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Sample records for group proteins antagonistically

  1. CHD3 proteins and polycomb group proteins antagonistically determine cell identity in Arabidopsis.

    PubMed

    Aichinger, Ernst; Villar, Corina B R; Farrona, Sara; Reyes, José C; Hennig, Lars; Köhler, Claudia

    2009-08-01

    Dynamic regulation of chromatin structure is of fundamental importance for modulating genomic activities in higher eukaryotes. The opposing activities of Polycomb group (PcG) and trithorax group (trxG) proteins are part of a chromatin-based cellular memory system ensuring the correct expression of specific transcriptional programs at defined developmental stages. The default silencing activity of PcG proteins is counteracted by trxG proteins that activate PcG target genes and prevent PcG mediated silencing activities. Therefore, the timely expression and regulation of PcG proteins and counteracting trxG proteins is likely to be of fundamental importance for establishing cell identity. Here, we report that the chromodomain/helicase/DNA-binding domain CHD3 proteins PICKLE (PKL) and PICKLE RELATED2 (PKR2) have trxG-like functions in plants and are required for the expression of many genes that are repressed by PcG proteins. The pkl mutant could partly suppress the leaf and flower phenotype of the PcG mutant curly leaf, supporting the idea that CHD3 proteins and PcG proteins antagonistically determine cell identity in plants. The direct targets of PKL in roots include the PcG genes SWINGER and EMBRYONIC FLOWER2 that encode subunits of Polycomb repressive complexes responsible for trimethylating histone H3 at lysine 27 (H3K27me3). Similar to mutants lacking PcG proteins, lack of PKL and PKR2 caused reduced H3K27me3 levels and, therefore, increased expression of a set of PcG protein target genes in roots. Thus, PKL and PKR2 are directly required for activation of PcG protein target genes and in roots are also indirectly required for repression of PcG protein target genes. Reduced PcG protein activity can lead to cell de-differentiation and callus-like tissue formation in pkl pkr2 mutants. Thus, in contrast to mammals, where PcG proteins are required to maintain pluripotency and to prevent cell differentiation, in plants PcG proteins are required to promote cell

  2. The CHD3 chromatin remodeler PICKLE and polycomb group proteins antagonistically regulate meristem activity in the Arabidopsis root.

    PubMed

    Aichinger, Ernst; Villar, Corina B R; Di Mambro, Riccardo; Sabatini, Sabrina; Köhler, Claudia

    2011-03-01

    The chromatin modifying Polycomb group (PcG) and trithorax group (trxG) proteins are central regulators of cell identity that maintain a tightly controlled balance between cell proliferation and cell differentiation. The opposing activities of PcG and trxG proteins ensure the correct expression of specific transcriptional programs at defined developmental stages. Here, we report that the chromatin remodeling factor PICKLE (PKL) and the PcG protein CURLY LEAF (CLF) antagonistically determine root meristem activity. Whereas loss of PKL function caused a decrease in meristematic activity, loss of CLF function increased meristematic activity. Alterations of meristematic activity in pkl and clf mutants were not connected with changes in auxin concentration but correlated with decreased or increased expression of root stem cell and meristem marker genes, respectively. Root stem cell and meristem marker genes are modified by the PcG-mediated trimethylation of histone H3 on lysine 27 (H3K27me3). Decreased expression levels of root stem cell and meristem marker genes in pkl correlated with increased levels of H3K27me3, indicating that root meristem activity is largely controlled by the antagonistic activity of PcG proteins and PKL.

  3. Antagonists of IAP proteins as cancer therapeutics.

    PubMed

    Dynek, Jasmin N; Vucic, Domagoj

    2013-05-28

    Inhibitor of apoptosis (IAP) proteins play pivotal roles in cellular survival by blocking apoptosis, modulating signal transduction, and affecting cellular proliferation. Through their interactions with inducers and effectors of apoptosis IAP proteins can effectively suppress apoptosis triggered by diverse stimuli including death receptor signaling, irradiation, chemotherapeutic agents, or growth factor withdrawal. Evasion of apoptosis, in part due to the action of IAP proteins, enhances resistance of cancer cells to treatment with chemotherapeutic agents and contributes to tumor progression. Additionally, IAP genes are known to be subject to amplification, mutation, and chromosomal translocation in human malignancies and autoimmune diseases. In this review we will discuss the role of IAP proteins in cancer and the development of antagonists targeting IAP proteins for cancer treatment.

  4. Nef proteins from simian immunodeficiency viruses are tetherin antagonists

    PubMed Central

    Zhang, Fengwen; Wilson, Sam J.; Langford, Wilmina; Virgen, Beatriz; Gregory, Devon; Johnson, Marc; Munch, Jan; Kirchhoff, Frank; Bieniasz, Paul D.; Hatziioannou, Theodora

    2010-01-01

    The tetherin/BST2/CD317 protein blocks the release of HIV-1 and other enveloped viruses by inducing tethering of nascent particles to infected cell surfaces. The HIV-1 Vpu protein antagonizes the antiviral activity of human but not monkey tetherins and many simian immunodeficiency viruses (SIVs) do not encode Vpu. Here, we show that the apparently ‘missing’ anti-tetherin activity in SIVs has been acquired by several SIV Nef proteins. Specifically, SIVMAC/SIVSMM, SIVAGM and SIVBLU Nef proteins can suppress tetherin activity. Notably, tetherin antagonism by SIV Nef proteins is species-specific, is genetically separable from other Nef activities and is most evident with simian rather than human tetherin proteins. Accordingly, a critical determinant of sensitivity to SIVMAC Nef in the tetherin cytoplasmic tail is variable in nonhuman primate tetherins and deleted in human tetherin, likely due to selective pressures imposed by viral antagonists, perhaps including Nef proteins. PMID:19501037

  5. G protein antagonists. A novel hydrophobic peptide competes with receptor for G protein binding.

    PubMed

    Mukai, H; Munekata, E; Higashijima, T

    1992-08-15

    A substance P (SP) analog, [D-Pro4,D-Trp7,9,10] SP4-11, is known to inhibit the actions of various structurally unrelated messenger molecules as well as SP. Our studies on the effects of this peptide on the regulation of purified G proteins by receptor showed that at least some of the biological effects of the peptide can be explained by the ability of the peptide to block the activation of G proteins by receptors. Here we report that a novel truncated SP-related peptide, pGlu-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2, inhibited the activation of G(i) or G(o) by M2 muscarinic cholinergic receptor (M2 mAChR) or of Gs by beta-adrenergic receptor in the reconstituted phospholipid vesicles, assayed by receptor-promoted GTP hydrolysis. The inhibition by the peptide was apparently reversible and competitive with respect to receptor binding to G proteins; the inhibition could be overcome by increasing the concentration of receptor in the vesicles and was not altered by changes in the concentration of G protein. The competing effects of the peptide were used to analyze the effect of agonist on receptor-G protein interaction. The concentration change of muscarinic agonist did not alter the inhibitory effects of the peptide on M2 mAChR-promoted GTPase by G(o), which is consistent with the idea that agonist increases the regulatory efficiency of the receptor but does not alter its affinity for G proteins. This new group of compounds (G protein antagonists) is a promising tool to study receptor-G protein interaction quantitatively.

  6. Proneurogenic Group II mGluR antagonist improves learning and reduces anxiety in Alzheimer Aβ oligomer mouse.

    PubMed

    Kim, S H; Steele, J W; Lee, S W; Clemenson, G D; Carter, T A; Treuner, K; Gadient, R; Wedel, P; Glabe, C; Barlow, C; Ehrlich, M E; Gage, F H; Gandy, S

    2014-11-01

    Proneurogenic compounds have recently shown promise in some mouse models of Alzheimer's pathology. Antagonists at Group II metabotropic glutamate receptors (Group II mGluR: mGlu2, mGlu3) are reported to stimulate neurogenesis. Agonists at those receptors trigger γ-secretase-inhibitor-sensitive biogenesis of Aβ42 peptides from isolated synaptic terminals, which is selectively suppressed by antagonist pretreatment. We have assessed the therapeutic potential of chronic pharmacological inhibition of Group II mGluR in Dutch APP (Alzheimer's amyloid precursor protein E693Q) transgenic mice that accumulate Dutch amyloid-β (Aβ) oligomers but never develop Aβ plaques. BCI-838 is a clinically well-tolerated, orally bioavailable, investigational prodrug that delivers to the brain BCI-632, the active Group II mGluR antagonist metabolite. Dutch Aβ-oligomer-forming APP transgenic mice (APP E693Q) were dosed with BCI-838 for 3 months. Chronic treatment with BCI-838 was associated with reversal of transgene-related amnestic behavior, reduction in anxiety, reduction in levels of brain Aβ monomers and oligomers, and stimulation of hippocampal neurogenesis. Group II mGluR inhibition may offer a unique package of relevant properties as an Alzheimer's disease therapeutic or prophylactic by providing both attenuation of neuropathology and stimulation of repair.

  7. A long-acting GH receptor antagonist through fusion to GH binding protein

    PubMed Central

    Wilkinson, Ian R.; Pradhananga, Sarbendra L.; Speak, Rowena; Artymiuk, Peter J.; Sayers, Jon R.; Ross, Richard J.

    2016-01-01

    Acromegaly is a human disease of growth hormone (GH) excess with considerable morbidity and increased mortality. Somatostatin analogues are first line medical treatment but the disease remains uncontrolled in up to 40% of patients. GH receptor (GHR) antagonist therapy is more effective but requires frequent high-dose injections. We have developed an alternative technology for generating a long acting potent GHR antagonist through translational fusion of a mutated GH linked to GH binding protein and tested three candidate molecules. All molecules had the amino acid change (G120R), creating a competitive GHR antagonist and we tested the hypothesis that an amino acid change in the GH binding domain (W104A) would increase biological activity. All were antagonists in bioassays. In rats all antagonists had terminal half-lives >20 hours. After subcutaneous administration in rabbits one variant displayed a terminal half-life of 40.5 hours. A single subcutaneous injection of the same variant in rabbits resulted in a 14% fall in IGF-I over 7 days. In conclusion: we provide proof of concept that a fusion of GHR antagonist to its binding protein generates a long acting GHR antagonist and we confirmed that introducing the W104A amino acid change in the GH binding domain enhances antagonist activity. PMID:27731358

  8. Structure-Based Design of a Periplasmic Binding Protein Antagonist that Prevents Domain Closure

    SciTech Connect

    Borrok, M. Jack; Zhu, Yimin; Forest, Katrina T.; Kiessling, Laura L.

    2009-07-31

    Many receptors undergo ligand-induced conformational changes to initiate signal transduction. Periplasmic binding proteins (PBPs) are bacterial receptors that exhibit dramatic conformational changes upon ligand binding. These proteins mediate a wide variety of fundamental processes including transport, chemotaxis, and quorum sensing. Despite the importance of these receptors, no PBP antagonists have been identified and characterized. In this study, we identify 3-O-methyl-D-glucose as an antagonist of glucose/galactose-binding protein and demonstrate that it inhibits glucose chemotaxis in E. coli. Using small-angle X-ray scattering and X-ray crystallography, we show that this antagonist acts as a wedge. It prevents the large-scale domain closure that gives rise to the active signaling state. Guided by these results and the structures of open and closed glucose/galactose-binding protein, we designed and synthesized an antagonist composed of two linked glucose residues. These findings provide a blueprint for the design of new bacterial PBP inhibitors. Given the key role of PBPs in microbial physiology, we anticipate that PBP antagonists will have widespread uses as probes and antimicrobial agents.

  9. Drug Discovery Toward Antagonists of Methyl-Lysine Binding Proteins

    PubMed Central

    Herold, J. Martin; Ingerman, Lindsey A; Gao, Cen; Frye, Stephen V

    2011-01-01

    The recognition of methyl-lysine and -arginine residues on both histone and other proteins by specific “reader” elements is important for chromatin regulation, gene expression, and control of cell-cycle progression. Recently the crucial role of these reader proteins in cancer development and dedifferentiation has emerged, owing to the increased interest among the scientific community. The methyl-lysine and -arginine readers are a large and very diverse set of effector proteins and targeting them with small molecule probes in drug discovery will inevitably require a detailed understanding of their structural biology and mechanism of binding. In the following review, the critical elements of methyl-lysine and -arginine recognition will be summarized with respect to each protein family and initial results in assay development, probe design, and drug discovery will be highlighted. PMID:22145013

  10. RNA Aptamers as Effective Protein Antagonists in a Multicellular Organism

    NASA Astrophysics Data System (ADS)

    Shi, Hua; Hoffman, Bryan E.; Lis, John T.

    1999-08-01

    RNA aptamers selected against proteins can be used to modulate specific protein function. Expression of such reagents in cells and whole organisms could provide a means of dissecting and controlling molecular mechanisms in vivo. We demonstrate that Drosophila B53 protein can be specifically inhibited in vitro and in vivo by a multivalent RNA aptamer. This inhibitory aptamer RNA binds B52 avidly and inhibits B52-stimulated pre-mRNA splicing. It can be expressed in cultured cells and whole animals in a stable form that accumulates up to 10% of total mRNA. It binds B52 in vivo and suppresses all phenotypes caused by B52 overexpression. The strategies presented here should prove general in design and expression of functional and therapeutic RNAs.

  11. A long-acting GH receptor antagonist through fusion to GH binding protein.

    PubMed

    Wilkinson, Ian R; Pradhananga, Sarbendra L; Speak, Rowena; Artymiuk, Peter J; Sayers, Jon R; Ross, Richard J

    2016-10-12

    Acromegaly is a human disease of growth hormone (GH) excess with considerable morbidity and increased mortality. Somatostatin analogues are first line medical treatment but the disease remains uncontrolled in up to 40% of patients. GH receptor (GHR) antagonist therapy is more effective but requires frequent high-dose injections. We have developed an alternative technology for generating a long acting potent GHR antagonist through translational fusion of a mutated GH linked to GH binding protein and tested three candidate molecules. All molecules had the amino acid change (G120R), creating a competitive GHR antagonist and we tested the hypothesis that an amino acid change in the GH binding domain (W104A) would increase biological activity. All were antagonists in bioassays. In rats all antagonists had terminal half-lives >20 hours. After subcutaneous administration in rabbits one variant displayed a terminal half-life of 40.5 hours. A single subcutaneous injection of the same variant in rabbits resulted in a 14% fall in IGF-I over 7 days.

  12. Oxytocin differentially modulates compromise and competitive approach but not withdrawal to antagonists from own vs. rivaling other groups.

    PubMed

    Ten Velden, Femke S; Baas, Matthijs; Shalvi, Shaul; Kret, Mariska E; De Dreu, Carsten K W

    2014-09-11

    In humans, oxytocin promotes cognitive and motivational tendencies that benefit the groups on which humans depend for their survival and prosperity. Here we examined decision making in an incentivized two-player poker game with either an in-group or out-group antagonist. Sixty nine healthy males received 24 IU oxytocin or matching placebo, and played four rounds of a simplified poker game. On each round they received either low or high value cards to create differences in competitive strength, and then responded to a bet placed by their (simulated) (in-group or out-group) antagonist. Under placebo, participants withdrew and competed depending on their own (low vs. high) competitive strength, regardless of their antagonist's group membership. Under oxytocin, however, participants settled more and competed less with an in-group as compared to an out-group antagonist; withdrawal was unaffected by group membership. We conclude that oxytocin sensitizes humans to the group membership of their interaction partner, rendering them relatively more benevolent and less competitive towards those seen as belonging to their own group. This article is part of a Special Issue entitled Oxytocin and Social Behav.

  13. Modulation of a 40-kDa catecholamine regulated protein by dopamine receptor antagonists.

    PubMed

    Sharan, N; Nair, V D; Mishra, R K

    2001-02-09

    Previous reports have shown that catecholamine regulated proteins (CRP) are central nervous system specific and covalently bind to catecholamines. In the present study, we report the subcellular localization and differential modulation of a 40-kDa catecholamine regulated protein (CRP40) by dopamine D1 and D2 receptor antagonists. CRP40 was found to be localized with nuclear and synaptosomal/mitochondrial and fractions. Chronic treatment with dopamine D2 receptor antagonist haloperidol in rats significantly increased the levels of CRP40 in the striatum, whereas, chronic R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH 23390) dopamine D1 receptor antagonist administration significantly decreased striatal CRP40 levels. Moreover, acute haloperidol treatment did not alter the levels of CRP40 in any of the brain regions. Despite a sequence homology with the heat shock protein 70 (HSP70), levels of HSP70 remained unchanged after either drug treatment, suggesting a distinct function of CRP40 than HSP70. These results further suggest that CRP40 play an important role in dopaminergic neuronal function and the dopamine D1 receptor-mediated signaling pathway may be involved in the regulation of CRP40.

  14. Screening bicyclic peptide libraries for protein-protein interaction inhibitors: discovery of a tumor necrosis factor-α antagonist.

    PubMed

    Lian, Wenlong; Upadhyaya, Punit; Rhodes, Curran A; Liu, Yusen; Pei, Dehua

    2013-08-14

    Protein-protein interactions represent a new class of exciting but challenging drug targets, because their large, flat binding sites lack well-defined pockets for small molecules to bind. We report here a methodology for chemical synthesis and screening of large combinatorial libraries of bicyclic peptides displayed on rigid small-molecule scaffolds. With planar trimesic acid as the scaffold, the resulting bicyclic peptides are effective for binding to protein surfaces such as the interfaces of protein-protein interactions. Screening of a bicyclic peptide library against tumor necrosis factor-α (TNFα) identified a potent antagonist that inhibits the TNFα-TNFα receptor interaction and protects cells from TNFα-induced cell death. Bicyclic peptides of this type may provide a general solution for inhibition of protein-protein interactions.

  15. Dimeric Macrocyclic Antagonists of Inhibitor of Apoptosis Proteins for the Treatment of Cancer

    PubMed Central

    2015-01-01

    A series of dimeric macrocyclic compounds were prepared and evaluated as antagonists for inhibitor of apoptosis proteins. The most potent analogue 11, which binds to XIAP and c-IAP proteins with high affinity and induces caspase-3 activation and ultimately cell apoptosis, inhibits growth of human melanoma and colorectal cell lines at low nanomolar concentrations. Furthermore, compound 11 demonstrated significant antitumor activity in the A875 human melanoma xenograft model at doses as low as 2 mg/kg on a q3d schedule. PMID:26191364

  16. Yeast mitochondrial fission proteins induce antagonistic Gaussian membrane curvatures to regulate apoptosis

    NASA Astrophysics Data System (ADS)

    Lee, Michelle; Hwee Lai, Ghee; Schmidt, Nathan; Xian, Wujing; Wong, Gerard C. L.

    2013-03-01

    Mitochondria form a dynamic and interconnected network, which disintegrates during apoptosis to generate numerous smaller mitochondrial fragments. This process is at present not well understood. Yeast mitochondrial fission machinery proteins, Dnm1 and Fis1, are believed to regulate programmed cell death in yeast. Yeast Dnm1 has been previously shown to promote mitochondrial fragmentation and degradation characteristic of apoptotic cells, while yeast Fis1 inhibits cell death by limiting the mitochondrial fission induced by Dnm1 [Fannjiang et al, Genes & Dev. 2004. 18: 2785-2797]. To better understand the mechanisms of these antagonistic fission proteins, we use synchrotron small angle x-ray scattering (SAXS) to investigate their interaction with model cell membranes. The relationship between each protein, Dnm1 and Fis1, and protein-induced changes in membrane curvature and topology is examined. Through the comparison of the membrane rearrangement and phase behavior induced by each protein, we will discuss their respective roles in the regulation of mitochondrial fission.

  17. TNF binding protein of variola virus acts as a TNF antagonist at epicutaneous application.

    PubMed

    Gileva, Irina P; Viazovaia, Elena A; Toporkova, Ludmila B; Tsyrendorzhiev, Dondok D; Shchelkunov, Sergei N; Orlovskaya, Irina A

    2015-01-01

    VARV-CrmB is a TNF binding protein of variola virus. VARV-CrmB protein was previously shown to be active as a TNF-antagonist in a number of in vivo and in vitro models. Here we investigated the epicutaneous effect of recombinant VARV-CrmB protein using an experimental model of muTNFinduced migration of skin leukocytes as well as colony forming activity of bone marrow cells (BMC). Epiсutaneous applications of muTNF enhanced the number of cells migrating from skin flaps of BALB/c mice, whereas subsequent applications of VARV-CrmB protein in 30 min after muTNF, abolished that effect. Epicutaneously applied muTNF influenced the activity of committed hematopoietic progenitors causing a reduction of erythroid (BFUe+CFUe) colonies and increase of granulocyte-macrophage (CFU-GM) colonies in the colony-forming tests. VARV-CrmB, applied in combination with muTNF, demonstrated an ability to reverse this effect, namely, to increase BFUe+CFUe and reduce CFU-GM back to the control levels. Taking together, these data demonstrate the TNF-blocking properties of VARV-CrmB in vivo at epicutaneous applications. As effective TNF antagonist VARV-CrmB protein might be conceded as a beneficial candidate for future research and development of therapeutic approaches in the field of inflammatory skin diseases.

  18. Candidate nematicidal proteins in a new Pseudomonas veronii isolate identified by its antagonistic properties against Xiphinema index.

    PubMed

    Canchignia, Hayron; Altimira, Fabiola; Montes, Christian; Sánchez, Evelyn; Tapia, Eduardo; Miccono, María; Espinoza, Daniel; Aguirre, Carlos; Seeger, Michael; Prieto, Humberto

    2017-03-17

    The nematode Xiphinema index affects grape vines and transmits important viruses associated with fanleaf degeneration. Pseudomonas spp. are an extensive bacterial group in which important biodegradation and/or biocontrol properties can occur for several strains in the group. The aim of this study was to identify new Pseudomonas isolates with antagonist activity against X. index. Forty bacterial isolates were obtained from soil and root samples from Chilean vineyards. Thirteen new fluorescent pseudomonads were found and assessed for their antagonistic capability. The nematicide Pseudomonas protegens CHA0 was used as a control. Challenges of nematode individuals in King's B semi-solid agar Petri dishes facilitated the identification of the Pseudomonas veronii isolate R4, as determined by a 16S rRNA sequence comparison. This isolate was as effective as CHA0 as an antagonist of X. index, although it had a different lethality kinetic. Milk-induced R4 cultures exhibited protease and lipase activities in cell supernatants using both gelatin/tributyrin Petri dish assays and zymograms. Three proteins with these activities were isolated and subjected to mass spectrometry. Amino acid partial sequences enabled the identification of a 49-kDa protease similar to metalloprotease AprA and two lipases of 50 kDa and 69 kDa similar to LipA and ExoU, respectively. Electron microscopy analyses of challenged nematodes revealed degraded cuticle after R4 supernatant treatment. These results represent a new and unexplored property in this species associated with the presence of secretable lipases and protease, similar to characterized enzymes present in biocontrol pseudomonads.

  19. Group II Introns and Their Protein Collaborators

    NASA Astrophysics Data System (ADS)

    Solem, Amanda; Zingler, Nora; Pyle, Anna Marie; Li-Pook-Than, Jennifer

    Group II introns are an abundant class of autocatalytic introns that excise themselves from precursor mRNAs. Although group II introns are catalytic RNAs, they require the assistance of proteins for efficient splicing in vivo. Proteins that facilitate splicing of organellar group II introns fall into two main categories: intron-encoded maturases and host-encoded proteins. This chapter will focus on the host proteins that group II introns recruited to ensure their function. It will discuss the great diversity of these proteins, define common features, and describe different strategies employed to achieve specificity. Special emphasis will be placed on DEAD-box ATPases, currently the best studied example of host-encoded proteins with a role in group II intron splicing. Since the exact mechanisms by which splicing is facilitated is not known for any of the host proteins, general mechanistic strategies for protein-mediated RNA folding are described and assessed for their potential role in group II intron splicing.

  20. The effects of sigma ligands on protein release from lacrimal acinar cells: a potential agonist/antagonist assay.

    PubMed

    Schoenwald, R D; Barfknecht, C F; Shirolkar, S; Xia, E

    1995-03-03

    Sigma receptor antagonists have been proposed as leading clinical candidates for use in various psychotic disorders. Prior to clinical testing, it is imperative that a new agent be correctly identified as an antagonist and not an agonist since the latter may worsen the psychosis. For sigma-ligands many behavioral and pharmacological assays have been developed in an attempt to classify agonist/antagonist activity. These assays evaluate a response or a behavior in an animal model that can be related to clinical efficacy. However, is the action by the presumed antagonist a consequence of sigma-receptor activity? Previously we have identified sigma-receptors in acinar cells of the main lacrimal gland of the New Zealand white rabbit and have measured protein release after the addition of various N,N-disubstituted phenylalkylamine derivatives known to be sigma-ligands by receptor binding studies. Although protein release from acinar cells has been attributed to either muscarinic or alpha-adrenergic stimulation, protein release from sigma-receptor stimulation was also confirmed. In the reported studies here, we isolated and incubated acinar cells with varying concentrations of known sigma-ligands and measured protein concentration. A knowledge of the receptor profile for the disubstituted phenylalkylamines permitted experiments to be designed in which various alpha, muscarinic, serotonergic, and dopaminergic antagonists could be added in equimolar concentrations. Under the conditions of these experiments, statistically significant increases in protein release for sigma-ligands could be attributed to stimulation of sigma-receptors. Haloperidol, an apparent sigma-antagonist, caused a statistically significant decrease in protein release and also inhibited protein release when tested with a known sigma-ligand, AF2975 [N,N-dimethyl-2-phenylethylamine]. In this system, stimulation and inhibition of protein release were defined as agonist and antagonist behavior, respectively

  1. All About the Protein Foods Group

    MedlinePlus

    ... Protein Foods Group? All foods made from meat, poultry, seafood, beans and peas, eggs, processed soy products, ... soy products, and nuts and seeds. Meat and poultry choices should be lean or low-fat. How ...

  2. A Single Amino Acid Dictates Protein Kinase R Susceptibility to Unrelated Viral Antagonists

    PubMed Central

    Esparo, Nicolle M.; Child, Stephanie J.; Geballe, Adam P.

    2016-01-01

    During millions of years of coevolution with their hosts, cytomegaloviruses (CMVs) have succeeded in adapting to overcome host-specific immune defenses, including the protein kinase R (PKR) pathway. Consequently, these adaptations may also contribute to the inability of CMVs to cross species barriers. Here, we provide evidence that the evolutionary arms race between the antiviral factor PKR and its CMV antagonist TRS1 has led to extensive differences in the species-specificity of primate CMV TRS1 proteins. Moreover, we identify a single residue in human PKR that when mutated to the amino acid present in African green monkey (Agm) PKR (F489S) is sufficient to confer resistance to HCMVTRS1. Notably, this precise molecular determinant of PKR resistance has evolved under strong positive selection among primate PKR alleles and is positioned within the αG helix, which mediates the direct interaction of PKR with its substrate eIF2α. Remarkably, this same residue also impacts sensitivity to K3L, a poxvirus-encoded pseudosubstrate that structurally mimics eIF2α. Unlike K3L, TRS1 has no homology to eIF2α, suggesting that unrelated viral genes have convergently evolved to target this critical region of PKR. Despite its functional importance, the αG helix exhibits extraordinary plasticity, enabling adaptations that allow PKR to evade diverse viral antagonists while still maintaining its critical interaction with eIF2α. PMID:27780231

  3. Target hopping as a useful tool for the identification of novel EphA2 protein-protein antagonists.

    PubMed

    Tognolini, Massimiliano; Incerti, Matteo; Pala, Daniele; Russo, Simonetta; Castelli, Riccardo; Hassan-Mohamed, Iftiin; Giorgio, Carmine; Lodola, Alessio

    2014-01-01

    Lithocholic acid (LCA), a physiological ligand for the nuclear receptor FXR and the G-protein-coupled receptor TGR5, has been recently described as an antagonist of the EphA2 receptor, a key member of the ephrin signalling system involved in tumour growth. Given the ability of LCA to recognize FXR, TGR5, and EphA2 receptors, we hypothesized that the structural requirements for a small molecule to bind each of these receptors might be similar. We therefore selected a set of commercially available FXR or TGR5 ligands and tested them for their ability to inhibit EphA2 by targeting the EphA2-ephrin-A1 interface. Among the selected compounds, the stilbene carboxylic acid GW4064 was identified as an effective antagonist of EphA2, being able to block EphA2 activation in prostate carcinoma cells, in the micromolar range. This finding proposes the "target hopping" approach as a new effective strategy to discover new protein-protein interaction inhibitors.

  4. Tenotomy of m.soleus antagonists prevents the changes in fiber type characteristics and sarcomeric cytoskeletal proteins in unloaded rats

    NASA Astrophysics Data System (ADS)

    Moukhina, Alexandra; Ardabievskaya, Anna; Vikhlyantsev, Ivan; Podlubnaya, Zoya; Nemirovskaya, Tatiana; Shenkman, Boris

    2005-08-01

    It is known that activity of postural extensors (m. soleus) decreases and activity of flexors (m. tibialis anterior) increases under unloading conditions. We have tested the hypothesis supposing that increased flexor activities during unloading exert suppressive influence on postural extensor activities and thus lead to dramatic changes in fiber size, MHC expression, sarcomeric proteins content in m.soleus. We have inactivated hindlimb flexor muscles (m.soleus antagonists) by bilateral tenotomy. 20 male Wistar rats were divided on 3 groups: cage control (C), hindlimb suspension for 14 days (HS), tenotomy of hindlimb flexor muscles with 14 days hindlimb suspension afterwards (HST). Several soleus muscle fiber characteristics decreased significantly in HS group (p<0.05) as compared with C group: cross sectional area (CSA) of type I muscle fibers, titin/MyHC ratio and nebulin/MyHC ratio. MyHC isoform pattern shifted slow-to-fast significantly. NFATc1 content increased in nuclear protein extract of m. soleus in HS group. None of these parameters was significantly different in HST group from those of C group. It has been concluded that the tenotomy of flexors under hindlimb suspension prevents atrophy of type I muscle fibers, decrease the degradation of titin and nebulin and prevent slow-to-fast shift of fiber MyHC isoform pattern, possibly through prevention of increase NFATc1 content in muscle fiber nuclear protein extract. Therefore, suppressive influence of increased flexor activity could be one of mechanisms that lead to the changes in m. soleus under unloading conditions. The work was supported by RFBR grants: 02-04-50025, 03- 04-48487 and the special program of RAS "Integration mechanisms of functional control in the living system".

  5. Purification and sequencing of radish seed calmodulin antagonists phosphorylated by calcium-dependent protein kinase.

    PubMed Central

    Polya, G M; Chandra, S; Condron, R

    1993-01-01

    A family of radish (Raphanus sativus) calmodulin antagonists (RCAs) was purified from seeds by extraction, centrifugation, batch-wise elution from carboxymethyl-cellulose, and high performance liquid chromatography (HPLC) on an SP5PW cation-exchange column. This RCA fraction was further resolved into three calmodulin antagonist polypeptides (RCA1, RCA2, and RCA3) by denaturation in the presence of guanidinium HCl and mercaptoethanol and subsequent reverse-phase HPLC on a C8 column eluted with an acetonitrile gradient in the presence of 0.1% trifluoroacetic acid. The RCA preparation, RCA1, RCA2, RCA3, and other radish seed proteins are phosphorylated by wheat embryo Ca(2+)-dependent protein kinase (CDPK). The RCA preparation contains other CDPK substrates in addition to RCA1, RCA2, and RCA3. The RCA preparation, RCA1, RCA2, and RCA3 inhibit chicken gizzard calmodulin-dependent myosin light chain kinase assayed with a myosin-light chain-based synthetic peptide substrate (fifty percent inhibitory concentrations of RCA2 and RCA3 are about 7 and 2 microM, respectively). N-terminal sequencing by sequential Edman degradation of RCA1, RCA2, and RCA3 revealed sequences having a high homology with the small subunit of the storage protein napin from Brassica napus and with related proteins. The deduced amino acid sequences of RCA1, RCA2, RCA3, and RCA3' (a subform of RCA3) have agreement with average molecular masses from electrospray mass spectrometry of 4537, 4543, 4532, and 4560 kD, respectively. The only sites for serine phosphorylation are near or at the C termini and hence adjacent to the sites of proteolytic precursor cleavage. PMID:8278508

  6. Transcriptional Regulation by Trithorax-Group Proteins

    PubMed Central

    Kingston, Robert E.; Tamkun, John W.

    2014-01-01

    The trithorax group of genes (trxG) was identified in mutational screens that examined developmental phenotypes and suppression of Polycomb mutant phenotypes. The protein products of these genes are primarily involved in gene activation, although some can also have repressive effects. There is no central function for these proteins. Some move nucleosomes about on the genome in an ATP-dependent manner, some covalently modify histones such as methylating lysine 4 of histone H3, and some directly interact with the transcription machinery or are a part of that machinery. It is interesting to consider why these specific members of large families of functionally related proteins have strong developmental phenotypes. PMID:25274705

  7. Structure based drug discovery of Rab38 protein- Identification of antagonists as cancer drug candidates.

    PubMed

    Vuruputuri, Uma; Abdelmonsef, Aboubakr Haredi; Dulapalli, Ramasree; Dasari, Thirupathi; Padmarao, Lavanya Souda; Mukkera, Thirupathi

    2016-10-26

    Cancer is responsible for one in eight deaths worldwide. The Rab family members are involved in important processes including membrane trafficking, cell growth and differentiation. It has been shown that Rab38 is located in melanosomes, and overexpressed at the RNA level in melanoma cancers. Rab38 represents a novel class of cellular modulators that can affect both initiation or progression of tumor cells in Homo sapiens. In the present work, the 3D structure of Rab38 (211 residues) was generated using homology modelling method; the structure shows the presence of 6 α- helices and 6 β- strands. The 3D structure was energy minimized and validated using standard protocols. The active site was identified using computational prediction tools like CASTp, efindsite and SiteMap, which show that the residues (Ser35 to Leu63) are important for binding. Protein-protein docking studies were carried out between the target protein and its natural substrate BLOC-3 using patchDock tool. Virtual screening studies were carried out with small ligand databases using glide Schrödinger suite. The ligands which are potential antagonists against the Rab38 protein were prioritized from the results of virtual screening based on glide score, glide energy and acceptable ADME properties.

  8. A Human Bone Morphogenetic Protein Antagonist Is Down-Regulated in Renal Cancer

    PubMed Central

    Blish, Kimberly Rose; Wang, Wei; Willingham, Mark C.; Du, Wei; Birse, Charles E.; Krishnan, Surekha R.; Brown, Julie C.; Hawkins, Gregory A.; Garvin, A. Julian; D'Agostino, Ralph B.; Torti, Frank M.

    2008-01-01

    We analyzed expression of candidate genes encoding cell surface or secreted proteins in normal kidney and kidney cancer. This screen identified a bone morphogenetic protein (BMP) antagonist, SOSTDC1 (sclerostin domain–containing-1) as down-regulated in kidney tumors. To confirm screening results, we probed cDNA dot blots with SOSTDC1. The SOSTDC1 message was decreased in 20/20 kidney tumors compared with normal kidney tissue. Immunohistochemistry confirmed significant decrease of SOSTDC1 protein in clear cell renal carcinomas relative to normal proximal renal tubule cells (p < 0.001). Expression of SOSTDC1 was not decreased in papillary and chromophobe kidney tumors. SOSTDC1 was abundantly expressed in podocytes, distal tubules, and transitional epithelia of the normal kidney. Transfection experiments demonstrated that SOSTDC1 is secreted and binds to neighboring cells and/or the extracellular matrix. SOSTDC1 suppresses both BMP-7–induced phosphorylation of R-Smads-1, -5, and -8 and Wnt-3a signaling. Restoration of SOSTDC1 in renal clear carcinoma cells profoundly suppresses proliferation. Collectively, these results demonstrate that SOSTDC1 is expressed in the human kidney and decreased in renal clear cell carcinoma. Because SOSTDC1 suppresses proliferation of renal carcinoma cells, restoration of SOSTDC1 signaling may represent a novel target in treatment of renal clear cell carcinoma. PMID:18032587

  9. Effect of interdomain linker length on an antagonistic folding-unfolding equilibrium between two protein domains.

    PubMed

    Cutler, Thomas A; Mills, Brandon M; Lubin, David J; Chong, Lillian T; Loh, Stewart N

    2009-02-27

    Fusion of one protein domain with another is a common event in both evolution and protein engineering experiments. When insertion is at an internal site (e.g., a surface loop or turn), as opposed to one of the termini, conformational strain can be introduced into both domains. Strain is manifested by an antagonistic folding-unfolding equilibrium between the two domains, which we previously showed can be parameterized by a coupling free-energy term (DeltaG(X)). The extent of strain is predicted to depend primarily on the ratio of the N-to-C distance of the guest protein to the distance between ends of the surface loop in the host protein. Here, we test that hypothesis by inserting ubiquitin (Ub) into the bacterial ribonuclease barnase (Bn), using peptide linkers from zero to 10 amino acids each. DeltaG(X) values are determined by measuring the extent to which Co(2+) binding to an engineered site on the Ub domain destabilizes the Bn domain. All-atom, unforced Langevin dynamics simulations are employed to gain structural insight into the mechanism of mechanically induced unfolding. Experimental and computational results find that the two domains are structurally and energetically uncoupled when linkers are long and that DeltaG(X) increases with decreasing linker length. When the linkers are fewer than two amino acids, strain is so great that one domain unfolds the other. However, the protein is able to refold as dimers and higher-order oligomers. The likely mechanism is a three-dimensional domain swap of the Bn domain, which relieves conformational strain. The simulations suggest that an effective route to mechanical unfolding begins with disruption of the hydrophobic core of Bn near the Ub insertion site.

  10. Exploiting Free-Energy Minima to Design Novel EphA2 Protein-Protein Antagonists: From Simulation to Experiment and Return.

    PubMed

    Russo, Simonetta; Callegari, Donatella; Incerti, Matteo; Pala, Daniele; Giorgio, Carmine; Brunetti, Jlenia; Bracci, Luisa; Vicini, Paola; Barocelli, Elisabetta; Capoferri, Luigi; Rivara, Silvia; Tognolini, Massimiliano; Mor, Marco; Lodola, Alessio

    2016-06-06

    The free-energy surface (FES) of protein-ligand binding contains information useful for drug design. Here we show how to exploit a free-energy minimum of a protein-ligand complex identified by metadynamics simulations to design a new EphA2 antagonist with improved inhibitory potency.

  11. Identification of a group of brominated flame retardants as novel androgen receptor antagonists and potential neuronal and endocrine disrupters.

    PubMed

    Kharlyngdoh, Joubert Banjop; Pradhan, Ajay; Asnake, Solomon; Walstad, Anders; Ivarsson, Per; Olsson, Per-Erik

    2015-01-01

    Brominated flame-retardants (BFRs) are used in industrial products to reduce the risk of fire. However, their continuous release into the environment is a concern as they are often persistent, bioaccumulating and toxic. Information on the impact these compounds have on human health and wildlife is limited and only a few of them have been identified to disrupt hormone receptor functions. In the present study we used in silico modeling to determine the interactions of selected BFRs with the human androgen receptor (AR). Three compounds were found to dock into the ligand-binding domain of the human AR and these were further tested using in vitro analysis. Allyl 2,4,6-tribromophenyl ether (ATE), 2-bromoallyl 2,4,6-tribromophenyl ether (BATE) and 2,3-dibromopropyl-2,4,6-tribromophenyl ether (DPTE) were observed to act as AR antagonists. These BFRs have recently been detected in the environment, in house dust and in aquatic animals. The compounds have been detected at high concentrations in both blubber and brain of seals and we therefore also assessed their impact on the expression of L-type amino acid transporter system (LAT) genes, that are needed for amino acid uptake across the blood-brain barrier, as disruption of LAT gene function has been implicated in several brain disorders. The three BFRs down-regulated the expression of AR target genes that encode for prostate specific antigen (PSA), 5α-reductases and β-microseminoprotein. The potency of PSA inhibition was of the same magnitude as the common prostate cancer drugs, demonstrating that these compounds are strong AR antagonists. Western blot analysis of AR protein showed that ATE, BATE and DPTE decreased the 5α-dihydrotestosterone-induced AR protein levels, further confirming that these BFRs act as AR antagonists. The transcription of the LAT genes was altered by the three BFRs, indicating an effect on amino-acid uptake across cellular membranes and blood-brain barrier. This study demonstrated that ATE, BATE

  12. Competition between antagonistic complement factors for a single protein on N. meningitidis rules disease susceptibility

    PubMed Central

    Caesar, Joseph JE; Lavender, Hayley; Ward, Philip N; Exley, Rachel M; Eaton, Jack; Chittock, Emily; Malik, Talat H; Goiecoechea De Jorge, Elena; Pickering, Matthew C; Tang, Christoph M; Lea, Susan M

    2014-01-01

    Genome-wide association studies have found variation within the complement factor H gene family links to host susceptibility to meningococcal disease caused by infection with Neisseria meningitidis (Davila et al., 2010). Mechanistic insights have been challenging since variation within this locus is complex and biological roles of the factor H-related proteins, unlike factor H, are incompletely understood. N. meningitidis subverts immune responses by hijacking a host-immune regulator, complement factor H (CFH), to the bacterial surface (Schneider et al., 2006; Madico et al., 2007; Schneider et al., 2009). We demonstrate that complement factor-H related 3 (CFHR3) promotes immune activation by acting as an antagonist of CFH. Conserved sequences between CFH and CFHR3 mean that the bacterium cannot sufficiently distinguish between these two serum proteins to allow it to hijack the regulator alone. The level of protection from complement attack achieved by circulating N. meningitidis therefore depends on the relative levels of CFH and CFHR3 in serum. These data may explain the association between genetic variation in both CFH and CFHR3 and susceptibility to meningococcal disease. DOI: http://dx.doi.org/10.7554/eLife.04008.001 PMID:25534642

  13. Transcriptional Silencing by Polycomb-Group Proteins

    PubMed Central

    Grossniklaus, Ueli; Paro, Renato

    2014-01-01

    Polycomb-group (PcG) genes encode chromatin proteins involved in stable and heritable transcriptional silencing. PcG proteins participate in distinct multimeric complexes that deposit, or bind to, specific histone modifications (e.g., H3K27me3 and H2AK119ub1) to prevent gene activation and maintain repressed chromatin domains. PcG proteins are evolutionary conserved and play a role in processes ranging from vernalization and seed development in plants, over X-chromosome inactivation in mammals, to the maintenance of stem cell identity. PcG silencing is medically relevant as it is often observed in human disorders, including cancer, and tissue regeneration, which involve the reprogramming of PcG-controlled target genes. PMID:25367972

  14. Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography

    SciTech Connect

    Mandal, Kalyaneswar; Uppalapati, Maruti; Ault-Riché, Dana; Kenney, John; Lowitz, Joshua; Sidhu, Sachdev S.; Kent, Stephen B.H.

    2012-10-23

    Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF{sub 165} to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {l_brace}D-protein antagonist + L-protein form of VEGF-A{r_brace}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 {angstrom}. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 {angstrom}{sup 2} in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2.

  15. Purinergic Receptor Antagonists Inhibit Odorant-Induced Heat Shock Protein 25 Induction in Mouse Olfactory Epithelium

    PubMed Central

    Hegg, Colleen C.; Lucero, Mary T.

    2010-01-01

    Heat shock proteins (HSPs) accumulate in cells exposed to a variety of physiological and environmental factors, such as heat shock, oxidative stress, toxicants, and odorants. Ischemic, stressed, and injured cells release ATP in large amounts. Our hypothesis is that noxious stimulation (in this case, strong odorant) evokes the release of ATP in the olfactory epithelium (OE). Extracellular ATP, a signal of cellular stress, induces the expression of HSPs via purinergic receptors. In the present study, in vivo odorant exposure (heptanal or r-carvone) led to a selective induction of HSP25 in glia-like sustentacular cells in the Swiss Webster mouse OE, as previously shown in rats (Carr et al., 2001). Furthermore, in vitro and in vivo administration of purinergic receptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) blocked the expression of HSP25 immunoreactivity in sustentacular cells. ATP released by acutely injured cells could act as an early signal of cell and tissue damage, causing HSP expression and initiating a stress signaling cascade to protect against further damage. Sustentacular cells have a high capacity to detoxify xenobiotics and thereby protect the olfactory epithelium from airborne pollutants. Thus, the robust, rapid induction of HSPs in sustentacular cells may help maintain the integrity of the OE during exposure to toxicants. PMID:16206165

  16. Control of hematopoietic stem cell emergence by antagonistic functions of ribosomal protein paralogs.

    PubMed

    Zhang, Yong; Duc, Anne-Cécile E; Rao, Shuyun; Sun, Xiao-Li; Bilbee, Alison N; Rhodes, Michele; Li, Qin; Kappes, Dietmar J; Rhodes, Jennifer; Wiest, David L

    2013-02-25

    It remains controversial whether the highly homologous ribosomal protein (RP) paralogs found in lower eukaryotes have distinct functions and this has not been explored in vertebrates. Here we demonstrate that despite ubiquitous expression, the RP paralogs, Rpl22 and Rpl22-like1 (Rpl22l1) play essential, distinct, and antagonistic roles in hematopoietic development. Knockdown of Rpl22 in zebrafish embryos selectively blocks the development of T lineage progenitors after they have seeded the thymus. In contrast, knockdown of the Rpl22 paralog, Rpl22l1, impairs the emergence of hematopoietic stem cells (HSC) in the aorta-gonad-mesonephros by abrogating Smad1 expression and the consequent induction of essential transcriptional regulator, Runx1. Indeed, despite the ability of both paralogs to bind smad1 RNA, Rpl22 and Rpl22l1 have opposing effects on Smad1 expression. Accordingly, circumstances that tip the balance of these paralogs in favor of Rpl22 (e.g., Rpl22l1 knockdown or Rpl22 overexpression) result in repression of Smad1 and blockade of HSC emergence.

  17. Control of hematopoietic stem cell emergence by antagonistic functions of ribosomal protein paralogs

    PubMed Central

    Zhang, Yong; Duc, Anne-Cécile E.; Rao, Shuyun; Sun, Xiao-Li; Bilbee, Alison N.; Rhodes, Michele; Li, Qin; Kappes, Dietmar J.; Rhodes, Jennifer; Wiest, David L.

    2013-01-01

    Summary It remains controversial whether the highly-homologous ribosomal protein (RP) paralogs found in lower eukaryotes have distinct functions and this has not been explored in vertebrates. Here we demonstrate that despite ubiquitous expression, the RP paralogs, Rpl22 and Rpl22-like1 (Rpl22l1) play essential, distinct, and antagonistic roles in hematopoietic development. Knockdown of rpl22 in zebrafish embryos selectively blocks the development of T lineage progenitors after they have seeded the thymus. In contrast, knockdown of the rpl22 paralog, rpl22l1, impairs the emergence of hematopoietic stem cells (HSC) in the aorta-gonad-mesonephros by abrogating Smad1 expression and the consequent induction of essential transcriptional regulator, Runx1. Indeed, despite the ability of both paralogs to bind Smad1 RNA, Rpl22 and Rpl22l1 have opposing effects on Smad1 expression. Accordingly, circumstances that tip the balance of these paralogs in favor of Rpl22 (e.g., Rpl22l1 knockdown or Rpl22 overexpression) result in repression of Smad1 and blockade of HSC emergence. PMID:23449473

  18. Genetic Evidence for an Interferon-Antagonistic Function of Rift Valley Fever Virus Nonstructural Protein NSs

    PubMed Central

    Bouloy, Michèle; Janzen, Christian; Vialat, Pierre; Khun, Huot; Pavlovic, Jovan; Huerre, Michel; Haller, Otto

    2001-01-01

    Rift Valley fever virus (RVFV), a phlebovirus of the family Bunyaviridae, is a major public health threat in Egypt and sub-Saharan Africa. The viral and host cellular factors that contribute to RVFV virulence and pathogenicity are still poorly understood. All pathogenic RVFV strains direct the synthesis of a nonstructural phosphoprotein (NSs) that is encoded by the smallest (S) segment of the tripartite genome and has an undefined accessory function. In this report, we show that MP12 and clone 13, two attenuated RVFV strains with mutations in the NSs gene, were highly virulent in IFNAR−/− mice lacking the alpha/beta interferon (IFN-α/β) receptor but remained attenuated in IFN-γ receptor-deficient mice. Both attenuated strains proved to be excellent inducers of early IFN-α/β production. In contrast, the virulent strain ZH548 failed to induce detectable amounts of IFN-α/β and replicated extensively in both IFN-competent and IFN-deficient mice. Clone 13 has a defective NSs gene with a large in-frame deletion. This defect in the NSs gene results in expression of a truncated protein which is rapidly degraded. To investigate whether the presence of the wild-type NSs gene correlated with inhibition of IFN-α/β production, we infected susceptible IFNAR−/− mice with S gene reassortant viruses. When the S segment of ZH548 was replaced by that of clone 13, the resulting reassortants became strong IFN inducers. When the defective S segment of clone 13 was exchanged with the wild-type S segment of ZH548, the reassortant virus lost the capacity to stimulate IFN-α/β production. These results demonstrate that the ability of RVFV to inhibit IFN-α/β production correlates with viral virulence and suggest that the accessory protein NSs is an IFN antagonist. PMID:11152510

  19. Biology of polycomb and trithorax group proteins.

    PubMed

    Breiling, Achim; Sessa, Luca; Orlando, Valerio

    2007-01-01

    Cellular phenotypes can be ascribed to different patterns of gene expression. Epigenetic mechanisms control the generation of different phenotypes from the same genotype. Thus differentiation is basically a process driven by changes in gene activity during development, often in response to transient factors or environmental stimuli. To keep the specific characteristics of cell types, tissue-specific gene expression patterns must be transmitted stably from one cell to the daughter cells, also in the absence of the early-acting determination factors. This heritability of patterns of active and inactive genes is enabled by epigenetic mechanisms that create a layer of information on top of the DNA sequence that ensures mitotic and sometimes also meiotic transmission of expression patterns. The proteins of the Polycomb and Trithorax group comprise such a cellular memory mechanism that preserves gene expression patterns through many rounds of cell division. This review provides an overview of the genetics and molecular biology of these maintenance proteins, concentrating mainly on mechanisms of Polycomb group-mediated repression.

  20. Effects of the calcium antagonists perhexiline and cinnarizine on vascular and cardiac contractile protein function.

    PubMed

    Silver, P J; Dachiw, J; Ambrose, J M; Pinto, P B

    1985-09-01

    The weakly basic, lipophilic Ca++ antagonists perhexiline and cinnarizine have been compared with the calmodulin inhibitor W-7 and the cardiotonics Vardax and APP-201-533 for the ability to modulate Ca++-dependent contractile protein interactions directly, as well as Ca++-calmodulin-mediated myosin light chain phosphorylation, in arterial actomyosin or cardiac myofibrils. Both perhexiline and cinnarizine inhibited arterial myosin P-light chain phosphorylation and superprecipitation of arterial actomyosin over the concentration range of 10 to 200 microM. Concomitant inhibition of arterial superprecipitation and phosphorylation by perhexiline (IC50 = 33 microM) and cinnarizine (IC50 = 60 microM) was similar to W-7 (IC50 = 35 microM), and was characterized by a rightward shift in the pCa superprecipitation and pCa-light chain phosphorylation relationships, depressed maximum activity and attenuation by 2 microM exogenous calmodulin. However, whereas inhibition of superprecipitation and P-light chain phosphorylation by W-7 was equal at different Mg++ concentrations, relatively greater inhibition with perhexiline and less inhibition with cinnarizine was apparent as the free Mg++ concentration was lowered. In cardiac myofibrils prepared from both bovine and canine ventricles, perhexiline stimulated Mg-adenosine triphosphatase (ATPase) activity and cinnarizine was without effect, whereas W-7 significantly depressed ATPase activity. Perhexiline was 10-fold more potent and 3-fold more efficacious than either Vardax or APP-201-533 in canine cardiac myofibrils. Whereas APP-201-533 increased Ca++ sensitivity and maximum ATPase activity (Vmax), perhexiline increased Ca++ sensitivity, but not Vmax, and W-7 depressed both parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. A Network of Genes Antagonistic to the LIN-35 Retinoblastoma Protein of Caenorhabditis elegans

    PubMed Central

    Polley, Stanley R. G.; Fay, David S.

    2012-01-01

    The Caenorhabditis elegans pRb ortholog, LIN-35, functions in a wide range of cellular and developmental processes. This includes a role of LIN-35 in nutrient utilization by the intestine, which it carries out redundantly with SLR-2, a zinc-finger protein. This and other redundant functions of LIN-35 were identified in genetic screens for mutations that display synthetic phenotypes in conjunction with loss of lin-35. To explore the intestinal role of LIN-35, we conducted a genome-wide RNA-interference-feeding screen for suppressors of lin-35; slr-2 early larval arrest. Of the 26 suppressors identified, 17 fall into three functional classes: (1) ribosome biogenesis genes, (2) mitochondrial prohibitins, and (3) chromatin regulators. Further characterization indicates that different categories of suppressors act through distinct molecular mechanisms. We also tested lin-35; slr-2 suppressors, as well as suppressors of the synthetic multivulval phenotype, to determine the spectrum of lin-35-synthetic phenotypes that could be suppressed following inhibition of these genes. We identified 19 genes, most of which are evolutionarily conserved, that can suppress multiple unrelated lin-35-synthetic phenotypes. Our study reveals a network of genes broadly antagonistic to LIN-35 as well as genes specific to the role of LIN-35 in intestinal and vulval development. Suppressors of multiple lin-35 phenotypes may be candidate targets for anticancer therapies. Moreover, screening for suppressors of phenotypically distinct synthetic interactions, which share a common altered gene, may prove to be a novel and effective approach for identifying genes whose activities are most directly relevant to the core functions of the shared gene. PMID:22542970

  2. Trithorax group proteins: switching genes on and keeping them active.

    PubMed

    Schuettengruber, Bernd; Martinez, Anne-Marie; Iovino, Nicola; Cavalli, Giacomo

    2011-11-23

    Cellular memory is provided by two counteracting groups of chromatin proteins termed Trithorax group (TrxG) and Polycomb group (PcG) proteins. TrxG proteins activate transcription and are perhaps best known because of the involvement of the TrxG protein MLL in leukaemia. However, in terms of molecular analysis, they have lived in the shadow of their more famous counterparts, the PcG proteins. Recent advances have improved our understanding of TrxG protein function and demonstrated that the heterogeneous group of TrxG proteins is of critical importance in the epigenetic regulation of the cell cycle, senescence, DNA damage and stem cell biology.

  3. Bone Morphogenetic Protein Antagonist Noggin Promotes Skin Tumorigenesis via Stimulation of the Wnt and Shh Signaling Pathways

    PubMed Central

    Sharov, Andrey A.; Mardaryev, Andrei N.; Sharova, Tatyana Y.; Grachtchouk, Marina; Atoyan, Ruzanna; Byers, H. Randolph; Seykora, John T.; Overbeek, Paul; Dlugosz, Andrzej; Botchkarev, Vladimir A.

    2009-01-01

    Bone morphogenetic proteins (BMPs) play pivotal roles in the regulation of skin development. To study the role of BMPs in skin tumorigenesis, BMP antagonist noggin was used to generate keratin 14-targeted transgenic mice. In contrast to wild-type mice, transgenic mice developed spontaneous hair follicle-derived tumors, which resemble human trichofolliculoma. Global gene expression profiles revealed that in contrast to anagen hair follicles of wild-type mice, tumors of transgenic mice showed stage-dependent increases in the expression of genes encoding the selected components of Wnt and Shh pathways. Specifically, expression of the Wnt ligands increased at the initiation stage of tumor formation, whereas expression of the Wnt antagonist and tumor suppressor Wnt inhibitory factor-1 decreased, as compared with fully developed tumors. In contrast, expression of the components of Shh pathway increased in fully developed tumors, as compared with the tumor placodes. Consistent with the expression data, pharmacological treatment of transgenic mice with Wnt and Shh antagonists resulted in the stage-dependent inhibition of tumor initiation, and progression, respectively. Furthermore, BMP signaling stimulated Wnt inhibitory factor-1 expression and promoter activity in cultured tumor cells and HaCaT keratinocytes, as well as inhibited Shh expression, as compared with the corresponding controls. Thus, tumor suppressor activity of the BMPs in skin epithelium depends on the local concentrations of noggin and is mediated at least in part via stage-dependent antagonizing of Wnt and Shh signaling pathways. PMID:19700758

  4. Genome Sequencing of Bacillus subtilis SC-8, Antagonistic to the Bacillus cereus Group, Isolated from Traditional Korean Fermented-Soybean Food

    PubMed Central

    Yeo, In-Cheol; Lee, Nam Keun

    2012-01-01

    Bacillus subtilis SC-8 is a Gram-positive bacterium displaying narrow antagonistic activity for the Bacillus cereus group. B. subtilis SC-8 was isolated from Korean traditional fermented-soybean food. Here we report the draft genome sequence of B. subtilis SC-8, including biosynthetic genes for antibiotics that may have beneficial effects for control of food-borne pathogens. PMID:22207744

  5. Death-associated protein kinase: A molecule with functional antagonistic duality and a potential role in inflammatory bowel disease (Review)

    PubMed Central

    STEINMANN, SARA; SCHEIBE, KRISTINA; ERLENBACH-WUENSCH, KATHARINA; NEUFERT, CLEMENS; SCHNEIDER-STOCK, REGINE

    2015-01-01

    The cytoskeleton-associated serine/threonine kinase death-associated protein kinase (DAPK) has been described as a cancer gene chameleon with functional antagonistic duality in a cell type and context specific manner. The broad range of interaction partners and substrates link DAPK to inflammatory processes especially in the gut. Herein we summarize our knowledge on the role of DAPK in different cell types that play a role under inflammatory conditions in the gut. Besides some promising experimental data suggesting DAPK as an interesting drug target in inflammatory bowel disease there are many open questions regarding direct evidence for a role of DAPK in intestinal inflammation. PMID:25963636

  6. Death-associated protein kinase: A molecule with functional antagonistic duality and a potential role in inflammatory bowel disease (Review).

    PubMed

    Steinmann, Sara; Scheibe, Kristina; Erlenbach-Wuensch, Katharina; Neufert, Clemens; Schneider-Stock, Regine

    2015-07-01

    The cytoskeleton-associated serine/threonine kinase death-associated protein kinase (DAPK) has been described as a cancer gene chameleon with functional antagonistic duality in a cell type and context specific manner. The broad range of interaction partners and substrates link DAPK to inflammatory processes especially in the gut. Herein we summarize our knowledge on the role of DAPK in different cell types that play a role under inflammatory conditions in the gut. Besides some promising experimental data suggesting DAPK as an interesting drug target in inflammatory bowel disease there are many open questions regarding direct evidence for a role of DAPK in intestinal inflammation.

  7. Testis dysfunction by isoproterenol is mediated by upregulating endothelin receptor A, leptin and protein kinase Cvarepsilon and is attenuated by an endothelin receptor antagonist CPU0213.

    PubMed

    Cheng, Yu-Si; Dai, De-Zai; Dai, Yin

    2010-07-01

    This study has examined whether upregulation of endothelin receptor A, leptin and phosphorylated protein kinase Cvarepsilon contributes to stress-induced testicular damaged and its possible reversal by endothelial (ET) antagonism. Adult male Sprague-Dawley rats were randomly divided into control and isoproterenol (ISO 1mg/kg, subcutaneous (s.c.), 10 days) groups, and intervened with the ET receptor antagonist CPU0213 (20mg/kg, s.c.), on days 6-10. In ISO group, testicular succinate dehydrogenase, lactate dehydrogenase, acid phosphotase, and gamma-glutamyl transpeptidase, and serum testosterone decreased, whereas FSH increased, relative to control. The seminiferous tubules were damaged in association with testicular upregulation of protein abundance of leptin and pPKCvarepsilon, and mRNA and protein expression of leptin receptor (OBRb) and ET(A). CPU0213 was effective in ameliorating these abnormalities. Over-expression of ET(A) and leptin accounting for the testis dysfunction is likely to be mediated by pPKCvarepsilon in the ISO treated rats. The upregulated ET pathway appears to be critical in pathologies of the testis.

  8. Vegetarian Choices in the Protein Foods Group

    MedlinePlus

    ... Health Benefits Tips to Help You Eat Vegetables Beans and Peas Are Unique Foods Food Gallery Grains ... Group for vegetarians include eggs (for ovo-vegetarians), beans and peas, nuts, nut butters, and soy products ( ...

  9. SET antagonist enhances the chemosensitivity of non-small cell lung cancer cells by reactivating protein phosphatase 2A.

    PubMed

    Hung, Man-Hsin; Wang, Cheng-Yi; Chen, Yen-Lin; Chu, Pei-Yi; Hsiao, Yung-Jen; Tai, Wei-Tien; Chao, Ting-Ting; Yu, Hui-Chuan; Shiau, Chung-Wai; Chen, Kuen-Feng

    2016-01-05

    SET is known as a potent PP2A inhibitor, however, its oncogenic role including its tumorigenic potential and involvement in the development of chemoresistance in non-small cell lung cancer (NSCLC) has not yet been fully discussed. In present study, we investigated the oncogenic role of SET by SET-knockdown and showed that SET silencing impaired cell growth rate, colony formation and tumor sphere formation in A549 cells. Notably, silencing SET enhanced the pro-apoptotic effects of paclitaxel, while ectopic expression of SET diminished the sensitivity of NSCLC cells to paclitaxel. Since the SET protein was shown to affect chemosensitivity, we next examined whether combining a novel SET antagonist, EMQA, sensitized NSCLC cells to paclitaxel. Both the in vitro and in vivo experiments suggested that EMQA and paclitaxel combination treatment was synergistic. Importantly, we found that downregulating p-Akt by inhibiting SET-mediated protein phosphatase 2A (PP2A) inactivation determined the pro-apoptotic effects of EMQA and paclitaxel combination treatment. To dissect the critical site for EMQA functioning, we generated several truncated SET proteins. By analysis of the effects of EMQA on the binding affinities of different truncated SET proteins to PP2A-catalytic subunits, we revealed that the 227-277 amino-acid sequence is critical for EMQA-induced SET inhibition. Our findings demonstrate the critical role of SET in NSCLC, particularly in the development of chemoresistance. The synergistic effects of paclitaxel and the SET antagonist shown in current study encourage further validation of the clinical potential of this combination.

  10. SET antagonist enhances the chemosensitivity of non-small cell lung cancer cells by reactivating protein phosphatase 2A

    PubMed Central

    Hung, Man-Hsin; Wang, Cheng-Yi; Chen, Yen-Lin; Chu, Pei-Yi; Hsiao, Yung-Jen; Tai, Wei-Tien; Chao, Ting-Ting; Yu, Hui-Chuan; Shiau, Chung-Wai; Chen, Kuen-Feng

    2016-01-01

    SET is known as a potent PP2A inhibitor, however, its oncogenic role including its tumorigenic potential and involvement in the development of chemoresistance in non-small cell lung cancer (NSCLC) has not yet been fully discussed. In present study, we investigated the oncogenic role of SET by SET-knockdown and showed that SET silencing impaired cell growth rate, colony formation and tumor sphere formation in A549 cells. Notably, silencing SET enhanced the pro-apoptotic effects of paclitaxel, while ectopic expression of SET diminished the sensitivity of NSCLC cells to paclitaxel. Since the SET protein was shown to affect chemosensitivity, we next examined whether combining a novel SET antagonist, EMQA, sensitized NSCLC cells to paclitaxel. Both the in vitro and in vivo experiments suggested that EMQA and paclitaxel combination treatment was synergistic. Importantly, we found that downregulating p-Akt by inhibiting SET-mediated protein phosphatase 2A (PP2A) inactivation determined the pro-apoptotic effects of EMQA and paclitaxel combination treatment. To dissect the critical site for EMQA functioning, we generated several truncated SET proteins. By analysis of the effects of EMQA on the binding affinities of different truncated SET proteins to PP2A-catalytic subunits, we revealed that the 227–277 amino-acid sequence is critical for EMQA-induced SET inhibition. Our findings demonstrate the critical role of SET in NSCLC, particularly in the development of chemoresistance. The synergistic effects of paclitaxel and the SET antagonist shown in current study encourage further validation of the clinical potential of this combination. PMID:26575017

  11. Discovery of Klotho peptide antagonists against Wnt3 and Wnt3a target proteins using combination of protein engineering, protein-protein docking, peptide docking and molecular dynamics simulations.

    PubMed

    Mirza, Shaher Bano; Ekhteiari Salmas, Ramin; Fatmi, M Qaiser; Durdagi, Serdar

    2017-12-01

    The Klotho is known as lifespan enhancing protein involved in antagonizing the effect of Wnt proteins. Wnt proteins are stem cell regulators, and uninterrupted exposure of Wnt proteins to the cell can cause stem and progenitor cell senescence, which may lead to aging. Keeping in mind the importance of Klotho in Wnt signaling, in silico approaches have been applied to study the important interactions between Klotho and Wnt3 and Wnt3a (wingless-type mouse mammary tumor virus (MMTV) integration site family members 3 and 3a). The main aim of the study is to identify important residues of the Klotho that help in designing peptides which can act as Wnt antagonists. For this aim, a protein engineering study is performed for Klotho, Wnt3 and Wnt3a. During the theoretical analysis of homology models, unexpected role of number of disulfide bonds and secondary structure elements has been witnessed in case of Wnt3 and Wnt3a proteins. Different in silico experiments were carried out to observe the effect of correct number of disulfide bonds on 3D protein models. For this aim, total of 10 molecular dynamics (MD) simulations were carried out for each system. Based on the protein-protein docking simulations of selected protein models of Klotho with Wnt3 and Wnt3a, different peptides derived from Klotho have been designed. Wnt3 and Wnt3a proteins have three important domains: Index finger, N-terminal domain and a patch of ∼10 residues on the solvent exposed surface of palm domain. Protein-peptide docking of designed peptides of Klotho against three important domains of palmitoylated Wnt3 and Wnt3a yields encouraging results and leads better understanding of the Wnt protein inhibition by proposed Klotho peptides. Further in vitro studies can be carried out to verify effects of novel designed peptides as Wnt antagonists.

  12. Apoptosis and the FLIP and NF-kappa B proteins as pharmacodynamic criteria for biosimilar TNF-alpha antagonists

    PubMed Central

    Urbano, Paulo César Martins; Soccol, Vanete Thomaz; Azevedo, Valderilio Feijó

    2014-01-01

    Various criteria are necessary to assess the efficacy and safety of biological medications in order to grant companies the right to register these medications with the appropriate bodies that regulate their sale. The imminent expiration of the patents on reference biological products which block the cytokine TNF-α (tumor necrosis factor-α) raises the possibility of bringing so-called biosimilars to the market (similar to the biologicals of reference products). This occurrence is inevitable, but criteria to adequately evaluate these medications are now needed. Even among controversy, there is a demand from publications correlating the pro-apoptotic mechanism of the original TNF-α antagonists (etanercept, infliximab, adalimumab, golimumab, and certolizumab pegol) in the treatment of rheumatoid arthritis and other diseases. In this article, the authors discuss the possibility of utilizing the pro-apoptotic effect correlated with the regulation of the anti-apoptotic proteins FLIP and NF-κB as new criteria for analyzing the pharmacodynamics of possible biosimilar TNF-α antagonists which should be submitted to regulatory agencies for evaluation. PMID:25114503

  13. Apoptosis and the FLIP and NF-kappa B proteins as pharmacodynamic criteria for biosimilar TNF-alpha antagonists.

    PubMed

    Urbano, Paulo César Martins; Soccol, Vanete Thomaz; Azevedo, Valderilio Feijó

    2014-01-01

    Various criteria are necessary to assess the efficacy and safety of biological medications in order to grant companies the right to register these medications with the appropriate bodies that regulate their sale. The imminent expiration of the patents on reference biological products which block the cytokine TNF-α (tumor necrosis factor-α) raises the possibility of bringing so-called biosimilars to the market (similar to the biologicals of reference products). This occurrence is inevitable, but criteria to adequately evaluate these medications are now needed. Even among controversy, there is a demand from publications correlating the pro-apoptotic mechanism of the original TNF-α antagonists (etanercept, infliximab, adalimumab, golimumab, and certolizumab pegol) in the treatment of rheumatoid arthritis and other diseases. In this article, the authors discuss the possibility of utilizing the pro-apoptotic effect correlated with the regulation of the anti-apoptotic proteins FLIP and NF-κB as new criteria for analyzing the pharmacodynamics of possible biosimilar TNF-α antagonists which should be submitted to regulatory agencies for evaluation.

  14. A calixpyrrole derivative acts as an antagonist to GPER, a G-protein coupled receptor: mechanisms and models

    PubMed Central

    Lappano, Rosamaria; Rosano, Camillo; Pisano, Assunta; Santolla, Maria Francesca; De Francesco, Ernestina Marianna; De Marco, Paola; Dolce, Vincenza; Ponassi, Marco; Felli, Lamberto; Cafeo, Grazia; Kohnke, Franz Heinrich; Abonante, Sergio; Maggiolini, Marcello

    2015-01-01

    ABSTRACT Estrogens regulate numerous pathophysiological processes, mainly by binding to and activating estrogen receptor (ER)α and ERβ. Increasing amounts of evidence have recently demonstrated that G-protein coupled receptor 30 (GPR30; also known as GPER) is also involved in diverse biological responses to estrogens both in normal and cancer cells. The classical ER and GPER share several features, including the ability to bind to identical compounds; nevertheless, some ligands exhibit opposed activity through these receptors. It is worth noting that, owing to the availability of selective agonists and antagonists of GPER for research, certain differential roles elicited by GPER compared with ER have been identified. Here, we provide evidence on the molecular mechanisms through which a calixpyrrole derivative acts as a GPER antagonist in different model systems, such as breast tumor cells and cancer-associated fibroblasts (CAFs) obtained from breast cancer patients. Our data might open new perspectives toward the development of a further class of selective GPER ligands in order to better dissect the role exerted by this receptor in different pathophysiological conditions. Moreover, calixpyrrole derivatives could be considered in future anticancer strategies targeting GPER in cancer cells. PMID:26183213

  15. Pseudomonas DING proteins as human transcriptional regulators and HIV-1 antagonists

    PubMed Central

    2013-01-01

    Background Anti-HIV-1 therapy depends upon multiple agents that target different phases of the viral replication cycle. Recent reports indicate that plant and human DING proteins are unique in targeting viral gene transcription as the basis of their anti-HIV-1 therapy. Methods Two cloned DING genes from Pseudomonas were transiently expressed in human cells, and effects on NFκB-mediated transcription, HIV-1 transcription, and HIV-1 production were measured. Results Both DING proteins elevated NFκB-mediated transcription. In microglial cells, one protein, from P. aeruginosa PA14, suppressed HIV-1 transcription; the other protein, from P. fluorescens SBW25, was inactive. The PA14DING protein also reduces HIV-1 production in microglial cells. Conclusions Structural differences between the two DING proteins highlight regions of the PA14DING protein essential to the anti-HIV-1 activity, and may guide the design of therapeutic agents. PMID:23855931

  16. Lloviu virus VP24 and VP35 proteins function as innate immune antagonists in human and bat cells.

    PubMed

    Feagins, Alicia R; Basler, Christopher F

    2015-11-01

    Lloviu virus (LLOV) is a new member of the filovirus family that also includes Ebola virus (EBOV) and Marburg virus (MARV). LLOV has not been cultured; however, its genomic RNA sequence indicates the coding capacity to produce homologs of the EBOV and MARV VP24, VP35, and VP40 proteins. EBOV and MARV VP35 proteins inhibit interferon (IFN)-alpha/beta production and EBOV VP35 blocks activation of the antiviral kinase PKR. The EBOV VP24 and MARV VP40 proteins inhibit IFN signaling, albeit by different mechanisms. Here we demonstrate that LLOV VP35 suppresses Sendai virus induced IFN regulatory factor 3 (IRF3) phosphorylation, IFN-α/β production, and PKR phosphorylation. Additionally, LLOV VP24 blocks tyrosine phosphorylated STAT1 binding to karyopherin alpha 5 (KPNA5), STAT1 nuclear accumulation, and IFN-induced gene expression. LLOV VP40 lacks detectable IFN antagonist function. These activities parallel EBOV IFN inhibitory functions. EBOV and LLOV VP35 and VP24 proteins also inhibit IFN responses in bat cells. These data suggest that LLOV infection will block innate immune responses in a manner similar to EBOV.

  17. Human eosinophil major basic protein is an endogenous allosteric antagonist at the inhibitory muscarinic M2 receptor.

    PubMed Central

    Jacoby, D B; Gleich, G J; Fryer, A D

    1993-01-01

    The effect of human eosinophil major basic protein (MBP) as well as other eosinophil proteins, on binding of [3H]N-methyl-scopolamine ([3H]NMS: 1 x 10(-10) M) to muscarinic M2 receptors in heart membranes and M3 receptors in submandibular gland membranes was studied. MBP inhibited specific binding of [3H]NMS to M2 receptors but not to M3 receptors. MBP also inhibited atropine-induced dissociation of [3H]NMS-receptor complexes in a dose-dependent fashion, demonstrating that the interaction of MBP with the M2 muscarinic receptor is allosteric. This effect of MBP suggests that it may function as an endogenous allosteric inhibitor of agonist binding to the M2 muscarinic receptor. Inhibition of [3H]NMS binding by MBP was reversible by treatment with heparin, which binds and neutralizes MBP. Eosinophil peroxidase (EPO) also inhibited specific binding of [3H]NMS to M2 receptors but not to M3 receptors and inhibited atropine-induced dissociation of [3H]NMS-receptor complexes. On a molar basis, EPO is less potent than MBP. Neither eosinophil cationic protein nor eosinophil-derived neurotoxin affected binding of [3H]NMS to M2 receptors. Thus both MBP and EPO are selective allosteric antagonists at M2 receptors. The effects of these proteins may be important causes of M2 receptor dysfunction and enhanced vagally mediated bronchoconstriction in asthma. Images PMID:8473484

  18. In vitro and in vivo protein phosphorylation in Avena sativa L. coleoptiles: effects of Ca2+, calmodulin antagonists, and auxin

    NASA Technical Reports Server (NTRS)

    Veluthambi, K.; Poovaiah, B. W.

    1986-01-01

    In vitro and in vivo protein phosphorylations in oat (Avena sativa L.) coleoptile segments were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by two-dimensional gel electrophoresis. In vitro phosphorylation of several polypeptides was distinctly promoted at 1 to 15 micromolar free Ca2+ concentrations. Ca2(+)-stimulated phosphorylation was markedly reduced by trifluoperazine, chlorpromazine, and naphthalene sulfonamide (W7). Two polypeptides were phosphorylated both under in vitro and in vivo conditions, but the patterns of phosphorylation of several other polypeptides were different under the two conditions indicating that the in vivo phosphorylation pattern of proteins is not truly reflected by in vitro phosphorylation studies. Trifluoperazine, W7, or ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) + calcium ionophore A23187 treatments resulted in reduced levels of in vivo protein phosphorylation of both control and auxin-treated coleoptile segments. Analysis by two-dimensional electrophoresis following in vivo phosphorylation revealed auxin-dependent changes of certain polypeptides. A general inhibition of phosphorylation by calmodulin antagonists suggested that both control and auxin-treated coleoptiles exhibited Ca2+, and calmodulin-dependent protein phosphorylation in vivo.

  19. Lloviu virus VP24 and VP35 proteins function as innate immune antagonists in human and bat cells

    SciTech Connect

    Feagins, Alicia R.; Basler, Christopher F.

    2015-11-15

    Lloviu virus (LLOV) is a new member of the filovirus family that also includes Ebola virus (EBOV) and Marburg virus (MARV). LLOV has not been cultured; however, its genomic RNA sequence indicates the coding capacity to produce homologs of the EBOV and MARV VP24, VP35, and VP40 proteins. EBOV and MARV VP35 proteins inhibit interferon (IFN)-alpha/beta production and EBOV VP35 blocks activation of the antiviral kinase PKR. The EBOV VP24 and MARV VP40 proteins inhibit IFN signaling, albeit by different mechanisms. Here we demonstrate that LLOV VP35 suppresses Sendai virus induced IFN regulatory factor 3 (IRF3) phosphorylation, IFN-α/β production, and PKR phosphorylation. Additionally, LLOV VP24 blocks tyrosine phosphorylated STAT1 binding to karyopherin alpha 5 (KPNA5), STAT1 nuclear accumulation, and IFN-induced gene expression. LLOV VP40 lacks detectable IFN antagonist function. These activities parallel EBOV IFN inhibitory functions. EBOV and LLOV VP35 and VP24 proteins also inhibit IFN responses in bat cells. These data suggest that LLOV infection will block innate immune responses in a manner similar to EBOV. - Highlights: • Lloviu virus (LLOV) is a new member of the filovirus family. • LLOV VP35 blocks IRF3 phosphorylation, IFN-α/β production and PKR phosphorylation. • LLOV VP24 inhibits IFN responses by targeting phospho-STAT1 KPNA interaction. • Infection by LLOV may block innate immune responses in a manner similar to EBOV.

  20. ACTH Antagonists

    PubMed Central

    Clark, Adrian John; Forfar, Rachel; Hussain, Mashal; Jerman, Jeff; McIver, Ed; Taylor, Debra; Chan, Li

    2016-01-01

    Adrenocorticotropin (ACTH) acts via a highly selective receptor that is a member of the melanocortin receptor subfamily of type 1 G protein-coupled receptors. The ACTH receptor, also known as the melanocortin 2 receptor (MC2R), is unusual in that it is absolutely dependent on a small accessory protein, melanocortin receptor accessory protein (MRAP) for cell surface expression and function. ACTH is the only known naturally occurring agonist for this receptor. This lack of redundancy and high degree of ligand specificity suggests that antagonism of this receptor could provide a useful therapeutic aid and a potential investigational tool. Clinical situations in which this could be useful include (1) Cushing’s disease and ectopic ACTH syndrome – especially while preparing for definitive treatment of a causative tumor, or in refractory cases, or (2) congenital adrenal hyperplasia – as an adjunct to glucocorticoid replacement. A case for antagonism in other clinical situations in which there is ACTH excess can also be made. In this article, we will explore the scientific and clinical case for an ACTH antagonist, and will review the evidence for existing and recently described peptides and modified peptides in this role. PMID:27547198

  1. Recombinant TNF-binding protein from variola virus as a novel potential TNF antagonist.

    PubMed

    Gileva, I P; Nepomnyashchikh, T S; Ryazankin, I A; Shchelkunov, S N

    2009-12-01

    Gel-filtration chromatographic separation of the lysate of Sf21 insect cells infected with recombinant baculovirus BVi67 containing the gene for TNF-binding protein (CrmB) of variola virus (VARV) revealed that hTNF-cytotoxicity neutralization activity is associated with a fraction corresponding mainly to high molecular weight proteins (above 500 kDa) and less with fractions corresponding to proteins of 270 or 90 kDa. The recombinant VARV-CrmB protein has been purified by affinity chromatography. Difference in the experimentally determined and estimated (according to amino acid composition) VARV-CrmB molecular weight is due to glycosylation of the recombinant protein expressed in the insect cells. VARV-CrmB neutralizes in vitro the cytotoxic effect of hTNF and hLTalpha, and its TNF-neutralizing activity is two to three orders of magnitude higher compared to the analogous effects of type I and II soluble TNF receptors, comparable with the activity of mAb MAK195, and somewhat lower than the effect of the commercial drug Remicade.

  2. Crystal structure of Bacillus subtilis anti-TRAP protein, an antagonist of TRAP/RNA interaction

    PubMed Central

    Shevtsov, Mikhail B.; Chen, Yanling; Gollnick, Paul; Antson, Alfred A.

    2005-01-01

    In Bacillus subtilis the anti-TRAP protein (AT) is produced in response to the accumulation of uncharged tRNATrp. AT regulates expression of genes involved in tryptophan biosynthesis and transport by binding to the tryptophan-activated trp RNA-binding attenuation protein (TRAP) and preventing its interaction with several mRNAs. Here, we report the x-ray structure of AT at 2.8 Å resolution, showing that the protein subunits assemble into tight trimers. Four such trimers are further associated into a 12-subunit particle in which individual trimers are related by twofold and threefold symmetry axes. Twelve DnaJ-like, cysteine-rich zinc-binding domains form spikes on the surface of the dodecamer. Available data suggest several possible ways for AT to interact with the 11-subunit TRAP. Interaction between the two symmetry-mismatching molecules could be assisted by the flexible nature of AT zinc-binding domains. PMID:16306262

  3. Enhanced Osteogenesis of Adipose-Derived Stem Cells by Regulating Bone Morphogenetic Protein Signaling Antagonists and Agonists

    PubMed Central

    Fan, Jiabing; Im, Choong Sung; Guo, Mian; Cui, Zhong-Kai; Fartash, Armita; Kim, Soyon; Patel, Nikhil; Bezouglaia, Olga; Wu, Benjamin M.; Wang, Cun-Yu

    2016-01-01

    Although adipose-derived stem cells (ASCs) are an attractive cell source for bone tissue engineering, direct use of ASCs alone has had limited success in the treatment of large bone defects. Although bone morphogenetic proteins (BMPs) are believed to be the most potent osteoinductive factors to promote osteogenic differentiation of ASCs, their clinical applications require supraphysiological dosage, leading to high medical burden and adverse side effects. In the present study, we demonstrated an alternative approach that can effectively complement the BMP activity to maximize the osteogenesis of ASCs without exogenous application of BMPs by regulating levels of antagonists and agonists to BMP signaling. Treatment of ASCs with the amiloride derivative phenamil, a positive regulator of BMP signaling, combined with gene manipulation to suppress the BMP antagonist noggin, significantly enhanced osteogenic differentiation of ASCs through increased BMP–Smad signaling in vitro. Furthermore, the combination approach of noggin suppression and phenamil stimulation enhanced the BMP signaling and bone repair in a mouse calvarial defect model by adding noggin knockdown ASCs to apatite-coated poly(lactic-coglycolic acid) scaffolds loaded with phenamil. These results suggest novel complementary osteoinductive strategies that could maximize activity of the BMP pathway in ASC bone repair while reducing potential adverse effects of current BMP-based therapeutics. Significance Although stem cell-based tissue engineering strategy offers a promising alternative to repair damaged bone, direct use of stem cells alone is not adequate for challenging healing environments such as in large bone defects. This study demonstrates a novel strategy to maximize bone formation pathways in osteogenic differentiation of mesenchymal stem cells and functional bone formation by combining gene manipulation with a small molecule activator toward osteogenesis. The findings indicate promising stem cell

  4. Identification of protein interaction antagonists using the repressed transactivator two-hybrid system.

    PubMed

    Joshi, Phalgun B; Hirst, Martin; Malcolm, Tom; Parent, Jennifer; Mitchell, David; Lund, Karen; Sadowski, Ivan

    2007-05-01

    The repressed transactivator (RTA) yeast two-hybrid system was developed to enable genetic identification of interactions with transcriptional activator proteins. We have devised modifications of this system that enable its use in screening for inhibitors of protein interactions from small molecule compound libraries. We show that inhibition of protein interactions can be measured by monitoring growth in selective medium containing 3-aminotriazole (3-AT) and using this assay have identified inhibitors of four independent protein interactions in screens with a 23,000 small molecule compound library. Compounds found to inhibit one of the tested interactions between FKBP12 and the transforming growth factor beta receptor (TGFbeta-R) were validated in vivo and found to inhibit calcineurin-dependent signaling in T cells. One of these compounds was also found to cause elevated basal expression of a TGFbeta-R/SMAD-dependent reporter gene. These results demonstrate the capability of the RTA small molecule screening assay for discovery of potentially novel therapeutic compounds.

  5. Molecular approach for the rapid detection of Bacillus and Pseudomonas genera--dominant antagonistic groups--from diverse ecological niches using colony multiplex PCR.

    PubMed

    Nair, Anusree V; Pradeep, M A; Vijayan, K K

    2014-07-01

    Bacillus and Pseudomonas are the dominant groups of bacteria known for their antagonistic potential against many plant and animal pathogens. Presently, exploration of these genera with antagonistic property for disease management of aquaculture system is gaining more importance to overcome the use of antibiotics and related resistance issues. Rapid screening and identification of these genera from diverse bacterial populations by conventional methods is laborious, cost-intensive, and time-consuming. To overcome these limiting factors, in the present study, a colony multiplex PCR (cmPCR) method was developed and evaluated for the rapid detection of Bacillus and Pseudomonas. The technique amplifies the partial 16S rRNA gene of Bacillus and Pseudomonas with a product size of ~1,100 and ~375 bp, respectively, using single forward (BSF2) and two reverse primers (PAGSR and BK1R). Reliability of the cmPCR method was confirmed by screening 472 isolates obtained from ten different eco-stations, of which 133 isolates belonged to Bacillus and 32 to Pseudomonas. The cmPCR method also helped to identify six different Pseudomonas spp. and 14 different Bacillus spp. from environmental samples. Of the total 472 isolates studied, 46 showed antagonistic activity, among which 63 % were Bacillus and 17.4 % were Pseudomonas. Thus, the newly developed molecular approach provides a quick, sensitive, and potential screening tool to detect novel, antagonistically important Bacillus and Pseudomonas genera for their use in aquaculture. Further, it can also act as a taxonomic tool to understand the distribution of these genera from wide ecological niches and their exploitation for diverse biotechnological applications.

  6. Hedgehog Proteins Consume Steroidal CYP17A1 Antagonists: Potential Therapeutic Significance in Advanced Prostate Cancer.

    PubMed

    Bordeau, Brandon M; Ciulla, Daniel A; Callahan, Brian P

    2016-09-20

    Abiraterone, a potent inhibitor of the human enzyme CYP17A1 (cytochrome P450c17), provides a last line of defense against ectopic androgenesis in advanced prostate cancer. Herein we report an unprecedented off-target interaction between abiraterone and oncogenic hedgehog proteins. Our experiments indicate that abiraterone and its structural congener, galeterone, can replace cholesterol as a substrate in a specialized biosynthetic event of hedgehog proteins, known as cholesterolysis. The off-target reaction generates covalent hedgehog-drug conjugates. Cell-based reporter assays indicate that these conjugates activate hedgehog signaling when present in the low nanomolar range. Because hedgehog signaling is implicated in prostate cancer progression, and abiraterone is administered to treat advanced stages of the disease, this off-target interaction may have therapeutic significance.

  7. Bone morphogenetic proteins and their antagonists: current and emerging clinical uses

    PubMed Central

    Ali, Imran H A; Brazil, Derek P

    2014-01-01

    Bone morphogenetic proteins (BMPs) are members of the TGFβ superfamily of secreted cysteine knot proteins that includes TGFβ1, nodal, activins and inhibins. BMPs were first discovered by Urist in the 1960s when he showed that implantation of demineralized bone into intramuscular tissue of rabbits induced bone and cartilage formation. Since this seminal discovery, BMPs have also been shown to play key roles in several other biological processes, including limb, kidney, skin, hair and neuronal development, as well as maintaining vascular homeostasis. The multifunctional effects of BMPs make them attractive targets for the treatment of several pathologies, including bone disorders, kidney and lung fibrosis, and cancer. This review will summarize current knowledge on the BMP signalling pathway and critically evaluate the potential of recombinant BMPs as pharmacological agents for the treatment of bone repair and tissue fibrosis in patients. PMID:24758361

  8. The protein interaction landscape of the human CMGC kinase group.

    PubMed

    Varjosalo, Markku; Keskitalo, Salla; Van Drogen, Audrey; Nurkkala, Helka; Vichalkovski, Anton; Aebersold, Ruedi; Gstaiger, Matthias

    2013-04-25

    Cellular information processing via reversible protein phosphorylation requires tight control of the localization, activity, and substrate specificity of protein kinases, which to a large extent is accomplished by complex formation with other proteins. Despite their critical role in cellular regulation and pathogenesis, protein interaction information is available for only a subset of the 518 human protein kinases. Here we present a global proteomic analysis of complexes of the human CMGC kinase group. In addition to subgroup-specific functional enrichment and modularity, the identified 652 high-confidence kinase-protein interactions provide a specific biochemical context for many poorly studied CMGC kinases. Furthermore, the analysis revealed a kinase-kinase subnetwork and candidate substrates for CMGC kinases. Finally, the presented interaction proteome uncovered a large set of interactions with proteins genetically linked to a range of human diseases, including cancer, suggesting additional routes for analyzing the role of CMGC kinases in controlling human disease pathways.

  9. Group G streptococcal M protein exhibits structural features analogous to those of class I M protein of group A streptococci.

    PubMed Central

    Collins, C M; Kimura, A; Bisno, A L

    1992-01-01

    We have previously studied a collection of group G streptococcal strains isolated from bacteremic human infections and demonstrated that such strains resist phagocytosis by human polymorphonuclear leukocytes but are type specifically opsonized by homologous antiserum. We have now performed Southern hybridization analysis on genomic DNA from eight blood isolates. All eight isolates showed DNA homology to a group A emm24 gene probe. The M-protein gene of one of the isolates, strain 1750, has now been isolated. This gene (emmG1) encodes a polypeptide of 67 kDa (MG1) which is reactive with antibodies to the partially purified M protein of the parent strain. The predicted amino acid structure of MG1 demonstrates significant identity with the carboxy terminus (C, D, and anchor domains) of M6 and M24 but only limited identity with the amino terminus (variable portion) of these group A M proteins. Southern hybridization of genomic DNA of the eight group G blood isolates with an emmG1 gene probe indicated there were at least four emm alleles associated with these strains. These studies indicate that M proteins of group G streptococci, like those of group A, are genetically heterogeneous. Moreover, MG1 appears to conform to the recently proposed class I structure of M-protein molecules and thus shares certain distinct structural features with the M proteins of well-established rheumatogenic group A streptococcal serotypes. Further comparison of the structures of group G and group A M proteins of throat and skin isolates may cast light on those configurations of the M protein molecules which are and are not critical for the expression of rheumatogenicity. Images PMID:1500178

  10. Patient-derived glioblastoma cells show significant heterogeneity in treatment responses to the inhibitor-of-apoptosis-protein antagonist birinapant

    PubMed Central

    Zakaria, Z; Tivnan, A; Flanagan, L; Murray, D W; Salvucci, M; Stringer, B W; Day, B W; Boyd, A W; Kögel, D; Rehm, M; O'Brien, D F; Byrne, A T; Prehn, J H M

    2016-01-01

    Background: Resistance to temozolomide (TMZ) greatly limits chemotherapeutic effectiveness in glioblastoma (GBM). Here we analysed the ability of the Inhibitor-of-apoptosis-protein (IAP) antagonist birinapant to enhance treatment responses to TMZ in both commercially available and patient-derived GBM cells. Methods: Responses to TMZ and birinapant were analysed in a panel of commercial and patient-derived GBM cell lines using colorimetric viability assays, flow cytometry, morphological analysis and protein expression profiling of pro- and antiapoptotic proteins. Responses in vivo were analysed in an orthotopic xenograft GBM model. Results: Single-agent treatment experiments categorised GBM cells into TMZ-sensitive cells, birinapant-sensitive cells, and cells that were insensitive to either treatment. Combination treatment allowed sensitisation to therapy in only a subset of resistant GBM cells. Cell death analysis identified three principal response patterns: Type A cells that readily activated caspase-8 and cell death in response to TMZ while addition of birinapant further sensitised the cells to TMZ-induced cell death; Type B cells that readily activated caspase-8 and cell death in response to birinapant but did not show further sensitisation with TMZ; and Type C cells that showed no significant cell death or moderately enhanced cell death in the combined treatment paradigm. Furthermore, in vivo, a Type C patient-derived cell line that was TMZ-insensitive in vitro and showed a strong sensitivity to TMZ and TMZ plus birinapant treatments. Conclusions: Our results demonstrate remarkable differences in responses of patient-derived GBM cells to birinapant single and combination treatments, and suggest that therapeutic responses in vivo may be greatly affected by the tumour microenvironment. PMID:26657652

  11. Insulin Stimulates S100B Secretion and These Proteins Antagonistically Modulate Brain Glucose Metabolism.

    PubMed

    Wartchow, Krista Minéia; Tramontina, Ana Carolina; de Souza, Daniela F; Biasibetti, Regina; Bobermin, Larissa D; Gonçalves, Carlos-Alberto

    2016-06-01

    Brain metabolism is highly dependent on glucose, which is derived from the blood circulation and metabolized by the astrocytes and other neural cells via several pathways. Glucose uptake in the brain does not involve insulin-dependent glucose transporters; however, this hormone affects the glucose influx to the brain. Changes in cerebrospinal fluid levels of S100B (an astrocyte-derived protein) have been associated with alterations in glucose metabolism; however, there is no evidence whether insulin modulates glucose metabolism and S100B secretion. Herein, we investigated the effect of S100B on glucose metabolism, measuring D-(3)H-glucose incorporation in two preparations, C6 glioma cells and acute hippocampal slices, and we also investigated the effect of insulin on S100B secretion. Our results showed that: (a) S100B at physiological levels decreases glucose uptake, through the multiligand receptor RAGE and mitogen-activated protein kinase/ERK signaling, and (b) insulin stimulated S100B secretion via PI3K signaling. Our findings indicate the existence of insulin-S100B modulation of glucose utilization in the brain tissue, and may improve our understanding of glucose metabolism in several conditions such as ketosis, streptozotocin-induced dementia and pharmacological exposure to antipsychotics, situations that lead to changes in insulin signaling and extracellular levels of S100B.

  12. The bone morphogenetic protein antagonist gremlin 1 is overexpressed in human cancers and interacts with YWHAH protein

    PubMed Central

    Namkoong, Hong; Shin, Seung Min; Kim, Hyun Kee; Ha, Seon-Ah; Cho, Goang Won; Hur, Soo Young; Kim, Tae Eung; Kim, Jin Woo

    2006-01-01

    Background Basic studies of oncogenesis have demonstrated that either the elevated production of particular oncogene proteins or the occurrence of qualitative abnormalities in oncogenes can contribute to neoplastic cellular transformation. The purpose of our study was to identify an unique gene that shows cancer-associated expression, and characterizes its function related to human carcinogenesis. Methods We used the differential display (DD) RT-PCR method using normal cervical, cervical cancer, metastatic cervical tissues, and cervical cancer cell lines to identify genes overexpressed in cervical cancers and identified gremlin 1 which was overexpressed in cervical cancers. We determined expression levels of gremlin 1 using Northern blot analysis and immunohistochemical study in various types of human normal and cancer tissues. To understand the tumorigenesis pathway of identified gremlin 1 protein, we performed a yeast two-hybrid screen, GST pull down assay, and immunoprecipitation to identify gremlin 1 interacting proteins. Results DDRT-PCR analysis revealed that gremlin 1 was overexpressed in uterine cervical cancer. We also identified a human gremlin 1 that was overexpressed in various human tumors including carcinomas of the lung, ovary, kidney, breast, colon, pancreas, and sarcoma. PIG-2-transfected HEK 293 cells exhibited growth stimulation and increased telomerase activity. Gremlin 1 interacted with homo sapiens tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, eta polypeptide (14-3-3 eta; YWHAH). YWHAH protein binding site for gremlin 1 was located between residues 61–80 and gremlin 1 binding site for YWHAH was found to be located between residues 1 to 67. Conclusion Gremlin 1 may play an oncogenic role especially in carcinomas of the uterine cervix, lung, ovary, kidney, breast, colon, pancreas, and sarcoma. Over-expressed gremlin 1 functions by interaction with YWHAH. Therefore, Gremlin 1 and its binding protein YWHAH could be good

  13. Antagonistic regulation of F-BAR protein assemblies controls actin polymerization during podosome formation.

    PubMed

    Tsujita, Kazuya; Kondo, Akihiro; Kurisu, Shusaku; Hasegawa, Junya; Itoh, Toshiki; Takenawa, Tadaomi

    2013-05-15

    FBP17, an F-BAR domain protein, has emerged as a crucial factor linking the plasma membrane to WASP-mediated actin polymerization. Although it is well established that FBP17 has a powerful self-polymerizing ability that promotes actin nucleation on membranes in vitro, knowledge of inhibitory factors that counteract this activity in vivo is limited. Here, we demonstrate that the assembly of FBP17 on the plasma membranes is antagonized by PSTPIP2, another F-BAR protein implicated in auto-inflammatory disorder. Knockdown of PSTPIP2 in macrophage promotes the assembly of FBP17 as well as subsequent actin nucleation at podosomes, resulting in an enhancement of matrix degradation. This phenotype is rescued by expression of PSTPIP2 in a manner dependent on its F-BAR domain. Time-lapse total internal reflection fluorescence (TIRF) microscopy observations reveal that the self-assembly of FBP17 at the podosomal membrane initiates actin polymerization, whereas the clustering of PSTPIP2 has an opposite effect. Biochemical analysis and live-cell imaging show that PSTPIP2 inhibits actin polymerization by competing with FBP17 for assembly at artificial as well as the plasma membrane. Interestingly, the assembly of FBP17 is dependent on WASP, and its dissociation by WASP inhibition strongly induces a self-organization of PSTPIP2 at podosomes. Thus, our data uncover a previously unappreciated antagonism between different F-BAR domain assemblies that determines the threshold of actin polymerization for the formation of functional podosomes and may explain how the absence of PSTPIP2 causes auto-inflammatory disorder.

  14. Genetic Variation in Toll-Interacting Protein Is Associated With Leprosy Susceptibility and Cutaneous Expression of Interleukin 1 Receptor Antagonist.

    PubMed

    Shah, Javeed A; Berrington, William R; Vary, James C; Wells, Richard D; Peterson, Glenna J; Kunwar, Chhatra B; Khadge, Saraswoti; Hagge, Deanna A; Hawn, Thomas R

    2016-04-01

    Leprosy is a chronic disease characterized by skin and peripheral nerve pathology and immune responses that fail to control Mycobacterium leprae. Toll-interacting protein (TOLLIP) regulates Toll-like receptor (TLR) and interleukin 1 receptor (IL-1R) signaling against mycobacteria. We analyzed messenger RNA (mRNA) expression of candidate immune genes in skin biopsy specimens from 85 individuals with leprosy. TOLLIP mRNA was highly and specifically correlated with IL-1R antagonist (IL-1Ra). In a case-control gene-association study with 477 cases and 1021 controls in Nepal, TOLLIP single-nucleotide polymorphism rs3793964 TT genotype was associated with increased susceptibility to leprosy (recessive, P = 1.4 × 10(-3)) and with increased skin expression of TOLLIP and IL-1Ra. Stimulation of TOLLIP-deficient monocytes with M. leprae produced significantly less IL-1Ra (P < .001), compared with control. These data suggest that M. leprae upregulates IL-1Ra by a TOLLIP-dependent mechanism. Inhibition of TOLLIP may decrease an individual's susceptibility to leprosy and offer a novel therapeutic target for IL-1-dependent diseases.

  15. Anti-stress effects of ONO-2952, a novel translocator protein 18 kDa antagonist, in rats.

    PubMed

    Mitsui, Katsukuni; Niwa, Tomohiro; Kawahara, Yuji; Morimoto, Noriko; Ohmoto, Kazuyuki; Kato, Masashi; Yamaura, Yoshiyuki; Yoshimoto, Naoki; Suna, Hideaki; Katsumata, Seishi

    2015-12-01

    Accumulating evidence has shown the pathophysiological significance of the translocator protein 18 kDa (TSPO) in the central nervous system. In this study, we evaluated the beneficial effects of ONO-2952, a novel TSPO antagonist in rat stress models. ONO-2952 potently bound both rat and human TSPO (Ki=0.330-9.30 nmol/L) with high selectivity over other receptors, transporters, ion channels and enzymes. ONO-2952 inhibited both neurosteroid accumulation and noradrenaline release in the brain of rats exposed to acute stress. The inhibitory effect of ONO-2952 on stress-induced noradrenaline release was attenuated by co-treatment with the TSPO agonist CB34 in a dose-dependent manner. ONO-2952, at 0.3 mg/kg or higher, dose-dependently suppressed restraint stress-induced defecation in rats with brain TSPO occupancy of more than 50%. In addition, ONO-2952, at 1 mg/kg or higher, suppressed conditioned fear stress-induced freezing behavior in rats with an efficacy equivalent to that of diazepam, given orally at 3 mg/kg. Results of the passive avoidance learning test revealed that ONO-2952, unlike diazepam, did not affect learning and memory even at doses 10 times higher than its effective doses in the stress models. The present findings indicate that ONO-2952 is a promising candidate for the treatment of stress-related disorders.

  16. Screening and Characterization of Hydrate Forms of T-3256336, a Novel Inhibitor of Apoptosis (IAP) Protein Antagonist.

    PubMed

    Takeuchi, Shoko; Kojima, Takashi; Hashimoto, Kentaro; Saito, Bunnai; Sumi, Hiroyuki; Ishikawa, Tomoyasu; Ikeda, Yukihiro

    2015-01-01

    Different crystal packing of hydrates from anhydrate crystals leads to different physical properties, such as solubility and stability. Investigation of the potential of varied hydrate formation, and understanding the stability in an anhydrous/hydrate system, are crucial to prevent an undesired transition during the manufacturing process and storage. Only one anhydrous form of T-3256336, a novel inhibitor of apoptosis (IAP) protein antagonist, was discovered during synthesis, and no hydrate form has been identified. In this study, we conducted hydrate screening such as dynamic water vapor sorption/desorption (DVS), and the slurry experiment, and characterized the solid-state properties of anhydrous/hydrate forms to determine the most desirable crystalline form for development. New hydrate forms, both mono-hydrate and hemi-hydrate forms, were discovered as a result of this hydrate screening. The characterization of two new hydrate forms was conducted, and the anhydrous form was determined to be the most desirable development form of T-3256336 in terms of solid-state stability. In addition, the stability of the anhydrous form was investigated using the water content and temperature controlled slurry experiment to obtain the desirable crystal form in the crystallization process. The water content regions of the stable phase of the desired form, the anhydrous form, were identified for the cooling crystallization process.

  17. Discovery of the First α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Antagonist Dependent upon Transmembrane AMPA Receptor Regulatory Protein (TARP) γ-8.

    PubMed

    Gardinier, Kevin M; Gernert, Douglas L; Porter, Warren J; Reel, Jon K; Ornstein, Paul L; Spinazze, Patrick; Stevens, F Craig; Hahn, Patric; Hollinshead, Sean P; Mayhugh, Daniel; Schkeryantz, Jeff; Khilevich, Albert; De Frutos, Oscar; Gleason, Scott D; Kato, Akihiko S; Luffer-Atlas, Debra; Desai, Prashant V; Swanson, Steven; Burris, Kevin D; Ding, Chunjin; Heinz, Beverly A; Need, Anne B; Barth, Vanessa N; Stephenson, Gregory A; Diseroad, Benjamin A; Woods, Tim A; Yu, Hong; Bredt, David; Witkin, Jeffrey M

    2016-05-26

    Transmembrane AMPA receptor regulatory proteins (TARPs) are a family of scaffolding proteins that regulate AMPA receptor trafficking and function. TARP γ-8 is one member of this family and is highly expressed within the hippocampus relative to the cerebellum. A selective TARP γ-8-dependent AMPA receptor antagonist (TDAA) is an innovative approach to modulate AMPA receptors in specific brain regions to potentially increase the therapeutic index relative to known non-TARP-dependent AMPA antagonists. We describe here, for the first time, the discovery of a noncompetitive AMPA receptor antagonist that is dependent on the presence of TARP γ-8. Three major iteration cycles were employed to improve upon potency, CYP1A2-dependent challenges, and in vivo clearance. An optimized molecule, compound (-)-25 (LY3130481), was fully protective against pentylenetetrazole-induced convulsions in rats without the motor impairment associated with non-TARP-dependent AMPA receptor antagonists. Compound (-)-25 could be utilized to provide proof of concept for antiepileptic efficacy with reduced motor side effects in patients.

  18. Diet-dependent modulation of hippocampal expression of endocannabinoid signaling-related proteins in cannabinoid antagonist-treated obese rats.

    PubMed

    Rivera, Patricia; Luque-Rojas, María Jesús; Pastor, Antoni; Blanco, Eduardo; Pavón, Francisco J; Serrano, Antonia; Crespillo, Ana; Vida, Margarita; Grondona, Jesús M; Cifuentes, Manuel; Bermúdez-Silva, Francisco J; de la Torre, Rafael; de Fonseca, Fernando Rodríguez; Suárez, Juan

    2013-01-01

    Diet-induced obesity produces changes in endocannabinoid signaling (ECS), influencing the regulation of energy homeostasis. Recently, we demonstrated that, in high-fat-diet-fed rats, blockade of CB1 receptor by AM251 not only reduced body weight but also increased adult neurogenesis in the hippocampus, suggesting an influence of diet on hippocampal cannabinoid function. To further explore the role of hippocampal ECS in high-fat-diet-induced obesity, we investigated whether the immunohistochemical expression of the enzymes that produce (diacylglycerol lipase alpha and N-acyl phosphatidylethanolamine phospholipase D) and degrade (monoacylglycerol lipase and fatty acid amino hydrolase) endocannabinoids may be altered in the hippocampus of AM251 (3 mg/kg)-treated rats fed three different diets: standard diet (normal chow), high-carbohydrate diet (70% carbohydrate) and high-fat diet (60% fat). Results indicated that AM251 reduced caloric intake and body weight gain, and induced a modulation of the expression of ECS-related proteins in the hippocampus of animals exposed to hypercaloric diets. These effects were differentially restricted to either the 2-arachinodoyl glycerol or anandamide signaling pathways, in a diet-dependent manner. AM251-treated rats fed the high-carbohydrate diet showed a reduction of the diacylglycerol lipase alpha : monoacylglycerol lipase ratio, whereas AM251-treated rats fed the high-fat diet showed a decrease of the N-acyl phosphatidylethanolamine phospholipase D : fatty acid amino hydrolase ratio. These results are consistent with the reduced levels of hippocampal endocannabinoids found after food restriction. Regarding the CB1 expression, AM251 induced specific changes focused in the CA1 stratum pyramidale of high-fat-diet-fed rats. These findings indicated that the cannabinoid antagonist AM251 modulates ECS-related proteins in the rat hippocampus in a diet-specific manner. Overall, these results suggest that the hippocampal ECS participates

  19. Small-molecule agonists and antagonists of F-box protein-substrate interactions in auxin perception and signaling.

    PubMed

    Hayashi, Ken-Ichiro; Tan, Xu; Zheng, Ning; Hatate, Tatsuya; Kimura, Yoshio; Kepinski, Stefan; Nozaki, Hiroshi

    2008-04-08

    The regulation of gene expression by the hormone auxin is a crucial mechanism in plant development. We have shown that the Arabidopsis F-box protein TIR1 is a receptor for auxin, and our recent structural work has revealed the molecular mechanism of auxin perception. TIR1 is the substrate receptor of the ubiquitin-ligase complex SCF(TIR1). Auxin binding enhances the interaction between TIR1 and its substrates, the Aux/IAA repressors, thereby promoting the ubiquitination and degradation of Aux/IAAs, altering the expression of hundreds of genes. TIR1 is the prototype of a new class of hormone receptor and the first example of an SCF ubiquitin-ligase modulated by a small molecule. Here, we describe the design, synthesis, and characterization of a series of auxin agonists and antagonists. We show these molecules are specific to TIR1-mediated events in Arabidopsis, and their mode of action in binding to TIR1 is confirmed by x-ray crystallographic analysis. Further, we demonstrate the utility of these probes for the analysis of TIR1-mediated auxin signaling in the moss Physcomitrella patens. Our work not only provides a useful tool for plant chemical biology but also demonstrates an example of a specific small-molecule inhibitor of F-box protein-substrate recruitment. Substrate recognition and subsequent ubiquitination by SCF-type ubiquitin ligases are central to many cellular processes in eukaryotes, and ubiquitin-ligase function is affected in several human diseases. Our work supports the idea that it may be possible to design small-molecule agents to modulate ubiquitin-ligase function therapeutically.

  20. Structural Isosteres of Phosphate Groups in the Protein Data Bank.

    PubMed

    Zhang, Yuezhou; Borrel, Alexandre; Ghemtio, Leo; Regad, Leslie; Boije Af Gennäs, Gustav; Camproux, Anne-Claude; Yli-Kauhaluoma, Jari; Xhaard, Henri

    2017-03-13

    We developed a computational workflow to mine the Protein Data Bank for isosteric replacements that exist in different binding site environments but have not necessarily been identified and exploited in compound design. Taking phosphate groups as examples, the workflow was used to construct 157 data sets, each composed of a reference protein complexed with AMP, ADP, ATP, or pyrophosphate as well other ligands. Phosphate binding sites appear to have a high hydration content and large size, resulting in U-shaped bioactive conformations recurrently found across unrelated protein families. A total of 16 413 replacements were extracted, filtered for a significant structural overlap on phosphate groups, and sorted according to their SMILES codes. In addition to the classical isosteres of phosphate, such as carboxylate, sulfone, or sulfonamide, unexpected replacements that do not conserve charge or polarity, such as aryl, aliphatic, or positively charged groups, were found.

  1. Classifying proteins into functional groups based on all-versus-all BLAST of 10 million proteins.

    PubMed

    Kolker, Natali; Higdon, Roger; Broomall, William; Stanberry, Larissa; Welch, Dean; Lu, Wei; Haynes, Winston; Barga, Roger; Kolker, Eugene

    2011-01-01

    To address the monumental challenge of assigning function to millions of sequenced proteins, we completed the first of a kind all-versus-all sequence alignments using BLAST for 9.9 million proteins in the UniRef100 database. Microsoft Windows Azure produced over 3 billion filtered records in 6 days using 475 eight-core virtual machines. Protein classification into functional groups was then performed using Hive and custom jars implemented on top of Apache Hadoop utilizing the MapReduce paradigm. First, using the Clusters of Orthologous Genes (COG) database, a length normalized bit score (LNBS) was determined to be the best similarity measure for classification of proteins. LNBS achieved sensitivity and specificity of 98% each. Second, out of 5.1 million bacterial proteins, about two-thirds were assigned to significantly extended COG groups, encompassing 30 times more assigned proteins. Third, the remaining proteins were classified into protein functional groups using an innovative implementation of a single-linkage algorithm on an in-house Hadoop compute cluster. This implementation significantly reduces the run time for nonindexed queries and optimizes efficient clustering on a large scale. The performance was also verified on Amazon Elastic MapReduce. This clustering assigned nearly 2 million proteins to approximately half a million different functional groups. A similar approach was applied to classify 2.8 million eukaryotic sequences resulting in over 1 million proteins being assign to existing KOG groups and the remainder clustered into 100,000 functional groups.

  2. Regulation by Polycomb and Trithorax Group Proteins in Arabidopsis

    PubMed Central

    Alvarez-Venegas, Raúl

    2010-01-01

    Polycomb group (PcG) and trithorax group (trxG) proteins are key regulators of homeotic genes and have crucial roles in cell proliferation, growth and development. PcG and trxG proteins form higher order protein complexes that contain SET domain proteins, with a histone methyltransferase (HMTase) activity, responsible for the different types of lysine methylation at the N-terminal tails of the core histone proteins. In recent years, genetic studies along with biochemical and cell biological analyses in Arabidopsis have enabled researchers to begin to understand how PcG and trxG proteins are recruited to chromatin and how they regulate their target genes and to elucidate their functions. This review focuses on the advances in our understanding of the biological roles of PcG and trxG proteins, their molecular mechanisms of action and further examines the role of histone marks in PcG and trxG regulation in Arabidopsis. PMID:22303254

  3. Kisspeptin antagonists.

    PubMed

    Roseweir, Antonia Kathryn; Millar, Robert P

    2013-01-01

    Kisspeptin is now known to be an important regulator of the hypothalamic--pituitary-gonadal axis and is the target of a range of regulators, such as steroid hormone feedback, nutritional and metabolic regulation. Kisspeptin binds to its cognate receptor, KISS1R (also called GPR54), on GnRH neurons and stimulates their activity, which in turn provides an obligatory signal for GnRH secretion-thus gating down-stream events supporting reproduction. The development of peripherally active kisspeptin antagonists could offer a unique therapeutic agent for treating hormone-dependent disorders of reproduction, including precocious puberty, endometriosis, and metastatic prostate cancer. The following chapter discusses the advances made in the search for both peptide and small molecule kisspeptin antagonists and their use in delineating the role of kisspeptin within the reproductive system. To date, four peptide antagonists and one small molecule antagonist have been designed.

  4. The zipper groups of the amyloid state of proteins

    SciTech Connect

    Stroud, James C.

    2013-04-01

    A formal derivation is provided of the 15 symmetry groups (zipper groups) available to the amyloid homosteric zipper. Fibrous proteins in the amyloid state are found both associated with numerous diseases and in the normal functions of cells. Amyloid fibers contain a repetitive spine, commonly built from a pair of β-sheets whose β-strands run perpendicular to the fiber direction and whose side chains interdigitate, much like the teeth of a zipper. In fiber spines known as homosteric zippers, identical protein segments sharing identical packing environments make the two β-sheets. In previous work based on atomic resolution crystal structures of homosteric zippers derived from a dozen proteins, the symmetries of homosteric zippers were categorized into eight classes. Here, it is shown through a formal derivation that each homosteric zipper class corresponds to a unique set of symmetry groups termed ‘zipper groups’. Furthermore, the eight previously identified classes do not account for all of the 15 possible zipper groups, which may be categorized into the complete set of ten classes. Because of their foundations in group theory, the 15 zipper groups provide a mathematically rigorous classification for homosteric zippers.

  5. An example of designed multiple ligands spanning protein classes: dual MCH-1R antagonists/DPPIV inhibitors.

    PubMed

    Gattrell, William T; Sambrook Smith, Colin P; Smith, Alun J

    2012-04-01

    A ligand-based approach to identify potential starting points for a dual MCH-1R antagonist/DPPIV inhibitor medicinal chemistry program was undertaken. Potential ligand pairs were identified by analysis of MCH-1R and DPPIV in vitro data. A highly targeted synthetic effort lead to the discovery of pyridone 11, a dual MCH-1R antagonist/DPPIV inhibitor with selectivity over DPP8 and DPP9.

  6. Combgap contributes to recruitment of Polycomb group proteins in Drosophila

    PubMed Central

    Ray, Payal; De, Sandip; Mitra, Apratim; Bezstarosti, Karel; Demmers, Jeroen A. A.; Pfeifer, Karl; Kassis, Judith A.

    2016-01-01

    Polycomb group (PcG) proteins are responsible for maintaining the silenced transcriptional state of many developmentally regulated genes. PcG proteins are organized into multiprotein complexes that are recruited to DNA via cis-acting elements known as “Polycomb response elements” (PREs). In Drosophila, PREs consist of binding sites for many different DNA-binding proteins, some known and others unknown. Identification of these DNA-binding proteins is crucial to understanding the mechanism of PcG recruitment to PREs. We report here the identification of Combgap (Cg), a sequence-specific DNA-binding protein that is involved in recruitment of PcG proteins. Cg can bind directly to PREs via GTGT motifs and colocalizes with the PcG proteins Pleiohomeotic (Pho) and Polyhomeotic (Ph) at the majority of PREs in the genome. In addition, Cg colocalizes with Ph at a number of targets independent of Pho. Loss of Cg leads to decreased recruitment of Ph at only a subset of sites; some of these sites are binding sites for other Polycomb repressive complex 1 (PRC1) components, others are not. Our data suggest that Cg can recruit Ph in the absence of PRC1 and illustrate the diversity and redundancy of PcG protein recruitment mechanisms. PMID:27001825

  7. Protein profiles and immunoreactivities of Acanthamoeba morphological groups and genotypes.

    PubMed

    Pumidonming, Wilawan; Koehsler, Martina; Leitsch, David; Walochnik, Julia

    2014-11-01

    Acanthamoeba is a free-living protozoan found in a wide variety of habitats. A classification of Acanthamoeba into currently eighteen genotypes (T1-T18) has been established, however, data on differences between genotypes on the protein level are scarce. The aim of this study was to compare protein and immunoreactivity profiles of Acanthamoeba genotypes. Thirteen strains, both clinical and non-clinical, from genotypes T4, T5, T6, T7, T9, T11 and T12, representing three morphological groups, were investigated for their protein profiles and IgG, IgM and IgA immunoreactivities. It was shown that protein and immunoreactivity profiles of Acanthamoeba genotypes T4, T5, T6, T7, T9, T11 and T12 are clearly distinct from each other, but the banding patterns correlate to the morphological groups. Normal human sera revealed anti-Acanthamoeba antibodies against isolates of all investigated genotypes, interestingly, however only very weak IgM and virtually no IgA immunoreactivity with T7 and T9, both representing morphological group I. The strongest IgG, IgM and IgA immunoreactivities were observed for genotypes T4, T5 and T6. Differences of both, protein and immunological patterns, between cytopathic and non-cytopathic strains, particularly within genotype T4, were not at the level of banding patterns, but rather in expression levels.

  8. The Meat and Protein Group. The Food Guide Pyramid.

    ERIC Educational Resources Information Center

    Frost, Helen

    This booklet for young children is part of a series that supports national science standards related to physical health and nutrition, describing and illustrating the importance of using the Food Guide Pyramid and eating from the meat and protein group. Colorful photographs support early readers in understanding the text. The repetition of words…

  9. Behaviour of protein carbonyl groups in juvenile myocardial infarction.

    PubMed

    Caimi, Gregorio; Canino, Baldassare; Incalcaterra, Egle; Ferrera, Eleonora; Montana, Maria; Lo Presti, Rosalia

    2013-01-01

    Acute myocardial infarction (AMI) is accompanied by oxidative stress, and protein oxidation is among the consequences of oxidative stress. We examined the plasma concentration of protein carbonyl groups (PC), a marker of protein oxidation, in a group of young subjects with AMI (45 men and 5 women; mean age 40.4 ± 4.8 yrs). We found a significant increase of PC (p < 0.001) in comparison with normal controls. No difference was observed between patients with AMI characterized by elevated ST segment and those without elevation of ST segment. There was no correlation between the ejection fraction and PC in the whole group nor in the subgroups of STEMI and non-STEMI patients. Subdividing the whole group of AMI patients according to the number of risk factors and the number of stenosed coronary vessels, the difference in PC level was not statistically significant among the subgroups. This study showed an increased protein oxidation in young subjects with recent AMI. Further investigation is needed to ascertain whether this can be a target of therapeutic intervention.

  10. Finding sequence motifs in groups of functionally related proteins.

    PubMed

    Smith, H O; Annau, T M; Chandrasegaran, S

    1990-01-01

    We have developed a method for rapidly finding patterns of conserved amino acid residues (motifs) in groups of functionally related proteins. All 3-amino acid patterns in a group of proteins of the type aa1 d1 aa2 d2 aa3, where d1 and d2 are distances that can be varied in a range up to 24 residues, are accumulated into an array. Segments of the proteins containing those patterns that occur most frequently are aligned on each other by a scoring method that obtains an average relatedness value for all the amino acids in each column of the aligned sequence block based on the Dayhoff relatedness odds matrix. The automated method successfully finds and displays nearly all of the sequence motifs that have been previously reported to occur in 33 reverse transcriptases, 18 DNA integrases, and 30 DNA methyltransferases.

  11. An inhibitor of apoptosis protein antagonist T-3256336 potentiates the antitumor efficacy of the Nedd8-activating enzyme inhibitor pevonedistat (TAK-924/MLN4924).

    PubMed

    Sumi, Hiroyuki; Inazuka, Masakazu; Morimoto, Megumi; Hibino, Ryosuke; Hashimoto, Kentaro; Ishikawa, Tomoyasu; Kuida, Keisuke; Smith, Peter G; Yoshida, Sei; Yabuki, Masato

    2016-11-18

    Inhibitors of apoptosis proteins (IAPs) are antiapoptotic regulators that block cell death, and are frequently overexpressed in several human cancers, where they facilitate evasion of apoptosis and promote cell survival. IAP antagonists are also known as second mitochondria-derived activator of caspase (SMAC)-mimetics, and have recently been considered as novel therapeutic agents for inducing apoptosis, alone and in combination with other anticancer drugs. In this study, we showed that T-3256336, the orally available IAP antagonist has synergistically enhances the antiproliferative effects of the NEDD8-activating enzyme (NAE) inhibitor pevonedistat (TAK-924/MLN4924), and these effects were attenuated by a TNFα-neutralizing antibody. In the present mechanistic analyses, pevonedistat induced TNFα mRNA and triggered IAP antagonist-dependent extrinsic apoptotic cell death in cancer cell lines. Furthermore, synergistic effects of the combination of T-3256336 and pevonedistat were demonstrated in a HL-60 mouse xenograft model. Our findings provide mechanistic evidence of the effects of IAP antagonists in combination with NAE inhibitors, and demonstrate the potential of a new combination therapy for cancer.

  12. Bone morphogenetic protein antagonist gremlin 1 is widely expressed by cancer-associated stromal cells and can promote tumor cell proliferation

    NASA Astrophysics Data System (ADS)

    Sneddon, Julie B.; Zhen, Hanson H.; Montgomery, Kelli; van de Rijn, Matt; Tward, Aaron D.; West, Robert; Gladstone, Hayes; Chang, Howard Y.; Morganroth, Greg S.; Oro, Anthony E.; Brown, Patrick O.

    2006-10-01

    Although tissue microenvironments play critical roles in epithelial development and tumorigenesis, the factors mediating these effects are poorly understood. In this work, we used a genomic approach to identify factors produced by cells in the microenvironment of basal cell carcinoma (BCC) of the skin, one of the most common human cancers. The global gene expression programs of stromal cell cultures derived from human BCCs showed consistent, systematic differences from those derived from nontumor skin. The gene most consistently expressed at a higher level in BCC tumor stromal cells compared with those from nontumor skin was GREMLIN 1, which encodes a secreted antagonist of the bone morphogenetic protein (BMP) pathway. BMPs and their antagonists are known to play a crucial role in stem and progenitor cell biology as regulators of the balance between expansion and differentiation. Consistent with the hypothesis that BMP antagonists might have a similar role in cancer, we found GREMLIN 1 expression in the stroma of human BCC tumors but not in normal skin in vivo. Furthermore, BMP 2 and 4 are expressed by BCC cells. Ex vivo, BMP inhibits, and Gremlin 1 promotes, proliferation of cultured BCC cells. We further found that GREMLIN 1 is expressed by stromal cells in many carcinomas but not in the corresponding normal tissue counterparts that we examined. Our data suggest that BMP antagonists may be important constituents of tumor stroma, providing a favorable microenvironment for cancer cell survival and expansion in many cancers. cancer biology | stem cell regulation | tissue microenvironment | tumor stroma

  13. Role of the alpha-amino group of protein in ubiquitin-mediated protein breakdown.

    PubMed Central

    Hershko, A; Heller, H; Eytan, E; Kaklij, G; Rose, I A

    1984-01-01

    Previous studies suggest that the conjugation of ubiquitin to NH2 groups of proteins is required for protein breakdown. We now show that the selective modification of NH2-terminal alpha-NH2 groups of globin and lysozyme prevents their degradation by the ubiquitin proteolytic system from reticulocytes. The conjugation by ubiquitin of epsilon-NH2 groups of lysine residues, usually seen in multiples, was also inhibited in alpha-NH2-blocked proteins. Naturally occurring N alpha-acetylated proteins are not degraded by the ubiquitin system at a significant rate, while their nonacetylated counterparts from other species are good substrates. This suggests that one function of N alpha-acetylation of cellular proteins is to prevent their degradation by the ubiquitin system. alpha-NH2-blocked proteins can have their activity as substrates for degradation increased by incorporation of alpha-NH2 groups through the introduction of polyalanine side chains. Proteins in which most epsilon-NH2 groups are blocked but the alpha-NH2 group is free are degraded by the ubiquitin system, but at a reduced rate. It is therefore suggested that the exposure of a free NH2 terminus of proteins is required for degradation and probably initiates the formation of ubiquitin conjugates committed for degradation. Images PMID:6095265

  14. Chromatin topology is coupled to Polycomb group protein subnuclear organization

    PubMed Central

    Wani, Ajazul H.; Boettiger, Alistair N.; Schorderet, Patrick; Ergun, Ayla; Münger, Christine; Sadreyev, Ruslan I.; Zhuang, Xiaowei; Kingston, Robert E.; Francis, Nicole J.

    2016-01-01

    The genomes of metazoa are organized at multiple scales. Many proteins that regulate genome architecture, including Polycomb group (PcG) proteins, form subnuclear structures. Deciphering mechanistic links between protein organization and chromatin architecture requires precise description and mechanistic perturbations of both. Using super-resolution microscopy, here we show that PcG proteins are organized into hundreds of nanoscale protein clusters. We manipulated PcG clusters by disrupting the polymerization activity of the sterile alpha motif (SAM) of the PcG protein Polyhomeotic (Ph) or by increasing Ph levels. Ph with mutant SAM disrupts clustering of endogenous PcG complexes and chromatin interactions while elevating Ph level increases cluster number and chromatin interactions. These effects can be captured by molecular simulations based on a previously described chromatin polymer model. Both perturbations also alter gene expression. Organization of PcG proteins into small, abundant clusters on chromatin through Ph SAM polymerization activity may shape genome architecture through chromatin interactions. PMID:26759081

  15. Longitudinal Profiles of Girls' Irritable, Defiant and Antagonistic Oppositional Symptoms: Evidence for Group Based Differences in Symptom Severity.

    PubMed

    Boylan, Khrista; Rowe, Richard; Duku, Eric; Waldman, Irwin; Stepp, Stephanie; Hipwell, Alison; Burke, Jeffrey

    2016-11-19

    Three subdimensions of ODD symptoms have been proposed -angry/irritable (IR), argumentative/defiant (DF) and antagonism (AN). This study tested whether longitudinal symptom trajectories could be identified by these subdimensions. Group-based trajectory analysis was used to identify developmental trajectories of IR, DF and AN symptoms. Multi-group trajectory analysis was then used to identify how subdimension trajectories were linked together over time. Data were drawn from the Pittsburgh Girls Study (PGS; N = 2450), an urban community sample of girls between the ages of five--eight at baseline. We included five waves of annual data across ages five-13 to model trajectories. Three trajectories were identified for each ODD subdimension: DF and AN were characterized by high, medium and low severity groups; IR was characterized by low, medium stable, and high increasing groups. Multi-trajectory analysis confirmed these subdimensions were best linked together based on symptom severity. We did not identify girls' trajectory groups that were characterized predominantly by a particular subdimension of ODD symptoms. Membership in more severe symptom groups was significantly associated with worse outcomes five years later. In childhood and early adolescence girls with high levels of ODD symptoms can be identified, and these youth are characterized by a persistently elevated profile of IR, DF and AN symptoms. Further studies in clinical samples are required to examine the ICD-10 proposal that ODD with irritability is a distinct or more severe form of ODD.

  16. The TRIM-NHL protein LIN-41 and the OMA RNA-binding proteins antagonistically control the prophase-to-metaphase transition and growth of Caenorhabditis elegans oocytes.

    PubMed

    Spike, Caroline A; Coetzee, Donna; Eichten, Carly; Wang, Xin; Hansen, Dave; Greenstein, David

    2014-12-01

    In many animals, oocytes enter meiosis early in their development but arrest in meiotic prophase I. Oocyte growth, which occurs during this arrest period, enables the acquisition of meiotic competence and the capacity to produce healthy progeny. Meiotic resumption, or meiotic maturation, involves the transition to metaphase I (M phase) and is regulated by intercellular signaling and cyclin-dependent kinase activation. Premature meiotic maturation would be predicted to diminish fertility as the timing of this event, which normally occurs after oocyte growth is complete, is crucial. In the accompanying article in this issue, we identify the highly conserved TRIM-NHL protein LIN-41 as a translational repressor that copurifies with OMA-1 and OMA-2, RNA-binding proteins redundantly required for normal oocyte growth and meiotic maturation. In this article, we show that LIN-41 enables the production of high-quality oocytes and plays an essential role in controlling and coordinating oocyte growth and meiotic maturation. lin-41 null mutants display a striking defect that is specific to oogenesis: pachytene-stage cells cellularize prematurely and fail to progress to diplotene. Instead, these cells activate CDK-1, enter M phase, assemble spindles, and attempt to segregate chromosomes. Translational derepression of the CDK-1 activator CDC-25.3 appears to contribute to premature M-phase entry in lin-41 mutant oocytes. Genetic and phenotypic analyses indicate that LIN-41 and OMA-1/2 exhibit an antagonistic relationship, and we suggest that translational regulation by these proteins could be important for controlling and coordinating oocyte growth and meiotic maturation.

  17. Blood Group Antigen Recognition via the Group A Streptococcal M Protein Mediates Host Colonization

    PubMed Central

    De Oliveira, David M. P.; Hartley-Tassell, Lauren; Everest-Dass, Arun; Day, Christopher J.; Dabbs, Rebecca A.; Ve, Thomas; Kobe, Bostjan; Nizet, Victor; Packer, Nicolle H.; Walker, Mark J.; Jennings, Michael P.

    2017-01-01

    ABSTRACT Streptococcus pyogenes (group A streptococcus [GAS]) is responsible for over 500,000 deaths worldwide each year. The highly virulent M1T1 GAS clone is one of the most frequently isolated serotypes from streptococcal pharyngitis and invasive disease. The oral epithelial tract is a niche highly abundant in glycosylated structures, particularly those of the ABO(H) blood group antigen family. Using a high-throughput approach, we determined that a strain representative of the globally disseminated M1T1 GAS clone 5448 interacts with numerous, structurally diverse glycans. Preeminent among GAS virulence factors is the surface-expressed M protein. M1 protein showed high affinity for several terminal galactose blood group antigen structures. Deletion mutagenesis shows that M1 protein mediates glycan binding via its B repeat domains. Association of M1T1 GAS with oral epithelial cells varied significantly as a result of phenotypic differences in blood group antigen expression, with significantly higher adherence to those cells expressing H antigen structures compared to cells expressing A, B, or AB antigen structures. These data suggest a novel mechanism for GAS attachment to host cells and propose a link between host blood group antigen expression and M1T1 GAS colonization. PMID:28119471

  18. Design, Synthesis, and Evaluation of Nonretinoid Retinol Binding Protein 4 Antagonists for the Potential Treatment of Atrophic Age-Related Macular Degeneration and Stargardt Disease

    PubMed Central

    2015-01-01

    Accumulation of lipofuscin in the retina is associated with pathogenesis of atrophic age-related macular degeneration and Stargardt disease. Lipofuscin bisretinoids (exemplified by N-retinylidene-N-retinylethanolamine) seem to mediate lipofuscin toxicity. Synthesis of lipofuscin bisretinoids depends on the influx of retinol from serum to the retina. Compounds antagonizing the retinol-dependent interaction of retinol-binding protein 4 (RBP4) with transthyretin in the serum would reduce serum RBP4 and retinol and inhibit bisretinoid formation. We recently showed that A1120 (3), a potent carboxylic acid based RBP4 antagonist, can significantly reduce lipofuscin bisretinoid formation in the retinas of Abca4–/– mice. As part of the NIH Blueprint Neurotherapeutics Network project we undertook the in vitro exploration to identify novel conformationally flexible and constrained RBP4 antagonists with improved potency and metabolic stability. We also demonstrate that upon acute and chronic dosing in rats, 43, a potent cyclopentyl fused pyrrolidine antagonist, reduced circulating plasma RBP4 protein levels by approximately 60%. PMID:25210858

  19. Design, synthesis, and evaluation of nonretinoid retinol binding protein 4 antagonists for the potential treatment of atrophic age-related macular degeneration and Stargardt disease.

    PubMed

    Cioffi, Christopher L; Dobri, Nicoleta; Freeman, Emily E; Conlon, Michael P; Chen, Ping; Stafford, Douglas G; Schwarz, Daniel M C; Golden, Kathy C; Zhu, Lei; Kitchen, Douglas B; Barnes, Keith D; Racz, Boglarka; Qin, Qiong; Michelotti, Enrique; Cywin, Charles L; Martin, William H; Pearson, Paul G; Johnson, Graham; Petrukhin, Konstantin

    2014-09-25

    Accumulation of lipofuscin in the retina is associated with pathogenesis of atrophic age-related macular degeneration and Stargardt disease. Lipofuscin bisretinoids (exemplified by N-retinylidene-N-retinylethanolamine) seem to mediate lipofuscin toxicity. Synthesis of lipofuscin bisretinoids depends on the influx of retinol from serum to the retina. Compounds antagonizing the retinol-dependent interaction of retinol-binding protein 4 (RBP4) with transthyretin in the serum would reduce serum RBP4 and retinol and inhibit bisretinoid formation. We recently showed that A1120 (3), a potent carboxylic acid based RBP4 antagonist, can significantly reduce lipofuscin bisretinoid formation in the retinas of Abca4(-/-) mice. As part of the NIH Blueprint Neurotherapeutics Network project we undertook the in vitro exploration to identify novel conformationally flexible and constrained RBP4 antagonists with improved potency and metabolic stability. We also demonstrate that upon acute and chronic dosing in rats, 43, a potent cyclopentyl fused pyrrolidine antagonist, reduced circulating plasma RBP4 protein levels by approximately 60%.

  20. ESG: extended similarity group method for automated protein function prediction

    PubMed Central

    Chitale, Meghana; Hawkins, Troy; Park, Changsoon; Kihara, Daisuke

    2009-01-01

    Motivation: Importance of accurate automatic protein function prediction is ever increasing in the face of a large number of newly sequenced genomes and proteomics data that are awaiting biological interpretation. Conventional methods have focused on high sequence similarity-based annotation transfer which relies on the concept of homology. However, many cases have been reported that simple transfer of function from top hits of a homology search causes erroneous annotation. New methods are required to handle the sequence similarity in a more robust way to combine together signals from strongly and weakly similar proteins for effectively predicting function for unknown proteins with high reliability. Results: We present the extended similarity group (ESG) method, which performs iterative sequence database searches and annotates a query sequence with Gene Ontology terms. Each annotation is assigned with probability based on its relative similarity score with the multiple-level neighbors in the protein similarity graph. We will depict how the statistical framework of ESG improves the prediction accuracy by iteratively taking into account the neighborhood of query protein in the sequence similarity space. ESG outperforms conventional PSI-BLAST and the protein function prediction (PFP) algorithm. It is found that the iterative search is effective in capturing multiple-domains in a query protein, enabling accurately predicting several functions which originate from different domains. Availability: ESG web server is available for automated protein function prediction at http://dragon.bio.purdue.edu/ESG/ Contact: cspark@cau.ac.kr; dkihara@purdue.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:19435743

  1. Purification and partial characterization of a novel calcium-binding protein from Bacillus cereus T spores and inhibition of germination by calmodulin antagonists

    SciTech Connect

    Shyu, Y.

    1989-01-01

    A novel calcium-binding protein has been purified from the dormant spores of Bacillus cereus T. B. cereus T spores were extensively washed, broken, and heated at 90{degree}C for 2 min. Using calcium-dependent hydrophobic interaction chromatography plus DEAE-cellulose and hydroxylapatite columns, a single protein was obtained which possessed calcium-binding capacity and some characteristics of calmodulin. This heat-stable protein was retained by hydrophobic matrices or a calmodulin antagonist in a calcium-dependent manner. The crude spore extract displaced bovine brain calmodulin from its antibody in a radioimmunoassay and the immunoreactive specific activity of the partially purified fraction which eluted from phenyl-Sepharose was ca. 200-fold greater than the crude spore extract. Purity of this protein was verified by sodium dodecyl sulfate-polyarcylamide gel electrophoresis and reversed-phase HPLC. Calcium-binding ability was verified with a competitive calcium binding assay using Chelex-100 resin and {sup 45}Ca autoradiography. SDS-PAGE and amino acid composition indicated the molecular weight of the protein was 24-kDa. The effects of two calmodulin antagonists, trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) on L-alanine-induced germination of Bacillus cereus T spores were examined by measuring commitment to germination, loss of heat resistance, release of calcium, decrease in optical density at 660 nm and phase-contrast microscopy.

  2. Defining MC1R regulation in human melanocytes by its agonist α-melanocortin and antagonists agouti signaling protein and β-defensin 3.

    PubMed

    Swope, Viki B; Jameson, Joshua A; McFarland, Kevin L; Supp, Dorothy M; Miller, William E; McGraw, Dennis W; Patel, Mira A; Nix, Matthew A; Millhauser, Glenn L; Babcock, George F; Abdel-Malek, Zalfa A

    2012-09-01

    The melanocortin 1 receptor (MC1R), a G(s) protein-coupled receptor, has an important role in human pigmentation. We investigated the regulation of expression and activity of the MC1R in primary human melanocyte cultures. Human β-defensin 3 (HBD3) acted as an antagonist for MC1R, inhibiting the α-melanocortin (α-melanocyte-stimulating hormone (α-MSH))-induced increase in the activities of adenylate cyclase and tyrosinase, the rate-limiting enzyme for melanogenesis. α-Melanocortin and forskolin, which activate adenylate cyclase, and 12-O-tetradecanoylphorbol-13-acetate, which activates protein kinase C, increased, whereas exposure to UV radiation reduced, MC1R gene and membrane protein expression. Brief treatment with α-MSH resulted in MC1R desensitization, whereas continuous treatment up to 3 hours caused a steady rise in cAMP, suggesting receptor recycling. Pretreatment with agouti signaling protein or HBD3 prohibited responsiveness to α-MSH, but not forskolin, suggesting receptor desensitization by these antagonists. Melanocytes from different donors expressed different levels of the G protein-coupled receptor kinases (GRKs) 2, 3, 5, and 6, as well as β-arrestin 1. Therefore, in addition to the MC1R genotype, regulation of MC1R expression and activity is expected to affect human pigmentation and the responses to UV.

  3. Banana Ovate Family Protein MaOFP1 and MADS-Box Protein MuMADS1 Antagonistically Regulated Banana Fruit Ripening

    PubMed Central

    Hu, Wei; Miao, Hongxia; Zhang, Jianbin; Jia, Caihong; Wang, Zhuo; Xu, Biyu; Jin, Zhiqiang

    2015-01-01

    The ovate family protein named MaOFP1 was identified in banana (Musa acuminata L.AAA) fruit by a yeast two-hybrid (Y2H) method using the banana MADS-box gene MuMADS1 as bait and a 2 day postharvest (DPH) banana fruit cDNA library as prey. The interaction between MuMADS1 and MaOFP1 was further confirmed by Y2H and Bimolecular Fluorescence Complementation (BiFC) methods, which showed that the MuMADS1 K domain interacted with MaOFP1. Real-time quantitative PCR evaluation of MuMADS1 and MaOFP1 expression patterns in banana showed that they are highly expressed in 0 DPH fruit, but present in low levels in the stem, which suggests that simultaneous but different expression patterns exist for both MuMADS1 and MaOFP1 in different tissues and developing fruits. Meanwhile, MuMADS1 and MaOFP1 expression was highly stimulated and greatly suppressed, respectively, by exogenous ethylene. In contrast, MaOFP1 expression was highly stimulated while MuMADS1 was greatly suppressed by the ethylene competitor 1-methylcyclopropene (1-MCP). These results indicate that MuMADS1 and MaOFP1 are antagonistically regulated by ethylene and might play important roles in postharvest banana fruit ripening. PMID:25886169

  4. The sorting of blood group active proteins during enucleation.

    PubMed

    Satchwell, Timothy J; Bell, Amanda J; Toye, Ashley M

    2015-04-01

    Enucleation represents the critical stage during red blood cell development when the nucleus is extruded from an orthochromatic erythroblast in order to generate a nascent immature reticulocyte. Extrusion of the nucleus results in loss of a proportion of the erythroblast plasma membrane, which surrounds the nucleus, the bulk of the endoplasmic reticulum and a small region of cytoplasm. For this reason enucleation provides an important point in erythroblast differentiation at which proteins not required for the function of the erythrocyte can be lost, whilst those that are important for the structure-function properties of the mature erythrocyte must be efficiently retained in the reticulocyte plasma membrane. Disturbances in protein distribution during enucleation are envisaged to occur during human diseases such as Hereditary Spherocytosis. This article will discuss the current knowledge of erythroblast enucleation in the context of retention and loss of proteins that display antigenic blood group sites and that exist within multiprotein complexes within the erythrocyte membrane.

  5. CARMIL is a potent capping protein antagonist: identification of a conserved CARMIL domain that inhibits the activity of capping protein and uncaps capped actin filaments.

    PubMed

    Uruno, Takehito; Remmert, Kirsten; Hammer, John A

    2006-04-14

    Acanthamoeba CARMIL was previously shown to co-purify with capping protein (CP) and to bind pure CP. Here we show that this interaction inhibits the barbed end-capping activity of CP. Even more strikingly, this interaction drives the uncapping of actin filaments previously capped with CP. These activities are CP-specific; CARMIL does not inhibit the capping activities of either gelsolin or CapG and does not uncap gelsolin-capped filaments. Although full-length (FL) CARMIL (residues 1-1121) possesses both anti-CP activities, C-terminal fragments like glutathione S-transferase (GST)-P (940-1121) that contain the CARMIL CP binding site are at least 10 times more active. We localized the full activities of GST-P to its C-terminal 51 residues (1071-1121). This sequence contains a stretch of 25 residues that is highly conserved in CARMIL proteins from protozoa, flies, worms, and vertebrates (CARMIL Homology domain 3; CAH3). Point mutations showed that the majority of the most highly conserved residues within CAH3 are critical for the anti-CP activity of GST-AP (862-1121). Finally, we found that GST-AP binds CP approximately 20-fold more tightly than does FL-CARMIL. This observation together with the elevated activities of C-terminal fragments relative to FL-CARMIL suggests that FL-CARMIL might exist primarily in an autoinhibited state. Consistent with this idea, proteolytic cleavage of FL-CARMIL with thrombin generated an approximately 14-kDa C-terminal fragment that expresses full anti-CP activities. We propose that, after some type of physiological activation event, FL-CARMIL could function in vivo as a potent CP antagonist. Given the pivotal role that CP plays in determining the global actin phenotype of cells, our results suggest that CARMIL may play an important role in the physiological regulation of actin assembly.

  6. Binding of fusion protein FLSC IgG1 to CCR5 is enhanced by CCR5 antagonist Maraviroc.

    PubMed

    Latinovic, Olga; Schneider, Kate; Szmacinski, Henryk; Lakowicz, Joseph R; Heredia, Alonso; Redfield, Robert R

    2014-12-01

    The CCR5 chemokine receptor is crucial for human immunodeficiency virus type 1 (HIV-1) infection, acting as the principal coreceptor for HIV-1 entry and transmission and is thus an attractive target for antiviral therapy. Studies have suggested that CCR5 surface density and its conformational changes subsequent to virion engagement are rate limiting for entry, and consequently, infection. Not all CCR5 antibodies inhibit HIV-1 infection, suggesting a need for more potent reagents. Here we evaluated full length single chain (FLSC) IgG1, a novel IgG-CD4-gp120(BAL) fusion protein with several characteristics that make it an attractive candidate for treatment of HIV-1 infections, including bivalency and a potentially increased serum half-life over FLSC, the parental molecule. FLSC IgG1 binds two domains on CCR5, the N-terminus and the second extracellular loop, lowering the levels of available CCR5 viral attachment sites. Furthermore, FLSC IgG1 synergizes with Maraviroc (MVC), the only licensed CCR5 antagonist. In this study, we used both microscopy and functional assays to address the mechanistic aspects of the interactions of FLSC IgG1 and MVC in the context of CCR5 conformational changes and viral infection. We used a novel stochastic optical reconstruction microscopy (STORM), based on high resolution localization of photoswitchable dyes to visualize direct contacts between FLSC IgG1 and CCR5. We compared viral entry inhibition by FLSC IgG1 with that of other CCR5 blockers and showed FLSC IgG1 to be the most potent. We also showed that lower CCR5 surface densities in HIV-1 infected primary cells result in lower FLSC IgG1 EC50 values. In addition, CCR5 binding by FLSC IgG1, but not CCR5 Ab 2D7, was significantly increased when cells were treated with MVC, suggesting MVC allosterically increases exposure of the FLSC IgG1 binding site. These data have implications for future antiviral therapy development.

  7. Interaction of Polycomb-group proteins controlling flowering in Arabidopsis.

    PubMed

    Chanvivattana, Yindee; Bishopp, Anthony; Schubert, Daniel; Stock, Christine; Moon, Yong-Hwan; Sung, Z Renee; Goodrich, Justin

    2004-11-01

    In Arabidopsis, the EMBYRONIC FLOWER2 (EMF2), VERNALISATION2 (VRN2) and FERTILISATION INDEPENDENT ENDOSPERM2 (FIS2) genes encode related Polycomb-group (Pc-G) proteins. Their homologues in animals act together with other Pc-G proteins as part of a multimeric complex, Polycomb Repressive Complex 2 (PRC2), which functions as a histone methyltransferase. Despite similarities between the fis2 mutant phenotype and those of some other plant Pc-G members, it has remained unclear how the FIS2/EMF2/VRN2 class Pc-G genes interact with the others. We have identified a weak emf2 allele that reveals a novel phenotype with striking similarity to that of severe mutations in another Pc-G gene, CURLY LEAF (CLF), suggesting that the two genes may act in a common pathway. Consistent with this, we demonstrate that EMF2 and CLF interact genetically and that this reflects interaction of their protein products through two conserved motifs, the VEFS domain and the C5 domain. We show that the full function of CLF is masked by partial redundancy with a closely related gene, SWINGER (SWN), so that null clf mutants have a much less severe phenotype than emf2 mutants. Analysis in yeast further indicates a potential for the CLF and SWN proteins to interact with the other VEFS domain proteins VRN2 and FIS2. The functions of individual Pc-G members may therefore be broader than single mutant phenotypes reveal. We suggest that plants have Pc-G protein complexes similar to the Polycomb Repressive Complex2 (PRC2) of animals, but the duplication and subsequent diversification of components has given rise to different complexes with partially discrete functions.

  8. The Antagonistic Effect of Selenium on Lead-Induced Inflammatory Factors and Heat Shock Protein mRNA Level in Chicken Cartilage Tissue.

    PubMed

    Zheng, Shufang; Song, Huanyu; Gao, Han; Liu, Chunpeng; Zhang, Ziwei; Fu, Jing

    2016-09-01

    Selenium (Se) is recognized as a necessary trace mineral in animal diets, including those of birds. Lead (Pb) is a toxic heavy metal and can damage organs in humans and animals. Complex antagonistic interactions between Se and heavy metals have been reported in previous studies. However, little is known regarding the effects of Se on Pb-induced toxicity and the expression of inflammatory factors and heat shock proteins (HSPs) in the cartilage of chickens. In this present study, we fed chickens either with Se or Pb or both Se and Pb supplement and later analyzed the mRNA expressions of inflammatory factors (inducible nitric oxide synthase (iNOS), nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2)) and HSPs (Hsp27, Hsp40, Hsp60, Hsp70, and Hsp90). The results showed that Se and Pb influenced the expression of inflammatory factors and HSP genes in the chicken cartilage tissues. Additionally, we also found that antagonistic interaction existed between Se and Pb supplementation. Our findings suggested that Se could exert a antagonistic effect on Pb in chicken cartilage tissues.

  9. Classification epitopes in groups based on their protein family

    PubMed Central

    2015-01-01

    Background The humoral immune system response is based on the interaction between antibodies and antigens for the clearance of pathogens and foreign molecules. The interaction between these proteins occurs at specific positions known as antigenic determinants or B-cell epitopes. The experimental identification of epitopes is costly and time consuming. Therefore the use of in silico methods, to help discover new epitopes, is an appealing alternative due the importance of biomedical applications such as vaccine design, disease diagnostic, anti-venoms and immune-therapeutics. However, the performance of predictions is not optimal been around 70% of accuracy. Further research could increase our understanding of the biochemical and structural properties that characterize a B-cell epitope. Results We investigated the possibility of linear epitopes from the same protein family to share common properties. This hypothesis led us to analyze physico-chemical (PCP) and predicted secondary structure (PSS) features of a curated dataset of epitope sequences available in the literature belonging to two different groups of antigens (metalloproteinases and neurotoxins). We discovered statistically significant parameters with data mining techniques which allow us to distinguish neurotoxin from metalloproteinase and these two from random sequences. After a five cross fold validation we found that PCP based models obtained area under the curve values (AUC) and accuracy above 0.9 for regression, decision tree and support vector machine. Conclusions We demonstrated that antigen's family can be inferred from properties within a single group of linear epitopes (metalloproteinases or neurotoxins). Also we discovered the characteristics that represent these two epitope groups including their similarities and differences with random peptides and their respective amino acid sequence. These findings open new perspectives to improve epitope prediction by considering the specific antigen

  10. (−)-Stepholidine is a potent pan-dopamine receptor antagonist of both G protein- and β-arrestin-mediated signaling

    PubMed Central

    Meade, Julie A.; Free, R. Benjamin; Miller, Nicole R.; Chun, Lani S.; Doyle, Trevor B.; Moritz, Amy E.; Conroy, Jennie L.; Watts, Val J.

    2017-01-01

    Rationale (−)-Stepholidine is a tetrahydroberberine alkaloid that is known to interact with dopamine receptors and has also been proposed as a novel antipsychotic agent. Its suggested novelty lies in the fact that it has been proposed to have D1-like receptor agonist and D2-like receptor antagonist properties. Thus, it might be effective in treating both positive and negative (cognition) symptoms of schizophrenia. However, its activity on specific dopamine receptor subtypes has not been clarified, especially with respect to its ability to activate D1-like receptors. Objectives We wished to examine the affinity and functional activity of (−)-stepholidine at each of the human dopamine receptor subtypes expressed in a defined cellular environment. Methods D1–D5 dopamine receptors were stably expressed in cell lines and their interactions with (−)-stepholidine were examined using radioligand binding and various functional signaling assays. Radioligand binding assays were also performed using bovine striatal membranes. Results (−)-Stepholidine exhibited high (nM) affinity for D1 and D5 receptors, somewhat lower (two- to four-fold) affinity for D2 and D3 receptors, and low micromolar affinity for D4 receptors. Functionally, (−)-stepholidine was ineffective in activating G protein-mediated signaling of D1-like and D2 receptors and was also ineffective in stimulating β-arrestin recruitment to any dopamine receptor subtype. It did, however, antagonize all of these responses. It also antagonized D1–D2 heteromer-mediated Ca2+ mobilization. Radioligand binding assays of D1-like receptors in brain membranes also indicated that (−)-stepholidine binds to the D1 receptor with antagonist-like properties. Conclusions (−)-Stepholidine is a pan-dopamine receptor antagonist and its in vivo effects are largely mediated through dopamine receptor blockade with potential cross-talk to other receptors or signaling proteins. PMID:25231919

  11. Protective Immunogenicity of Group A Streptococcal M-Related Proteins

    PubMed Central

    Niedermeyer, Shannon E.; Agbaosi, Tina; Hysmith, Nicholas D.; Penfound, Thomas A.; Hohn, Claudia M.; Pullen, Matthew; Bright, Michael I.; Murrell, Daniel S.; Shenep, Lori E.; Courtney, Harry S.

    2015-01-01

    Many previous studies have focused on the surface M proteins of group A streptococci (GAS) as virulence determinants and protective antigens. However, the majority of GAS isolates express M-related protein (Mrp) in addition to M protein, and both have been shown to be required for optimal virulence. In the current study, we evaluated the protective immunogenicity of Mrp to determine its potential as a vaccine component that may broaden the coverage of M protein-based vaccines. Sequence analyses of 33 mrp genes indicated that there are three families of structurally related Mrps (MrpI, MrpII, and MrpIII). N-terminal peptides of Mrps were cloned, expressed, and purified from M type 2 (M2) (MrpI), M4 (MrpII), and M49 (MrpIII) GAS. Rabbit antisera against the Mrps reacted at high titers with the homologous Mrp, as determined by enzyme-linked immunosorbent assay, and promoted bactericidal activity against GAS emm types expressing Mrps within the same family. Mice passively immunized with rabbit antisera against MrpII were protected against challenge infections with M28 GAS. Assays for Mrp antibodies in serum samples from 281 pediatric subjects aged 2 to 16 indicated that the Mrp immune response correlated with increasing age of the subjects. Affinity-purified human Mrp antibodies promoted bactericidal activity against a number of GAS representing different emm types that expressed an Mrp within the same family but showed no activity against emm types expressing an Mrp from a different family. Our results indicate that Mrps have semiconserved N-terminal sequences that contain bactericidal epitopes which are immunogenic in humans. These findings may have direct implications for the development of GAS vaccines. PMID:25630406

  12. Increased Uterine NK cell numbers and perforin expression during the implantation phase in IVF Cycles with GnRH Antagonist Protocol

    PubMed Central

    Xu, Bufang; Wang, Jingwen; Xia, Lan; Zhang, Dan; Wu, Xian; Zhang, Aijun

    2017-01-01

    GnRH antagonist negatively affects endometrial receptivity in in vitro fertilization (IVF) cycles, however, its underlying mechanism remains unclear. To explore its target molecules, we studied endometria in the window phase of fixed GnRH antagonist, low-dose flexible GnRH antagonist, GnRH agonist long protocol, and untreated control groups. There were 384 differentially expressed genes (DEGs) in the fixed antagonist group with greater than twofold expression change compared with the control group and 197 DEGs between the fixed antagonist and agonist groups, the majority of which were associated with the natural killer (NK) cell-mediated cytotoxicity pathway. We then analysed the PRF1 and FASLG protein levels. The perforin level were significantly higher in both the antagonist groups than in other two groups, and was higher in the fixed antagonist group. Similarly, the uNK cell numbers were higher in the antagonist groups, and the highest uNK cell number occurred in the fixed group (p < 0.05). No significant differences existed in the Fas ligand levels and apoptosis rates among the three treatment groups, but were higher in the treatment groups than the control group. Together, these data indicate that GnRH antagonist may increase the uNK cell numbers and perforin expression, and this effect may be dose-dependent. PMID:28045093

  13. Tamoxifen resistant breast cancer: coregulators determine the direction of transcription by antagonist-occupied steroid receptors.

    PubMed

    Takimoto, G S; Graham, J D; Jackson, T A; Tung, L; Powell, R L; Horwitz, L D; Horwitz, K B

    1999-01-01

    Pharmacological antagonists of steroid receptor action had been thought to exert their effects by a passive mechanism driven principally by the ability of the antagonist to compete with agonist for the ligand binding site. However, recent analyses of antagonist-occupied receptor function suggest a more complex picture. Antagonists can be subdivided into two groups, type I, or pure antagonists, and type II, or mixed antagonists that can have variable transcriptional activity based upon differential dimerization and DNA binding properties. This led us to propose that receptor antagonism may not simply be a passive competition for the ligand binding site, but may, in some cases, involve active recruitment of corepressor or coactivator proteins to produce a mixed transcriptional phenotype. We used a yeast two-hybrid screen to identify proteins that interact specifically with antagonist-occupied receptors. Two proteins have been characterized: L7/SPA, a ribosome-associated protein that is localized in both the cytoplasm and nucleus, but with no known extranucleolar nuclear function; and hN-CoR, the human homolog of the mouse thyroid receptor corepressor mN-CoR. In in vivo transcription assays we show that L7/SPA enhances the partial agonist activity of type II mixed antagonists, and that N-CoR and the related corepressor, SMRT, suppresses it. The coregulators do not affect agonists or pure antagonists. Moreover, the net agonist activity seen with mixed antagonists is a function of the ratio of coactivator to corepressor. Based upon these results, we proposed that in breast tumors the inappropriate agonist activity seen with therapeutic antagonists such as tamoxifen is responsible for the hormone-resistant state. To confirm this, we are quantitating coactivator/corepressor ratios in breast tumor cells lines and clinical breast cancers. Results should provide new insights into the mechanisms underlying the progression of breast cancer to hormone resistance, and may

  14. Post-blast treatment with Nociceptin/Orphanin FQ peptide (NOP) receptor antagonist reduces brain injury-induced hypoxia and signaling proteins in vestibulomotor-related brain regions.

    PubMed

    Awwad, Hibah O; Durand, Cindy D; Gonzalez, Larry P; Tompkins, Paul; Zhang, Yong; Lerner, Megan R; Brackett, Daniel J; Sherry, David M; Awasthi, Vibhudutta; Standifer, Kelly M

    2016-10-25

    Mild traumatic brain injury (mTBI) diagnoses have increased due to aggressive sports and blast-related injuries, but the cellular mechanisms and pathology underlying mTBI are not completely understood. Previous reports indicate that Nociceptin Orphanin/FQ (N/OFQ), an endogenous neuropeptide, contributes to post-injury ischemia following mechanical brain injury, yet its specific role in cerebral hypoxia, vestibulomotor function and injury marker expression following blast-induced TBI is not known. This study is the first to identify a direct association of N/OFQ and its N/OFQ peptide (NOP) receptor with TBI-induced changes following a single 80psi head blast exposure in male rats. N/OFQ and NOP receptor expression increased in brain tissue and plasma following TBI, concurrent with vestibular dysfunction but preceding hypoxia and appearance of injury markers compared to sham rats. A single post-blast treatment with the NOP receptor antagonist, SB-612111, transiently improved acute vestibulomotor performance. It also prevented increases in markers of TBI-induced hypoxia, pro-apoptotic proteins and injury seen 8-10days post-blast. This study reveals an apparent role for the N/OFQ-NOP receptor system in blast TBI and suggests potential therapeutic utility of NOP receptor antagonists for mTBI.

  15. Antigenic and immunogenic analysis of group A and group B respiratory syncytial virus G proteins expressed from recombinant baculoviruses.

    PubMed

    Sullender, W M; Britt, W J

    1996-04-01

    The attachment glycoprotein G plays a major role in the antigenic variability of respiratory syncytial (RS) virus. We have expressed from recombinant baculoviruses antigenic group A and group B RS virus G proteins (designated bacAG for the group A and bacBG for the group B virus G protein). The insect cell-produced G proteins migrated more rapidly in SDS-PAGE as compared to HEp-2 cell derived G proteins owing to glycosylation differences. Antigenicity was tested by immunofluorescence; five or five group cross-reactive, five or six group A-specific, and six of six group B-specific MAbs reacted appropriately with bacAG and/or bacBG. In addition, bacAG and bacBG reacted with human polyclonal antibodies to RS virus. Cotton rats were immunized with bacAG, bacBG or a control lysate and challenged intranasally with a group A RS virus. The bacAG-immunized group had a statistically significant reduction in viral replication in the lungs (lung titres as mean log10 p.f.u./g +/- SD, bacAG = 3.1 +/- 1.2; control = 4.8 +/- 0.6, P = 0.013). The bacBG-immunized group showed less reduction in viral titres (bacBG lung titres = 4.1 +/- 0.6, P = 0.13 for bacBG compared to control). Thus, as expected, homologous protein (bacAG) immunization provided more protection against viral replication than immunization with the heterologous protein (bacBG). The G protein of RS virus expressed in insect cells had antigenic and immunogenic features which were similar to that of the G protein expressed in mammalian cells. The baculovirus-expressed G proteins should be useful for the study of immune responses to RS viruses.

  16. Locating overlapping dense subgraphs in gene (protein) association networks and predicting novel protein functional groups among these subgraphs

    NASA Astrophysics Data System (ADS)

    Palla, Gergely; Derenyi, Imre; Farkas, Illes J.; Vicsek, Tamas

    2006-03-01

    Most tasks in a cell are performed not by individual proteins, but by functional groups of proteins (either physically interacting with each other or associated in other ways). In gene (protein) association networks these groups show up as sets of densely connected nodes. In the yeast, Saccharomyces cerevisiae, known physically interacting groups of proteins (called protein complexes) strongly overlap: the total number of proteins contained by these complexes by far underestimates the sum of their sizes (2750 vs. 8932). Thus, most functional groups of proteins, both physically interacting and other, are likely to share many of their members with other groups. However, current algorithms searching for dense groups of nodes in networks usually exclude overlaps. With the aim to discover both novel functions of individual proteins and novel protein functional groups we combine in protein association networks (i) a search for overlapping dense subgraphs based on the Clique Percolation Method (CPM) (Palla, G., et.al. Nature 435, 814-818 (2005), http://angel.elte.hu/clustering), which explicitly allows for overlaps among the groups, and (ii) a verification and characterization of the identified groups of nodes (proteins) with the help of standard annotation databases listing known functions.

  17. The down-regulation of IL-6-stimulated fibrinogen steady state mRNA and protein levels by human recombinant IL-1 is not PGE2-dependent: effects of IL-1 receptor antagonist (IL-1RA).

    PubMed

    Conti, P; Bartle, L; Barbacane, R C; Reale, M; Sipe, J D

    1995-01-26

    Infections, trauma and inflammatory processes induce a host response with increases in a large group of structurally and functionally diverse plasma proteins. Parental administration of foreign proteins also induce an increase in plasma fibrinogen. Interleukin-6 (IL-6) is a monocyte-derived mediator and has regulatory effects on acute phase protein genes which result in the induction of fibrinogen synthesis in primary hepatocytes, while the addition of interleukin-1 (IL-1) exerts a negative modulating influence on the IL-6-stimulated fibrinogen. In order to understand the mechanisms by which IL-1 inhibits IL-6-stimulated fibrinogen transcription and translation, and since IL-1 is believed to act through PGE2 stimulation, we have studied the influence of PGE2 in IL-6 or IL-1, alone and in combination, on Fg mRNA expression (by Northern blot analysis) and the influence of PGE2, indomethacin, and arachidonic acid on Fg secretion. Moreover, since human recombinant interleukin-1 receptor antagonist (hrIL-1ra) is a strong inhibitor of IL-1 induced IL-1 transcription and translation and has an inhibitory effect on PGE2, we have studied the effects of IL-1ra on the down-regulation of IL-6 stimulated fibrinogen by IL-1, using an Fg ELISA method.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. An efficient synthesis of a rationally designed 1,5 disubstituted imidazole AT1 Angiotensin II receptor antagonist: reorientation of imidazole pharmacophore groups in losartan reserves high receptor affinity and confirms docking studies

    NASA Astrophysics Data System (ADS)

    Agelis, George; Roumelioti, Panagiota; Resvani, Amalia; Durdagi, Serdar; Androutsou, Maria-Eleni; Kelaidonis, Konstantinos; Vlahakos, Demetrios; Mavromoustakos, Thomas; Matsoukas, John

    2010-09-01

    A new 1,5 disubstituted imidazole AT1 Angiotensin II (AII) receptor antagonist related to losartan with reversion of butyl and hydroxymethyl groups at the 2-, 5-positions of the imidazole ring was synthesized and evaluated for its antagonist activity ( V8). In vitro results indicated that the reorientation of butyl and hydroxymethyl groups on the imidazole template of losartan retained high binding affinity to the AT1 receptor concluding that the spacing of the substituents at the 2,5- positions is of primary importance. The docking studies are confirmed by binding assay results which clearly show a comparable binding score of the designed compound V8 with that of the prototype losartan. An efficient, regioselective and cost effective synthesis renders the new compound as an attractive candidate for advanced toxicological evaluation and a drug against hypertension.

  19. GnRH antagonists may affect endometrial receptivity

    PubMed Central

    Rackow, Beth W.; Kliman, Harvey J.; Taylor, Hugh S.

    2009-01-01

    Study objective HOXA10 is an essential regulator of endometrial receptivity. To determine the effect of gonadotropin releasing hormone (GnRH) antagonists on endometrial receptivity we assessed endometrial HOXA10 expression in GnRH antagonist, GnRH agonist, and natural cycles. Design Prospective case-control study Setting University academic medical center Patients Nineteen subjects were included: 12 subjects underwent controlled ovarian hyperstimulation (COH) with recombinant follicle stimulating hormone (rFSH) and used either a GnRH antagonist or a GnRH agonist; 7 control subjects underwent natural cycles. Interventions Pipelle endometrial biopsies were obtained 11 days after human chorionic gonadotropin (hCG) administration or spontaneous luteinizing hormone (LH) surge in untreated cycles, respectively. Immunohistochemistry was used to assess HOXA10 protein expression in endometrial glands and stroma. Main outcome measure(s) Endometrial HOXA10 protein expression Results HOXA10 expression was significantly decreased in endometrial stromal cells in GnRH antagonist treated cycles compared with GnRH agonist treated cycles or natural cycle controls. There was no significant difference in glandular cell HOXA10 expression among the three groups. Conclusions Use of GnRH antagonists may be associated with impaired HOXA10 expression in endometrial stromal cells, and thus may affect endometrial receptivity. PMID:18410932

  20. Generation of Mutant Uukuniemi Viruses Lacking the Nonstructural Protein NSs by Reverse Genetics Indicates that NSs Is a Weak Interferon Antagonist

    PubMed Central

    Rezelj, Veronica V.; Överby, Anna K.

    2015-01-01

    ABSTRACT Uukuniemi virus (UUKV) is a tick-borne member of the Phlebovirus genus (family Bunyaviridae) and has been widely used as a safe laboratory model to study aspects of bunyavirus replication. Recently, a number of new tick-borne phleboviruses have been discovered, some of which, like severe fever with thrombocytopenia syndrome virus and Heartland virus, are highly pathogenic in humans. UUKV could now serve as a useful comparator to understand the molecular basis for the different pathogenicities of these related viruses. We established a reverse-genetics system to recover UUKV entirely from cDNA clones. We generated two recombinant viruses, one in which the nonstructural protein NSs open reading frame was deleted from the S segment and one in which the NSs gene was replaced with green fluorescent protein (GFP), allowing convenient visualization of viral infection. We show that the UUKV NSs protein acts as a weak interferon antagonist in human cells but that it is unable to completely counteract the interferon response, which could serve as an explanation for its inability to cause disease in humans. IMPORTANCE Uukuniemi virus (UUKV) is a tick-borne phlebovirus that is apathogenic for humans and has been used as a convenient model to investigate aspects of phlebovirus replication. Recently, new tick-borne phleboviruses have emerged, such as severe fever with thrombocytopenia syndrome virus in China and Heartland virus in the United States, that are highly pathogenic, and UUKV will now serve as a comparator to aid in the understanding of the molecular basis for the virulence of these new viruses. To help such investigations, we have developed a reverse-genetics system for UUKV that permits manipulation of the viral genome. We generated viruses lacking the nonstructural protein NSs and show that UUKV NSs is a weak interferon antagonist. In addition, we created a virus that expresses GFP and thus allows convenient monitoring of virus replication. These new tools

  1. Discovery of pyrazine carboxamide CB1 antagonists: the introduction of a hydroxyl group improves the pharmaceutical properties and in vivo efficacy of the series.

    PubMed

    Ellsworth, Bruce A; Wang, Ying; Zhu, Yeheng; Pendri, Annapurna; Gerritz, Samuel W; Sun, Chongqing; Carlson, Kenneth E; Kang, Liya; Baska, Rose A; Yang, Yifan; Huang, Qi; Burford, Neil T; Cullen, Mary Jane; Johnghar, Susan; Behnia, Kamelia; Pelleymounter, Mary Ann; Washburn, William N; Ewing, William R

    2007-07-15

    Structure-activity relationships for a series of pyrazine carboxamide CB1 antagonists are reported. Pharmaceutical properties of the series are improved via inclusion of hydroxyl-containing sidechains. This structural modification sufficiently improved ADME properties of an orally inactive series such that food intake reduction was achieved in rat feeding models. Compound 35 elicits a 46% reduction in food intake in ad libidum fed rats 4-h post-dose.

  2. The binding of the bone morphogenetic protein antagonist gremlin to kidney heparan sulfate: Such binding is not essential for BMP antagonism.

    PubMed

    Tatsinkam, Arnold Junior; Rune, Naomi; Smith, Joy; Norman, Jill T; Mulloy, Barbara; Rider, Christopher C

    2017-02-01

    Gremlin-1, a bone morphogenetic protein (BMP) antagonist, has essential roles in kidney and limb bone development, and is important in chronic diseases including tissue fibrosis. It also functions as an activating ligand of the vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2), and binds strongly to the sulfated polysaccharide, heparin. Here we investigated the extent to which gremlin binds to the related polysaccharide heparan sulfate (HS), which unlike heparin is widely distributed spread within tissues. We determined that both highly sulfated HS and kidney HS are able to partially compete for the binding of heparin to gremlin, whereas low sulfated HS is a poor competitor. In further investigations of the interaction between gremlin and HS, we found that wild-type gremlin is able to bind broadly across the various regions of kidney in an HS-dependent manner, with particularly intense binding to tubular structures in the renal cortex. In a model of chronic kidney disease, fibrotic changes in the kidney result in a loss of gremlin binding sites. Gremlin mutants with reduced affinity for heparin showed negligible binding under the same conditions. These mutants nonetheless remain functional as BMP antagonists on C2C12 myoblastic cells transfected with a Smad 1 reporter gene construct. Overall our findings indicate that on secretion, gremlin will bind to HS structures on the cell surface and in the extracellular matrix, thus providing for a localised reservoir which can modulate BMP activity in a temporospatially restricted manner. Although binding of heparin/HS to gremlin has been shown elsewhere to be necessary for gremlin activation of VEGFR2, this does not appear to be essential for BMP antagonism by gremlin. Thus these sulfated polysaccharides differentially regulate the activities of gremlin.

  3. A CRM domain protein functions dually in group I and group II intron splicing in land plant chloroplasts.

    PubMed

    Asakura, Yukari; Barkan, Alice

    2007-12-01

    The CRM domain is a recently recognized RNA binding domain found in three group II intron splicing factors in chloroplasts, in a bacterial protein that associates with ribosome precursors, and in a family of uncharacterized proteins in plants. To elucidate the functional repertoire of proteins with CRM domains, we studied CFM2 (for CRM Family Member 2), which harbors four CRM domains. RNA coimmunoprecipitation assays showed that CFM2 in maize (Zea mays) chloroplasts is associated with the group I intron in pre-trnL-UAA and group II introns in the ndhA and ycf3 pre-mRNAs. T-DNA insertions in the Arabidopsis thaliana ortholog condition a defective-seed phenotype (strong allele) or chlorophyll-deficient seedlings with impaired splicing of the trnL group I intron and the ndhA, ycf3-int1, and clpP-int2 group II introns (weak alleles). CFM2 and two previously described CRM proteins are bound simultaneously to the ndhA and ycf3-int1 introns and act in a nonredundant fashion to promote their splicing. With these findings, CRM domain proteins are implicated in the activities of three classes of catalytic RNA: group I introns, group II introns, and 23S rRNA.

  4. Agonistic and antagonistic effects of C5a-chimera bearing S19 ribosomal protein tail portion on the C5a receptor of monocytes and neutrophils, respectively.

    PubMed

    Oda, Yuuichiro; Tokita, Kazutaka; Ota, Yoshihiko; Li, Ying; Taniguchi, Keisuke; Nishino, Norikazu; Takagi, Katsumasa; Yamamoto, Tetsuro; Nishiura, Hiroshi

    2008-09-01

    C-terminus of S19 ribosomal protein (RP S19) endows the cross-linked homodimer with a dual effect on the C5a receptor in leucocyte chemoattraction; agonistic effect on the monocyte receptor, and antagonistic effect on the neutrophil receptor. C5a exhibits the uniform agonistic effect on this receptor of both cell types. We have currently prepared a recombinant C5a-chimeric protein bearing the C-terminus of RP S19 (C5a/RP S19 chimera) to be used as a substitute of the RP S19 dimer. In vitro, this chimera similarly inhibited the intracellular Ca(2+) mobilization of neutrophils induced by C5a to the RP S19 dimer did. In the guinea pig skin, 10(-7) M C5a/RP S19 chimera exhibited an inhibitory capacity to the neutrophil infiltration induced by 3 x 10(-7) M C5a without enhancing monocyte infiltration. In reverse passive Arthus reaction, the neutrophil infiltration associated with plasma extravasation was significantly reduced by the simultaneous administration of 10(-7) M C5a/RP S19 chimera with antibodies. The C5a/RP S19 chimera is a useful tool not only to examine the molecular mechanism that underlies the functional difference of the C5a receptor between monocytes and neutrophils, but also to prevent C5a-mediated hyper-response of neutrophils in acute inflammation.

  5. Inhibitor of apoptosis proteins (IAPs) and their antagonists regulate spontaneous and tumor necrosis factor (TNF)-induced proinflammatory cytokine and chemokine production.

    PubMed

    Kearney, Conor J; Sheridan, Clare; Cullen, Sean P; Tynan, Graham A; Logue, Susan E; Afonina, Inna S; Vucic, Domagoj; Lavelle, Ed C; Martin, Seamus J

    2013-02-15

    Inhibitor of apoptosis proteins (IAPs) play a major role in determining whether cells undergo apoptosis in response to TNF as well as other stimuli. However, TNF is also highly proinflammatory through its ability to trigger the secretion of multiple inflammatory cytokines and chemokines, which is arguably the most important role of TNF in vivo. Indeed, deregulated production of TNF-induced cytokines is a major driver of inflammation in several autoimmune conditions such as rheumatoid arthritis. Here, we show that IAPs are required for the production of multiple TNF-induced proinflammatory mediators. Ablation or antagonism of IAPs potently suppressed TNF- or RIPK1-induced proinflammatory cytokine and chemokine production. Surprisingly, IAP antagonism also led to spontaneous production of chemokines, particularly RANTES, in vitro and in vivo. Thus, IAPs play a major role in influencing the production of multiple inflammatory mediators, arguing that these proteins are important regulators of inflammation in addition to apoptosis. Furthermore, small molecule IAP antagonists can modulate spontaneous as well as TNF-induced inflammatory responses, which may have implications for use of these agents in therapeutic settings.

  6. High Mobility Group A proteins in esophageal carcinomas.

    PubMed

    Palumbo Júnior, Antonio; Da Costa, Nathalia Meireles; Esposito, Francesco; Fusco, Alfredo; Pinto, Luis Felipe Ribeiro

    2016-09-16

    We have recently shown that HMGA2 is overexpressed in esophageal squamous cell carcinoma (ESCC) and its detection allows to discriminate between cancer and normal surrounding tissue proposing HMGA2 as a novel diagnostic marker. Interestingly, esophageal adenocarcinoma shows an opposite behavior with the overexpression of HMGA1 but not HMGA2. Moreover, we show that the suppression of HMGA2 in 2 ESCC cell lines reduces the malignant phenotype. Then, this paper highlights a differential induction of the HMGA proteins, depending on the cancer histological type, and reinforces the perspective of an innovative esophageal cancer therapy based on the suppression of the HMGA protein function and/or expression.

  7. Differential Control of BST2 Restriction and Plasmacytoid Dendritic Cell Antiviral Response by Antagonists Encoded by HIV-1 Group M and O Strains.

    PubMed

    Bego, Mariana G; Cong, Lijun; Mack, Katharina; Kirchhoff, Frank; Cohen, Éric A

    2016-11-15

    BST2/tetherin is a type I interferon (IFN-I)-stimulated host factor that restricts the release of HIV-1 by entrapping budding virions at the cell surface. This membrane-associated protein can also engage and activate the plasmacytoid dendritic cell (pDC)-specific immunoglobulin-like transcript 7 (ILT7) inhibitory receptor to downregulate the IFN-I response by pDCs. Pandemic HIV-1 group M uses Vpu (M-Vpu) to counteract the two BST2 isoforms (long and short) that are expressed in human cells. M-Vpu efficiently downregulates surface long BST2, while it displaces short BST2 molecules away from viral assembly sites. We recently found that this attribute is used by M-Vpu to activate the BST2/ILT7-dependent negative-feedback pathway and to suppress pDC IFN-I responses during sensing of infected cells. However, whether this property is conserved in endemic HIV-1 group O, which has evolved Nef (O-Nef) to counteract specifically the long BST2 isoform, remains unknown. In the present study, we validated that O-Nefs have the capacity to downregulate surface BST2 and enhance HIV-1 particle release although less efficiently than M-Vpu. In contrast to M-Vpu, O-Nef did not efficiently enhance viral spread in T cell culture or displace short BST2 from viral assembly sites to prevent its occlusion by tethered HIV-1 particles. Consequently, O-Nef impairs the ability of BST2 to activate negative ILT7 signaling to suppress the IFN-I response by pDC-containing peripheral blood mononuclear cells (PBMCs) during sensing of infected cells. These distinctive features of BST2 counteraction by O-Nefs may in part explain the limited spread of HIV-1 group O in the human population.

  8. Role of bacterial infection in the epigenetic regulation of Wnt antagonist WIF1 by PRC2 protein EZH2

    PubMed Central

    Roy, Badal C.; Subramaniam, Dharmalingam; Ahmed, Ishfaq; Jala, Venkatakrishna R.; Hester, Christina; Greiner, K. Allen; Haribabu, Bodduluri; Anant, Shrikant; Umar, Shahid

    2014-01-01

    The Enhancer of Zeste Homolog-2 (EZH2) represses gene transcription through histone H3 lysine-27-trimethylation (H3K27me3). Citrobacter rodentium (CR) promotes crypt hyperplasia and tumorigenesis by aberrantly regulating Wnt/β-catenin signaling. We aimed at investigating EZH2’s role in epigenetically regulating Wnt/β-catenin signaling following bacterial infection. NIH:Swiss outbred and ApcMin/+ mice were infected with CR (108cfu); BLT1−/−ApcMin/+ mice, AOM/DSS-treated mice and de-identified human adenocarcinoma samples were models of colon cancer. Following infection with wild type but not mutant CR, elevated EZH2 levels in the crypt at days-6 and 12 (peak hyperplasia) coincided with increases in H3K27me3 and β-catenin levels, respectively. Chromatin immunoprecipitation revealed EZH2 and H3K27me3’s occupancy on WIF1 (Wnt Inhibitory Factor-1) promoter resulting in reduced WIF1 mRNA and protein expression. Following EZH2 knockdown via siRNA or EZH2-inhibitor DZNep either alone or in combination with HDAC inhibitor SAHA, WIF1 promoter activity increased significantly while overexpression of EZH2 attenuated WIF1-reporter activity. Ectopic overexpression of SET domain mutant (F681Y) almost completely rescued WIF1 reporter activity and partially rescued WIF1 protein levels while H3K27me3 levels were significantly attenuated suggesting that an intact methyltransferases activity is required for EZH2-dependent effects. Interestingly, while β-catenin levels were lower in EZH2-knocked-down cells, F681Y mutants exhibited only partial reduction in β-catenin levels. Besides EZH2, increases in miR-203 expression in the crypts at days-6 and 12 post-infection correlated with reduced levels of its target WIF1; overexpression of miR-203 in primary colonocytes decreased WIF1 mRNA and protein levels. Elevated levels of EZH2 and β-catenin with concomitant decrease in WIF1 expression in the polyps of CR-infected ApcMin/+ mice paralleled changes recorded in BLT1

  9. Associations of erythrocyte membrane fatty acids with the concentrations of C-reactive protein, interleukin 1 receptor antagonist and adiponectin in 1373 men.

    PubMed

    Takkunen, M J; de Mello, V D F; Schwab, U S; Ågren, J J; Kuusisto, J; Uusitupa, M I J

    2014-10-01

    Dietary and endogenous fatty acids could play a role in low-grade inflammation. In this cross-sectional study the proportions of erythrocyte membrane fatty acids (EMFA) and the concentrations of C-reactive protein (CRP), interleukin-1 receptor antagonist (IL-1Ra) and adiponectin were measured and their confounder-adjusted associations examined in 1373 randomly selected Finnish men aged 45-70 years participating in the population based Metsim study in Eastern Finland. The sum of n-6 EMFAs, without linoleic acid (LA), was positively associated with concentrations of CRP and IL-1Ra (r partial=0.139 and r partial=0.115, P<0.001). These associations were especially strong among lean men (waist circumference <94 cm; r partial=0.156 and r partial=0.189, P<0.001). Total n-3 EMFAs correlated inversely with concentrations of CRP (r partial=-0.098, P<0.001). Palmitoleic acid (16:1n-7) correlated positively with CRP (r partial=0.096, P<0.001). Cis-vaccenic acid (18:1n-7) was associated with high concentrations of adiponectin (r partial=0.139, P<0.001). In conclusion, n-6 EMFAs, except for LA, correlated positively with the inflammatory markers. Palmitoleic acid was associated with CRP, whereas, interestingly, its elongation product, cis-vaccenic acid, associated with anti-inflammatory adiponectin.

  10. The F-box-containing protein UFO and AGAMOUS participate in antagonistic pathways governing early petal development in Arabidopsis.

    PubMed

    Durfee, Tim; Roe, Judith L; Sessions, R Allen; Inouye, Carla; Serikawa, Kyle; Feldmann, Kenneth A; Weigel, Detlef; Zambryski, Patricia C

    2003-07-08

    The UNUSUAL FLORAL ORGANS (UFO) gene is required for multiple processes in the developing Arabidopsis flower, including the proper patterning and identity of both petals and stamens. The gene encodes an F-box-containing protein, UFO, which interacts physically and genetically with the Skp1 homolog, ASK1. In this report, we describe four ufo alleles characterized by the absence of petals, which uncover another role for UFO in promoting second whorl development. This UFO-dependent pathway is required regardless of the second whorl organ to be formed, arguing that it affects a basic process acting in parallel with those establishing organ identity. However, the pathway is dispensable in the absence of AGAMOUS (AG), a known inhibitor of petal development. In situ hybridization results argue that AG is not transcribed in the petal region, suggesting that it acts non-cell-autonomously to inhibit second whorl development in ufo mutants. These results are combined into a genetic model explaining early second whorl initiation/proliferation, in which UFO functions to inhibit an AG-dependent activity.

  11. The F-box-containing protein UFO and AGAMOUS participate in antagonistic pathways governing early petal development in Arabidopsis

    PubMed Central

    Durfee, Tim; Roe, Judith L.; Sessions, R. Allen; Inouye, Carla; Serikawa, Kyle; Feldmann, Kenneth A.; Weigel, Detlef; Zambryski, Patricia C.

    2003-01-01

    The UNUSUAL FLORAL ORGANS (UFO) gene is required for multiple processes in the developing Arabidopsis flower, including the proper patterning and identity of both petals and stamens. The gene encodes an F-box-containing protein, UFO, which interacts physically and genetically with the Skp1 homolog, ASK1. In this report, we describe four ufo alleles characterized by the absence of petals, which uncover another role for UFO in promoting second whorl development. This UFO-dependent pathway is required regardless of the second whorl organ to be formed, arguing that it affects a basic process acting in parallel with those establishing organ identity. However, the pathway is dispensable in the absence of AGAMOUS (AG), a known inhibitor of petal development. In situ hybridization results argue that AG is not transcribed in the petal region, suggesting that it acts non-cell-autonomously to inhibit second whorl development in ufo mutants. These results are combined into a genetic model explaining early second whorl initiation/proliferation, in which UFO functions to inhibit an AG-dependent activity. PMID:12826617

  12. Cross-interactions of two p38 mitogen-activated protein (MAP) kinase inhibitors and two cholecystokinin (CCK) receptor antagonists with the CCK1 receptor and p38 MAP kinase.

    PubMed

    Morel, Caroline; Ibarz, Géraldine; Oiry, Catherine; Carnazzi, Eric; Bergé, Gilbert; Gagne, Didier; Galleyrand, Jean-Claude; Martinez, Jean

    2005-06-03

    Although SB202190 and SB203580 are described as specific p38 MAP kinase inhibitors, several reports have indicated that other enzymes are also sensitive to SB203580. Using a pharmacological approach, we report for the first time that compounds SB202190 and SB203580 were able to directly and selectively interact with a G-protein-coupled receptor, namely the cholecystokinin receptor subtype CCK1, but not with the CCK2 receptor. We demonstrated that these compounds were non-competitive antagonists of the CCK1 receptor at concentrations typically used to inhibit protein kinases. By chimeric construction of the CCK2 receptor, we determined the involvement of two CCK1 receptor intracellular loops in the binding of SB202190 and SB203580. We also showed that two CCK antagonists, L364,718 and L365,260, were able to regulate p38 mitogen-activated protein (MAP) kinase activity. Using a reporter gene strategy and immunoblotting experiments, we demonstrated that both CCK antagonists inhibited selectively the enzymatic activity of p38 MAP kinase. Kinase assays suggested that this inhibition resulted from a direct interaction with both CCK antagonists. Molecular modeling simulations suggested that this interaction occurs in the ATP binding pocket of p38 MAP kinase. These results suggest that SB202190 and SB203580 bind to the CCK1 receptor and, as such, these compounds should be used with caution in models that express this receptor. We also found that L364,718 and L365,260, two CCK receptor antagonists, directly interacted with p38 MAP kinase and inhibited its activity. These findings suggest that the CCK1 receptor shares structural analogies with the p38 MAP kinase ATP binding site. They open the way to potential design of either a new family of MAP kinase inhibitors from CCK1 receptor ligand structures or new CCK1 receptor ligands based on p38 MAP kinase inhibitor structures.

  13. Cholinergic antagonists in a solitary wasp venom.

    PubMed

    Piek, T; Mantel, P

    1986-01-01

    The venom of the solitary wasp Philanthus triangulum contains a cholinergic antagonist of the nicotinic receptor of the rectus abdominis muscle of the frog, Xenopus laevis. The venom of African P. triangulum contains two different cholinergic factors, a competitive and a non-competitive antagonist. The venom of the European P. triangulum may not contain a competitive antagonist of the nicotinic receptor of X. laevis, but only a very strong non-competitive antagonist. The possible non-synonymity of both groups of P. triangulum is discussed.

  14. Quantitative evaluation of interaction force between functional groups in protein and polymer brush surfaces.

    PubMed

    Sakata, Sho; Inoue, Yuuki; Ishihara, Kazuhiko

    2014-03-18

    To understand interactions between polymer surfaces and different functional groups in proteins, interaction forces were quantitatively evaluated by force-versus-distance curve measurements using atomic force microscopy with a functional-group-functionalized cantilever. Various polymer brush surfaces were systematically prepared by surface-initiated atom transfer radical polymerization as well-defined model surfaces to understand protein adsorption behavior. The polymer brush layers consisted of phosphorylcholine groups (zwitterionic/hydrophilic), trimethylammonium groups (cationic/hydrophilic), sulfonate groups (anionic/hydrophilic), hydroxyl groups (nonionic/hydrophilic), and n-butyl groups (nonionic/hydrophobic) in their side chains. The interaction forces between these polymer brush surfaces and different functional groups (carboxyl groups, amino groups, and methyl groups, which are typical functional groups existing in proteins) were quantitatively evaluated by force-versus-distance curve measurements using atomic force microscopy with a functional-group-functionalized cantilever. Furthermore, the amount of adsorbed protein on the polymer brush surfaces was quantified by surface plasmon resonance using albumin with a negative net charge and lysozyme with a positive net charge under physiological conditions. The amount of proteins adsorbed on the polymer brush surfaces corresponded to the interaction forces generated between the functional groups on the cantilever and the polymer brush surfaces. The weakest interaction force and least amount of protein adsorbed were observed in the case of the polymer brush surface with phosphorylcholine groups in the side chain. On the other hand, positive and negative surfaces generated strong forces against the oppositely charged functional groups. In addition, they showed significant adsorption with albumin and lysozyme, respectively. These results indicated that the interaction force at the functional group level might be

  15. Macroevolutionary trends of atomic composition and related functional group proportion in eukaryotic and prokaryotic proteins.

    PubMed

    Zhang, Yu-Juan; Yang, Chun-Lin; Hao, You-Jin; Li, Ying; Chen, Bin; Wen, Jian-Fan

    2014-01-25

    To fully explore the trends of atomic composition during the macroevolution from prokaryote to eukaryote, five atoms (oxygen, sulfur, nitrogen, carbon, hydrogen) and related functional groups in prokaryotic and eukaryotic proteins were surveyed and compared. Genome-wide analysis showed that eukaryotic proteins have more oxygen, sulfur and nitrogen atoms than prokaryotes do. Clusters of Orthologous Groups (COG) analysis revealed that oxygen, sulfur, carbon and hydrogen frequencies are higher in eukaryotic proteins than in their prokaryotic orthologs. Furthermore, functional group analysis demonstrated that eukaryotic proteins tend to have higher proportions of sulfhydryl, hydroxyl and acylamino, but lower of sulfide and carboxyl. Taken together, an apparent trend of increase was observed for oxygen and sulfur atoms in the macroevolution; the variation of oxygen and sulfur compositions and their related functional groups in macroevolution made eukaryotic proteins carry more useful functional groups. These results will be helpful for better understanding the functional significances of atomic composition evolution.

  16. Screening for inhibitors of low-affinity epigenetic peptide-protein interactions: an AlphaScreen-based assay for antagonists of methyl-lysine binding proteins.

    PubMed

    Wigle, Tim J; Herold, J Martin; Senisterra, Guillermo A; Vedadi, Masoud; Kireev, Dmitri B; Arrowsmith, Cheryl H; Frye, Stephen V; Janzen, William P

    2010-01-01

    The histone code comprises many posttranslational modifications that occur mainly in histone tail peptides. The identity and location of these marks are read by a variety of histone-binding proteins that are emerging as important regulators of cellular differentiation and development and are increasingly being implicated in numerous disease states. The authors describe the development of the first high-throughput screening assay for the discovery of inhibitors of methyl-lysine binding proteins that will be used to initiate a full-scale discovery effort for this broad target class. They focus on the development of an AlphaScreen-based assay for malignant brain tumor (MBT) domain-containing proteins, which bind to the lower methylation states of lysine residues present in histone tail peptides. This assay takes advantage of the avidity of the AlphaScreen beads to clear the hurdle to assay development presented by the low micromolar binding constants of the histone binding proteins for their cognate peptides. The assay is applicable to other families of methyl-lysine binding proteins, and it has the potential to be used in screening efforts toward the discovery of novel small molecules with utility as research tools for cellular reprogramming and ultimately drug discovery.

  17. CSNK1A1 and Gli2 as Novel Targets Identified Through an Integrative Analysis of Gene Expression Data, Protein-Protein Interaction and Pathways Networks in Glioblastoma Tumors: Can These Two Be Antagonistic Proteins?

    PubMed

    Mishra, Seema

    2014-01-01

    Glioblastoma (GBM) is the malignant form of glioma, and the interplay of different pathways working in concert in GBM development and progression needs to be fully understood. Wnt signaling and sonic hedgehog (SHH) signaling pathways, having basic similarities, are among the major pathways aberrantly activated in GBM, and hence, need to be targeted. It becomes imperative, therefore, to explore the functioning of these pathways in context of each other in GBM. An integrative approach may help provide new biological insights, as well as solve the problem of identifying common drug targets for simultaneous targeting of these pathways. The beauty of this approach is that it can recapitulate several known facts, as well as decipher new emerging patterns, identifying those targets that could be missed when relying on one type of data at a time. This approach can be easily extended to other systems to discover key patterns in the functioning of signaling molecules. Studies were designed to assess the relationship between significant differential expression of genes of the Wnt (Wnt/β-catenin canonical and Wnt non-canonical) and SHH signaling pathways and their connectivity patterns in interaction and signaling networks. Further, the aim was to decipher underlying mechanistic patterns that may be involved in a more specific way and to generate a ranked list of genes that can be used as markers or drug targets. These studies predict that Wnt pathway plays a relatively more pro-active role than the SHH pathway in GBM. Further, CTNNB1, CSNK1A1, and Gli2 proteins may act as key drug targets common to these pathways. While CTNNB1 is a widely studied molecule in the context of GBM, the likely roles of CSNK1A1 and Gli2 are found to be relatively novel. It is surmised that Gli2 may be antagonistic to CSNK1A1, preventing the phosphorylation of CTNNB1 and SMO proteins in Wnt and SHH signaling pathway, respectively, by CSNK1A1, and thereby, aberrant activation. New insights into the

  18. CSNK1A1 and Gli2 as Novel Targets Identified Through an Integrative Analysis of Gene Expression Data, Protein-Protein Interaction and Pathways Networks in Glioblastoma Tumors: Can These Two Be Antagonistic Proteins?

    PubMed Central

    Mishra, Seema

    2014-01-01

    Glioblastoma (GBM) is the malignant form of glioma, and the interplay of different pathways working in concert in GBM development and progression needs to be fully understood. Wnt signaling and sonic hedgehog (SHH) signaling pathways, having basic similarities, are among the major pathways aberrantly activated in GBM, and hence, need to be targeted. It becomes imperative, therefore, to explore the functioning of these pathways in context of each other in GBM. An integrative approach may help provide new biological insights, as well as solve the problem of identifying common drug targets for simultaneous targeting of these pathways. The beauty of this approach is that it can recapitulate several known facts, as well as decipher new emerging patterns, identifying those targets that could be missed when relying on one type of data at a time. This approach can be easily extended to other systems to discover key patterns in the functioning of signaling molecules. Studies were designed to assess the relationship between significant differential expression of genes of the Wnt (Wnt/β-catenin canonical and Wnt non-canonical) and SHH signaling pathways and their connectivity patterns in interaction and signaling networks. Further, the aim was to decipher underlying mechanistic patterns that may be involved in a more specific way and to generate a ranked list of genes that can be used as markers or drug targets. These studies predict that Wnt pathway plays a relatively more pro-active role than the SHH pathway in GBM. Further, CTNNB1, CSNK1A1, and Gli2 proteins may act as key drug targets common to these pathways. While CTNNB1 is a widely studied molecule in the context of GBM, the likely roles of CSNK1A1 and Gli2 are found to be relatively novel. It is surmised that Gli2 may be antagonistic to CSNK1A1, preventing the phosphorylation of CTNNB1 and SMO proteins in Wnt and SHH signaling pathway, respectively, by CSNK1A1, and thereby, aberrant activation. New insights into the

  19. Programmed cell death 4 protein (Pdcd4) and homeodomain-interacting protein kinase 2 (Hipk2) antagonistically control translation of Hipk2 mRNA.

    PubMed

    Ohnheiser, Johanna; Ferlemann, Eva; Haas, Astrid; Müller, Jan P; Werwein, Eugen; Fehler, Olesja; Biyanee, Abhiruchi; Klempnauer, Karl-Heinz

    2015-07-01

    The tumor suppressor protein programmed cell death 4 (Pdcd4) is a highly conserved RNA-binding protein that inhibits the translation of specific mRNAs. Here, we have identified the homeobox-interacting protein kinase-2 (Hipk2) mRNA as a novel translational target of Pdcd4. Unlike most other protein kinases Hipk2 is constitutively active after being synthesized by the ribosome and its expression and activity are thought to be mainly controlled by modulation of the half-life of the kinase. Our work provides the first evidence that Hipk2 expression is also controlled on the level of translation. We show that Hipk2 stimulates the translation of its own mRNA and that Pdcd4 suppresses the translation of Hipk2 mRNA by interfering with this auto-regulatory feedback mechanism. We also show that the translation of the related kinase Hipk1 is controlled by a similar feedback loop and that Hipk2 also stimulates the translation of Hipk1 mRNA. Taken together, our work describes a novel mechanism of translational suppression by Pdcd4 and shows for the first time that Hipk2 controls its own synthesis by an auto-regulatory feedback mechanism. Furthermore, the effect of Hipk2 on the translation of Hipk1 RNA suggests that Hipk2 and Pdcd4 can act in similar manner to control the translation of other mRNAs.

  20. Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor

    PubMed Central

    Kudryavtsev, Denis; Bychkov, Maxim L.; Kulbatskii, Dmitrii S.; Kasheverov, Igor E.; Astapova, Maria V.; Feofanov, Alexey V.; Thomsen, Morten S.; Mikkelsen, Jens D.; Shenkarev, Zakhar O.; Tsetlin, Victor I.; Dolgikh, Dmitry A.; Kirpichnikov, Mikhail P.

    2016-01-01

    SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,—non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that

  1. Competitive labelling, a method for determining the reactivity of individual groups in proteins. The amino groups of porcine elastase

    PubMed Central

    Kaplan, H.; Stevenson, K. J.; Hartley, B. S.

    1971-01-01

    1. A method is described for determining the ionization constants and reactivities of individual amino groups in proteins. The principle is that in the presence of a trace amount of radioactive label, the various reactive groups in a protein molecule will compete for the label and the amount incorporated into any one group will be determined by its nucleophilicity, pK and micro-environment. The relative amounts of label incorporated into various groups will be proportional to their second-order rate constants and by comparing these rate constants with those expected on the basis of a linear free-energy relationship obtained with a series of standard compounds, the micro-environment can be defined for a particular amino group. 2. The method consists of treating a protein and an internal standard with a limiting amount of radioactive reagent and then with an excess of unlabelled reagent to yield a chemically homogeneous but heterogeneously labelled compound. After appropriate enzymic digestion peptides containing each labelled group are isolated and their rates of reaction, relative to the internal standard, are determined from their specific radioactivities. The entire procedure is repeated at several pH values. 3. When the method was applied to the amino groups of porcine elastase by using tritiated acetic anhydride as the labelling reagent, the N-terminus was found to have pKa 9.7 and a much lower than normal reactivity. Lysine-87 and lysine-224 were found to have pKa 10.3 and normal reactivities. At pH values greater than 10.5 there are discontinuities in all the titration curves, indicating that the entire molecule is undergoing a structural reorganization. ImagesPLATE 3PLATE 1PLATE 2 PMID:5158490

  2. An IgM-kappa rat monoclonal antibody specific for the type 1 sphingosine 1-phosphate G protein-coupled receptor with antagonist and agonist activities.

    PubMed

    Goetzl, Edward J; Dembrow, Dale; Van Brocklyn, James R; Gráler, Markus; Huang, Mei-Chuan

    2004-04-30

    Sphingosine 1-phosphate (S1P) type 1G protein-coupled receptors (S1P1 GPCRs) are specific high-affinity transducers for this lipid growth factor and cellular mediator. S1P1 GPCRs are widely-expressed and physiologically critical in the cardiovascular and immune systems. Functional rat monoclonal antibodies (MoAbs) have been generated against human S1P1 GPCRs expressed in rat null-cell transductants to provide bioavailable agents capable of stimulating or suppressing the S1P-S1P1 GPCR axis. The rat IgM-kappa anti-S1P1 GPCR MoAb designated 4B5.2 binds specifically to native human or mouse S1P1 GPCRs in cell membranes, but not to solubilized and denatured S1P1 GPCRs. Specific binding of 32P-S1P to cellular S1P1 GPCRs is not blocked by 4B5.2. T cell chemotactic responses to S1P and S1P suppression of T cell chemotaxis to chemokines both are inhibited selectively by 4B5.2. In contrast, generation of gamma-interferon by stimulated T cells is diminished by 4B5.2 as by S1P. T cell S1P1 GPCR-selective antagonist and agonist effects of 4B5.2 in vivo may alter immune responses as distinctively as the available poly-S1P GPCR-directed pharmacological agents, without the undesirable side-effects attributable to actions of these agents on other S1P GPCRs.

  3. Protein-sparing therapy after major abdominal surgery: lack of clinical effects. Protein-Sparing Therapy Study Group.

    PubMed Central

    Doglietto, G B; Gallitelli, L; Pacelli, F; Bellantone, R; Malerba, M; Sgadari, A; Crucitti, F

    1996-01-01

    OBJECTIVE: A prospective multicenter randomized trial was designed to evaluate the clinical efficacy of postoperative protein-sparing therapy. SUMMARY BACKGROUND DATA: The metabolic effect of postoperative protein-sparing therapy has been shown by several studies, but the clinical utility of this treatment has not been investigated by large prospective trials. METHODS: Six hundred seventy-eight patients undergoing major elective abdominal surgery were randomly assigned to receive either protein-sparing therapy after surgery (protein-sparing therapy group) or conventional therapy (control group). The patients were monitored for postoperative complications and mortality. RESULTS: The rate of major postoperative complications was similar in both groups (protein-sparing therapy group, 19.5%; control group, 20.9%; p=0.66) as were the overall postoperative mortality rates (4.7% and 3.5%, respectively; p=0.43). CONCLUSIONS: The present study indicates that routine protein-sparing therapy for patients normonourished or mildly malnourished undergoing major abdominal surgery is not clinically justified. PMID:8633913

  4. Successful Conversion of the Bacillus subtilis BirA Group II Biotin Protein Ligase into a Group I Ligase

    PubMed Central

    Henke, Sarah K.; Cronan, John E.

    2014-01-01

    Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that allows transcriptional regulation of biotin biosynthetic and transport genes whereas Group I BPLs lack this N-terminal domain. The Bacillus subtilis BPL, BirA, is classified as a Group II BPL based on sequence predictions of an N-terminal helix-turn-helix motif and mutational alteration of its regulatory properties. We report evidence that B. subtilis BirA is a Group II BPL that regulates transcription at three genomic sites: bioWAFDBI, yuiG and yhfUTS. Moreover, unlike the paradigm Group II BPL, E. coli BirA, the N-terminal DNA binding domain can be deleted from Bacillus subtilis BirA without adverse effects on its ligase function. This is the first example of successful conversion of a Group II BPL to a Group I BPL with retention of full ligase activity. PMID:24816803

  5. Isotope-Encoded Carboxyl Group Footprinting for Mass Spectrometry-Based Protein Conformational Studies

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; Liu, Haijun; Blankenship, Robert E.; Gross, Michael L.

    2016-01-01

    We report an isotope-encoding method coupled with carboxyl-group footprinting to monitor protein conformational changes. The carboxyl groups of aspartic/glutamic acids and of the C-terminus of proteins can serve as reporters for protein conformational changes when labeled with glycine ethyl ester (GEE) mediated by carbodiimide. In the new development, isotope-encoded "heavy" and "light" GEE are used to label separately the two states of the orange carotenoid protein (OCP) from cyanobacteria. Two samples are mixed (1:1 ratio) and analyzed by a single LC-MS/MS experiment. The differences in labeling extent between the two states are represented by the ratio of the "heavy" and "light" peptides, providing information about protein conformational changes. Combining isotope-encoded MS quantitative analysis and carboxyl-group footprinting reduces the time of MS analysis and improves the sensitivity of GEE and other footprinting.

  6. Cotranscriptional splicing of a group I intron is facilitated by the Cbp2 protein

    SciTech Connect

    Lewin, A.S.; Thomas, J. Jr.; Tirupati, H.K.

    1995-12-01

    This report investigates the coupling between transcription and splicing of a mitochondrial group I intron in Saccharomyces cerevisiae and the effect of the Cbp2 protein on splicing. 65 refs., 7 figs.

  7. Pho85p, a cyclin-dependent protein kinase, and the Snf1p protein kinase act antagonistically to control glycogen accumulation in Saccharomyces cerevisiae.

    PubMed Central

    Huang, D; Farkas, I; Roach, P J

    1996-01-01

    In Saccharomyces cerevisiae, nutrient levels control multiple cellular processes. Cells lacking the SNF1 gene cannot express glucose-repressible genes and do not accumulate the storage polysaccharide glycogen. The impaired glycogen synthesis is due to maintenance of glycogen synthase in a hyperphosphorylated, inactive state. In a screen for second site suppressors of the glycogen storage defect of snf1 cells, we identified a mutant gene that restored glycogen accumulation and which was allelic with PHO85, which encodes a member of the cyclin-dependent kinase family. In cells with disrupted PHO85 genes, we observed hyperaccumulation of glycogen, activation of glycogen synthase, and impaired glycogen synthase kinase activity. In snf1 cells, glycogen synthase kinase activity was elevated. Partial purification of glycogen synthase kinase activity from yeast extracts resulted in the separation of two fractions by phenyl-Sepharose chromatography, both of which phosphorylated and inactivated glycogen synthase. The activity of one of these, GPK2, was inhibited by olomoucine, which potently inhibits cyclin-dependent protein kinases, and contained an approximately 36-kDa species that reacted with antibodies to Pho85p. Analysis of Ser-to-Ala mutations at the three potential Gsy2p phosphorylation sites in pho85 cells implicated Ser-654 and/or Thr-667 in PHO85 control of glycogen synthase. We propose that Pho85p is a physiological glycogen synthase kinase, possibly acting downstream of Snf1p. PMID:8754836

  8. Regulation of Autophagy-Related Protein and Cell Differentiation by High Mobility Group Box 1 Protein in Adipocytes

    PubMed Central

    Feng, Huanhuan; Zhang, Guojun; Liu, Guoyan; Yang, Can

    2016-01-01

    High mobility group box 1 protein (HMGB1) is a molecule related to the development of inflammation. Autophagy is vital to maintain cellular homeostasis and protect against inflammation of adipocyte injury. Our recent work focused on the relationship of HMGB1 and autophagy in 3T3-L1 cells. In vivo experimental results showed that, compared with the normal-diet group, the high-fat diet mice displayed an increase in adipocyte size in the epididymal adipose tissues. The expression levels of HMGB1 and LC3II also increased in epididymal adipose tissues in high-fat diet group compared to the normal-diet mice. The in vitro results indicated that HMGB1 protein treatment increased LC3II formation in 3T3-L1 preadipocytes in contrast to that in the control group. Furthermore, LC3II formation was inhibited through HMGB1 knockdown by siRNA. Treatment with the HMGB1 protein enhanced LC3II expression after 2 and 4 days but decreased the expression after 8 and 10 days among various differentiation stages of adipocytes. By contrast, FABP4 expression decreased on the fourth day and increased on the eighth day. Hence, the HMGB1 protein modulated autophagy-related proteins and lipid-metabolism-related genes in adipocytes and could be a new target for treatment of obesity and related metabolic diseases. PMID:27843198

  9. Conserved patterns hidden within group A Streptococcus M protein hypervariability recognize human C4b-binding protein

    SciTech Connect

    Buffalo, Cosmo Z.; Bahn-Suh, Adrian J.; Hirakis, Sophia P.; Biswas, Tapan; Amaro, Rommie E.; Nizet, Victor; Ghosh, Partho

    2016-09-05

    No vaccine exists against group A Streptococcus (GAS), a leading cause of worldwide morbidity and mortality. A severe hurdle is the hypervariability of its major antigen, the M protein, with >200 different M types known. Neutralizing antibodies typically recognize M protein hypervariable regions (HVRs) and confer narrow protection. In stark contrast, human C4b-binding protein (C4BP), which is recruited to the GAS surface to block phagocytic killing, interacts with a remarkably large number of M protein HVRs (apparently ~90%). Such broad recognition is rare, and we discovered a unique mechanism for this through the structure determination of four sequence-diverse M proteins in complexes with C4BP. The structures revealed a uniform and tolerant ‘reading head’ in C4BP, which detected conserved sequence patterns hidden within hypervariability. Our results open up possibilities for rational therapies that target the M–C4BP interaction, and also inform a path towards vaccine design.

  10. The antitumor agent doxorubicin binds to Fanconi anemia group F protein.

    PubMed

    Kusayanagi, Tomoe; Tsukuda, Senko; Shimura, Satomi; Manita, Daisuke; Iwakiri, Kanako; Kamisuki, Shinji; Takakusagi, Yoichi; Takeuchi, Toshifumi; Kuramochi, Kouji; Nakazaki, Atsuo; Sakaguchi, Kengo; Kobayashi, Susumu; Sugawara, Fumio

    2012-11-01

    Doxorubicin, a commonly used cancer chemotherapy agent, elicits several potent biological effects, including synergistic-antitumor activity in combination with cisplatin. However, the mechanism of this synergism remains obscure. Here, we employed an improved T7 phage display screening method to identify Fanconi anemia group F protein (FANCF) as a doxorubicin-binding protein. The FANCF-doxorubicin interaction was confirmed by pull-down assay and SPR analysis. FANCF is a component of the Fanconi anemia complex, which monoubiquitinates D2 protein of Fanconi anemia group as a cellular response against DNA cross-linkers such as cisplatin. We observed that the monoubiquitination was inhibited by doxorubicin treatment.

  11. Membrane cofactor protein (CD46) is a keratinocyte receptor for the M protein of the group A streptococcus.

    PubMed Central

    Okada, N; Liszewski, M K; Atkinson, J P; Caparon, M

    1995-01-01

    The pathogenic Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) is the causative agent of numerous suppurative diseases of human skin. The M protein of S. pyogenes mediates the adherence of the bacterium to keratinocytes, the most numerous cell type in the epidermis. In this study, we have constructed and analyzed a series of mutant M proteins and have shown that the C repeat domain of the M molecule is responsible for cell recognition. The binding of factor H, a serum regulator of complement activation, to the C repeat region of M protein blocked bacterial adherence. Factor H is a member of a large family of complement regulatory proteins that share a homologous structural motif termed the short consensus repeat. Membrane cofactor protein (MCP), or CD46, is a short consensus repeat-containing protein found on the surface of keratinocytes, and purified MCP could competitively inhibit the adherence of S. pyogenes to these cells. Furthermore, the M protein was found to bind directly to MCP, whereas mutant M proteins that lacked the C repeat domain did not bind MCP, suggesting that recognition of MCP plays an important role in the ability of the streptococcus to adhere to keratinocytes. Images Fig. 1 Fig. 2 Fig. 3 PMID:7708671

  12. High-mobility group box-1 protein determination in postmortem samples.

    PubMed

    Palmiere, Cristian; Augsburger, Marc; Mangin, Patrice

    2014-06-01

    The aims of this study were to assess whether high-mobility group box-1 protein can be determined in biological fluids collected during autopsy and evaluate the diagnostic potential of high-mobility group box-1 protein in identifying sepsis-related deaths. High-mobility group box-1 protein was measured in serum collected during hospitalization as well as in undiluted and diluted postmortem serum and pericardial fluid collected during autopsy in a group of sepsis-related deaths and control cases with noninfectious causes of death. Inclusion criteria consisted of full biological sample availability and postmortem interval not exceeding 6h. The preliminary results indicate that high-mobility group box-1 protein levels markedly increase after death. Concentrations beyond the upper limit of the calibration curve were obtained in undiluted postmortem serum in septic and traumatic control cases. In pericardial fluid, concentrations beyond the upper limit of the calibration curve were found in all cases. These findings suggest that the diagnostic potential of high-mobility group box-1 protein in the postmortem setting is extremely limited due to molecule release into the bloodstream after death, rendering antemortem levels difficult or impossible to estimate even after sample dilution.

  13. Identification and Characterization of a Novel Secreted Immunoglobulin Binding Protein from Group A Streptococcus

    PubMed Central

    Fagan, Peter K.; Reinscheid, Dieter; Gottschalk, Birgit; Chhatwal, Gursharan S.

    2001-01-01

    Immunoglobulin binding proteins are one of several pathogenicity factors which have been associated with invasive disease caused by group A streptococci. The surface-bound M and M-like proteins of Streptococcus pyogenes are the most characterized of these immunoglobulin binding proteins, and in most cases they bind only a single antibody class. Here we report the identification of a novel non-M-type secreted protein, designated SibA (for secreted immunoglobulin binding protein from group A streptococcus), which binds all immunoglobulin G (IgG) subclasses, the Fc and Fab fragments, and also IgA and IgM. SibA has no significant sequence homology to any M-related proteins, is not found in the vir regulon, and contains none of the characteristic M-protein regions, such as the A or C repeats. Like M proteins, however, SibA does have relatively high levels of alanine, lysine, glutamic acid, leucine, and glycine. SibA and M proteins also share an alpha-helical N-terminal secondary structure which has been previously implicated in immunoglobulin binding in M proteins. Evidence presented here indicates that this is also the case for SibA. SibA also has regions of local similarity with other coiled-coil proteins such as Listeria monocytogenes P45 autolysin, human myosin heavy chain, macrogolgin, and Schistoma mansoni paramyosin, some of which are of potential significance since cross-reactive antibodies between myosin proteins and M proteins have been implicated in the development of the autoimmune sequelae of streptococcal disease. PMID:11447160

  14. Quantification of protein group coherence and pathway assignment using functional association

    PubMed Central

    2011-01-01

    Background Genomics and proteomics experiments produce a large amount of data that are awaiting functional elucidation. An important step in analyzing such data is to identify functional units, which consist of proteins that play coherent roles to carry out the function. Importantly, functional coherence is not identical with functional similarity. For example, proteins in the same pathway may not share the same Gene Ontology (GO) terms, but they work in a coordinated fashion so that the aimed function can be performed. Thus, simply applying existing functional similarity measures might not be the best solution to identify functional units in omics data. Results We have designed two scores for quantifying the functional coherence by considering association of GO terms observed in two biological contexts, co-occurrences in protein annotations and co-mentions in literature in the PubMed database. The counted co-occurrences of GO terms were normalized in a similar fashion as the statistical amino acid contact potential is computed in the protein structure prediction field. We demonstrate that the developed scores can identify functionally coherent protein sets, i.e. proteins in the same pathways, co-localized proteins, and protein complexes, with statistically significant score values showing a better accuracy than existing functional similarity scores. The scores are also capable of detecting protein pairs that interact with each other. It is further shown that the functional coherence scores can accurately assign proteins to their respective pathways. Conclusion We have developed two scores which quantify the functional coherence of sets of proteins. The scores reflect the actual associations of GO terms observed either in protein annotations or in literature. It has been shown that they have the ability to accurately distinguish biologically relevant groups of proteins from random ones as well as a good discriminative power for detecting interacting pairs of

  15. Trapping Open and Closed Forms of FitE-A Group III Periplasmic Binding Protein

    SciTech Connect

    Shi, R.; Proteau, A; Wagner, J; Cui, Q; Purisima, E; Matte, A; Cygler, M

    2009-01-01

    Periplasmic binding proteins (PBPs) are essential components of bacterial transport systems, necessary for bacterial growth and survival. The two-domain structures of PBPs are topologically classified into three groups based on the number of crossovers or hinges between the globular domains: group I PBPs have three connections, group II have two, and group III have only one. Although a large number of structures for group I or II PBPs are known, fewer group III PBPs have been structurally characterized. Group I and II PBPs exhibit significant domain motions during transition from the unbound to ligand-bound form, however, no large conformational changes have been observed to date in group III PBPs. We have solved the crystal structure of a periplasmic binding protein FitE, part of an iron transport system, fit, recently identified in a clinical E. coli isolate. The structure, determined at 1.8 {angstrom} resolution, shows that FitE is a group III PBP containing a single {alpha}-helix bridging the two domains. Among the individual FitE molecules present in two crystal forms we observed three different conformations (open, closed, intermediate). Our crystallographic and molecular dynamics results strongly support the notion that group III PBPs also adopt the same Venus flytrap mechanism as do groups I and II PBPs. Unlike other group III PBPs, FitE forms dimers both in solution and in the crystals. The putative siderophore binding pocket is lined with arginine residues, suggesting an anionic nature of the iron-containing siderophore.

  16. UV damage-specific DNA-binding protein in xeroderma pigmentosum complementation group E

    SciTech Connect

    Kataoka, H.; Fujiwara, Y. )

    1991-03-29

    The gel mobility shift assay method revealed a specifically ultraviolet (UV) damage recognizing, DNA-binding protein in nuclear extracts of normal human cells. The resulted DNA/protein complexes caused the two retarded mobility shifts. Four xeroderma pigmentosum complementation group E (XPE) fibroblast strains derived from unrelated Japanese families were not deficient in such a DNA damage recognition/binding protein because of the normal complex formation and gel mobility shifts, although we confirmed the reported lack of the protein in the European XPE (XP2RO and XP3RO) cells. Thus, the absence of this binding protein is not always commonly observed in all the XPE strains, and the partially repair-deficient and intermediately UV-hypersensitive phenotype of XPE cells are much similar whether or not they lack the protein.

  17. Desolvation penalty for burying hydrogen-bonded peptide groups in protein folding.

    PubMed

    Baldwin, Robert L

    2010-12-16

    A novel analysis of the enthalpy of protein unfolding is proposed and used to test for a desolvation penalty when hydrogen-bonded peptide groups are desolvated via folding. The unfolding enthalpy has three components, (1) the change when peptide hydrogen bonds are broken and the exposed -CO and -NH groups are solvated, (2) the change when protein-protein van der Waals interactions are broken and replaced by protein-water van der Waals interactions, and (3) the change produced by the hydrophobic interaction when nonpolar groups in the protein interior (represented as a liquid hydrocarbon) are transferred to water. A key feature of the analysis is that the enthalpy change from the hydrophobic interaction goes through 0 at 22 °C according to the liquid hydrocarbon model. Protein unfolding enthalpies are smaller at 22 °C than the enthalpy change for unfolding an alanine peptide helix. Data in the literature indicate that the van der Waals contribution to the unfolding enthalpy is considerably larger than the unfolding enthalpy itself at 22 °C, and therefore, a sizable desolvation penalty is predicted. Such a desolvation penalty was predicted earlier from electrostatic calculations of a stabilizing interaction between water and the hydrogen-bonded peptide group.

  18. A Semiautomated Assignment Protocol for Methyl Group Side Chains in Large Proteins.

    PubMed

    Kim, Jonggul; Wang, Yingjie; Li, Geoffrey; Veglia, Gianluigi

    2016-01-01

    The developments of biosynthetic specific labeling strategies for side-chain methyl groups have allowed structural and dynamic characterization of very large proteins and protein complexes. However, the assignment of the methyl-group resonances remains an Achilles' heel for NMR, as the experiments designed to correlate side chains to the protein backbone become rather insensitive with the increase of the transverse relaxation rates. In this chapter, we outline a semiempirical approach to assign the resonances of methyl-group side chains in large proteins. This method requires a crystal structure or an NMR ensemble of conformers as an input, together with NMR data sets such as nuclear Overhauser effects (NOEs) and paramagnetic relaxation enhancements (PREs), to be implemented in a computational protocol that provides a probabilistic assignment of methyl-group resonances. As an example, we report the protocol used in our laboratory to assign the side chains of the 42-kDa catalytic subunit of the cAMP-dependent protein kinase A. Although we emphasize the labeling of isoleucine, leucine, and valine residues, this method is applicable to other methyl group side chains such as those of alanine, methionine, and threonine, as well as reductively methylated cysteine side chains.

  19. Kicking against the PRCs – A Domesticated Transposase Antagonises Silencing Mediated by Polycomb Group Proteins and Is an Accessory Component of Polycomb Repressive Complex 2

    PubMed Central

    Perera, Pumi; Mora-García, Santiago; de Leau, Erica; Thornton, Harry; de Alves, Flavia Lima; Rapsilber, Juri; Yang, Suxin; James, Geo Velikkakam; Schneeberger, Korbinian; Finnegan, E. Jean; Turck, Franziska; Goodrich, Justin

    2015-01-01

    The Polycomb group (PcG) and trithorax group (trxG) genes play crucial roles in development by regulating expression of homeotic and other genes controlling cell fate. Both groups catalyse modifications of chromatin, particularly histone methylation, leading to epigenetic changes that affect gene activity. The trxG antagonizes the function of PcG genes by activating PcG target genes, and consequently trxG mutants suppress PcG mutant phenotypes. We previously identified the ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN1 (ALP1) gene as a genetic suppressor of mutants in the Arabidopsis PcG gene LIKE HETEROCHROMATIN PROTEIN1 (LHP1). Here, we show that ALP1 interacts genetically with several other PcG and trxG components and that it antagonizes PcG silencing. Transcriptional profiling reveals that when PcG activity is compromised numerous target genes are hyper-activated in seedlings and that in most cases this requires ALP1. Furthermore, when PcG activity is present ALP1 is needed for full activation of several floral homeotic genes that are repressed by the PcG. Strikingly, ALP1 does not encode a known chromatin protein but rather a protein related to PIF/Harbinger class transposases. Phylogenetic analysis indicates that ALP1 is broadly conserved in land plants and likely lost transposase activity and acquired a novel function during angiosperm evolution. Consistent with this, immunoprecipitation and mass spectrometry (IP-MS) show that ALP1 associates, in vivo, with core components of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a widely conserved PcG protein complex which functions as a H3K27me3 histone methyltransferase. Furthermore, in reciprocal pulldowns using the histone methyltransferase CURLY LEAF (CLF), we identify not only ALP1 and the core PRC2 components but also plant-specific accessory components including EMBRYONIC FLOWER 1 (EMF1), a transcriptional repressor previously associated with PRC1-like complexes. Taken together our data suggest that ALP1 inhibits Pc

  20. Chemical Interactions of Polyethylene Glycols (PEG) and Glycerol with Protein Functional Groups: Applications to PEG, Glycerol Effects on Protein Processes

    PubMed Central

    Knowles, DB; Shkel, Irina A; Phan, Noel M; Sternke, Matt; Lingeman, Emily; Cheng, Xian; Cheng, Lixue; O’Connor, Kevin; Record, M. Thomas

    2015-01-01

    Here we obtain the data needed to predict chemical interactions of polyethylene glycols (PEGs) and glycerol with proteins and related organic compounds, and thereby interpret or predict chemical effects of PEGs on protein processes. To accomplish this we determine interactions of glycerol and tetraEG with >30 model compounds displaying the major C, N, and O functional groups of proteins. Analysis of these data yields coefficients (α-values) quantifying interactions of glycerol, tetraEG and PEG end (-CH2OH) and interior (-CH2OCH2-) groups with these groups, relative to interactions with water. TetraEG (strongly) and glycerol (weakly) interact favorably with aromatic C, amide N, and cationic N, but unfavorably with amide O, carboxylate O and salt ions. Strongly unfavorable O and salt anion interactions help make both small and large PEGs effective protein precipitants. Interactions of tetraEG and PEG interior groups with aliphatic C are quite favorable, while interactions of glycerol and PEG end groups with aliphatic C are not. Hence tetraEG and PEG 300 favor unfolding of the DNA-binding domain of lac repressor (lacDBD) while glycerol, di- and mono-ethylene glycol are stabilizers. Favorable interactions with aromatic and aliphatic C explain why PEG400 greatly increases the solubility of aromatic hydrocarbons and steroids. PEG400-steroid interactions are unusually favorable, presumably because of simultaneous interactions of multiple PEG interior groups with the fused ring system of the steroid. Using α-values reported here, chemical contributions to PEG m-values can be predicted or interpreted in terms of changes in water-accessible surface area (ΔASA), and separated from excluded volume effects. PMID:25962980

  1. Single-molecule studies of high-mobility group B architectural DNA bending proteins.

    PubMed

    Murugesapillai, Divakaran; McCauley, Micah J; Maher, L James; Williams, Mark C

    2017-02-01

    Protein-DNA interactions can be characterized and quantified using single molecule methods such as optical tweezers, magnetic tweezers, atomic force microscopy, and fluorescence imaging. In this review, we discuss studies that characterize the binding of high-mobility group B (HMGB) architectural proteins to single DNA molecules. We show how these studies are able to extract quantitative information regarding equilibrium binding as well as non-equilibrium binding kinetics. HMGB proteins play critical but poorly understood roles in cellular function. These roles vary from the maintenance of chromatin structure and facilitation of ribosomal RNA transcription (yeast high-mobility group 1 protein) to regulatory and packaging roles (human mitochondrial transcription factor A). We describe how these HMGB proteins bind, bend, bridge, loop and compact DNA to perform these functions. We also describe how single molecule experiments observe multiple rates for dissociation of HMGB proteins from DNA, while only one rate is observed in bulk experiments. The measured single-molecule kinetics reveals a local, microscopic mechanism by which HMGB proteins alter DNA flexibility, along with a second, much slower macroscopic rate that describes the complete dissociation of the protein from DNA.

  2. Localization of xeroderma pigmentosum group A protein and replication protein A on damaged DNA in nucleotide excision repair

    PubMed Central

    Krasikova, Yuliya S.; Rechkunova, Nadejda I.; Maltseva, Ekaterina A.; Petruseva, Irina O.; Lavrik, Olga I.

    2010-01-01

    The interaction of xeroderma pigmentosum group A protein (XPA) and replication protein A (RPA) with damaged DNA in nucleotide excision repair (NER) was studied using model dsDNA and bubble-DNA structure with 5-{3-[6-(carboxyamido-fluoresceinyl)amidocapromoyl]allyl}-dUMP lesions in one strand and containing photoreactive 5-iodo-dUMP residues in defined positions. Interactions of XPA and RPA with damaged and undamaged DNA strands were investigated by DNA–protein photocrosslinking and gel shift analysis. XPA showed two maximums of crosslinking intensities located on the 5′-side from a lesion. RPA mainly localized on undamaged strand of damaged DNA duplex and damaged bubble-DNA structure. These results presented for the first time the direct evidence for the localization of XPA in the 5′-side of the lesion and suggested the key role of XPA orientation in conjunction with RPA binding to undamaged strand for the positioning of the NER preincision complex. The findings supported the mechanism of loading of the heterodimer consisting of excision repair cross-complementing group 1 and xeroderma pigmentosum group F proteins by XPA on the 5′-side from the lesion before damaged strand incision. Importantly, the proper orientation of XPA and RPA in the stage of preincision was achieved in the absence of TFIIH and XPG. PMID:20693538

  3. Generation of a Potent Low Density Lipoprotein Receptor-related Protein 1 (LRP1) Antagonist by Engineering a Stable Form of the Receptor-associated Protein (RAP) D3 Domain.

    PubMed

    Prasad, Joni M; Migliorini, Mary; Galisteo, Rebeca; Strickland, Dudley K

    2015-07-10

    The low density lipoprotein receptor-related protein 1 (LRP1) is a member of the low density lipoprotein receptor family and plays important roles in a number of physiological and pathological processes. Expression of LRP1 requires the receptor-associated protein (RAP), a molecular chaperone that binds LRP1 and other low density lipoprotein receptor family members in the endoplasmic reticulum and traffics with them to the Golgi where the acidic environment causes its dissociation. Exogenously added RAP is a potent LRP1 antagonist and binds to LRP1 on the cell surface, preventing ligands from binding. Following endocytosis, RAP dissociates in the acidic endosome, allowing LRP1 to recycle back to the cell surface. The acid-induced dissociation of RAP is mediated by its D3 domain, a relatively unstable three-helical bundle that denatures at pH <6.2 due to protonation of key histidine residues on helices 2 and 3. To develop an LRP1 inhibitor that does not dissociate at low pH, we introduced a disulfide bond between the second and third helices in the RAP D3 domain. By combining this disulfide bond with elimination of key histidine residues, we generated a stable RAP molecule that is resistant to both pH- and heat-induced denaturation. This molecule bound to LRP1 with high affinity at both neutral and acidic pH and proved to be a potent inhibitor of LRP1 function both in vitro and in vivo, suggesting that our stable RAP molecule may be useful in multiple pathological settings where LRP1 blockade has been shown to be effective.

  4. The trithorax-group protein Lid is a histone H3 trimethyl-Lys4 demethylase.

    PubMed

    Lee, Nara; Zhang, Junyu; Klose, Robert J; Erdjument-Bromage, Hediye; Tempst, Paul; Jones, Richard S; Zhang, Yi

    2007-04-01

    Recent studies have demonstrated that histone methylation can be dynamically regulated through active demethylation. However, no demethylase specific to histone H3 trimethyl-Lys4 (H3K4me3) has been identified. Here we report that the Drosophila melanogaster protein 'little imaginal discs' (Lid), a JmjC domain-containing trithorax group protein, can demethylate H3K4me3. Consistent with its genetic classification, Lid positively regulates Hox gene expression in S2 cells.

  5. Long-term treatment with l-DOPA and an mGlu5 receptor antagonist prevents changes in brain basal ganglia dopamine receptors, their associated signaling proteins and neuropeptides in parkinsonian monkeys.

    PubMed

    Morin, Nicolas; Jourdain, Vincent A; Morissette, Marc; Grégoire, Laurent; Di Paolo, Thérèse

    2014-04-01

    Brain glutamate overactivity is well documented in Parkinson's disease (PD) and antiglutamatergic drugs decrease L-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesias (LID); the implication of dopamine neurotransmission is not documented in this anti-LID activity. Therefore, we evaluated changes of dopamine receptors, their associated signaling proteins and neuropeptides mRNA, in normal control monkeys, in saline-treated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys and in L-DOPA-treated MPTP monkeys, without or with an adjunct treatment to reduce the development of LID: 2-methyl-6-(phenylethynyl)pyridine (MPEP), the prototypal metabotropic glutamate 5 (mGlu5) receptor antagonist. All de novo treatments were administered for 1 month and the animals were sacrificed thereafter. MPTP monkeys treated with l-DOPA + MPEP developed significantly less LID than MPTP monkeys treated with l-DOPA alone. [(3)H]SCH-23390 specific binding to D1 receptors of all MPTP monkeys was decreased as compared to controls in the basal ganglia and no difference was observed between all MPTP groups, while striatal D1 receptor mRNA levels remained unchanged. [(3)H]raclopride specific binding to striatal D2 receptors and mRNA levels of D2 receptors were increased in MPTP monkeys compared to controls; l-DOPA treatment reduced this binding in MPTP monkeys while it remained elevated with the l-DOPA + MPEP treatment. Striatal [(3)H]raclopride specific binding correlated positively with D2 receptor mRNA levels of all MPTP-lesioned monkeys. Striatal preproenkephalin/preprodynorphin mRNA levels and phosphorylated ERK1/2 and Akt/GSK3β levels increased only in L-DOPA-treated MPTP monkeys as compared to controls, saline treated-MPTP and l-DOPA + MPEP treated MPTP monkeys. Hence, reduction of development of LID with MPEP was associated with changes in D2 receptors, their associated signaling proteins and neuropeptides.

  6. Polycomb Group (PcG) Proteins and Human Cancers: Multifaceted Functions and Therapeutic Implications

    PubMed Central

    Wang, Wei; Qin, Jiang-Jiang; Voruganti, Sukesh; Nag, Subhasree; Zhou, Jianwei; Zhang, Ruiwen

    2016-01-01

    Polycomb group (PcG) proteins are transcriptional repressors that regulate several crucial developmental and physiological processes in the cell. More recently, they have been found to play important roles in human carcinogenesis and cancer development and progression. The deregulation and dysfunction of PcG proteins often lead to blocking or inappropriate activation of developmental pathways, enhancing cellular proliferation, inhibiting apoptosis, and increasing the cancer stem cell population. Genetic and molecular investigations of PcG proteins have long been focused on their PcG functions. However, PcG proteins have recently been shown to exert non-polycomb functions, contributing to the regulation of diverse cellular functions. We and others have demonstrated that PcG proteins regulate the expression and function of several oncogenes and tumor suppressor genes in a PcG-independent manner, and PcG proteins are associated with the survival of patients with cancer. In this review, we summarize the recent advances in the research on PcG proteins, including both the polycomb-repressive and non-polycomb functions. We specifically focus on the mechanisms by which PcG proteins play roles in cancer initiation, development, and progression. Finally, we discuss the potential value of PcG proteins as molecular biomarkers for the diagnosis and prognosis of cancer, and as molecular targets for cancer therapy. PMID:26227500

  7. Structure-based design of broadly protective group a streptococcal M protein-based vaccines

    SciTech Connect

    Dale, James B.; Smeesters, Pierre R.; Courtney, Harry S.; Penfound, Thomas A.; Hohn, Claudia M.; Smith, Jeremy C.; Baudry, Jerome Y.

    2016-11-24

    Here, a major obstacle to the development of broadly protective M protein-based group A streptococcal (GAS) vaccines is the variability within the N-terminal epitopes that evoke potent bactericidal antibodies. The concept of M type-specific protective immune responses has recently been challenged based on the observation that multivalent M protein vaccines elicited cross-reactive bactericidal antibodies against a number of non-vaccine M types of GAS. Additionally, a new “cluster-based” typing system of 175 M proteins identified a limited number of clusters containing closely related M proteins. In the current study, we used the emm cluster typing system, in combination with computational structure-based peptide modeling, as a novel approach to the design of potentially broadly protective M protein-based vaccines.

  8. Structure-based design of broadly protective group a streptococcal M protein-based vaccines

    DOE PAGES

    Dale, James B.; Smeesters, Pierre R.; Courtney, Harry S.; ...

    2016-11-24

    Here, a major obstacle to the development of broadly protective M protein-based group A streptococcal (GAS) vaccines is the variability within the N-terminal epitopes that evoke potent bactericidal antibodies. The concept of M type-specific protective immune responses has recently been challenged based on the observation that multivalent M protein vaccines elicited cross-reactive bactericidal antibodies against a number of non-vaccine M types of GAS. Additionally, a new “cluster-based” typing system of 175 M proteins identified a limited number of clusters containing closely related M proteins. In the current study, we used the emm cluster typing system, in combination with computational structure-basedmore » peptide modeling, as a novel approach to the design of potentially broadly protective M protein-based vaccines.« less

  9. Functional Gene Group Analysis Indicates No Role for Heterotrimeric G Proteins in Cognitive Ability

    PubMed Central

    Davies, Gail; Liewald, David Cherry McLachlan; Payton, Anthony; Craig, Leone C. A.; Whalley, Lawrence J.; Horan, Mike; Ollier, William; Starr, John M.; Pendleton, Neil; Posthuma, Danielle; Bates, Timothy C.; Deary, Ian J.

    2014-01-01

    Previous functional gene group analyses implicated common single nucleotide polymorphisms (SNPs) in heterotrimeric G protein coding genes as being associated with differences in human intelligence. Here, we sought to replicate this finding using five independent cohorts of older adults including current IQ and childhood IQ, and using both gene- and SNP-based analytic strategies. No significant associations were found between variation in heterotrimeric G protein genes and intelligence in any cohort at either of the two time points. These results indicate that, whereas G protein systems are important in cognition, common genetic variation in these genes is unlikely to be a substantial influence on human intelligence differences. PMID:24626473

  10. Ethrel-stimulated prolongation of latex flow in the rubber tree (Hevea brasiliensis Muell. Arg.): an Hev b 7-like protein acts as a universal antagonist of rubber particle aggregating factors from lutoids and C-serum.

    PubMed

    Shi, Min-Jing; Cai, Fu-Ge; Tian, Wei-Min

    2016-02-01

    Ethrel is the most effective stimuli in prolonging the latex flow that consequently increases yield per tapping. This effect is largely ascribed to the enhanced lutoid stability, which is associated with the decreased release of initiators of rubber particle (RP) aggregation from lutoid bursting. However, the increase in both the bursting index of lutoids and the duration of latex flow after applying ethrel or ethylene gas in high concentrations suggests that a new mechanism needs to be introduced. In this study, a latex allergen Hev b 7-like protein in C-serum was identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF MS). In vitro analysis showed that the protein acted as a universal antagonist of RP aggregating factors from lutoids and C-serum. Ethrel treatment obviously weakened the effect of C-serum on RP aggregation, which was closely associated with the increase in the level of the Hev b 7-like protein and the decrease in the level of the 37 kDa protein, as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting analysis and antibody neutralization. Thus, the increase of the Hev b 7-like protein level or the ratio of the Hev b 7-like protein to the 37 kDa protein in C-serum should be primarily ascribed to the ethrel-stimulated prolongation of latex flow duration.

  11. A domain shared by the Polycomb group proteins Scm and ph mediates heterotypic and homotypic interactions.

    PubMed

    Peterson, A J; Kyba, M; Bornemann, D; Morgan, K; Brock, H W; Simon, J

    1997-11-01

    The Sex comb on midleg (Scm) and polyhomeotic (ph) proteins are members of the Polycomb group (PcG) of transcriptional repressors. PcG proteins maintain differential patterns of homeotic gene expression during development in Drosophila flies. The Scm and ph proteins share a homology domain with 38% identity over a length of 65 amino acids, termed the SPM domain, that is located at their respective C termini. Using the yeast two-hybrid system and in vitro protein-binding assays, we show that the SPM domain mediates direct interaction between Scm and ph. Binding studies with isolated SPM domains from Scm and ph show that the domain is sufficient for these protein interactions. These studies also show that the Scm-ph and Scm-Scm domain interactions are much stronger than the ph-ph domain interaction, indicating that the isolated domain has intrinsic binding specificity determinants. Analysis of site-directed point mutations identifies residues that are important for SPM domain function. These binding properties, predicted alpha-helical secondary structure, and conservation of hydrophobic residues prompt comparisons of the SPM domain to the helix-loop-helix and leucine zipper domains used for homotypic and heterotypic protein interactions in other transcriptional regulators. In addition to in vitro studies, we show colocalization of the Scm and ph proteins at polytene chromosome sites in vivo. We discuss the possible roles of the SPM domain in the assembly or function of molecular complexes of PcG proteins.

  12. Identification of isp, a locus encoding an immunogenic secreted protein conserved among group A streptococci.

    PubMed Central

    McIver, K S; Subbarao, S; Kellner, E M; Heath, A S; Scott, J R

    1996-01-01

    The protein Mga (mga), which is required for transcription of several virulence genes of group A streptococci (GAS), including the antiphagocytic M protein, was suggested to act as the response regulator element of a bacterial two-component pathway. To investigate whether a gene encoding a cognate sensor protein is located upstream of mga, 3.1 kb of DNA 5' of the mga translational start site was cloned from serotype M6 GAS strain JRS4. Sequence analysis of this region revealed two adjacent open reading frames, a previously described orf and a new locus, isp (immunogenic secreted protein), which could encode proteins of 9 and 59 kDa, respectively. Inactivation of either open reading frame had no significant effect on transcription of the gene encoding M protein (emm) under normal growth conditions, suggesting that neither isp nor orf is involved in the Mga regulatory circuit. A protein migrating at an apparent molecular weight of 65,000 was produced when isp was transcribed and translated in vitro. The predicted isp product (Isp) contains an amino-terminal signal sequence region homologous to that found in bacterial secreted proteins, and expression of isp in Escherichia coli resulted in the presence of Isp in the periplasmic fraction. Convalescent-phase serum from a patient with an active GAS infection recognized forms of Isp both from the periplasm of E. coli and the supernatant of a GAS strain. Both isp and orf are highly conserved among strains of GAS, as shown by hybridization analyses. PMID:8698478

  13. Muscarinic Receptor Antagonists.

    PubMed

    Matera, Maria Gabriella; Cazzola, Mario

    2017-01-01

    Parasympathetic activity is increased in patients with chronic obstructive pulmonary disease (COPD) and asthma and appears to be the major reversible component of airway obstruction. Therefore, treatment with muscarinic receptor antagonists is an effective bronchodilator therapy in COPD and also in asthmatic patients. In recent years, the accumulating evidence that the cholinergic system controls not only contraction by airway smooth muscle but also the functions of inflammatory cells and airway epithelial cells has suggested that muscarinic receptor antagonists could exert other effects that may be of clinical relevance when we must treat a patient suffering from COPD or asthma. There are currently six muscarinic receptor antagonists licenced for use in the treatment of COPD, the short-acting muscarinic receptor antagonists (SAMAs) ipratropium bromide and oxitropium bromide and the long-acting muscarinic receptor antagonists (LAMAs) aclidinium bromide, tiotropium bromide, glycopyrronium bromide and umeclidinium bromide. Concerns have been raised about possible associations of muscarinic receptor antagonists with cardiovascular safety, but the most advanced compounds seem to have an improved safety profile. Further beneficial effects of SAMAs and LAMAs are seen when added to existing treatments, including LABAs, inhaled corticosteroids and phosphodiesterase 4 inhibitors. The importance of tiotropium bromide in the maintenance treatment of COPD, and likely in asthma, has spurred further research to identify new LAMAs. There are a number of molecules that are being identified, but only few have reached the clinical development.

  14. Qualitative and quantitative evaluation of derivatization reagents for different types of protein-bound carbonyl groups.

    PubMed

    Bollineni, Ravi Chand; Fedorova, Maria; Hoffmann, Ralf

    2013-09-07

    Mass spectrometry (MS) of 'carbonylated proteins' often involves derivatization of reactive carbonyl groups to facilitate their enrichment, identification and quantification. Among the many reported reagents, 2,4-dinitrophenylhydrazine (DNPH), biotin hydrazide (BHZ) and O-(biotinylcarbazoylmethyl) hydroxylamine (ARP) are the most frequently used. Despite their common use in carbonylation research, their reactivity towards protein-bound carbonyls has not been quantitatively evaluated in detail, to the best of our knowledge. Thus we studied the reactivity and specificity of these reagents towards different classes of reactive carbonyl groups (e.g. aldehydes, ketones and lactams), each being represented by a synthetic peptide carrying an accordingly modified residue. All three tagging reagents were selective for aliphatic aldehydes and ketones. Lactams and carbonyl-containing tryptophan oxidation products, however, were labelled only at low levels or not at all. Whereas DNPH derivatization was efficient under the published standard conditions, the derivatization conditions for BHZ and ARP had to be altered. Acidic conditions provided quantitative labelling yields for ARP. Peptides derivatized with DNPH, BHZ and ARP fragmented efficiently in tandem mass spectrometry, when the experimental conditions were chosen carefully for each reagent. Importantly, the tested carbonylated peptides did not cross-react with amino groups in other proteins present during sample preparations or enzymatic digestion. Thus, it appears favourable to digest proteins first and then derivatise the reactive carbonyl groups more efficiently at the peptide level under acidic conditions. The carbonylated model peptides used in this study might be valid internal standards for carbonylation proteomics.

  15. Characterization of New Members of the Group 3 Outer Membrane Protein Family of Brucella spp.

    PubMed Central

    Salhi, Imed; Boigegrain, Rose-Anne; Machold, Jan; Weise, Christoph; Cloeckaert, Axel; Rouot, Bruno

    2003-01-01

    Impairment of the omp25 gene in Brucella spp. leads to attenuated strains and confers protection to the host. Omp25 and Omp31, whose functions remain unknown, were the first characterized members of group 3 outer membrane proteins (Omps) (25 to 34 kDa). Recently, genomic and proteomic approaches identified five new putative members of this family, some of which are produced in B. melitensis or B. abortus. In the present study, using protein microsequencing, we identified new members of group 3 Omps proteins produced in B. suis. Since several monoclonal antibodies (MAbs) against Omp25 cross-reacted with other members of group 3 Omps, we also performed Western immunoblotting to compare wild-type B. suis with mutants systematically having B. suis omp25-related genes knocked out. We demonstrate the production of three paralogs of Omp31 and/or Omp25 in B. suis, and the existence of a common site of signal peptide cleavage (AXAAD), which is very similar to that present in the five homologous Omps of Bartonella quintana. The seven group 3 Omps were classified in four-subgroups on the basis of percentage amino acid sequence identities: Omp25 alone, the Omp25b-Omp25c-Omp25d cluster, the Omp31/31b subgroup, and the less related Omp22 protein (also called Omp3b). Together with previous data, our results demonstrate that all new members of group 3 Omps are produced in B. suis or in other Brucella species and we propose a nomenclature that integrates all of these proteins to facilitate the understanding of future Brucella interspecies study results. PMID:12874309

  16. Protein dynamics and thermodynamics crossover at 10 °C: Different roles of hydration at hydrophilic and hydrophobic groups

    NASA Astrophysics Data System (ADS)

    Chong, Yuan; Kleinhammes, Alfred; Wu, Yue

    2016-11-01

    Water at hydrophilic and hydrophobic groups interact differently with proteins. Particularly, hydration properties at hydrophobic groups undergo qualitative changes as temperature decreases below 10 °C. The influence of such interfacial changes on protein dynamics and thermodynamics remains largely unexplored. Here, nanosecond to microsecond protein dynamics and the free energy, enthalpy, and entropy of protein hydration are investigated by in-situ NMR as a function of hydration level and temperature. A crossover at 10 °C in protein dynamics and thermodynamics is revealed. The influence of water at hydrophilic groups shows little temperature dependence, whereas water at hydrophobic groups has stronger effect above 10 °C.

  17. Identification of plasma protein profiles associated with risk groups of prostate cancer patients

    PubMed Central

    Nordström, Malin; Wingren, Christer; Rose, Carsten; Bjartell, Anders; Becker, Charlotte; Lilja, Hans; Borrebaeck, Carl AK

    2015-01-01

    Purpose Early detection of prostate cancer (PC) using prostate-specific antigen (PSA) in blood reduces PC-death among unscreened men. However, due to modest specificity of PSA at commonly used cut-offs, there are urgent needs for additional biomarkers contributing enhanced risk classification among men with modestly elevated PSA. Experimental design Recombinant antibody microarrays were applied for protein expression profiling of 80 plasma samples from routine PSA-measurements, a priori divided into four risk groups, based on levels of total and %free PSA. Results The results demonstrated that plasma protein profiles could be identified that pinpointed PC (a malignant biomarker signature) and most importantly that showed moderate to high correlation with biochemically defined PC risk groups. Notably, the data also implied that the risk group with mid-range PSA and low %free PSA, a priori known to be heterogeneous, could be further stratified into two subgroups, more resembling the lowest and highest risk groups, respectively. Conclusions and clinical relevance In this pilot study, we have shown that plasma protein biomarker signatures, associated with risk groups of PC, could be identified from crude plasma samples using affinity proteomics. This approach could in the longer perspective provide novel opportunities for improved risk classification of PC patients. PMID:25196118

  18. Conformational Properties of Helical Protein Polymers with Varying Densities of Chemically Reactive Groups.

    PubMed

    Farmer, Robin S; Argust, Lindsey M; Sharp, Jared D; Kiick, Kristi L

    2006-01-01

    Protein engineering strategies have proven valuable for the production of a variety of well-defined macromolecular materials with controlled properties that have enabled their use in a range of materials and biological applications. In this work, such biosynthetic strategies have been employed in the production of monodisperse alanine-rich, helical protein polymers with the sequences [AAAQEAAAAQAAAQAEAAQAAQ](3) and [AAAQAAQAQAAAEAAAQAAQAQ](6). The composition of these protein polymers is similar to that of a previously reported family of alanine-rich protein polymers, but the density and placement of chemically reactive residues has been varied to facilitate the future use of these macromolecules in elucidating polymeric structure-function relationships in biological recognition events. Both protein polymers are readily expressed from E. coli and purified to homogeneity; characterization of their conformational behavior via circular dichroic spectroscopy (CD) indicates that they adopt highly helical conformations under a range of solution conditions. Differential scanning calorimetry, in concert with CD, demonstrates that the conformational transition from helix to coil in these macromolecules can be well-defined, with helicity, conformational transitions, T(m) values, and calorimetric enthalpies that vary with the molecular weight of the protein polymers. A combination of infrared spectroscopy and CD also reveals that the macromolecules can adopt beta-sheet structures at elevated temperatures and concentrations and that the existence and kinetics of this conformational transition appear to be related to the density of charged groups on the protein polymer.

  19. Conformational responses to changes in the state of ionization of titrable groups in proteins

    NASA Astrophysics Data System (ADS)

    Richman, Daniel Eric

    Electrostatic energy links the structural properties of proteins with some of their important biological functions, including catalysis, energy transduction, and binding and recognition. Accurate calculation of electrostatic energy is essential for predicting and for analyzing function from structure. All proteins have many ionizable residues at the protein-water interface. These groups tend to have ionization equilibria (pK a values) shifted slightly relative to their values in water. In contrast, groups buried in the hydrophobic interior usually have highly anomalous p Ka values. These shifts are what structure-based calculations have to reproduce to allow examination of contributions from electrostatics to stability, solubility and interactions of proteins. Electrostatic energies are challenging to calculate accurately because proteins are heterogeneous dielectric materials. Any individual ionizable group can experience very different local environments with different dielectric properties. The studies in this thesis examine the hypothesis that proteins reorganize concomitant with changes in their state of ionization. It appears that the pKa value measured experimentally reflects the average of pKa values experienced in the different electrostatic environments corresponding to different conformational microstates. Current computational models fail to sample conformational reorganization of the backbone correctly. Staphyloccocal nuclease (SNase) was used as a model protein in nuclear magnetic resonance (NMR) spectroscopy studies to characterize the conformational rearrangements of the protein coupled to changes in the ionization state of titrable groups. One set of experiments tests the hypothesis that proton binding to surface Asp and Glu side chains drives local unfolding by stabilizing less-native, more water-solvated conformations in which the side chains have normalized pKa values. Increased backbone flexibility in the ps-ns timescale, hydrogen bond (H

  20. Effect of Hofmeister ions on protein thermal stability: roles of ion hydration and peptide groups?

    PubMed

    Sedlák, Erik; Stagg, Loren; Wittung-Stafshede, Pernilla

    2008-11-01

    We have systematically explored the Hofmeister effects of cations and anions (0.3-1.75 M range) for acidic Desulfovibrio desulfuricans apoflavodoxin (net charge -19, pH 7) and basic horse heart cytochrome c (net charge +17, pH 4.5). The Hofmeister effect of the ions on protein thermal stability was assessed by the parameter dT trs/d[ion] (T trs; thermal midpoint). We show that dT trs/d[ion] correlates with ion partition coefficients between surface and bulk water and ion surface tension effects: this suggests direct interactions between ions and proteins. Surprisingly, the stability effects of the different ions on the two model proteins are similar, implying a major role of the peptide backbone, instead of charged groups, in mediation of the interactions. Upon assessing chemical/physical properties of the ions responsible for the Hofmeister effects on protein stability, ion charge density was identified as most important. Taken together, our study suggests key roles for ion hydration and the peptide group in facilitating interactions between Hofmeister ions and proteins.

  1. Hydrogen bonding motifs of protein side chains: descriptions of binding of arginine and amide groups.

    PubMed Central

    Shimoni, L.; Glusker, J. P.

    1995-01-01

    The modes of hydrogen bonding of arginine, asparagine, and glutamine side chains and of urea have been examined in small-molecule crystal structures in the Cambridge Structural Database and in crystal structures of protein-nucleic acid and protein-protein complexes. Analysis of the hydrogen bonding patterns of each by graph-set theory shows three patterns of rings (R) with one or two hydrogen bond acceptors and two donors and with eight, nine, or six atoms in the ring, designated R2(2)(8), R2(2)(9), and R1(2)(6). These three patterns are found for arginine-like groups and for urea, whereas only the first two patterns R2(2)(8) and R2(2)(9) are found for asparagine- and glutamine-like groups. In each case, the entire system is planar within 0.7 A or less. On the other hand, in macromolecular crystal structures, the hydrogen bonding patterns in protein-nucleic acid complexes between the nucleic acid base and the protein are all R2(2)(9), whereas hydrogen bonding between Watson-Crick-like pairs of nucleic acid bases is R2(2)(8). These two hydrogen bonding arrangements [R2(2)(9)] and R2(2)(8)] are predetermined by the nature of the groups available for hydrogen bonding. The third motif identified, R1(2)(6), involves hydrogen bonds that are less linear than in the other two motifs and is found in proteins. PMID:7773178

  2. Group B Streptococcus surface proteins as major determinants for meningeal tropism.

    PubMed

    Tazi, Asmaa; Bellais, Samuel; Tardieux, Isabelle; Dramsi, Shaynoor; Trieu-Cuot, Patrick; Poyart, Claire

    2012-02-01

    Streptococcus agalactiae (group B Streptococcus, GBS), a normal constituent of the intestinal microbiota is the major cause of human neonatal infections and a worldwide spread 'hypervirulent' clone, GBS ST-17, is strongly associated with neonatal meningitis. Adhesion to epithelial and endothelial cells constitutes a key step of the infectious process. Therefore GBS surface-anchored proteins are obvious potential adhesion mediators of barrier crossing and determinant of hypervirulence. This review addresses the most recent molecular insights gained from studies on GBS surface proteins proven to be involved in the crossing of the brain-blood barrier and emphasizes on the specificity of a hypervirulent clone that displays meningeal tropism.

  3. Epigenetic regulation of cellular memory by the Polycomb and Trithorax group proteins.

    PubMed

    Ringrose, Leonie; Paro, Renato

    2004-01-01

    During the development of multicellular organisms, cells become different from one another by changing their genetic program in response to transient stimuli. Long after the stimulus is gone, "cellular memory" mechanisms enable cells to remember their chosen fate over many cell divisions. The Polycomb and Trithorax groups of proteins, respectively, work to maintain repressed or active transcription states of developmentally important genes through many rounds of cell division. Here we review current ideas on the protein and DNA components of this transcriptional memory system and how they interact dynamically with each other to orchestrate cellular memory for several hundred genes.

  4. Mathematical Characterization of Protein Sequences Using Patterns as Chemical Group Combinations of Amino Acids.

    PubMed

    Das, Jayanta Kumar; Das, Provas; Ray, Korak Kumar; Choudhury, Pabitra Pal; Jana, Siddhartha Sankar

    2016-01-01

    Comparison of amino acid sequence similarity is the fundamental concept behind the protein phylogenetic tree formation. By virtue of this method, we can explain the evolutionary relationships, but further explanations are not possible unless sequences are studied through the chemical nature of individual amino acids. Here we develop a new methodology to characterize the protein sequences on the basis of the chemical nature of the amino acids. We design various algorithms for studying the variation of chemical group transitions and various chemical group combinations as patterns in the protein sequences. The amino acid sequence of conventional myosin II head domain of 14 family members are taken to illustrate this new approach. We find two blocks of maximum length 6 aa as 'FPKATD' and 'Y/FTNEKL' without repeating the same chemical nature and one block of maximum length 20 aa with the repetition of chemical nature which are common among all 14 members. We also check commonality with another motor protein sub-family kinesin, KIF1A. Based on our analysis we find a common block of length 8 aa both in myosin II and KIF1A. This motif is located in the neck linker region which could be responsible for the generation of mechanical force, enabling us to find the unique blocks which remain chemically conserved across the family. We also validate our methodology with different protein families such as MYOI, Myosin light chain kinase (MLCK) and Rho-associated protein kinase (ROCK), Na+/K+-ATPase and Ca2+-ATPase. Altogether, our studies provide a new methodology for investigating the conserved amino acids' pattern in different proteins.

  5. Mathematical Characterization of Protein Sequences Using Patterns as Chemical Group Combinations of Amino Acids

    PubMed Central

    Choudhury, Pabitra Pal; Jana, Siddhartha Sankar

    2016-01-01

    Comparison of amino acid sequence similarity is the fundamental concept behind the protein phylogenetic tree formation. By virtue of this method, we can explain the evolutionary relationships, but further explanations are not possible unless sequences are studied through the chemical nature of individual amino acids. Here we develop a new methodology to characterize the protein sequences on the basis of the chemical nature of the amino acids. We design various algorithms for studying the variation of chemical group transitions and various chemical group combinations as patterns in the protein sequences. The amino acid sequence of conventional myosin II head domain of 14 family members are taken to illustrate this new approach. We find two blocks of maximum length 6 aa as ‘FPKATD’ and ‘Y/FTNEKL’ without repeating the same chemical nature and one block of maximum length 20 aa with the repetition of chemical nature which are common among all 14 members. We also check commonality with another motor protein sub-family kinesin, KIF1A. Based on our analysis we find a common block of length 8 aa both in myosin II and KIF1A. This motif is located in the neck linker region which could be responsible for the generation of mechanical force, enabling us to find the unique blocks which remain chemically conserved across the family. We also validate our methodology with different protein families such as MYOI, Myosin light chain kinase (MLCK) and Rho-associated protein kinase (ROCK), Na+/K+-ATPase and Ca2+-ATPase. Altogether, our studies provide a new methodology for investigating the conserved amino acids’ pattern in different proteins. PMID:27930687

  6. Do DEAD-box proteins promote group II intron splicing without unwinding RNA?

    PubMed

    Del Campo, Mark; Tijerina, Pilar; Bhaskaran, Hari; Mohr, Sabine; Yang, Quansheng; Jankowsky, Eckhard; Russell, Rick; Lambowitz, Alan M

    2007-10-12

    The DEAD-box protein Mss116p promotes group II intron splicing in vivo and in vitro. Here we explore two hypotheses for how Mss116p promotes group II intron splicing: by using its RNA unwinding activity to act as an RNA chaperone or by stabilizing RNA folding intermediates. We show that an Mss116p mutant in helicase motif III (SAT/AAA), which was reported to stimulate splicing without unwinding RNA, retains ATP-dependent unwinding activity and promotes unfolding of a structured RNA. Its unwinding activity increases sharply with decreasing duplex length and correlates with group II intron splicing activity in quantitative assays. Additionally, we show that Mss116p can promote ATP-independent RNA unwinding, presumably via single-strand capture, also potentially contributing to DEAD-box protein RNA chaperone activity. Our findings favor the hypothesis that DEAD-box proteins function in group II intron splicing as in other processes by using their unwinding activity to act as RNA chaperones.

  7. Modification of Hypoxic Respiratory Response by Protein Tyrosine Kinase in Brainstem Ventral Respiratory Neuron Group

    PubMed Central

    Wang, Hui; Huai, Ruituo; Yang, Junqing; Li, Yanchun

    2016-01-01

    Protein tyrosine kinase (PTK) mediated the tyrosine phosphorylation modification of neuronal receptors and ion channels. Whether such modification resulted in changes of physiological functions was not sufficiently studied. In this study we examined whether the hypoxic respiratory response—which is the enhancement of breathing in hypoxic environment could be affected by the inhibition of PTK at brainstem ventral respiratory neuron column (VRC). Experiments were performed on urethane anesthetized adult rabbits. Phrenic nerve discharge was recorded as the central respiratory motor output. Hypoxic respiratory response was produced by ventilating the rabbit with 10% O2-balance 90% N2 for 5 minutes. The responses of phrenic nerve discharge to hypoxia were observed before and after microinjecting PTK inhibitor genistein, AMPA receptor antagonist CNQX, or inactive PTK inhibitor analogue daidzein at the region of ambiguus nucleus (NA) at levels 0–2 mm rostral to obex where the inspiratory subgroup of VRC were recorded. Results were as follows: 1. the hypoxic respiratory response was significantly attenuated after microinjection of genistein and/or CNQX, and no additive effect (i.e., further attenuation of hypoxic respiratory response) was observed when genistein and CNQX were microinjected one after another at the same injection site. Microinjection of daidzein had no effect on hypoxic respiratory response. 2. Fluorescent immunostaining showed that hypoxia significantly increased the number of phosphotyrosine immunopositive neurons in areas surrounding NA and most of these neurons were also immunopositive to glutamate AMPA receptor subunit GluR1. These results suggested that PTK played an important role in regulating the hypoxic respiratory response, possibly through the tyrosine phosphorylation modification of glutamate AMPA receptors on the respiratory neurons of ventral respiratory neuron column. PMID:27798679

  8. Genetic linkage of capsid protein-encoding RNA segments in group A equine rotaviruses.

    PubMed

    Miño, Samuel; Barrandeguy, María; Parreño, Viviana; Parra, Gabriel I

    2016-04-01

    Rotavirus virions are formed by three concentric protein layers that enclose the 11 dsRNA genome segments and the viral proteins VP1 and VP3. Interactions amongst the capsid proteins (VP2, VP6, VP7 and VP4) have been described to play a major role in viral fitness, whilst restricting the reassortment of the genomic segments during co-infection with different rotavirus strains. In this work we describe and characterize the linkage between VP6 and VP7 proteins based on structural and genomic analyses of group A rotavirus strains circulating in Argentinean horses. Strains with the VP7 genotype G3 showed a strong association with the VP6 genotype I6, whilst strains with G14 were associated with the I2 genotype. Most of the differences on the VP6 and VP7 proteins were observed in interactive regions between the two proteins, suggesting that VP6 : VP7 interactions may drive the co-evolution and co-segregation of their respective gene segments.

  9. Synthesis of a select group of proteins by Neisseria gonorrhoeae in response to thermal stress.

    PubMed

    Woods, M L; Bonfiglioli, R; McGee, Z A; Georgopoulos, C

    1990-03-01

    We report the thermal conditions that induce the heat shock response in Neisseria gonorrhoeae. Under conditions of thermal stress, Neisseria gonorrhoeae synthesizes heat shock proteins (hsps), which differ quantitatively from conventionally studied gonococcal proteins. Gonococci accelerate the rate of synthesis of the hsps as early as 5 min after the appropriate stimulus is applied, with synthesis continuing for 30 min, as demonstrated by in vivo labeling experiments with L-[35S]methionine. Two of the gonococcal hsps are immunologically cross-reactive with the hsps of Escherichia coli, DnaK and GroEL, as demonstrated by Western blot (immunoblot) analysis. Ten hsps can be identified on two-dimensional autoradiograms of whole gonococci (total protein). Four hsps can be identified on two-dimensional autoradiograms of 1% N-lauroylsarcosine (sodium salt) (Sarkosyl)-insoluble membrane fractions. Two of the hsps from the 1% Sarkosyl-insoluble fraction are found exclusively in this fraction, suggesting that they are membrane proteins. The identification of this group of proteins will facilitate further study of the function of these proteins and provide insight into the possible role of hsps in disease pathogenesis.

  10. Xeroderma pigmentosum complementation group A protein acts as a processivity factor.

    PubMed

    Lambert, M W; Yang, L

    2000-05-19

    We have previously shown that endonucleases present in a protein complex, which has specificity for cyclobutane pyrimidine dimers, locate sites of damage in DNA by a processive mechanism of action in normal human lymphoblastoid cells. In contrast, the endonucleases present in this complex from xeroderma pigmentosum complementation group A (XPA) cells locate damage sites by a distributive or significantly less processive mechanism. Since the XPA protein has been shown to be responsible for the DNA repair defect in XPA cells, this protein was examined for involvement in the mechanism of target site location of these endonucleases. A recombinant XPA protein, produced by expression of the normal XPA cDNA in E. coli, was isolated and purified. The results show that the recombinant XPA protein was able to correct the defect in ability of the XPA endonucleases to act by a processive mechanism of action on UVC irradiated DNA. These studies indicate that the XPA protein, in addition to a role in damage recognition or damage verification, may function as a processivity factor.

  11. Differential carbonylation of cytoskeletal proteins in blood group O erythrocytes: potential role in protection against severe malaria.

    PubMed

    Méndez, Darío; Hernáez, María L; Kamali, Ali N; Diez, Amalia; Puyet, Antonio; Bautista, José M

    2012-12-01

    The molecular basis for the prevalence of blood group O in regions where malaria is endemic remains unclear. In some genetic backgrounds oxidative modifications have been linked to a reduced susceptibility to severe malaria disease. Through redox proteomics, we detected differences in carbonylated membrane proteins among the different blood groups, both in Plasmodium-infected and uninfected erythrocytes (RBC). Carbonylation profiles of RBC membrane proteins revealed that group O blood shows a reduced protein oxidation pattern compared to groups A, B and AB. Upon infection with Plasmodium falciparum Dd2, erythrocytes of all blood groups showed increased oxidation of membrane proteins. By examining 4-hydroxy-2-nonenal (4-HNE) modified proteins by LC-MS/MS (liquid chromatography/mass spectrometry) we observed that, upon malaria infection, the protein components of lipid rafts and cytoskeleton were the main targets of 4-HNE carbonylation in all blood groups. Ankyrins and protein bands 4.2 and 4.1 were differentially carbonylated in group O as compared to A and B groups. During trophozoite maturation in group O erythrocytes, a steady increase was observed in the number of 4-HNE-modified proteins, suggesting a parasite-driven 4-HNE-carbonylation process. Our findings indicate a possible correlation between the protection against severe malaria in blood group O individuals and a specific pattern of 4-HNE-carbonylation of cytoskeleton proteins.

  12. A novel integrin {alpha}5{beta}1 antagonistic peptide, A5-1, screened by Protein Chip system as a potent angiogenesis inhibitor

    SciTech Connect

    Kim, Eung-Yoon; Bang, Ji Young; Chang, Soo-Ik; Kang, In-Cheol

    2008-12-26

    Integrin {alpha}5{beta}1 immobilized on a ProteoChip was used to screen new antagonistic peptides from multiple hexapeptide sub-libraries of the positional scanning synthetic peptide combinatorial library (PS-SPCL). The integrin {alpha}5{beta}1-Fibronectin interaction was demonstrated on the chip. A novel peptide ligand, A5-1 (VILVLF), with high affinity to integrin {alpha}5{beta}1 was identified from the hexapeptide libraries with this chip-based screening method on the basis of a competitive inhibition assay. A5-1 inhibits the integrin-fibronectin interaction in a dose-dependent manner (IC{sub 50}; 1.56 {+-} 0.28 {mu}M. In addition, it inhibits human umbilical vein endothelial cell proliferation, migration, adhesion, tubular network formation, and bFGF-induced neovascularization in a chick chorioallantoic membrane. These results suggest that A5-1 will be a potent inhibitor of neovascularization.

  13. The selective glucocorticoid receptor antagonist ORG 34116 decreases immobility time in the forced swim test and affects cAMP-responsive element-binding protein phosphorylation in rat brain.

    PubMed

    Bachmann, Cornelius G; Bilang-Bleuel, Alicia; De Carli, Sonja; Linthorst, Astrid C E; Reul, Johannes M H M

    2005-01-01

    Glucocorticoid receptor (GR) antagonists can block the retention of the immobility response in the forced swimming test. Recently, we showed that forced swimming evokes a distinct spatiotemporal pattern of cAMP-responsive element-binding protein (CREB) phosphorylation in the dentate gyrus (DG) and neocortex. In the present study, we found that chronic treatment of rats with the selective GR antagonist ORG 34116 decreased the immobility time in the forced swim test, increased baseline levels of phosphorylated CREB (P-CREB) in the DG and neocortex and affected the forced swimming-induced changes in P-CREB levels in a time- and site-specific manner. Overall, we observed that, in control rats, forced swimming evoked increases in P-CREB levels in the DG and neocortex, whereas in ORG 34116-treated animals a major dephosphorylation of P-CREB was observed. These observations underscore an important role of GRs in the control of the phosphorylation state of CREB which seems to be of significance for the immobility response in the forced swim test and extend the molecular mechanism of action of GRs in the brain.

  14. Group I Metabotropic Glutamate Receptor Interacting Proteins: Fine-Tuning Receptor Functions in Health and Disease

    PubMed Central

    Kalinowska, Magdalena; Francesconi, Anna

    2016-01-01

    Group I metabotropic glutamate receptors mediate slow excitatory neurotransmission in the central nervous system and are critical to activity-dependent synaptic plasticity, a cellular substrate of learning and memory. Dysregulated receptor signaling is implicated in neuropsychiatric conditions ranging from neurodevelopmental to neurodegenerative disorders. Importantly, group I metabotropic glutamate receptor signaling functions can be modulated by interacting proteins that mediate receptor trafficking, expression and coupling efficiency to signaling effectors. These interactions afford cell- or pathway-specific modulation to fine-tune receptor function, thus representing a potential target for pharmacological interventions in pathological conditions. PMID:27296642

  15. Localization of a bacterial group II intron-encoded protein in human cells.

    PubMed

    Reinoso-Colacio, Mercedes; García-Rodríguez, Fernando Manuel; García-Cañadas, Marta; Amador-Cubero, Suyapa; García Pérez, José Luis; Toro, Nicolás

    2015-08-05

    Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells.

  16. Localization of a bacterial group II intron-encoded protein in human cells

    PubMed Central

    Reinoso-Colacio, Mercedes; García-Rodríguez, Fernando Manuel; García-Cañadas, Marta; Amador-Cubero, Suyapa; Pérez, José Luis García; Toro, Nicolás

    2015-01-01

    Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells. PMID:26244523

  17. Development of selective agonists and antagonists of P2Y receptors

    PubMed Central

    Ivanov, Andrei A.; de Castro, Sonia; Harden, T. Kendall; Ko, Hyojin

    2008-01-01

    Although elucidation of the medicinal chemistry of agonists and antagonists of the P2Y receptors has lagged behind that of many other members of group A G protein-coupled receptors, detailed qualitative and quantitative structure–activity relationships (SARs) were recently constructed for several of the subtypes. Agonists selective for P2Y1, P2Y2, and P2Y6 receptors and nucleotide antagonists selective for P2Y1 and P2Y12 receptors are now known. Selective nonnucleotide antagonists were reported for P2Y1, P2Y2, P2Y6, P2Y11, P2Y12, and P2Y13 receptors. At the P2Y1 and P2Y12 receptors, nucleotide agonists (5′-diphosphate derivatives) were converted into antagonists of nanomolar affinity by altering the phosphate moieties, with a focus particularly on the ribose conformation and substitution pattern. Nucleotide analogues with conformationally constrained ribose-like rings were introduced as selective receptor probes for P2Y1 and P2Y6 receptors. Screening chemically diverse compound libraries has begun to yield new lead compounds for the development of P2Y receptor antagonists, such as competitive P2Y12 receptor antagonists with antithrombotic activity. Selective agonists for the P2Y4, P2Y11, and P2Y13 receptors and selective antagonists for P2Y4 and P2Y14 receptors have not yet been identified. The P2Y14 receptor appears to be the most restrictive of the class with respect to modification of the nucleobase, ribose, and phosphate moieties. The continuing process of ligand design for the P2Y receptors will aid in the identification of new clinical targets. PMID:18600475

  18. Interaction of the Arabidopsis polycomb group proteins FIE and MEA mediates their common phenotypes.

    PubMed

    Spillane, C; MacDougall, C; Stock, C; Köhler, C; Vielle-Calzada, J P; Nunes, S M; Grossniklaus, U; Goodrich, J

    2000-11-30

    Genes of the FERTILISATION INDEPENDENT SEED (FIS) class regulate cell proliferation during reproductive development in Arabidopsis [1-5]. The FIS genes FERTILISATION INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA) encode homologs of animal Polycomb group (Pc-G) proteins, transcriptional regulators that modify chromatin structure and are thought to form multimeric complexes [3-11]. To test whether similarities in fis mutant phenotypes reflect interactions between their protein products, we characterised FIE RNA and protein localisation in vivo, and FIE protein interactions in yeast and in vitro. Expression of FIE mRNA overlaps with that of MEA during embryo sac and seed development and is unaffected in mea mutants. Results from the yeast two-hybrid system and an in vitro pull-down assay indicate that MEA and FIE proteins interact. The relevance of this interaction in vivo is supported by the finding that FIE and MEA co-localise in the nucleus in transfected plant cells. Interaction of MEA and FIE is mediated by the amino-terminal region of MEA. Despite sequence divergence in this domain, MEA can interact with its corresponding animal partner Extrasexcombs (ESC) in the yeast two-hybrid system. We conclude that FIE and MEA act together as part of a multimeric complex and that this accounts for the similarities in mutant phenotypes. We propose that an ancient mechanism for chromatin modification has been independently recruited to different developmental processes in the two kingdoms.

  19. Xeroderma Pigmentosum Group A Protein Loads as a Separate Factor onto DNA Lesions

    PubMed Central

    Rademakers, Suzanne; Volker, Marcel; Hoogstraten, Deborah; Nigg, Alex L.; Moné, Martijn J.; van Zeeland, Albert A.; Hoeijmakers, Jan H. J.; Houtsmuller, Adriaan B.; Vermeulen, Wim

    2003-01-01

    Nucleotide excision repair (NER) is the main DNA repair pathway in mammals for removal of UV-induced lesions. NER involves the concerted action of more than 25 polypeptides in a coordinated fashion. The xeroderma pigmentosum group A protein (XPA) has been suggested to function as a central organizer and damage verifier in NER. How XPA reaches DNA lesions and how the protein is distributed in time and space in living cells are unknown. Here we studied XPA in vivo by using a cell line stably expressing physiological levels of functional XPA fused to green fluorescent protein and by applying quantitative fluorescence microscopy. The majority of XPA moves rapidly through the nucleoplasm with a diffusion rate different from those of other NER factors tested, arguing against a preassembled XPA-containing NER complex. DNA damage induced a transient (∼5-min) immobilization of maximally 30% of XPA. Immobilization depends on XPC, indicating that XPA is not the initial lesion recognition protein in vivo. Moreover, loading of replication protein A on NER lesions was not dependent on XPA. Thus, XPA participates in NER by incorporation of free diffusing molecules in XPC-dependent NER-DNA complexes. This study supports a model for a rapid consecutive assembly of free NER factors, and a relatively slow simultaneous disassembly, after repair. PMID:12897146

  20. Biophysical characterization of G protein ectodomain of group B human respiratory syncytial virus from E. coli.

    PubMed

    Khan, Wajihul Hasan; Srungaram, V L N Raghuram; Islam, Asimul; Beg, Ilyas; Haider, Md Shakir H; Ahmad, Faizan; Broor, Shobha; Parveen, Shama

    2016-07-03

    Human respiratory syncytial virus (hRSV) is an important pathogen of acute respiratory tract infection. The G protein of hRSV is a transmembrane glycoprotein that is a neutralizing antigen and is thus a vaccine candidate. In this study, synthetic codon optimized ectodomain G protein [G(ΔTM)] of BA genotype of group B hRSV was cloned, expressed, and characterized using biophysical techniques. The molar absorption coefficient and mean residue ellipticity at 222 nm ([θ]222) of G (ΔTM) was found to be 7950 M(-1) cm(-1) and -19701.7 deg cm(2) dmol(-1) respectively. It was concluded that G(ΔTM) mainly consist of α-helix (74.9%) with some amount of β-sheet (4%). The protein was stable up to 85°C without any transition curve. However, heat-induced denaturation of G(ΔTM) resulted in total loss of β-sheet whereas not much change was observed in the α-helix part of the secondary structure. It was concluded that G(ΔTM) is an α-helical protein and it is highly stable at high temperature, but could be easily denatured using high concentrations of GdmCl/urea or acidic condition. This is the first investigation of cloning, expression, and characterization of G(ΔTM) of BA viruses from India. Structural characterization of G protein will assist in drug designing and vaccine development for hRSV.

  1. High Mobility Group B Proteins, Their Partners, and Other Redox Sensors in Ovarian and Prostate Cancer

    PubMed Central

    Barreiro-Alonso, Aida; Lamas-Maceiras, Mónica; Rodríguez-Belmonte, Esther; Vizoso-Vázquez, Ángel; Quindós, María; Cerdán, M. Esperanza

    2016-01-01

    Cancer cells try to avoid the overproduction of reactive oxygen species by metabolic rearrangements. These cells also develop specific strategies to increase ROS resistance and to express the enzymatic activities necessary for ROS detoxification. Oxidative stress produces DNA damage and also induces responses, which could help the cell to restore the initial equilibrium. But if this is not possible, oxidative stress finally activates signals that will lead to cell death. High mobility group B (HMGB) proteins have been previously related to the onset and progressions of cancers of different origins. The protein HMGB1 behaves as a redox sensor and its structural changes, which are conditioned by the oxidative environment, are associated with different functions of the protein. This review describes recent advances in the role of human HMGB proteins and other proteins interacting with them, in cancerous processes related to oxidative stress, with special reference to ovarian and prostate cancer. Their participation in the molecular mechanisms of resistance to cisplatin, a drug commonly used in chemotherapy, is also revised. PMID:26682011

  2. Phosphorylation and subcellular redistribution of high mobility group proteins 14 and 17, analyzed by mass spectrometry.

    PubMed Central

    Louie, D. F.; Gloor, K. K.; Galasinski, S. C.; Resing, K. A.; Ahn, N. G.

    2000-01-01

    High mobility group (HMG) proteins 14 and 17 are nonhistone nuclear proteins that have been implicated in control of transcription and chromatin structure. To examine the posttranslational modifications of HMG-14 and -17 in vivo, HMG proteins were prepared from nuclear vs. cytosolic fractions of human K562 cells treated with 12-O-tetradecanoylphorbol 13-acetate (TPA) or okadaic acid (OA) and examined by electrospray mass spectrometry. Analysis of full-length masses demonstrated mono-, di-, and triphosphorylation of HMG-14 and mono- and diphosphorylation of HMG-17 from OA treated cells, whereas HMG-14 and -17 from TPA treated cells were monophosphorylated. Peptide mass and sequence analysis showed major and minor phosphorylation sites, respectively, at Ser24 and Ser28 in HMG-17, and Ser20 and Ser24 in HMG-14. These sites were found in the consensus sequence RRSARLSAK, within the nucleosomal binding domain of each protein. A third phosphorylation site in HMG-14 was located at either Ser6 or Ser7. Interestingly, the proportion of HMG-14 and -17 found in cytosolic pools increased significantly after 1 h of treatment compared to control cells and showed preferential phosphorylation compared with proteins from nuclear fractions. These results suggest that phosphorylation of HMG-14 and -7 interferes with nuclear localization mechanisms in a manner favoring release from nuclei. PMID:10739259

  3. Characterization of TcHMGB, a high mobility group B family member protein from Trypanosoma cruzi.

    PubMed

    Cribb, Pamela; Perozzi, Marina; Villanova, Gabriela Vanina; Trochine, Andrea; Serra, Esteban

    2011-09-01

    High mobility group B (HMGB) proteins are highly abundant non-histone chromatin proteins that play important roles in the execution and control of many nuclear functions. Based on homology searches, we identified the coding sequence for the TcHMGB protein, an HMGB family member from Trypanosoma cruzi. TcHMGB has two HMG box domains, similar to mammalian HMGBs, but lacks the typical C-terminal acidic tail. Instead, it contains a 110 amino acid long N-terminal domain. The TcHMGB N-terminal domain is conserved between the TriTryp sequences (70-80% similarity) and seems to be characteristic of kinetoplastid HMGBs. Despite these differences, TcHMGB maintains HMG box architectural functions: we demonstrated that the trypanosomatid HMGB binds distorted DNA structures such as cruciform DNA in gel shift assays. TcHMGB is also able to bend linear DNA as determined by T4 ligase circularization assays, similar to other HMGB family members. Immunofluorescence and western blot assays showed that TcHMGB is a nuclear protein expressed in all life cycle stages. Protein levels, however, seem to vary throughout the life cycle, which may be related to previously described changes in heterochromatin distribution and transcription rates.

  4. Opioid Antagonist Impedes Exposure.

    ERIC Educational Resources Information Center

    Merluzzi, Thomas V.; And Others

    1991-01-01

    Thirty spider-phobic adults underwent exposure to 17 phobic-related, graded performance tests. Fifteen subjects were assigned to naltrexone, an opioid antagonist, and 15 were assigned to placebo. Naltrexone had a significant effect on exposure, with naltrexone subjects taking significantly longer to complete first 10 steps of exposure and with…

  5. Structural Properties of 2/2 Hemoglobins: The Group III Protein from Helicobacter hepaticus

    PubMed Central

    Nothnagel, Henry J.; Winer, Benjamin Y.; Vuletich, David A.; Pond, Matthew P.; Lecomte, Juliette T.J.

    2011-01-01

    Summary The ε-proteobacterium Helicobacter hepaticus (Hh) contains a gene coding for a hemoglobin (Hb). The protein belongs to the 2/2 Hb lineage and is representative of Group III, a set of Hbs about which little is known. An expression and purification procedure was developed for Hh Hb. Electronic absorption and NMR spectra were used to characterize ligation states of the ferric and ferrous protein. The pKa of the acid/alkaline transition of ferric Hh Hb was 7.3, an unusually low value. NMR analysis of the cyanomet complex showed the orientation of the heme group to be reversed compared to most Group I and II 2/2 Hbs. Ferrous Hh Hb formed a stable cyanide complex that yielded NMR spectra similar to those of the carbonmonoxy complex. All forms of Hh Hb self-associated at NMR concentrations. Comparison was made to the related Campylobacter jejuni 2/2 Hb (Ctb), and the amino acid conservation pattern of Group III was re-inspected to help in the generalization of structure–function relationships. PMID:21445851

  6. The Structure of the Human Centrin 2-Xeroderma Pigmentosum Group C Protein Complex

    SciTech Connect

    Thompson,J.; Ryan, Z.; Salisbury, J.; Kumar, R.

    2006-01-01

    Human centrin-2 plays a key role in centrosome function and stimulates nucleotide excision repair by binding to the xeroderma pigmentosum group C protein. To determine the structure of human centrin-2 and to develop an understanding of molecular interactions between centrin and xeroderma pigmentosum group C protein, we characterized the crystal structure of calcium-loaded full-length centrin-2 complexed with a xeroderma pigmentosum group C peptide. Our structure shows that the carboxyl-terminal domain of centrin-2 binds this peptide and two calcium atoms, whereas the amino-terminal lobe is in a closed conformation positioned distantly by an ordered {alpha}-helical linker. A stretch of the amino-terminal domain unique to centrins appears disordered. Two xeroderma pigmentosum group C peptides both bound to centrin-2 also interact to form an {alpha}-helical coiled-coil. The interface between centrin-2 and each peptide is predominantly nonpolar, and key hydrophobic residues of XPC have been identified that lead us to propose a novel binding motif for centrin.

  7. A novel protein modification generating an aldehyde group in sulfatases: its role in catalysis and disease.

    PubMed

    von Figura, K; Schmidt, B; Selmer, T; Dierks, T

    1998-06-01

    In multiple sulfatase deficiency, a rare human lysosomal storage disorder, all known sulfatases are synthesized as catalytically poorly active polypeptides. Analysis of the latter has shown that they lack a protein modification that was detected in all members of the sulfatase family. This novel protein modification generates a 2-amino-3-oxopropanoic acid (C alpha-formylglycine) residue by oxidation of the thiol group of a cysteine that is conserved among all eukaryotic sulfatases. The oxidation occurs in the endoplasmic reticulum at a stage when the nascent polypeptide is not yet folded. The aldehyde is part of the catalytic site and is likely to act as an aldehyde hydrate. One of the geminal hydroxyl groups accepts the sulfate during sulfate ester cleavage leading to the formation of a covalently sulfated enzyme intermediate. The other hydroxyl is required for the subsequent elimination of the sulfate and regeneration of the aldehyde group. In some prokaryotic members of the sulfatase gene family, the DNA sequence predicts a serine residue, and not a cysteine. Analysis of one of these prokaryotic sulfatases, however, revealed the presence of the C alpha-formylglycine indicating that the aldehyde group is essential for all members of the sulfatase family and that it can be generated from either cysteine or serine.

  8. Entropy Loss of Hydroxyl Groups of Balanol upon Binding to Protein Kinase A

    NASA Astrophysics Data System (ADS)

    Gidofalvi, Gergely; Wong, Chung F.; McCammon, J. Andrew

    2002-09-01

    This article describes a short project for an undergraduate to learn several techniques for computer-aided drug design. The project involves estimating the loss of the rotational entropy of the hydroxyl groups of balanol upon its binding to the enzyme protein kinase A (PKA), as the entropy loss can significantly influence PKA balanol binding affinity. This work employs semiempirical quantum mechanical techniques for estimating the potential energy curves for the rotation of the hydroxyl groups of balanol in vacuum and in PKA, and solves the Poisson equation to correct the potential energy curves for hydration effects. Statistical mechanical principles are then applied to estimate the desired entropy loss from the potential energy curves. The analysis examines the influence of hydration effects on the rotational preference of the hydroxyl groups and the significance of the rotational entropy in determining binding affinity.

  9. Phylogenomics and signature proteins for the alpha Proteobacteria and its main groups

    PubMed Central

    Gupta, Radhey S; Mok, Amy

    2007-01-01

    Background Alpha proteobacteria are one of the largest and most extensively studied groups within bacteria. However, for these bacteria as a whole and for all of its major subgroups (viz. Rhizobiales, Rhodobacterales, Rhodospirillales, Rickettsiales, Sphingomonadales and Caulobacterales), very few or no distinctive molecular or biochemical characteristics are known. Results We have carried out comprehensive phylogenomic analyses by means of Blastp and PSI-Blast searches on the open reading frames in the genomes of several α-proteobacteria (viz. Bradyrhizobium japonicum, Brucella suis, Caulobacter crescentus, Gluconobacter oxydans, Mesorhizobium loti, Nitrobacter winogradskyi, Novosphingobium aromaticivorans, Rhodobacter sphaeroides 2.4.1, Silicibacter sp. TM1040, Rhodospirillum rubrum and Wolbachia (Drosophila) endosymbiont). These studies have identified several proteins that are distinctive characteristics of all α-proteobacteria, as well as numerous proteins that are unique repertoires of all of its main orders (viz. Rhizobiales, Rhodobacterales, Rhodospirillales, Rickettsiales, Sphingomonadales and Caulobacterales) and many families (viz. Rickettsiaceae, Anaplasmataceae, Rhodospirillaceae, Acetobacteraceae, Bradyrhiozobiaceae, Brucellaceae and Bartonellaceae). Many other proteins that are present at different phylogenetic depths in α-proteobacteria provide important information regarding their evolution. The evolutionary relationships among α-proteobacteria as deduced from these studies are in excellent agreement with their branching pattern in the phylogenetic trees and character compatibility cliques based on concatenated sequences for many conserved proteins. These studies provide evidence that the major groups within α-proteobacteria have diverged in the following order: (Rickettsiales(Rhodospirillales (Sphingomonadales (Rhodobacterales (Caulobacterales-Parvularculales (Rhizobiales)))))). We also describe two conserved inserts in DNA Gyrase B and RNA

  10. Expression of xeroderma pigmentosum complementation group C protein predicts cisplatin resistance in lung adenocarcinoma patients.

    PubMed

    Lai, Tan-Chen; Chow, Kuan-Chih; Fang, Hsin-Yuan; Cho, Hsin-Ching; Chen, Chih-Yi; Lin, Tze-Yi; Chiang, I-Ping; Ho, Shu-Peng

    2011-05-01

    DNA repair has been suggested to be a major cause of spontaneous drug resistance in patients with lung adenocarcinomas (LADC). Among the DNA repair-related proteins, excision repair cross-complementation group 1 (ERCC1) has been shown to be essential for repairing cisplatin-induced interstrand cross-linkage. However, the role of other DNA repair-related proteins in drug resistance has not been clearly elucidated. In this study, we used suppression subtractive hybridization and microarray analysis to identify the DNA repair-related genes associated with cisplatin resistance. We focused on the association of XPC protein expression, which plays a pivotal role in the earliest response to global genomic repair, with the survival of LADC patients. Using suppression subtractive hybridization and a microarray analysis to identify drug resistance-associated DNA repair-related genes, we found that the mRNA levels of ERCC1, MSH-3, MSH-6 and XPC were significantly increased in LADC patients. Since the results of ERCC1 mRNA expression corresponded well with those in previous reports, in this study we focused on the clinical correlation between XPC expression and patient survival. The level of XPC protein was determined by immunohistochemical and immunoblotting analyses. We detected the XPC protein in 46 (43%) of 107 pathological LADC samples. XPC protein expression correlated with tumor stage, cigarette smoking and poor survival. In the in vitro experiments with LADC cell lines, increased XPC expression was associated with elevated drug resistance, and silencing of XPC expression reduced cisplatin resistance. Our results suggest that XPC expression predicts drug resistance in LADC.

  11. Accumulation of Group 3 Late Embryogenesis Abundant Proteins in Zea mays Embryos 1

    PubMed Central

    Thomann, Estela B.; Sollinger, John; White, Constance; Rivin, Carol J.

    1992-01-01

    Several different types of proteins that are modulated by abscisic acid (ABA) accumulate in developing embryos of maize (Zea mays L.). Some of these proteins are specific to the developing seed, such as the storage globulin, GLB1, whereas others are involved in general responses to water deficit. Here we describe a maize protein family of this second type, a Group 3 late embryogenesis abundant (MLG3). Like other proteins of this class, MLG3 polypeptides are ABA-responsive. They are found in maturing seeds and in dehydrating plant tissues. Antigenically related proteins are found in other cereals. To distinguish the regulation of developmentally programmed ABA responses from those that are environmentally induced, we compared the ontological pattern and accumulation requirements of MLG3 polypeptides with those we previously described for GLB1. GLB1 accumulation begins early in the maturation phase and specifically requires high levels of ABA and the participation of the Viviparous-1 (Vp1) gene product. Vp1 is required for other ABA-modulated events in maize seed development as well. In experiments using vp1 mutants and mutants deficient in ABA synthesis (vp5 mutation), we show that MLG3 accumulation also is dependent upon ABA, but it shows striking differences from GLB1. MLG3 accumulates much later in embryogenesis, coincident with the onset of dehydration. In contrast to GLB1, MLG3 proteins can be induced by de novo ABA synthesis in response to culturing in high osmoticum. Unlike GLB1, MLG3 has no specific requirement for the Vp1 gene product. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6Figure 7Figure 8 PMID:16668930

  12. Two CRM protein subfamilies cooperate in the splicing of group IIB introns in chloroplasts.

    PubMed

    Asakura, Yukari; Bayraktar, Omer Ali; Barkan, Alice

    2008-11-01

    Chloroplast genomes in angiosperms encode approximately 20 group II introns, approximately half of which are classified as subgroup IIB. The splicing of all but one of the subgroup IIB introns requires a heterodimer containing the peptidyl-tRNA hydrolase homolog CRS2 and one of two closely related proteins, CAF1 or CAF2, that harbor a recently recognized RNA binding domain called the CRM domain. Two CRS2/CAF-dependent introns require, in addition, a CRM domain protein called CFM2 that is only distantly related to CAF1 and CAF2. Here, we show that CFM3, a close relative of CFM2, associates in vivo with those CRS2/CAF-dependent introns that are not CFM2 ligands. Mutant phenotypes in rice and Arabidopsis support a role for CFM3 in the splicing of most of the introns with which it associates. These results show that either CAF1 or CAF2 and either CFM2 or CFM3 simultaneously bind most chloroplast subgroup IIB introns in vivo, and that the CAF and CFM subunits play nonredundant roles in splicing. These results suggest that the expansion of the CRM protein family in plants resulted in two subfamilies that play different roles in group II intron splicing, with further diversification within a subfamily to accommodate multiple intron ligands.

  13. Identification of a group of Haemophilus influenzae penicillin-binding proteins that may have complementary physiological roles

    SciTech Connect

    Malouin, F.; Parr, T.R. Jr.; Bryan, L.E. )

    1990-02-01

    (35S)penicillin bound to different Haemophilus influenzae proteins in assays performed at 20, 37, or 42{degrees}C. Penicillin-binding proteins 3a, 3b, 4, and 4' formed a group characterized by their affinity for moxalactam, cefotaxime, and piperacillin. Penicillin-binding protein 4' showed specific properties that may reflect its complementary role in septation.

  14. Induction of human gamma interferon by structurally defined polypeptide fragments of group A streptococcal M protein.

    PubMed Central

    Weigent, D A; Beachey, E H; Huff, T; Peterson, J W; Stanton, G J; Baron, S

    1984-01-01

    The presence of interferon (IFN) has been demonstrated previously (i) in fluids obtained from the middle ears of children with Streptococcus pneumoniae infections, (ii) from the serum of mice injected intraperitoneally with either S. pneumoniae or Streptococcus pyogenes, and (iii) from human lymphoid cell cultures treated with a variety of bacteria. In this study, we showed that highly purified peptic extracts of three different serotypes of group A streptococcal M protein (pep M5, pep M6, and pep M24) stimulated human peripheral leukocytes to produce IFN. IFN production was apparent by 10 h and peaked 24 h after exposure. Dose-response experiments indicated that IFN could be detected in cultures treated with concentrations of M protein as low as 6 micrograms/ml, whereas maximum IFN production occurred at a concentration of 200 micrograms/ml. The IFN had antigenic and physicochemical characteristics of IFN-gamma. Preliminary leukocyte fractionation studies revealed that the IFN-producing cell was a nonadherent lymphocyte with receptors for sheep erythrocytes (T cell). Rabbit antisera specific for these structurally defined polypeptide fragments of streptococcal M protein (pep M5, pep M6, and pep M24) blocked IFN induction by each of the polypeptides. The data suggest that the different serotypes of streptococcal M protein may induce IFN by a common structural determinant shared by each of the polypeptide fragments tested. PMID:6418655

  15. SUMOylation of xeroderma pigmentosum group C protein regulates DNA damage recognition during nucleotide excision repair

    PubMed Central

    Akita, Masaki; Tak, Yon-Soo; Shimura, Tsutomu; Matsumoto, Syota; Okuda-Shimizu, Yuki; Shimizu, Yuichiro; Nishi, Ryotaro; Saitoh, Hisato; Iwai, Shigenori; Mori, Toshio; Ikura, Tsuyoshi; Sakai, Wataru; Hanaoka, Fumio; Sugasawa, Kaoru

    2015-01-01

    The xeroderma pigmentosum group C (XPC) protein complex is a key factor that detects DNA damage and initiates nucleotide excision repair (NER) in mammalian cells. Although biochemical and structural studies have elucidated the interaction of XPC with damaged DNA, the mechanism of its regulation in vivo remains to be understood in more details. Here, we show that the XPC protein undergoes modification by small ubiquitin-related modifier (SUMO) proteins and the lack of this modification compromises the repair of UV-induced DNA photolesions. In the absence of SUMOylation, XPC is normally recruited to the sites with photolesions, but then immobilized profoundly by the UV-damaged DNA-binding protein (UV-DDB) complex. Since the absence of UV-DDB alleviates the NER defect caused by impaired SUMOylation of XPC, we propose that this modification is critical for functional interactions of XPC with UV-DDB, which facilitate the efficient damage handover between the two damage recognition factors and subsequent initiation of NER. PMID:26042670

  16. Mallory bodies: lesions of hepatocytes containing proteins of the keratin-myosin-epidermin group.

    PubMed

    Meloan, S N; Puchtler, H

    1982-01-01

    Mallory's alcoholic hyalin in hepatocytes was found also in other diseases and is now referred to as Mallory bodies. Data concerning their histochemical, immuno and electron microscopic properties are partly contradictory. In this study, early stages of Mallory bodies reacted strongly with configurational technics for myosins; affinity tended to decrease when material with the properties of keratohyalin and the matrix of stratum corneum was formed. Thus, many Mallory bodies contained histochemically distinct myoid and keratin-like proteins. Electron microscopists demonstrated thick and thin filaments resembling contractile systems in Mallory bodies; the failure of immunologists to visualize actomyosin may be due to the heterogeneity of these proteins. The currently popular term prekeratin has been applied to a variety of substances extracted from epidermis, hoof and hair under different conditions. The prekeratin of recent immunofluorescence studies seems to contain mainly epidermin and low molecular matrix proteins; both were studied extensively by chemists. Epithelial filaments, including tonofibrils and contractile fibrils regarded as a subgroup of myofibrils, were well known half a century ago, but were banished by electron microscopy. Observations in this study and data on epidermal actomyosin indicate that different proteins of the k-m-e-f group can indeed coexist in epithelial cells. The formation and resolution of Mallory bodies can be regarded as an example of the well known shifts of epithelial cells between secretory and keratinizing states.

  17. Calcium antagonists and atherosclerosis protection in hypertension.

    PubMed

    Hernández, Rafael Hernández; Armas-Hernández, María José; Velasco, Manuel; Israili, Zafar H; Armas-Padilla, María Cristina

    2003-01-01

    Calcium antagonists are effective in hypertensive patients of all ethnic groups, irrespective of age, dietary salt intake, salt-sensitivity status or plasma renin activity profile. Some prospective studies show that the calcium antagonists, nifedipine GITS and nitrendipine, reduce cardiovascular morbidity and mortality at least to the same extent as the diuretics. Other prospective studies are in progress to evaluate the effect of calcium antagonists on cardiovascular morbidity and mortality, and the progression of atherosclerosis in hypertensive patients. Calcium antagonists, especially the highly lipophilic amlodipine, lacidipine and nisoldipine, are shown to possess antioxidant properties. These drugs reduce the oxidation of LDL and its influx into the arterial wall, and reduce atherosclerotic lesions in animals. Platelet production of malondialdehyde, a marker of oxygen free radical formation, is suppressed by amlodipine, lacidipine or nifedipine in hypertensive patients. New evidence from long-term clinical trials of calcium antagonists indicates that these drugs can reduce the rate of progression of atherosclerosis in hypertensive and coronary heart disease patients. In the Regression Growth Evaluation Statin Study (REGRESS), co-administration of calcium antagonist, amlodipine or nifedipine with pravasatin caused a significant reduction in the appearance of new angiographic lesions. In the Verapamil in Hypertension and Atherosclerosis Study (VHAS), verapamil was more effective than chlorthalidone in promoting regression of thicker carotid lesions in parallel with a reduction in the incidence of cardiovascular events. In the Prospective Randomized Evaluation of the Vascular Effects of Norvasc Trial (PREVENT), amlodipine slowed the progression of early coronary atherosclerosis in patients with coronary artery disease. In a subprotocol of the Intervention as a Goal in the Hypertension Treatment (INSIGHT) study, nifedipine GITS significantly decreased intima

  18. CF45-1, a Secreted Protein Which Participates in Dictyostelium Group Size Regulation

    PubMed Central

    Brock, Debra A.; Hatton, R. Diane; Giurgiutiu, Dan-Victor; Scott, Brenton; Jang, Wonhee; Ammann, Robin; Gomer, Richard H.

    2003-01-01

    Developing Dictyostelium cells aggregate to form fruiting bodies containing typically 2 × 104 cells. To prevent the formation of an excessively large fruiting body, streams of aggregating cells break up into groups if there are too many cells. The breakup is regulated by a secreted complex of polypeptides called counting factor (CF). Countin and CF50 are two of the components of CF. Disrupting the expression of either of these proteins results in cells secreting very little detectable CF activity, and as a result, aggregation streams remain intact and form large fruiting bodies, which invariably collapse. We find that disrupting the gene encoding a third protein present in crude CF, CF45-1, also results in the formation of large groups when cells are grown with bacteria on agar plates and then starve. However, unlike countin− and cf50− cells, cf45-1− cells sometimes form smaller groups than wild-type cells when the cells are starved on filter pads. The predicted amino acid sequence of CF45-1 has some similarity to that of lysozyme, but recombinant CF45-1 has no detectable lysozyme activity. In the exudates from starved cells, CF45-1 is present in a ∼450-kDa fraction that also contains countin and CF50, suggesting that it is part of a complex. Recombinant CF45-1 decreases group size in colonies of cf45-1− cells with a 50% effective concentration (EC50) of ∼8 ng/ml and in colonies of wild-type and cf50− cells with an EC50 of ∼40 ng/ml. Like countin− and cf50− cells, cf45-1− cells have high levels of cytosolic glucose, high cell-cell adhesion, and low cell motility. Together, the data suggest that CF45-1 participates in group size regulation in Dictyostelium. PMID:12912898

  19. Indirect protein quantification of drug-transforming enzymes using peptide group-specific immunoaffinity enrichment and mass spectrometry

    PubMed Central

    Weiß, Frederik; Schnabel, Anke; Planatscher, Hannes; van den Berg, Bart H. J.; Serschnitzki, Bettina; Nuessler, Andreas K.; Thasler, Wolfgang E.; Weiss, Thomas S.; Reuss, Matthias; Stoll, Dieter; Templin, Markus F.; Joos, Thomas O.; Marcus, Katrin; Poetz, Oliver

    2015-01-01

    Immunoaffinity enrichment of proteotypic peptides, coupled with selected reaction monitoring, enables indirect protein quantification. However the lack of suitable antibodies limits its widespread application. We developed a method in which multi-specific antibodies are used to enrich groups of peptides, thus facilitating multiplexed quantitative protein assays. We tested this strategy in a pharmacokinetic experiment by targeting a group of homologous drug transforming proteins in human hepatocytes. Our results indicate the generic applicability of this method to any biological system. PMID:25737130

  20. Advantages of an antagonist: bicuculline and other GABA antagonists

    PubMed Central

    Johnston, Graham AR

    2013-01-01

    The convulsant alkaloid bicuculline continues to be investigated more than 40 years after the first publication of its action as an antagonist of receptors for the inhibitory neurotransmitter GABA. This historical perspective highlights key aspects of the discovery of bicuculline as a GABA antagonist and the sustained interest in this and other GABA antagonists. The exciting advances in the molecular biology, pharmacology and physiology of GABA receptors provide a continuing stimulus for the discovery of new antagonists with increasing selectivity for the myriad of GABA receptor subclasses. Interesting GABA antagonists not structurally related to bicuculline include gabazine, salicylidene salicylhydrazide, RU5135 and 4-(3-biphenyl-5-(4-piperidyl)-3-isoxazole. Bicuculline became the benchmark antagonist for what became known as GABAA receptors, but not all ionotropic GABA receptors are susceptible to bicuculline. In addition, not all GABAA receptor antagonists are convulsants. Thus there are still surprises in store as the study of GABA receptors evolves. PMID:23425285

  1. Thermodynamic characterization of the biocompatible ionic liquid effects on protein model compounds and their functional groups.

    PubMed

    Attri, Pankaj; Venkatesu, Pannuru

    2011-04-14

    The stability of proteins under co-solvent conditions is dependant on the nature of the co-solvent; the co-solvent can alter a protein's properties and structural effects through bimolecular interactions between its functional groups and co-solvent particles. Ionic liquids (ILs) represent a rather diverse class of co-solvents that are combinations of different ions, which are liquids at or close to room temperature. To quantify the bimolecular interactions of protein functional groups with biocompatible ILs, we report the systematic and quantitative apparent transfer free energies (ΔG'(tr)) of a homologous series of cyclic dipeptides (CDs) from water to aqueous solutions of ILs through solubility measurements, as a function of IL concentration at 25 °C under atmospheric pressure. The materials investigated in the present work included the CDs of cyclo(Gly-Gly), cyclo(Ala-Gly), cyclo(Ala-Ala), cyclo(Leu-Ala), and cyclo(Val-Val). The ILs used such as diethylammonium acetate ([Et(2)NH][CH(3)COO], DEAA), triethylammonium acetate ([Et(3)NH][CH(3)COO], TEAA), diethylammonium dihydogen phosphate ([Et(3)NH][H(2)PO(4)], DEAP), triethylammonium dihydogen phosphate ([Et(3)NH][H(2)PO(4)], TEAP), diethylammonium sulfate ([Et(3)NH][HSO(4)], DEAS) and triethylammonium sulfate ([Et(3)NH][HSO(4)], TEAS). We observed positive values of ΔG'(tr) for CDs from water to ILs, indicating that interactions between ILs and CDs are unfavourable, which leads to stabilization of the native structure of CDs. The experimental results were further used for estimating the transfer free energies (Δg'(tr)) of the peptide bond (-CONH-), the peptide backbone unit (-CH(2)C=ONH-), and various functional groups from water to IL solutions. Our results explicitly elucidate that a series of all ammonium ILs act as stabilizers for tested model compounds through the exclusion of ILs from CDs surface.

  2. Opioid antagonists for smoking cessation

    PubMed Central

    David, Sean P; Lancaster, Tim; Stead, Lindsay F; Evins, A. Eden; Prochaska, Judith J

    2014-01-01

    Background The reinforcing properties of nicotine may be mediated through release of various neurotransmitters both centrally and systemically. People who smoke report positive effects such as pleasure, arousal, and relaxation as well as relief of negative affect, tension, and anxiety. Opioid (narcotic) antagonists are of particular interest to investigators as potential agents to attenuate the rewarding effects of cigarette smoking. Objectives To evaluate the efficacy of opioid antagonists in promoting long-term smoking cessation. The drugs include naloxone and the longer-acting opioid antagonist naltrexone. Search methods We searched the Cochrane Tobacco Addiction Group Specialised Register for trials of naloxone, naltrexone and other opioid antagonists and conducted an additional search of MEDLINE using ’Narcotic antagonists’ and smoking terms in April 2013. We also contacted investigators, when possible, for information on unpublished studies. Selection criteria We considered randomised controlled trials comparing opioid antagonists to placebo or an alternative therapeutic control for smoking cessation. We included in the meta-analysis only those trials which reported data on abstinence for a minimum of six months. We also reviewed, for descriptive purposes, results from short-term laboratory-based studies of opioid antagonists designed to evaluate psycho-biological mediating variables associated with nicotine dependence. Data collection and analysis We extracted data in duplicate on the study population, the nature of the drug therapy, the outcome measures, method of randomisation, and completeness of follow-up. The main outcome measure was abstinence from smoking after at least six months follow-up in patients smoking at baseline. Abstinence at end of treatment was a secondary outcome. We extracted cotinine- or carbon monoxide-verified abstinence where available. Where appropriate, we performed meta-analysis, pooling risk ratios using a Mantel

  3. Identification of non-heme diiron proteins that catalyze triple bond and epoxy group formation.

    PubMed

    Lee, M; Lenman, M; Banaś, A; Bafor, M; Singh, S; Schweizer, M; Nilsson, R; Liljenberg, C; Dahlqvist, A; Gummeson, P O; Sjödahl, S; Green, A; Stymne, S

    1998-05-08

    Acetylenic bonds are present in more than 600 naturally occurring compounds. Plant enzymes that catalyze the formation of the Delta12 acetylenic bond in 9-octadecen-12-ynoic acid and the Delta12 epoxy group in 12,13-epoxy-9-octadecenoic acid were characterized, and two genes, similar in sequence, were cloned. When these complementary DNAs were expressed in Arabidopsis thaliana, the content of acetylenic or epoxidated fatty acids in the seeds increased from 0 to 25 or 15 percent, respectively. Both enzymes have characteristics similar to the membrane proteins containing non-heme iron that have histidine-rich motifs.

  4. Role of the Methoxy Group in Immune Responses to mPEG-Protein Conjugates

    PubMed Central

    2012-01-01

    Anti-PEG antibodies have been reported to mediate the accelerated clearance of PEG-conjugated proteins and liposomes, all of which contain methoxyPEG (mPEG). The goal of this research was to assess the role of the methoxy group in the immune responses to mPEG conjugates and the potential advantages of replacing mPEG with hydroxyPEG (HO-PEG). Rabbits were immunized with mPEG, HO-PEG, or t-butoxyPEG (t-BuO-PEG) conjugates of human serum albumin, human interferon-α, or porcine uricase as adjuvant emulsions. Assay plates for enzyme-linked immunosorbent assays (ELISAs) were coated with mPEG, HO-PEG, or t-BuO-PEG conjugates of the non-cross-reacting protein, porcine superoxide dismutase (SOD). In sera from rabbits immunized with HO-PEG conjugates of interferon-α or uricase, the ratio of titers of anti-PEG antibodies detected on mPEG-SOD over HO-PEG-SOD (“relative titer”) had a median of 1.1 (range 0.9–1.5). In contrast, sera from rabbits immunized with mPEG conjugates of three proteins had relative titers with a median of 3.0 (range 1.1–20). Analyses of sera from rabbits immunized with t-BuO-PEG-albumin showed that t-butoxy groups are more immunogenic than methoxy groups. Adding Tween 20 or Tween 80 to buffers used to wash the assay plates, as is often done in ELISAs, greatly reduced the sensitivity of detection of anti-PEG antibodies. Competitive ELISAs revealed that the affinities of antibodies raised against mPEG-uricase were c. 70 times higher for 10 kDa mPEG than for 10 kDa PEG diol and that anti-PEG antibodies raised against mPEG conjugates of three proteins had >1000 times higher affinities for albumin conjugates with c. 20 mPEGs than for analogous HO-PEG-albumin conjugates. Overall, these results are consistent with the hypothesis that antibodies with high affinity for methoxy groups contribute to the loss of efficacy of mPEG conjugates, especially if multiply-PEGylated. Using monofunctionally activated HO-PEG instead of mPEG in preparing conjugates for

  5. Functional group placement in protein binding sites: a comparison of GRID and MCSS

    NASA Astrophysics Data System (ADS)

    Bitetti-Putzer, Ryan; Joseph-McCarthy, Diane; Hogle, James M.; Karplus, Martin

    2001-10-01

    One approach to combinatorial ligand design begins by determining optimal locations (i.e., local potential energy minima) for functional groups in the binding site of a target macromolecule. MCSS and GRID are two methods, based on significantly different algorithms, which are used for this purpose. A comparison of the two methods for the same functional groups is reported. Calculations were performed for nonpolar and polar functional groups in the internal hydrophobic pocket of the poliovirus capsid protein, and on the binding surface of the src SH3 domain. The two approaches are shown to agree qualitatively; i.e., the global characteristics of the functional group maps generated by MCSS and GRID are similar. However, there are significant differences in the relative interaction energies of the two sets of minima, a consequence of the different functional form used to evaluate polar interactions (electrostatics and hydrogen bonding) in the two methods. The single sphere representation used by GRID affords only positional information, supplemented by the identification of hydrogen bonding interactions. By contrast, the multi-atom representation of most MCSS groups yields in both positional and orientational information. The two methods are most similar for small functional groups, while for larger functional groups MCSS yields results consistent with GRID but superior in detail. These results are in accord with the somewhat different purposes for which the two methods were developed. GRID has been used mainly to introduce functionalities at specific positions in lead compounds, in which case the orientation is predetermined by the structure of the latter. The orientational information provided by MCSS is important for its use in the de novo design of large, multi-functional ligands, as well as for improving lead compounds.

  6. alpha2-Adrenoreceptor antagonists.

    PubMed

    Mayer, P; Imbert, T

    2001-06-01

    A review of the literature relating to the therapeutic potential of alpha2-adrenoceptor antagonists published between 1990 and 2000 is presented. Although extensively studied since the early 1970s in a wide spectrum of therapeutic applications, the distinction of alpha2-adrenoceptor subtypes and some emerging evidence concerning new applications in neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases, obesity and schizophrenia, have refreshed an interest in this class of agents.

  7. Trivalent M-related protein as a component of next generation group A streptococcal vaccines

    PubMed Central

    2017-01-01

    Purpose There is a need to broaden protective coverage of M protein–based vaccines against group A streptococci (GAS) because coverage of the current 30-valent M protein vaccine does not extend to all emm types. An additional GAS antigen and virulence factor that could potentially extend vaccine coverage is M-related protein (Mrp). Previous work indicated that there are three structurally related families of Mrp (MrpI, MrpII, and MrpIII) and peptides of all three elicited bactericidal antibodies against multiple emm types. The purpose of this study was to determine if a recombinant form containing Mrp from the three families would evoke bactericidal antiserum and to determine if this antiserum could enhance the effectiveness of antisera to the 30-valent M protein vaccine. Materials and Methods A trivalent recombinant Mrp (trMrp) protein containing N-terminal fragments from the three families (trMrp) was constructed, purified and used to immunize rabbits. Anti-trMrp sera contained high titers of antibodies against the trMrp immunogen and recombinant forms representing MrpI, MrpII, and MrpIII. Results The antisera opsonized emm types of GAS representing each Mrp family and also opsonized emm types not covered by the 30-valent M protein–based vaccine. Importantly, a combination of trMrp and 30-valent M protein antiserum resulted in higher levels of opsonization of GAS than either antiserum alone. Conclusion These findings suggest that trMrp may be an effective addition to future constructs of GAS vaccines. PMID:28168173

  8. A protocol for protein estimation that gives a nearly constant color yield with simple proteins and nullifies the effects of four known interfering agents: microestimation of peptide groups.

    PubMed

    Raghupathi, R N; Diwan, A M

    1994-06-01

    A modified version of the Lowry protocol for protein measurement is being introduced in which the increment in color due to copper-protein interactions can be directly equated to the concentration of peptide groups (the CO-NH moieties) present in the sample. The method provides an accurate estimation of protein concentrations in the range of 5-40 micrograms, with a sensitivity similar to that of the original assay procedure. It yields hardly any protein-to-protein variation with simple proteins and eliminates the interference by four compounds that commonly affect the Lowry assay. A numerical factor has been calculated that allows conversion of micromoles of peptide groups into actual micrograms of protein present in the sample.

  9. Prokaryotic Virus Orthologous Groups (pVOGs): a resource for comparative genomics and protein family annotation

    PubMed Central

    Grazziotin, Ana Laura; Koonin, Eugene V.; Kristensen, David M.

    2017-01-01

    Viruses are the most abundant and diverse biological entities on earth, and while most of this diversity remains completely unexplored, advances in genome sequencing have provided unprecedented glimpses into the virosphere. The Prokaryotic Virus Orthologous Groups (pVOGs, formerly called Phage Orthologous Groups, POGs) resource has aided in this task over the past decade by using automated methods to keep pace with the rapid increase in genomic data. The uses of pVOGs include functional annotation of viral proteins, identification of genes and viruses in uncharacterized DNA samples, phylogenetic analysis, large-scale comparative genomics projects, and more. The pVOGs database represents a comprehensive set of orthologous gene families shared across multiple complete genomes of viruses that infect bacterial or archaeal hosts (viruses of eukaryotes will be added at a future date). The pVOGs are constructed within the Clusters of Orthologous Groups (COGs) framework that is widely used for orthology identification in prokaryotes. Since the previous release of the POGs, the size has tripled to nearly 3000 genomes and 300 000 proteins, and the number of conserved orthologous groups doubled to 9518. User-friendly webpages are available, including multiple sequence alignments and HMM profiles for each VOG. These changes provide major improvements to the pVOGs database, at a time of rapid advances in virus genomics. The pVOGs database is hosted jointly at the University of Iowa at http://dmk-brain.ecn.uiowa.edu/pVOGs and the NCBI at ftp://ftp.ncbi.nlm.nih.gov/pub/kristensen/pVOGs/home.html. PMID:27789703

  10. Alternative splicing of a group II intron in a surface layer protein gene in Clostridium tetani

    PubMed Central

    McNeil, Bonnie A.; Simon, Dawn M.; Zimmerly, Steven

    2014-01-01

    Group II introns are ribozymes and retroelements found in bacteria, and are thought to have been the ancestors of nuclear pre-mRNA introns. Whereas nuclear introns undergo prolific alternative splicing in some species, group II introns are not known to carry out equivalent reactions. Here we report a group II intron in the human pathogen Clostridium tetani, which undergoes four alternative splicing reactions in vivo. Together with unspliced transcript, five mRNAs are produced, each encoding a distinct surface layer protein isoform. Correct fusion of exon reading frames requires a shifted 5′ splice site located 8 nt upstream of the canonical boundary motif. The shifted junction is accomplished by an altered IBS1-EBS1 pairing between the intron and 5′ exon. Growth of C. tetani under a variety of conditions did not result in large changes in alternative splicing levels, raising the possibility that alternative splicing is constitutive. This work demonstrates a novel type of gene organization and regulation in bacteria, and provides an additional parallel between group II and nuclear pre-mRNA introns. PMID:24214997

  11. Alternative splicing of a group II intron in a surface layer protein gene in Clostridium tetani.

    PubMed

    McNeil, Bonnie A; Simon, Dawn M; Zimmerly, Steven

    2014-02-01

    Group II introns are ribozymes and retroelements found in bacteria, and are thought to have been the ancestors of nuclear pre-mRNA introns. Whereas nuclear introns undergo prolific alternative splicing in some species, group II introns are not known to carry out equivalent reactions. Here we report a group II intron in the human pathogen Clostridium tetani, which undergoes four alternative splicing reactions in vivo. Together with unspliced transcript, five mRNAs are produced, each encoding a distinct surface layer protein isoform. Correct fusion of exon reading frames requires a shifted 5' splice site located 8 nt upstream of the canonical boundary motif. The shifted junction is accomplished by an altered IBS1-EBS1 pairing between the intron and 5' exon. Growth of C. tetani under a variety of conditions did not result in large changes in alternative splicing levels, raising the possibility that alternative splicing is constitutive. This work demonstrates a novel type of gene organization and regulation in bacteria, and provides an additional parallel between group II and nuclear pre-mRNA introns.

  12. Group 1 LEA proteins contribute to the desiccation and freeze tolerance of Artemia franciscana embryos during diapause.

    PubMed

    Toxopeus, Jantina; Warner, Alden H; MacRae, Thomas H

    2014-11-01

    Water loss either by desiccation or freezing causes multiple forms of cellular damage. The encysted embryos (cysts) of the crustacean Artemia franciscana have several molecular mechanisms to enable anhydrobiosis-life without water-during diapause. To better understand how cysts survive reduced hydration, group 1 late embryogenesis abundant (LEA) proteins, hydrophilic unstructured proteins that accumulate in the stress-tolerant cysts of A. franciscana, were knocked down using RNA interference (RNAi). Embryos lacking group 1 LEA proteins showed significantly lower survival than control embryos after desiccation and freezing, or freezing alone, demonstrating a role for group 1 LEA proteins in A. franciscana tolerance of low water conditions. In contrast, regardless of group 1 LEA protein presence, cysts responded similarly to hydrogen peroxide (H2O2) exposure, indicating little to no function for these proteins in diapause termination. This is the first in vivo study of group 1 LEA proteins in an animal and it contributes to the fundamental understanding of these proteins. Knowing how LEA proteins protect A. franciscana cysts from desiccation and freezing may have applied significance in aquaculture, where Artemia is an important feed source, and in the cryopreservation of cells for therapeutic applications.

  13. Requirement for sex comb on midleg protein interactions in Drosophila polycomb group repression.

    PubMed

    Peterson, Aidan J; Mallin, Daniel R; Francis, Nicole J; Ketel, Carrie S; Stamm, Joyce; Voeller, Rochus K; Kingston, Robert E; Simon, Jeffrey A

    2004-07-01

    The Drosophila Sex Comb on Midleg (SCM) protein is a transcriptional repressor of the Polycomb group (PcG). Although genetic studies establish SCM as a crucial PcG member, its molecular role is not known. To investigate how SCM might link to PcG complexes, we analyzed the in vivo role of a conserved protein interaction module, the SPM domain. This domain is found in SCM and in another PcG protein, Polyhomeotic (PH), which is a core component of Polycomb repressive complex 1 (PRC1). SCM-PH interactions in vitro are mediated by their respective SPM domains. Yeast two-hybrid and in vitro binding assays were used to isolate and characterize >30 missense mutations in the SPM domain of SCM. Genetic rescue assays showed that SCM repressor function in vivo is disrupted by mutations that impair SPM domain interactions in vitro. Furthermore, overexpression of an isolated, wild-type SPM domain produced PcG loss-of-function phenotypes in flies. Coassembly of SCM with a reconstituted PRC1 core complex shows that SCM can partner with PRC1. However, gel filtration chromatography showed that the bulk of SCM is biochemically separable from PH in embryo nuclear extracts. These results suggest that SCM, although not a core component of PRC1, interacts and functions with PRC1 in gene silencing.

  14. Group A Streptococcal M1 Protein Provides Resistance against the Antimicrobial Activity of Histones

    PubMed Central

    Döhrmann, Simon; LaRock, Christopher N.; Anderson, Ericka L.; Cole, Jason N.; Ryali, Brinda; Stewart, Chelsea; Nonejuie, Poochit; Pogliano, Joe; Corriden, Ross; Ghosh, Partho; Nizet, Victor

    2017-01-01

    Histones are essential elements of chromatin structure and gene regulation in eukaryotes. An unexpected attribute of these nuclear proteins is their antimicrobial activity. A framework for histone release and function in host defense in vivo was revealed with the discovery of neutrophil extracellular traps, a specialized cell death process in which DNA-based structures containing histones are extruded to ensnare and kill bacteria. Investigating the susceptibility of various Gram-positive pathogens to histones, we found high-level resistance by one leading human pathogen, group A Streptococcus (GAS). A screen of isogenic mutants revealed that the highly surface-expressed M1 protein, a classical GAS virulence factor, was required for high-level histone resistance. Biochemical and microscopic analyses revealed that the N-terminal domain of M1 protein binds and inactivates histones before they reach their cell wall target of action. This finding illustrates a new pathogenic function for this classic GAS virulence factor, and highlights a potential innate immune evasion strategy that may be employed by other bacterial pathogens. PMID:28220899

  15. A Polycomb Group Protein Is Retained at Specific Sites on Chromatin in Mitosis

    PubMed Central

    Follmer, Nicole E.; Wani, Ajazul H.; Francis, Nicole J.

    2012-01-01

    Epigenetic regulation of gene expression, including by Polycomb Group (PcG) proteins, may depend on heritable chromatin states, but how these states can be propagated through mitosis is unclear. Using immunofluorescence and biochemical fractionation, we find PcG proteins associated with mitotic chromosomes in Drosophila S2 cells. Genome-wide sequencing of chromatin immunoprecipitations (ChIP–SEQ) from mitotic cells indicates that Posterior Sex Combs (PSC) is not present at well-characterized PcG targets including Hox genes in mitosis, but does remain at a subset of interphase sites. Many of these persistent sites overlap with chromatin domain borders described by Sexton et al. (2012), which are genomic regions characterized by low levels of long range contacts. Persistent PSC binding sites flank both Hox gene clusters. We hypothesize that disruption of long-range chromatin contacts in mitosis contributes to PcG protein release from most sites, while persistent binding at sites with minimal long-range contacts may nucleate re-establishment of PcG binding and chromosome organization after mitosis. PMID:23284300

  16. A novel role for high-mobility group a proteins in cellular senescence and heterochromatin formation.

    PubMed

    Narita, Masashi; Narita, Masako; Krizhanovsky, Valery; Nuñez, Sabrina; Chicas, Agustin; Hearn, Stephen A; Myers, Michael P; Lowe, Scott W

    2006-08-11

    Cellular senescence is a stable state of proliferative arrest that provides a barrier to malignant transformation and contributes to the antitumor activity of certain chemotherapies. Senescent cells can accumulate senescence-associated heterochromatic foci (SAHFs), which may provide a chromatin buffer that prevents activation of proliferation-associated genes by mitogenic transcription factors. Surprisingly, we show that the High-Mobility Group A (HMGA) proteins, which can promote tumorigenesis, accumulate on the chromatin of senescent fibroblasts and are essential structural components of SAHFs. HMGA proteins cooperate with the p16(INK4a) tumor suppressor to promote SAHF formation and proliferative arrest and stabilize senescence by contributing to the repression of proliferation-associated genes. These antiproliferative activities are canceled by coexpression of the HDM2 and CDK4 oncogenes, which are often coamplified with HMGA2 in human cancers. Our results identify a component of the senescence machinery that contributes to heterochromatin formation and imply that HMGA proteins also act in tumor suppressor networks.

  17. Dichotomous Life of DNA Binding High Mobility Group Box1 Protein in Human Health and Disease.

    PubMed

    Lohani, Neelam; Rajeswari, Moganty R

    2016-01-01

    The High mobility group box 1 (HMGB1) protein is an extremely versatile, highly conserved nuclear protein, with its unique intracellular and extracellular functions mediated by its relatively simple domain structure. Within the nucleus, HMGB1 binds to DNA minor groove in a nonspecific manner and causes bends in the double helix thus helps in recruiting a number of DNA binding protein and transcription factors, to facilitate transcription of various genes. HMGB1 also helps in DNA repair, chromatin remodeling, V (D) J recombination, and assembly of nucleosome on the chromatin. On contrary, under pathological conditions HMGB1 displays inflammatory response by interaction with specific cell surface receptors like RAGE, TLR-4, TLR9, and TLR2 and activates NF-kB downstream signaling pathways. The upregulation of HMGB1 is directly associated with the pathogenesis of cancer, sepsis, ischemia, hemorrhagic shock, anorexia, rheumatic disease, periodontal disease etc. Therefore, HMGB1 has been considered as a promising target in the treatment of various human diseases. The interest in HMGB1 is evident and reflected in the exponential increase in the recent publications, and therefore there is a need for an update on the understanding of the role of HMGB1 in pathogenesis and its potential application of HMGB1 as a therapeutic target in a number of human diseases.

  18. Requirement for sex comb on midleg protein interactions in Drosophila polycomb group repression.

    PubMed Central

    Peterson, Aidan J; Mallin, Daniel R; Francis, Nicole J; Ketel, Carrie S; Stamm, Joyce; Voeller, Rochus K; Kingston, Robert E; Simon, Jeffrey A

    2004-01-01

    The Drosophila Sex Comb on Midleg (SCM) protein is a transcriptional repressor of the Polycomb group (PcG). Although genetic studies establish SCM as a crucial PcG member, its molecular role is not known. To investigate how SCM might link to PcG complexes, we analyzed the in vivo role of a conserved protein interaction module, the SPM domain. This domain is found in SCM and in another PcG protein, Polyhomeotic (PH), which is a core component of Polycomb repressive complex 1 (PRC1). SCM-PH interactions in vitro are mediated by their respective SPM domains. Yeast two-hybrid and in vitro binding assays were used to isolate and characterize >30 missense mutations in the SPM domain of SCM. Genetic rescue assays showed that SCM repressor function in vivo is disrupted by mutations that impair SPM domain interactions in vitro. Furthermore, overexpression of an isolated, wild-type SPM domain produced PcG loss-of-function phenotypes in flies. Coassembly of SCM with a reconstituted PRC1 core complex shows that SCM can partner with PRC1. However, gel filtration chromatography showed that the bulk of SCM is biochemically separable from PH in embryo nuclear extracts. These results suggest that SCM, although not a core component of PRC1, interacts and functions with PRC1 in gene silencing. PMID:15280237

  19. The implication and potential applications of high-mobility group box 1 protein in breast cancer

    PubMed Central

    Sohun, Moonindranath

    2016-01-01

    High-mobility group box 1 protein (HMGB1) is a highly conserved, non-histone and ubiquitous chromosomal protein found enriched in active chromatin forming part of the high mobility group family of proteins and is encoded by the HMGB1 gene (13q12) in human beings. It has various intranuclear and extracellular functions. It plays an important role in the pathogenesis of many diseases including cancer. In 2012, there was approximately 1.67 million new breast cancer cases diagnosed which makes it the second most frequent cancer in the world after lung cancer (25% of all cancers) and the commonest cancer among women. Both pre-clinical and clinical studies have suggested that HMGB1 might be a useful target in the management of breast cancer. This review summarises the structure and functions of HMGB1 and its dual role in carcinogenesis both as a pro-tumorigenic and anti-tumorigenic factor. It also sums up evidence from in vitro and in vivo studies using breast cancer cell lines and samples which demonstrate its influence in radiotherapy, chemotherapy and hormonal therapy in breast cancer. It may have particular importance in HER2 positive and metastatic breast cancer. It might pave the way for new breast cancer treatments through development of novel drugs, use of microRNAs (miRNAs), targeting breast cancer stem cells (CSCs) and breast cancer immunotherapy. It may also play a role in determining breast cancer prognosis. Thus HMGB1 may open up novel avenues in breast cancer management. PMID:27386491

  20. Antagonistic effects of beta-site amyloid precursor protein-cleaving enzymes 1 and 2 on beta-amyloid peptide production in cells.

    PubMed

    Basi, Guriqbal; Frigon, Normand; Barbour, Robin; Doan, Tam; Gordon, Grace; McConlogue, Lisa; Sinha, Sukanto; Zeller, Michelle

    2003-08-22

    The deposition of extracellular beta-amyloid peptide (A beta) in the brain is a pathologic feature of Alzheimer's disease. The beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), an integral membrane aspartyl protease responsible for cleavage of amyloid precursor protein (APP) at the beta-site, promotes A beta production. A second integral membrane aspartyl protease related to BACE1, referred to as beta-site amyloid precursor protein cleaving enzyme 2 (BACE2) has also been demonstrated to cleave APP at the beta-cleavage site in transfected cells. The role of endogenous BACE2 in A beta production remains unresolved. We investigated the role of endogenous BACE2 in A beta production in cells by selective inactivation of its transcripts using RNA interference. We are able to reduce steady state levels for mRNA for each enzyme by >85%, and protein amounts by 88-94% in cells. Selective inactivation of BACE1 by RNA interference results in decreased beta-cleaved secreted APP and A beta peptide secretion from cells, as expected. Selective inactivation of BACE2 by RNAi results in increased beta-cleaved secreted APP and A beta peptide secretion from cells. Simultaneous targeting of both enzymes by RNA interference does not have any net effect on A beta released from cells. Our observations of changes in APP metabolism and A beta are consistent with a role of BACE2 in suppressing A beta production in cells that co-express both enzymes.

  1. Decreased organ failure in patients with severe SIRS and septic shock treated with the platelet-activating factor antagonist TCV-309: a prospective, multicenter, double-blind, randomized phase II trial. TCV-309 Septic Shock Study Group.

    PubMed

    Poeze, M; Froon, A H; Ramsay, G; Buurman, W A; Greve, J W

    2000-10-01

    Sepsis and organ failure remain the main cause of death on the ICU. Sepsis is characterized by a severe inflammatory response, in which platelet-activating factor (PAF) is considered to play an important role. This study investigated whether treatment with the PAF-antagonist TCV-309 reduces morbidity and mortality in patients with septic shock. The study was conducted as a double-blind, randomized, placebo controlled multicenter study. The included patients had to fulfill the SIRS criteria with a clinical suspicion of infection, an admission APACHE II score greater than 15, and shock, defined as a mean arterial pressure <70 mmHg and/or a decrease > or =40 mmHg despite adequate fluid resuscitation. Patients received 1.0 mg/kg TCV-309 or placebo, twice daily, intravenously during 14 days. The prospectively set goals were MOF score, recovery from shock, mortality, and assessment of the safety of the medication. A total of 98 patients were included of which 97 were analyzed on an intention-to-treat basis. The overall survival at day 56 of TCV-309 treated patients was similar compared to placebo treated patients (51.0% vs. 41.7%, P = 0.47). In contrast, the mean percentage of failed organs per patient present after 14 days in the TCV-309 treated patients was significantly lower compared to the placebo treated patients (11.9% vs. 25.1%, P = 0.04), leading to a reduced need for vasopressors, dialysis, and ventilatory support. Furthermore, the mean APACHE-II score during treatment with TCV-309 was significantly lower and the number of patients recovered from shock after day 14 was significantly higher in the TCV-309 treated patient group (2/32 vs. 9/29, P = 0.01). The number of adverse events was not significantly different between the TCV-309 and placebo treated patients. TCV-309 did not change overall mortality of septic shock, however a substantial reduction in organ dysfunction and morbidity, frequently associated with septic shock was achieved, without significant

  2. Photoaffinity analogues of methotrexate as folate antagonist binding probes. 2. Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from murine L1210 cells

    SciTech Connect

    Price, E.M.; Freisheim, J.H.

    1987-07-28

    A membrane-derived component of the methotrexate/one-carbon-reduced folate transport system in murine L1210 cells has been identified by using a photoaffinity analogue of methotrexate. The compound, a radioiodinated 4-azidosalicylyl derivative of the lysine analogue of methotrexate, is transported into murine L1210 cells in a temperature-dependent, sulfhydryl reagent inhibitable manner with a K/sub t/ of 506 +/- 79 nM and a V/sub max/ of 17.9 +/- 4.2 pmol min/sup -1/ (mg of total cellular protein)/sup -1/. Uptake of the iodinated compound at 200 nM is inhibited by low amounts of methotrexate. The parent compounds of the iodinated photoprobe inhibit (/sup 3/H)methotrexate uptake, with the uniodinated 4-azidosalicylyl derivative exhibiting a K/sub i/ of 66 +/- 21 nM. UV irradiation, at 4 /sup 0/C, of a cell suspension that had been incubated with the probe results in the covalent modification of a 46K-48K protein. This can be demonstrated when the plasma membranes from the labeled cells are analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Labeling of this protein occurs half-maximally at a reagent concentration that correlates with the K/sub t/ for transport of the iodinated compound. Protection against labeling of this protein by increasing amounts of methotrexate parallels the concentration dependence of inhibition of photoprobe uptake by methotrexate. Evidence that, in the absence of irradiation and at 37/sup 0/C, the iodinated probe is actually internalized is demonstrated by the labeling of two soluble proteins (M/sub r/ 38K and 21K) derived from the cell homogenate supernatant.

  3. The influence of glutamatergic receptor antagonists on biochemical and ultrastructural changes in myelin membranes of rats subjected to experimental autoimmune encephalomyelitis.

    PubMed

    Dąbrowska-Bouta, Beata; Strużyńska, Lidia; Chalimoniuk, Małgorzata; Frontczak-Baniewicz, Małgorzata; Sulkowski, Grzegorz

    2015-01-01

    Elevated extracellular glutamate in the synaptic cleft causes overactivation of glutamate receptors and kills neurons by an excitotoxic mechanism. Recent studies have shown that glutamate can also lead to toxic injury of white matter oligodendrocytes in myelin sheaths and consequently to axon demyelination. The present study was performed using the rodent model of multiple sclerosis known as experimental autoimmune encephalomyelitis (EAE). The aim of the study was to test the effects of the glutamatergic receptor antagonists amantadine and memantine (antagonists of NMDA receptors), LY 367384 (an antagonist of mGluR1), and MPEP (an mGluR5 antagonist) on the development of neurological symptoms in immunized animals, morphological changes in cerebral myelin, and expression of mRNA of the principal myelin proteins PLP, MBP, MOG, MAG, and CNPase. Pharmacological inhibition of NMDA receptors by amantadine and memantine was found to suppress neurological symptoms in EAE rats, whereas antagonists of the group I metabotropic glutamate receptors (mGluRs G I) did not function positively. In the symptomatic phase of the disease we observed destruction of myelin sheaths via electron microscopy and decreased levels of mRNA for all of the principal myelin proteins. The results reveal that glutamate receptor antagonists have a positive effect on the expression of mRNA MBP and glycoproteins MAG and MOG but not on myelin ultrastructure.

  4. Polycomb group proteins are required to couple seed coat initiation to fertilization

    PubMed Central

    Roszak, Pawel; Köhler, Claudia

    2011-01-01

    Seed development in flowering plants is initiated after a double fertilization event leading to the formation of zygotic embryo and endosperm tissues surrounded by the maternally derived seed coat. Although the seed coat does not take part in the fertilization process it develops immediately after fertilization, implicating a signaling mechanism from zygotic tissues to the surrounding maternal tissues. We addressed the question of the underlying mechanisms repressing seed coat development before fertilization and initiating seed coat development after fertilization by analyzing combinations of mutants that initiate seed development in the absence of fertilization. We discovered that seed coat development is actively repressed before fertilization by dosage-sensitive Polycomb group proteins acting in maternal tissues surrounding the female gametophyte. This repression is relieved after fertilization by a signal that is formed by the sexual endosperm. Fertilization is required for signal formation, as asexually formed endosperm fails to effectively initiate seed coat development in mutants with uncompromised maternal Polycomb group function. Mutants for the MADS-box transcription factor AGL62 initiate embryo and endosperm formation but fail to develop a seed coat, implicating AGL62 expression in the endosperm as a requirement for signal initiation. Together, our results provide evidence that fertilization of the central cell generates a signal that relieves Polycomb group-mediated repression in the surrounding maternal tissues to initiate seed coat formation. PMID:22143805

  5. Contrasting evolutionary patterns of spore coat proteins in two Bacillus species groups are linked to a difference in cellular structure

    PubMed Central

    2013-01-01

    Background The Bacillus subtilis-group and the Bacillus cereus-group are two well-studied groups of species in the genus Bacillus. Bacteria in this genus can produce a highly resistant cell type, the spore, which is encased in a complex protective protein shell called the coat. Spores in the B. cereus-group contain an additional outer layer, the exosporium, which encircles the coat. The coat in B. subtilis spores possesses inner and outer layers. The aim of this study is to investigate whether differences in the spore structures influenced the divergence of the coat protein genes during the evolution of these two Bacillus species groups. Results We designed and implemented a computational framework to compare the evolutionary histories of coat proteins. We curated a list of B. subtilis coat proteins and identified their orthologs in 11 Bacillus species based on phylogenetic congruence. Phylogenetic profiles of these coat proteins show that they can be divided into conserved and labile ones. Coat proteins comprising the B. subtilis inner coat are significantly more conserved than those comprising the outer coat. We then performed genome-wide comparisons of the nonsynonymous/synonymous substitution rate ratio, dN/dS, and found contrasting patterns: Coat proteins have significantly higher dN/dS in the B. subtilis-group genomes, but not in the B. cereus-group genomes. We further corroborated this contrast by examining changes of dN/dS within gene trees, and found that some coat protein gene trees have significantly different dN/dS between the B subtilis-clade and the B. cereus-clade. Conclusions Coat proteins in the B. subtilis- and B. cereus-group species are under contrasting selective pressures. We speculate that the absence of the exosporium in the B. subtilis spore coat effectively lifted a structural constraint that has led to relaxed negative selection pressure on the outer coat. PMID:24283940

  6. High Mobility Group Box Protein-1 correlates with renal function in chronic kidney disease (CKD).

    PubMed

    Bruchfeld, Annette; Qureshi, Abdul Rashid; Lindholm, Bengt; Barany, Peter; Yang, Lihong; Stenvinkel, Peter; Tracey, Kevin J

    2008-01-01

    Chronic kidney disease (CKD) is associated with inflammation and malnutrition and carries a markedly increased risk of cardiovascular disease (CVD). High Mobility Group Box Protein-1 (HMGB-1) is a 30-kDa nuclear and cytosolic protein known as a transcription and growth factor, recently identified as a proinflammatory mediator of tissue injury. Recent data implicates HMGB-1 in endotoxin lethality, rheumatoid arthritis, and atherosclerosis. The aim of this post-hoc, cross-sectional study was to determine whether HMGB-1 serum levels are elevated in CKD patients. The study groups were categorized as follows: 110 patients starting dialysis defined as CKD 5; 67 patients with moderately to severely reduced renal function or CKD 3-4; and 48 healthy controls. High-sensitivity C-reactive-protein (hs-CRP), interleukin-6 (IL-6), tumor necrosis factor (TNF), serum-albumin (S-albumin), hemoglobin A(1c) (HbA(1c)), hemoglobin, subjective global nutritional assessment (SGA), and glomerular filtration rate (GFR) were analyzed. Kruskal-Wallis test was used to compare groups and Spearman's rank correlation test was used for continuous variables. HMGB-1, measured by Western blot, was significantly (P < 0.001) elevated in CKD 5 (146.7 +/- 58.6 ng/mL) and CKD 3-4 (85.6 +/- 31.8) compared with controls (10.9 +/- 10.5). HMGB-1 levels were correlated positively with TNF (Rho = 0.52; P < 0.001), hs-CRP (Rho = 0.38; P < 0.001), IL-6 (Rho = 0.30; P < 0.001), HbA(1c) (Rho = 0.14; P = 0.02) and SGA (Rho = 0.21; P = 0.002) and negatively correlated with GFR (Rho = -0.69; P = 0.0001), Hb (Rho = -0.60; P < 0.001), S-albumin (Rho = -0.31; P < 0.001). The current study has revealed that HMGB-1 is elevated significantly in CKD patients and correlates with GFR as well as markers of inflammation and malnutrition. Future studies may delineate whether HMGB-1 is also a marker of disease activity and severity as well as a predictor of outcome in CKD.

  7. Adamantane-based amphiphiles (ADAs) for membrane protein study: importance of a detergent hydrophobic group in membrane protein solubilisation.

    PubMed

    Chae, Pil Seok; Bae, Hyoung Eun; Das, Manabendra

    2014-10-21

    We prepared adamantane-containing amphiphiles and evaluated them using a large membrane protein complex in terms of protein solubilisation and stabilization efficacy. These agents were superior to conventional detergents, especially in terms of the membrane protein solubilisation efficiency, implying a new detergent structure-property relationship.

  8. Antagonist-Elicited Cannabis Withdrawal in Humans

    PubMed Central

    Gorelick, David A.; Goodwin, Robert S.; Schwilke, Eugene; Schwope, David M.; Darwin, William D.; Kelly, Deanna L.; McMahon, Robert P.; Liu, Fang; Ortemann-Renon, Catherine; Bonnet, Denis; Huestis, Marilyn A.

    2013-01-01

    Cannabinoid CB1 receptor antagonists have potential therapeutic benefits, but antagonist-elicited cannabis withdrawal has not been reported in humans. Ten male daily cannabis smokers received 8 days of increasingly frequent 20-mg oral Δ9-tetrahydrocannabinol (THC) dosages (40–120 mg/d) around-the-clock to standardize cannabis dependence while residing on a closed research unit. On the ninth day, double-blind placebo or 20- (suggested therapeutic dose) or 40-mg oral rimonabant, a CB1-cannabinoid receptor antagonist, was administered. Cannabis withdrawal signs and symptoms were assessed before and for 23.5 hours after rimonabant. Rimonabant, THC, and 11-hydroxy-THC plasma concentrations were quantified by mass spectrometry. The first 6 subjects received 20-mg rimonabant (1 placebo); the remaining 4 subjects received 40-mg rimonabant (1 placebo). Fourteen subjects enrolled; 10 completed before premature termination because of withdrawal of rimonabant from clinical development. Three of 5 subjects in the 20-mg group, 1 of 3 in the 40-mg group, and none of 2 in the placebo group met the prespecified withdrawal criterion of 150% increase or higher in at least 3 visual analog scales for cannabis withdrawal symptoms within 3 hours of rimonabant dosing. There were no significant associations between visual analog scale, heart rate, or blood pressure changes and peak rimonabant plasma concentration, area-under-the-rimonabant-concentration-by-time curve (0–8 hours), or peak rimonabant/THC or rimonabant/(THC + 11-hydroxy-THC) plasma concentration ratios. In summary, prespecified criteria for antagonist-elicited cannabis withdrawal were not observed at the 20- or 40-mg rimonabant doses. These data do not preclude antagonist-elicited withdrawal at higher rimonabant doses. PMID:21869692

  9. Antagonist-elicited cannabis withdrawal in humans.

    PubMed

    Gorelick, David A; Goodwin, Robert S; Schwilke, Eugene; Schwope, David M; Darwin, William D; Kelly, Deanna L; McMahon, Robert P; Liu, Fang; Ortemann-Renon, Catherine; Bonnet, Denis; Huestis, Marilyn A

    2011-10-01

    Cannabinoid CB1 receptor antagonists have potential therapeutic benefits, but antagonist-elicited cannabis withdrawal has not been reported in humans. Ten male daily cannabis smokers received 8 days of increasingly frequent 20-mg oral Δ⁹-tetrahydrocannabinol (THC) dosages (40-120 mg/d) around-the-clock to standardize cannabis dependence while residing on a closed research unit. On the ninth day, double-blind placebo or 20- (suggested therapeutic dose) or 40-mg oral rimonabant, a CB1-cannabinoid receptor antagonist, was administered. Cannabis withdrawal signs and symptoms were assessed before and for 23.5 hours after rimonabant. Rimonabant, THC, and 11-hydroxy-THC plasma concentrations were quantified by mass spectrometry. The first 6 subjects received 20-mg rimonabant (1 placebo); the remaining 4 subjects received 40-mg rimonabant (1 placebo). Fourteen subjects enrolled; 10 completed before premature termination because of withdrawal of rimonabant from clinical development. Three of 5 subjects in the 20-mg group, 1 of 3 in the 40-mg group, and none of 2 in the placebo group met the prespecified withdrawal criterion of 150% increase or higher in at least 3 visual analog scales for cannabis withdrawal symptoms within 3 hours of rimonabant dosing. There were no significant associations between visual analog scale, heart rate, or blood pressure changes and peak rimonabant plasma concentration, area-under-the-rimonabant-concentration-by-time curve (0-8 hours), or peak rimonabant/THC or rimonabant/(THC + 11-hydroxy-THC) plasma concentration ratios. In summary, prespecified criteria for antagonist-elicited cannabis withdrawal were not observed at the 20- or 40-mg rimonabant doses. These data do not preclude antagonist-elicited withdrawal at higher rimonabant doses.

  10. PPARα/γ antagonists reverse the ameliorative effects of osthole on hepatic lipid metabolism and inflammatory response in steatohepatitic rats.

    PubMed

    Zhao, Xi; Wang, Feng; Zhou, Ruijun; Zhu, Zengyan; Xie, Meilin

    2017-02-25

    Our previous studies have indicated that osthole may ameliorate the hepatic lipid metabolism and inflammatory response in nonalcoholic steatohepatitic rats, but the underlying mechanisms remain unclear. This study aimed to determine whether the effects of osthole were mediated by the activation of hepatic peroxisome proliferator-activated receptor α/γ (PPARα/γ). A rat model with steatohepatitis was induced by orally feeding high-fat and high-sucrose emulsion for 6 weeks. These experimental rats were then treated with osthole (20 mg/kg), PPARα antagonist MK886 (1 mg/kg) plus osthole (20 mg/kg), PPARγ antagonist GW9662 (1 mg/kg) plus osthole (20 mg/kg) and MK886 (1 mg/kg) plus GW9662 (1 mg/kg) plus osthole (20 mg/kg) for 4 weeks. The results showed that after osthole treatment, the hepatic triglycerides, free fatty acids, tumor necrosis factor-α, monocyte chemotactic protein-1, interleukin-6 (IL-6), IL-8 and liver index decreased by 52.3, 31.0, 32.4, 28.9, 36.3, 29.3 and 29.9%, respectively, and the score of steatohepatitis also decreased by 70.0%, indicating that osthole improved the hepatic steatosis and inflammation. However, these effects of osthole were reduced or abrogated after simultaneous addition of the specific PPARα antagonist MK886 or/and the PPARγ antagonist GW9662, especially in the co-PPARα/γ antagonists-treated group. Importantly, the osthole-induced hepatic expressions of PPARα/γ proteins were decreased, and the osthole-regulated hepatic expressions of lipogenic and inflammatory gene proteins were also reversed by PPARα/γ antagonist treatment. These findings demonstrated that the ameliorative effect of osthole on nonalcoholic steatohepatitis was mediated by PPARα/γ activation, and osthole might be a natural dual PPARα/γ activator.

  11. The Investigation and Characterization of the Group 3 [Nickel-Iron]-Hydrogenases Using Protein Film Electrochemistry

    NASA Astrophysics Data System (ADS)

    McIntosh, Chelsea Lee

    Hydrogenases, the enzymes that reversibly convert protons and electrons to hydrogen, are used in all three domains of life. [NiFe]-hydrogenases are considered best suited for biotechnological applications because of their reversible inactivation with oxygen. Phylogenetically, there are four groups of [NiFe]-hydrogenases. The best characterized group, "uptake" hydrogenases, are membrane-bound and catalyze hydrogen oxidation in vivo. In contrast, the group 3 [NiFe]-hydrogenases are heteromultimeric, bifunctional enzymes that fulfill various cellular roles. In this dissertation, protein film electrochemistry (PFE) is used to characterize the catalytic properties of two group 3 [NiFe]-hydrogenases: HoxEFUYH from Synechocystsis sp. PCC 6803 and SHI from Pyrococcus furiosus. First, HoxEFUYH is shown to be biased towards hydrogen production. Upon exposure to oxygen, HoxEFUYH inactivates to two states, both of which can be reactivated on the timescale of seconds. Second, we show that PfSHI is the first example of an oxygen tolerant [NiFe]-hydrogenase that produces two inactive states upon exposure to oxygen. Both inactive states are analogous to those characterized for HoxEFUYH, but oxygen exposed PfSHI produces a greater fraction that reactivates at high potentials, enabling hydrogen oxidation in the presence of oxygen. Third, it is shown that removing the NAD(P)-reducing subunits from PfSHI leads to a decrease in bias towards hydrogen oxidation and renders the enzyme oxygen sensitive. Both traits are likely due to impaired intramolecular electron transfer. Mechanistic hypotheseses for these functional differences are considered.

  12. Methyl group turnover on methyl-accepting chemotaxis proteins during chemotaxis by Bacillus subtilis

    SciTech Connect

    Thoelke, M.S.; Casper, J.M.; Ordal, G.W. )

    1990-02-05

    The addition of attractant to Bacillus subtilis briefly exposed to radioactive methionine causes an increase of labeling of the methyl-accepting chemotaxis proteins. The addition of attractant to cells radiolabeled for longer times shows no change in the extent of methylation. Therefore, the increase in labeling for the briefly labeled cells is due to an increased turnover of methyl groups caused by attractant. All amino acids gave enhanced turnover. This turnover lasted for a prolonged time, probably spanning the period of smooth swimming caused by the attractant addition. Repellent did not affect the turnover when added alone or simultaneously with attractant. Thus, for amino acid attractants, the turnover is probably the excitatory signal, which is seen to extend long into or throughout the adaptation period, not just at the start of it.

  13. A summary of the measured pK values of the ionizable groups in folded proteins

    PubMed Central

    Grimsley, Gerald R; Scholtz, J Martin; Pace, C Nick

    2009-01-01

    We tabulated 541 measured pK values reported in the literature for the Asp, Glu, His, Cys, Tyr, and Lys side chains, and the C and N termini of 78 folded proteins. The majority of these values are for the Asp, Glu, and His side chains. The average pK values are Asp 3.5 ± 1.2 (139); Glu 4.2 ± 0.9 (153); His 6.6 ± 1.0 (131); Cys 6.8 ± 2.7 (25); Tyr 10.3 ± 1.2 (20); Lys 10.5 ± 1.1 (35); C-terminus 3.3 ± 0.8 (22) and N-terminus 7.7 ± 0.5 (16). We compare these results with the measured pK values of these groups in alanine pentapeptides, and comment on our overall findings. PMID:19177368

  14. The Dimerization State of the Mammalian High Mobility Group Protein AT-Hook 2 (HMGA2)

    PubMed Central

    Frost, Lorraine; Baez, Maria A. M.; Harrilal, Christopher; Garabedian, Alyssa; Fernandez-Lima, Francisco; Leng, Fenfei

    2015-01-01

    The mammalian high mobility group protein AT-hook 2 (HMGA2) is a chromosomal architectural transcription factor involved in cell transformation and oncogenesis. It consists of three positively charged “AT-hooks” and a negatively charged C-terminus. Sequence analyses, circular dichroism experiments, and gel-filtration studies showed that HMGA2, in the native state, does not have a defined secondary or tertiary structure. Surprisingly, using combined approaches of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemical cross-linking, analytical ultracentrifugation, fluorescence resonance energy transfer (FRET), and mass spectrometry, we discovered that HMGA2 is capable of self-associating into homodimers in aqueous buffer solution. Our results showed that electrostatic interactions between the positively charged “AT-hooks” and the negatively charged C-terminus greatly contribute to the homodimer formation. PMID:26114780

  15. Polycomb group protein ezh2 controls actin polymerization and cell signaling.

    PubMed

    Su, I-hsin; Dobenecker, Marc-Werner; Dickinson, Ephraim; Oser, Matthew; Basavaraj, Ashwin; Marqueron, Raphael; Viale, Agnes; Reinberg, Danny; Wülfing, Christoph; Tarakhovsky, Alexander

    2005-05-06

    Polycomb group protein Ezh2, one of the key regulators of development in organisms from flies to mice, exerts its epigenetic function through regulation of histone methylation. Here, we report the existence of the cytosolic Ezh2-containing methyltransferase complex and tie the function of this complex to regulation of actin polymerization in various cell types. Genetic evidence supports the essential role of cytosolic Ezh2 in actin polymerization-dependent processes such as antigen receptor signaling in T cells and PDGF-induced dorsal circular ruffle formation in fibroblasts. Revealed function of Ezh2 points to a broader usage of lysine methylation in regulation of both nuclear and extra-nuclear signaling processes.

  16. Tree ferns: monophyletic groups and their relationships as revealed by four protein-coding plastid loci.

    PubMed

    Korall, Petra; Pryer, Kathleen M; Metzgar, Jordan S; Schneider, Harald; Conant, David S

    2006-06-01

    Tree ferns are a well-established clade within leptosporangiate ferns. Most of the 600 species (in seven families and 13 genera) are arborescent, but considerable morphological variability exists, spanning the giant scaly tree ferns (Cyatheaceae), the low, erect plants (Plagiogyriaceae), and the diminutive endemics of the Guayana Highlands (Hymenophyllopsidaceae). In this study, we investigate phylogenetic relationships within tree ferns based on analyses of four protein-coding, plastid loci (atpA, atpB, rbcL, and rps4). Our results reveal four well-supported clades, with genera of Dicksoniaceae (sensu ) interspersed among them: (A) (Loxomataceae, (Culcita, Plagiogyriaceae)), (B) (Calochlaena, (Dicksonia, Lophosoriaceae)), (C) Cibotium, and (D) Cyatheaceae, with Hymenophyllopsidaceae nested within. How these four groups are related to one other, to Thyrsopteris, or to Metaxyaceae is weakly supported. Our results show that Dicksoniaceae and Cyatheaceae, as currently recognised, are not monophyletic and new circumscriptions for these families are needed.

  17. High mobility group 1 (HMG1) protein in mouse preimplantation embryos.

    PubMed

    Spada, F; Brunet, A; Mercier, Y; Renard, J P; Bianchi, M E; Thompson, E M

    1998-08-01

    High mobility group 1 protein (HMG1) has traditionally been considered a structural component of chromatin, possibly similar in function to histone H1. In fact, at the onset of Xenopus and Drosophila development, HMG1 appears to substitute for histone H1: HMG1 is abundant when histone H1 is absent after the midblastula transition histone H1 largely replaces HMG1. We show that in early mouse embryos the expression patterns of HMG1 and histone H1 are not complementary. Instead, HMG1 content increases after zygotic genome activation at the same time as histone H1. HMG1 does not remain associated to mitotic chromosomes either in embryos or somatic cells. These results argue against a shared structural role for HMG1 and histone H1 in mammalian chromatin.

  18. Correlates of Protection for M Protein-Based Vaccines against Group A Streptococcus

    PubMed Central

    Smeesters, Pierre R.; Frost, Hannah R. C.; Steer, Andrew C.

    2015-01-01

    Group A streptococcus (GAS) is known to cause a broad spectrum of illness, from pharyngitis and impetigo, to autoimmune sequelae such as rheumatic heart disease, and invasive diseases. It is a significant cause of infectious disease morbidity and mortality worldwide, but no efficacious vaccine is currently available. Progress in GAS vaccine development has been hindered by a number of obstacles, including a lack of standardization in immunoassays and the need to define human correlates of protection. In this review, we have examined the current immunoassays used in both GAS and other organisms, and explored the various challenges in their implementation in order to propose potential future directions to identify a correlate of protection and facilitate the development of M protein-based vaccines, which are currently the main GAS vaccine candidates. PMID:26101780

  19. Polycomb Group Protein Ezh2 Regulates Hepatic Progenitor Cell Proliferation and Differentiation in Murine Embryonic Liver

    PubMed Central

    Ueno, Yasuharu; Nakata, Susumu; Obana, Yuta; Sekine, Keisuke; Zheng, Yun-Wen; Takebe, Takanori; Isono, Kyoichi; Koseki, Haruhiko; Taniguchi, Hideki

    2014-01-01

    In embryonic liver, hepatic progenitor cells are actively proliferating and generate a fundamental cellular pool for establishing parenchymal components. However, the molecular basis for the expansion of the progenitors maintaining their immature state remains elusive. Polycomb group proteins regulate gene expression throughout the genome by modulating of chromatin structure and play crucial roles in development. Enhancer of zeste homolog 2 (Ezh2), a key component of polycomb group proteins, catalyzes tri-methylation of lysine 27 of histone H3 (H3K27me3), which trigger the gene suppression. In the present study, we investigated a role of Ezh2 in the regulation of the expanding hepatic progenitor population in vivo. We found that Ezh2 is highly expressed in the actively proliferating cells at the early developmental stage. Using a conditional knockout mouse model, we show that the deletion of the SET domain of Ezh2, which is responsible for catalytic induction of H3K27me3, results in significant reduction of the total liver size, absolute number of liver parenchymal cells, and hepatic progenitor cell population in size. A clonal colony assay in the hepatic progenitor cells directly isolated from in vivo fetal livers revealed that the bi-potent clonogenicity was significantly attenuated by the Ezh2 loss of function. Moreover, a marker expression based analysis and a global gene expression analysis showed that the knockout of Ezh2 inhibited differentiation to hepatocyte with reduced expression of a number of liver-function related genes. Taken together, our results indicate that Ezh2 is required for the hepatic progenitor expansion in vivo, which is essential for the functional maturation of embryonic liver, through its activity for catalyzing H3K27me3. PMID:25153170

  20. Nonhistone nuclear high mobility group proteins 14 and 17 stabilize nucleosome core particles

    SciTech Connect

    Paton, A.E.; Wilkinson-Singley, E.; Olins, D.W.

    1983-11-10

    Nucleosome core particles form well defined complexes with the nuclear nonhistone proteins HMG 14 or 17. The binding of HMG 14 or 17 to nucleosomes results in greater stability of the nucleosomal DNA as shown by circular dichroism and thermal denaturation. Under appropriate conditions the binding is cooperative, and cooperativity is ionic strength dependent. The specificity and cooperative transitions of high mobility group (HMG) binding are preserved in 1 M urea. Specificity is lost in 4 M urea. Thermal denaturation and circular dichroism show a dramatic reversal of the effects of urea on nucleosomes when HMG 14 or 17 is bound, indicating stabilization of the nucleosome by HMG proteins. Complexes formed between reconstructed nucleosomes containing purified inner histones plus poly (dA-dT) and HMG 14 or 17 demonstrate that the HMG binding site requires only DNA and histones. Electron microscopy reveals no major structural alterations in the nucleosome upon binding of HMG 14 or 17. Cross-linking the nucleosome extensively with formaldehyde under cooperative HMG binding conditions does not prevent the ionic strength-dependent shift to noncooperative binding. This suggests mechanisms other than internal nucleosome conformational changes may be involved in cooperative HMG binding.

  1. High mobility group protein 1: A collaborator in nucleosome dynamics and estrogen-responsive gene expression

    PubMed Central

    Scovell, William M

    2016-01-01

    High mobility group protein 1 (HMGB1) is a multifunctional protein that interacts with DNA and chromatin to influence the regulation of transcription, DNA replication and repair and recombination. We show that HMGB1 alters the structure and stability of the canonical nucleosome (N) in a nonenzymatic, adenosine triphosphate-independent manner. As a result, the canonical nucleosome is converted to two stable, physically distinct nucleosome conformers. Although estrogen receptor (ER) does not bind to its consensus estrogen response element within a nucleosome, HMGB1 restructures the nucleosome to facilitate strong ER binding. The isolated HMGB1-restructured nucleosomes (N’ and N’’) remain stable and exhibit a number of characteristics that are distinctly different from the canonical nucleosome. These findings complement previous studies that showed (1) HMGB1 stimulates in vivo transcriptional activation at estrogen response elements and (2) knock down of HMGB1 expression by siRNA precipitously reduced transcriptional activation. The findings indicate that a major facet of the mechanism of HMGB1 action involves a restructuring of aspects of the nucleosome that appear to relax structural constraints within the nucleosome. The findings are extended to reveal the differences between ER and the other steroid hormone receptors. A working proposal outlines mechanisms that highlight the multiple facets that HMGB1 may utilize in restructuring the nucleosome. PMID:27247709

  2. Pluripotency Factors and Polycomb Group Proteins Repress Aryl Hydrocarbon Receptor Expression in Murine Embryonic Stem Cells

    PubMed Central

    Ko, Chia-I; Wang, Qin; Fan, Yunxia; Xia, Ying; Puga, Alvaro

    2013-01-01

    The aryl hydrocarbon receptor (AHR) is a transcription factor and environmental sensor that regulates expression of genes involved in drug-metabolism and cell cycle regulation. Chromatin immunoprecipitation analyses, Ahr ablation in mice and studies with orthologous genes in invertebrates suggest that AHR may also play a significant role in embryonic development. To address this hypothesis, we studied the regulation of Ahr expression in mouse embryonic stem cells and their differentiated progeny. In ES cells, interactions between OCT3/4, NANOG, SOX2 and Polycomb Group proteins at the Ahr promoter repress AHR expression, which can also be repressed by ectopic expression of reprogramming factors in hepatoma cells. In ES cells, unproductive RNA polymerase II binds at the Ahr transcription start site and drives the synthesis of short abortive transcripts. Activation of Ahr expression during differentiation follows from reversal of repressive marks in Ahr promoter chromatin, release of pluripotency factors and PcG proteins, binding of Sp factors, establishment of histone marks of open chromatin, and engagement of active RNAPII to drive full-length RNA transcript elongation. Our results suggest that reversible Ahr repression in ES cells holds the gene poised for expression and allows for a quick switch to activation during embryonic development. PMID:24316986

  3. Antagonistic control of the turnover pathway for the global regulatory sRNA CsrB by the CsrA and CsrD proteins

    PubMed Central

    Vakulskas, Christopher A.; Leng, Yuanyuan; Abe, Hazuki; Amaki, Takumi; Okayama, Akihiro; Babitzke, Paul; Suzuki, Kazushi; Romeo, Tony

    2016-01-01

    The widely conserved protein CsrA (carbon storage regulator A) globally regulates bacterial gene expression at the post-transcriptional level. In many species, CsrA activity is governed by untranslated sRNAs, CsrB and CsrC in Escherichia coli, which bind to multiple CsrA dimers, sequestering them from lower affinity mRNA targets. Both the synthesis and turnover of CsrB/C are regulated. Their turnover requires the housekeeping endonuclease RNase E and is activated by the presence of a preferred carbon source via the binding of EIIAGlc of the glucose transport system to the GGDEF-EAL domain protein CsrD. We demonstrate that the CsrB 3′ segment contains the features necessary for CsrD-mediated decay. RNase E cleavage in an unstructured segment located immediately upstream from the intrinsic terminator is necessary for subsequent degradation to occur. CsrA stabilizes CsrB against RNase E cleavage by binding to two canonical sites adjacent to the necessary cleavage site, while CsrD acts by overcoming CsrA-mediated protection. Our genetic, biochemical and structural studies establish a molecular framework for sRNA turnover by the CsrD-RNase E pathway. We propose that CsrD evolution was driven by the selective advantage of decoupling Csr sRNA decay from CsrA binding, connecting it instead to the availability of a preferred carbon source. PMID:27235416

  4. Identification of two auto-cleavage products of nonstructural protein 1 (nsp1) in porcine reproductive and respiratory syndrome virus infected cells: nsp1 function as interferon antagonist

    SciTech Connect

    Chen, Z.; Lawson, S.; Sun, Z.; Zhou, X.; Guan, X.; Christopher-Hennings, J.; Nelson, E.A.; Fang, Y.

    2010-03-01

    The porcine reproductive and respiratory syndrome virus nsp1 is predicted to be auto-cleaved from the replicase polyprotein into nsp1alpha and nsp1beta subunits. In infected cells, we detected the actual existence of nsp1alpha and nsp1beta. Cleavage sites between nsp1alpha/nsp1beta and nsp1beta/nsp2 were identified by protein microsequencing analysis. Time course study showed that nsp1alpha and nsp1beta mainly localize into the cell nucleus after 10 h post infection. Further analysis revealed that both proteins dramatically inhibited IFN-beta expression. The nsp1beta was observed to significantly inhibit expression from an interferon-stimulated response element promoter after Sendai virus infection or interferon treatment. It was further determined to inhibit nuclear translocation of STAT1 in the JAK-STAT signaling pathway. These results demonstrated that nsp1beta has ability to inhibit both interferon synthesis and signaling, while nsp1alpha alone strongly inhibits interferon synthesis. These findings provide important insights into mechanisms of nsp1 in PRRSV pathogenesis and its impact in vaccine development.

  5. Equine arteritis virus does not induce interferon production in equine endothelial cells: identification of nonstructural protein 1 as a main interferon antagonist.

    PubMed

    Go, Yun Young; Li, Yanhua; Chen, Zhenhai; Han, Mingyuan; Yoo, Dongwan; Fang, Ying; Balasuriya, Udeni B R

    2014-01-01

    The objective of this study was to investigate the effect of equine arteritis virus (EAV) on type I interferon (IFN) production. Equine endothelial cells (EECs) were infected with the virulent Bucyrus strain (VBS) of EAV and expression of IFN-β was measured at mRNA and protein levels by quantitative real-time RT-PCR and IFN bioassay using vesicular stomatitis virus expressing the green fluorescence protein (VSV-GFP), respectively. Quantitative RT-PCR results showed that IFN-β mRNA levels in EECs infected with EAV VBS were not increased compared to those in mock-infected cells. Consistent with quantitative RT-PCR, Sendai virus- (SeV-) induced type I IFN production was inhibited by EAV infection. Using an IFN-β promoter-luciferase reporter assay, we subsequently demonstrated that EAV nsps 1, 2, and 11 had the capability to inhibit type I IFN activation. Of these three nsps, nsp1 exhibited the strongest inhibitory effect. Taken together, these data demonstrate that EAV has the ability to suppress the type I IFN production in EECs and nsp1 may play a critical role to subvert the equine innate immune response.

  6. Multiple Targeting Approaches on Histamine H3 Receptor Antagonists

    PubMed Central

    Khanfar, Mohammad A.; Affini, Anna; Lutsenko, Kiril; Nikolic, Katarina; Butini, Stefania; Stark, Holger

    2016-01-01

    With the very recent market approval of pitolisant (Wakix®), the interest in clinical applications of novel multifunctional histamine H3 receptor antagonists has clearly increased. Since histamine H3 receptor antagonists in clinical development have been tested for a variety of different indications, the combination of pharmacological properties in one molecule for improved pharmacological effects and reduced unwanted side-effects is rationally based on the increasing knowledge on the complex neurotransmitter regulations. The polypharmacological approaches on histamine H3 receptor antagonists on different G-protein coupled receptors, transporters, enzymes as well as on NO-signaling mechanism are described, supported with some lead structures. PMID:27303254

  7. Multiple Targeting Approaches on Histamine H3 Receptor Antagonists.

    PubMed

    Khanfar, Mohammad A; Affini, Anna; Lutsenko, Kiril; Nikolic, Katarina; Butini, Stefania; Stark, Holger

    2016-01-01

    With the very recent market approval of pitolisant (Wakix®), the interest in clinical applications of novel multifunctional histamine H3 receptor antagonists has clearly increased. Since histamine H3 receptor antagonists in clinical development have been tested for a variety of different indications, the combination of pharmacological properties in one molecule for improved pharmacological effects and reduced unwanted side-effects is rationally based on the increasing knowledge on the complex neurotransmitter regulations. The polypharmacological approaches on histamine H3 receptor antagonists on different G-protein coupled receptors, transporters, enzymes as well as on NO-signaling mechanism are described, supported with some lead structures.

  8. The trithorax group proteins Kismet and ASH1 promote H3K36 dimethylation to counteract Polycomb group repression in Drosophila

    PubMed Central

    Dorighi, Kristel M.; Tamkun, John W.

    2013-01-01

    Members of the Polycomb group of repressors and trithorax group of activators maintain heritable states of transcription by modifying nucleosomal histones or remodeling chromatin. Although tremendous progress has been made toward defining the biochemical activities of Polycomb and trithorax group proteins, much remains to be learned about how they interact with each other and the general transcription machinery to maintain on or off states of gene expression. The trithorax group protein Kismet (KIS) is related to the SWI/SNF and CHD families of chromatin remodeling factors. KIS promotes transcription elongation, facilitates the binding of the trithorax group histone methyltransferases ASH1 and TRX to active genes, and counteracts repressive methylation of histone H3 on lysine 27 (H3K27) by Polycomb group proteins. Here, we sought to clarify the mechanism of action of KIS and how it interacts with ASH1 to antagonize H3K27 methylation in Drosophila. We present evidence that KIS promotes transcription elongation and counteracts Polycomb group repression via distinct mechanisms. A chemical inhibitor of transcription elongation, DRB, had no effect on ASH1 recruitment or H3K27 methylation. Conversely, loss of ASH1 function had no effect on transcription elongation. Mutations in kis cause a global reduction in the di- and tri-methylation of histone H3 on lysine 36 (H3K36) - modifications that antagonize H3K27 methylation in vitro. Furthermore, loss of ASH1 significantly decreases H3K36 dimethylation, providing further evidence that ASH1 is an H3K36 dimethylase in vivo. These and other findings suggest that KIS antagonizes Polycomb group repression by facilitating ASH1-dependent H3K36 dimethylation. PMID:24004944

  9. Proline/arginine-rich end leucine-rich repeat protein N-terminus is a novel osteoclast antagonist that counteracts bone loss.

    PubMed

    Rucci, Nadia; Capulli, Mattia; Ventura, Luca; Angelucci, Adriano; Peruzzi, Barbara; Tillgren, Viveka; Muraca, Maurizio; Heinegård, Dick; Teti, Anna

    2013-09-01

    (hbd) PRELP is a peptide corresponding to the N-terminal heparin binding domain of the matrix protein proline/arginine-rich end leucine-rich repeat protein (PRELP). (hbd) PRELP inhibits osteoclastogenesis entering pre-fusion osteoclasts through a chondroitin sulfate- and annexin 2-dependent mechanism and reducing the nuclear factor-κB transcription factor activity. In this work, we hypothesized that (hbd) PRELP could have a pharmacological relevance, counteracting bone loss in a variety of in vivo models of bone diseases induced by exacerbated osteoclast activity. In healthy mice, we demonstrated that the peptide targeted the bone and increased trabecular bone mass over basal level. In mice treated with retinoic acid to induce an acute increase of osteoclast formation, the peptide consistently antagonized osteoclastogenesis and prevented the increase of the serum levels of the osteoclast-specific marker tartrate-resistant acid phosphatase. In ovariectomized mice, in which osteoclast activity was chronically enhanced by estrogen deficiency, (hbd) PRELP counteracted exacerbated osteoclast activity and bone loss. In mice carrying osteolytic bone metastases, in which osteoclastogenesis and bone resorption were enhanced by tumor cell-derived factors, (hbd) PRELP reduced the incidence of osteolytic lesions, both preventively and curatively, with mechanisms involving impaired tumor cell homing to bone and tumor growth in the bone microenvironment. Interestingly, in tumor-bearing mice, (hbd) PRELP also inhibited breast tumor growth in orthotopic sites and development of metastatic disease in visceral organs, reducing cachexia and improving survival especially when administered preventively. (hbd) PRELP was retained in the tumor tissue and appeared to affect tumor growth by interacting with the microenvironment rather than by directly affecting the tumor cells. Because safety studies and high-dose treatments revealed no adverse effects, (hbd) PRELP could be employed as a

  10. Hydrophobic tendency of polar group hydration as a major force in type I antifreeze protein recognition.

    PubMed

    Yang, Cheng; Sharp, Kim A

    2005-05-01

    The random network model of water quantitatively describes the different hydration heat capacities of polar and apolar solutes in terms of distortions of the water-water hydrogen bonding angle in the first hydration shell (Gallagher and Sharp, JACS 2003;125:9853). The distribution of this angle in pure water is bimodal, with a low-angle population and high-angle population. Polar solutes increase the high-angle population while apolar solutes increase the low-angle population. The ratio of the two populations quantifies the hydrophobicity of the solute and provides a sensitive measure of water structural distortions. This method of analysis is applied to study hydration of type I thermal hysteresis protein (THP) from winter flounder and three quadruple mutants of four threonine residues at positions 2, 13, 24, and 35. Wild-type and two mutants (VVVV and AAAA) have antifreeze (thermal hysteresis) activity, while the other mutant (SSSS) has no activity. The analysis reveals significant differences in the hydration structure of the ice-binding site. For the SSSS mutant, polar groups have a typical polar-like hydration, that is, more high-angle H-bonds than bulk water. For the wild-type and active mutants, polar groups have unusual, very apolar-like hydration, that is, more low-angle H-bonds than bulk water. This pattern of hydration was seen previously in the structurally distinct type III THPs (Yang & Sharp Biophys Chem 2004;109:137), suggesting for the first time a general mechanism for different THP classes. The specific shape, residue size, and clustering of both polar and apoler groups are essential for an active ice binding surface.

  11. Preoperative prognostic values of α-fetoprotein (AFP) and protein induced by vitamin K absence or antagonist-II (PIVKA-II) in patients with hepatocellular carcinoma for living donor liver transplantation

    PubMed Central

    Kim, Seok-Hwan; Kim, Wan-Joon; Kang, Woo-Hyoung; Kwon, Jae Hyun; Jwa, Eun Kyung; Cho, Hwui-Dong; Ha, Su-Min; Chung, Yong-Kyu; Lee, Sung-Gyu

    2016-01-01

    Background Adult living donor liver transplantation (LDLT) is one of the best treatments for hepatocellular carcinoma (HCC). However, when recurrence of HCC after LDLT occurs, the prognosis is poor because of rapid progression. Preoperative level of α-fetoprotein (AFP) and protein induced by vitamin K antagonist-II (PIVKA-II) reportedly correlate with recurrence of HCC after LDLT. Methods We examined AFP and PIVKA-II preoperatively as predictors of HCC recurrence in 461 patients who underwent LDLT using right liver graft for HCC from May 2007 to December 2013. Results Among these, 77 patients (16.7%) who experienced recurrence were retrospectively reviewed. Multivariate analysis revealed tumor size >5 cm, AFP >150 nag/mol and PIVKA-II >100 maul/mol as significant independent risk factors for recurrence. The median time to recurrence was 10 months. The median survival time after recurrence was 26 months, and the 1-, 3- and 5-year survival rates after recurrence were 80.5%, 58%, and 28.3% respectively. Conclusions Preoperatively, not only morphology of the tumor but also AFP and PIVKA-II levels can offers important information for the recurrence after LDLT for HCC. Thus, combination of tumor markers might be used for expansion of pre-existing strict selection criteria of liver transplantation for HCC. PMID:28124000

  12. Fc-receptor and M-protein genes of group A streptococci are products of gene duplication.

    PubMed Central

    Heath, D G; Cleary, P P

    1989-01-01

    The partial nucleotide sequence for an Fc-receptor gene from an M-type 76 group A streptococcus was determined. DNA sequence analysis revealed considerable sequence similarity between the Fc-receptor and M-protein genes in their proposed promoter regions, signal sequences, and 3' termini. Additional analysis indicated that the deduced Fc-receptor protein contains a proline-rich region and membrane anchor region highly similar to that of M protein. In view of these results, we postulated that Fc-receptor and M-protein genes of group A streptococci are the products of gene duplication from a common ancestral gene. It is proposed that DNA sequence similarity between these two genes may allow for extragenic homologous recombination as a means of generating antigenic diversity in these two surface proteins. PMID:2660147

  13. Heterogeneity of high-mobility-group protein 2. Enrichment of a rapidly migrating form in testis.

    PubMed Central

    Bucci, L R; Brock, W A; Meistrich, M L

    1985-01-01

    A determination of the absolute amounts of high-mobility-group proteins 1 and 2 (HMG1 and HMG2) in rat tissues demonstrated that amounts of HMG2 were low in non-proliferating tissues, somewhat higher in proliferating and lymphoid tissues, but were extremely elevated in the testis. This increase was due to a germ-cell-specific form of HMG2 with increased mobility relative to somatic HMG2 on acid/urea/polyacrylamide-gel electrophoresis. To determine if the findings in the rat were a general feature of spermatogenesis, testis (germinal), spleen (lymphoid), and liver (non-proliferating) tissues from various vertebrate species were examined for their relative amounts of HMG1 and HMG2, and for HMG2 heterogeneity. Bull, chimpanzee, cynomologus monkey, dog, gopher, guinea pig, hamster, mouse, opossum, rabbit, rat, rhesus monkey, squirrel and toad (Xenopus) tissues were analysed. Nearly all species showed relatively high contents of HMG2 in testis tissue, whereas HMG1 contents were similar in all species and tissues. Ten of thirteen species showed a rapidly migrating HMG2 subtype in testis tissue, separable by acid/urea/polyacrylamide-gel electrophoresis. Xenopus, which lacks HMG2 in somatic tissues, showed an HMG2-like protein in testis tissue. Although the rapidly migrating HMG2 subtype in species other than rat was not testis-specific, it was always enriched in the testis. This study indicates that increased amounts of HMG2 and the enrichment of a rapidly migrating HMG2 subtype are general features of spermatogenic cells. Images Fig. 1. Fig. 2. Fig. 3. PMID:4038257

  14. Crystal Structure of the N-terminal Domain of the Group B Streptococcus Alpha C Protein

    SciTech Connect

    Auperin,T.; Bolduc, G.; Baron, M.; Heroux, A.; Filman, D.; Madoff, L.; Hogle, J.

    2005-01-01

    Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis, and meningitis among neonates and an important cause of morbidity among pregnant women and immunocompromised adults. Invasive diseases due to GBS are attributed to the ability of the pathogen to translocate across human epithelial surfaces. The alpha C protein (ACP) has been identified as an invasin that plays a role in internalization and translocation of GBS across epithelial cells. The soluble N-terminal domain of ACP (NtACP) blocks the internalization of GBS. We determined the 1.86-{angstrom} resolution crystal structure of NtACP comprising residues Ser{sup 52} through Leu{sup 225} of the full-length ACP. NtACP has two domains, an N-terminal {beta}-sandwich and a C-terminal three-helix bundle. Structural and topological alignments reveal that the {beta}-sandwich shares structural elements with the type III fibronectin fold (FnIII), but includes structural elaborations that make it unique. We have identified a potential integrin-binding motif consisting of Lys-Thr-Asp{sup 146}, Arg{sup 110}, and Asp{sup 118}. A similar arrangement of charged residues has been described in other invasins. ACP shows a heparin binding activity that requires NtACP. We propose a possible heparin-binding site, including one surface of the three-helix bundle, and nearby portions of the sandwich and repeat domains. We have validated this prediction using assays of the heparin binding and cell-adhesion properties of engineered fragments of ACP. This is the first crystal structure of a member of the highly conserved Gram-positive surface alpha-like protein family, and it will enable the internalization mechanism of GBS to be dissected at the atomic level.

  15. [Protein phosphatases: structure and function].

    PubMed

    Bulanova, E G; Budagian, V M

    1994-01-01

    The process of protein and enzyme systems phosphorylation is necessary for cell growth, differentiation and preparation for division and mitosis. The conformation changes of protein as a result of phosphorylation lead to increased enzyme activity and enhanced affinity to substrates. A large group of enzymes--protein kinases--is responsible for phosphorylation process in cell, which are divided into tyrosine- and serine-threonine-kinases depending on their ability to phosphorylate appropriate amino acid residues. In this review has been considered the functional importance and structure of protein phosphatases--enzymes, which are functional antagonists of protein kinases.

  16. Temperature, pressure, and electrochemical constraints on protein speciation: Group additivity calculation of the standard molal thermodynamic properties of ionized unfolded proteins

    NASA Astrophysics Data System (ADS)

    Dick, J. M.; Larowe, D. E.; Helgeson, H. C.

    2006-07-01

    Thermodynamic calculations can be used to quantify environmental constraints on the speciation of proteins, such as the pH and temperature dependence of ionization state, and the relative chemical stabilities of proteins in different biogeochemical settings. These calculations depend in part on values of the standard molal Gibbs energies of proteins and their ionization reactions as a function of temperature and pressure. Because these values are not generally available, we calculated values of the standard molal thermodynamic properties at 25°C and 1 bar as well as the revised Helgeson-Kirkham-Flowers equations of state parameters of neutral and charged zwitterionic reference model compounds including aqueous amino acids, polypeptides, and unfolded proteins. The experimental calorimetric and volumetric data for these species taken from the literature were combined with group additivity algorithms to calculate the properties and parameters of neutral and ionized sidechain and backbone groups in unfolded proteins. The resulting set of group contributions enables the calculation of the standard molal Gibbs energy, enthalpy, entropy, isobaric heat capacity, volume, and isothermal compressibility of unfolded proteins in a range of proton ionization states to temperatures and pressures exceeding 100°C and 1000 bar. This approach provides a useful frame of reference for thermodynamic studies of protein folding and complexation reactions. It can also be used to assign provisional values of the net charge and Gibbs energy of ionized proteins as a function of temperature and pH. Using these values, an Eh-pH diagram for a reaction representing the speciation of extracellular proteins from Pyrococcus furiosus and Bacillus subtilis was generated. The predicted predominance limits of these proteins correspond with the different electrochemical conditions of hydrothermal vents and soils. More comprehensive calculations of this kind may reveal pervasive chemical potential

  17. Development of Kappa Opioid Receptor Antagonists

    PubMed Central

    Carroll, F. Ivy; Carlezon, William A.

    2013-01-01

    Kappa opioid receptors (KORs) belong to the G-protein coupled class of receptors (GPCRs). They are activated by the endogenous opioid peptide dynorphin (DYN) and expressed at particularly high levels within brain areas implicated in modulation of motivation, emotion, and cognitive function. Chronic activation of KORs in animal models has maladaptive effects including increases in behaviors that reflect depression, the propensity to engage in drug-seeking behavior, and drug craving. The fact that KOR activation has such a profound influence on behaviors often triggered by stress has led to interest in selective KOR antagonists as potential therapeutic agents. This perspective provides a description of preclinical research conducted in the development of several different classes of selective KOR antagonists, a summary of the clinical studies conducted thus far, and recommendations for the type of work needed in the future to determine if these agents would be useful as pharmacotherapies for neuropsychiatric illness. PMID:23360448

  18. THE SMALL ACID SOLUBLE PROTEINS (SASP α and SASP β) OF BACILLUS WEIHENSTEPHANENSIS AND B. MYCOIDES GROUP 2 ARE THE MOST DISTINCT AMONG THE B. CEREUS GROUP

    PubMed Central

    Callahan, Courtney; Fox, Karen; Fox, Alvin

    2009-01-01

    The Bacillus cereus group includes Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides and Bacillus weihenstephanensis. The small acid-soluble spore protein (SASP) β has been previously demonstrated to be among the biomarkers differentiating B. anthracis and B. cereus; SASP β of B. cereus most commonly exhibits one or two amino acid substitutions when compared to B. anthracis. SASP α is conserved in sequence among these two species. Neither SASP α nor β for B. thuringiensis, B. mycoides and B. weihenstephanensis have been previously characterized as taxonomic discriminators. In the current work molecular weight (MW) variation of these SASPs were determined by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) for representative strains of the 5 species within the B. cereus group. The measured MWs also correlate with calculated MWs of translated amino acid sequences generated from whole genome sequencing projects. SASP α and β demonstrated consistent MW among B. cereus, B. thuringiensis, and B. mycoides strains (group 1). However B. mycoides (group 2) and B. weihenstephanensis SASP α and β were quite distinct making them unique among the B. cereus group. Limited sequence changes were observed in SASP α (at most 3 substitutions and 2 deletions) indicating it is a more conserved protein than SASP β (up to 6 substitutions and a deletion). Another even more conserved SASP, SASP α-β type, was described here for the first time. PMID:19616612

  19. Small Molecule CXCR3 Antagonists.

    PubMed

    Andrews, Stephen P; Cox, Rhona J

    2016-04-14

    Chemokines and their receptors are known to play important roles in disease. More than 40 chemokine ligands and 20 chemokine receptors have been identified, but, to date, only two small molecule chemokine receptor antagonists have been approved by the FDA. The chemokine receptor CXCR3 was identified in 1996, and nearly 20 years later, new areas of CXCR3 disease biology continue to emerge. Several classes of small molecule CXCR3 antagonists have been developed, and two have shown efficacy in preclinical models of inflammatory disease. However, only one CXCR3 antagonist has been evaluated in clinical trials, and there remain many opportunities to further investigate known classes of CXCR3 antagonists and to identify new chemotypes. This Perspective reviews the known CXCR3 antagonists and considers future opportunities for the development of small molecules for clinical evaluation.

  20. Topology of membrane sulfhydryl groups in the human erythrocyte. Demonstration of a non-reactive population in intrinsic proteins.

    PubMed

    Haest, C W; Kamp, D; Deuticke, B

    1981-05-06

    A major fraction of the protein sulfhydryl groups of human erythrocyte membranes can be oxidized to disulfide bonds by the lipid soluble reagent, diamide, and the hydrophilic reagent, tetrathionate. Furthermore, the same fraction also reacts with the monofunctional reagent, N-ethylmaleimide. About 20% of the SH groups, however, do not react with any of these agents even upon prolonged treatment and increased concentrations. These 'non-reacting' SH groups were now localized by a procedure involving blockage of the accessible SH groups by non-labeled N-ethylmaleimide or by diamide, subsequent isolation and solubilization of the membranes in SDS and labelling of the now accessible, residual SH groups with N-[ethyl-2-3H]ethylmaleimide. The distribution of the radioactivity over the peptide fractions shows that the non-reacting SH groups are mainly localized in the intrinsic proteins, while essentially all of the SH groups of the extrinsic protein, spectrin, are reactive. After solubilization of the membranes with Triton X-100 the non-reacting SH groups became reactive towards N-ethylmaleimide. It is proposed that lack of reaction of SH groups in the native membranes is due to their localization within the hydrophobic core of the membrane.

  1. Characterization of the spore surface and exosporium proteins of Clostridium sporogenes; implications for Clostridium botulinum group I strains.

    PubMed

    Janganan, Thamarai K; Mullin, Nic; Tzokov, Svetomir B; Stringer, Sandra; Fagan, Robert P; Hobbs, Jamie K; Moir, Anne; Bullough, Per A

    2016-10-01

    Clostridium sporogenes is a non-pathogenic close relative and surrogate for Group I (proteolytic) neurotoxin-producing Clostridium botulinum strains. The exosporium, the sac-like outermost layer of spores of these species, is likely to contribute to adhesion, dissemination, and virulence. A paracrystalline array, hairy nap, and several appendages were detected in the exosporium of C. sporogenes strain NCIMB 701792 by EM and AFM. The protein composition of purified exosporium was explored by LC-MS/MS of tryptic peptides from major individual SDS-PAGE-separated protein bands, and from bulk exosporium. Two high molecular weight protein bands both contained the same protein with a collagen-like repeat domain, the probable constituent of the hairy nap, as well as cysteine-rich proteins CsxA and CsxB. A third cysteine-rich protein (CsxC) was also identified. These three proteins are also encoded in C. botulinum Prevot 594, and homologues (75-100% amino acid identity) are encoded in many other Group I strains. This work provides the first insight into the likely composition and organization of the exosporium of Group I C. botulinum spores.

  2. Group B streptococcal serine-rich repeat proteins promote interaction with fibrinogen and vaginal colonization.

    PubMed

    Wang, Nai-Yu; Patras, Kathryn A; Seo, Ho Seong; Cavaco, Courtney K; Rösler, Berenice; Neely, Melody N; Sullam, Paul M; Doran, Kelly S

    2014-09-15

    Group B streptococcus (GBS) can cause severe disease in susceptible hosts, including newborns, pregnant women, and the elderly. GBS serine-rich repeat (Srr) surface glycoproteins are important adhesins/invasins in multiple host tissues, including the vagina. However, exact molecular mechanisms contributing to their importance in colonization are unknown. We have recently determined that Srr proteins contain a fibrinogen-binding region (BR) and hypothesize that Srr-mediated fibrinogen binding may contribute to GBS cervicovaginal colonization. In this study, we observed that fibrinogen enhanced wild-type GBS attachment to cervical and vaginal epithelium, and that this was dependent on Srr1. Moreover, purified Srr1-BR peptide bound directly to host cells, and peptide administration in vivo reduced GBS recovery from the vaginal tract. Furthermore, a GBS mutant strain lacking only the Srr1 "latching" domain exhibited decreased adherence in vitro and decreased persistence in a mouse model of GBS vaginal colonization, suggesting the importance of Srr-fibrinogen interactions in the female reproductive tract.

  3. [Social aspects of energy and protein consumption in a group of pregnant women].

    PubMed

    Quiroz, S E; Casanueva, E; Avila, H; García, D; Arroyo, P

    1981-03-01

    This paper presents the results of a dietary survey (weighed intake/24 hr) carried out in 64 pregnant women living in Huamantla, Tlaxcala, Mexico and its proximities, during November 1973. The primary objective of the study was to identify social indicators with predictive value in relation to food consumption patterns, in order to detect groups at risk of having an inadequate intake. A subgroup of the women (15 cases) had adequate levels of energy and protein intake (E/P intake) when compared to the recommendations established by FAO/OMS, 1973, although they consumed a diet with relatively few variations which was also low in fat content. Family organization was the socioanthropological variable which had the greatest relationship with E/P intake. The extended families presented a better E/P ratio than the nuclear families. Interestingly, the per capita expenditure did not show a significant association with nutritional indicators. These data suggest, therefore, that in the population studied the E/P ratio is more closely associated with family organization than with its income level.

  4. Conformational study of the binding of a high mobility group protein with chromatin

    SciTech Connect

    Sasi, R.; Huvoes, P.E.; Fasman, G.D.

    1982-10-10

    The nature of the binding of a high mobility group protein (HMG 17) to native and H1-H5-depleted chicken erythrocyte chromatin was studied, as a function of ionic strength, using circular dichroism and thermal denaturation techniques. The circular dichroism properties of the HMG 17-reconstituted whole chromatin and H1-H5-depleted chromatin structure occurred upon HMG 17 binding at low ionic strength. Thermal denaturation profiles confirmed this change in the structure of chromatin induced by HMG 17. Thermal denaturation profiles were resolved into three-component transitions. These results indicate that the binding sites of HMG 17 are situated in the linker regions immediately adjacent to the core. The nature of the interaction of HMG 17 at higher ionic strength with whole chromatin and H1-H5-depleted chromatin was found to be different. These observations suggest that HMG 17 does not loosen chromatin structure but produces an overall stabilization and condensation of structure. The implications of these results to the currently accepted models of transcriptionally active chromatin are discussed.

  5. High-mobility group box 1 protein and its role in severe acute pancreatitis

    PubMed Central

    Shen, Xiao; Li, Wei-Qin

    2015-01-01

    The high mobility group box 1 (HMGB1), which belongs to the subfamily of HMG-1/-2, is a highly conserved single peptide chain consisting of 215 amino acid residues with a molecular weight of approximately 24894 Da. HMGB1 is a ubiquitous nuclear protein in mammals and plays a vital role in inflammatory diseases. Acute pancreatitis is one of the most common causes of acute abdominal pain with a poor prognosis. Acute pancreatitis is an acute inflammatory process of the pancreas (duration of less than six months), for which the severe form is called severe acute pancreatitis (SAP). More and more studies have shown that HMGB1 has a bidirectional effect in the pathogenesis of SAP. Extracellular HMGB1 can aggravate the pancreatic inflammatory process, whereas intracellular HMGB1 has a protective effect against pancreatitis. The mechanism of HMGB1 is multiple, mainly through the nuclear factor-κB pathway. Receptors for advanced glycation end-products and toll-like receptors (TLR), especially TLR-2 and TLR-4, are two major types of receptors mediating the inflammatory process triggered by HMGB1 and may be also the main mediators in the pathogenesis of SAP. HMGB1 inhibitors, such as ethyl pyruvate, pyrrolidine dithiocarbamate and Scolopendra subspinipes mutilans, can decrease the level of extracellular HMGB1 and are the promising targets in the treatment of SAP. PMID:25663762

  6. Polycomb group protein gene silencing, non-coding RNA, stem cells, and cancer.

    PubMed

    Gieni, Randall S; Hendzel, Michael J

    2009-10-01

    Epigenetic programming is an important facet of biology, controlling gene expression patterns and the choice between developmental pathways. The Polycomb group proteins (PcGs) silence gene expression, allowing cells to both acquire and maintain identity. PcG silencing is important for stemness, X chromosome inactivation (XCI), genomic imprinting, and the abnormally silenced genes in cancers. Stem and cancer cells commonly share gene expression patterns, regulatory mechanisms, and signalling pathways. Many microRNA species have oncogenic or tumor suppressor activity, and disruptions in these networks are common in cancer; however, long non-coding (nc)RNA species are also important. Many of these directly guide PcG deposition and gene silencing at the HOX locus, during XCI, and in examples of genomic imprinting. Since inappropriate HOX expression and loss of genomic imprinting are hallmarks of cancer, disruption of long ncRNA-mediated PcG silencing likely has a role in oncogenesis. Aberrant silencing of coding and non-coding loci is critical for both the genesis and progression of cancers. In addition, PcGs are commonly abnormally overexpressed years prior to cancer pathology, making early PcG targeted therapy an option to reverse tumor formation, someday replacing the blunt instrument of eradication in the cancer therapy arsenal.

  7. A new transgenic mouse model for conditional overexpression of the Polycomb Group protein EZH2.

    PubMed

    Koppens, Martijn A J; Tanger, Ellen; Nacerddine, Karim; Westerman, Bart; Song, Ji-Ying; van Lohuizen, Maarten

    2017-04-01

    The Polycomb Group protein EZH2 is upregulated in most prostate cancers, and its overexpression is associated with poor prognosis. Most insights into the functional role of EZH2 in prostate cancer have been gained using cell lines and EZH2 inactivation studies. However, the question remains whether overexpression of EZH2 can initiate prostate tumourigenesis or drive tumour progression. Appropriate transgenic mouse models that are required to answer such questions are lacking. We developed one such transgenic mouse model for conditional overexpression of Ezh2. In this transgene, Ezh2 and Luciferase are transcribed from a single open reading frame. The latter gene enables intravital bioluminescent imaging of tissues expressing this transgene, allowing the detection of tumour outgrowth and potential metastatic progression over time. Prostate-specific Ezh2 overexpression by crossbreeding with Probasin-Cre mice led to neoplastic prostate lesions at low incidence and with a long latency. Compounding a previously described Bmi1-transgene and Pten-deficiency prostate cancer mouse model with the Ezh2 transgene did not enhance tumour progression or drive metastasis formation. In conclusion, we here report the generation of a wildtype Ezh2 overexpression mouse model that allows for intravital surveillance of tissues with activated transgene. This model will be an invaluable tool for further unravelling the role of EZH2 in cancer.

  8. Piwi maintains germline stem cells and oogenesis in Drosophila through negative regulation of Polycomb group proteins.

    PubMed

    Peng, Jamy C; Valouev, Anton; Liu, Na; Lin, Haifan

    2016-03-01

    The Drosophila melanogaster Piwi protein regulates both niche and intrinsic mechanisms to maintain germline stem cells, but its underlying mechanism remains unclear. Here we report that Piwi interacts with Polycomb group complexes PRC1 and PRC2 in niche and germline cells to regulate ovarian germline stem cells and oogenesis. Piwi physically interacts with the PRC2 subunits Su(z)12 and Esc in the ovary and in vitro. Chromatin coimmunoprecipitation of Piwi, the PRC2 enzymatic subunit E(z), histone H3 trimethylated at lysine 27 (H3K27me3) and RNA polymerase II in wild-type and piwi mutant ovaries demonstrates that Piwi binds a conserved DNA motif at ∼ 72 genomic sites and inhibits PRC2 binding to many non-Piwi-binding genomic targets and H3K27 trimethylation. Moreover, Piwi influences RNA polymerase II activities in Drosophila ovaries, likely via inhibiting PRC2. We hypothesize that Piwi negatively regulates PRC2 binding by sequestering PRC2 in the nucleoplasm, thus reducing PRC2 binding to many targets and influencing transcription during oogenesis.

  9. A bridging model for persistence of a polycomb group protein complex through DNA replication in vitro.

    PubMed

    Lo, Stanley M; Follmer, Nicole E; Lengsfeld, Bettina M; Madamba, Egbert V; Seong, Samuel; Grau, Daniel J; Francis, Nicole J

    2012-06-29

    Epigenetic regulation may involve heritable chromatin states, but how chromatin features can be inherited through DNA replication is incompletely understood. We address this question using cell-free replication of chromatin. Previously, we showed that a Polycomb group complex, PRC1, remains continuously associated with chromatin through DNA replication. Here we investigate the mechanism of persistence. We find that a single PRC1 subunit, Posterior sex combs (PSC), can reconstitute persistence through DNA replication. PSC binds nucleosomes and self-interacts, bridging nucleosomes into a stable, oligomeric structure. Within these structures, individual PSC-chromatin contacts are dynamic. Stable association of PSC with chromatin, including through DNA replication, depends on PSC-PSC interactions. Our data suggest that labile individual PSC-chromatin contacts allow passage of the DNA replication machinery while PSC-PSC interactions prevent PSC from dissociating, allowing it to rebind to replicated chromatin. This mechanism may allow inheritance of chromatin proteins including PRC1 through DNA replication to maintain chromatin states.

  10. Protein NMR Studies of substrate binding to human blood group A and B glycosyltransferases.

    PubMed

    Peters, Thomas; Grimm, Lena Lisbeth; Weissbach, Sophie; Flügge, Friedemann; Begemann, Nora; Palcic, Monica

    2017-03-03

    Donor and acceptor substrate binding to human blood group A and B glycosyltransferases (GTA, GTB) has been studied by a variety of protein NMR experiments. Prior crystallographic studies have shown these enzymes to adopt an open conformation in the absence of substrates. Binding of either the donor substrate UDP-Gal, or of UDP induces a semi-closed conformation. In the presence of both, donor- and acceptor substrates, the enzymes shift towards a closed conformation with ordering of an internal loop and the C-terminal residues, which then completely cover the donor-binding pocket. Chemical shift titrations of uniformly 2H,15N labeled GTA or GTB with UDP affected about 20% of all cross peaks in 1H,15N-TROSY-HSQC spectra reflecting substantial plasticity of the enzymes. On the other hand, it is this conformational flexibility that impedes NH backbone assignments. Chemical shift perturbation experiments using 1-13C-methyl Ile labeled samples revealed two Ile residues, Ile123 at the bottom of the UDP binding pocket, and Ile192 as part of the internal loop that were significantly disturbed upon stepwise addition of UDP and H-disaccharide, also revealing long-range perturbations. Finally, methyl TROSY based relaxation dispersion experiments do not reveal s to ms time scale motions. Although this study reveals substantial conformational plasticity of GTA and GTB it remains enigmatic how binding of substrates shifts the enzymes into catalytically competent states.

  11. Associations of High Sensitivity C-Reactive Protein Levels in Schizophrenia and Comparison Groups

    PubMed Central

    Joseph, Jamie; Depp, Colin; Martin, Averria Sirkin; Daly, Rebecca; Glorioso, Danielle K; Palmer, Barton W; Jeste, Dilip V

    2015-01-01

    Schizophrenia is characterized by physical (mainly metabolic and cardiovascular) comorbidity and shortened lifespan. High sensitivity C- reactive protein (hs-CRP), an inflammatory marker of hepatic origin linked to metabolic and cardiovascular diseases and mortality in the general population, has been reported to be elevated in people with schizophrenia. However, the relationship of hs-CRP to psychiatric and medical risk factors, after controlling for potentially confounding variables such as smoking, is not well established in schizophrenia. We assessed hs-CRP levels along with various demographic, psychiatric, and metabolic measures in 88 clinically stable outpatients with schizophrenia or schizoaffective disorder and 71 age epoch-matched comparison subjects with no history of a major psychiatric illness. hs-CRP levels were significantly higher in individuals with schizophrenia than in comparison subjects. Higher hs-CRP levels in schizophrenia group were associated with female gender, more severe negative symptoms, greater medical comorbidity, and worse metabolic risk factors including BMI, fasting glucose, and hemoglobin A1c levels. hs-CRP was not related to age, race, education, smoking status, antipsychotic dosage, or cognitive impairment. Longitudinal studies are needed to investigate the relationship between hs-CRP and long-term health outcomes including metabolic syndrome, cardiovascular disease, and mortality in schizophrenia. PMID:26341579

  12. Conformation of a group 2 late embryogenesis abundant protein from soybean. Evidence of poly (L-proline)-type II structure.

    PubMed

    Soulages, Jose L; Kim, Kangmin; Arrese, Estela L; Walters, Christina; Cushman, John C

    2003-03-01

    Late embryogenesis abundant (LEA) proteins are members of a large group of hydrophilic, glycine-rich proteins found in plants, algae, fungi, and bacteria known collectively as hydrophilins that are preferentially expressed in response to dehydration or hyperosmotic stress. Group 2 LEA (dehydrins or responsive to abscisic acid) proteins are postulated to stabilize macromolecules against damage by freezing, dehydration, ionic, or osmotic stress. However, the structural and physicochemical properties of group 2 LEA proteins that account for such functions remain unknown. We have analyzed the structural properties of a recombinant form of a soybean (Glycine max) group 2 LEA (rGmDHN1). Differential scanning calorimetry of purified rGmDHN1 demonstrated that the protein does not display a cooperative unfolding transition upon heating. Ultraviolet absorption and circular dichroism spectroscopy revealed that the protein is in a largely hydrated and unstructured conformation in solution. However, ultraviolet absorption and circular dichroism measurements collected at different temperatures showed that the protein exists in equilibrium between two extended conformational states: unordered and left-handed extended helical or poly (L-proline)-type II structures. It is estimated that 27% of the residues of rGmDHN1 adopt or poly (L-proline)-type II-like helical conformation at 12 degrees C. The content of extended helix gradually decreases to 15% as the temperature is increased to 80 degrees C. Studies of the conformation of the protein in solution in the presence of liposomes, trifluoroethanol, and sodium dodecyl sulfate indicated that rGmDHN1 has a very low intrinsic ability to adopt alpha-helical structure and to interact with phospholipid bilayers through amphipathic alpha-helices. The ability of the protein to remain in a highly extended conformation at low temperatures could constitute the basis of the functional role of GmDHN1 in the prevention of freezing, desiccation

  13. A group 6 late embryogenesis abundant protein from common bean is a disordered protein with extended helical structure and oligomer-forming properties.

    PubMed

    Rivera-Najera, Lucero Y; Saab-Rincón, Gloria; Battaglia, Marina; Amero, Carlos; Pulido, Nancy O; García-Hernández, Enrique; Solórzano, Rosa M; Reyes, José L; Covarrubias, Alejandra A

    2014-11-14

    Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also accumulate in response to water deficit in vegetative tissues, which leads to a remarkable association between their presence and low water availability conditions. A major sub-group of these proteins, also known as typical LEA proteins, shows high hydrophilicity and a high percentage of glycine and other small amino acid residues, distinctive physicochemical properties that predict a high content of structural disorder. Although all typical LEA proteins share these characteristics, seven groups can be distinguished by sequence similarity, indicating structural and functional diversity among them. Some of these groups have been extensively studied; however, others require a more detailed analysis to advance in their functional understanding. In this work, we report the structural characterization of a group 6 LEA protein from a common bean (Phaseolus vulgaris L.) (PvLEA6) by circular dichroism and nuclear magnetic resonance showing that it is a disordered protein in aqueous solution. Using the same techniques, we show that despite its unstructured nature, the addition of trifluoroethanol exhibited an intrinsic potential in this protein to gain helicity. This property was also promoted by high osmotic potentials or molecular crowding. Furthermore, we demonstrate that PvLEA6 protein is able to form soluble homo-oligomeric complexes that also show high levels of structural disorder. The association between PvLEA6 monomers to form dimers was shown to occur in plant cells by bimolecular fluorescence complementation, pointing to the in vivo functional relevance of this association.

  14. A Group 6 Late Embryogenesis Abundant Protein from Common Bean Is a Disordered Protein with Extended Helical Structure and Oligomer-forming Properties*

    PubMed Central

    Rivera-Najera, Lucero Y.; Saab-Rincón, Gloria; Battaglia, Marina; Amero, Carlos; Pulido, Nancy O.; García-Hernández, Enrique; Solórzano, Rosa M.; Reyes, José L.; Covarrubias, Alejandra A.

    2014-01-01

    Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also accumulate in response to water deficit in vegetative tissues, which leads to a remarkable association between their presence and low water availability conditions. A major sub-group of these proteins, also known as typical LEA proteins, shows high hydrophilicity and a high percentage of glycine and other small amino acid residues, distinctive physicochemical properties that predict a high content of structural disorder. Although all typical LEA proteins share these characteristics, seven groups can be distinguished by sequence similarity, indicating structural and functional diversity among them. Some of these groups have been extensively studied; however, others require a more detailed analysis to advance in their functional understanding. In this work, we report the structural characterization of a group 6 LEA protein from a common bean (Phaseolus vulgaris L.) (PvLEA6) by circular dichroism and nuclear magnetic resonance showing that it is a disordered protein in aqueous solution. Using the same techniques, we show that despite its unstructured nature, the addition of trifluoroethanol exhibited an intrinsic potential in this protein to gain helicity. This property was also promoted by high osmotic potentials or molecular crowding. Furthermore, we demonstrate that PvLEA6 protein is able to form soluble homo-oligomeric complexes that also show high levels of structural disorder. The association between PvLEA6 monomers to form dimers was shown to occur in plant cells by bimolecular fluorescence complementation, pointing to the in vivo functional relevance of this association. PMID:25271167

  15. The Drosophila Hrb87F gene encodes a new member of the A and B hnRNP protein group.

    PubMed Central

    Haynes, S R; Johnson, D; Raychaudhuri, G; Beyer, A L

    1991-01-01

    Nascent premessenger RNA transcripts are packaged into heterogeneous nuclear ribonucleoprotein (hnRNP) complexes containing specific nuclear proteins, the hnRNP proteins. The A and B group proteins constitute a major class of small basic proteins found in mammalian hnRNP complexes. We have previously characterized the Drosophila melanogaster Hrb98DE gene, which is alternatively spliced to encode four protein isoforms closely related to the A and B proteins. We report here that the Drosophila genome contains a family of genes related to the Hrb98DE gene. One member of the family, Hrb87F, is very homologous to Hrb98DE in both sequence and structure. The Hrb87F transcripts (1.7 and 2.2 kb) utilize two alternative polyadenylation sites, are abundant in ovaries and early embryos, and are present in lesser amounts throughout development. In one wildtype strain of Drosophila there is a naturally-occurring polymorphism in this gene due to the insertion of a 412 transposable element in the 3' untranslated region. The larger transcript is not produced in these files and thus is not required for viability. Sequence identities among the Drosophila Hrb proteins and the vertebrate A and B hnRNP proteins suggest that these proteins may form a distinct subfamily within the larger family of related RNA binding proteins. Images PMID:1849257

  16. GnRH agonist and GnRH antagonist protocols in ovarian stimulation: differential regulation pathway of aromatase expression in human granulosa cells.

    PubMed

    Khalaf, Mohamad; Mittre, Hervé; Levallet, Jérôme; Hanoux, Vincent; Denoual, Christine; Herlicoviez, Michel; Bonnamy, Pierre-Jacques; Benhaim, Annie

    2010-07-01

    Gonadotrophin-releasing hormone (GnRH) agonists and antagonists have been widely used to prevent premature LH surge during ovarian stimulation. However, studies have shown a significantly lower serum oestradiol concentration on the day of human chorionic gonadotrophin administration for cycles using GnRH antagonist. This study compared aromatase gene expression in granulosa lutein cells from 50 women randomly assigned to receive either GnRH agonist (group 1, n=28) or GnRH antagonist (group 2, n=22). The cellular mechanism involved in the observed effects was also investigated. GnRH antagonist treatment significantly affected serum oestradiol concentration (1894+/-138 versus 1074+/-63 pg/ml; P < or = 0.001), follicular-fluid oestradiol concentration in large follicles (18,565+/-2467 versus 10,184+/-1993 pg/ml; P < or = 0.05), aromatase activity (9600+/-1179 versus 5376+/-997 fmol/10(6) cells/h; P < or = 0.05) and mRNA aromatase/mRNA glyceraldehyde 3-phosphate dehydrogenase (15+/-3 versus 6+/-1; P < 0.05). Protein kinase C (PKC) activity in granulosa lutein cells from the GnRH antagonist group was 2.5-fold higher than in the GnRH agonist group. In-vitro experiments showed that selective down-regulation of PKC was only observed in GnRH-desensitized granulosa lutein cells. This report suggests that, in granulosa lutein cells, the modulation of the FSH-induced protein kinase A pathway by PKC was different in agonist versus antagonist cycles.

  17. Functionalized Congeners of P2Y1 Receptor Antagonists:

    SciTech Connect

    de Castro, Sonia; Maruoka, Hiroshi; Hong, Kunlun; Kilbey, II, S Michael; Costanzi, Stefano; Hechler, Béatrice; Gachet, Christian; Harden, T. Kendall; Jacobson, Kenneth A.

    2010-01-01

    The P2Y{sub 1} receptor is a prothrombotic G protein-coupled receptor (GPCR) activated by ADP. Preference for the North (N) ring conformation of the ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of the P2Y{sub 1} receptor was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute. A series of covalently linkable N{sup 6}-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphates containing extended 2-alkynyl chains was designed, and binding affinity at the human (h) P2Y{sub 1} receptor determined. The chain of these functionalized congeners contained hydrophilic moieties, a reactive substituent, or biotin, linked via an amide. Variation of the chain length and position of an intermediate amide group revealed high affinity of carboxylic congener 8 (K{sub i} 23 nM) and extended amine congener 15 (K{sub i} 132 nM), both having a 2-(1-pentynoyl) group. A biotin conjugate 18 containing an extended {epsilon}-aminocaproyl spacer chain exhibited higher affinity than a shorter biotinylated analogue. Alternatively, click coupling of terminal alkynes of homologous 2-dialkynyl nucleotide derivatives to alkyl azido groups produced triazole derivatives that bound to the P2Y{sub 1} receptor following deprotection of the bisphosphate groups. The preservation of receptor affinity of the functionalized congeners was consistent with new P2Y{sub 1} receptor modeling and ligand docking. Attempted P2Y{sub 1} antagonist conjugation to PAMAM dendrimer carriers by amide formation or palladium-catalyzed reaction between an alkyne on the dendrimer and a 2-iodopurine-derivatized nucleotide was unsuccessful. A dialkynyl intermediate containing the chain length favored in receptor binding was conjugated to an azide-derivatized dendrimer, and the conjugate inhibited ADP-promoted human platelet aggregation. This is the first example of attaching a strategically functionalized P2Y receptor antagonist to a PAMAM dendrimer to

  18. Use of fluorescein hydrazide and fluorescein thiosemicarbazide reagents for the fluorometric determination of protein carbonyl groups and for the detection of oxidized protein on polyacrylamide gels.

    PubMed

    Ahn, B; Rhee, S G; Stadtman, E R

    1987-03-01

    Highly fluorescent thiosemicarbazide and hydrazide prepared by reaction of fluorescein isothiocyanate with hydrazine or adipic acid dihydrazide have been used to monitor the presence of carbonyl groups in oxidatively modified proteins. After oxidation, proteins react with these reagents under anaerobic conditions in the dark to yield fluorescent protein conjugates (presumably thiosemicarbazones or hydrazones) which can be visualized as fluorescent bands following electrophoresis (0-4 degrees C) on lithium dodecyl sulfate-polyacrylamide gels. These reagents do not react with unoxidized proteins. The conjugates formed dissociate readily at room temperature but are fairly stable at pH 6-9, 0 degrees C. Current data suggest that these reagents will be useful in the detection and quantitation of oxidatively modified proteins in biological systems.

  19. Conserved patterns hidden within group A Streptococcus M protein hypervariability are responsible for recognition of human C4b-binding protein

    PubMed Central

    Buffalo, Cosmo Z.; Bahn-Suh, Adrian J.; Hirakis, Sophia P.; Biswas, Tapan; Amaro, Rommie E.; Nizet, Victor; Ghosh, Partho

    2016-01-01

    No vaccine exists against group A Streptococcus (GAS), a leading cause of worldwide morbidity and mortality. A severe hurdle is the hypervariability of its major antigen, the M protein, with >200 different M types known. Neutralizing antibodies typically recognize M protein hypervariable regions (HVRs) and confer narrow protection. In stark contrast, human C4b-binding protein (C4BP), which is recruited to the GAS surface to block phagocytic killing, interacts with a remarkably large number of M protein HVRs (apparently ~90%). Such broad recognition is rare, and we discovered a unique mechanism for this through structure determination of four sequence-diverse M proteins in complex with C4BP. The structures revealed a uniform and tolerant ‘reading head’ in C4BP, which detected conserved sequence patterns hidden within hypervariability. Our results open up possibilities for rational therapies targeting the M-C4BP interaction, and also inform a path towards vaccine design. PMID:27595425

  20. Differentiation of species in the Bacillus brevis group and the Bacillus aneurinolyticus group based on the electrophoretic whole-cell protein pattern.

    PubMed

    Shida, O; Takagi, H; Kadowaki, K; Yano, H; Komagata, K

    1996-07-01

    Ninety strains of eleven Bacillus species in the Bacillus brevis group and the Bacillus aneurinolyticus group were compared with the electrophoretic whole-cell protein pattern. The strains were separated into two clusters at the similarity of 55%. One cluster (cluster 1) was consisted of strains from the B. brevis group, and another cluster (cluster 2) was composed of strains from the B. aneurinolyticus group. The cluster 1 was separated into eight subclusters. Out of eight subclusters, seven subclusters contain strains from B. brevis, B. laterosporus, B. agri, B. reuszeri, B. choshinensis, B. formosus, and B. borstelensis. Another subcluster was further separated into two related clusters, which corresponded to B. centrosporus and B. parabrevis, and they were fused at the similarity of 76%. Cluster 2 was separated into two subclusters, which corresponded to B. aneurinolyticus and B. migulanus. The above eleven species showed characteristic patterns distinct from one another, and this correlated well with the published DNA relatedness data. The comparison of the electrophoretic whole-cell protein pattern proved to be useful for evaluation of taxonomic relationships among these taxa.

  1. CXCR2 receptor antagonists: a medicinal chemistry perspective.

    PubMed

    Dwyer, Michael P; Yu, Younong

    2014-01-01

    Dysregulated leukocyte recruitment is believed to be a key contributor to various acute and chronic inflammatory disorders which can lead to serious pathological consequences. Chemokines are small molecular weight proteins that have been shown to be imperative in the direction of leukocytes to the sites of inflammation. In humans, several of these chemokines (CXCL8 and CXCL1) are elevated in inflammatory disorders such as asthma, arthritis, and chronic obstructive pulmonary disease (COPD). These chemokines modulate their downstream effects thru G-protein coupled receptors, such as CXCR2, making the identification of small-molecule antagonists of this receptor attractive towards developing novel therapies to treat inflammatory conditions. Since the first report of a CXCR2 receptor antagonist in 1998, there has been a considerable effort conducted mainly in the pharmaceutical industry to identify novel classes of CXCR2 receptor antagonists. Over a dozen distinct classes of CXCR2 receptor antagonists have been reported in the literature to date with a number of these compounds having reached mid-stage clinical trials. This review will provide a broad overview the medicinal chemistry efforts over the past 15 years towards the identification of CXCR2 receptor antagonists. The discussion will focus upon the early preclinical space covering the structure activity relationships (SAR), pharmacology, as well in preclinical in vivo evaluation for the different series of CXCR2 receptor antagonists. In addition, the available clinical data for the most advanced compounds in the clinic will be discussed and along with a perspective of the area moving forward.

  2. High-mobility group box protein 1 promotes the survival of myeloid-derived suppressor cells by inducing autophagy.

    PubMed

    Parker, Katherine H; Horn, Lucas A; Ostrand-Rosenberg, Suzanne

    2016-09-01

    Myeloid-derived suppressor cells are immune-suppressive cells that are elevated in most individuals with cancer, where their accumulation and suppressive activity are driven by inflammation. As myeloid-derived suppressor cells inhibit anti-tumor immunity and promote tumor progression, we are determining how their viability is regulated. Previous studies have established that the damage-associated molecular pattern molecule high-mobility group box protein 1 drives myeloid-derived suppressor cell accumulation and suppressive potency and is ubiquitously present in the tumor microenvironment. As high-mobility group box protein 1 also facilitates tumor cell survival by inducing autophagy, we sought to determine if high-mobility group box protein 1 regulates myeloid-derived suppressor cell survival through induction of autophagy. Inhibition of autophagy increased the quantity of apoptotic myeloid-derived suppressor cells, demonstrating that autophagy extends the survival and increases the viability of myeloid-derived suppressor cells. Inhibition of high-mobility group box protein 1 similarly increased the level of apoptotic myeloid-derived suppressor cells and reduced myeloid-derived suppressor cell autophagy, demonstrating that in addition to inducing the accumulation of myeloid-derived suppressor cells, high-mobility group box protein 1 sustains myeloid-derived suppressor cell viability. Circulating myeloid-derived suppressor cells have a default autophagic phenotype, and tumor-infiltrating myeloid-derived suppressor cells are more autophagic, consistent with the concept that inflammatory and hypoxic conditions within the microenvironment of solid tumors contribute to tumor progression by enhancing immune-suppressive myeloid-derived suppressor cells. Overall, these results demonstrate that in addition to previously recognized protumor effects, high-mobility group box protein 1 contributes to tumor progression by increasing myeloid-derived suppressor cell viability by

  3. Capsaicin- and mustard oil-induced extracellular signal-regulated protein kinase phosphorylation in sensory neurons in vivo: effects of neurokinins 1 and 2 receptor antagonists and of a nitric oxide synthase inhibitor.

    PubMed

    Donnerer, Josef; Liebmann, Ingrid; Schuligoi, Rufina

    2009-01-01

    Stimulation of primary sensory neurons with capsaicin or mustard oil leads to phosphorylation of extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) via activation of transient receptor potential V1 (TRPV1) or TRPA1, respectively. p-ERK1/2 was determined by Western immunoblotting in the dorsal root ganglia and in the sciatic nerve of rats following either systemic or perineural capsaicin treatment, or mustard oil application to the hind paw skin. To investigate the possible involvement of neurokinin 1 (NK(1)) and NK(2) receptors as well as of nitric oxide, the selective antagonists, SR140333 for NK(1) and SR48968 for NK(2), and the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), were employed. The increase of p-ERK1/2 after systemic capsaicin treatment was markedly attenuated by SR140333, while only the increase in the dorsal root ganglia was impaired by SR48968; in contrast, inhibition of nitric oxide synthase had no effect. Perineural capsaicin induced an increase in p-ERK1/2 in the ipsilateral sciatic nerve and in the dorsal root ganglia. This effect was not influenced by SR140333 or L-NAME. We found for the first time that mustard oil application to the hind paw skin caused an increase in p-ERK1/2 in the sciatic nerve and in the dorsal root ganglia and only the phosphorylation in the latter was attenuated by SR140333 while L-NAME showed no effect. From the present results, it may be assumed that capsaicin- or mustard oil-induced p-ERK1/2 in sensory neurons is not solely directly linked to TRPV1 or TRPA1 channels, but under certain conditions NK(1)- and NK(2)-mediated mechanisms are involved.

  4. Effects of Group I metabotropic glutamate receptor antagonists on sensitivity to reinforcer magnitude and delayed reinforcement in a delay-discounting task in rats: Contribution of delay presentation order.

    PubMed

    Yates, Justin R; Rogers, Katherine K; Gunkel, Benjamin T; Prior, Nicholas A; Hughes, Mallory N; Sharpe, Sara M; Campbell, Hunter L; Johnson, Anthony B; Keller, Margaret G; Breitenstein, Kerry A; Shults, Hansen N

    2017-03-30

    Metabotropic glutamate receptor 1 (mGluR1) blockade has been shown to decrease impulsive choice, as measured in delay discounting. However, several variables are known to influence an animal's discounting, including sensitivity to delayed reinforcement and sensitivity to reinforcer magnitude. The goal of this experiment was to determine the effects of mGluR1, as well as mGluR5, antagonism on these parameters. Forty Sprague Dawley rats were trained in delay discounting, in which consistently choosing a small, immediate reward reflects impulsive choice. For half of the rats, the delay to the large reinforcer increased across blocks of trials, whereas the delay decreased across the session for half of the rats. Following training, half of the rats received injections of the mGluR1 antagonist JNJ 16259685 (JNJ; 0, 0.1, 0.3, or 1.0mg/kg; i.p), and half received injections of the mGluR5 antagonist MPEP (0, 1.0, 3.0, or 10.0mg/kg; i.p.). Administration of JNJ increased sensitivity to delayed reinforcement (i.e., promoted impulsive choice), regardless of which schedule was used. However, the order in which delays were presented modulated the effects of JNJ on sensitivity to reinforcer magnitude. Specifically, JNJ decreased sensitivity to reinforcer magnitude in rats trained on the descending schedule only. MPEP did not alter sensitivity to reinforcer magnitude or sensitivity to delayed reinforcement. These results show that mGluR1 is an important mediator of impulsive choice, and they provide further evidence that delay order presentation is an important variable that influences drug effects in delay discounting.

  5. Application of encoded library technology (ELT) to a protein-protein interaction target: discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists.

    PubMed

    Kollmann, Christopher S; Bai, Xiaopeng; Tsai, Ching-Hsuan; Yang, Hongfang; Lind, Kenneth E; Skinner, Steven R; Zhu, Zhengrong; Israel, David I; Cuozzo, John W; Morgan, Barry A; Yuki, Koichi; Xie, Can; Springer, Timothy A; Shimaoka, Motomu; Evindar, Ghotas

    2014-04-01

    The inhibition of protein-protein interactions remains a challenge for traditional small molecule drug discovery. Here we describe the use of DNA-encoded library technology for the discovery of small molecules that are potent inhibitors of the interaction between lymphocyte function-associated antigen 1 and its ligand intercellular adhesion molecule 1. A DNA-encoded library with a potential complexity of 4.1 billion compounds was exposed to the I-domain of the target protein and the bound ligands were affinity selected, yielding an enriched small-molecule hit family. Compounds representing this family were synthesized without their DNA encoding moiety and found to inhibit the lymphocyte function-associated antigen 1/intercellular adhesion molecule-1 interaction with submicromolar potency in both ELISA and cell adhesion assays. Re-synthesized compounds conjugated to DNA or a fluorophore were demonstrated to bind to cells expressing the target protein.

  6. Novel conserved group A streptococcal proteins identified by the antigenome technology as vaccine candidates for a non-M protein-based vaccine.

    PubMed

    Fritzer, Andrea; Senn, Beatrice M; Minh, Duc Bui; Hanner, Markus; Gelbmann, Dieter; Noiges, Birgit; Henics, Tamás; Schulze, Kai; Guzman, Carlos A; Goodacre, John; von Gabain, Alexander; Nagy, Eszter; Meinke, Andreas L

    2010-09-01

    Group A streptococci (GAS) can cause a wide variety of human infections ranging from asymptomatic colonization to life-threatening invasive diseases. Although antibiotic treatment is very effective, when left untreated, Streptococcus pyogenes infections can lead to poststreptococcal sequelae and severe disease causing significant morbidity and mortality worldwide. To aid the development of a non-M protein-based prophylactic vaccine for the prevention of group A streptococcal infections, we identified novel immunogenic proteins using genomic surface display libraries and human serum antibodies from donors exposed to or infected by S. pyogenes. Vaccine candidate antigens were further selected based on animal protection in murine lethal-sepsis models with intranasal or intravenous challenge with two different M serotype strains. The nine protective antigens identified are highly conserved; eight of them show more than 97% sequence identity in 13 published genomes as well as in approximately 50 clinical isolates tested. Since the functions of the selected vaccine candidates are largely unknown, we generated deletion mutants for three of the protective antigens and observed that deletion of the gene encoding Spy1536 drastically reduced binding of GAS cells to host extracellular matrix proteins, due to reduced surface expression of GAS proteins such as Spy0269 and M protein. The protective, highly conserved antigens identified in this study are promising candidates for the development of an M-type-independent, protein-based vaccine to prevent infection by S. pyogenes.

  7. Study of model systems to test the potential function of Artemia group 1 late embryogenesis abundant (LEA) proteins.

    PubMed

    Warner, Alden H; Guo, Zhi-hao; Moshi, Sandra; Hudson, John W; Kozarova, Anna

    2016-01-01

    Embryos of the brine shrimp, Artemia franciscana, are genetically programmed to develop either ovoviparously or oviparously depending on environmental conditions. Shortly upon their release from the female, oviparous embryos enter diapause during which time they undergo major metabolic rate depression while simultaneously synthesize proteins that permit them to tolerate a wide range of stressful environmental events including prolonged periods of desiccation, freezing, and anoxia. Among the known stress-related proteins that accumulate in embryos entering diapause are the late embryogenesis abundant (LEA) proteins. This large group of intrinsically disordered proteins has been proposed to act as molecular shields or chaperones of macromolecules which are otherwise intolerant to harsh conditions associated with diapause. In this research, we used two model systems to study the potential function of the group 1 LEA proteins from Artemia. Expression of the Artemia group 1 gene (AfrLEA-1) in Escherichia coli inhibited growth in proportion to the number of 20-mer amino acid motifs expressed. As well, clones of E. coli, transformed with the AfrLEA-1 gene, expressed multiple bands of LEA proteins, either intrinsically or upon induction with isopropyl-β-thiogalactoside (IPTG), in a vector-specific manner. Expression of AfrLEA-1 in E. coli did not overcome the inhibitory effects of high concentrations of NaCl and KCl but modulated growth inhibition resulting from high concentrations of sorbitol in the growth medium. In contrast, expression of the AfrLEA-1 gene in Saccharomyces cerevisiae did not alter the growth kinetics or permit yeast to tolerate high concentrations of NaCl, KCl, or sorbitol. However, expression of AfrLEA-1 in yeast improved its tolerance to drying (desiccation) and freezing. Under our experimental conditions, both E. coli and S. cerevisiae appear to be potentially suitable hosts to study the function of Artemia group 1 LEA proteins under environmentally

  8. Interactions of Freshwater Cyanobacteria with Bacterial Antagonists

    PubMed Central

    Beier, Sara; Grabherr, Manfred

    2017-01-01

    ABSTRACT Cyanobacterial and algal mass development, or blooms, have severe effects on freshwater and marine systems around the world. Many of these phototrophs produce a variety of potent toxins, contribute to oxygen depletion, and affect water quality in several ways. Coexisting antagonists, such as cyanolytic bacteria, hold the potential to suppress, or even terminate, such blooms, yet the nature of this interaction is not well studied. We isolated 31 cyanolytic bacteria affiliated with the genera Pseudomonas, Stenotrophomonas, Acinetobacter, and Delftia from three eutrophic freshwater lakes in Sweden and selected four phylogenetically diverse bacterial strains with strong-to-moderate lytic activity. To characterize their functional responses to the presence of cyanobacteria, we performed RNA sequencing (RNA-Seq) experiments on coculture incubations, with an initial predator-prey ratio of 1:1. Genes involved in central cellular pathways, stress-related heat or cold shock proteins, and antitoxin genes were highly expressed in both heterotrophs and cyanobacteria. Heterotrophs in coculture expressed genes involved in cell motility, signal transduction, and putative lytic activity. l,d-Transpeptidase was the only significantly upregulated lytic gene in Stenotrophomonas rhizophila EK20. Heterotrophs also shifted their central metabolism from the tricarboxylic acid cycle to the glyoxylate shunt. Concurrently, cyanobacteria clearly show contrasting antagonistic interactions with the four tested heterotrophic strains, which is also reflected in the physical attachment to their cells. In conclusion, antagonistic interactions with cyanobacteria were initiated within 24 h, and expression profiles suggest varied responses for the different cyanobacteria and studied cyanolytes. IMPORTANCE Here, we present how gene expression profiles can be used to reveal interactions between bloom-forming freshwater cyanobacteria and antagonistic heterotrophic bacteria. Species

  9. Novel function of the Fanconi anemia group J or RECQ1 helicase to disrupt protein-DNA complexes in a replication protein A-stimulated manner.

    PubMed

    Sommers, Joshua A; Banerjee, Taraswi; Hinds, Twila; Wan, Bingbing; Wold, Marc S; Lei, Ming; Brosh, Robert M

    2014-07-18

    Understanding how cellular machinery deals with chromosomal genome complexity is an important question because protein bound to DNA may affect various cellular processes of nucleic acid metabolism. DNA helicases are at the forefront of such processes, yet there is only limited knowledge how they remodel protein-DNA complexes and how these mechanisms are regulated. We have determined that representative human RecQ and Fe-S cluster DNA helicases are potently blocked by a protein-DNA interaction. The Fanconi anemia group J (FANCJ) helicase partners with the single-stranded DNA-binding protein replication protein A (RPA) to displace BamHI-E111A bound to duplex DNA in a specific manner. Protein displacement was dependent on the ATPase-driven function of the helicase and unique properties of RPA. Further biochemical studies demonstrated that the shelterin proteins TRF1 and TRF2, which preferentially bind the telomeric repeat found at chromosome ends, effectively block FANCJ from unwinding the forked duplex telomeric substrate. RPA, but not the Escherichia coli single-stranded DNA-binding protein or shelterin factor Pot1, stimulated FANCJ ejection of TRF1 from the telomeric DNA substrate. FANCJ was also able to displace TRF2 from the telomeric substrate in an RPA-dependent manner. The stimulation of helicase-catalyzed protein displacement is also observed with the DNA helicase RECQ1, suggesting a conserved functional interaction of RPA-interacting helicases. These findings suggest that partnerships between RPA and interacting human DNA helicases may greatly enhance their ability to dislodge proteins bound to duplex DNA, an activity that is likely to be highly relevant to their biological roles in DNA metabolism.

  10. Endothelial nitric oxide synthase-enhancing G-protein coupled receptor antagonist inhibits pulmonary artery hypertension by endothelin-1-dependent and endothelin-1-independent pathways in a monocrotaline model.

    PubMed

    Liu, Chung-Pin; Dai, Zen-Kong; Huang, Chein-Heng; Yeh, Jwu-Lai; Wu, Bin-Nan; Wu, Jiunn-Ren; Chen, Ing-Jun

    2014-06-01

    This study investigates whether endothelin-1 (ET-1) mediates monocrotaline (MCT)-induced pulmonary artery hypertension (PAH) and right ventricular hypertrophy (RVH), and if so, whether the G-protein coupled receptor antagonist KMUP-1 (7-{2-[4-(2-chlorobenzene)piperazinyl]ethyl}-1,3-dimethylxanthine) inhibits ET-1-mediated PA constriction and the aforementioned pathological changes. In a chronic rat model, intraperitoneal MCT (60 mg/kg) induced PAH and increased PA medial wall thickening and RV/left ventricle + septum weight ratio on Day 21 after MCT injection. Treatment with sublingual KMUP-1 (2.5 mg/kg/day) for 21 days prevented these changes and restored vascular endothelial nitric oxide synthase (eNOS) immunohistochemical staining of lung tissues. Western blotting analysis demonstrated that KMUP-1 enhanced eNOS, soluble guanylate cyclase, and protein kinase G levels, and reduced ET-1 expression and inactivated Rho kinase II (ROCKII) in MCT-treated lung tissue over long-term administration. In MCT-treated rats, KMUP-1 decreased plasma ET-1 on Day 21. KMUP-1 (3.6 mg/kg) maximally appeared at 0.25 hours in the plasma and declined to basal levels within 24 hours after sublingual administration. In isolated PA of MCT-treated rats, compared with control and pretreatment with l-NG-nitroarginine methyl ester (100 μM), KMUP-1 (0.1-100 μM) inhibited ET-1 (0.01 μM)-induced vasoconstriction. Endothelium-denuded PA sustained higher contractility in the presence of KMUP-1. In a 24-hour culture of smooth muscle cells (i.e., PA smooth muscle cells or PASMCs), KMUP-1 (0.1-10 μM) inhibited RhoA- and ET-1-induced RhoA activation. KMUP-1 prevented MCT-induced PAH, PA wall thickening, and RVH by enhancing eNOS and suppressing ET-1/ROCKII expression. In vitro, KMUP-1 inhibited ET-1-induced PA constriction and ET-1-dependent/independent RhoA activation of PASMCs. In summary, KMUP-1 attenuates ET-1-induced/ET-1-mediated PA constriction, and could thus aid in the treatment of PAH

  11. Immunization with a streptococcal multiple-epitope recombinant protein protects mice against invasive group A streptococcal infection

    PubMed Central

    Kuo, Chih-Feng; Tsao, Nina; Hsieh, I-Chen; Lin, Yee-Shin; Wu, Jiunn-Jong; Hung, Yu-Ting

    2017-01-01

    Streptococcus pyogenes (group A Streptococcus; GAS) causes clinical diseases, including pharyngitis, scarlet fever, impetigo, necrotizing fasciitis and streptococcal toxic shock syndrome. A number of group A streptococcus vaccine candidates have been developed, but only one 26-valent recombinant M protein vaccine has entered clinical trials. Differing from the design of a 26-valent recombinant M protein vaccine, we provide here a vaccination using the polyvalence epitope recombinant FSBM protein (rFSBM), which contains four different epitopes, including the fibronectin-binding repeats domain of streptococcal fibronectin binding protein Sfb1, the C-terminal immunogenic segment of streptolysin S, the C3-binding motif of streptococcal pyrogenic exotoxin B, and the C-terminal conserved segment of M protein. Vaccination with the rFSBM protein successfully prevented mortality and skin lesions caused by several emm strains of GAS infection. Anti-FSBM antibodies collected from the rFSBM-immunized mice were able to opsonize at least six emm strains and can neutralize the hemolytic activity of streptolysin S. Furthermore, the internalization of GAS into nonphagocytic cells is also reduced by anti-FSBM serum. These findings suggest that rFSBM can be applied as a vaccine candidate to prevent different emm strains of GAS infection. PMID:28355251

  12. Phylogeny of replication initiator protein TrfA reveals a highly divergent clade of incompatibility group P1 plasmids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Incompatibility group P-1 (incP-1) includes broad host range plasmids of Gram negative bacteria and are classified into five subgroups (alpha, beta, gamma, delta, and epsilon). The incP-1 replication module consists of the trfA gene, encoding the replication initiator protein TrfA, and the origin o...

  13. Molecular basis determining inhibition/activation of nociceptive receptor TRPA1 protein: a single amino acid dictates species-specific actions of the most potent mammalian TRPA1 antagonist.

    PubMed

    Banzawa, Nagako; Saito, Shigeru; Imagawa, Toshiaki; Kashio, Makiko; Takahashi, Kenji; Tominaga, Makoto; Ohta, Toshio

    2014-11-14

    The transient receptor potential ankyrin 1 (TRPA1) is a Ca(2+)-permeable, nonselective cation channel mainly expressed in a subset of nociceptive neurons. TRPA1 functions as a cellular sensor detecting mechanical, chemical, and thermal stimuli. Because TRPA1 is considered to be a key player in nociception and inflammatory pain, TRPA1 antagonists have been developed as analgesic agents. Recently, by utilizing species differences, we identified the molecular basis of the antagonistic action of A967079, one of the most potent mammalian TRPA1 antagonists. Here, we show a unique effect of A967079 on TRPA1 from diverse vertebrate species, i.e. it acts as an agonist but not as an antagonist for chicken and frog TRPA1s. By characterizing chimeric channels of human and chicken TRPA1s, as well as point mutants, we found that a single specific amino acid residue located within the putative fifth transmembrane domain was involved in not only the stimulatory but also the inhibitory actions of A967079. AP18, structurally related to A967079, exerted similar pharmacological properties to A967079. Our findings and previous reports on species differences in the sensitivity to TRPA1 antagonists supply useful information in the search for novel analgesic medicines targeting TRPA1.

  14. Medicinal chemistry of competitive kainate receptor antagonists.

    PubMed

    Larsen, Ann M; Bunch, Lennart

    2011-02-16

    Kainic acid (KA) receptors belong to the group of ionotropic glutamate receptors and are expressed throughout in the central nervous system (CNS). The KA receptors have been shown to be involved in neurophysiological functions such as mossy fiber long-term potentiation (LTP) and synaptic plasticity and are thus potential therapeutic targets in CNS diseases such as schizophrenia, major depression, neuropathic pain and epilepsy. Extensive effort has been made to develop subtype-selective KA receptor antagonists in order to elucidate the physiological function of each of the five subunits known (GluK1-5). However, to date only selective antagonists for the GluK1 subunit have been discovered, which underlines the strong need for continued research in this area. The present review describes the structure-activity relationship and pharmacological profile for 10 chemically distinct classes of KA receptor antagonists comprising, in all, 45 compounds. To the medicinal chemist this information will serve as reference guidance as well as an inspiration for future effort in this field.

  15. Medicinal Chemistry of Competitive Kainate Receptor Antagonists

    PubMed Central

    2010-01-01

    Kainic acid (KA) receptors belong to the group of ionotropic glutamate receptors and are expressed throughout in the central nervous system (CNS). The KA receptors have been shown to be involved in neurophysiological functions such as mossy fiber long-term potentiation (LTP) and synaptic plasticity and are thus potential therapeutic targets in CNS diseases such as schizophrenia, major depression, neuropathic pain and epilepsy. Extensive effort has been made to develop subtype-selective KA receptor antagonists in order to elucidate the physiological function of each of the five subunits known (GluK1−5). However, to date only selective antagonists for the GluK1 subunit have been discovered, which underlines the strong need for continued research in this area. The present review describes the structure−activity relationship and pharmacological profile for 10 chemically distinct classes of KA receptor antagonists comprising, in all, 45 compounds. To the medicinal chemist this information will serve as reference guidance as well as an inspiration for future effort in this field. PMID:22778857

  16. Identification of Proteins Interacting with Cytoplasmic High-Mobility Group Box 1 during the Hepatocellular Response to Ischemia Reperfusion Injury

    PubMed Central

    Zhang, Tianjiao; Wei, Weiwei; Dirsch, Olaf; Krüger, Thomas; Kan, Chunyi; Xie, Chichi; Kniemeyer, Olaf; Fang, Haoshu; Settmacher, Utz; Dahmen, Uta

    2017-01-01

    Ischemia/reperfusion injury (IRI) occurs inevitably in liver transplantations and frequently during major resections, and can lead to liver dysfunction as well as systemic disorders. High-mobility group box 1 (HMGB1) plays a pathogenic role in hepatic IRI. In the normal liver, HMGB1 is located in the nucleus of hepatocytes; after ischemia reperfusion, it translocates to the cytoplasm and it is further released to the extracellular space. Unlike the well-explored functions of nuclear and extracellular HMGB1, the role of cytoplasmic HMGB1 in hepatic IRI remains elusive. We hypothesized that cytoplasmic HMGB1 interacts with binding proteins involved in the hepatocellular response to IRI. In this study, binding proteins of cytoplasmic HMGB1 during hepatic IRI were identified. Liver tissues from rats with warm ischemia reperfusion (WI/R) injury and from normal rats were subjected to cytoplasmic protein extraction. Co-immunoprecipitation using these protein extracts was performed to enrich HMGB1-protein complexes. To separate and identify the immunoprecipitated proteins in eluates, 2-dimensional electrophoresis and subsequent mass spectrometry detection were performed. Two of the identified proteins were verified using Western blotting: betaine–homocysteine S-methyltransferase 1 (BHMT) and cystathionine γ-lyase (CTH). Therefore, our results revealed the binding of HMGB1 to BHMT and CTH in cytoplasm during hepatic WI/R. This finding may help to better understand the cellular response to IRI in the liver and to identify novel molecular targets for reducing ischemic injury. PMID:28275217

  17. Euglena gracilis chloroplast ribosomal protein operon: a new chloroplast gene for ribosomal protein L5 and description of a novel organelle intron category designated group III.

    PubMed Central

    Christopher, D A; Hallick, R B

    1989-01-01

    We describe the structure (3840 bp) of a novel Euglena gracilis chloroplast ribosomal protein operon that encodes the five genes rpl16-rpl14-rpl5-rps8-rpl36. The gene organization resembles the spc and the 3'-end of the S10 ribosomal protein operons of E. coli. The rpl5 is a new chloroplast gene not previously reported for any chloroplast genome to date and also not described as a nuclear-encoded, chloroplast protein gene. The operon contains at least 7 introns. We present evidence from primer extension analysis of chloroplast RNA for the correct in vivo splicing of five of the introns. Two of the introns within the rps8 gene flank an 8 bp exon, the smallest exon yet characterized in a chloroplast gene. Three introns resemble the classical group II introns of organelle genomes. The remaining 4 introns appear to be unique to the Euglena chloroplast DNA. They are uniform in size (95-109 nt), share common features with each other and are distinct from both group I and group II introns. We designate this new intron category as 'group III'. Images PMID:2477800

  18. Protein intake and exercise for optimal muscle function with aging: recommendations from the ESPEN Expert Group.

    PubMed

    Deutz, Nicolaas E P; Bauer, Jürgen M; Barazzoni, Rocco; Biolo, Gianni; Boirie, Yves; Bosy-Westphal, Anja; Cederholm, Tommy; Cruz-Jentoft, Alfonso; Krznariç, Zeljko; Nair, K Sreekumaran; Singer, Pierre; Teta, Daniel; Tipton, Kevin; Calder, Philip C

    2014-12-01

    The aging process is associated with gradual and progressive loss of muscle mass along with lowered strength and physical endurance. This condition, sarcopenia, has been widely observed with aging in sedentary adults. Regular aerobic and resistance exercise programs have been shown to counteract most aspects of sarcopenia. In addition, good nutrition, especially adequate protein and energy intake, can help limit and treat age-related declines in muscle mass, strength, and functional abilities. Protein nutrition in combination with exercise is considered optimal for maintaining muscle function. With the goal of providing recommendations for health care professionals to help older adults sustain muscle strength and function into older age, the European Society for Clinical Nutrition and Metabolism (ESPEN) hosted a Workshop on Protein Requirements in the Elderly, held in Dubrovnik on November 24 and 25, 2013. Based on the evidence presented and discussed, the following recommendations are made (a) for healthy older people, the diet should provide at least 1.0-1.2 g protein/kg body weight/day, (b) for older people who are malnourished or at risk of malnutrition because they have acute or chronic illness, the diet should provide 1.2-1.5 g protein/kg body weight/day, with even higher intake for individuals with severe illness or injury, and (c) daily physical activity or exercise (resistance training, aerobic exercise) should be undertaken by all older people, for as long as possible.

  19. Protein intake and exercise for optimal muscle function with aging: Recommendations from the ESPEN Expert Group

    PubMed Central

    Deutz, Nicolaas E. P.; Bauer, Jurgen M.; Barazzoni, Rocco; Biolo, Gianni; Boirie, Yves; Bosy-Westphal, Anja; Cederholm, Tommy; Cruz-Jentoft, Alfonso; Krznaric, Zeljko; Nair, K. Sreekumaran; Singer, Pierre; Teta, Daniel; Tipton, Kevin; Calder, Philip C.

    2014-01-01

    The aging process is associated with gradual and progressive loss of muscle mass along with lowered strength and physical endurance. This condition, sarcopenia, has been widely observed with aging in sedentary adults. Regular aerobic and resistance exercise programs have been shown to counteract most aspects of sarcopenia. In addition, good nutrition, especially adequate protein and energy intake, can help limit and treat age-related declines in muscle mass, strength, and functional abilities. Protein nutrition in combination with exercise is considered optimal for maintaining muscle function. With the goal of providing recommendations for health care professionals to help older adults sustain muscle strength and function into older age, the European Society for Clinical Nutrition and Metabolism (ESPEN) hosted a Workshop on Protein Requirements in the Elderly, held in Dubrovnik on November 24 and 25, 2013. Based on the evidence presented and discussed, the following recommendations are made: (1) for healthy older people, the diet should provide at least 1.0 to 1.2 g protein/kg body weight/day (2) for older people who are malnourished or at risk of malnutrition because they have acute or chronic illness, the diet should provide 1.2 to 1.5 g protein/kg body weight/day, with even higher intake for individuals with severe illness or injury, and (3) daily physical activity or exercise (resistance training, aerobic exercise) should be undertaken by all older people, for as long as possible. PMID:24814383

  20. Synthesis and characterization of a 'fluorous' (fluorinated alkyl) affinity reagent that labels primary amine groups in proteins/peptides.

    PubMed

    Qian, Jiang; Cole, Richard B; Cai, Yang

    2011-01-01

    Strong non-covalent interactions such as biotin-avidin affinity play critical roles in protein/peptide purification. A new type of 'fluorous' (fluorinated alkyl) affinity approach has gained popularity due especially to its low level of non-specific binding to proteins/peptides. We have developed a novel water-soluble fluorous labeling reagent that is reactive (via an active sulfo-N-hydroxylsuccinimidyl ester group) to primary amine groups in proteins/peptides. After fluorous affinity purification, the bulky fluorous tag moiety and the long oligoethylene glycol (OEG) spacer of this labeling reagent can be trimmed via the cleavage of an acid labile linker. Upon collision-induced dissociation, the labeled peptide ion yields a characteristic fragment that can be retrieved from the residual portion of the fluorous affinity tag, and this fragment ion can serve as a marker to indicate that the relevant peptide has been successfully labeled. As a proof of principle, the newly synthesized fluorous labeling reagent was evaluated for peptide/protein labeling ability in phosphate-buffered saline (PBS). Results show that both the aqueous environment protein/peptide labeling and the affinity enrichment/separation process were highly efficient.

  1. Influence of surface groups of proteins on water studied by freezing/thawing hysteresis and infrared spectroscopy.

    PubMed

    Zelent, Bogumil; Bryan, Michael A; Sharp, Kim A; Vanderkooi, Jane M

    2009-05-01

    The influence of proteins and solutes on hysteresis of freezing and melting of water was measured by infrared (IR) spectroscopy. Of the solutes examined, poly-L-arginine and flounder antifreeze protein produced the largest freezing point depression of water, with little effect on the melting temperature. Poly-L-lysine, poly-L-glutamate, cytochrome c and bovine serum albumin had less effect on the freezing of water. Small compounds used to mimic non-polar (trimethylamine N-oxide, methanol), positively charged (guanidinium chloride, NH(4)Cl, urea) and negatively charged (Na acetate) groups on protein surfaces were also examined. These molecules and ions depress water's freezing point and the melting profiles became broad. Since infrared absorption measures both bulk solvent and solvent bound to the solutes, this result is consistent with solutes interacting with liquid water. The amide I absorption bands of antifreeze protein and poly-L-arginine do not detectably change with the phase transition of water. An interpretation is that the antifreeze protein and poly-L-arginine order liquid water such that the water around the group is ice-like.

  2. Psip1/Ledgf p75 restrains Hox gene expression by recruiting both trithorax and polycomb group proteins

    PubMed Central

    Pradeepa, Madapura M.; Grimes, Graeme R.; Taylor, Gillian C.A.; Sutherland, Heidi G.; Bickmore, Wendy A.

    2014-01-01

    Trithorax and polycomb group proteins are generally thought to antagonize one another. The trithorax family member MLL (myeloid/lymphoid or mixed-lineage leukemia) is presumed to activate Hox expression, counteracting polycomb-mediated repression. PC4 and SF2 interacting protein 1 (PSIP1)/p75, also known as LEDGF, whose PWWP domain binds to H3K36me3, interacts with MLL and tethers MLL fusion proteins to HOXA9 in leukaemias. Here we show, unexpectedly, that Psip1/p75 regulates homeotic genes by recruiting not only MLL complexes, but also the polycomb group protein Bmi1. In Psip1−/− cells binding of Mll1/2, Bmi1 and the co-repressor Ctbp1 at Hox loci are all abrogated and Hoxa and Hoxd mRNA expression increased. Our data not only reveal a potential mechanism of action for Psip1 in the regulation of Hox genes but also suggest an unexpected interplay between proteins usually considered as transcriptional activators and repressors. PMID:25056311

  3. Group 3 LEA Protein, ZmLEA3, Is Involved in Protection from Low Temperature Stress

    PubMed Central

    Liu, Yang; Liang, Jianan; Sun, Liping; Yang, Xinghong; Li, Dequan

    2016-01-01

    Late embryogenesis abundant (LEA) proteins are a family of small highly hydrophilic proteins that accumulate at the onset of seed desiccation and in response to adverse conditions such as drought, salinity, low temperature, or water deficit. In previous studies, we demonstrated that ZmLEA3 could enhance the transgenic tobacco tolerance to osmotic and oxidative stresses. Here, we demonstrated that the transcription of ZmLEA3 in the maize stems could be significantly induced by low temperature and osmotic stresses and by treatment with abscisic acid (ABA) and H2O2. Further study indicated that ZmLEA3 is a single copy gene in the maize genome. The ZmLEA3 protein could protect lactate dehydrogenase (LDH) activity at low temperatures. The overexpression of ZmLEA3 conferred tolerance to low-temperature stress to transgenic tobacco, yeast (GS115) and E. coli (BL21). PMID:27471509

  4. Breast Cancer Stem-Like Cells Are Inhibited by Diosgenin, a Steroidal Saponin, by the Attenuation of the Wnt β-Catenin Signaling via the Wnt Antagonist Secreted Frizzled Related Protein-4

    PubMed Central

    Bhuvanalakshmi, G.; Basappa; Rangappa, Kanchugarakoppal S.; Dharmarajan, Arun; Sethi, Gautam; Kumar, Alan P.; Warrier, Sudha

    2017-01-01

    Background: Identification of breast cancer stem cells as the chemo-resistant and tumor-initiating population represents an important milestone in approaching anticancer therapies. Targeting this minor subpopulation of chemo- and radio-resistant stem-like cells, termed as the cancer stem cells (CSCs) and their eradication could significantly enhance clinical outcomes. Most of the presently administered chemotherapeutics target the tumor bulk but are ineffective against the CSCs. We report here that diosgenin (DG), a naturally occurring steroidal saponin, could effectively inhibit CSCs from three breast cancer cell lines, MCF7, T47D and MDA-MB-231, by inducing apoptosis and inhibiting the CSC associated phenotypes. Methods: CSCs were enriched in these cells lines, characterized for CSC traits by immunocytochemistry and flow cytometry. Proliferation and apoptosis assays were performed in these breast CSCs in the presence of DG to obtain the inhibitory concentration. Apoptosis was confirmed with gene expression analysis, Western blotting and propidium iodide staining. TCF-LEF reporter assay, sFRP overexpression and RNAi silencing studies were performed to study regulation of the Wnt pathway. Statistical significance was evaluated by a two-sided Student’s t-test. Results: Using the TCF-LEF reporter system, we show the effect of DG on CSCs is predominantly through the network regulating CSC self renewal, the Wnt β-catenin pathway. Specifically, the Wnt antagonist, the secreted frizzled related protein 4, (sFRP4), had a defining role in the action of DG. Gain-of-function of sFRP4 in CSCs could improve the response to DG wherein CSC mediators were inhibited, β-catenin was down regulated and the effectors of epithelial to mesenchymal transition and pro-invasive markers were repressed. Conversely, the loss-of-function of sFRP4 had a reverse effect on the CSC population which therein became enriched, their response to DG treatment was modest, β-catenin levels increased

  5. Breast Cancer Stem-Like Cells Are Inhibited by Diosgenin, a Steroidal Saponin, by the Attenuation of the Wnt β-Catenin Signaling via the Wnt Antagonist Secreted Frizzled Related Protein-4.

    PubMed

    Bhuvanalakshmi, G; Basappa; Rangappa, Kanchugarakoppal S; Dharmarajan, Arun; Sethi, Gautam; Kumar, Alan P; Warrier, Sudha

    2017-01-01

    Background: Identification of breast cancer stem cells as the chemo-resistant and tumor-initiating population represents an important milestone in approaching anticancer therapies. Targeting this minor subpopulation of chemo- and radio-resistant stem-like cells, termed as the cancer stem cells (CSCs) and their eradication could significantly enhance clinical outcomes. Most of the presently administered chemotherapeutics target the tumor bulk but are ineffective against the CSCs. We report here that diosgenin (DG), a naturally occurring steroidal saponin, could effectively inhibit CSCs from three breast cancer cell lines, MCF7, T47D and MDA-MB-231, by inducing apoptosis and inhibiting the CSC associated phenotypes. Methods: CSCs were enriched in these cells lines, characterized for CSC traits by immunocytochemistry and flow cytometry. Proliferation and apoptosis assays were performed in these breast CSCs in the presence of DG to obtain the inhibitory concentration. Apoptosis was confirmed with gene expression analysis, Western blotting and propidium iodide staining. TCF-LEF reporter assay, sFRP overexpression and RNAi silencing studies were performed to study regulation of the Wnt pathway. Statistical significance was evaluated by a two-sided Student's t-test. Results: Using the TCF-LEF reporter system, we show the effect of DG on CSCs is predominantly through the network regulating CSC self renewal, the Wnt β-catenin pathway. Specifically, the Wnt antagonist, the secreted frizzled related protein 4, (sFRP4), had a defining role in the action of DG. Gain-of-function of sFRP4 in CSCs could improve the response to DG wherein CSC mediators were inhibited, β-catenin was down regulated and the effectors of epithelial to mesenchymal transition and pro-invasive markers were repressed. Conversely, the loss-of-function of sFRP4 had a reverse effect on the CSC population which therein became enriched, their response to DG treatment was modest, β-catenin levels increased

  6. Effect of Oxygen-containing Functional Groups on Protein Stability in Ionic Liquid Solutions

    NASA Technical Reports Server (NTRS)

    Turner, Megan B.; Holbrey, John D.; Spear, Scott K.; Pusey, Marc L.; Rogers, Robin D.

    2004-01-01

    The ability of functionalized ionic liquids (ILs) to provide an environment of increased stability for biomolecules has been studied. Serum albumin is an inexpensive, widely available protein that contributes to the overall colloid osmotic blood pressure within the vascular system. Albumin is used in the present study as a marker of biomolecular stability in the presence of various ILs in a range of concentrations. The incorporation of hydroxyl functionality into the methylimidazolium-based cation leads to increased protein stability detected by fluorescence spectroscopy and circular dichroic (CD) spectrometry.

  7. Zebrafish phenotypic screen identifies novel Notch antagonists.

    PubMed

    Velaithan, Vithya; Okuda, Kazuhide Shaun; Ng, Mei Fong; Samat, Norazwana; Leong, Sze Wei; Faudzi, Siti Munirah Mohd; Abas, Faridah; Shaari, Khozirah; Cheong, Sok Ching; Tan, Pei Jean; Patel, Vyomesh

    2017-04-01

    Zebrafish represents a powerful in vivo model for phenotype-based drug discovery to identify clinically relevant small molecules. By utilizing this model, we evaluated natural product derived compounds that could potentially modulate Notch signaling that is important in both zebrafish embryogenesis and pathogenic in human cancers. A total of 234 compounds were screened using zebrafish embryos and 3 were identified to be conferring phenotypic alterations similar to embryos treated with known Notch inhibitors. Subsequent secondary screens using HEK293T cells overexpressing truncated Notch1 (HEK293TΔE) identified 2 compounds, EDD3 and 3H4MB, to be potential Notch antagonists. Both compounds reduced protein expression of NOTCH1, Notch intracellular domain (NICD) and hairy and enhancer of split-1 (HES1) in HEK293TΔE and downregulated Notch target genes. Importantly, EDD3 treatment of human oral cancer cell lines demonstrated reduction of Notch target proteins and genes. EDD3 also inhibited proliferation and induced G0/G1 cell cycle arrest of ORL-150 cells through inducing p27(KIP1). Our data demonstrates the utility of the zebrafish phenotypic screen and identifying EDD3 as a promising Notch antagonist for further development as a novel therapeutic agent.

  8. Barnidipine, a novel calcium antagonist for once-daily treatment of hypertension: a multicenter, double-blind, placebo-controlled, dose-ranging study. Dutch Barnidipine Multicenter Study Group.

    PubMed

    Hart, W; Holwerda, N J

    1997-11-01

    The antihypertensive effects and tolerance of once-daily barnidipine, a novel dihydropyridine calcium antagonist, were evaluated. A total of 190 patients with a sitting diastolic blood pressure (DBP) of 95-114 mmHg were investigated in this multicenter, double-blind, placebo-controlled, dose-ranging study. After a 4-week single-blind placebo run-in period, patients were randomized to placebo or barnidipine (10 mg, 20 mg, or 30 mg modified release capsules) once daily for 6 weeks. Nonresponders (sitting DBP > or =90 mmHg and a decrease of < 10 mmHg) were treated for an additional 6 weeks with a dose increase of 10 mg. At each clinic visit, sitting and standing blood pressure and heart rate were measured approximately 24 hours after the last dose of study drug was taken. Compared with placebo, barnidipine lowered blood pressure, with a trend toward a dose-response relationship over the dose range 10-30 mg. A dose increment of 10 mg in nonresponders resulted in additional reductions in blood pressure. At the end of the active treatment period, the responder rates were 41% and 57% for 10 mg and 20 mg barnidipine, respectively. Heart rate in both sitting and standing positions was not affected by barnidipine. Treatment with barnidipine was well tolerated, and the incidence of adverse events was dose related and consistent with vasodilatation. In conclusion, barnidipine (10-30 mg) administered once daily is well tolerated and reduces blood pressure in patients with mild to moderate hypertension.

  9. Partially redundant functions of two SET-domain polycomb-group proteins in controlling initiation of seed development in Arabidopsis.

    PubMed

    Wang, Dongfang; Tyson, Mark D; Jackson, Shawn S; Yadegari, Ramin

    2006-08-29

    In Arabidopsis, a complex of Polycomb-group (PcG) proteins functions in the female gametophyte to control the initiation of seed development. Mutations in the PcG genes, including MEDEA (MEA) and FERTILIZATION-INDEPENDENT SEED 2 (FIS2), produce autonomous seeds where endosperm proliferation occurs in the absence of fertilization. By using a yeast two-hybrid screen, we identified MEA and a related protein, SWINGER (SWN), as SET-domain partners of FIS2. Localization data indicated that all three proteins are present in the female gametophyte. Although single-mutant swn plants did not show any defects, swn mutations enhanced the mea mutant phenotype in producing autonomous seeds. Thus, MEA and SWN perform partially redundant functions in controlling the initiation of endosperm development before fertilization in Arabidopsis.

  10. Phylogenetic relationships and protein modelling revealed two distinct subfamilies of group II HKT genes between crop and model grasses.

    PubMed

    Ariyarathna, H A Chandima K; Francki, Michael G

    2016-07-01

    Molecular evolution of large protein families in closely related species can provide useful insights on structural functional relationships. Phylogenetic analysis of the grass-specific group II HKT genes identified two distinct subfamilies, I and II. Subfamily II was represented in all species, whereas subfamily I was identified only in the small grain cereals and possibly originated from an ancestral gene duplication post divergence from the coarse grain cereal lineage. The core protein structures were highly analogous despite there being no more than 58% amino acid identity between members of the two subfamilies. Distinctly variable regions in known functional domains, however, indicated functional divergence of the two subfamilies. The subsets of codons residing external to known functional domains predicted signatures of positive Darwinian selection potentially identifying new domains of functional divergence and providing new insights on the structural function and relationships between protein members of the two subfamilies.

  11. Correction of the DNA repair defect in xeroderma pigmentosum group E by injection of a DNA damage-binding protein

    SciTech Connect

    Keeney, S.; Brody, T.; Linn, S.; Eker, A.P.M.; Vermeulen, W.; Bootsma, D.; Hoeijmakers, J.H.J.

    1994-04-26

    Cells from a subset of patients with the DNA-repair-defective disease xeroderma pigmentosum complementation group E (XP-E) are known to lack a DNA damage-binding (DDB) activity. Purified human DDB protein was injected into XP-E cells to test whether the DNA-repair defect in these cells is caused by a defect in DDB activity. Injected DDB protein stimulated DNA repair to normal levels in those strains that lack the DDB activity but did not stimulate repair in cells from other xeroderma pigmentosum groups or in XP-E cells that contain the activity. These results provide direct evidence that defective DDB activity causes the repair defect in a subset of XP-E patients, which in turn establishes a role for this activity in nucleotide-excision repair in vivo.

  12. Insights into the evolution of Archaea and eukaryotic protein modifier systems revealed by the genome of a novel archaeal group

    PubMed Central

    Nunoura, Takuro; Takaki, Yoshihiro; Kakuta, Jungo; Nishi, Shinro; Sugahara, Junichi; Kazama, Hiromi; Chee, Gab-Joo; Hattori, Masahira; Kanai, Akio; Atomi, Haruyuki; Takai, Ken; Takami, Hideto

    2011-01-01

    The domain Archaea has historically been divided into two phyla, the Crenarchaeota and Euryarchaeota. Although regarded as members of the Crenarchaeota based on small subunit rRNA phylogeny, environmental genomics and efforts for cultivation have recently revealed two novel phyla/divisions in the Archaea; the ‘Thaumarchaeota’ and ‘Korarchaeota’. Here, we show the genome sequence of Candidatus ‘Caldiarchaeum subterraneum’ that represents an uncultivated crenarchaeotic group. A composite genome was reconstructed from a metagenomic library previously prepared from a microbial mat at a geothermal water stream of a sub-surface gold mine. The genome was found to be clearly distinct from those of the known phyla/divisions, Crenarchaeota (hyperthermophiles), Euryarchaeota, Thaumarchaeota and Korarchaeota. The unique traits suggest that this crenarchaeotic group can be considered as a novel archaeal phylum/division. Moreover, C. subterraneum harbors an ubiquitin-like protein modifier system consisting of Ub, E1, E2 and small Zn RING finger family protein with structural motifs specific to eukaryotic system proteins, a system clearly distinct from the prokaryote-type system recently identified in Haloferax and Mycobacterium. The presence of such a eukaryote-type system is unprecedented in prokaryotes, and indicates that a prototype of the eukaryotic protein modifier system is present in the Archaea. PMID:21169198

  13. Insights into the evolution of Archaea and eukaryotic protein modifier systems revealed by the genome of a novel archaeal group.

    PubMed

    Nunoura, Takuro; Takaki, Yoshihiro; Kakuta, Jungo; Nishi, Shinro; Sugahara, Junichi; Kazama, Hiromi; Chee, Gab-Joo; Hattori, Masahira; Kanai, Akio; Atomi, Haruyuki; Takai, Ken; Takami, Hideto

    2011-04-01

    The domain Archaea has historically been divided into two phyla, the Crenarchaeota and Euryarchaeota. Although regarded as members of the Crenarchaeota based on small subunit rRNA phylogeny, environmental genomics and efforts for cultivation have recently revealed two novel phyla/divisions in the Archaea; the 'Thaumarchaeota' and 'Korarchaeota'. Here, we show the genome sequence of Candidatus 'Caldiarchaeum subterraneum' that represents an uncultivated crenarchaeotic group. A composite genome was reconstructed from a metagenomic library previously prepared from a microbial mat at a geothermal water stream of a sub-surface gold mine. The genome was found to be clearly distinct from those of the known phyla/divisions, Crenarchaeota (hyperthermophiles), Euryarchaeota, Thaumarchaeota and Korarchaeota. The unique traits suggest that this crenarchaeotic group can be considered as a novel archaeal phylum/division. Moreover, C. subterraneum harbors an ubiquitin-like protein modifier system consisting of Ub, E1, E2 and small Zn RING finger family protein with structural motifs specific to eukaryotic system proteins, a system clearly distinct from the prokaryote-type system recently identified in Haloferax and Mycobacterium. The presence of such a eukaryote-type system is unprecedented in prokaryotes, and indicates that a prototype of the eukaryotic protein modifier system is present in the Archaea.

  14. Isolation of heart- and kidney-binding protein from group A streptococci.

    PubMed Central

    Stinson, M W; Bergey, E J

    1982-01-01

    Tritium-labeled, water-soluble components of Streptococcus pyogenes type M6 absorbed to cardiac tissue in vitro. Tissue binding was time dependent, saturable, and reversible. Chromatography of the crude bacterial extract on Bio-Gel P-300 indicated a molecular weight greater than 300,000 for the heart-binding component. Sodium dodecyl sulfate (SDS) dissociated this aggregate into a protein of 18,000 to 20,000 daltons as determined by Sephacryl S-200 chromatography and SDS-polyacrylamide disc gel electrophoresis. The tissue-binding protein was also purified from streptococcal extracts by absorption to immobilized heart components. SDS-polyacrylamide gel electrophoresis of the protein desorbed from tissue revealed a radioactive band of 19,000 daltons. Indirect immunofluorescence tests on cardiac tissue treated with streptococcal extract showed an accumulation of a bacterial antigen on the sarcolemmal sheaths. Streptococcal components also adsorbed to basement membranes of kidney. Antisera prepared to isolated cytoplasmic membranes and water-soluble extracts of S. pyogenes type M6 were the most sensitive reagents for the detection of bacterial components bound to tissue. Antisera prepared to isolated cell walls and to intact bacteria were weakly reactive in these assays. Images PMID:7033140

  15. Investigation on quantitative structure activity relationships and pharmacophore modeling of a series of mGluR2 antagonists.

    PubMed

    Zhang, Meng-Qi; Zhang, Xiao-Le; Li, Yan; Fan, Wen-Jia; Wang, Yong-Hua; Hao, Ming; Zhang, Shu-Wei; Ai, Chun-Zhi

    2011-01-01

    MGluR2 is G protein-coupled receptor that is targeted for diseases like anxiety, depression, Parkinson's disease and schizophrenia. Herein, we report the three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of a series of 1,3-dihydrobenzo[ b][1,4]diazepin-2-one derivatives as mGluR2 antagonists. Two series of models using two different activities of the antagonists against rat mGluR2, which has been shown to be very similar to the human mGluR2, (activity I: inhibition of [(3)H]-LY354740; activity II: mGluR2 (1S,3R)-ACPD inhibition of forskolin stimulated cAMP.) were derived from datasets composed of 137 and 69 molecules respectively. For activity I study, the best predictive model obtained from CoMFA analysis yielded a Q(2) of 0.513, R(2) (ncv) of 0.868, R(2) (pred) = 0.876, while the CoMSIA model yielded a Q(2) of 0.450, R(2) (ncv) = 0.899, R(2) (pred) = 0.735. For activity II study, CoMFA model yielded statistics of Q(2) = 0.5, R(2) (ncv) = 0.715, R(2) (pred) = 0.723. These results prove the high predictability of the models. Furthermore, a combined analysis between the CoMFA, CoMSIA contour maps shows that: (1) Bulky substituents in R(7), R(3) and position A benefit activity I of the antagonists, but decrease it when projected in R(8) and position B; (2) Hydrophilic groups at position A and B increase both antagonistic activity I and II; (3) Electrostatic field plays an essential rule in the variance of activity II. In search for more potent mGluR2 antagonists, two pharmacophore models were developed separately for the two activities. The first model reveals six pharmacophoric features, namely an aromatic center, two hydrophobic centers, an H-donor atom, an H-acceptor atom and an H-donor site. The second model shares all features of the first one and has an additional acceptor site, a positive N and an aromatic center. These models can be used as guidance for the development of new mGluR2 antagonists of high activity and selectivity

  16. Imaging and three-dimensional reconstruction of chemical groups inside a protein complex using atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Kim, Duckhoe; Sahin, Ozgur

    2015-03-01

    Scanning probe microscopes can be used to image and chemically characterize surfaces down to the atomic scale. However, the localized tip-sample interactions in scanning probe microscopes limit high-resolution images to the topmost atomic layer of surfaces, and characterizing the inner structures of materials and biomolecules is a challenge for such instruments. Here, we show that an atomic force microscope can be used to image and three-dimensionally reconstruct chemical groups inside a protein complex. We use short single-stranded DNAs as imaging labels that are linked to target regions inside a protein complex, and T-shaped atomic force microscope cantilevers functionalized with complementary probe DNAs allow the labels to be located with sequence specificity and subnanometre resolution. After measuring pairwise distances between labels, we reconstruct the three-dimensional structure formed by the target chemical groups within the protein complex using simple geometric calculations. Experiments with the biotin-streptavidin complex show that the predicted three-dimensional loci of the carboxylic acid groups of biotins are within 2 Å of their respective loci in the corresponding crystal structure, suggesting that scanning probe microscopes could complement existing structural biological techniques in solving structures that are difficult to study due to their size and complexity.

  17. Invasion of protein coding genes by green algal ribosomal group I introns.

    PubMed

    McManus, Hilary A; Lewis, Louise A; Fučíková, Karolina; Haugen, Peik

    2012-01-01

    The spread of group I introns depends on their association with intron-encoded homing endonucleases. Introns that encode functional homing endonuclease genes (HEGs) are highly invasive, whereas introns that only encode the group I ribozyme responsible for self-splicing are generally stably inherited (i.e., vertical inheritance). A number of recent case studies have provided new knowledge on the evolution of group I introns, however, there are still large gaps in understanding of their distribution on the tree of life, and how they have spread into new hosts and genic sites. During a larger phylogenetic survey of chlorophyceaen green algae, we found that 23 isolates contain at least one group I intron in the rbcL chloroplast gene. Structural analyses show that the introns belong to one of two intron lineages, group IA2 intron-HEG (GIY-YIG family) elements inserted after position 462 in the rbcL gene, and group IA1 introns inserted after position 699. The latter intron type sometimes encodes HNH homing endonucleases. The distribution of introns was analyzed on an exon phylogeny and patterns were recovered that are consistent with vertical inheritance and possible horizontal transfer. The rbcL 462 introns are thus far reported only within the Volvocales, Hydrodictyaceae and Bracteacoccus, and closely related isolates of algae differ in the presence of rbcL introns. Phylogenetic analysis of the intron conserved regions indicates that the rbcL699 and rbcL462 introns have distinct evolutionary origins. The rbcL699 introns were likely derived from ribosomal RNA L2449 introns, whereas the rbcL462 introns form a close relationship with psbA introns.

  18. XPA gene mutations resulting in subtle truncation of protein in xeroderma pigmentosum group A patients with mild skin symptoms.

    PubMed

    Takahashi, Yoshito; Endo, Yoko; Sugiyama, Yoshinori; Inoue, Shintaro; Iijima, Masahiro; Tomita, Yasushi; Kuru, Satoshi; Takigawa, Masahiro; Moriwaki, Shinichi

    2010-10-01

    Comparisons of the clinical manifestations with gene mutations in patients with xeroderma pigmentosum group A (XPA) have suggested that those with mutations closer to the C-terminal coding region of the XPA gene have milder neurological and cutaneous symptoms. Here we report on four middle-aged, newly diagnosed Japanese XPA patients whose unusually mild symptoms, especially those affecting the skin, implicate a reduced association of a subtle defect in the C-terminus of XPA protein with skin lesions. All patients had a heterozygous G → C transversion at the splice acceptor site of XPA intron 3. We identified previously unreported heterozygous mutations in exon 6: a single-base insertion (690insT) in one patient and a four-base insertion (779insTT and 780insTT) in the other patients. These mutations led to the frameshift that created new premature termination codons, resulting in the production of truncated XPA proteins. They were longer than any previously reported truncated XPA protein, suggesting that the minimal cutaneous symptoms in these patients are due to a higher residual level of XPA protein activity and that the subtle defect in the C-terminus of XPA protein is more closely related to neurological impairment than to cutaneous abnormalities.

  19. Identification, structural, and biochemical characterization of a group of large Csn2 proteins involved in CRISPR-mediated bacterial immunity.

    PubMed

    Lee, Kwang-Hoon; Lee, Seong-Gyu; Eun Lee, Kyung; Jeon, Hyesung; Robinson, Howard; Oh, Byung-Ha

    2012-11-01

    Many prokaryotic organisms acquire immunity against foreign genetic material by incorporating a short segment of foreign DNA called spacer into chromosomal loci, termed clustered regularly interspaced short palindromic repeats (CRISPRs). The encoded RNAs are processed into small fragments that guide the silencing of the invading genetic elements. The CRISPR-associated (Cas) proteins are the main executioners of these processes. Herein, we report the crystal structure of Stu0660 of Streptococcus thermophilus, a Cas protein involved in the acquisition of new spacers. By homotetramerization, Stu0660 forms a central channel which is decorated with basic amino acids and binds linear double-stranded DNA (dsDNA), but not circular dsDNA. Despite undetectably low sequence similarity, two N-terminal domains of Stu0660 are similar to the entire structure of an Enterococcus faecalis Csn2 protein, which also forms a homotetramer and binds dsDNA. Thus, this work identifies a previously unknown group of Stu0660-like Csn2 proteins (∼350 residues), which are larger than the known canonical Csn2 proteins (∼220 residues) by containing an extra C-terminal domain. The commonly present central channel in the two subgroups appears as a design to selectively interact with linear dsDNA.

  20. Biochemical and biological properties of the binding of human fibrinogen to M protein in group A streptococci

    SciTech Connect

    Whitnack, E.; Beachey, E.H.

    1985-10-01

    Fibrinogen is known to bind to group A streptococci and precipitate with extracts containing streptococcal M protein. The authors have previously shown that the binding of fibrinogen to M-positive streptococci prevents opsonization by complement and protects that organism from phagocytosis in nonimmune blood. In the present study, they used TH-labeled fibrinogen, a highly purified peptide fragment of type 24 M protein (pep M24), and anti-pep M sera to show that fibrinogen binds to M-positive streptococci with high affinity; occupation of the high-affinity binding sites suffices to protect the organism from phagocytosis; proteolytic treatments that remove M protein from streptococcal cells abolish binding; binding is competitively inhibited by anti-pep M sera; pep M24 precipitates fibrinogen; and binding to type 24 cells is inhibited by pep M24. They conclude that M protein is the cell surface structure principally responsible for binding fibrinogen on the surface of M-positive streptococci and that this binding contributes to the known antiopsonic property of M proteins.

  1. SUMO modification is required for in vivo Hox gene regulation by the Caenorhabditis elegans Polycomb group protein SOP-2.

    PubMed

    Zhang, Hong; Smolen, Gromoslaw A; Palmer, Rachel; Christoforou, Andrea; van den Heuvel, Sander; Haber, Daniel A

    2004-05-01

    Post-translational modification of proteins by the ubiquitin-like molecule SUMO (sumoylation) regulates their subcellular localization and affects their functional properties in vitro, but the physiological function of sumoylation in multicellular organisms is largely unknown. Here, we show that the C. elegans Polycomb group (PcG) protein SOP-2 interacts with the SUMO-conjugating enzyme UBC-9 through its evolutionarily conserved SAM domain. Sumoylation of SOP-2 is required for its localization to nuclear bodies in vivo and for its physiological repression of Hox genes. Global disruption of sumoylation phenocopies a sop-2 mutation by causing ectopic Hox gene expression and homeotic transformations. Chimeric constructs in which the SOP-2 SAM domain is replaced with that derived from fruit fly or mammalian PcG proteins, but not those in which the SOP-2 SAM domain is replaced with the SAM domains of non-PcG proteins, confer appropriate in vivo nuclear localization and Hox gene repression. These observations indicate that sumoylation of PcG proteins, modulated by their evolutionarily conserved SAM domain, is essential to their physiological repression of Hox genes.

  2. Antagonistic functional duality of cancer genes.

    PubMed

    Stepanenko, A A; Vassetzky, Y S; Kavsan, V M

    2013-10-25

    Cancer evolution is a stochastic process both at the genome and gene levels. Most of tumors contain multiple genetic subclones, evolving in either succession or in parallel, either in a linear or branching manner, with heterogeneous genome and gene alterations, extensively rewired signaling networks, and addicted to multiple oncogenes easily switching with each other during cancer progression and medical intervention. Hundreds of discovered cancer genes are classified according to whether they function in a dominant (oncogenes) or recessive (tumor suppressor genes) manner in a cancer cell. However, there are many cancer "gene-chameleons", which behave distinctly in opposite way in the different experimental settings showing antagonistic duality. In contrast to the widely accepted view that mutant NADP(+)-dependent isocitrate dehydrogenases 1/2 (IDH1/2) and associated metabolite 2-hydroxyglutarate (R)-enantiomer are intrinsically "the drivers" of tumourigenesis, mutant IDH1/2 inhibited, promoted or had no effect on cell proliferation, growth and tumorigenicity in diverse experiments. Similar behavior was evidenced for dozens of cancer genes. Gene function is dependent on genetic network, which is defined by the genome context. The overall changes in karyotype can result in alterations of the role and function of the same genes and pathways. The diverse cell lines and tumor samples have been used in experiments for proving gene tumor promoting/suppressive activity. They all display heterogeneous individual karyotypes and disturbed signaling networks. Consequently, the effect and function of gene under investigation can be opposite and versatile in cells with different genomes that may explain antagonistic duality of cancer genes and the cell type- or the cellular genetic/context-dependent response to the same protein. Antagonistic duality of cancer genes might contribute to failure of chemotherapy. Instructive examples of unexpected activity of cancer genes and

  3. Characterization of protoberberine analogs employed as novel human P2X{sub 7} receptor antagonists

    SciTech Connect

    Lee, Ga Eun; Lee, Won-Gil; Lee, Song-Yi; Lee, Cho-Rong; Park, Chul-Seung; Chang, Sunghoe; Park, Sung-Gyoo; Song, Mi-Ryoung; Kim, Yong-Chul

    2011-04-15

    The P2X{sub 7} receptor (P2X{sub 7}R), a member of the ATP-gated ion channel family, is regarded as a promising target for therapy of immune-related diseases including rheumatoid arthritis and chronic pain. A group of novel protoberberine analogs (compounds 3-5), discovered by screening of chemical libraries, was here investigated with respect to their function as P2X{sub 7}R antagonists. Compounds 3-5 non-competitively inhibited BzATP-induced ethidium ion influx into hP2X{sub 7}-expressing HEK293 cells, with IC{sub 50} values of 100-300 nM. This antagonistic action on the channel further confirmed that both BzATP-induced inward currents and Ca{sup 2+} influx were strongly inhibited by compounds 3-5 in patch-clamp and Ca{sup 2+} influx assays. The antagonists also effectively suppressed downstream signaling of P2X{sub 7} receptors including IL-1{beta} release and phosphorylation of ERK1/2 and p38 proteins in hP2X{sub 7}-expressing HEK293 cells or in differentiated human monocytes (THP-1 cells). Moreover, IL-2 secretion from CD3/CD28-stimulated Jurkat T cell was also dramatically inhibited by the antagonist. These results imply that novel protoberberine analogs may modulate P2X{sub 7} receptor-mediated immune responses by allosteric inhibition of the receptor. - Graphical abstract: Display Omitted

  4. Towards understanding the first genome sequence of a crenarchaeon by genome annotation using clusters of orthologous groups of proteins (COGs)

    PubMed Central

    Natale, Darren A; Shankavaram, Uma T; Galperin, Michael Y; Wolf, Yuri I; Aravind, L; Koonin, Eugene V

    2000-01-01

    Background: Standard archival sequence databases have not been designed as tools for genome annotation and are far from being optimal for this purpose. We used the database of Clusters of Orthologous Groups of proteins (COGs) to reannotate the genomes of two archaea, Aeropyrum pernix, the first member of the Crenarchaea to be sequenced, and Pyrococcus abyssi. Results: A. pernix and P. abyssi proteins were assigned to COGs using the COGNITOR program; the results were verified on a case-by-case basis and augmented by additional database searches using the PSI-BLAST and TBLASTN programs. Functions were predicted for over 300 proteins from A. pernix, which could not be assigned a function using conventional methods with a conservative sequence similarity threshold, an approximately 50% increase compared to the original annotation. A. pernix shares most of the conserved core of proteins that were previously identified in the Euryarchaeota. Cluster analysis or distance matrix tree construction based on the co-occurrence of genomes in COGs showed that A. pernix forms a distinct group within the archaea, although grouping with the two species of Pyrococci, indicative of similar repertoires of conserved genes, was observed. No indication of a specific relationship between Crenarchaeota and eukaryotes was obtained in these analyses. Several proteins that are conserved in Euryarchaeota and most bacteria are unexpectedly missing in A. pernix, including the entire set of de novo purine biosynthesis enzymes, the GTPase FtsZ (a key component of the bacterial and euryarchaeal cell-division machinery), and the tRNA-specific pseudouridine synthase, previously considered universal. A. pernix is represented in 48 COGs that do not contain any euryarchaeal members. Many of these proteins are TCA cycle and electron transport chain enzymes, reflecting the aerobic lifestyle of A. pernix. Conclusions: Special-purpose databases organized on the basis of phylogenetic analysis and carefully

  5. Protein adsorption of dialdehyde cellulose-crosslinked chitosan with high amino group contents.

    PubMed

    Kim, Ung-Jin; Lee, Yeong Ro; Kang, Tong Ho; Choi, Joon Weon; Kimura, Satoshi; Wada, Masahisa

    2017-05-01

    Crosslinked chitosan was prepared by Schiff base formation between the aldehyde groups of dialdehyde cellulose (DAC) and the amino groups of chitosan and a subsequent reduction. DAC was obtained through periodate oxidation of cellulose and solubilization in hot water at 100°C for 1h. Three grades of DAC-crosslinked chitosan were prepared by adding various amounts DAC. The degrees of crosslinking as determined by amino group content were 3.8, 8.3, and 12.1%, respectively. DAC-crosslinked chitosan showed higher stability in the pH 2-9 range and no cytotoxicity was identified over the course of a 21-day long-term stability test. Also, DAC-crosslinked chitosan showed remarkably high bovine serum albumin (BSA) adsorption capacity at pH 5.5 as a result of the increased amino group content, due to the reaction between DAC and chitosan molecular chains occurring at multiple points even though DAC-crosslinked chitosan showed a lower degree of crosslinking.

  6. Purification and characterization of the SegA protein of bacteriophage T4, an endonuclease related to proteins encoded by group I introns.

    PubMed Central

    Sharma, M; Hinton, D M

    1994-01-01

    Although not encoded by an intron, the bacteriophage T4 SegA protein shares common amino acid motifs with a family of proteins found within mobile group I introns present in fungi and phage. Each of these intron-encoded proteins is thought to initiate the homing of its own intron by cleaving the intronless DNA at or near the site of insertion. Previously, we have found that SegA also cleaves DNA. In this report, we have purified the SegA protein and characterized this endonuclease activity extensively. SegA protein cleaved circular and linear plasmids, DNA containing unmodified cytosines, and wild-type T4 DNA containing hydroxymethylated, glucosylated cytosines. In all cases, certain sites on the DNA were highly preferred for cleavage, but with increasing protein concentration or time of incubation, cleavage occurred at many sites. SegA cleaving activity was stimulated by the presence of ATP or ATP gamma S. Sequence analysis of three highly preferred cleavage sites did not reveal a simple consensus sequence, suggesting that even among highly preferred sites, SegA tolerates many different sequences. A T4 segA amber mutant that we constructed had no phenotype, and PCR analyses indicated that several T-even-related phages lack the segA gene. Taken together, our results show that SegA is an endonuclease with a hierarchy of site specificity, and these results are consistent with the insertion of segA DNA into the T4 genome some time after the divergence of the closely consistent with the insertion of segA DNA into the T4 genome some time after the divergence of the closely related T-even phages. Images PMID:7961394

  7. Evidence for methyl group transfer between the methyl-accepting chemotaxis proteins in Bacillus subtilis

    SciTech Connect

    Bedale, W.A.; Nettleton, D.O.; Sopata, C.S.; Thoelke, M.S.; Ordal, G.W.

    1988-01-01

    The authors present evidence for methyl (as methyl or methoxy) transfer from the methyl-accepting chemotaxis proteins H1 and possibly H3 of Bacillus subtilis to the methyl-accepting chemotaxis protein H2. This methyl transfer, which has been observed in vitro was strongly stimulated by the chemoattractant aspartate and thus may plan an important role in the sensory processing system of this organism. Although radiolabeling of H1 and H3 began at once after the addition of (/sup 3/H) methionine, radiolabeling of H2 showed a lag. Furthermore, the addition of excess nonradioactive methionine caused immediate exponential delabeling of H1 and H3 while labeling of H2 continued to increase. Methylation of H2 required the chemotactic methyltransferase, probably to first methylate H1 and H3. Aspartate caused increased labeling of H2 and strongly decreased labeling of H1 and H3 after the addition of nonradioactive methionine. Without the addition of nonradioactive methionine, aspartate caused demethylation of H1 and to a lesser extent H3, with an approximately equal increase of methylation of H2.

  8. Evidence for methyl group transfer between the methyl-accepting chemotaxis proteins in Bacillus subtilis.

    PubMed Central

    Bedale, W A; Nettleton, D O; Sopata, C S; Thoelke, M S; Ordal, G W

    1988-01-01

    We present evidence for methyl (as methyl or methoxy) transfer from the methyl-accepting chemotaxis proteins H1 and possibly H3 of Bacillus subtilis to the methyl-accepting chemotaxis protein H2. This methyl transfer, which has been observed in vitro (D. J. Goldman and G. W. Ordal, Biochemistry 23:2600-2606, 1984), was strongly stimulated by the chemoattractant aspartate and thus may play an important role in the sensory processing system of this organism. Although radiolabeling of H1 and H3 began at once after the addition of [3H]methionine, radiolabeling of H2 showed a lag. Furthermore, the addition of excess nonradioactive methionine caused immediate exponential delabeling of H1 and H3 while labeling of H2 continued to increase. Methylation of H2 required the chemotactic methyltransferase, probably to first methylate H1 and H3. Aspartate caused increased labeling of H2 and strongly decreased labeling of H1 and H3 after the addition of nonradioactive methionine. Without the addition of nonradioactive methionine, aspartate caused demethylation of H1 and to a lesser extent H3, with an approximately equal increase of methylation of H2. Images PMID:3121584

  9. A Quantitative Study on the in-vitro and in-vivo Acetylation of High Mobility Group A1 Proteins

    PubMed Central

    Zhang, Qingchun; Zhang, Kangling; Zou, Yan; Perna, Avi; Wang, Yinsheng

    2007-01-01

    High mobility group (HMG) A1 proteins are subject to a number of post-translational modifications, which may regulate their function in gene transcription and other cellular processes. We examined, by using mass spectrometry, the acetylation of HMGA1a and HMGA1b proteins induced by histone acetyltransferases p300 and PCAF in vitro and in PC-3 human prostate cancer cells in vivo. It turned out that five lysine residues in HMGA1a, i.e., Lys-14, Lys-64, Lys-66, Lys-70, and Lys-73, could be acetylated by both p300 and PCAF. We further quantified the level of acetylation by analyzing, with LC-MS/MS, the proteolytic peptides of the in-vitro or in-vivo acetylated HMGA1 proteins where the unmodified lysine residues were chemically derivatized with a perdeuterated acetyl group. Quantification results revealed that p300 and PCAF exhibited different site preferences for the acetylation; the preference of p300 acetylation followed the order of Lys-64~Lys-70 > Lys-66 > Lys-14~Lys73, whereas the selectivity of PCAF acetylation followed the sequence of Lys-70~Lys-73 > Lys-64~Lys-66 > Lys-14. HMGA1b was acetylated in a very similar fashion as HMGA1a. We also demonstrated that C-terminal phosphorylation of HMGA1 proteins did not affect the in-vitro acetylation of the two proteins by either p300 or PCAF. Moreover, we examined the acetylation of lysine residues in HMGA1a and HMGA1b isolated from PC-3 human prostate cancer cells. Our results showed that all the above five lysine residues were also acetylated in vivo, with Lys-64, Lys-66 and Lys-70 in HMGA1a exhibiting higher levels of acetylation than Lys-14 and Lys-73. PMID:17627840

  10. Discrimination of Psychrotrophic and Mesophilic Strains of the Bacillus cereus Group by PCR Targeting of Major Cold Shock Protein Genes

    PubMed Central

    Francis, Kevin P.; Mayr, Ralf; von Stetten, Felix; Stewart, Gordon S. A. B.; Scherer, Siegfried

    1998-01-01

    Detection of psychrotrophic strains (those able to grow at or below 7°C) of the Bacillus cereus group (Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides) in food products is at present extremely slow with conventional microbiology. This is due to an inability to discriminate these cold-adapted strains from their mesophilic counterparts (those able to grow only above 7°C) by means other than growth at low temperature, which takes 5 to 10 days for detection. Here we report the development of a single PCR assay that, using major cold shock protein-specific primers and appropriate annealing temperatures, is capable of both rapidly identifying bacteria of the B. cereus group and discriminating between psychrotrophic and mesophilic strains. It is intended that this development help to more accurately predict the shelf life of refrigerated pasteurized food and dairy products and to reduce the incidence of food poisoning by psychrotrophic strains of the B. cereus group. PMID:9726910

  11. Discovery of the improved antagonistic prolactin variants by library screening.

    PubMed

    Liu, Yun; Gong, Wei; Breinholt, Jens; Nørskov-Lauritsen, Leif; Zhang, Jinchao; Ma, Qinhong; Chen, Jianhe; Panina, Svetlana; Guo, Wei; Li, Tengkun; Zhang, Jingyuan; Kong, Meng; Liu, Zibing; Mao, Jingjing; Christensen, Leif; Hu, Sean; Wang, Lingyun

    2011-11-01

    Prolactin (PRL), a potent growth stimulator of the mammary epithelium, has been suggested to be a factor contributing to the development and progression of breast and prostate cancer. Several PRL receptor (PRLR) antagonists have been identified in the past decades, but their in vivo growth inhibitory potency was restricted by low receptor affinity, rendering them pharmacologically unattractive for clinical treatment. Thus, higher receptor affinity is essential for the development of improved PRLR antagonistic variants with improved in vivo potency. In this study, we generated Site 1 focused protein libraries of human G129R-PRL mutants and screened for those with increased affinity to the human PRLR. By combining the mutations with enhanced affinities for PRLR, we identified a novel G129R-PRL variant with mutations at Site 1 that render nearly 50-fold increase in the antagonistic potency in vitro.

  12. Cohesion group approach for evolutionary analysis of TyrA, a protein family with wide-ranging substrate specificities.

    PubMed

    Bonner, Carol A; Disz, Terrence; Hwang, Kaitlyn; Song, Jian; Vonstein, Veronika; Overbeek, Ross; Jensen, Roy A

    2008-03-01

    Many enzymes and other proteins are difficult subjects for bioinformatic analysis because they exhibit variant catalytic, structural, regulatory, and fusion mode features within a protein family whose sequences are not highly conserved. However, such features reflect dynamic and interesting scenarios of evolutionary importance. The value of experimental data obtained from individual organisms is instantly magnified to the extent that given features of the experimental organism can be projected upon related organisms. But how can one decide how far along the similarity scale it is reasonable to go before such inferences become doubtful? How can a credible picture of evolutionary events be deduced within the vertical trace of inheritance in combination with intervening events of lateral gene transfer (LGT)? We present a comprehensive analysis of a dehydrogenase protein family (TyrA) as a prototype example of how these goals can be accomplished through the use of cohesion group analysis. With this approach, the full collection of homologs is sorted into groups by a method that eliminates bias caused by an uneven representation of sequences from organisms whose phylogenetic spacing is not optimal. Each sufficiently populated cohesion group is phylogenetically coherent and defined by an overall congruence with a distinct section of the 16S rRNA gene tree. Exceptions that occasionally are found implicate a clearly defined LGT scenario whereby the recipient lineage is apparent and the donor lineage of the gene transferred is localized to those organisms that define the cohesion group. Systematic procedures to manage and organize otherwise overwhelming amounts of data are demonstrated.

  13. Cohesion Group Approach for Evolutionary Analysis of TyrA, a Protein Family with Wide-Ranging Substrate Specificities

    PubMed Central

    Bonner, Carol A.; Disz, Terrence; Hwang, Kaitlyn; Song, Jian; Vonstein, Veronika; Overbeek, Ross; Jensen, Roy A.

    2008-01-01

    Summary: Many enzymes and other proteins are difficult subjects for bioinformatic analysis because they exhibit variant catalytic, structural, regulatory, and fusion mode features within a protein family whose sequences are not highly conserved. However, such features reflect dynamic and interesting scenarios of evolutionary importance. The value of experimental data obtained from individual organisms is instantly magnified to the extent that given features of the experimental organism can be projected upon related organisms. But how can one decide how far along the similarity scale it is reasonable to go before such inferences become doubtful? How can a credible picture of evolutionary events be deduced within the vertical trace of inheritance in combination with intervening events of lateral gene transfer (LGT)? We present a comprehensive analysis of a dehydrogenase protein family (TyrA) as a prototype example of how these goals can be accomplished through the use of cohesion group analysis. With this approach, the full collection of homologs is sorted into groups by a method that eliminates bias caused by an uneven representation of sequences from organisms whose phylogenetic spacing is not optimal. Each sufficiently populated cohesion group is phylogenetically coherent and defined by an overall congruence with a distinct section of the 16S rRNA gene tree. Exceptions that occasionally are found implicate a clearly defined LGT scenario whereby the recipient lineage is apparent and the donor lineage of the gene transferred is localized to those organisms that define the cohesion group. Systematic procedures to manage and organize otherwise overwhelming amounts of data are demonstrated. PMID:18322033

  14. High mobility group box protein 1 in complex with lipopolysaccharide or IL-1 promotes an increased inflammatory phenotype in synovial fibroblasts

    PubMed Central

    2011-01-01

    Introduction In addition to its direct proinflammatory activity, extracellular high mobility group box protein 1 (HMGB1) can strongly enhance the cytokine response evoked by other proinflammatory molecules, such as lipopolysaccharide (LPS), CpG-DNA and IL-1β, through the formation of complexes. Extracellular HMGB1 is abundant in arthritic joint tissue where it is suggested to promote inflammation as intra-articular injections of HMGB1 induce synovitis in mice and HMGB1 neutralizing therapy suppresses development of experimental arthritis. The aim of this study was to determine whether HMGB1 in complex with LPS, interleukin (IL)-1α or IL-1β has enhancing effects on the production of proinflammatory mediators by rheumatoid arthritis synovial fibroblasts (RASF) and osteoarthritis synovial fibroblasts (OASF). Furthermore, we examined the toll-like receptor (TLR) 4 and IL-1RI requirement for the cytokine-enhancing effects of the investigated HMGB1-ligand complexes. Methods Synovial fibroblasts obtained from rheumatoid arthritis (RA) and osteoarthritis (OA) patients were stimulated with HMGB1 alone or in complex with LPS, IL-1α or IL-1β. Tumour necrosis factor (TNF) production was determined by enzyme-linked immunospot assay (ELISPOT) assessment. Levels of IL-10, IL-1-β, IL-6 and IL-8 were measured using Cytokine Bead Array and matrix metalloproteinase (MMP) 3 production was determined by ELISA. Results Stimulation with HMGB1 in complex with LPS, IL-1α or IL-1β enhanced production of TNF, IL-6 and IL-8. HMGB1 in complex with IL-1β increased MMP production from both RASF and OASF. The cytokine production was inhibited by specific receptor blockade using detoxified LPS or IL-1 receptor antagonist, indicating that the synergistic effects were mediated through the partner ligand-reciprocal receptors TLR4 and IL-1RI, respectively. Conclusions HMGB1 in complex with LPS, IL-1α or IL-1β boosted proinflammatory cytokine- and MMP production in synovial fibroblasts from

  15. Deregulated Expression of the Polycomb-Group Protein SUZ12 Target Genes Characterizes Mantle Cell Lymphoma

    PubMed Central

    Martín-Pérez, Daniel; Sánchez, Esther; Maestre, Lorena; Suela, Javier; Vargiu, Pierfrancesco; Di Lisio, Lorena; Martínez, Nerea; Alves, Javier; Piris, Miguel A.; Sánchez-Beato, Margarita

    2010-01-01

    Polycomb proteins are known to be of great importance in human cancer pathogenesis. SUZ12 is a component of the Polycomb PRC2 complex that, along with EZH2, is involved in embryonic stem cell differentiation. EZH2 plays an essential role in many cancer types, but an equivalent involvement of SUZ12 has not been as thoroughly demonstrated. Here we show that SUZ12 is anomalously expressed in human primary tumors, especially in mantle cell lymphoma (MCL), pulmonary carcinomas and melanoma, and is associated with gene locus amplification in some cases. Using MCL as a model, functional and genomic studies demonstrate that SUZ12 loss compromises cell viability, increases apoptosis, and targets genes involved in central oncogenic pathways associated with MCL pathogenesis. Our results support the hypothesis that the abnormal expression of SUZ12 accounts for some of the unexplained features of MCL, such as abnormal DNA repair and increased resistance to apoptosis. PMID:20558579

  16. SpyAD, a moonlighting protein of group A Streptococcus contributing to bacterial division and host cell adhesion.

    PubMed

    Gallotta, Marilena; Gancitano, Giovanni; Pietrocola, Giampiero; Mora, Marirosa; Pezzicoli, Alfredo; Tuscano, Giovanna; Chiarot, Emiliano; Nardi-Dei, Vincenzo; Taddei, Anna Rita; Rindi, Simonetta; Speziale, Pietro; Soriani, Marco; Grandi, Guido; Margarit, Immaculada; Bensi, Giuliano

    2014-07-01

    Group A streptococcus (GAS) is a human pathogen causing a wide repertoire of mild and severe diseases for which no vaccine is yet available. We recently reported the identification of three protein antigens that in combination conferred wide protection against GAS infection in mice. Here we focused our attention on the characterization of one of these three antigens, Spy0269, a highly conserved, surface-exposed, and immunogenic protein of unknown function. Deletion of the spy0269 gene in a GAS M1 isolate resulted in very long bacterial chains, which is indicative of an impaired capacity of the knockout mutant to properly divide. Confocal microscopy and immunoprecipitation experiments demonstrated that the protein was mainly localized at the cell septum and could interact in vitro with the cell division protein FtsZ, leading us to hypothesize that Spy0269 is a member of the GAS divisome machinery. Predicted structural domains and sequence homologies with known streptococcal adhesins suggested that this antigen could also play a role in mediating GAS interaction with host cells. This hypothesis was confirmed by showing that recombinant Spy0269 could bind to mammalian epithelial cells in vitro and that Lactococcus lactis expressing Spy0269 on its cell surface could adhere to mammalian cells in vitro and to mice nasal mucosa in vivo. On the basis of these data, we believe that Spy0269 is involved both in bacterial cell division and in adhesion to host cells and we propose to rename this multifunctional moonlighting protein as SpyAD (Streptococcus pyogenes Adhesion and Division protein).

  17. A human ortholog of archaeal DNA repair protein Hef is defective in Fanconi anemia complementation group M.

    PubMed

    Meetei, Amom Ruhikanta; Medhurst, Annette L; Ling, Chen; Xue, Yutong; Singh, Thiyam Ramsing; Bier, Patrick; Steltenpool, Jurgen; Stone, Stacie; Dokal, Inderjeet; Mathew, Christopher G; Hoatlin, Maureen; Joenje, Hans; de Winter, Johan P; Wang, Weidong

    2005-09-01

    Fanconi anemia is a genetic disease characterized by genomic instability and cancer predisposition. Nine genes involved in Fanconi anemia have been identified; their products participate in a DNA damage-response network involving BRCA1 and BRCA2 (refs. 2,3). We previously purified a Fanconi anemia core complex containing the FANCL ubiquitin ligase and six other Fanconi anemia-associated proteins. Each protein in this complex is essential for monoubiquitination of FANCD2, a key reaction in the Fanconi anemia DNA damage-response pathway. Here we show that another component of this complex, FAAP250, is mutant in individuals with Fanconi anemia of a new complementation group (FA-M). FAAP250 or FANCM has sequence similarity to known DNA-repair proteins, including archaeal Hef, yeast MPH1 and human ERCC4 or XPF. FANCM can dissociate DNA triplex, possibly owing to its ability to translocate on duplex DNA. FANCM is essential for monoubiquitination of FANCD2 and becomes hyperphosphorylated in response to DNA damage. Our data suggest an evolutionary link between Fanconi anemia-associated proteins and DNA repair; FANCM may act as an engine that translocates the Fanconi anemia core complex along DNA.

  18. The conserved lymphokine element-0 in the IL5 promoter binds to a high mobility group-1 protein.

    PubMed

    Marrugo, J; Marsh, D G; Ghosh, B

    1996-10-01

    The conserved lymphokine elements-0 (CLE0) in the IL5 promoter is essential for the expression of IL-5. Here, we report the cloning and expression of a cDNA encoding a novel CLE0-binding protein, CLEBP-1 from a mouse Th2 clone, D10.G4.1. Interestingly, it was found that the CLEBP1 cDNA sequence was almost identical to the sequences of known high mobility group-1 (HMG1) cDNAs. When expressed as a recombinant fusion protein in Escherichia coli, CLEBP-1 was shown to bind to the IL5-CLE0 element in electrophoretic mobility-shift assays (EMSA) and southwestern blot analysis. The CLEBP-1 fusion protein cross-reacts with and-HMG-1/2 in Western blot analysis. It also binds to the CLE0 elements of IL4, GMCSF and GCSF genes. CLEBP-1 and closely related HMG-1 and HMG-2 proteins may play key roles in facilitating the expression of the lymphokine genes that contain CLE0 elements.

  19. Identification of a gag protein epitope conserved among all four groups of primate immunodeficiency viruses by using monoclonal antibodies.

    PubMed

    Otteken, A; Nick, S; Bergter, W; Voss, G; Faisst, A C; Stahl-Hennig, C; Hunsmann, G

    1992-10-01

    Five monoclonal antibodies (MAbs) were raised against the gag proteins of simian immunodeficiency virus (SIV) from African green monkey (SIVagmTYO-7). Two MAbs reacted with the matrix protein p17 and the other three with the core protein p24. Studies on the cross-reactivity of the MAbs revealed that the anti-p24 MAbs detected an epitope shared by the viruses belonging to the human immunodeficiency virus type 2 (HIV-2)/SIVmac group and SIVagmTYO-7 and SIVagmTYO-5. The anti-p17 MAbs recognized an epitope present on all these viruses and on SIVagmTYO-1, HIV-1 and SIVmnd. This finding demonstrates for the first time that the matrix protein, p17 or p18, respectively, of all nine HIV and SIV isolates tested in this study expresses at least one conserved immunogenic epitope recognized serologically. By using synthetic peptides, this epitope was identified at the N terminus of p17. Furthermore, this epitope was analysed by multiple sequence alignments of the peptide with homologous sequences of HIV and SIV p17.

  20. Group A streptococcus cell-associated pathogenic proteins as revealed by growth in hyaluronic acid-enriched media.

    PubMed

    Zhang, Meng; McDonald, Fiona M; Sturrock, Shane S; Charnock, Simon J; Humphery-Smith, Ian; Black, Gary W

    2007-05-01

    Group A streptococcus (GAS), also know as Streptococcus pyogenes, is a human pathogen and can cause several fatal invasive diseases such as necrotising fasciitis, the so-called flesh-eating disease, and toxic shock syndrome. The destruction of connective tissue and the hyaluronic acid (HA) therein, is a key element of GAS pathogenesis. We therefore propagated GAS in HA-enriched growth media in an attempt to create a simple biological system that could reflect some elements of GAS pathogenesis. Our results show that several recognised virulence factors were up-regulated in HA-enriched media, including the M1 protein, a collagen-like surface protein and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, which has been shown to play important roles in streptococcal pathogenesis. Interestingly, two hypothetical proteins of unknown function were also up-regulated and detailed bioinformatics analysis showed that at least one of these hypothetical proteins is likely to be involved in pathogenesis. It was therefore concluded that this simple biological system provided a valuable tool for the identification of potential GAS virulence factors.

  1. Tl(+) showed negligible interaction with inner membrane sulfhydryl groups of rat liver mitochondria, but formed complexes with matrix proteins.

    PubMed

    Korotkov, Sergey M; Brailovskaya, Irina V; Kormilitsyn, Boris N; Furaev, Viktor V

    2014-04-01

    The effects of Tl(+) on protein sulfhydryl (SH) groups, swelling, and respiration of rat liver mitochondria (RLM) were studied in a medium containing TlNO3 and sucrose, or TlNO3 and KNO3 as well as glutamate plus malate, or succinate plus rotenone. Detected with Ellman's reagent, an increase in the content of the SH groups was found in the inner membrane fraction, and a simultaneous decline was found in the content of the matrix-soluble fraction for RLM, incubated and frozen in 25-75 mM TlNO3 . This increase was greater in the medium containing KNO3 regardless of the presence of Ca(2+) . It was eliminated completely for RLM injected in the medium containing TlNO3 and then washed and frozen in the medium containing KNO3 . Calcium-loaded RLM showed increased swelling and decreased respiration. These results suggest that a ligand interaction of Tl(+) with protein SH groups, regardless of the presence of calcium, may underlie the mechanism of thallium toxicity.

  2. Antagonistic effects of abscisic acid and jasmonates on salt stress-inducible transcripts in rice roots.

    PubMed Central

    Moons, A; Prinsen, E; Bauw, G; Van Montagu, M

    1997-01-01

    Abscisic acid (ABA) and jasmonates have been implicated in responses to water deficit and wounding. We compared the molecular and physiological effects of jasmonic acid (JA) (< or = 10 microM), ABA, and salt stress in roots of rice. JA markedly induced a cationic peroxidase, two novel 32- and 28-kD proteins, acidic PR-1 and PR-10 pathogenesis-related proteins, and the salt stress-responsive SalT protein in roots. Most JA-responsive proteins (JIPs) from roots also accumulated when plants were subjected to salt stress. None of the JIPs accumulated when plants were treated with ABA. JA did not induce an ABA-responsive group 3 late-embryogenesis abundant (LEA) protein. Salt stress and ABA but not JA induced oslea3 transcript accumulation. By contrast, JA, ABA, and salt stress induced transcript accumulation of salT and osdrr, which encodes a rice PR-10 protein. However, ABA also negatively affected salT transcript accumulation, whereas JA negatively affected ABA-induced oslea3 transcript levels. Endogenous root ABA and methyl jasmonate levels showed a differential increase with the dose and the duration of salt stress. The results indicate that ABA and jasmonates antagonistically regulated the expression of salt stress-inducible proteins associated with water deficit or defense responses. PMID:9437865

  3. The N-terminal domain of thrombomodulin sequesters high-mobility group-B1 protein, a novel antiinflammatory mechanism

    PubMed Central

    Abeyama, Kazuhiro; Stern, David M.; Ito, Yuji; Kawahara, Ko-ichi; Yoshimoto, Yasushi; Tanaka, Motoyuki; Uchimura, Tomonori; Ida, Nobuo; Yamazaki, Yoshiaki; Yamada, Shingo; Yamamoto, Yasuhiko; Yamamoto, Hiroshi; Iino, Satoshi; Taniguchi, Noboru; Maruyama, Ikuro

    2005-01-01

    Thrombomodulin (TM) is an endothelial anticoagulant cofactor that promotes thrombin-mediated formation of activated protein C (APC). We have found that the N-terminal lectin-like domain (D1) of TM has unique antiinflammatory properties. TM, via D1, binds high-mobility group-B1 DNA-binding protein (HMGB1), a factor closely associated with necrotic cell damage following its release from the nucleus, thereby preventing in vitro leukocyte activation, in vivo UV irradiation–induced cutaneous inflammation, and in vivo lipopolysaccharide-induced lethality. Our data also demonstrate antiinflammatory properties of a peptide spanning D1 of TM and suggest its therapeutic potential. These findings highlight a novel mechanism, i.e., sequestration of mediators, through which an endothelial cofactor, TM, suppresses inflammation quite distinctly from its anticoagulant cofactor activity, thereby preventing the interaction of these mediators with cell surface receptors on effector cells in the vasculature. PMID:15841214

  4. The Brown Algae Pl.LSU/2 Group II Intron-Encoded Protein Has Functional Reverse Transcriptase and Maturase Activities

    PubMed Central

    Zerbato, Madeleine; Holic, Nathalie; Moniot-Frin, Sophie; Ingrao, Dina; Galy, Anne; Perea, Javier

    2013-01-01

    Group II introns are self-splicing mobile elements found in prokaryotes and eukaryotic organelles. These introns propagate by homing into precise genomic locations, following assembly of a ribonucleoprotein complex containing the intron-encoded protein (IEP) and the spliced intron RNA. Engineered group II introns are now commonly used tools for targeted genomic modifications in prokaryotes but not in eukaryotes. We speculate that the catalytic activation of currently known group II introns is limited in eukaryotic cells. The brown algae Pylaiella littoralis Pl.LSU/2 group II intron is uniquely capable of in vitro ribozyme activity at physiological level of magnesium but this intron remains poorly characterized. We purified and characterized recombinant Pl.LSU/2 IEP. Unlike most IEPs, Pl.LSU/2 IEP displayed a reverse transcriptase activity without intronic RNA. The Pl.LSU/2 intron could be engineered to splice accurately in Saccharomyces cerevisiae and splicing efficiency was increased by the maturase activity of the IEP. However, spliced transcripts were not expressed. Furthermore, intron splicing was not detected in human cells. While further tool development is needed, these data provide the first functional characterization of the PI.LSU/2 IEP and the first evidence that the Pl.LSU/2 group II intron splicing occurs in vivo in eukaryotes in an IEP-dependent manner. PMID:23505475

  5. The brown algae Pl.LSU/2 group II intron-encoded protein has functional reverse transcriptase and maturase activities.

    PubMed

    Zerbato, Madeleine; Holic, Nathalie; Moniot-Frin, Sophie; Ingrao, Dina; Galy, Anne; Perea, Javier

    2013-01-01

    Group II introns are self-splicing mobile elements found in prokaryotes and eukaryotic organelles. These introns propagate by homing into precise genomic locations, following assembly of a ribonucleoprotein complex containing the intron-encoded protein (IEP) and the spliced intron RNA. Engineered group II introns are now commonly used tools for targeted genomic modifications in prokaryotes but not in eukaryotes. We speculate that the catalytic activation of currently known group II introns is limited in eukaryotic cells. The brown algae Pylaiella littoralis Pl.LSU/2 group II intron is uniquely capable of in vitro ribozyme activity at physiological level of magnesium but this intron remains poorly characterized. We purified and characterized recombinant Pl.LSU/2 IEP. Unlike most IEPs, Pl.LSU/2 IEP displayed a reverse transcriptase activity without intronic RNA. The Pl.LSU/2 intron could be engineered to splice accurately in Saccharomyces cerevisiae and splicing efficiency was increased by the maturase activity of the IEP. However, spliced transcripts were not expressed. Furthermore, intron splicing was not detected in human cells. While further tool development is needed, these data provide the first functional characterization of the PI.LSU/2 IEP and the first evidence that the Pl.LSU/2 group II intron splicing occurs in vivo in eukaryotes in an IEP-dependent manner.

  6. Sleep duration during the school week is associated with C-reactive protein Risk Groups in healthy adolescents

    PubMed Central

    Hall, Martica H.; Lee, Laisze; Matthews, Karen A.

    2015-01-01

    Background The prevalence of short sleep duration in adolescence and the relevance of early risk factors to cardiovascular disease in adulthood suggest that adolescence is an opportune time to evaluate links between sleep duration and cardiovascular disease risk. We examined associations among actigraphy-assessed sleep duration and sleep debt with elevated C-Reactive Protein (CRP), a known risk factor for cardiovascular disease. Methods Participants were 244 (137 Blacks, 116 males) healthy high school students, each of whom wore wrist actigraphs for one week and provided a fasting blood draw. CRP was examined as both a continuous and categorical outcome, with CRP > 3 mg/L identifying a High Risk Group. Results Sleep duration and sleep debt were significantly associated with CRP High Risk Group in covariate-adjusted analyses. Shorter sleep duration on school nights was associated with a greater likelihood of being in the High Risk CRP Group. Likelihood of being in the High Risk CRP Group was doubled in students who obtained an average of two or more hours of “catch up” sleep on weekend nights. Conclusions Reduced weekday sleep duration and sleep debt were both associated with CRP Risk Group in adolescence. That these relationships may be observed prior to the onset of clinical disease suggests that adolescence may provide an opportune period for disease prevention. PMID:25468623

  7. Cockayne syndrome group B (CSB) protein: at the crossroads of transcriptional networks.

    PubMed

    Vélez-Cruz, Renier; Egly, Jean-Marc

    2013-01-01

    Cockayne syndrome (CS) is a rare genetic disorder characterized by a variety of growth and developmental defects, photosensitivity, cachectic dwarfism, hearing loss, skeletal abnormalities, progressive neurological degeneration, and premature aging. CS arises due to mutations in the CSA and CSB genes. Both gene products are required for the transcription-coupled (TC) branch of the nucleotide excision repair (NER) pathway, however, the severe phenotype of CS patients is hard to reconcile with a sole defect in TC-NER. Studies using cells from patients and mouse models have shown that the CSB protein is involved in a variety of cellular pathways and plays a major role in the cellular response to stress. CSB has been shown to regulate processes such as the transcriptional recovery after DNA damage, the p53 transcriptional response, the response to hypoxia, the response to insulin-like growth factor-1 (IGF-1), transactivation of nuclear receptors, transcription of housekeeping genes and the transcription of rDNA. Some of these processes are also affected in combined XP/CS patients. These new advances in the function(s) of CSB shed light onto the etiology of the clinical features observed in CS patients and could potentially open therapeutic avenues for these patients in the future. Moreover, the study of CS could further our knowledge of the aging process.

  8. Cockayne syndrome group B protein regulates DNA double-strand break repair and checkpoint activation.

    PubMed

    Batenburg, Nicole L; Thompson, Elizabeth L; Hendrickson, Eric A; Zhu, Xu-Dong

    2015-05-12

    Mutations of CSB account for the majority of Cockayne syndrome (CS), a devastating hereditary disorder characterized by physical impairment, neurological degeneration and segmental premature aging. Here we report the generation of a human CSB-knockout cell line. We find that CSB facilitates HR and represses NHEJ. Loss of CSB or a CS-associated CSB mutation abrogating its ATPase activity impairs the recruitment of BRCA1, RPA and Rad51 proteins to damaged chromatin but promotes the formation of 53BP1-Rif1 damage foci in S and G2 cells. Depletion of 53BP1 rescues the formation of BRCA1 damage foci in CSB-knockout cells. In addition, knockout of CSB impairs the ATM- and Chk2-mediated DNA damage responses, promoting a premature entry into mitosis. Furthermore, we show that CSB accumulates at sites of DNA double-strand breaks (DSBs) in a transcription-dependent manner. The kinetics of DSB-induced chromatin association of CSB is distinct from that of its UV-induced chromatin association. These results reveal novel, important functions of CSB in regulating the DNA DSB repair pathway choice as well as G2/M checkpoint activation.

  9. Cockayne syndrome group B protein regulates DNA double-strand break repair and checkpoint activation

    PubMed Central

    Batenburg, Nicole L; Thompson, Elizabeth L; Hendrickson, Eric A; Zhu, Xu-Dong

    2015-01-01

    Mutations of CSB account for the majority of Cockayne syndrome (CS), a devastating hereditary disorder characterized by physical impairment, neurological degeneration and segmental premature aging. Here we report the generation of a human CSB-knockout cell line. We find that CSB facilitates HR and represses NHEJ. Loss of CSB or a CS-associated CSB mutation abrogating its ATPase activity impairs the recruitment of BRCA1, RPA and Rad51 proteins to damaged chromatin but promotes the formation of 53BP1-Rif1 damage foci in S and G2 cells. Depletion of 53BP1 rescues the formation of BRCA1 damage foci in CSB-knockout cells. In addition, knockout of CSB impairs the ATM- and Chk2-mediated DNA damage responses, promoting a premature entry into mitosis. Furthermore, we show that CSB accumulates at sites of DNA double-strand breaks (DSBs) in a transcription-dependent manner. The kinetics of DSB-induced chromatin association of CSB is distinct from that of its UV-induced chromatin association. These results reveal novel, important functions of CSB in regulating the DNA DSB repair pathway choice as well as G2/M checkpoint activation. PMID:25820262

  10. Deformations of the Heme Group of Different Ferrocytochrome c Proteins Probed by Resonance Raman Spectroscopy

    SciTech Connect

    Hagarman, Andrew; Schweitzer-Stenner, Reinhard; Wallace, Carmichael; Laberge, Monique

    2008-11-14

    We measured the low-frequency polarized resonance Raman spectra of horse heart, chicken, and yeast(C102T) ferrocytochromes c with Soret excitation. We examined the out-of-plane deformations of the heme groups by determining the relative intensities and depolarization ratios of a variety of out-of-plane and in-plane Raman active bands. Analysis of relative Raman intensities shows differences in non-planarity of the heme groups of yeast(C102T), horse heart and chicken cytochrome c. Cytochrome c has been shown to have a dominant ruffling (B{sub 1u}) deformation by means of normal coordinate structural decomposition (NSD) analysis of the heme group in crystal structures. The presence and intensity of B{sub 1u} modes, {gamma}{sub 10}-{gamma}{sub 12}, support the indication of ruffling being the major contribution to the non-planar deformations in cytochrome c. Other types of non-planar deformations like doming (A{sub 2U}) and waving (E{sub g}) can be deduced from the Raman activity of {gamma}{sub 5} (A{sub 2u}), {gamma}{sub 21} and {gamma}{sub 22} (E{sub g}). The depolarization ratios of {gamma}{sub 5}, {gamma}{sub 10}, {gamma}{sub 11} and {gamma}{sub 12} are larger than 0.125, indicating the presence of other deformations such as saddling (B{sub 2u}) and propellering (A{sub 1u}), which is again in agreement with the crystal structures of horse heart and yeast ferrocytochrome c. An analysis of the intensities and depolarization ratios of out-of-plane modes revealed that ruffling is comparable in yeast and horse heart cytochrome c, saddling is larger and doming as well as propellering are lower in yeast cytochrome c. With respect to doming and ruffling our results contradict values obtained from the NSD analysis of the corresponding crystal structures. With respect to saddling, our data are in agreement with the crystal structure. The NSD analysis of heme structures resulting from MD simulations did not correlate very well with the spectroscopically obtained results

  11. Preclinical immunogenicity and safety of a Group A streptococcal M protein-based vaccine candidate.

    PubMed

    Batzloff, Michael R; Fane, Anne; Gorton, Davina; Pandey, Manisha; Rivera-Hernandez, Tania; Calcutt, Ainslie; Yeung, Grace; Hartas, Jon; Johnson, Linda; Rush, Catherine M; McCarthy, James; Ketheesan, Natkunam; Good, Michael F

    2016-12-01

    Streptococcus pyogenes (group A streptococcus, GAS) causes a wide range of clinical manifestations ranging from mild self-limiting pyoderma to invasive diseases such as sepsis. Also of concern are the post-infectious immune-mediated diseases including rheumatic heart disease. The development of a vaccine against GAS would have a large health impact on populations at risk of these diseases. However, there is a lack of suitable models for the safety evaluation of vaccines with respect to post-infectious complications. We have utilized the Lewis Rat model for cardiac valvulitis to evaluate the safety of the J8-DT vaccine formulation in parallel with a rabbit toxicology study. These studies demonstrated that the vaccine did not induce abnormal pathology. We also show that in mice the vaccine is highly immunogenic but that 3 doses are required to induce protection from a GAS skin challenge even though 2 doses are sufficient to induce a high antibody titer.

  12. High mobility group box protein-1 (HMGB-1) as a new diagnostic marker in patients with acute appendicitis

    PubMed Central

    2011-01-01

    Background The aim of this prospective study was therefore to evaluate the diagnostic value of preoperative serum High Mobility Group Box Protein-1 (HMGB-1) levels in patients with Acute Appendicitis (AA) who show normal white blood cell count (WBC) counts. Method Our study was carried out from October 2010 through November 2010 and included 20 healthy control group participants and 60 patients who presented at the emergency department of Erzurum Training and Research Hospital in Turkey with acute abdominal pain complaints, who were pathologically diagnosed with AA after laparotomy, and who agreed to participate in the study. Results Of the 60 patients who underwent appendectomies, 36 were male and 24 were female, and of the healthy group, 12 were male and 8 female. The age averages of the patients in Groups 1, 2 and 3 were, respectively, 31.3+15.4, 34.0+16.3 and 31.0+13.1 years. The WBC averages of Groups 1, 2 and 3 were, respectively, 7.41+2.02 (x109/L), 15.71+2.85 (x109/L) and 8.51+1.84 (x109/L). The HMGB-1 levels for Groups 1 (healthy persons), 2 (AA patients with high WBC counts ) and 3 (AA patients with normal WBC counts) were, respectively, 21.71 ± 11.36, 37.28+13.37 and 36.5 ± 17.73 ng/ml. The average HMGB-1 level of the patients with AA was 36.92 ± 15.43 ng/ml while the average HMGB-1 value of the healthy group was 21.71 ± 11.36 ng/ml. Conclusion The significantly higher levels of HMGB-1 in AA patients compared to healthy persons infer that HMGB-1 might be useful in the diagnosis of AA. Use of HMGB-1, especially in patients with normal WBC counts, will reduce the number of unnecessary explorations. PMID:21507210

  13. Effect of subdomain interactions on methyl group dynamics in the hydrophobic core of villin headpiece protein

    PubMed Central

    Vugmeyster, Liliya; Do, Tien; Ostrovsky, Dmitry; Fu, Riqianq

    2014-01-01

    Thermostable villin headpiece protein (HP67) consists of the N-terminal subdomain (residues 10–41) and the autonomously folding C-terminal subdomain (residues 42–76) which pack against each other to form a structure with a unified hydrophobic core. The X-ray structures of the isolated C-terminal subdomain (HP36) and its counterpart in HP67 are very similar for the hydrophobic core residues. However, fine rearrangements of the free energy landscape are expected to occur because of the interactions between the two subdomains. We detect and characterize these changes by comparing the µs-ms time scale dynamics of the methyl-bearing side chains in isolated HP36 and in HP67. Specifically, we probe three hydrophobic side chains at the interface of the two subdomains (L42, V50, and L75) as well as at two residues far from the interface (L61 and L69). Solid-state deuteron NMR techniques are combined with computational modeling for the detailed characterization of motional modes in terms of their kinetic and thermodynamic parameters. The effect of interdomain interactions on side chain dynamics is seen for all residues but L75. Thus, changes in dynamics because of subdomain interactions are not confined to the site of perturbation. One of the main results is a two-to threefold increase in the value of the activation energies for the rotameric mode of motions in HP67 compared with HP36. Detailed analysis of configurational entropies and heat capacities complement the kinetic view of the degree of the disorder in the folded state. PMID:24243806

  14. A Comprehensive Analysis of RALF Proteins in Green Plants Suggests There Are Two Distinct Functional Groups

    PubMed Central

    Campbell, Liam; Turner, Simon R.

    2017-01-01

    Rapid Alkalinization Factors (RALFs) are small, cysteine-rich peptides known to be involved in various aspects of plant development and growth. Although RALF peptides have been identified within many species, a single wide-ranging phylogenetic analysis of the family across the plant kingdom has not yet been undertaken. Here, we identified RALF proteins from 51 plant species that represent a variety of land plant lineages. The inferred evolutionary history of the 795 identified RALFs suggests that the family has diverged into four major clades. We found that much of the variation across the family exists within the mature peptide region, suggesting clade-specific functional diversification. Clades I, II, and III contain the features that have been identified as important for RALF activity, including the RRXL cleavage site and the YISY motif required for receptor binding. In contrast, members of clades IV that represent a third of the total dataset, is highly diverged and lacks these features that are typical of RALFs. Members of clade IV also exhibit distinct expression patterns and physico-chemical properties. These differences suggest a functional divergence of clades and consequently, we propose that the peptides within clade IV are not true RALFs, but are more accurately described as RALF-related peptides. Expansion of this RALF–related clade in the Brassicaceae is responsible for the large number of RALF genes that have been previously described in Arabidopsis thaliana. Future experimental work will help to establish the nature of the relationship between the true RALFs and the RALF-related peptides, and whether they function in a similar manner. PMID:28174582

  15. Coiled-coil destabilizing residues in the group A Streptococcus M1 protein are required for functional interaction

    PubMed Central

    Stewart, Chelsea M.; Buffalo, Cosmo Z.; Valderrama, J. Andrés; Henningham, Anna; Cole, Jason N.; Nizet, Victor; Ghosh, Partho

    2016-01-01

    The sequences of M proteins, the major surface-associated virulence factors of the widespread bacterial pathogen group A Streptococcus, are antigenically variable but have in common a strong propensity to form coiled coils. Paradoxically, these sequences are also replete with coiled-coil destabilizing residues. These features are evident in the irregular coiled-coil structure and thermal instability of M proteins. We present an explanation for this paradox through studies of the B repeats of the medically important M1 protein. The B repeats are required for interaction of M1 with fibrinogen (Fg) and consequent proinflammatory activation. The B repeats sample multiple conformations, including intrinsically disordered, dissociated, as well as two alternate coiled-coil conformations: a Fg-nonbinding register 1 and a Fg-binding register 2. Stabilization of M1 in the Fg-nonbinding register 1 resulted in attenuation of Fg binding as expected, but counterintuitively, so did stabilization in the Fg-binding register 2. Strikingly, these register-stabilized M1 proteins gained the ability to bind Fg when they were destabilized by a chaotrope. These results indicate that M1 stability is antithetical to Fg interaction and that M1 conformational dynamics, as specified by destabilizing residues, are essential for interaction. A “capture-and-collapse” model of association accounts for these observations, in which M1 captures Fg through a dynamic conformation and then collapses into a register 2-coiled coil as a result of stabilization provided by binding energy. Our results support the general conclusion that destabilizing residues are evolutionarily conserved in M proteins to enable functional interactions necessary for pathogenesis. PMID:27512043

  16. Group 3 late embryogenesis abundant proteins from embryos of Artemia franciscana: structural properties and protective abilities during desiccation.

    PubMed

    Boswell, Leaf C; Menze, Michael A; Hand, Steven C

    2014-01-01

    Group 3 late embryogenesis abundant (LEA) proteins are highly hydrophilic, and their expression is associated with desiccation tolerance in both plants and animals. Here we show that two LEA proteins from embryos of Artemia franciscana, AfrLEA2 and AfrLEA3m, are intrinsically disordered in solution but upon desiccation gain secondary structure, as measured by circular dichroism. Trifluoroethanol and sodium dodecyl sulfate are both shown to induce α-helical structure in AfrLEA2 and AfrLEA3m. Bioinformatic predictions of secondary-structure content for both proteins correspond most closely to conformations measured in the dry state. Because some LEA proteins afford protection to desiccation-sensitive proteins during drying and subsequent rehydration, we tested for this capacity in AfrLEA2 and AfrLEA3m. The protective capacities vary, depending on the target enzyme. For the cytoplasmic enzyme lactate dehydrogenase, neither AfrLEA2 nor AfrLEA3m, with or without trehalose present, was able to afford protection better than that provided by bovine serum albumin (BSA) under the same conditions. However, for another cytoplasmic enzyme, phosphofructokinase, both AfrLEA2 and AfrLEA3m in the presence of trehalose were able to afford protection far greater than that provided by BSA with trehalose. Finally, for the mitochondrial enzyme citrate synthase, 400-μg/mL AfrLEA3m without trehalose provided significantly more protection than the same concentration of either AfrLEA2 or BSA.

  17. Nucleosome remodeling by the SWI/SNF complex is enhanced by yeast High Mobility Group Box (HMGB) proteins

    PubMed Central

    Hepp, Matias I.; Alarcon, Valentina; Dutta, Arnob; Workman, Jerry L.; Gutiérrez, José L.

    2014-01-01

    Regulation of gene expression at the level of transcription involves the concerted action of several proteins and protein complexes committed to dynamically alter the surrounding chromatin environment of a gene being activated or repressed. ATP-dependent chromatin remodeling complexes are key actors in chromatin remodeling, and the SWI/SNF complex is the founding member. While many studies have linked the action of these complexes to specific transcriptional regulation of a large number of genes and much is known about their catalytic activity, less is known about the nuclear elements that can enhance or modulate their activity. A number of studies have found that certain High Mobility Group (HMG) proteins are able to stimulate ATP-dependent chromatin remodeling activity, but their influence on the different biochemical outcomes of this activity is still unknown. In this work we studied the influence of the yeast Nhp6A, Nhp6B and Hmo1 proteins (HMGB family members) on different biochemical outcomes of yeast SWI/SNF remodeling activity. We found that all these HMG proteins stimulate the sliding activity of ySWI/SNF, while transient exposure of nucleosomal DNA and octamer transfer catalyzed by this complex are only stimulated by Hmo1. Consistently, only Hmo1 stimulates SWI/SNF binding to the nucleosome. Additionally, the sliding activity of another chromatin remodeling complex, ISW1a, is only stimulated by Hmo1. Further analyses show that these differential stimulatory effects of Hmo1 are dependent on the presence of its C-terminal tail, which contains a stretch of acidic and basic residues. PMID:24972368

  18. Boosting Adaptive Immunity: A New Role for PAFR Antagonists

    PubMed Central

    Koga, Marianna M.; Bizzarro, Bruna; Sá-Nunes, Anderson; Rios, Francisco J.; Jancar, Sonia

    2016-01-01

    We have previously shown that the Platelet-Activating Factor Receptor (PAFR) engagement in murine macrophages and dendritic cells (DCs) promotes a tolerogenic phenotype reversed by PAFR-antagonists treatment in vitro. Here, we investigated whether a PAFR antagonist would modulate the immune response in vivo. Mice were subcutaneously injected with OVA or OVA with PAFR-antagonist WEB2170 on days 0 and 7. On day 14, OVA–specific IgG2a and IgG1 were measured in the serum. The presence of WEB2170 during immunization significantly increased IgG2a without affecting IgG1 levels. When WEB2170 was added to OVA in complete Freund’s adjuvant, enhanced IgG2a but not IgG1 production was also observed, and CD4+ FoxP3+ T cell frequency in the spleen was reduced compared to mice immunized without the antagonist. Similar results were observed in PAFR-deficient mice, along with increased Tbet mRNA expression in the spleen. Additionally, bone marrow-derived DCs loaded with OVA were transferred into naïve mice and their splenocytes were co-cultured with fresh OVA-loaded DCs. CD4+ T cell proliferation was higher in the group transferred with DCs treated with the PAFR-antagonist. We propose that the activation of PAFR by ligands present in the site of immunization is able to fine-tune the adaptive immune response. PMID:27966635

  19. Dynamic renormalisation group reveals sequential mechanism of the secondary nucleation of proteins

    NASA Astrophysics Data System (ADS)

    Michaels, Thomas; Arosio, Paolo; Knowles, Tuomas

    2014-03-01

    Secondary nucleation has emerged as a key process in the self-assembly of amyloid fibrils associated with a number of neurodegenerative disorders. Secondary nucleation conceptually involves both aggregates and monomers, but a variety of ways exist, in which this process may occur. Elucidation of this complex mechanism using experimental data represents a theoretical challenge. A systematic coarse-graining procedure inspired by the renormalisation group is used to bridge the length- and timescale gaps between detailed microscopic descriptions and the processes observed in experiments. Various mechanisms of secondary nucleation are discussed at different levels of coarse graining and compact terms in the master equation are generated, that provide a single-step description of this process. This treatment is general and allows to test assumptions regarding mechanisms at the microscopic level and to filter their effect on the kinetics at the macroscopic scale. By analysing data from the polymerisation of amylin, we conclude that pre-critical nuclei in islet amyloid polypeptides stay attached to the aggregates during the process of secondary nucleation.

  20. Inactivation of glycogen synthase kinase-3beta and up-regulation of LINGO-1 are involved in LINGO-1 antagonist regulated survival of cerebellar granular neurons.

    PubMed

    Zhao, Xiang-Hui; Jin, Wei-Lin; Wu, Jiang; Mi, Sha; Ju, Gong

    2008-08-01

    LINGO-1 has been critically implicated in the central regulation of CNS axon regeneration and oligodendrocyte maturation. We have recently demonstrated that pretreatment with LINGO-1 antagonist (LINGO-1-Fc) inhibited low potassium-induced cerebellar granular neurons (CGNs) apoptosis. In the present study, we examined the neuroprotective mechanism of LINGO-1-Fc by Western blot and in situ GST pull-down assay. CGN cultures were preincubated in medium with LINGO-1-Fc or control protein at the concentration of 10 mug/ml for 2 h and then switched to low potassium medium in the presence of corresponding proteins. Cultures were harvested at indicated time intervals for successive analysis. Several apoptosis-associated signaling factors, GSK-3beta, ERK1/2, and Rho GTPases, were observed to be activated in response to potassium deprivation and the activation/dephosphorylation of GSK-3beta was suppressed by LINGO-1-Fc pretreatment compared with control group. Besides, the endogenous LINGO-1 expression level of CGN cultures was augmented by low potassium stimuli and restrained by LINGO-1 antagonist treatment. Although the protein level of p75(NTR) and Nogo-A were down-regulated in different patterns during apoptosis, neither of them was affected by LINGO-1-Fc application. Taken together, these results suggest a new mechanism of LINGO-1 antagonist regulated neuronal survival involving protein synthesis of LINGO-1 and inactivation of GSK-3 pathway.

  1. Molecular Epidemiology and Distribution of Serotypes, Surface Proteins, and Antibiotic Resistance among Group B Streptococci in Italy▿

    PubMed Central

    Gherardi, Giovanni; Imperi, Monica; Baldassarri, Lucilla; Pataracchia, Marco; Alfarone, Giovanna; Recchia, Simona; Orefici, Graziella; Dicuonzo, Giordano; Creti, Roberta

    2007-01-01

    Group B streptococci (GBS) comprising three different sets of isolates (31 invasive, 36 noninvasive, and 24 colonizing isolates) were collected in Italy during the years 2002 to 2005. Clonal groups were established by pulsed-field gel electrophoresis (PFGE), and selected isolates were studied by multilocus sequence typing (MLST). GBS isolates were also characterized by classical and molecular techniques for serotyping and protein gene and antibiotic resistance profiling. Some serotypes were significantly associated with a particular isolate population: serotype Ia more frequently corresponded to invasive strains than other strains, serotype V was more frequently encountered among noninvasive strains, and nontypeable strains were more common among isolates from carriers. Four major clonal groups accounted for 52.7% of all isolates: PFGE type 1/clonal complex 1 (CC1) comprised mainly serotype V isolates carrying the alp3 gene, PFGE type 2/CC23 encompassed serotype Ia isolates with the alp1 or alpha gene, PFGE type 3/CC17 comprised serotype III isolates carrying the rib gene, and PFGE type 4/CC19 consisted mainly of serotype II isolates possessing the rib gene. The same serotypes were shared by isolates of different clonal groups, and conversely, isolates belonging to the same clonal groups were found to be of different serotypes, presumably due to capsular switching by the horizontal transfer of capsular genes. Erythromycin resistance (prevalence, 16.5%; 15 resistant isolates of 91) was restricted to strains isolated from patients with noninvasive infections and carriers, while tetracycline resistance was evenly distributed (prevalence, 68.1%; 62 resistant isolates of 91). Most erythromycin-resistant GBS strains were of serotype V, were erm(B) positive, and belonged to the PFGE type 1/CC1 group, suggesting that macrolide resistance may have arisen both by clonal dissemination and by the horizontal transfer of resistance genes. PMID:17634303

  2. The cannabinoid 1-receptor silent antagonist O-2050 attenuates preference for high-fat diet and activated astrocytes in mice.

    PubMed

    Higuchi, Sei; Irie, Keiichi; Mishima, Shohei; Araki, Maiko; Ohji, Makiko; Shirakawa, Atsunori; Akitake, Yoshiharu; Matsuyama, Kiyoshi; Mishima, Kenji; Mishima, Kenichi; Iwasaki, Katsunori; Fujiwara, Michihiro

    2010-01-01

    Endocannabinoids have been shown to activate reward-related feeding and to promote astrocytic differentiation. We investigated whether high-fat diet (HFD) intake produced a preference for HFD via an endocannabinoid-dependent mechanism. In the conditioned place preference test, the 2-week HFD-intake group showed preference for HFD and had increased expression of a marker for reactive astrocytes, glial fibrillary acid protein (GFAP), in the hypothalamus. The cannabinoid CB(1)-receptor antagonist O-2050 reduced the preference for HFD and expression of GFAP in the hypothalamus. These results suggested that HFD intake led to the development of a preference for HFD via astrocytic CB(1) receptors in the hypothalamus.

  3. Hematopoietic stem cell fate decisions are regulated by Wnt antagonists: comparisons and current controversies.

    PubMed

    Cain, Corey J; Manilay, Jennifer O

    2013-01-01

    Wingless and int (Wnt) proteins are secreted proteins that are important for regulating hematopoietic stem cell self-renewal and differentiation in the bone marrow microenvironment in mice. The mechanisms by which Wnt signaling regulates these hematopoietic cell fate decisions are not fully understood. Secreted Wnt antagonists, which are expressed in bone and bone marrow stromal cells, either bind to Wnt ligands directly or block Wnt receptors and co-receptors to halt Wnt-mediated signal transduction in both osteolineage and hematopoietic cell types. Secreted frizzled related proteins-1 and -2, Wnt inhibitory factor-1, Dickkopf-1, and Sclerostin are Wnt antagonists that influence hematopoietic cell fate decisions in the bone marrow niche. In this review, we compare and contrast the roles of these Wnt antagonists and their effects on hematopoietic development in mice, and also discuss the clinical significance of targeting Wnt antagonists within the context of hematopoietic disease.

  4. Xeroderma pigmentosum complementation group A protein is driven to nucleotide excision repair sites by the electrostatic potential of distorted DNA.

    PubMed

    Camenisch, Ulrike; Dip, Ramiro; Vitanescu, Mirela; Naegeli, Hanspeter

    2007-12-01

    The presumed DNA-binding cleft of xeroderma pigmentosum group A (XPA) protein, a key regulatory subunit of the eukaryotic nucleotide excision repair complex, displays a distinctive array of 6 positively charged amino acid side chains. Here, the molecular function of these closely spaced electropositive residues has been tested by systematic site-directed mutagenesis. After the introduction of single amino acid substitutions, the mutants were probed for protein-DNA interactions in electrophoretic mobility shift and photochemical crosslinking assays. This analysis led to the identification of a critical hot-spot for DNA substrate recognition composed of two neighboring lysines at codons 141 and 179 of the human XPA sequence. The replacement of other basic side chains in the DNA interaction domain conferred more moderate defects of substrate binding. When the function of XPA was tested as a fusion product with either mCherry or green-fluorescent protein, a glutamate substitution of one of the positively charged residues at positions 141 and 179 was sufficient to decrease DNA repair activity in human fibroblasts. Thus, the removal of a single cationic side chain abolished DNA-binding activity and significant excision repair defects could be induced by single charge inversions on the XPA surface, indicating that this molecular sensor participates in substrate recognition by monitoring the electrostatic potential of distorted DNA repair sites.

  5. The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-induced Lysine Acetylation of Mitochondrial Proteins

    PubMed Central

    Davies, Michael N.; Kjalarsdottir, Lilja; Thompson, J. Will; Dubois, Laura G.; Stevens, Robert D.; Ilkayeva, Olga R.; Brosnan, M. Julia; Rolph, Timothy P.; Grimsrud, Paul A.; Muoio, Deborah M.

    2016-01-01

    Lysine acetylation (AcK), a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acetyltransferase (CrAT), an enzyme that buffers the mitochondrial acetyl-CoA pool by converting short-chain acyl-CoAs to their membrane permeant acylcarnitine counterparts. CrAT deficiency increased tissue acetyl-CoA levels and susceptibility to diet-induced AcK of broad-ranging mitochondrial proteins, coincident with diminished whole body glucose control. Sub-compartment acetylproteome analyses of muscles from obese mice and humans showed remarkable overrepresentation of mitochondrial matrix proteins. These findings reveal roles for CrAT and L-carnitine in modulating the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK. PMID:26748706

  6. [Relationship of food groups intake and C-reactive protein in healthy adults from Mexicali, Baja California, México].

    PubMed

    Ruiz-Esparza, Josefina; Robinson-Navarro, Octavio; Ortega-Vélez, María Isabel; Diaz-Molina, Raúl; Carrillo-Cedillo, Eugenia Gabriela; Soria-Rodriguez, Carmen G

    2013-09-01

    The high sensitivity C-reactive protein (hs-CRP) is an important biomarker in inflammatory processes. The objective was to analyze the relationship between the concentrations of hs-CRP in adults from a northern Mexico region with their typical food intake patterns. A sample of 72 university professors underwent clinical and anthropometric assessments and their hs-CRP levels were quantified with an immunoenzymometric assay. Additionally, they filled out a food intake frequency questionnaire, from which the servings of different food groups were obtained with the ESHA software. The average age of participants was 49.75 +/- 10.05 years and the average hs-CRP concentration was 1.66 (0.97, 3.52) mg/L. The value of the association between fruit consumption and hs-CRP level was protective, according to the logistic regression analysis, being the Odds Ratio (OR) 0.23 (95% CI: 0.05, 1.03); while for vegetables the OR was 0.66 (95% CI: 0.12, 3.68). Furthermore, high protein content foods, dairy products, oils and fats were associated with elevated levels of hs-CRP. In conclusion, in our study, the intake of some food groups like fruits and vegetables, and to a lesser extent cereals, were associated with low values of hs-PCR.

  7. Group 1 metabotropic glutamate receptors 1 and 5 form a protein complex in mouse hippocampus and cortex

    PubMed Central

    Pandya, Nikhil J.; Klaassen, Remco V.; van der Schors, Roel C.; Slotman, Johan A.; Houtsmuller, Adriaan; Smit, August B.

    2016-01-01

    The group 1 metabotropic glutamate receptors 1 and 5 (mGluR1/5) have been implicated in mechanisms of synaptic plasticity and may serve as potential therapeutic targets in autism spectrum disorders. The interactome of group 1 mGluRs has remained largely unresolved. Using a knockout‐controlled interaction proteomics strategy we examined the mGluR5 protein complex in two brain regions, hippocampus and cortex, and identified mGluR1 as its major interactor in addition to the well described Homer proteins. We confirmed the presence of mGluR1/5 complex by (i) reverse immunoprecipitation using an mGluR1 antibody to pulldown mGluR5 from hippocampal tissue, (ii) coexpression in HEK293 cells followed by coimmunoprecipitation to reveal the direct interaction of mGluR1 and 5, and (iii) superresolution microscopy imaging of hippocampal primary neurons to show colocalization of the mGluR1/5 in the synapse. PMID:27392515

  8. [Effect of dexmedetomidine on high-mobility group box 1 protein in rats with sepsis].

    PubMed

    Li, Xin; Li, Jinghui

    2015-09-01

    目的:探讨右美托咪定(dexmedetomidine,DEX)对脓毒症大鼠血液和脾组织中高迁移率族蛋白B1(high-mobility group box 1 protein,HMGB1)的作用及其可能的机制。方法:将100只雄性Wistar大鼠随机分为对照组、模型组、DEX组、α-银环蛇毒素(α-bungarotoxin-tetramethylrhodamine,α-BGT)+DEX组和α-BGT组,每组20只。对照组静脉注射等量的生理盐水,其余各组均通过静注内毒素(lipopolysaccharide,LPS)构建脓毒症模型,并给予不同的处理,48 h后处死大鼠,留取血液及脾组织标本。应用ELISA分别检测血液中TNF-α和晚期炎症介质HMGB1的水平,Western印迹检测脾组织中HMGB1的含量。结果:5组大鼠间病死率、血清TNF-α水平、HMGB1的血清水平和脾组织含量差异均有统计学意义(均P<0.05),且HMGB1的血清水平和脾组织含量存在显著相关性(r=0.863,P<0.05)。结论:DEX可明显降低脓毒症大鼠血液及脾组织中晚期炎症介质HMGB1的含量,这种作用可能是通过激活胆碱能抗炎通路而实现的。.

  9. Thermodynamic analysis of antagonist and agonist interactions with dopamine receptors.

    PubMed

    Duarte, E P; Oliveira, C R; Carvalho, A P

    1988-03-01

    The binding of [3H]spiperone to dopamine D-2 receptors and its inhibition by antagonists and agonists were examined in microsomes derived from the sheep caudate nucleus, at temperatures between 37 and 1 degree C, and the thermodynamic parameters of the binding were evaluated. The affinity of the receptor for the antagonists, spiperone and (+)-butaclamol, decreased as the incubation temperature decreased; the affinity for haloperidol did not further decrease at temperatures below 15 degrees C. The binding of the antagonists was associated with very large increases in entropy, as expected for hydrophobic interactions. The enthalpy and entropy changes associated with haloperidol binding were dependent on temperature, in contrast to those associated with spiperone and (+)-butaclamol. The magnitude of the entropy increase associated with the specific binding of the antagonists did not correlate with the degree of lipophilicity of these drugs. The data suggest that, in addition to hydrophobic forces, other forces are also involved in the antagonist-dopamine receptor interactions, and that a conformational change of the receptor could occur when the antagonist binds. Agonist binding data are consistent with a two-state model of the receptor, a high-affinity state (RH) and a low-affinity state (RL). The affinity of dopamine binding to the RH decreased with decreasing temperatures below 20 degrees C, whereas the affinity for the RL increased at low temperatures. In contrast, the affinity of apomorphine for both states of receptor decreased as the temperature decreased from 30 to 8 degrees C. A clear distinction between the energetics of high-affinity and low-affinity agonist binding was observed. The formation of the high-affinity complex was associated with larger increases in enthalpy and entropy than the interaction with the low-affinity state was. The results suggest that the interaction of the receptor with the G-proteins, induced or stabilized by the binding of

  10. Alternative splicing for members of human mosaic domain superfamilies. I. The CH and LIM domains containing group of proteins.

    PubMed

    Friedberg, Felix

    2009-05-01

    In this paper we examine (restricted to homo sapiens) the products resulting from gene duplication and the subsequent alternative splicing for the members of a multidomain group of proteins which possess the evolutionary conserved calponin homology CH domain, i.e. an "actin binding domain", as a singlet and which, in addition, contain the conserved cysteine rich double Zn finger possessing Lim domain, also as a singlet. Seven genes, resulting from gene duplications, were identified that code for seven group members for which pre-mRNAs appear to have undergone multiple alternative splicing: Mical 1, 2 and 3 are located on chromosomes 6q21, 11p15 and 22q11, respectively. The LMO7 gene is present on chromosome 13q22 and the LIMCH1 gene on chromosome 4p13. Micall1 is mapped to chromosome 22q13 and Micall2 to chromosome 7p22. Translated Gen/Bank ESTs suggest the existence of multiple products alternatively spliced from the pre-mRNAs encoded by these genes. Characteristic indicators of such splicing among the proteins derived from one gene must include containment of some common extensive 100% identical regions. In some instances only one exon might be partly or completely eliminated. Sometimes alternative splicing is also associated with an increased frequency of creation of an exon or part of an exon from an intron. Not only coding regions for the body of the protein but also for its N- or -C ends could be affected by the splicing. If created forms are merely beginning at different starting points but remain identical in sequence thereafter, their existence as products of alternate splicing must be questioned. In the splicings, described in this paper, multiple isoforms rather than a single isoform appear as products during the gene expression.

  11. Interleukin-1 receptor antagonist gene polymorphism and mortality in patients with severe sepsis

    PubMed Central

    ARNALICH, F; LÓPEZ-MADERUELO, D; CODOCEO, R; LOPEZ, J; SOLIS-GARRIDO, L M; CAPISCOL, C; FERNANDEZ-CAPITÁN, C; MADERO, R; MONTIEL, C

    2002-01-01

    This study aims to determine the influence of the polymorphism within the intron 2 of the interleukin-1 receptor antagonist gene (IL-1RN*) on the outcome of severe sepsis, and to assess its functional significance by correlating this polymorphism with the total production of interleukin-1 receptor antagonist (IL-1Ra) protein determined in stimulated peripheral blood mononuclear cells (PBMC). A group of 78 patients with severe sepsis (51 survivors and 27 nonsurvivors) was compared with a healthy control group of 130 blood donors, and 56 patients with uncomplicated pneumonia. We found a significant association between IL-1RN* polymorphism and survival. Thus, after adjusting for age and APACHE II score, multiple logistic regression analysis showed that patients homozygotes for the allele *2 had a 6·47-fold increased risk of death (95% CI 1·01–41·47, P = 0·04). Besides, compared with patients homozygous or heterozygous for the allele *1, IL-1RN*2 homozygotes produced significantly lower levels of IL-1Ra from their PBMC. Our results suggest that insufficient production of this cytokine might contribute, among other factors, to the higher mortality rate found in severe sepsis patients with the IL-1RN*2 homozygous genotype. PMID:11876758

  12. Fabrication of Nanometer- and Micrometer-Scale Protein Structures by Site-Specific Immobilization of Histidine-Tagged Proteins to Aminosiloxane Films with Photoremovable Protein-Resistant Protecting Groups

    PubMed Central

    2016-01-01

    The site-specific immobilization of histidine-tagged proteins to patterns formed by far-field and near-field exposure of films of aminosilanes with protein-resistant photolabile protecting groups is demonstrated. After deprotection of the aminosilane, either through a mask or using a scanning near-field optical microscope, the amine terminal groups are derivatized first with glutaraldehyde and then with N-(5-amino-1-carboxypentyl)iminodiacetic acid to yield a nitrilo-triacetic-acid-terminated surface. After complexation with Ni2+, this surface binds histidine-tagged GFP and CpcA-PEB in a site-specific fashion. The chemistry is simple and reliable and leads to extensive surface functionalization. Bright fluorescence is observed in fluorescence microscopy images of micrometer- and nanometer-scale patterns. X-ray photoelectron spectroscopy is used to study quantitatively the efficiency of photodeprotection and the reactivity of the modified surfaces. The efficiency of the protein binding process is investigated quantitatively by ellipsometry and by fluorescence microscopy. We find that regions of the surface not exposed to UV light bind negligible amounts of His-tagged proteins, indicating that the oligo(ethylene glycol) adduct on the nitrophenyl protecting group confers excellent protein resistance; in contrast, exposed regions bind His-GFP very effectively, yielding strong fluorescence that is almost completely removed on treatment of the surface with imidazole, confirming a degree of site-specific binding in excess of 90%. This simple strategy offers a versatile generic route to the spatially selective site-specific immobilization of proteins at surfaces. PMID:26820378

  13. CysLT(1)R antagonists inhibit tumor growth in a xenograft model of colon cancer.

    PubMed

    Savari, Sayeh; Liu, Minghui; Zhang, Yuan; Sime, Wondossen; Sjölander, Anita

    2013-01-01

    The expression of the inflammatory G-protein coupled receptor CysLT1R has been shown to be upregulated in colon cancer patients and associated with poor prognosis. The present study investigated the correlation between CysLT1R and colon cancer development in vivo using CysLT1R antagonists (ZM198,615 or Montelukast) and the nude mouse xenograft model. Two drug administration regimens were established. The first regimen was established to investigate the importance of CysLT1R in tumor initiation. Nude mice were inoculated with 50 µM CysLT1R antagonist-pretreated HCT-116 colon cancer cells and received continued treatment (5 mg/kg/day, intraperitoneally). The second regimen aimed to address the role of CysLT1R in tumor progression. Nude mice were inoculated with non-pretreated HCT-116 cells and did not receive CysLT1R antagonist treatment until recordable tumor appearance. Both regimens resulted in significantly reduced tumor size, attributed to changes in proliferation and apoptosis as determined by reduced Ki-67 levels and increased levels of p21(WAF/Cip1) (P<0.01), cleaved caspase 3, and the caspase-cleaved product of cytokeratin 18. Decreased levels of VEGF (P<0.01) and reduced vessel size (P<0.05) were also observed, the latter only in the ZM198,615-pretreatment group. Furthermore, we performed a series of in vitro studies using the colon cancer cell line HCT-116 and CysLT1R antagonists. In addition to significant reductions in cell proliferation, adhesion and colony formation, we observed induction of cell cycle arrest and apoptosis in a dose-dependent manner. The ability of Montelukast to inhibit growth of human colon cancer xenograft was further validated by using two additional colon cancer cell lines, SW-480 and HT-29. Our results demonstrate that CysLT1R antagonists inhibit growth of colon cancer xenografts primarily by reducing proliferation and inducing apoptosis of the tumor cells.

  14. CysLT1R Antagonists Inhibit Tumor Growth in a Xenograft Model of Colon Cancer

    PubMed Central

    Savari, Sayeh; Liu, Minghui; Zhang, Yuan; Sime, Wondossen; Sjölander, Anita

    2013-01-01

    The expression of the inflammatory G-protein coupled receptor CysLT1R has been shown to be upregulated in colon cancer patients and associated with poor prognosis. The present study investigated the correlation between CysLT1R and colon cancer development in vivo using CysLT1R antagonists (ZM198,615 or Montelukast) and the nude mouse xenograft model. Two drug administration regimens were established. The first regimen was established to investigate the importance of CysLT1R in tumor initiation. Nude mice were inoculated with 50 µM CysLT1R antagonist-pretreated HCT-116 colon cancer cells and received continued treatment (5 mg/kg/day, intraperitoneally). The second regimen aimed to address the role of CysLT1R in tumor progression. Nude mice were inoculated with non-pretreated HCT-116 cells and did not receive CysLT1R antagonist treatment until recordable tumor appearance. Both regimens resulted in significantly reduced tumor size, attributed to changes in proliferation and apoptosis as determined by reduced Ki-67 levels and increased levels of p21WAF/Cip1 (P<0.01), cleaved caspase 3, and the caspase-cleaved product of cytokeratin 18. Decreased levels of VEGF (P<0.01) and reduced vessel size (P<0.05) were also observed, the latter only in the ZM198,615-pretreatment group. Furthermore, we performed a series of in vitro studies using the colon cancer cell line HCT-116 and CysLT1R antagonists. In addition to significant reductions in cell proliferation, adhesion and colony formation, we observed induction of cell cycle arrest and apoptosis in a dose-dependent manner. The ability of Montelukast to inhibit growth of human colon cancer xenograft was further validated by using two additional colon cancer cell lines, SW-480 and HT-29. Our results demonstrate that CysLT1R antagonists inhibit growth of colon cancer xenografts primarily by reducing proliferation and inducing apoptosis of the tumor cells. PMID:24039952

  15. Genetic enhancement of visual learning by activation of protein kinase C pathways in small groups of rat cortical neurons.

    PubMed

    Zhang, Guo-Rong; Wang, Xiaodan; Kong, Lingxin; Lu, Xiu-Gui; Lee, Brian; Liu, Meng; Sun, Mei; Franklin, Corinna; Cook, Robert G; Geller, Alfred I

    2005-09-14

    Although learning and memory theories hypothesize that memories are encoded by specific circuits, it has proven difficult to localize learning within a cortical area. Neural network theories predict that activation of a small fraction of the neurons in a circuit can activate that circuit. Consequently, altering the physiology of a small group of neurons might potentiate a specific circuit and enhance learning, thereby localizing learning to that circuit. In this study, we activated protein kinase C (PKC) pathways in small groups of neurons in rat postrhinal (POR) cortex. We microinjected helper virus-free herpes simplex virus vectors that expressed a constitutively active PKC into POR cortex. This PKC was expressed predominantly in glutamatergic and GABAergic neurons in POR cortex. This intervention increased phosphorylation of five PKC substrates that play critical roles in neurotransmitter release (GAP-43 and dynamin) or glutamatergic neurotransmission (specific subunits of AMPA or NMDA receptors and myristoylated alanine-rich C kinase substrate). Additionally, activation of PKC pathways in cultured cortical neurons supported activation-dependent increases in release of glutamate and GABA. This intervention enhanced the learning rate and accuracy of visual object discriminations. In individual rats, the numbers of transfected neurons positively correlated with this learning. During learning, neuronal activity was increased in neurons proximal to the transfected neurons. These results demonstrate that potentiating small groups of glutamatergic and GABAergic neurons in POR cortex enhances visual object learning. More generally, these results suggest that learning can be mediated by specific cortical circuits.

  16. Origin of abrupt rise in deuteron NMR longitudinal relaxation times of protein methyl groups below 90 K.

    PubMed

    Vugmeyster, Liliya; Ostrovsky, Dmitry; Lipton, Andrew S

    2013-05-23

    In order to examine the origin of the abrupt change in the temperature dependence of (2)H NMR longitudinal relaxation times observed previously for methyl groups of L69 in the hydrophobic core of villin headpiece protein at around 90 K (Vugmeyster et al. J. Am. Chem. Soc. 2010, 132, 4038-4039), we extended the measurements to several other methyl groups in the hydrophobic core. We show that, for all methyl groups, relaxation times experience a dramatic jump several orders of magnitude around this temperature. Theoretical modeling supports the conclusion that the origin of the apparent transition in the relaxation times is due to the existence of the distribution of conformers distinguished by their activation energy for methyl three-site hops. It is also crucial to take into account the differential contribution of individual conformers into overall signal intensity. When a particular conformer approaches the regime at which its three-site hop rate constant is on the order of the quadrupolar coupling interaction constant, the intensity of the signal due to this conformer experiences a sharp drop, thus changing the balance of the contributions of different conformers into the overall signal. As a result, the observed apparent transition in the relaxation rates can be explained without the assumption of an underlying transition in the rate constants. This work in combination with earlier results also shows that the model based on the distribution of conformers explains the relaxation behavior in the entire temperature range between 300 and 70 K.

  17. Origin of Abrupt Rise in Deuteron NMR Longitudinal Relaxation Times of Protein Methyl Groups Below 90 K

    SciTech Connect

    Vugmeyster, Liliya; Ostrovsky, Dmitry; Lipton, Andrew S.

    2013-05-23

    In order to examine the origin of the abrupt change in the temperature dependence of NMR longitudinal relaxation times observed earlier for methyl groups of L69 in the hydrophobic core of villin headpiece protein at around 90 K (Vugmeyster et al. J. Am. Chem. Soc. 2010, 132, 4038), we extended the measurements to several other methyl groups in the hydrophobic core. We show that for all methyl groups, relaxation times experience a dramatic jump several orders of magnitude around this temperature. Theoretical modeling supports the conclusion that the origin of the apparent transition in the relaxation times is due to the existence of the distribution of conformers distinguished by their activation energy for methyl three-site hops. It is also crucial to take into account the differential contribution of individual conformers into overall signal intensity. When a particular conformer approaches the regime at which its three-site hops rate constant is on the order of the quadrupolar coupling interaction constant, the intensity of the signal due to this conformer experiences a sharp drop, thus changing the balance of the contributions of different conformers into the overall signal. As a result, the observed apparent transition in the relaxation rates can be explained without the assumption of an underlying transition in the rate constants. This work in combination with earlier results also shows that the model based on the distribution of conformers explains the relaxation behavior in the entire temperature range between 300-70 K.

  18. Mineralcorticoid antagonists in heart failure.

    PubMed

    D'Elia, Emilia; Krum, Henry

    2014-10-01

    Mineralocorticoid receptor antagonists (MRAs) have become mandated therapy in patients with reduced ejection fraction (systolic) heart failure (HF) across all symptom classes. These agents should also be prescribed in the early post-myocardial infarction setting in those with reduced ejection fraction and either HF symptoms or diabetes. This article explores the pathophysiological role of aldosterone, an endogenous ligand for the mineralcorticoid receptor (MR), and summarizes the clinical data supporting guideline recommendations for these agents in systolic HF. The use of MRAs in novel areas beyond systolic HF ejection is also explored. Finally, the current status of newer agents will be examined.

  19. NK-1 Antagonists and Itch.

    PubMed

    Ständer, Sonja; Luger, Thomas A

    2015-01-01

    Substance P (SP) is an important mediator of pro-inflammatory mechanisms in the skin. It targets multiple cells such as keratinocytes, mast cells, and fibroblasts which are involved in the cutaneous generation of pruritus. This suggests that SP is an interesting target for therapy. In fact, in recent case reports and case series, SP antagonists demonstrated a significant antipruritic effect in acute and chronic pruritus such as drug-induced pruritus, paraneoplastic pruritus, prurigo nodularis, cutaneous T-cell lymphoma, and brachioradial pruritus.

  20. The active gene that encodes human High Mobility Group 1 protein (HMG1) contains introns and maps to chromosome 13

    SciTech Connect

    Ferrari, S.; Finelli, P.; Rocchi, M.

    1996-07-15

    The human genome contains a large number of sequences related to the cDNA for High Mobility Group 1 protein (HMG1), which so far has hampered the cloning and mapping of the active HMG1 gene. We show that the human HMG1 gene contains introns, while the HMG1-related sequences do not and most likely are retrotransposed pseudogenes. We identified eight YACs from the ICI and CEPH libraries that contain the human HMG1 gene. The HMG1 gene is similar in structure to the previously characterized murine homologue and maps to human chromosome 13 and q12, as determined by in situ hybridization. The mouse Hmg1 gene maps to the telomeric region of murine Chromosome 5, which is syntenic to the human 13q12 band. 18 refs., 3 figs.

  1. Monoclonal Anti-HMGB1 (High Mobility Group Box Chromosomal Protein 1) Antibody Protection in Two Experimental Arthritis Models

    PubMed Central

    Schierbeck, Hanna; Lundbäck, Peter; Palmblad, Karin; Klevenvall, Lena; Erlandsson-Harris, Helena; Andersson, Ulf; Ottosson, Lars

    2011-01-01

    High mobility group box chromosomal protein 1 (HMGB1) is a DNA-binding nuclear protein that can be released from dying cells and activated myeloid cells. Extracellularly, HMGB1 promotes inflammation. Experimental studies demonstrate HMGB1 to be a pathogenic factor in many inflammatory conditions including arthritis. HMGB1-blocking therapies in arthritis models alleviate disease and confer significant protection against cartilage and bone destruction. So far, the most successful HMGB1-targeted therapies have been demonstrated with HMGB1-specific polyclonal antibodies and with recombinant A box protein, a fragment of HMGB1. The present study is the first to evaluate the potential of a monoclonal anti-HMGB1 antibody (2G7, mouse IgG2b) to ameliorate arthritis. Effects of repeated injections of this antibody have now been studied in two conceptually different models of arthritis: collagen type II–induced arthritis (CIA) in DBA/1 mice and in a spontaneous arthritis disease in mice with combined deficiencies for genes encoding for the enzyme DNase type II and interferon type I receptors. These mice are unable to degrade phagocytozed DNA in macrophages and develop chronic, destructive polyarthritis. Therapeutic intervention in CIA and prophylactic administration of anti-HMGB1 monoclonal antibody (mAb) in the spontaneous arthritis model significantly ameliorated the clinical courses. Anti-HMGB1 mAb therapy also partially prevented joint destruction, as demonstrated by histological examination. The beneficial antiarthritic effects by the anti-HMGB1 mAb in two diverse models of arthritis represent additional proof-of-concept, indicating that HMGB1 may be a valid target molecule to consider for development of future clinical therapy. PMID:21666956

  2. Peptide-Recombinant VP6 Protein Based Enzyme Immunoassay for the Detection of Group A Rotaviruses in Multiple Host Species

    PubMed Central

    Sircar, Subhankar; Saurabh, Sharad; Gulati, Baldev R.; Singh, Neeraj; Singh, Arvind Kumar; Joshi, Vinay G.; Banyai, Krisztian; Dhama, Kuldeep

    2016-01-01

    We developed a novel enzyme immunoassay for the detection of group A rotavirus (RVA) antigen in fecal samples of multiple host species. The assay is based on the detection of conserved VP6 protein using anti-recombinant VP6 antibodies as capture antibodies and anti-multiple antigenic peptide (identified and constructed from highly immunodominant epitopes within VP6 protein) antibodies as detector antibodies. The clinical utility of the assay was evaluated using a panel of 914 diarrhoeic fecal samples from four different host species (bovine, porcine, poultry and human) collected from diverse geographical locations in India. Using VP6- based reverse transcription-polymerase chain reaction (RT-PCR) as the gold standard, we found that the diagnostic sensitivity (DSn) and specificity (DSp) of the new assay was high [bovine (DSn = 94.2% & DSp = 100%); porcine (DSn = 94.6% & DSp = 93.3%); poultry (DSn = 74.2% & DSp = 97.7%) and human (DSn = 82.1% & DSp = 98.7%)]. The concordance with RT-PCR was also high [weighted kappa (k) = 0.831–0.956 at 95% CI = 0.711–1.0] as compared to RNA-polyacrylamide gel electrophoresis (RNA-PAGE). The performance characteristics of the new immunoassay were comparable to those of the two commercially available ELISA kits. Our results suggest that this peptide-recombinant protein based assay may serve as a preliminary assay for epidemiological surveillance of RVA antigen and for evaluation of vaccine effectiveness especially in low and middle income settings. PMID:27391106

  3. Effect on proliferation and apoptosis of retinoblastoma cell by RNA inhibiting high mobility group protein box-1 expression

    PubMed Central

    Wang, Li-Lun; Feng, Yan-Qin; Cheng, Yu-Hong

    2017-01-01

    AIM To investigate the effect of high mobility group protein box-1 (HMGB1) siRNA on proliferation and apoptosis of retinoblastoma (Rb) cells. METHODS The expression of HMGB1 in Rb cells were detected by real-time polymerase chain reaction (RT-PCR) and Western blot. Chemically synthesized HMGB1 siRNA was transfected into Y79 cells. The inhibitory rate was also examined by RT-PCR and Western blot. After HMGB1 siRNA transfection, the cell proliferation was analyzed by MTT, and cell apoptosis was detected by Caspase-3 active detection kit. Cell cycle distribution and apoptosis were detected by flow cytometry. RESULTS The expression of HMGB1 significantly elevated in Rb cells (P<0.01). After transfected by siRNA, the HMGB1 protein level of Y79 cells was significantly reduced (P<0.01). After siRNA interference HMGB1, the proportion of proliferating cells reduced, and the proportion of quiescent cells increased (P<0.05). In addition, apoptosis rate of Y79 cells increased from 2.03% to 9.10% after interfering with HMGB1 siRNA (P<0.05). CONCLUSION Specific HMGB1 siRNA can inhibit the expression of HMGB1. The effect may be attributed to inhibit the proliferation and promote cell apoptosis. PMID:28149773

  4. Spike Protein VP8* of Human Rotavirus Recognizes Histo-Blood Group Antigens in a Type-Specific Manner

    PubMed Central

    Huang, Pengwei; Xia, Ming; Zhong, Weiming; Wei, Chao; Wang, Leyi; Morrow, Ardythe

    2012-01-01

    Rotaviruses (RVs), an important cause of severe diarrhea in children, have been found to recognize sialic acid as receptors for host cell attachment. While a few animal RVs (of P[1], P[2], P[3], and P[7]) are sialidase sensitive, human RVs and the majority of animal RVs are sialidase insensitive. In this study, we demonstrated that the surface spike protein VP8* of the major P genotypes of human RVs interacts with the secretor histo-blood group antigens (HBGAs). Strains of the P[4] and P[8] genotypes shared reactivity with the common antigens of Lewis b (Leb) and H type 1, while strains of the P[6] genotype bound the H type 1 antigen only. The bindings between recombinant VP8* and human saliva, milk, or synthetic HBGA oligosaccharides were demonstrated, which was confirmed by blockade of the bindings by monoclonal antibodies (MAbs) specific to Leb and/or H type 1. In addition, specific binding activities were observed when triple-layered particles of a P[8] (Wa) RV were tested. Our results suggest that the spike protein VP8* of RVs is involved in the recognition of human HBGAs that may function as ligands or receptors for RV attachment to host cells. PMID:22345472

  5. Temperature-Induced Extended Helix/Random Coil Transitions in a Group 1 Late Embryogenesis-Abundant Protein from Soybean1

    PubMed Central

    Soulages, Jose L.; Kim, Kangmin; Walters, Christina; Cushman, John C.

    2002-01-01

    Group 1 late embryogenesis-abundant (LEA) proteins are a subset of hydrophilins that are postulated to play important roles in protecting plant macromolecules from damage during freezing, desiccation, or osmotic stress. To better understand the putative functional roles of group 1 LEA proteins, we analyzed the structure of a group 1 LEA protein from soybean (Glycine max). Differential scanning calorimetry of the purified, recombinant protein demonstrated that the protein assumed a largely unstructured state in solution. In the presence of trifluoroethanol (50% [w/v]), the protein acquired a 30% α-helical content, indicating that the polypeptide is highly restricted to adopt α-helical structures. In the presence of sodium dodecyl sulfate (1% [w/v]), 8% of the polypeptide chain adopted an α-helical structure. However, incubation with phospholipids showed no effect on the protein structure. Ultraviolet absorption and circular dichroism spectroscopy revealed that the protein existed in equilibrium between two conformational states. Ultraviolet absorption spectroscopy studies also showed that the protein became more hydrated upon heating. Furthermore, circular dichroism spectral measurements indicated that a minimum of 14% of amino acid residues existed in a solvent-exposed, left-handed extended helical or poly (l-proline)-type (PII) conformation at 20°C with the remainder of the protein being unstructured. The content of PII-like structure increased as temperature was lowered. We hypothesize that by favoring the adoption of PII structure, instead of the formation of α-helical or β-sheet structures, group 1 LEA proteins retain a high content of surface area available for interaction with the solvent. This feature could constitute the basis of a potential role of LEA proteins in preventing freezing, desiccation, or osmotic stress damage. PMID:11891239

  6. Vitamin K antagonists: beyond bleeding.

    PubMed

    Krüger, Thilo; Floege, Jürgen

    2014-01-01

    Warfarin is the most widely used oral anticoagulant in clinical use today. Indications range from prosthetic valve replacement to recurrent thromboembolic events due to antiphospholipid syndrome. In hemodialysis (HD) patients, warfarin use is even more frequent than in the nonrenal population due to increased cardiovascular comorbidities. The use of warfarin in dialysis patients with atrial fibrillation requires particular caution because side effects may outweigh the assumed benefit of reduced stroke rates. Besides increased bleeding risk, coumarins exert side effects which are not in the focus of clinical routine, yet they deserve special consideration in dialysis patients and should influence the decision of whether or not to prescribe vitamin K antagonists in cases lacking clear guidelines. Issues to be taken into consideration in HD patients are the induction or acceleration of cardiovascular calcifications, a 10-fold increased risk of calciphylaxis and problems related to maintaining a target INR range. New anticoagulants like direct thrombin inhibitors are promising but have not yet been approved for ESRD patients. Here, we summarize the nontraditional side effects of coumarins and give recommendations about the use of vitamin K antagonists in ESRD patients.

  7. Members of the high mobility group B protein family are dynamically expressed in embryonic neural stem cells

    PubMed Central

    2013-01-01

    Neural Stem Cells (NSCs) are a distinct group of cells present in the embryonic and adult mammalian central nervous system (CNS) that are able to differentiate into neurons, astrocytes and oligodendrocytes. As NSC proliferation declines with age, factors that regulate this process need to be defined. To search for NSC regulatory factors, we performed a quantitative shotgun proteomics study that revealed that members of the High Mobility Group B (HMGB) family are highly expressed in NSCs. Using a neurosphere assay, we report the differential expression of HMGB 1, 2, 3, and 4 mRNAs in proliferating NSCs isolated from various time points during embryonic development, as well as the dynamic expression of HMGB1 and B2 mRNAs and proteins in differentiating embryonic NSCs. Expression of HMGB2 underwent the most dramatic changes during the developmental ages examined; as a result, we assessed its role in NSC proliferation and differentiation. We report the predominance of small diameter HMGB2-/- neurospheres in comparison to wild-type, which correlated with increased proliferation in these smaller HMGB2-/- neurospheres. Our data suggest that HMGB2 plays a regulatory role in NSC cell proliferation and maintenance pathways. PMID:23621913

  8. Structure of the polycomb group protein PCGF1 in complex with BCOR reveals basis for binding selectivity of PCGF homologs.

    PubMed

    Junco, Sarah E; Wang, Renjing; Gaipa, John C; Taylor, Alexander B; Schirf, Virgil; Gearhart, Micah D; Bardwell, Vivian J; Demeler, Borries; Hart, P John; Kim, Chongwoo A

    2013-04-02

    Polycomb-group RING finger homologs (PCGF1, PCGF2, PCGF3, PCGF4, PCGF5, and PCGF6) are critical components in the assembly of distinct Polycomb repression complex 1 (PRC1)-related complexes. Here, we identify a protein interaction domain in BCL6 corepressor, BCOR, which binds the RING finger- and WD40-associated ubiquitin-like (RAWUL) domain of PCGF1 (NSPC1) and PCGF3 but not of PCGF2 (MEL18) or PCGF4 (BMI1). Because of the selective binding, we have named this domain PCGF Ub-like fold discriminator (PUFD). The structure of BCOR PUFD bound to PCGF1 reveals that (1) PUFD binds to the same surfaces as observed for a different Polycomb group RAWUL domain and (2) the ability of PUFD to discriminate among RAWULs stems from the identity of specific residues within these interaction surfaces. These data show the molecular basis for determining the binding preference for a PCGF homolog, which ultimately helps determine the identity of the larger PRC1-like assembly.

  9. Different positioning of the ligand-binding domain helix 12 and the F domain of the estrogen receptor accounts for functional differences between agonists and antagonists.

    PubMed Central

    Nichols, M; Rientjes, J M; Stewart, A F

    1998-01-01

    The estrogen receptor is capable of binding a diverse set of ligands that are broadly categorized as agonists or antagonists, depending on their abilities to induce or interfere with transcriptional responsiveness. We show, using a fusion protein assay for ligand-binding which does not rely on transcriptional responsiveness, that agonists and antagonists differently position the C-terminus of the ligand-binding domain (helix 12) and the F domain. Upon antagonist binding, the F domain interferes with the fusion protein activity. Mutational disruption of helix 12 alters the position of the F domain, imposing interference after agonist or antagonist binding. Genetically selected inversion mutations where only agonists, but not antagonists, induce interference are similarly reliant on helix 12 and F domain positioning. Our results demonstrate that agonists and antagonists differently position helix 12 and implicate the F domain in mechanisms of antagonist action. PMID:9451001

  10. Functional analysis of the group 4 late embryogenesis abundant proteins reveals their relevance in the adaptive response during water deficit in Arabidopsis.

    PubMed

    Olvera-Carrillo, Yadira; Campos, Francisco; Reyes, José Luis; Garciarrubio, Alejandro; Covarrubias, Alejandra A

    2010-09-01

    Late-Embryogenesis Abundant (LEA) proteins accumulate to high levels during the last stages of seed development, when desiccation tolerance is acquired, and in vegetative and reproductive tissues under water deficit, leading to the hypothesis that these proteins play a role in the adaptation of plants to this stress condition. In this work, we obtained the accumulation patterns of the Arabidopsis (Arabidopsis thaliana) group 4 LEA proteins during different developmental stages and plant organs in response to water deficit. We demonstrate that overexpression of a representative member of this group of proteins confers tolerance to severe drought in Arabidopsis plants. Moreover, we show that deficiency of LEA proteins in this group leads to susceptible phenotypes upon water limitation, during germination, or in mature plants after recovery from severe dehydration. Upon recovery from this stress condition, mutant plants showed a reduced number of floral and axillary buds when compared with wild-type plants. The lack of these proteins also correlates with a reduced seed production under optimal irrigation, supporting a role in fruit and/or seed development. A bioinformatic analysis of group 4 LEA proteins from many plant genera showed that there are two subgroups, originated through ancient gene duplication and a subsequent functional specialization. This study represents, to our knowledge, the first genetic evidence showing that one of the LEA protein groups is directly involved in the adaptive response of higher plants to water deficit, and it provides data indicating that the function of these proteins is not redundant to that of the other LEA proteins.

  11. Bradykinin antagonists and thiazolidinone derivatives as new potential anti-cancer compounds.

    PubMed

    Avdieiev, Stanislav; Gera, Lajos; Havrylyuk, Dmytro; Hodges, Robert S; Lesyk, Roman; Ribrag, Vincent; Vassetzky, Yegor; Kavsan, Vadym

    2014-08-01

    Glioblastoma (GB), the most aggressive brain tumour, and mantle cell lymphoma (MCL), a rare but very aggressive type of lymphoma, are highly resistant to chemotherapy. GB and MCL chemotherapy gives very modest results, the vast majority of patients experience recurrent disease. To find out the new treatment modality for drug-resistant GB and MCL cells, combining of bradykinin (BK) antagonists with conventional temozolomide (TMZ) treatment, and screening of thiazolidinones derivatives were the main objectives of this work. As it was revealed here, BKM-570 was the lead compound among BK antagonists under investigation (IC50 was 3.3 μM) in human GB cells. It strongly suppressed extracellular signal-regulated kinases 1/2 (ERK1/2) and protein kinase B (AKT) phosphorylation. BK antagonists did not decrease the viability of MCL cells, thus showing the cell-specific mode, while thiazolidinone derivatives, a novel group of promising anti-tumour compounds inhibited proliferation of MCL cells: IC₅₀ of ID 4526 and ID 4527 compounds were 0.27 μM and 0.16 μM, correspondingly. However, single agents are often not effective in clinic due to activation of collateral pathways in tumour cells. We demonstrated a strong synergistic effect after combinatorial treatment by BKM-570 together with TMZ that drastically increased cytotoxic action of this drug in rat and human glioma cells. Small proportion of cells was still viable after such treatment that could be explained by presence of TMZ-resistant cells in the population. It is possible to expect that the combined therapy aimed simultaneously at different elements of tumourigenesis will be more effective with lower drug concentrations than the first-line drug temozolomide used alone in clinics.

  12. Single exposure of dopamine D1 antagonist prevents and D2 antagonist attenuates methylphenidate effect

    PubMed Central

    Claussen, Catherine M; Witte, Lindsey J; Dafny, Nachum

    2015-01-01

    Methylphenidate (MPD) is a readily prescribed drug for the treatment of attention deficit hyperactivity disorder (ADHD) and moreover is used illicitly by youths for its cognitive-enhancing effects and recreation. MPD exposure in rodents elicits increased locomotor activity. Repetitive MPD exposure leads to further augmentation of their locomotor activity. This behavioral response is referred to as behavioral sensitization. Behavioral sensitization is used as an experimental marker for a drug’s ability to elicit dependence. There is evidence that dopamine (DA) is a key player in the acute and chronic MPD effect; however, the role of DA in the effects elicited by MPD is still debated. The objective of this study was to investigate the role of D1 and/or D2 DA receptors in the acute and chronic effect of MPD on locomotor activity. The study lasted for 12 consecutive days. Seven groups of male Sprague Dawley® rats were used. A single D1 or D2 antagonist was given before and after acute and chronic MPD administration. Single injection of D1 DA antagonist was able to significantly attenuate the locomotor activity when given prior to the initial MPD exposure and after repetitive MPD exposure, while the D2 DA antagonist partially attenuated the locomotor activity only when given before the second MPD exposure. The results show the role, at least in part, of the D1 DA receptor in the mechanism of behavioral sensitization, whereas the D2 DA receptor only partially modulates the response to acute and chronic MPD. PMID:27186140

  13. The Unstructured N-terminal Region of Arabidopsis Group 4 Late Embryogenesis Abundant (LEA) Proteins Is Required for Folding and for Chaperone-like Activity under Water Deficit.

    PubMed

    Cuevas-Velazquez, Cesar L; Saab-Rincón, Gloria; Reyes, José Luis; Covarrubias, Alejandra A

    2016-05-13

    Late embryogenesis abundant (LEA) proteins are a conserved group of proteins widely distributed in the plant kingdom that participate in the tolerance to water deficit of different plant species. In silico analyses indicate that most LEA proteins are structurally disordered. The structural plasticity of these proteins opens the question of whether water deficit modulates their conformation and whether these possible changes are related to their function. In this work, we characterized the secondary structure of Arabidopsis group 4 LEA proteins. We found that they are disordered in aqueous solution, with high intrinsic potential to fold into α-helix. We demonstrate that complete dehydration is not required for these proteins to sample ordered structures because milder water deficit and macromolecular crowding induce high α-helix levels in vitro, suggesting that prevalent conditions under water deficit modulate their conformation. We also show that the N-terminal region, conserved across all group 4 LEA proteins, is necessary and sufficient for conformational transitions and that their protective function is confined to this region, suggesting that folding into α-helix is required for chaperone-like activity under water limitation. We propose that these proteins can exist as different conformers, favoring functional diversity, a moonlighting property arising from their structural dynamics.

  14. A new alcohol antagonist: Phaclofen

    SciTech Connect

    Allan, A.M. ); Harris, R.A. )

    1989-01-01

    The ability of the GABA{sub B} receptor antagonist, phaclofen to alter behavioral effects of ethanol was evaluated by loss of righting reflex (sleep time), motor incoordination (bar holding), spontaneous locomotion (open field activity) and hypothermia. Pretreatment with phaclofen significantly decreased the effects of ethanol on motor incoordination, locomotor activity and hypothermia. However, phaclofen had no effect on either pentobarbital- or diazepam-induced motor incoordination. Phaclofen slightly increased the ED{sub 50} for loss of the righting reflex but did not alter either the duration of reflex loss produced by ethanol or blood ethanol levels at awakening. Our results suggest phaclofen is rapidly inactivated resulting in difficulty in observing antagonism of long duration ethanol effects. These findings suggest that the GABA{sub B} system may play a role in mediating several important actions of ethanol.

  15. The high mobility group box 1 protein of Sciaenops ocellatus is a secreted cytokine that stimulates macrophage activation.

    PubMed

    Zhao, Lu; Hu, Yong-Hua; Sun, Jin-Sheng; Sun, Li

    2011-10-01

    High mobility group box 1 protein (HMGB1) is a chromatin-associated nonhistone protein that is involved in nucleosome formation and transcriptional regulation. In addition, HMGB1 is also known as an extracellular cytokine that triggers inflammation and immune responses. HMGB1-like sequences have been identified in a number of fish species, however, the function of piscine HMGB1 remains uninvestigated. In this study, we reported the identification and analysis of SoHMGB1, an HMGB1 homologue from red drum (Sciaenops ocellatus). SoHMGB1 is 206 residues in length and contains two basic HMG boxes and a highly acidic C-terminal domain. SoHMGB1 shares 71-87% overall sequence identities with the HMGB1 counterparts from human, rat, and several fish species. Quantitative real time RT-PCR analysis showed that constitutive SoHMGB1 expression was detected in various tissues, with the lowest and highest levels found in kidney and muscle respectively. Bacterial challenge upregulated SoHMGB1 expression in head kidney (HK) and HK macrophages and induced extracellular secretion of SoHMGB1 by the activated macrophages. Recombinant SoHMGB1 (rSoHMGB1) purified from yeast exhibited no direct antimicrobial effect but was significantly stimulatory on the proliferation, activation, and bactericidal activity of HK macrophages. Taken together, these results indicate for the first time that a fish HMGB1, SoHMGB1, can function as a secreted cytokine in the event of bacterial infection and promote innate defense through the activation of macrophages.

  16. The novel inflammatory cytokine high mobility group box protein 1 (HMGB1) is expressed by human term placenta

    PubMed Central

    Holmlund, Ulrika; Wähämaa, Heidi; Bachmayer, Nora; Bremme, Katarina; Sverremark-Ekström, Eva; Palmblad, Karin

    2007-01-01

    High mobility group box protein 1 (HMGB1) was previously considered a strict nuclear protein, but lately data are accumulating on its extranuclear functions. In addition to its potent proinflammatory capacities, HMGB1 has a prominent role in a number of processes of specific interest for the placenta. Our overall aim was to investigate the expression of HMGB1 in human term placenta and elucidate a potential difference in HMGB1 expression comparing vaginal deliveries with elective Caesarean sections. In addition, placentas from normal pregnancies were compared with placentas from pregnancies complicated by pre-eclampsia. Twenty-five placentas, 12 from normal term pregnancies and 13 from pregnancies complicated by pre-eclampsia were analysed with immunohistochemistry for HMGB1 and its putative receptors; receptor for advanced glycation end-products (RAGE), Toll-like receptor 2 (TLR2) and TLR4. We present the novel finding that in addition to a strong nuclear HMGB1 expression in almost all cells in investigated placentas, an individual variation of cytoplasmic HMGB1 expression was detected in the syncytiotrophoblast covering the peripheral chorionic villi, by cells in the decidua and in amnion. Production of HMGB1 was confirmed by in situ hybridization. Although labour can be described as a controlled inflammatory-like process no differences in HMGB1 expression could be observed comparing active labour and elective Caesarean sections. However, a tendency towards a higher expression of cytoplasmic HMGB1 in the decidua from women with pre-eclampsia was demonstrated. The abundant expression of the receptors RAGE, TLR2 and TLR4 implicates a local capability to respond to HMGB1, although the precise role in the placenta remains to be elucidated. PMID:17617154

  17. Complementary Activities of TELOMERE REPEAT BINDING Proteins and Polycomb Group Complexes in Transcriptional Regulation of Target Genes[OPEN

    PubMed Central

    Hartwig, Benjamin; James, Geo Velikkakam

    2016-01-01

    In multicellular organisms, Polycomb Repressive Complex 1 (PRC1) and PRC2 repress target genes through histone modification and chromatin compaction. Arabidopsis thaliana mutants strongly compromised in the pathway cannot develop differentiated organs. LIKE HETEROCHROMATIN PROTEIN1 (LHP1) is so far the only known plant PRC1 component that directly binds to H3K27me3, the histone modification set by PRC2, and also associates genome-wide with trimethylation of lysine 27 of histone H3 (H3K27me3). Surprisingly, lhp1 mutants show relatively mild phenotypic alterations. To explain this paradox, we screened for genetic enhancers of lhp1 mutants to identify novel components repressing target genes together with, or in parallel to, LHP1. Two enhancing mutations were mapped to TELOMERE REPEAT BINDING PROTEIN1 (TRB1) and its paralog TRB3. We show that TRB1 binds to thousands of genomic sites containing telobox or related cis-elements with a significant increase of sites and strength of binding in the lhp1 background. Furthermore, in combination with lhp1, but not alone, trb1 mutants show increased transcription of LHP1 targets, such as floral meristem identity genes, which are more likely to be bound by TRB1 in the lhp1 background. By contrast, expression of a subset of LHP1-independent TRB1 target genes, many involved in primary metabolism, is decreased in the absence of TRB1 alone. Thus, TRB1 is a bivalent transcriptional modulator that maintains downregulation of Polycomb Group (PcG) target genes in lhp1 mutants, while it sustains high expression of targets that are regulated independently of PcG. PMID:26721861

  18. The High Mobility Group Protein 1 Is a Coactivator of Herpes Simplex Virus ICP4 In Vitro

    PubMed Central

    Carrozza, Michael J.; DeLuca, Neal

    1998-01-01

    ICP4 is an activator of herpes simplex virus early and late gene transcription during infection and in vitro can efficiently activate the transcription of a core promoter template containing only a TATA box and an initiator element. In this study, we noted that the extent of activation by ICP4 in vitro was highly dependent on the purity of TFIID when recombinant TFIIB, TFIIE, and TFIIF were used as sources of these factors. ICP4 efficiently activated transcription with a crude TFIID fraction. However, when immunoaffinity-purified TFIID was used in place of the less pure TFIID, ICP4 activated transcription to a significantly lesser extent. This finding indicated that the crude TFIID fraction may contain additional factors that serve as coactivators of ICP4. To test this hypothesis, the crude TFIID preparation was further fractionated by gel filtration chromatography. The TFIID that eluted from the column lacked the hypothesized coactivator activity. A fraction well separated from TFIID contained an activity that when added with the TFIID fraction resulted in higher levels of transcription in the presence ICP4. Further purification of the coactivator-containing fraction resulted in the isolation of a single 30-kDa polypeptide (p30). p30 was also shown to serve as a coactivator of ICP4 with immunoaffinity-purified TFIID; however, p30 had no effect on basal transcription. Amino acid sequence analysis revealed that p30 was the high mobility group protein 1, which has been shown to facilitate the formation of higher-order DNA-protein complexes. PMID:9658123

  19. Interchangeability of meningococcal group C conjugate vaccines with different carrier proteins in the United Kingdom infant immunisation schedule.

    PubMed

    Ladhani, Shamez N; Andrews, Nick J; Waight, Pauline; Hallis, Bassam; Matheson, Mary; England, Anna; Findlow, Helen; Bai, Xilian; Borrow, Ray; Burbidge, Polly; Pearce, Emma; Goldblatt, David; Miller, Elizabeth

    2015-01-29

    An open, non-randomised study was undertaken in England during 2011-12 to evaluate vaccine antibody responses in infants after completion of the routine primary infant immunisation schedule, which included two doses of meningococcal group C (MenC) conjugate (MCC) vaccine at 3 and 4 months. Any of the three licensed MCC vaccines could be used for either dose, depending on local availability. Healthy term infants registered at participating general practices (GPs) in Hertfordshire and Gloucestershire, UK, were recruited prospectively to provide a single blood sample four weeks after primary immunisation, which was administered by the GP surgery. Vaccination history was obtained at blood sampling. MenC serum bactericidal antibody (SBA) and IgG antibodies against Haemophilus influenzae b (Hib), pertussis toxin (PT), diphtheria toxoid (DT), tetanus toxoid (TT) and thirteen pneumococcal serotypes were analysed according to MCC vaccines received. MenC SBA responses differed significantly (P<0.001) according to MCC vaccine schedule as follows: MenC SBA geometric mean titres (GMTs) were significantly lower in infants receiving a diphtheria cross-reacting material-conjugated MCC (MCC-CRM) vaccine followed by TT-conjugated MCC (MCC-TT) vaccine (82.0; 95% CI, 39-173; n=14) compared to those receiving two MCC-CRM (418; 95% CI, 325-537; n=82), two MCC-TT (277; 95% CI, 223-344; n=79) or MCC-TT followed by MCC-CRM (553; 95% CI, 322-949; n=18). The same group also had the lowest Hib geometric mean concentrations (0.60 μg/mL, 0.27-1.34) compared to 1.85 μg/mL (1.23-2.78), 2.86 μg/mL (2.02-4.05) and 4.26 μg/mL (1.94-9.36), respectively. Our results indicate that MCC vaccines with different carrier proteins are not interchangeable. When several MCC vaccines are available, children requiring more than one dose should receive MCC vaccines with the same carrier protein or, alternatively, receive MCC-TT first wherever possible.

  20. Crystal structure of the adenosine A2A receptor bound to an antagonist reveals a potential allosteric pocket

    PubMed Central

    Sun, Bingfa; Bachhawat, Priti; Chu, Matthew Ling-Hon; Wood, Martyn; Ceska, Tom; Sands, Zara A.; Mercier, Joel; Lebon, Florence; Kobilka, Tong Sun; Kobilka, Brian K.

    2017-01-01

    The adenosine A2A receptor (A2AR) has long been implicated in cardiovascular disorders. As more selective A2AR ligands are being identified, its roles in other disorders, such as Parkinson’s disease, are starting to emerge, and A2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A2A receptor bound to compound 1 (Cmpd-1), a novel A2AR/N-methyl d-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A2A receptor with a cytochrome b562-RIL (BRIL) fusion (A2AR–BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phase diffusion. Whereas A2AR–BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1–bound A2AR–BRIL prevented formation of the lattice observed with the ZM241385–bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr91.35 and Tyr2717.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A2AR-selective compounds for the treatment of Parkinson’s disease. PMID:28167788

  1. Potent and orally efficacious benzothiazole amides as TRPV1 antagonists.

    PubMed

    Besidski, Yevgeni; Brown, William; Bylund, Johan; Dabrowski, Michael; Dautrey, Sophie; Harter, Magali; Horoszok, Lucy; Hu, Yin; Johnson, Dean; Johnstone, Shawn; Jones, Paul; Leclerc, Sandrine; Kolmodin, Karin; Kers, Inger; Labarre, Maryse; Labrecque, Denis; Laird, Jennifer; Lundström, Therese; Martino, John; Maudet, Mickaël; Munro, Alexander; Nylöf, Martin; Penwell, Andrea; Rotticci, Didier; Slaitas, Andis; Sundgren-Andersson, Anna; Svensson, Mats; Terp, Gitte; Villanueva, Huascar; Walpole, Christopher; Zemribo, Ronald; Griffin, Andrew M

    2012-10-01

    Benzothiazole amides were identified as TRPV1 antagonists from high throughput screening using recombinant human TRPV1 receptor and structure-activity relationships were explored to pinpoint key pharmacophore interactions. By increasing aqueous solubility, through the attachment of polar groups to the benzothiazole core, and enhancing metabolic stability, by blocking metabolic sites, the drug-like properties and pharmokinetic profiles of benzothiazole compounds were sufficiently optimized such that their therapeutic potential could be verified in rat pharmacological models of pain.

  2. Client Perceptions of Two Antagonist Programs.

    ERIC Educational Resources Information Center

    Capone, Thomas A.; And Others

    1980-01-01

    Reports results of a questionnaire administered to participants in an antagonist drug outpatient clinic and an antagonist drug work-release program to obtain awareness of acceptance of the program participants. Naltrexone patients recommended an alternative method of administering the drug and changing the money system to award deserving inmates…

  3. A screen for new trithorax group genes identified little imaginal discs, the Drosophila melanogaster homologue of human retinoblastoma binding protein 2.

    PubMed Central

    Gildea, J J; Lopez, R; Shearn, A

    2000-01-01

    The proteins encoded by two groups of conserved genes, the Polycomb and trithorax groups, have been proposed to maintain, at the level of chromatin structure, the expression pattern of homeotic genes during Drosophila development. To identify new members of the trithorax group, we screened a collection of deficiencies for intergenic noncomplementation with a mutation in ash1, a trithorax group gene. Five of the noncomplementing deletions uncover genes previously classified as members of the Polycomb group. This evidence suggests that there are actually three groups of genes that maintain the expression pattern of homeotic genes during Drosophila development. The products of the third group appear to be required to maintain chromatin in both transcriptionally inactive and active states. Six of the noncomplementing deficiencies uncover previously unidentified trithorax group genes. One of these deficiencies removes 25D2-3 to 26B2-5. Within this region, there are two, allelic, lethal P-insertion mutations that identify one of these new trithorax group genes. The gene has been called little imaginal discs based on the phenotype of mutant larvae. The protein encoded by the little imaginal discs gene is the Drosophila homologue of human retinoblastoma binding protein 2. PMID:11014813

  4. Indirect readout of DNA sequence by p22 repressor: roles of DNA and protein functional groups in modulating DNA conformation.

    PubMed

    Harris, Lydia-Ann; Watkins, Derrick; Williams, Loren Dean; Koudelka, Gerald B

    2013-01-09

    The repressor of bacteriophage P22 (P22R) discriminates between its various DNA binding sites by sensing the identity of non-contacted base pairs at the center of its binding site. The "indirect readout" of these non-contacted bases is apparently based on DNA's sequence-dependent conformational preferences. The structures of P22R-DNA complexes indicate that the non-contacted base pairs at the center of the binding site are in the B' state. This finding suggests that indirect readout and therefore binding site discrimination depend on P22R's ability to either sense and/or impose the B' state on the non-contacted bases of its binding sites. We show here that the affinity of binding sites for P22R depends on the tendency of the central bases to assume the B'-DNA state. Furthermore, we identify functional groups in the minor groove of the non-contacted bases as the essential modulators of indirect readout by P22R. In P22R-DNA complexes, the negatively charged E44 and E48 residues are provocatively positioned near the negatively charged DNA phosphates of the non-contacted nucleotides. The close proximity of the negatively charged groups on protein and DNA suggests that electrostatics may play a key role in the indirect readout process. Changing either of two negatively charged residues to uncharged residues eliminates the ability of P22R to impose structural changes on DNA and to recognize non-contacted base sequence. These findings suggest that these negatively charged amino acids function to force the P22R-bound DNA into the B' state and therefore play a key role in indirect readout by P22R.

  5. Cytoplasmic translocation of high-mobility group box-1 protein is induced by diabetes and high glucose in retinal pericytes

    PubMed Central

    Kim, Junghyun; Kim, Chan-Sik; Sohn, Eunjin; Kim, Jin Sook

    2016-01-01

    The aim of the present study was to assess the involvement of the high-mobility group box-1 (HMGB1) protein, receptor for advanced glycation end products (RAGE) and nuclear factor (NF)-κB signaling pathway in the development of diabetic retinopathy. Rat primary retinal pericytes were exposed to 25 mmol/l D-glucose for 48 h. Diabetic retinal vessels were prepared from streptozotocin-induced diabetic rats 12 weeks following the induction of diabetes. The expression of HMGB1 was detected using immunofluorescence staining. The expression of RAGE and the activity of NF-κB were analyzed using western blot and electrophoretic mobility shift assays, respectively. The results showed that HMGB1 was translocated to the cytoplasm of the high glucose-treated pericytes and diabetic retinal pericytes, whereas, in the control cells and the normal retinas, HMGB1 was expressed in the cell nuclei only. The expression of RAGE, a potential receptor for HMGB1, and the activity of NF-κB were also increased in the high glucose-treated pericytes, compared with the normal control cells. In addition, high glucose increased the binding of NF-κB to the RAGE promoter. These findings suggested that the cytoplasmic translocation of HMGB1 may be caused by diabetes and high glucose in retinal pericytes, and that the pathogenic role of HMGB1 may be dependent on the expression of RAGE and activation of NF-κB. PMID:27599553

  6. The expansion of thymopoiesis in neonatal mice is dependent on expression of high mobility group a 2 protein (Hmga2).

    PubMed

    Berent-Maoz, Beata; Montecino-Rodriguez, Encarnacion; Fice, Michael; Casero, David; Seet, Christopher S; Crooks, Gay M; Lowry, William; Dorshkind, Kenneth

    2015-01-01

    Cell number in the mouse thymus increases steadily during the first two weeks after birth. It then plateaus and begins to decline by seven weeks after birth. The factors governing these dramatic changes in cell production are not well understood. The data herein correlate levels of High mobility group A 2 protein (Hmga2) expression with these temporal changes in thymopoiesis. Hmga2 is expressed at high levels in murine fetal and neonatal early T cell progenitors (ETP), which are the most immature intrathymic precursors, and becomes almost undetectable in these progenitors after 5 weeks of age. Hmga2 expression is critical for the initial, exponential expansion of thymopoiesis, as Hmga2 deficient mice have a deficit of ETPs within days after birth, and total thymocyte number is repressed compared to wild type littermates. Finally, our data raise the possibility that similar events occur in humans, because Hmga2 expression is high in human fetal thymic progenitors and falls in these cells during early infancy.

  7. The Expansion of Thymopoiesis in Neonatal Mice Is Dependent on Expression of High Mobility Group A 2 Protein (Hmga2)

    PubMed Central

    Fice, Michael; Casero, David; Seet, Christopher S.; Crooks, Gay M.; Lowry, William; Dorshkind, Kenneth

    2015-01-01

    Cell number in the mouse thymus increases steadily during the first two weeks after birth. It then plateaus and begins to decline by seven weeks after birth. The factors governing these dramatic changes in cell production are not well understood. The data herein correlate levels of High mobility group A 2 protein (Hmga2) expression with these temporal changes in thymopoiesis. Hmga2 is expressed at high levels in murine fetal and neonatal early T cell progenitors (ETP), which are the most immature intrathymic precursors, and becomes almost undetectable in these progenitors after 5 weeks of age. Hmga2 expression is critical for the initial, exponential expansion of thymopoiesis, as Hmga2 deficient mice have a deficit of ETPs within days after birth, and total thymocyte number is repressed compared to wild type littermates. Finally, our data raise the possibility that similar events occur in humans, because Hmga2 expression is high in human fetal thymic progenitors and falls in these cells during early infancy. PMID:25933067

  8. A Multi-Serotype Approach Clarifies the Catabolite Control Protein A Regulon in the Major Human Pathogen Group A Streptococcus

    PubMed Central

    DebRoy, Sruti; Saldaña, Miguel; Travisany, Dante; Montano, Andrew; Galloway-Peña, Jessica; Horstmann, Nicola; Yao, Hui; González, Mauricio; Maass, Alejandro; Latorre, Mauricio; Shelburne, Samuel A.

    2016-01-01

    Catabolite control protein A (CcpA) is a highly conserved, master regulator of carbon source utilization in gram-positive bacteria, but the CcpA regulon remains ill-defined. In this study we aimed to clarify the CcpA regulon by determining the impact of CcpA-inactivation on the virulence and transcriptome of three distinct serotypes of the major human pathogen Group A Streptococcus (GAS). CcpA-inactivation significantly decreased GAS virulence in a broad array of animal challenge models consistent with the idea that CcpA is critical to gram-positive bacterial pathogenesis. Via comparative transcriptomics, we established that the GAS CcpA core regulon is enriched for highly conserved CcpA binding motifs (i.e. cre sites). Conversely, strain-specific differences in the CcpA transcriptome seems to consist primarily of affected secondary networks. Refinement of cre site composition via analysis of the core regulon facilitated development of a modified cre consensus that shows promise for improved prediction of CcpA targets in other medically relevant gram-positive pathogens. PMID:27580596

  9. Overexpression of OsEm1 encoding a group I LEA protein confers enhanced drought tolerance in rice.

    PubMed

    Yu, Jing; Lai, Yongmin; Wu, Xi; Wu, Gang; Guo, Changkui

    2016-09-16

    Drought is the greatest threat for crops, including rice. In an effort to identify rice genes responsible for drought tolerance, a drought-responsive gene OsEm1 encoding a group I LEA protein, was chosen for this study. OsEm1 was shown at vegetative stages to be responsive to various abiotic stresses, including drought, salt, cold and the hormone ABA. In this study, we generated OsEm1-overexpressing rice plants to explore the function of OsEm1 under drought conditions. Overexpression of OsEm1 increases ABA sensitivity and enhances osmotic tolerance in rice. Compared with wild type, the OsEm1-overexpressing rice plants showed enhanced plant survival ratio at the vegetative stage; moreover, over expression of OsEm1 in rice increased the expression of other LEA genes, including RAB16A, RAB16C, RAB21, and LEA3, likely protecting organ integrity against harsh environments. Interestingly, the elevated level of OsEm1 had no different phenotype compared with wild type under normal condition. Our findings suggest that OsEm1 is a positive regulator of drought tolerance and is potentially promising for engineering drought tolerance in rice.

  10. Structural, signalling and regulatory properties of the group I metabotropic glutamate receptors: prototypic family C G-protein-coupled receptors.

    PubMed Central

    Hermans, E; Challiss, R A

    2001-01-01

    In 1991 a new type of G-protein-coupled receptor (GPCR) was cloned, the type 1a metabotropic glutamate (mGlu) receptor, which, despite possessing the defining seven-transmembrane topology of the GPCR superfamily, bore little resemblance to the growing number of other cloned GPCRs. Subsequent studies have shown that there are eight mammalian mGlu receptors that, together with the calcium-sensing receptor, the GABA(B) receptor (where GABA is gamma-aminobutyric acid) and a subset of pheromone, olfactory and taste receptors, make up GPCR family C. Currently available data suggest that family C GPCRs share a number of structural, biochemical and regulatory characteristics, which differ markedly from those of the other GPCR families, most notably the rhodopsin/family A GPCRs that have been most widely studied to date. This review will focus on the group I mGlu receptors (mGlu1 and mGlu5). This subgroup of receptors is widely and differentially expressed in neuronal and glial cells within the brain, and receptor activation has been implicated in the control of an array of key signalling events, including roles in the adaptative changes needed for long-term depression or potentiation of neuronal synaptic connectivity. In addition to playing critical physiological roles within the brain, the mGlu receptors are also currently the focus of considerable attention because of their potential as drug targets for the treatment of a variety of neurological and psychiatric disorders. PMID:11672421

  11. Polycomb-Group Proteins and FLOWERING LOCUS T Maintain Commitment to Flowering in Arabidopsis thaliana[C][W][OPEN

    PubMed Central

    Müller-Xing, Ralf; Clarenz, Oliver; Pokorny, Lena; Goodrich, Justin; Schubert, Daniel

    2014-01-01

    The switch from vegetative to reproductive growth is extremely stable even if plants are only transiently exposed to environmental stimuli that trigger flowering. In the photoperiodic pathway, a mobile signal, florigen, encoded by FLOWERING LOCUS T (FT) in Arabidopsis thaliana, induces flowering. Because FT activity in leaves is not maintained after transient photoperiodic induction, the molecular basis for stable floral commitment is unclear. Here, we show that Polycomb-group (Pc-G) proteins, which mediate epigenetic gene regulation, maintain the identity of inflorescence and floral meristems after floral induction. Thus, plants with reduced Pc-G activity show a remarkable increase of cauline leaves under noninductive conditions and floral reversion when shifted from inductive to noninductive conditions. These phenotypes are almost completely suppressed by loss of FLOWERING LOCUS C (FLC) and SHORT VEGETATIVE PHASE, which both delay flowering and promote vegetative shoot identity. Upregulation of FLC in Pc-G mutants leads to a strong decrease of FT expression in inflorescences. We find that this activity of FT is needed to prevent floral reversion. Collectively, our results reveal that floral meristem identity is at least partially maintained by a daylength-independent role of FT whose expression is indirectly sustained by Pc-G activity. PMID:24920331

  12. The Polycomb Group Protein Pcgf1 Is Dispensable in Zebrafish but Involved in Early Growth and Aging

    PubMed Central

    Le Bourhis, Xuefen; Angrand, Pierre-Olivier

    2016-01-01

    Polycomb Repressive Complex (PRC) 1 regulates the control of gene expression programs via chromatin structure reorganization. Through mutual exclusion, different PCGF members generate a variety of PRC1 complexes with potentially distinct cellular functions. In this context, the molecular function of each of the PCGF family members remains elusive. The study of PCGF family member expression in zebrafish development and during caudal fin regeneration reveals that the zebrafish pcgf genes are subjected to different regulations and that all PRC1 complexes in terms of Pcgf subunit composition are not always present in the same tissues. To unveil the function of Pcgf1 in zebrafish, a mutant line was generated using the TALEN technology. Mutant pcgf1-/- fish are viable and fertile, but the growth rate at early developmental stages is reduced in absence of pcgf1 gene function and a significant number of pcgf1-/- fish show signs of premature aging. This first vertebrate model lacking Pcgf1 function shows that this Polycomb Group protein is involved in cell proliferation during early embryogenesis and establishes a link between epigenetics and aging. PMID:27442247

  13. High Mobility Group Box-1 Protein and Outcomes in Critically Ill Surgical Patients Requiring Open Abdominal Management

    PubMed Central

    Malig, Michelle S.; Jenne, Craig N.; Ball, Chad G.; Roberts, Derek J.; Xiao, Zhengwen

    2017-01-01

    Background. Previous studies assessing various cytokines in the critically ill/injured have been uninformative in terms of translating to clinical care management. Animal abdominal sepsis work suggests that enhanced intraperitoneal (IP) clearance of Damage-Associated Molecular Patterns (DAMPs) improves outcome. Thus measuring the responses of DAMPs offers alternate potential insights and a representative DAMP, High Mobility Group Box-1 protein (HMGB-1), was considered. While IP biomediators are being recognized in critical illness/trauma, HMGB-1 behaviour has not been examined in open abdomen (OA) management. Methods. A modified protocol for HMGB-1 detection was used to examine plasma/IP fluid samples from 44 critically ill/injured OA patients enrolled in a randomized controlled trial comparing two negative pressure peritoneal therapies (NPPT): Active NPPT (ANPPT) and Barker's Vacuum Pack NPPT (BVP). Samples were collected and analyzed at the time of laparotomy and at 24 and 48 hours after. Results. There were no statistically significant differences in survivor versus nonsurvivor HMGB-1 plasma or IP concentrations at baseline, 24 hours, or 48 hours. However, plasma HMGB-1 levels tended to increase continuously in the BVP cohort. Conclusions. HMGB-1 appeared to behave differently between NPPT cohorts. Further studies are needed to elucidate the relationship of HMGB-1 and outcomes in septic/injured patients. PMID:28286376

  14. FK520 interacts with the discrete intrahelical amino acids of multidrug transporter Cdr1 protein and acts as antagonist to selectively chemosensitize azole-resistant clinical isolates of Candida albicans.

    PubMed

    Nim, Shweta; Rawal, Manpreet K; Prasad, Rajendra

    2014-06-01

    FK520, a homolog of antifungal FK506, displays fungicidal synergism with azoles in Candida albicans and inhibits drug efflux mediated by ABC multidrug transporter. This study establishes the molecular basis of interaction of FK520 with Cdr1 protein, which is one of the major ABC multidrug transporters of C. albicans. For this, we have exploited an in-house library of Cdr1 protein consisting of 252 mutant variants where the entire primary structure of the two transmembrane domains comprising of 12 transmembrane helices was subjected to alanine scanning. With these mutant variants of Cdr1 protein, we could identify the critical amino acids of the transporter protein, which if replaced with alanine, not only abrogated FK520-dependent competitive inhibition of drug efflux but simultaneously decreased susceptibility to azoles. Notably, the replacement of most of the residues with alanine was inconsequential; however, there were close to 13% mutant variants, which showed abrogation of drug efflux and reversal of fungicidal synergy with azoles. Of note, all the intrahelical residues of Cdr1 protein, which abrogated inhibitor's ability to block the efflux and reversed fungicidal synergy, were common. Taken together, our results provide evidence of cross-talk of FK520 with Cdr1 by interacting with the select intrahelical residues of the protein to chemosensitize isolates of Candida.

  15. Classification of mouse VK groups based on the partial amino acid sequence to the first invariant tryptophan: impact of 14 new sequences from IgG myeloma proteins.

    PubMed

    Potter, M; Newell, J B; Rudikoff, S; Haber, E

    1982-12-01

    Fourteen new VK sequences derived from BALB/c IgG myeloma proteins were determined to the first invariant tryptophan (Trp 35). These partial sequences were compared with 65 other published VK sequences using a computer program. The 79 sequences were organized according to the length of the sequence from the amino terminus to the first invariant tryptophan (Trp 35), into seven groups (33, 34, 35, 36, 39, 40 and 41aa). A distance matrix of all 79 sequences was then computed, i.e. the number of amino acid substitutions necessary to convert one sequence to another was determined. From these data a dendrogram was constructed. Most of the VK sequences fell into clusters or closely related groups. The definition of a sequence group is arbitrary but facilitates the classification of VK proteins. We used 12 substitutions as the basis for defining a sequence group based on the known number of substitutions that are found in the VK21 proteins. By this criterion there were 18 groups in the Trp 35 dendrogram. Twelve of the 14 new sequences fell into one of these sequence groups; two formed new sequence groups. Collective amino acid sequencing is still encountering new VK structures indicating more sequences will be required to attain an accurate estimate of the total number of VK groups. Updated dendrograms can be quickly generated to include newly generated sequences.

  16. [Sulglycotide combined with H2-antagonists in the prevention of duodenal ulcer recurrence. Multicenter study].

    PubMed

    Ciaco, A; Papi, C; Capurso, L

    1992-01-01

    The aim of the present study was to evaluate the role of sulglycotide, a molecule with gastroprotective properties, in monotherapy and in association with H2-antagonists in the maintenance treatment of duodenal ulcer. The study was performed using a fully randomized experimental design. Following endoscopic confirmation, 626 patients with healed duodenal ulcer were treated for 6 months with sulglycotide 200 mg tid (293 patients) or sulglycotide + H2-antagonists (333 patients). After 2, 4 and 6 months patients underwent a clinical control whereas an endoscopic control was performed after 6 months. The cumulative percentage of recidivation was 3.6% in the sulglycotide + H2-antagonist treated group, whereas the group treated with sulglycotide alone showed a recidivation rate of 15.4% (p < 0.001). These findings suggest the utility of combined sulglycotide and H2-antagonist treatment in the maintenance therapy for duodenal ulcer.

  17. Dual regulation of fragile X mental retardation protein by group I metabotropic glutamate receptors controls translation-dependent epileptogenesis in the hippocampus.

    PubMed

    Zhao, Wangfa; Chuang, Shih-Chieh; Bianchi, Riccardo; Wong, Robert K S

    2011-01-12

    Group I metabotropic glutamate receptors (mGluRs) stimulation activates translation-dependent epileptogenesis in the hippocampus. This translation is regulated by repressors, including BC1 RNA and fragile X mental retardation protein (FMRP). Recent data indicate that group I mGluR stimulation exerts bidirectional control over FMRP level by activating translation and ubiquitin-proteasome system (UPS)-dependent proteolysis for the up- and downregulation of the protein, respectively. At present, the temporal relationship of translation and proteolysis on FMRP and their interplay for group I mGluR-mediated translation and epileptogenesis are unknown. We addressed these issues by using mouse hippocampal slices. Agonist [(S)-3,5-dihydroxyphenylglycine (DHPG)] stimulation of group I mGluRs caused a biphasic change in FMRP level. An initial decrease (within 10 min) was followed by an increase at 30 min. When slices were pretreated with translation inhibitor (anisomycin or cycloheximide), group I mGluRs elicited a sustained decrease in FMRP. This decrease was prevented by a proteasome inhibitor [Z-Leu-Leu-Leu-CHO (MG-132)]. When slices were pretreated with MG-132 alone, DHPG no longer elicited any change in FMRP. MG-132 also suppressed increase in other proteins, including postsynaptic density-95 and α-calcium/calmodulin-dependent protein kinase II, normally elicited by group I mGluR stimulation. Physiological experiments showed that proteasome inhibitor suppressed group I mGluR-induced prolonged synchronized discharges. However, proteasome inhibitor did not affect group I mGluR-induced prolonged synchronized discharges in Fmr1(-/-) preparations, where functional FMRP is absent. The results suggest that constitutive FMRP in hippocampal cells acts as a brake on group I mGluR-mediated translation and epileptogenesis. FMRP downregulation via UPS removes this brake enabling group I mGluR-mediated translation and epileptogenesis.

  18. Neuroprotection Profile of the High Affinity NMDA Receptor Antagonist Conantokin-G

    DTIC Science & Technology

    2002-01-01

    ABSTRACT Conantokin-G (Con-G or CGX-1007), a potent NR2B subunit selective NMDA receptor antagonist, was evaluated for its neuroprotective properties...protection against staurosporine-induced apoptotic injury (Pɘ.01, n = 12/group), which was linked to the NR2B subunit. For in vivo brain injury...CGX-1007), a potent NR2B subunit selective NMDA receptor antagonist, was evaluated for its neuroprotective properties in experimental models of

  19. Antianginal Actions of Beta-Adrenoceptor Antagonists

    PubMed Central

    2007-01-01

    Angina pectoris is usually the first clinical sign of underlying myocardial ischemia, which results from an imbalance between oxygen supply and oxygen demand in the heart. This report describes the pharmacology of β-adrenoceptor antagonists as it relates to the treatment of angina. The β-adrenoceptor antagonists are widely used in long-term maintenance therapy to prevent acute ischemic episodes in patients with chronic stable angina. Beta-adrenoceptor antagonists competitively inhibit the binding of endogenous catecholamines to β1-adrenoceptors in the heart. Their anti-ischemic effects are due primarily to a reduction in myocardial oxygen demand. By decreasing heart rate, myocardial contractility and afterload, β-adrenoceptor antagonists reduce myocardial workload and oxygen consumption at rest as well as during periods of exertion or stress. Predictable adverse effects include bradycardia and cardiac depression, both of which are a direct result of the blockade of cardiac β1-adrenoceptors, but adverse effects related to the central nervous system (eg, lethargy, sleep disturbances, and depression) may also be bothersome to some patients. Beta-adrenoceptor antagonists must be used cautiously in patients with diabetes mellitus, peripheral vascular disease, heart failure, and asthma or other obstructive airway diseases. Beta-adrenoceptor antagonists may be used in combination with nitrates or calcium channel blockers, which takes advantage of the diverse mechanisms of action of drugs from each pharmacologic category. Moreover, concurrent use of β-adrenoceptor antagonists may alleviate the reflex tachycardia that sometimes occurs with other antianginal agents. PMID:17998992

  20. Preparation of ultrathin, robust protein microcapsules through template-mediated interfacial reaction between amine and catechol groups.

    PubMed

    Wang, Xiaoli; Shi, Jiafu; Jiang, Zhongyi; Li, Zheng; Zhang, Wenyan; Song, Xiaokai; Ai, Qinghong; Wu, Hong

    2013-11-11

    A novel approach to the synthesis of protein microcapsules is developed through template-mediated interfacial reaction. Protein-doped CaCO3 templates are first synthetized via coprecipitation and then coated with a catechol-containing alginate (AlgDA) layer. Afterward, the templates are exposed to ethylenediamine tetraacetic acid disodium (EDTA) solution to dissolve CaCO3. During CaCO3 dissolution, the generated CO2 gas pushes protein molecules moving to the AlgDA layer, and thereby Michael addition and Schiff base reactions proceed, forming the shell of protein microcapsules. Three kinds of proteins, namely, bovine serum albumin, catalase, and protamine sulfate, are utilized. The shell thickness of microcapsule varies from 25 to 82 nm as the doping amount of protein increased from 2 to 6 mg per 66 mg CaCO3. The protein microcapsules have a robust but flexible shell and can be reversibly deformed upon exposure to osmotic pressure. The bioactivity of protein microcapsules is demonstrated through enzymatic catalysis experiments. The protein microcapsules remain about 80% enzymatic activity of the equivalent free protein. Hopefully, our approach could be extended to many other applications such as drug/gene delivery, tissue scaffolds, and catalysis due to the universality of Michael reaction and Schiff base reactions.

  1. Antagonistic coevolution accelerates molecular evolution

    PubMed Central

    Paterson, Steve; Vogwill, Tom; Buckling, Angus; Benmayor, Rebecca; Spiers, Andrew J.; Thomson, Nicholas R.; Quail, Mike; Smith, Frances; Walker, Danielle; Libberton, Ben; Fenton, Andrew; Hall, Neil; Brockhurst, Michael A.

    2013-01-01

    The Red Queen hypothesis proposes that coevolution of interacting species (such as hosts and parasites) should drive molecular evolution through continual natural selection for adaptation and counter-adaptation1–3. Although the divergence observed at some host-resistance4–6 and parasite-infectivity7–9 genes is consistent with this, the long time periods typically required to study coevolution have so far prevented any direct empirical test. Here we show, using experimental populations of the bacterium Pseudomonas fluorescens SBW25 and its viral parasite, phage Φ2 (refs 10, 11), that the rate of molecular evolution in the phage was far higher when both bacterium and phage coevolved with each other than when phage evolved against a constant host genotype. Coevolution also resulted in far greater genetic divergence between replicate populations, which was correlated with the range of hosts that coevolved phage were able to infect. Consistent with this, the most rapidly evolving phage genes under coevolution were those involved in host infection. These results demonstrate, at both the genomic and phenotypic level, that antagonistic coevolution is a cause of rapid and divergent evolution, and is likely to be a major driver of evolutionary change within species. PMID:20182425

  2. Group additivity calculation of the standard molal thermodynamic properties of aqueous amino acids, polypeptides and unfolded proteins as a function of temperature, pressure and ionization state

    NASA Astrophysics Data System (ADS)

    Dick, J. M.; Larowe, D. E.; Helgeson, H. C.

    2005-10-01

    Thermodynamic calculation of the chemical speciation of proteins and the limits of protein metastability affords a quantitative understanding of the biogeochemical constraints on the distribution of proteins within and among different organisms and chemical environments. These calculations depend on accurate determination of the ionization states and standard molal Gibbs free energies of proteins as a function of temperature and pressure, which are not generally available. Hence, to aid predictions of the standard molal thermodynamic properties of ionized proteins as a function of temperature and pressure, calculated values are given below of the standard molal thermodynamic properties at 25°C and 1 bar and the revised Helgeson-Kirkham-Flowers equations of state parameters of the structural groups comprising amino acids, polypeptides and unfolded proteins. Group additivity and correlation algorithms were used to calculate contributions by ionized and neutral sidechain and backbone groups to the standard molal Gibbs free energy (Δ G°), enthalpy (Δ H°), entropy (S°), isobaric heat capacity (C°P), volume (V°) and isothermal compressibility (κ°T) of multiple reference model compounds. Experimental values of C°P, V° and κ°T at high temperature were taken from the recent literature, which ensures an internally consistent revision of the thermodynamic properties and equations of state parameters of the sidechain and backbone groups of proteins, as well as organic groups. As a result, Δ G°, Δ H°, S° C°P, V° and κ°T of unfolded proteins in any ionization state can be calculated up to T~-300°C and P~-5000 bars. In addition, the ionization states of unfolded proteins as a function of not only pH, but also temperature and pressure can be calculated by taking account of the degree of ionization of the sidechain and backbone groups present in the sequence. Calculations of this kind represent a first step in the prediction of chemical affinities of many

  3. Polycomb Group Proteins Bind an engrailed PRE in Both the “ON” and “OFF” Transcriptional States of engrailed

    PubMed Central

    Langlais, Kristofor K.; Brown, J. Lesley; Kassis, Judith A.

    2012-01-01

    Polycomb group (PcG) and trithorax Group (trxG) proteins maintain the “OFF” and “ON” transcriptional states of HOX genes and other targets by modulation of chromatin structure. In Drosophila, PcG proteins are bound to DNA fragments called Polycomb group response elements (PREs). The prevalent model holds that PcG proteins bind PREs only in cells where the target gene is “OFF”. Another model posits that transcription through PREs disrupts associated PcG complexes, contributing to the establishment of the “ON” transcriptional state. We tested these two models at the PcG target gene engrailed. engrailed exists in a gene complex with invected, which together have 4 well-characterized PREs. Our data show that these PREs are not transcribed in embryos or larvae. We also examined whether PcG proteins are bound to an engrailed PRE in cells where engrailed is transcribed. By FLAG-tagging PcG proteins and expressing them specifically where engrailed is “ON” or “OFF”, we determined that components of three major PcG protein complexes are present at an engrailed PRE in both the “ON” and “OFF” transcriptional states in larval tissues. These results show that PcG binding per se does not determine the transcriptional state of engrailed. PMID:23139817

  4. Molecular cloning, genomic structure, and tissue distribution of EW135, a novel chicken egg white protein with group B scavenger receptor cysteine-rich domains.

    PubMed

    Yoo, Whayoung; Nakamura, Tomohiro; Asanuma, Hideki; Matsushita, Misao

    2013-11-01

    Approximately 80 proteins are reported to be present in chicken egg white. The major function of egg white proteins isolated so far is to defend the egg yolk against infections. We recently isolated a novel protein termed EW135 from chicken egg white. In this paper, we have determined the complete amino acid sequence of EW135 based on cDNA cloning. EW135 consists of 970 amino acids with a putative signal peptide of 17 amino acids. It is composed exclusively of tandem repeats of nine group B scavenger receptor cysteine-rich (SRCR) domains separated by eight seven-amino acid peptides. The features of consensus sequences found in the group B SRCR domain were well conserved in EW135. The EW135 gene consists of putative 11 exons, with each SRCR domain being encoded by a single exon. Reverse transcription PCR showed that EW135 is expressed in only the oviduct among the 11 types of tissues tested. EW135 is a second soluble protein belonging to the group B SRCR domain superfamily identified in chickens. One of the important functions of proteins belonging to the group B SRCR domain superfamily is to recognize pathogens in innate immunity. It is, therefore, conceivable that EW135 could be involved in host defense in egg white.

  5. Effects of high mobility group protein box 1 and toll like receptor 4 pathway on warts caused by human papillomavirus.

    PubMed

    Weng, Hui; Liu, Hongbo; Deng, Yunhua; Xie, Yuyan; Shen, Guanxin

    2014-10-01

    Accumulative evidence has demonstrated that inflammation has an important role in human papillomavirus (HPV) oncogenicity. However, the effects of high mobility group protein box 1 (HMGB1)-toll like receptor 4 (TLR4) signaling pathway associated inflammation on epidermal warts caused by HPV remain unclear. The present study investigated the HMGB1, TLR4 and nuclear factor-κB p65 expression in condyloma acuminatum (CA) and verruca vulgaris (VV). Immunohistochemistry and western blot analysis revealed that p65 expression in epithelial nuclei in VV and CA was significantly higher than in normal skin (NS) (P<0.01), and p65 in CA was higher than in VV but this difference was not significant. The level of extracellular HMGB1 increased significantly and progressively from NS to VV to CA (P<0.05). The level of TLR4 on the surface of epithelial membranes in the CA samples was significantly higher than in NS (P<0.01), and TLR4 in VV samples was significantly lower than in NS (P<0.01). There was a positive correlation between p65 expression in the epithelial nuclei and HMGB1 in the epithelial intercellular spaces (r=0.5199, P<0.01). These findings indicate that inflammation is intensified in warts caused by HPV. HMGB1-TLR4 pathway-associated inflammation may therefore have a pivotal role in CA. HMGB1, rather than TLR4, may be a vital mediator of inflammation in VV. Therapies targeting HMGB1 may be a potential strategy for the treatment of HPV-associated warts.

  6. Impact of serum high-mobility group box 1 protein elevation on oxygenation impairment after thoracic aortic aneurysm repair.

    PubMed

    Kohno, Takashi; Anzai, Toshihisa; Shimizu, Hideyuki; Kaneko, Hidehiro; Sugano, Yasuo; Yamada, Shingo; Yoshikawa, Tsutomu; Ishizaka, Akitoshi; Yozu, Ryohei; Ogawa, Satoshi

    2011-05-01

    High-mobility group box 1 protein (HMGB1) is a late mediator of inflammatory responses that can cause acute lung injury. We examined the significance of serum HMGB1 elevation in the development of systemic inflammatory response syndrome (SIRS) and lung oxygenation impairment (LOI) after thoracic aortic aneurysm (TAA) repair. Serial measurements of the serum HMGB1 level and SIRS score for 7 days after surgery were determined in 20 patients with TAA who underwent surgical repair. Arterial oxygen tension was measured serially for at least 4 days after surgery, and LOI was defined as the lowest PaO(2)/FiO(2) ratio ≤ 200 mmHg. The serum HMGB1 level was markedly increased after surgery, peaking on day 2, and remained significantly elevated on day 7. Peak HMGB1 level positively correlated with SIRS duration and the cumulative SIRS score during postoperative days 1-7 (P = 0.0013 and P = 0.0004, respectively). Peak HMGB1 level and cumulative SIRS score were higher in patients with LOI than in those without (P = 0.01 and P = 0.044, respectively). Peak HMGB1 level was negatively correlated with the lowest PaO(2)/FiO(2) ratio (P = 0.0077) and positively correlated with postoperative length of hospitalization (P = 0.042). A greater serum HMGB1 elevation after TAA repair was associated with more severe SIRS and a higher incidence of LOI. HMGB1 might play a key role in the pathogenesis of SIRS and LOI after surgical TAA repair.

  7. Proinflammatory effect of high-mobility group protein B1 on keratinocytes: an autocrine mechanism underlying psoriasis development.

    PubMed

    Zhang, Weigang; Guo, Sen; Li, Bing; Liu, Lin; Ge, Rui; Cao, Tianyu; Wang, Huina; Gao, Tianwen; Wang, Gang; Li, Chunying

    2017-02-01

    Psoriasis is an autoimmune skin disease, in which keratinocytes play a crucial pathogenic role. High-mobility group protein B1 (HMGB1) is an inflammatory factor that can be released from keratinocyte nuclei in psoriatic lesions. We aimed to investigate the proinflammatory effect of HMGB1 on keratinocytes and the contribution of HMGB1 to psoriasis development. Normal human keratinocytes were treated with recombinant human HMGB1, and the production of inflammatory factors and the intermediary signalling pathways were examined. Furthermore, the imiquimod-induced psoriasis-like mouse model was used to investigate the role of HMGB1 in psoriasis development in vivo. A total of 11 inflammatory factors were shown to be upregulated by HMGB1 in keratinocytes, among which interleukin (IL)-18 showed the greatest change. We then found that activation of the nuclear factor-κB signalling pathway and inflammasomes accounted for HMGB1-induced IL-18 expression and secretion. Moreover, HMGB1 and downstream IL-18 contributed to the development of psoriasiform dermatitis in the imiquimod-treated mice. In addition, T-helper 17 immune response in the psoriasis-like mouse model could be inhibited by both HMGB1 and IL-18 blockade. Our findings indicate that HMGB1 secreted from keratinocytes can facilitate the production and secretion of inflammatory factors such as IL-18 in keratinocytes in an autocrine way, thus promoting the development of psoriasis. Blocking the proinflammatory function of the HMGB1-IL-18 axis may be useful for psoriasis treatment in the future. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  8. Antagonists of the kappa opioid receptor.

    PubMed

    Urbano, Mariangela; Guerrero, Miguel; Rosen, Hugh; Roberts, Edward

    2014-05-01

    The research community has increasingly focused on the development of OPRK antagonists as pharmacotherapies for the treatment of depression, anxiety, addictive disorders and other psychiatric conditions produced or exacerbated by stress. Short-acting OPRK antagonists have been recently developed as a potential improvement over long-acting prototypic ligands including nor-BNI and JDTic. Remarkably the short-acting LY2456302 is undergoing phase II clinical trials for the augmentation of the antidepressant therapy in treatment-resistant depression. This Letter reviews relevant chemical and pharmacological advances in the identification and development of OPRK antagonists.

  9. μ Opioid receptor: novel antagonists and structural modeling

    PubMed Central

    Kaserer, Teresa; Lantero, Aquilino; Schmidhammer, Helmut; Spetea, Mariana; Schuster, Daniela

    2016-01-01

    The μ opioid receptor (MOR) is a prominent member of the G protein-coupled receptor family and the molecular target of morphine and other opioid drugs. Despite the long tradition of MOR-targeting drugs, still little is known about the ligand-receptor interactions and structure-function relationships underlying the distinct biological effects upon receptor activation or inhibition. With the resolved crystal structure of the β-funaltrexamine-MOR complex, we aimed at the discovery of novel agonists and antagonists using virtual screening tools, i.e. docking, pharmacophore- and shape-based modeling. We suggest important molecular interactions, which active molecules share and distinguish agonists and antagonists. These results allowed for the generation of theoretically validated in silico workflows that were employed for prospective virtual screening. Out of 18 virtual hits evaluated in in vitro pharmacological assays, three displayed antagonist activity and the most active compound significantly inhibited morphine-induced antinociception. The new identified chemotypes hold promise for further development into neurochemical tools for studying the MOR or as potential therapeutic lead candidates. PMID:26888328

  10. μ Opioid receptor: novel antagonists and structural modeling

    NASA Astrophysics Data System (ADS)

    Kaserer, Teresa; Lantero, Aquilino; Schmidhammer, Helmut; Spetea, Mariana; Schuster, Daniela

    2016-02-01

    The μ opioid receptor (MOR) is a prominent member of the G protein-coupled receptor family and the molecular target of morphine and other opioid drugs. Despite the long tradition of MOR-targeting drugs, still little is known about the ligand-receptor interactions and structure-function relationships underlying the distinct biological effects upon receptor activation or inhibition. With the resolved crystal structure of the β-funaltrexamine-MOR complex, we aimed at the discovery of novel agonists and antagonists using virtual screening tools, i.e. docking, pharmacophore- and shape-based modeling. We suggest important molecular interactions, which active molecules share and distinguish agonists and antagonists. These results allowed for the generation of theoretically validated in silico workflows that were employed for prospective virtual screening. Out of 18 virtual hits evaluated in in vitro pharmacological assays, three displayed antagonist activity and the most active compound significantly inhibited morphine-induced antinociception. The new identified chemotypes hold promise for further development into neurochemical tools for studying the MOR or as potential therapeutic lead candidates.

  11. Selection and differentiation of Bacillus spp. Antagonistic to Fusarium oxysporum f.sp. lycopersici and Al