of carbon nanotubes is very challenging for their applications. The paper here describes a new method to functionalize multi-walled carbon nanotubes (MWCNTs) as specific affinity adsorbents. MWCNTs were acid purified and pretreated with (3-aminopropyl)-triethoxysilane (APTES) in order to introduce abundant amino groups on the surface of MWCNTs. After the conversion of amino groups to carboxyl groups by succinic acid anhydride, MWCNTs were attached to protein A or aminodextran using 1-ethyl-3,3' (dimethylamion)-propylcarbodiimide as a biofunctional crosslinker. The incorporation of aminodextran as a spacer arm noticeably increased the binding capacity of the APTES-modified MWCNTs for protein A. The application of affinity MWCNTs for purification of immunoglobulin G was then evaluated. The affinity of MWCNTs with AMD spacer exhibited a high adsorption capacity of {approx}361 {mu}g IgG/mg MWCNT (wet basis). About 75% of ...

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Abstract. Objective: Lapatinib (Tykerb, GW572016), a potent inhibitor of the catalytic activities of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) (ErbB2), inhibits population growth of selected EGFR and HER2 overexpressing cell lines. Previous studies with a small number of cell lines suggest a correlation between overexpression of EGFR and/or HER2 and sensitivity to growth inhibition by lapatinib; however, the precise determinants of lapatinib selectivity for tumour and/or other cells remain unclear. Materials and methods: To clarify the determinants of its selectivity in cultured cells, lapatinib-induced cell population growth inhibition and relative EGFR and HER2 protein expression were quantified in 61 different human tumour cell lines fro...

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Shewanella oneidensis MR-1 is a model environmental organism that possesses diverse respiratory capacities, including the ability to reduce soluble Cr(VI) to sparingly soluble, less toxic Cr(III). Effective bioremediation of Cr-contaminated sites requires knowledge of the molecular mechanisms and regulation of heavy metal resistance and biotransformation by dissimilatory metal-reducing bacteria. Towards this goal, our ERSP-funded work is focused on the identification and functional analysis of genes/proteins comprising the response pathways for chromate detoxification and/or reduction. Previous transcriptomic profiling and whole-cell proteomic analyses implicated the involvement of a functionally undefined DNA-binding response regulator (SO2426) and a putative azoreductase (SO3585) in the chromate stress response of MR-1. Here we describe a detailed functional analysis of SO2426 and SO3585 in order to begin to understand the role of these ...

British Library Electronic Table of Contents (United Kingdom)

Cholestatic liver injury induced by alpha-naphthylisothiocyanate (ANIT) is provoked by injury to intrahepatic bile ducts and the progression of hepatic necrosis requires the procoagulant protein tissue factor (TF) and extrahepatic cells including neutrophils. Recent studies have shown that myeloid cell TF contributes to neutrophil activation. We tested the hypothesis that myeloid cell TF contributes to neutrophil activation in ANIT-treated mice. TF activity in liver homogenates increased significantly in TF^f^l^o^x^/^f^l^o^x mice treated with ANIT, but not in TF^f^l^o^x^/^f^l^o^x/LysMCre mice (TFD^M^y^e^l^o^i^d mice), which have reduced TF expression in monocytes/macrophages and neutrophils. Myeloid cell-specific TF deficiency did not alter expression of the chemokines KC or MIP-2 but redu...

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Spot 14 (S14) is a protein that is abundantly expressed in lipogenic tissues and is regulated in a manner similar to other enzymes involved in fatty acid synthesis. Deletion of S14 in mice decreased lipid synthesis in lactating mammary tissue, but the mechanism of S14's action is unknown. Here we present the crystal structure of S14 to 2.65 {angstrom} and biochemical data showing that S14 can form heterodimers with MIG12. MIG12 modulates fatty acid synthesis by inducing the polymerization and activity of acetyl-CoA carboxylase, the first committed enzymatic reaction in the fatty acid synthesis pathway. Coexpression of S14 and MIG12 leads to heterodimers and reduced acetyl-CoA carboxylase polymerization and activity. The structure of S14 suggests a mechanism whereby heterodimer formation with MIG12 attenuates the ability of MIG12 to activate ACC.

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Vascular endothelial growth factor (VEGF) is a mitogen that is critically involved in vasculogenesis, angiogenesis, and hematopoiesis. However, what and how transcription factors participate in the regulation of vegf gene expression are not fully understood. Here we report the cloning and sequencing of the zebrafish vegf promoter which revealed that the promoter contains a number of bone morphogenetic protein (BMP)-activated Smad binding elements (SBE), implicating Smad1 and Smad5 in the regulation of BMP-induced expression of vegf. Electrophoretic mobility shift assays of adding recombinant Smad proteins to the SBE-containing DNA oligonucleotides that represent portions of zebrafish vegf promoter resulted in mobility shift of the oligonucleotides. These changes demonstrate potential interactions between Smad1/5 and the vegf promoter. Reporter activity assays using the wild-type or SBE-deleted vegf ...

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Tungsten, in the form of tungstate, polymerizes with phosphate, and as extensive polymerization occurs, cellular phosphorylation and dephosphorylation reactions may be disrupted, resulting in negative effects on cellular functions. A series of studies were conducted to evaluate the effect of tungsten on several phosphate-dependent intracellular functions, including energy cycling (ATP), regulation of enzyme activity (cytosolic protein tyrosine kinase [cytPTK] and tyrosine phosphatase), and intracellular secondary messengers (cyclic adenosine monophosphate [cAMP]). Rat noncancerous hepatocyte (Clone-9), rat cancerous hepatocyte (H4IIE), and human cancerous hepatocyte (HepG2) cells were exposed to 1-1000 mg/l tungsten (in the form of sodium tungstate) for 24 h, lysed, and analyzed for the ab...

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Aims: Rapamycin (sirolimus) is an antibiotic that inhibits protein synthesis through mammalian targeting of rapamycin (mTOR) signaling, and is used as an immunosuppressant in the treatment of organ rejection in transplant recipients. Rapamycin confers preconditioning-like protection against ischemic-reperfusion injury in isolated mouse heart cultures. Our aim was to further define the role of rapamycin in intracellular Ca^2^+ homeostasis and to investigate the mechanism by which rapamycin protects cardiomyocytes from hypoxic damage. Main methods: We demonstrate here that rapamycin protects rat heart cultures from hypoxic-reoxygenation (H/R) damage, as revealed by assays of lactate dehydrogenase (LDH) and creatine kinase (CK) leakage to the medium, by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-d...

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Background. A 19-year-old girl was diagnosed with systemic lupus erythematosus, based on findings of arthritis, malar rash, positive antinuclear antibody test and high levels of antibodies to double-stranded DNA. Two months after diagnosis, the patient presented with a sudden drop in blood hemoglobin level. Several days later, she developed bloody sputum, rapidly progressive dyspnea and hypoxemia. High-resolution CT showed diffuse alveolar infiltrates in both lung fields.Investigations. Physical examination, complete blood count, erythrocyte sedimentation rate, urinalysis, 24-h urine protein excretion, fecal occult blood test, d-dimer test, acid hemolysis test, activated partial thromboplastin time and prothrombin time, direct and indirect Coombs tests, bone marrow smear, arterial blood ga...

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Manganese toxicity can cause a neurodegenerative disorder affecting cortical and basal ganglia structures with a neurological presentation resembling features of Parkinson's disease. Children are more sensitive to Mn-induced neurological dysfunction than adults, and recent studies from our laboratory revealed a marked sensitivity of male juvenile mice to neuroinflammatory injury from Mn, relative to females. To determine the role of estrogen (E2) in mediating sex-dependent vulnerability to Mn-induced neurotoxicity, we exposed transgenic mice expressing an NF-kB-driven enhanced green fluorescent protein (EGFP) reporter construct (NF-kB-EGFP mice) to Mn, postulating that supplementing male mice with E2 during juvenile development would attenuate neuroinflammatory changes associated with glia...

British Library Electronic Table of Contents (United Kingdom)

PP2B is a Ca2+/calmodulin-dependent protein phosphatase that is ubiquitously expressed in mammals. Among other actions, it is an effector mechanism in NMDA-mediated glutamate neurotransmission as well as a regulator of GSK3? and MAPK signaling cascades. Because all of these mechanisms have demonstrable roles in the control of circadian rhythyms, we hypothesized that PP2B would be a key regulator of rhythm generation and entrainment, and that through inhibition of its phosphatase activity, the circadian system would be affected by immunosuppressant drug therapy. We report here that immunosuppressant drugs (cyclosporin A, FK506) (1) block the circadian responses to light that underlie photic entrainment; (2) produce circadian phase shifts with a characteristic nonphotic profile; and (3) disr...

British Library Electronic Table of Contents (United Kingdom)

Objective: : Evaluate expression of inducible negative regulators of JAK/STAT pathway and their target proteins during the course of ligature-induced experimental periodontal disease in rats. Design: : Rats were sacrificed 07, 15 and 30days after disease induction for histological evaluation of periodontal inflammation and macroscopic analysis of alveolar bone loss. SOCS expression and the activation status of STAT1 and STAT3 were evaluated in gingival biopsies by real time PCR and Western blot. Results: : Ligature-induced model presented significant progressive bone loss from 7 to 30days. Inflammation was evident and similar for 07 and 15days; however, a decrease on severity at the end of the experimental period was observed. There was a significant (p<0.05) increase on SOCS1 and SOCS3 ge...

British Library Electronic Table of Contents (United Kingdom)

The chemopreventive efficacy of turmeric has been established in experimental systems. However, its mechanism(s) of action are not fully elucidated in vivo. The present study investigates the mechanism of turmeric-mediated chemoprevention in 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis at 2, 4, 6, 10 and 12?weeks. Dietary turmeric (1%) led to decrease in DMBA-induced tumor burden and multiplicity, and enhanced the latency period in parallel, to its modulatory effects on oncogene products and various cellular responses during HBP tumorigenesis. DMBA-induced expression of ras oncogene product, p21 and downstream target, the mitogen-activated protein kinases were significantly decreased by turmeric during HBP carcinogenesis. Turmeric also diminished ...

British Library Electronic Table of Contents (United Kingdom)

Apelin is a peptide known as the ligand of the G-protein-coupled receptor APJ. Several active apelin forms exist such as apelin-36, apelin-17, apelin-13, and the pyroglutamated form of apelin-13. Apelin and APJ are expressed in the central nervous system, particularly in the hypothalamus and in many peripheral tissues. Apelin has been shown to be involved in the regulation of cardiovascular and fluid homeostasis, food intake, cell proliferation, and angiogenesis. In addition to be an ubiquitous peptide, apelin is also produced and secreted by adipocytes and thus considered as an adipokine. This has opened a new field of investigation establishing a link between apelin and metabolic disorders (obesity, type 2 diabetes, etc.) which is the focus of the present review. Several studies, but not...

Science.gov (United States)

Sida cordifolia is a plant belonging to the Malvaceae family used in many ayurvedic preparations. This study aimed at assessing the effects of ethanolic extract of Sida cordifolia root on quinolinic acid (QUIN) induced neurotoxicity and to compare its effect with the standard drug deprenyl in rat brain. Rats were divided into six groups: (1) control group (2) QUIN (55 microg/100 g bwt/day) (3) 50% ethanolic plant extract treated group (50 mg/100 g bwt/day) (4) Deprenyl (100 microg/100 g bwt/day) (5) QUIN (55 microg/100 g bwt/day) + 50% ethanolic plant extract treated group (50 mg/100 g bwt/day) (6) QUIN (55 microg/100 g bwt/day) + Deprenyl (100 microg/100 g bwt/day). At the end of the experimental period a status of lipid peroxidation products, protein peroxidation product, activities of the scavenging enzymes and the activities of the inflammatory markers were analyzed. Results revealed that the lipid peroxidation products ...

DEFF Research Database (Denmark)

Aldosterone has been suggested to elicit vessel contraction via a nongenomic mechanism. We tested this proposal in microdissected, perfused rabbit renal afferent arterioles. Aldosterone had no effect on internal diameter in concentrations from 10(-10) to 10(-5) mol/L, but aldosterone abolished the ability of 100 mmol/L KCl to induce vascular contraction. The inhibitory effect of aldosterone was observed from 1 pmol/L. The inhibitory effect was significant after 5 minutes and maximal after 20 minutes and was fully reversible. Actinomycin D (10(-6) mol/L) prolonged the effect of aldosterone. The effect was abolished by the mineralocorticoid receptor antagonist spironolactone (10(-7) mol/L) but not by the glucocorticoid receptor antagonist mifepristone (10(-6) mol/L). The K+-mediated increase of intracellular calcium concentration in afferent arterioles was not affected by aldosterone. Mineralocorticoid receptor was detected by reverse transcription-polymerase chain reaction and ...

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The herpes simplex virus type 1 thymidine kinase (HSV1-TK) reporter system is being used to directly and indirectly monitor therapeutic gene expression, immune cell trafficking and protein-protein interactions in various living animals. However, the issues of HSV1-TK enzyme stability in living cells and whether this reporter system is optimal for dynamic studies of gene expression events in genetic imaging have not be addressed. The purpose of the present study was to evaluate the application of this reporter system in dynamic studies of transcriptional gene regulation. To achieve this purpose, we established two tetracycline-inducible murine sarcoma cell lines, tetracycline-turn-off HSV1-tk-expressing cell line (NG4TL4/tet-off-HSV1-tk) and tetracycline-turn-off Luc-expressing cell line (NG4TL4/tet-off-Luc), to create an artificially regulated gene expression model in vitro. The dynamic transcriptional events mediating a series of doxycycline (Dox) inductions were ...

Science.gov (United States)

Poncirin, flavanone glycoside, isolated from the fruit of Poncirus trifoliata, has anti-bacterial and anti-inflammatory activities. In this study, the effects of poncirin on the differentiation of mesenchymal stem cells were investigated. The C3H10T1/2 mesenchymal stem cells and primary bone marrow mesenchymal stem cells were studied. In the C3H10T1/2 cells, poncirin prevented adipocyte differentiation, as demonstrated by inhibition of cytoplasm lipid droplet accumulation and peroxisome proliferator-activating receptor-? (PPAR-?) and CCAAT-enhancer-binding protein-? (C/EBP-?) mRNA expression. By contrast, poncirin enhanced the expression of the key osteogenic transcription factors, runt-related transcription factor 2 (Runx2) and transcriptional coactivator with PDZ-binding motif (TAZ). Poncirin also enhanced expression of the osteogenic marker genes including alkaline phosphatase (ALP) and osteocalcin (OC). Poncirin ...

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Sheep red blood cells (SRBCs) were labelled with a concanavalin A-luminol-bovine serum albumin conjugate specific for the transmembrane anion transport protein (Band 3) and exposed to 2450-MHz continuous-wave microwave radiation at an average specific absorption rate of 91W/kg for 10 min. The temperature was held constant at 25, 37, 40, 42, or 45C with an airflow heat-exchange system. Following exposure to microwave or air heating, the decrease in residual base-activated chemiluminescence (CL) of the SRBCs was measured as an indication of infield oxidase activity. Air heating resulted in a significant decrease in residual CL at temperatures above 37C (74% decrease at 45C). Microwave radiation inhibited the decline in residual CL above 37C. At 45C the inhibition was 40%. The results suggest microwave radiation either reversibly altered the thermodynamics of oxygen binding to haemoglobin or failed to energize a significant ...

Science.gov (United States)

Tau is a microtubule-associated protein that promotes microtubule assembly and stability. In Alzheimer's disease and related tauopathies, tau fibrillizes and aggregates into neurofibrillary tangles. Recently, oleocanthal isolated from extra virgin olive oil was found to display non-steroidal anti-inflammatory activity similar to ibuprofen. Since our unpublished data indicates an inhibitory effect of oleocanthal on A? fibrillization, we reasoned that it might inhibit tau fibrillization as well. Herein we demonstrate that oleocanthal abrogates fibrillization of tau by locking tau into the naturally unfolded state. Using PHF6 consisting of the amino acid residues VQIVYK, a hexapeptide within the third repeat of tau that is essential for fibrillization, we show that oleocanthal forms an adduct with the lysine via initial Schiff base formation. Structure and function studies demonstrate that the two aldehyde groups of oleocanthal are required for ...

International Nuclear Information System (INIS)

Astrocyte dysfunction is implicated in course of various age-related neurodegenerative diseases. Chronic injection of D-galactose can cause a progressive deterioration in learning and memory capacity and serve as an animal model of aging. To investigate the involvement of astrocytes in this model, oxidative stress biomarkers, biochemical and pathological changes of astrocytes were examined in the hippocampus of the rats with six weeks of D-galactose injection. D-galactose-injected rats displayed impaired antioxidant systems, an increase in nitric oxide levels, and a decrease in reduced glutathione levels. Consistently, western blotting and immunostaining of glial fibrillary acidic protein showed extensive activation of astrocytes. Double-immunofluorescent staining further showed activated astrocytes highly expressed inducible nitric oxide synthase. Electron microscopy demonstrated the degeneration of astrocytes, especially ...

International Nuclear Information System (INIS)

An assay for the microsomal hydroxylation of lauric acid (LA), based on HPLC with flow-through radiochemical detection, has been developed. Conditions were optimized for resolution and quantitation of three microsomal metabolites of "1"4C-LA, one of which has not been reported previously as a metabolite of LA in mammalian microsomal incubations. These products, 12-(omega)-hydroxy-LA, 11-(omega-1)-hydroxy-LA, and a novel metabolite, 10-(omega-2)-hydroxy-LA, were isolated by HPLC and identified by gas chromatography/mass spectrometry. In the presence of NADPH, the formation of all three metabolites was linear with time and microsomal protein concentration. Hydrogen peroxide also supported the microsomal metabolism of LA, although the ratio of metabolites was substantially different than that produced by NADPH-supported microsomes. Several biochemical probes (metyrapone, #alpha#-naphthoflavone, 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride, and 10-undecynoic ...

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The ''biotechnology 2000'' project is supervised by the project sponsor ''biology, energy, ecology'' at Forschungszentrum Juelich on behalf of the Federal Ministry for Research and Technology. The project activities cover the development of techniques and methods, cell biology, gene structure and gene regulation, photosynthetic production processes, biological hydrogen production, synthetic biology, protein design, neurobiological research, biosystems, plant breeding, phytomedicine and plant protection, the biology of waste disposal methods, research into methods which replace animal experiments, biological safety research, technology risk assessment, and ethical aspects. A general survey introduces the promoted projects, and standardized data sheets briefly introduce the individual activities. The appendix gives the project indices, the indices of ...

Science.gov (United States)

From the dried fruiting bodies of the mushroom Armillaria luteo-virens, a dimeric lectin with a molecular mass of 29.4 kDa has been isolated. The purification procedure involved (NH{sub 4}){sub 2}SO{sub 4} precipitation, ion exchange chromatography on DEAE-cellulose, CM-cellulose, and Q-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The hemagglutinating activity of the lectin could not be inhibited by simple sugars but was inhibited by the polysaccharide inulin. The activity was stable up to 70 {sup o}C but was acid- and alkali-labile. Salts including FeCl{sub 3}, AlCl{sub 3}, and ZnCl{sub 2} inhibited the activity whereas MgCl{sub 2}, MnCl{sub 2}, and CaCl{sub 2} did not. The lectin stimulated mitogenic response of mouse splenocytes with the maximal response achieved by 1 {mu}M lectin. Proliferation of tumor cells including MBL2 cells, HeLa cells, and L1210 cells ...

Science.gov (United States)

We previously reported that the peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) mRNA in rat epitrochlearis muscle was increased after swimming exercise training. In the present study, we demonstrated further that PGC-1 mRNA expression in the epitrochlearis muscle of 4-5-week-old male Sprague-Dawley rats was increased after a 6-h acute bout of low-intensity swimming exercise. With this increase, the expression level was approximately 8-fold of control and immersion group rats that stayed for 6-h in warm water, maintained at the identical temperature of the swimming barrel (35 degrees C) (p<0.01). Second, PGC-1 mRNA expression in the muscle was found to have increased 6-h after 30 10-s tetani contractions were induced by in vitro electrical stimulation. Finally, PGC-1 mRNA expression in the muscle incubated for 18-h with 0.5mM 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR: a 5' AMP-activated ...

UK PubMed Central (United Kingdom)

Known vertebrate GATA proteins contain two zinc fingers and are required in development, whereas invertebrates express a class of essential proteins containing one GATA-type zinc finger. We isolated...Full Text Available

UK PubMed Central (United Kingdom)

The fusogenic orthoreoviruses express nonstructural fusion-associated small transmembrane (FAST) proteins that induce cell-cell fusion and syncytium formation. It has been speculated that the FAST proteins...Full Text Available

UK PubMed Central (United Kingdom)

The changes in protein species synthesized during early Drosophila embryogenesis were characterized by two-dimensional electrophoresis. Of the 261 proteins scored, 68 (26%) show dramatic changes in...Full Text Available

UK PubMed Central (United Kingdom)

A protein evolution strategy is described by which double-stranded DNA fragments encoding defined E. coli protein secondary structural elements (α-helices, β-strands...Full Text Available

UK PubMed Central (United Kingdom)

We have used the two-electrode voltage clamp technique and the patch clamp technique to investigate the regulation of ROMK1 channels by protein-tyrosine phosphatase (PTP) and protein-tyrosine...Full Text Available

UK PubMed Central (United Kingdom)

A heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)propionate, was synthesized. Its N-hydroxysuccinimide ester group reacts with amino groups and the 2-pyridyl disulphide structure reacts...Full Text Available

UK PubMed Central (United Kingdom)

Protein modifications such as phosphorylation are often studied by two-dimensional gel electrophoresis since the perturbation in the protein’s pI value is readily detected by this method....Full Text Available

UK PubMed Central (United Kingdom)

The nucleocapsid protein (NC) of retroviruses plays a major role in genomic RNA packaging, and some evidence has implicated the matrix protein (MA) of certain retroviruses in viral RNA binding. To further...Full Text Available

UK PubMed Central (United Kingdom)

In this study, we investigated on a systems level how complex protein interactions underlying cell polarity in yeast determine the dynamic association of proteins with the polar cortical domain (PCD)...Full Text Available

UK PubMed Central (United Kingdom)

Many genetic processes depend on proteins interacting with specific sequences on DNA. Despite the large excess of nonspecific DNA in the cell, proteins can locate their targets rapidly. After initial...Full Text Available

UK PubMed Central (United Kingdom)

Computer-assisted analysis revealed a striking sequence similarity between the putative 24-kDa protein (p24) encoded by open reading frame (ORF) 5 of beet yellows closterovirus and the coat protein...Full Text Available

UK PubMed Central (United Kingdom)

Tooth enamel biomineralization is mediated by enamel proteins synthesized by ameloblast cells. Two classes of proteins have been described: enamelins and amelogenins. In lower vertebrates the absence...Full Text Available

UK PubMed Central (United Kingdom)

BackgroundThe fast growing Protein Data Bank contains the three-dimensional description of more than 45000 protein- and nucleic-acid structures today. The large majority of the data...Full Text Available

UK PubMed Central (United Kingdom)

Alpha-hemoglobin-stabilizing protein (AHSP) is an erythroid-specific protein that acts as a molecular chaperone for the free α chains of hemoglobin. Evidence strongly suggests...Full Text Available

Science.gov (United States)

Using the phasmid vector pSL5, the genomic DNA fragment of T. aquaticus YT1 which contained the thermostable DNA polymerase (Taq-polymerase) gene was cloned. The BglII fragment of this genome locus was subcloned in the BamHI site of the pUC19 plasmid. To optimize the Taq-polymerase gene expression in E. coli cells, the gene was cloned in the correct reading frame regarding the initiation ATG codon of the pPR-TGATG-1 expression vector. The gene expression in this vector was controlled by the phage lambda PR promoter and the temperature-sensitive phage lambda repressor. We used PCR to amplify the short 5'-end fragment of the Taq-polymerase gene coding for the part into which an artificial SacI site was introduced. This site has been used for cloning the PCR product into the pPR-TGATG-1 vector, and the missing gene part was cloned into the KpnI site of the PCR product from the natural cloned gene. The cells of the E. coli PVG-A1 strain, which was obtained in the end, expressed efficiently ...

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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{sub 3}) receptors. Promoters containing the palindromic T{sub 3} response element TCAGGTCA TGACCTGA are positively regulated by the T{sub 3} receptor after the administration of T{sub 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{sub 3} or estrogen receptors on the estrogen or T{sub 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 ...

UK PubMed Central (United Kingdom)

The receptor-associated protein (RAP) is a molecular chaperone that binds tightly to certain newly synthesized LDL receptor family members in the endoplasmic reticulum (ER) and facilitates their delivery...Full Text Available

British Library Electronic Table of Contents (United Kingdom)

Summary: Protein features are often displayed along the linear sequence of amino acids that make up that protein, but in reality these features occupy a position in the folded proteins 3D space. Mapping sequence features to known or predicted protein structures is useful when trying to deduce the function of those features and when evaluating sequence or structural predictions. To facilitate this goal, we developed PDBpaint, a simple tool that displays protein sequence features gathered from bioinformatics resources on top of protein structures, which are displayed in an interactive window (using the Jmol Java viewer). PDBpaint can be used either with existing protein structures or with novel structures provided by the user. The current version of PDBpaint allows the visualization of annot...

UK PubMed Central (United Kingdom)

We identify new organelles associated with the vacuolar system in plant cells. These organelles are defined biochemically by their internal content of three integral membrane proteins: a chimeric reporter...Full Text Available

Science.gov (United States)

Protein isolated from hen egg-white and functions as a bacteriostatic enzyme by degrading bacterial cell walls. First enzyme ever characterized by protein ...

International Nuclear Information System (INIS)

The present study was designed to assess the radioprotective effects of amifostine on ovarian follicles. Three week-old female mice with or without pretreatment of amifostine were irradiated with 6.42 Gy of #gamma# -ray. Ovaries were collected 0 and 6h after irradiation. DNA fragmentation pattern and expression of genes and activity of proteins related with apoptosis were investigated by means of RT-PCR and Western blot. Proliferation of granulosa cells was reduced and incidence rate of follicular atresia was increased in ovarian follicles in #gamma# -ray irradiated mice compared to those in control or amifostine-treated group. DNA fragmentation was increased in time-dependent manner in granulosa cells of all irradiated groups. However, no difference between amifostine pre-treated group and irradiated groups was found and the expression of p53 as tumor suppressor gene and Bax as one of pro-apoptotic Bcl-2 family was increased in irradiated mice ...

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The maintenance of a balance between cell gain and cell loss is essential for proper liver function. The exact role of aryl hydrocarbon receptor (AhR) in regulating cell proliferation and apoptosis of liver cells remains unclear, since ligand-dependent activation of AhR has been shown to induce cell cycle arrest, proliferation, differentiation or apoptosis, depending on the cellular model used. AhR can directly interact with retinoblastoma protein in hepatic cells, forming protein complexes that can efficiently block cell cycle progression by inducing G1 arrest, or to induce the expression of inhibitors of cyclin-dependent kinases, such as p271. On the other hand, it has been suggested that AhR could play a stimulatory role in cell proliferation, either directly or by mediating a release from contact inhibition. It is now generally accepted that progenitor cells exist in the liver, are activated in ...

UK PubMed Central (United Kingdom)

Pseudomonas syringae is a gram-negative bacterial plant pathogen that is dependent on a type III protein secretion system (TTSS) and the effector proteins it translocates into plant...Full Text Available

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Biological cells of the nitrogen fixating bacteria Xanthobacter autotrophicus GZ 29 have been cultivated with 57-Fe, and from the nitrogenase of these cells the 57-Fe marked molybdenum-iron-protein is extracted. The Moessbauer spectra of this enzyme show that the structure of the iron cluster is similar to the structure in the molybdenum-iron-proteins of Azotobacter vinelandii, Klebsiella pneumoniae or Clostridium pasteurianum.

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UK PubMed Central (United Kingdom)

Protein bodies within the endosperm of castor bean (Ricinus communis L. cv. Hale) seeds arise from numerous small vacuoles which progressively become filled with storage protein, of...Full Text Available

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Science.gov (United States)

Several examples of agonist-directed trafficking of receptor signalling at 5-HT2A and 5-HT2C receptors have been reported that involve independent downstream transduction pathways. We now report the functional selectivity of a series of chemically diverse agonists at human (h)5-HT2A, h5-HT2B and h5-HT2C-VSV by examining two related responses, the upstream activation of Gq/11 proteins in comparison with its associated cascade of calcium mobilisation. At the h5-HT2A receptor, d-lysergic acid diethylamide (LSD) and the antiparkinsonian agents lisuride, bromocriptine and pergolide exhibit a higher potency for Gq/11 activation than calcium release in contrast with all the other tested ligands such as 5-HT, mCPP and BW723C86, that show an opposite preference of signalling pathway. Comparable observations are made at h5-HT2B and h5-HT2C-VSV receptors, suggesting a similar mechanism of functional selectivity for the three serotonin ...

British Library Electronic Table of Contents (United Kingdom)

Assembly of icosahedral capsids of proper size and symmetry is not understood. Residue F170 in bacteriophage P22 coat protein is critical for conformational switching during assembly. Substitutions at this site cause assembly of tubes of hexamerically arranged coat protein. Intragenic suppressors of the ts phenotype of F170A and F170K coat protein mutants were isolated. Suppressors were repeatedly found in the coat protein telokin-like domain at position 285, which caused coat protein to assemble into petite procapsids and capsids. Petite capsid assembly strongly correlated to the side chain volume of the substituted amino acid. We hypothesize that larger side chains at position 285 torque the telokin-like domain, changing flexibility of the subunit and intercapsomer contacts. Thus, a sing...

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White spot syndrome virus (WSSV) is the causative agent of the white spot disease of shrimp. Penaeus monodon were captured from Muttukadu Estuary in Chennai, India, transported to the laboratory and maintained in an aerated system with continuous water circulation-biofiltration. WSSV-free P. monodon were challenged by feeding them only once with WSSV-infected tissues of P. monodon. Cumulative mortality (100%) of the infected individuals was determined. Tissues from infected and uninfected shrimp such as muscles, hepatopancreas, heart, gills and eye tissues (100mg of each) and haemolymph (50 microl) were subjected to SDS-PAGE. In infected muscle tissue, six newly expressed proteins were detected. In infected haemolymph, four new proteins and three intensely expressed high molecular weight proteins were observed. Three intensely expressed high molecular weight proteins were detected in infected heart ...

Science.gov (United States)

The steroidogenic factor 1 (SF-1, also known as NR5A1) is a transcription factor belonging to the nuclear receptor superfamily. Whereas most of the members of this family have been extensively characterized, the therapeutic potential and pharmacology of SF-1 still remains elusive. Described here is the identification and characterization of selective inhibitory chemical probes of SF-1 by a rational ultra-high-throughput screening (uHTS) strategy. A set of 64,908 compounds from the National Institute of Health's Molecular Libraries Small Molecule Repository was screened in a transactivation cell-based assay employing a chimeric SF-1 construct. Two analogous isoquinolinones, ethyl 2-[2-[2-(2,3-dihydro-1,4-benzodioxin-7-ylamino)-2-oxoethyl]-1-oxoisoquinolin-5-yl]oxypropanoate (SID7969543) and ethyl 2-[2-[2-(1,3-benzodioxol-5-ylmethylamino)-2-oxoethyl]-1-oxoisoquinolin-5-yl]oxypropanoate and (SID7970631), were identified as potent submicromolar inhibitors, yielding IC(50) values of 760 and ...

International Nuclear Information System (INIS)

In view of the anticipated increased use of atomic energy in industry, the possible long-term effects of chronic radiation exposure were studied in five generations of rats. Female rats (F_0) were given tritiated drinking water ("3HOH;3#mu#Ci/ml) from adolescence (60 days) until and throughout pregnancy. A separate study showed that the maximum radioactivity in the urine is reached in 30 days, and in the blood in 42 days. In the newborns, the highest specific activity was in the nucleic acid fraction, but total radioactivity was mainly due to the water (body fluids) compartment. No signs of radiation illness or increase in cataract formation in the mothers were observed. The food and water intake and body weight changes before pregnancy were normal. The course and the outcome of pregnancy were also normal. However, 60% of the newborns (F_1) exhibited hematomas, edemas, and subdural hemorrhages, which disappeared at 30 days of age. Bollod analysis of the F_1 ...

International Nuclear Information System (INIS)

Microdamage naturally occurs in bone tissue as a result of cyclic loading placed on the body from normal daily activities. While it is usually repaired through the bone turnover process, accumulation of microdamage may result in reduced bone quality and increased fracture risk. It is unclear whether certain areas of bone are more susceptible to microdamage than others due to compositional differences. This study examines whether areas of microdamaged bone are chemically different than undamaged areas of bone. Bone samples (L3 vertebrae) were harvested from 15 dogs. Samples were stained with basic fuchsin, embedded in poly-methylmethacrylate, and cut into 5-(micro)m-thick sections. Fuchsin staining was used to identify regions of microdamage, and synchrotron infrared microspectroscopic imaging was used to determine the local bone composition. Results showed that microdamaged areas of bone were chemically different than the surrounding undamaged areas. Specifically, ...

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We previously reported the identification of a new family of plant methyltransferases (MTs), named the SABATH family, that use S-adenosyl-l-methionine (SAM) to methylate a carboxyl moiety or a nitrogen-containing functional group on a diverse array of plant compounds. The Arabidopsis genome alone contains 24 distinct SABATH genes. To identify the catalytic specificities of members of this protein family in Arabidopsis, we screened recombinantly expressed and purified enzymes with a large number of potential substrates. Here, we report that the Arabidopsis thaliana gene At3g44860 encodes a protein with high catalytic specificity towards farnesoic acid (FA). Under steady-state conditions, this farnesoic acid carboxyl methyltransferase (FAMT) exhibits K{sub M} values of 41 and 71 {mu}M for FA and SAM, respectively. A three-dimensional model of FAMT constructed based upon similarity to the experimentally determined structure of Clarkia breweri ...

Science.gov (United States)

Four major natriuretic peptides have been isolated: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and Dendroaspis-type natriuretic peptide (DNP). Natriuretic peptides play an important role in the regulation of cardiovascular homeostasis maintaining blood pressure and extracellular fluid volume. The classical endocrine effects of natriuretic peptides to modulate fluid and electrolyte balance and vascular smooth muscle tone are complemented by autocrine and paracrine actions that include regulation of coronary blood flow and, therefore, myocardial perfusion; modulation of proliferative responses during myocardial and vascular remodeling; and cytoprotective anti-ischemic effects. The actions of natriuretic peptides are mediated by the specific binding of these peptides to three cell surface receptors: type A natriuretic peptide receptor (NPR-A), type B natriuretic peptide receptor (NPR-B), and type C natriuretic peptide receptor ...

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This study aimed to correlate the efficiency of enzymatic hydrolysis of the cellulose contained in a sugarcane bagasse sample pretreated with dilute H(2)SO(4) with the levels of independent variables such as initial content of solids and loadings of enzymes and surfactant (Tween 20), for two cellulolytic commercial preparations. The preparations, designated cellulase I and cellulase II, were characterized regarding the activities of total cellulases, endoglucanase, cellobiohydrolase, cellobiase, ?-glucosidase, xylanase, and phenoloxidases (laccase, manganese and lignin peroxidases), as well as protein contents. Both extracts showed complete cellulolytic complexes and considerable activities of xylanases, without activities of phenoloxidases. For the enzymatic hydrolyses, two 2(3) central composite full factorial designs were employed to evaluate the effects caused by the initial content of solids ...

Science.gov (United States)

The adipocytokine adiponectin is released by adipocytes upon activation of the peroxisome proliferator-activated receptor gamma (PPAR gamma). PPAR gamma has binding sites for thiazolidinediones and free fatty acids (FFAs). To evaluate if adiponectin serum concentrations are synergistically regulated by FFAs and thiazolidinediones IN VIVO plasma FFAs were acutely elevated in healthy subjects pre-treated with rosiglitazone or placebo. Sixteen healthy male subjects (23-37 years) were included in this double-blind, randomized, placebo-controlled parallel-group study. Rosiglitazone 8 mg or placebo was administered daily for 21 days. On the last day plasma FFA concentrations were increased by an intravenous triglyceride/heparin infusion. Blood for determination of adiponectin, C-reactive protein (CRP), leptin, resistin, FFAs, glucose, and insulin was drawn at baseline and on day 21 before and after 5 hours of triglyceride/heparin ...

DEFF Research Database (Denmark)

The heavy chain (HC) subunits of the bikunin proteins are covalently attached to a single chondroitin sulfate (CS) chain originating from bikunin and can be transferred to different hyaluronan (HA) molecules by TSG-6/HC2. In the present study, we demonstrate that HCs transferred to HA may function as HC donors in subsequent transfer reactions, and we show that the CS of bikunin may serve as an HC acceptor, analogous to HA. Our data suggest that TSG-6/HC2 link HCs randomly on the CS chain of bikunin, in contrast to the ordered attachment observed during the biosynthesis. Moreover, the results show that the transfer activity is indifferent to the new HC position, and the relocated HCs are thus prone to further TSG-6/HC2-induced transfer reactions. The data suggest that HCs may be transferred directly from HA to HA without the involvement of the bikunin CS chain. The results demonstrate reversibility of the interactions between HCs and ...

British Library Electronic Table of Contents (United Kingdom)

We hypothesized that I kappa B kinase (IKK)-mediated nuclear factor kappa B and forkhead BoxO3a phosphorylation will be associated with age-related endothelial dysfunction. Endothelium-dependent dilation and aortic protein expression/phosphorylation were determined in young and old male B6D2F1 mice and old mice treated with the IKK inhibitor, salicylate. IKK activation was greater in old mice and was associated with greater nitrotyrosine and cytokines. Endothelium-dependent dilation, nitric oxide (NO), and endothelial NO synthase phosphorylation were lower in old mice. Endothelium-dependent dilation and NO bioavailability were restored by a superoxide dismutase mimetic. Nuclear factor kappa B and forkhead BoxO3a phosphorylation were greater in old and were associated with increased express...

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Radioadapted chicken embryo cells (X-irradiation in ovo with 10 cGy at the 14th day of development with priming periods of 24 h) were treated in vitro by challenge doses of 14 different DNA- and/or chromatin-interactive agents, including X-rays. A decrease in the cellular damage, as measured by scheduled DNA synthesis, was only observed with X-irradiation. Sedimentation of nucleoids as well as viscosity of alkaline lysates from ethidium bromide (0.35-400 {mu}g/ml)-, vovobiocin (125-1800 {mu}g/ml)-, and hyperthermia (30 min at 43 and 45 )-treated cells suggest a higher tendency of radioadapted cells to undergo positive DNA supercoiling. When DNA from adapted and non-adapted chicken embryo cells was used as substrate, neither its digestion by DNase I nor the inhibition of DNase I activity by various DNA-interactive agents was changed in primed cells. From the previous investigations as well as from the present results it is concluded that an increase of tightening of ...