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Sample records for polymerization promoting protein

  1. Tubulin Binding and Polymerization Promoting Properties of Tubulin Polymerization Promoting Proteins Are Evolutionarily Conserved.

    PubMed

    Oláh, Judit; Szénási, Tibor; Szabó, Adél; Kovács, Kinga; Lőw, Péter; Štifanić, Mauro; Orosz, Ferenc

    2017-02-21

    Tubulin polymerization promoting proteins (TPPPs) constitute a eukaryotic protein family. There are three TPPP paralogs in the human genome, denoted as TPPP1-TPPP3. TPPP1 and TPPP3 are intrinsically unstructured proteins (IUPs) that bind and polymerize tubulin and stabilize microtubules, but TPPP2 does not. Vertebrate TPPPs originated from the ancient invertebrate TPPP by two-round whole-genome duplication; thus, whether the tubulin/microtubule binding function of TPPP1 and TPPP3 is a newly acquired property or was present in the invertebrate orthologs (generally one TPPP per species) has been an open question. To answer this question, we investigated a TPPP from a simple and early branching animal, the sponge Suberites domuncula. Bioinformatics, biochemical, immunochemical, spectroscopic, and electron microscopic data showed that the properties of the sponge protein correspond to those of TPPP1; namely, it is an IUP that strongly binds tubulin and induces its polymerization, proving that these features of animal TPPPs have been evolutionarily conserved.

  2. Protein specific polymeric immunomicrospheres

    NASA Technical Reports Server (NTRS)

    Rembaum, Alan (Inventor); Yen, Shiao-Ping S. (Inventor); Dreyer, William J. (Inventor)

    1980-01-01

    Small, round, bio-compatible microspheres capable of covalently bonding proteins and having a uniform diameter below about 3500 A are prepared by substantially instantaneously initiating polymerization of an aqueous emulsion containing no more than 35% total monomer including an acrylic monomer substituted with a covalently bondable group such as hydroxyl, amino or carboxyl and a minor amount of a cross-linking agent.

  3. Direct polymerization of proteins.

    PubMed

    Albayrak, Cem; Swartz, James R

    2014-06-20

    We report the synthesis of active polymers of superfolder green fluorescent protein (sfGFP) in one step using Click chemistry. Up to six copies of the non-natural amino acids (nnAAs) p-azido-l-phenylalanine (pAzF) or p-propargyloxy-l-phenylalanine (pPaF) were site-specifically inserted into sfGFP by cell-free protein synthesis (CFPS). sfGFP containing two or three copies of these nnAAs were coupled by copper-catalyzed azide-alkyne cycloaddition to synthesize linear or branched protein polymers, respectively. The protein polymers retained ≥63% of their specific activity (i.e., fluorescence) after coupling. Polymerization of a concentrated solution of triply substituted sfGFP resulted in fluorescent macromolecular particles. Our method can be generalized to synthesize polymers of a protein or copolymers of any two or more proteins, and the conjugation sites can be determined exactly by standard genetic manipulation. Polymers of proteins and small molecules can also be created with this technology to make a new class of scaffolds or biomaterials.

  4. Natively unfolded tubulin polymerization promoting protein TPPP/p25 is a common marker of alpha-synucleinopathies.

    PubMed

    Kovács, Gábor G; László, Lajos; Kovács, János; Jensen, Poul Henning; Lindersson, Evo; Botond, Gergo; Molnár, Tamás; Perczel, András; Hudecz, Ferenc; Mezo, Gábor; Erdei, Anna; Tirián, László; Lehotzky, Attila; Gelpi, Ellen; Budka, Herbert; Ovádi, Judit

    2004-11-01

    The novel basic, heat-stable tubulin polymerization promoting protein TPPP/p25 is associated with microtubules in vitro and can induce the formation of aberrant microtubule assemblies. We show by 1H-NMR spectroscopy that TPPP/p25 is natively unfolded. Antisera against peptide 186GKGKAGRVDLVDESG200NH2 (186-200) are highly specific to TPPP/p25. Immunohistochemistry and confocal microscopy demonstrates that TPPP/p25 is enriched in filamentous alpha-synuclein bearing Lewy bodies of Parkinson's (PD) and diffuse Lewy body disease (DLBD), as well as glial inclusions of multiple system atrophy (MSA). There is a correlation between TPPP/p25 and alpha-synuclein immunoreactivity in Western blot. In contrast, TPPP/p25 is not associated with abnormally phosphorylated tau in various inclusions of Pick's disease (PiD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). However, electron microscopy confirms clusters of TPPP/p25 immunoreactivity along filaments of unstructured but not compact neurofibrillary tangles in Alzheimer's disease (AD). TPPP/p25 seems to be a novel marker of alpha-synucleinopathies.

  5. Tubulin polymerization promoting protein (TPPP/p25) as a marker for oligodendroglial changes in multiple sclerosis.

    PubMed

    Höftberger, Romana; Fink, Stephanie; Aboul-Enein, Fahmy; Botond, Gergö; Olah, Judit; Berki, Timea; Ovadi, Judit; Lassmann, Hans; Budka, Herbert; Kovacs, Gabor G

    2010-11-15

    Multiple sclerosis (MS) is an idiopathic chronic inflammatory demyelinating disease of the central nervous system with variable extent of remyelination. Remyelination originates from oligodendrocyte (OG) precursor cells, which migrate and differentiate into mature OG. Tubulin polymerization promoting protein (TPPP/p25) is located in mature OG and aggregates in oligodendroglial cytoplasmic inclusions in multiple system atrophy. We developed a novel monoclonal anti-TPPP/p25 antibody to quantify OG in different subtypes and disease stages of MS, and possible degenerative changes in OG. We evaluated autopsy material from 25 MS cases, including acute, primary progressive, secondary progressive, relapsing remitting MS, and five controls. Demyelinated lesions revealed loss of TPPP/p25-positive OG within the plaques. In remyelination, TPPP/p25 was first expressed in OG cytoplasms and later became positive in myelin sheaths. We observed increased numbers of TPPP/p25 immunoreactive OG in the normal appearing white matter (NAWM) in MS patients. In MS cases, the cytoplasmic area of TPPP/p25 immunoreactivity in the OG was higher in the periplaque area when compared with NAWM and the plaque, and TPPP/p25 immunoreactive OG cytoplasmic area inversely correlated with the disease duration. There was a lack of phospho-TDP-43, phospho-tau, α-synuclein, and ubiquitin immunoreactivity in OG with enlarged cytoplasm. Our data suggest impaired differentiation, migration, and activation capacity of OG in later disease stages of MS. Upregulation of TPPP/p25 in the periplaque white matter OG without evidence for inclusion body formation might reflect an activation state. Distinct and increased expression of TPPP/p25 in MS renders it a potential prognostic and diagnostic marker of MS.

  6. Transglutaminase polymerization of peanut proteins.

    PubMed

    Clare, D A; Gharst, G; Sanders, T H

    2007-01-24

    Transglutaminase promotes protein cross-linking reactions through an acyl transferase mechanism involving protein-bound glutaminyl residues and primary amines including the epsilon-amino group of lysine residues in soy, myosin, gluten, oat globulin, casein, and whey. Herein, we present a first report of exogenous transglutaminase catalysis of several peanut protein fractions, including purified Ara h 1. In most cases, SDS-PAGE banding patterns revealed the formation of high molecular weight polymers while catalysis of Ara h 1 resulted in distinct dimer formation. Cross-linking effects were accomplished in the presence and absence of the reducing reagent, dithiothreitol. Ortho-phthaldialdehyde assays, used to quantify the degree of polymerization, indicated approximately 21% and approximately 30% coupling over a similar time interval, using either cold hexane extracted peanut protein fractions or lightly roasted flour dispersions, respectively. Rheological measurements established that transglutaminase-modified peanut extracts exhibited lowered viscosity readings compared to nontreated dispersions. Peanut protein polymers and glycoprotein conjugates, created by covalent linkage between protein substrates and monosaccharide amino sugars, exhibited similar IgE binding activity, compared to control solutions. These results suggested that potential allergic responses were not enhanced after enzymatic modification. Ultimately, these approaches may provide novel peanut-based food ingredients with unique functional characteristics for expanded applications within the world marketplace.

  7. Caspase-11 and caspase-1 differentially modulate actin polymerization via RhoA and Slingshot proteins to promote bacterial clearance

    PubMed Central

    Caution, Kyle; Gavrilin, Mikhail A.; Tazi, Mia; Kanneganti, Apurva; Layman, Daniel; Hoque, Sheshadri; Krause, Kathrin; Amer, Amal O.

    2015-01-01

    Inflammasomes are multiprotein complexes that include members of the NOD-like receptor family and caspase-1. Caspase-1 is required for the fusion of the Legionella vacuole with lysosomes. Caspase-11, independently of the inflammasome, also promotes phagolysosomal fusion. However, it is unclear how these proteases alter intracellular trafficking. Here, we show that caspase-11 and caspase-1 function in opposing manners to phosphorylate and dephosphorylate cofilin, respectively upon infection with Legionella. Caspase-11 targets cofilin via the RhoA GTPase, whereas caspase-1 engages the Slingshot phosphatase. The absence of either caspase-11 or caspase-1 maintains actin in the polymerized or depolymerized form, respectively and averts the fusion of pathogen-containing vacuoles with lysosomes. Therefore, caspase-11 and caspase-1 converge on the actin machinery with opposing effects to promote vesicular trafficking. PMID:26686473

  8. C-Terminal Region of MAP7 Domain Containing Protein 3 (MAP7D3) Promotes Microtubule Polymerization by Binding at the C-Terminal Tail of Tubulin

    PubMed Central

    Yadav, Saroj; Verma, Paul J.; Panda, Dulal

    2014-01-01

    MAP7 domain containing protein 3 (MAP7D3), a newly identified microtubule associated protein, has been shown to promote microtubule assembly and stability. Its microtubule binding region has been reported to consist of two coiled coil motifs located at the N-terminus. It possesses a MAP7 domain near the C-terminus and belongs to the microtubule associated protein 7 (MAP7) family. The MAP7 domain of MAP7 protein has been shown to bind to kinesin-1; however, the role of MAP7 domain in MAP7D3 remains unknown. Based on the bioinformatics analysis of MAP7D3, we hypothesized that the MAP7 domain of MAP7D3 may have microtubule binding activity. Indeed, we found that MAP7 domain of MAP7D3 bound to microtubules as well as enhanced the assembly of microtubules in vitro. Interestingly, a longer fragment MDCT that contained the MAP7 domain (MD) with the C-terminal tail (CT) of the protein promoted microtubule polymerization to a greater extent than MD and CT individually. MDCT stabilized microtubules against dilution induced disassembly. MDCT bound to reconstituted microtubules with an apparent dissociation constant of 3.0±0.5 µM. An immunostaining experiment showed that MDCT localized along the length of the preassembled microtubules. Competition experiments with tau indicated that MDCT shares its binding site on microtubules with tau. Further, we present evidence indicating that MDCT binds to the C-terminal tail of tubulin. In addition, MDCT could bind to tubulin in HeLa cell extract. Here, we report a microtubule binding region in the C-terminal region of MAP7D3 that may have a role in regulating microtubule assembly dynamics. PMID:24927501

  9. Knockdown of Tubulin Polymerization Promoting Protein Family Member 3 Suppresses Proliferation and Induces Apoptosis in Non-Small-Cell Lung Cancer

    PubMed Central

    Li, Yintao; Xu, Yali; Ye, Kuanping; Wu, Nan; Li, Junfeng; Liu, Naijia; He, Min; Lu, Bin; Zhou, Wenbai; Hu, Renming

    2016-01-01

    Our previous studies demonstrated that depletion of tubulin polymerization promoting protein family member 3 (TPPP3) inhibits proliferation and induces apoptosis of HeLa cells. However, the expression and roles of TPPP3 in cancers remain largely unknown. In this study, we investigated the expression of TPPP3 in clinicopathological correlations in non-small-cell lung cancer (NSCLC) samples by immunohistochemistry. TPPP3 expression was significantly upregulated in NSCLC tissues, and high TPPP3 expression was positively associated with tumor size, lymph node metastasis, clinical stage, and poor survival. Furthermore, knockdown of TPPP3 by shRNA significantly inhibited cell proliferation and induced cell apoptosis and cell cycle arrest in vitro. In addition, depletion of TPPP3 inhibited lung cancer growth in vivo in the xenografts of H1299 cells; this effect was accompanied by the suppression of Ki67 expression. Our data suggested that TPPP3 might act as an oncogene in NSCLC. TPPP3 warrants consideration as a therapeutic candidate with anti-tumor potential. PMID:27390593

  10. Tubulin polymerization promoting protein 1 (Tppp1) phosphorylation by Rho-associated coiled-coil kinase (rock) and cyclin-dependent kinase 1 (Cdk1) inhibits microtubule dynamics to increase cell proliferation.

    PubMed

    Schofield, Alice V; Gamell, Cristina; Suryadinata, Randy; Sarcevic, Boris; Bernard, Ora

    2013-03-15

    Tubulin polymerization promoting protein 1 (Tppp1) regulates microtubule (MT) dynamics via promoting MT polymerization and inhibiting histone deacetylase 6 (Hdac6) activity to increase MT acetylation. Our results reveal that as a consequence, Tppp1 inhibits cell proliferation by delaying the G1/S-phase and the mitosis to G1-phase transitions. We show that phosphorylation of Tppp1 by Rho-associated coiled-coil kinase (Rock) prevents its Hdac6 inhibitory activity to enable cells to enter S-phase. Whereas, our analysis of the role of Tppp1 during mitosis revealed that inhibition of its MT polymerizing and Hdac6 regulatory activities were necessary for cells to re-enter the G1-phase. During this investigation, we also discovered that Tppp1 is a novel Cyclin B/Cdk1 (cyclin-dependent kinase) substrate and that Cdk phosphorylation of Tppp1 inhibits its MT polymerizing activity. Overall, our results show that dual Rock and Cdk phosphorylation of Tppp1 inhibits its regulation of the cell cycle to increase cell proliferation.

  11. Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization*

    PubMed Central

    Hien, Tran Thi; Turczyńska, Karolina M.; Dahan, Diana; Ekman, Mari; Grossi, Mario; Sjögren, Johan; Nilsson, Johan; Braun, Thomas; Boettger, Thomas; Garcia-Vaz, Eliana; Stenkula, Karin; Swärd, Karl; Gomez, Maria F.; Albinsson, Sebastian

    2016-01-01

    Both type 1 and type 2 diabetes are associated with increased risk of cardiovascular disease. This is in part attributed to the effects of hyperglycemia on vascular endothelial and smooth muscle cells, but the underlying mechanisms are not fully understood. In diabetic animal models, hyperglycemia results in hypercontractility of vascular smooth muscle possibly due to increased activation of Rho-kinase. The aim of the present study was to investigate the regulation of contractile smooth muscle markers by glucose and to determine the signaling pathways that are activated by hyperglycemia in smooth muscle cells. Microarray, quantitative PCR, and Western blot analyses revealed that both mRNA and protein expression of contractile smooth muscle markers were increased in isolated smooth muscle cells cultured under high compared with low glucose conditions. This effect was also observed in hyperglycemic Akita mice and in diabetic patients. Elevated glucose activated the protein kinase C and Rho/Rho-kinase signaling pathways and stimulated actin polymerization. Glucose-induced expression of contractile smooth muscle markers in cultured cells could be partially or completely repressed by inhibitors of advanced glycation end products, L-type calcium channels, protein kinase C, Rho-kinase, actin polymerization, and myocardin-related transcription factors. Furthermore, genetic ablation of the miR-143/145 cluster prevented the effects of glucose on smooth muscle marker expression. In conclusion, these data demonstrate a possible link between hyperglycemia and vascular disease states associated with smooth muscle contractility. PMID:26683376

  12. Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization.

    PubMed

    Hien, Tran Thi; Turczyńska, Karolina M; Dahan, Diana; Ekman, Mari; Grossi, Mario; Sjögren, Johan; Nilsson, Johan; Braun, Thomas; Boettger, Thomas; Garcia-Vaz, Eliana; Stenkula, Karin; Swärd, Karl; Gomez, Maria F; Albinsson, Sebastian

    2016-02-12

    Both type 1 and type 2 diabetes are associated with increased risk of cardiovascular disease. This is in part attributed to the effects of hyperglycemia on vascular endothelial and smooth muscle cells, but the underlying mechanisms are not fully understood. In diabetic animal models, hyperglycemia results in hypercontractility of vascular smooth muscle possibly due to increased activation of Rho-kinase. The aim of the present study was to investigate the regulation of contractile smooth muscle markers by glucose and to determine the signaling pathways that are activated by hyperglycemia in smooth muscle cells. Microarray, quantitative PCR, and Western blot analyses revealed that both mRNA and protein expression of contractile smooth muscle markers were increased in isolated smooth muscle cells cultured under high compared with low glucose conditions. This effect was also observed in hyperglycemic Akita mice and in diabetic patients. Elevated glucose activated the protein kinase C and Rho/Rho-kinase signaling pathways and stimulated actin polymerization. Glucose-induced expression of contractile smooth muscle markers in cultured cells could be partially or completely repressed by inhibitors of advanced glycation end products, L-type calcium channels, protein kinase C, Rho-kinase, actin polymerization, and myocardin-related transcription factors. Furthermore, genetic ablation of the miR-143/145 cluster prevented the effects of glucose on smooth muscle marker expression. In conclusion, these data demonstrate a possible link between hyperglycemia and vascular disease states associated with smooth muscle contractility. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Degradable PEGylated Protein Conjugates Utilizing RAFT Polymerization.

    PubMed

    Decker, Caitlin G; Maynard, Heather D

    2015-04-01

    Poly(ethylene glycol) (PEG)-protein therapeutics exhibit enhanced pharmacokinetics, but have drawbacks including decreased protein activities and polymer accumulation in the body. Therefore a major aim for second-generation polymer therapeutics is to introduce degradability into the backbone. Herein we describe the synthesis of poly(poly(ethylene glycol methyl ether methacrylate)) (pPEGMA) degradable polymers with protein-reactive end-groups via reversible addition-fragmentation chain transfer (RAFT) polymerization, and the subsequent covalent attachment to lysozyme through a reducible disulfide linkage. RAFT copolymerization of cyclic ketene acetal (CKA) monomer 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) with PEGMA yielded two polymers with number-average molecular weight (Mn ) (GPC) of 10.9 and 20.9 kDa and molecular weight dispersities (Ð) of 1.34 and 1.71, respectively. Hydrolytic degradation of the polymers was analyzed by (1)H-NMR and GPC under basic and acidic conditions. The reversible covalent attachment of these polymers to lysozyme, as well as the hydrolytic and reductive cleavage of the polymer from the protein, was analyzed by gel electrophoresis and mass spectrometry. Following reductive cleavage of the polymer, an increase in activity was observed for both conjugates, with the released protein having full activity. This represents a method to prepare PEGylated proteins, where the polymer is readily cleaved from the protein and the main chain of the polymer is degradable.

  14. Relocation of p25α/tubulin polymerization promoting protein from the nucleus to the perinuclear cytoplasm in the oligodendroglia of sporadic and COQ2 mutant multiple system atrophy.

    PubMed

    Ota, Kiyobumi; Obayashi, Masato; Ozaki, Kokoro; Ichinose, Shizuko; Kakita, Akiyoshi; Tada, Mari; Takahashi, Hitoshi; Ando, Noboru; Eishi, Yoshinobu; Mizusawa, Hidehiro; Ishikawa, Kinya

    2014-09-11

    p25α/tubulin polymerization promoting protein (TPPP) is an oligodendroglial protein that plays crucial roles including myelination, and the stabilization of microtubules. In multiple system atrophy (MSA), TPPP is suggested to relocate from the myelin sheath to the oligodendroglial cell body, before the formation of glial cytoplasmic inclusions (GCIs), the pathologic hallmark of MSA. However, much is left unknown about the re-distribution of TPPP in MSA. We generated new antibodies against the N- and C-terminus of TPPP, and analyzed control and MSA brains, including the brain of a familial MSA patient carrying homozygous mutations in the coenzyme Q2 gene (COQ2). In control brain tissues, TPPP was localized not only in the cytoplasmic component of the oligodendroglia including perinuclear cytoplasm and peripheral processes in the white matter, but also in the nucleus of a fraction (62.4%) of oligodendroglial cells. Immunoelectron microscopic analysis showed TPPP in the nucleus and mitochondrial membrane of normal oligodendroglia, while western blot also supported its nuclear and mitochondrial existence. In MSA, the prevalence of nuclear TPPP was 48.6% in the oligodendroglia lacking GCIs, whereas it was further decreased to 19.6% in the oligodendroglia with phosphorylated α-synuclein (pα-syn)-positive GCIs, both showing a significant decrease compared to controls (62.4%). In contrast, TPPP accumulated in the perinuclear cytoplasm where mitochondrial membrane (TOM20 and cytochrome C) and fission (DRP1) proteins were often immunoreactive. We conclude that in MSA-oligodendroglia, TPPP is reduced, not only in the peripheral cytoplasm, but also in the nucleus and relocated to the perinuclear cytoplasm.

  15. Protein Kinase D Controls Actin Polymerization and Cell Motility through Phosphorylation of Cortactin*

    PubMed Central

    Eiseler, Tim; Hausser, Angelika; De Kimpe, Line; Van Lint, Johan; Pfizenmaier, Klaus

    2010-01-01

    We here identify protein kinase D (PKD) as an upstream regulator of the F-actin-binding protein cortactin and the Arp actin polymerization machinery. PKD phosphorylates cortactin in vitro and in vivo at serine 298 thereby generating a 14-3-3 binding motif. In vitro, a phosphorylation-deficient cortactin-S298A protein accelerated VCA-Arp-cortactin-mediated synergistic actin polymerization and showed reduced F-actin binding, indicative of enhanced turnover of nucleation complexes. In vivo, cortactin co-localized with the nucleation promoting factor WAVE2, essential for lamellipodia extension, in the actin polymerization zone in Heregulin-treated MCF-7 cells. Using a 3-dye FRET-based approach we further demonstrate that WAVE2-Arp and cortactin prominently interact at these structures. Accordingly, cortactin-S298A significantly enhanced lamellipodia extension and directed cell migration. Our data thus unravel a previously unrecognized mechanism by which PKD controls cancer cell motility. PMID:20363754

  16. Promiscuous stimulation of ParF protein polymerization by heterogeneous centromere binding factors.

    PubMed

    Machón, Cristina; Fothergill, Timothy J G; Barillà, Daniela; Hayes, Finbarr

    2007-11-16

    The segrosome is the nucleoprotein complex that mediates accurate segregation of bacterial plasmids. The segrosome of plasmid TP228 comprises ParF and ParG proteins that assemble on the parH centromere. ParF, which exemplifies one clade of the ubiquitous ParA superfamily of segregation proteins, polymerizes extensively in response to ATP binding. Polymerization is modulated by the ParG centromere binding factor (CBF). The segrosomes of plasmids pTAR, pVT745 and pB171 include ParA homologues of the ParF subgroup, as well as diverse homodimeric CBFs with no primary sequence similarity to ParG, or each other. Centromere binding by these analogues is largely specific. Here, we establish that the ParF homologues of pTAR and pB171 filament modestly with ATP, and that nucleotide hydrolysis is not required for this polymerization, which is more prodigious when the cognate CBF is also present. By contrast, the ParF homologue of plasmid pVT745 did not respond appreciably to ATP alone, but polymerized extensively in the presence of both its cognate CBF and ATP. The co-factors also stimulated nucleotide-independent polymerization of cognate ParF proteins. Moreover, apart from the CBF of pTAR, the disparate ParG analogues promoted polymerization of non-cognate ParF proteins suggesting that filamentation of the ParF proteins is enhanced by a common mechanism. Like ParG, the co-factors may be modular, possessing a centromere-specific interaction domain linked to a flexible region containing determinants that promiscuously stimulate ParF polymerization. The CBFs appear to function as bacterial analogues of formins, microtubule-associated proteins or related ancillary factors that regulate eucaryotic cytoskeletal dynamics.

  17. Actin Polymerization is Stimulated by Actin Crosslinking Protein Palladin

    PubMed Central

    Gurung, Ritu; Yadav, Rahul; Brungardt, Joseph G.; Orlova, Albina; Egelman, Edward H.; Beck, Moriah R.

    2016-01-01

    The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the coordinated regulation of actin dynamics. However, the potential effect of palladin on actin dynamics has remained elusive. Here we show that the actin binding immunoglobulin-like domain of palladin, which is directly responsible for both actin binding and bundling, also stimulates actin polymerization in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation step of actin polymerization. Furthermore, palladin dramatically reduced depolymerization, slightly enhanced the elongation rate, and did not alter the critical concentration. Microscopy and in vitro crosslinking assays reveal differences in actin bundle architecture when palladin is incubated with actin before or after polymerization. These results suggest a model whereby palladin stimulates a polymerization-competent form of G-actin, akin to metal ions, either through charge neutralization or conformational changes. PMID:26607837

  18. Polymeric penetration enhancers promote humoral immune responses to mucosal vaccines.

    PubMed

    Klein, Katja; Mann, Jamie F S; Rogers, Paul; Shattock, Robin J

    2014-06-10

    Protective mucosal immune responses are thought best induced by trans-mucosal vaccination, providing greater potential to generate potent local immune responses than conventional parenteral vaccination. However, poor trans-mucosal permeability of large macromolecular antigens limits bioavailability to local inductive immune cells. This study explores the utility of polymeric penetration enhancers to promote trans-mucosal bioavailability of insulin, as a biomarker of mucosal absorption, and two vaccine candidates: recombinant HIV-1 envelope glycoprotein (CN54gp140) and tetanus toxoid (TT). Responses to vaccinating antigens were assessed by measurement of serum and the vaginal humoral responses. Polyethyleneimine (PEI), Dimethyl-β-cyclodextrin (DM-β-CD) and Chitosan enhanced the bioavailability of insulin following intranasal (IN), sublingual (SL), intravaginal (I.Vag) and intrarectal (IR) administration. The same penetration enhancers also increased antigen-specific IgG and IgA antibody responses to the model vaccine antigens in serum and vaginal secretions following IN and SL application. Co-delivery of both antigens with PEI or Chitosan showed the highest increase in systemic IgG and IgA responses following IN or SL administration. However the highest IgA titres in vaginal secretions were achieved after IN immunisations with PEI and Chitosan. None of the penetration enhancers were able to increase antibody responses to gp140 after I.Vag immunisations, while in contrast PEI and Chitosan were able to induce TT-specific systemic IgG levels following I.Vag administration. In summary, we present supporting data that suggest appropriate co-formulation of vaccine antigens with excipients known to influence mucosal barrier functions can increase the bioavailability of mucosally applied antigens promoting the induction of mucosal and systemic antibody responses. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Polymeric microspheres as protein transduction reagents.

    PubMed

    Nagel, David; Behrendt, Jonathan M; Chimonides, Gwen F; Torr, Elizabeth E; Devitt, Andrew; Sutherland, Andrew J; Hine, Anna V

    2014-06-01

    Discovering the function of an unknown protein, particularly one with neither structural nor functional correlates, is a daunting task. Interaction analyses determine binding partners, whereas DNA transfection, either transient or stable, leads to intracellular expression, though not necessarily at physiologically relevant levels. In theory, direct intracellular protein delivery (protein transduction) provides a conceptually simpler alternative, but in practice the approach is problematic. Domains such as HIV TAT protein are valuable, but their effectiveness is protein specific. Similarly, the delivery of intact proteins via endocytic pathways (e.g. using liposomes) is problematic for functional analysis because of the potential for protein degradation in the endosomes/lysosomes. Consequently, recent reports that microspheres can deliver bio-cargoes into cells via a non-endocytic, energy-independent pathway offer an exciting and promising alternative for in vitro delivery of functional protein. In order for such promise to be fully exploited, microspheres are required that (i) are stably linked to proteins, (ii) can deliver those proteins with good efficiency, (iii) release functional protein once inside the cells, and (iv) permit concomitant tracking. Herein, we report the application of microspheres to successfully address all of these criteria simultaneously, for the first time. After cellular uptake, protein release was autocatalyzed by the reducing cytoplasmic environment. Outside of cells, the covalent microsphere-protein linkage was stable for ≥90 h at 37 °C. Using conservative methods of estimation, 74.3% ± 5.6% of cells were shown to take up these microspheres after 24 h of incubation, with the whole process of delivery and intracellular protein release occurring within 36 h. Intended for in vitro functional protein research, this approach will enable study of the consequences of protein delivery at physiologically relevant levels, without recourse to

  20. Polymeric Nanoparticles for Pulmonary Protein and DNA Delivery

    PubMed Central

    Menon, Jyothi U.; Ravikumar, Priya; Pise, Amruta; Gyawali, Dipendra; Hsia, Connie C.W.; Nguyen, Kytai T.

    2014-01-01

    Polymeric nanoparticles (NPs) are promising carriers of biological agents to lung due to advantages including biocompatibility, ease of surface modification, localized action and reduced systemic toxicity. However, there have been no studies extensively characterizing and comparing the behavior of polymeric NPs for pulmonary protein/DNA delivery both in vitro and in vivo. We screened six polymeric NPs: gelatin, chitosan, alginate, poly lactic-co-glycolic acid (PLGA), PLGA-chitosan, and PLGA-polyethylene glycol (PEG), for inhalational protein/ DNA delivery. All NPs except PLGA-PEG and alginate were <300 nm in size with bi-phasic core compound release profile. Gelatin, PLGA NPs and PLGA-PEG NPs remained stable in deionized water, serum, saline and simulated lung fluid (Gamble’s solution) over 5 days. PLGA-based NPs and natural polymer NPs exhibited highest cytocompatibility and dose-dependent in vitro uptake respectively by human alveolar type-1 epithelial cells. Based on these profiles, gelatin and PLGA NPs were used to encapsulate a) plasmid DNA encoding yellow fluorescent protein (YFP) or b) rhodamine-conjugated erythropoietin (EPO) for inhalational delivery to rats. Following a single inhalation, widespread pulmonary EPO distribution persisted for up to 10 days while increasing YFP expression was observed for at least 7 days for both NPs. The overall results support both PLGA and gelatin NPs as promising carriers for pulmonary protein/DNA delivery. PMID:24512977

  1. Chimeric protein repair of laminin polymerization ameliorates muscular dystrophy phenotype.

    PubMed

    McKee, Karen K; Crosson, Stephanie C; Meinen, Sarina; Reinhard, Judith R; Rüegg, Markus A; Yurchenco, Peter D

    2017-03-01

    Mutations in laminin α2-subunit (Lmα2, encoded by LAMA2) are linked to approximately 30% of congenital muscular dystrophy cases. Mice with a homozygous mutation in Lama2 (dy2J mice) express a nonpolymerizing form of laminin-211 (Lm211) and are a model for ambulatory-type Lmα2-deficient muscular dystrophy. Here, we developed transgenic dy2J mice with muscle-specific expression of αLNNd, a laminin/nidogen chimeric protein that provides a missing polymerization domain. Muscle-specific expression of αLNNd in dy2J mice resulted in strong amelioration of the dystrophic phenotype, manifested by the prevention of fibrosis and restoration of forelimb grip strength. αLNNd also restored myofiber shape, size, and numbers to control levels in dy2J mice. Laminin immunostaining and quantitation of tissue extractions revealed increased Lm211 expression in αLNNd-transgenic dy2J mice. In cultured myotubes, we determined that αLNNd expression increased myotube surface accumulation of polymerization-deficient recombinant laminins, with retention of collagen IV, reiterating the basement membrane (BM) changes observed in vivo. Laminin LN domain mutations linked to several of the Lmα2-deficient muscular dystrophies are predicted to compromise polymerization. The data herein support the hypothesis that engineered expression of αLNNd can overcome polymerization deficits to increase laminin, stabilize BM structure, and substantially ameliorate muscular dystrophy.

  2. Oligomeric and polymeric aggregates formed by proteins containing expanded polyglutamine

    PubMed Central

    Iuchi, S.; Hoffner, G.; Verbeke, P.; Djian, P.; Green, H.

    2003-01-01

    Neurological diseases resulting from proteins containing expanded polyglutamine (polyQ) are characteristically associated with insoluble neuronal inclusions, usually intranuclear, and neuronal death. We describe here oligomeric and polymeric aggregates formed in cells by expanded polyQ. These aggregates are not dissociated by concentrated formic acid, an extremely effective solvent for otherwise insoluble proteins. Perinuclear inclusions formed in cultured cells by expanded polyQ can be completely dissolved in concentrated formic acid, but a soluble protein oligomer containing the expanded polyQ and released by the formic acid is not dissociated to monomer. In Huntington's disease, a formic acid-resistant oligomer is present in cerebral cortex, but not in cerebellum. Cortical nuclei contain a polymeric aggregate of expanded polyQ that is insoluble in formic acid, does not enter polyacrylamide gels, but is retained on filters. This finding shows that the process of polymerization is more advanced in the cerebral cortex than in cultured cells. The resistance of oligomer and polymer to formic acid suggests the participation of covalent bonds in their stabilization. PMID:12591956

  3. Polymeric-based particulate systems for delivery of therapeutic proteins.

    PubMed

    Akash, Muhammad Sajid Hamid; Rehman, Kanwal; Chen, Shuqing

    2016-01-01

    Polymeric-based particulate systems have been intensively developed to increase the short biological half-life and prevent enzymatic degradation of therapeutic proteins. These techniques demonstrate the useful characteristics for the delivery of therapeutic proteins and peptides to the targeted site of application and prevent the interaction of encapsulated drug with the normal cells. In this article, we have described the in depth of different pharmaceutical-based techniques that are currently being practiced for efficient delivery of therapeutic proteins and peptides. A comprehensive English literature was searched using different electronic search databases including PubMed, Science Direct, Web of Science, google scholar and library search. Different search terms and advanced search were made by combining all the search fields in abstract, keywords and/or titles. Findings of various studies that have been discussed in this article clearly indicate that polymeric-based techniques can significantly increase the therapeutic potentials of incorporated proteins with no known toxic effects. These techniques have shown to maintain the stability and retain biological activity of protein therapeutics. Hence it can be suggested that pharmaceutical-based techniques are promising drug carriers for efficient delivery of therapeutic proteins.

  4. 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.

  5. Interfacial Polymerization for Colorimetric Labeling of Protein Expression in Cells

    PubMed Central

    Lilly, Jacob L.; Sheldon, Phillip R.; Hoversten, Liv J.; Romero, Gabriela; Balasubramaniam, Vivek; Berron, Brad J.

    2014-01-01

    Determining the location of rare proteins in cells typically requires the use of on-sample amplification. Antibody based recognition and enzymatic amplification is used to produce large amounts of visible label at the site of protein expression, but these techniques suffer from the presence of nonspecific reactivity in the biological sample and from poor spatial control over the label. Polymerization based amplification is a recently developed alternative means of creating an on-sample amplification for fluorescence applications, while not suffering from endogenous labels or loss of signal localization. This manuscript builds upon polymerization based amplification by developing a stable, archivable, and colorimetric mode of amplification termed Polymer Dye Labeling. The basic concept involves an interfacial polymer grown at the site of protein expression and subsequent staining of this polymer with an appropriate dye. The dyes Evans Blue and eosin were initially investigated for colorimetric response in a microarray setting, where both specifically stained polymer films on glass. The process was translated to the staining of protein expression in human dermal fibroblast cells, and Polymer Dye Labeling was specific to regions consistent with desired protein expression. The labeling is stable for over 200 days in ambient conditions and is also compatible with modern mounting medium. PMID:25536421

  6. Block copolymer adhesion promoters via ring-opening metathesis polymerization

    DOEpatents

    Kent, Michael S.; Saunders, Randall

    1997-01-01

    Coupling agents based on functionalized block copolymers for bonding thermoset polymers to solid materials. These are polymers which possess at least two types of functional groups, one which is able to attach to and react with solid surfaces, and another which can react with a thermoset resin, which are incorporated as pendant groups in monomers distributed in blocks (typically two) along the backbone of the chain. The block copolymers in this invention are synthesized by living ring-opening metathesis polymerization.

  7. Block copolymer adhesion promoters via ring-opening metathesis polymerization

    DOEpatents

    Kent, M.S.; Saunders, R.

    1997-02-18

    Coupling agents are disclosed based on functionalized block copolymers for bonding thermoset polymers to solid materials. These are polymers which possess at least two types of functional groups, one which is able to attach to and react with solid surfaces, and another which can react with a thermoset resin, which are incorporated as pendant groups in monomers distributed in blocks (typically two) along the backbone of the chain. The block copolymers in this invention are synthesized by living ring-opening metathesis polymerization. 18 figs.

  8. Electrospray deposition in vacuum as method to create functionally active protein immobilization on polymeric substrates.

    PubMed

    Fornari, Enzo; Roberts, Clive J; Temperton, Robert H; O'Shea, James N

    2015-09-01

    We demonstrate in this work the deposition of a large biological molecule (fibronectin) on polymeric substrates in a high vacuum environment using an electrospray deposition system. Fibronectin was deposited and its distribution and structure investigated and retention of function (ability to promote cell adhesion) on return to liquid environment is shown. AFM was used to monitor changes in the morphology of the surface before and after fibronectin deposition, whilst the biological activity of the deposited protein is assessed through a quantitative analysis of the biomolecular adhesion and migration of fibroblast cells to the modified surfaces. For the first time we have demonstrated that using high vacuum electrospray deposition it is possible to deposit large protein molecules on polymeric surfaces whilst maintaining the protein activity. The deposition of biological molecules such as proteins with the retention of their activity onto clean well-controlled surfaces under vacuum condition, offers the possibility for future studies utilizing high resolution vacuum based techniques at the atomic and molecular scale providing a greater understanding of protein-surface interface behaviour of relevance to a wide range of applications such as in sensors, diagnostics and tissue engineering.

  9. Protein adsorption resistance of PVP-modified polyurethane film prepared by surface-initiated atom transfer radical polymerization

    NASA Astrophysics Data System (ADS)

    Yuan, Huihui; Qian, Bin; Zhang, Wei; Lan, Minbo

    2016-02-01

    An anti-fouling surface of polyurethane (PU) film grafted with Poly(N-vinylpyrrolidone) (PVP) was prepared through surface-initiated atom transfer radical polymerization (SI-ATRP). And the polymerization time was investigated to obtain PU films with PVP brushes of different lengths. The surface properties and protein adsorption of modified PU films were evaluated. The results showed that the hydrophilicity of PU-PVP films were improved with the increase of polymerization time, which was not positive correlation with the surface roughness due to the brush structure. Additionally, the protein resistance performance was promoted when prolonging the polymerization time. The best antifouling PU-PVP (6.0 h) film reduced the adsoption level of bovine serum albumin (BSA), lysozyme (LYS), and brovin serum fibrinogen (BFG) by 93.4%, 68.3%, 85.6%, respectively, compared to the unmodified PU film. The competitive adsorption of three proteins indicated that LYS preferentially adsorbed on the modified PU film, while BFG had the lowest adsorption selectivity. And the amount of BFG on PU-PVP (6.0 h) film reduced greatly to 0.08 μg/cm2, which was almost one-tenth of its adsorption from the single-protein system. Presented results suggested that both hydrophilicity and surface roughness might be the important factors in all cases of protein adsorption, and the competitive or selective adsorption might be related to the size of the proteins, especially on the non-charged films.

  10. Polymeric ADAM protein mimics interrogate mammalian sperm-egg binding.

    PubMed

    Lee, Younjoo; Sampson, Nicole S

    2009-03-23

    The sperm proteins ADAM2 and ADAM3, members of the ADAM family of proteins, have been implicated in mammalian sperm-egg binding. However, elucidating their roles is complex because of the interdependence of ADAM protein expression in the testis. Hence, multivalent probes containing the three-amino acid binding sequence of ADAM2, glutamate-cysteine-aspartate (ECD), and ADAM3, glutamine-cysteine-aspartate (QCD), were designed, synthesized, and tested to investigate gamete interactions. In this work, ECD polymer mimics were synthesized by ring-opening metathesis polymerization with a faster initiating ruthenium catalyst than previously used. Polymers containing 100 copies of the ECD peptide mimic were found to be the best inhibitors of fertilization. The multivalent QCD polymers were also tested as inhibitors of fertilization. The structure-activity profile was the same as ECD polymers, but the overall potency was lower. Both ECD and QCD polymers require the presence of beta(1) integrin to inhibit fertilization. Next, triblock ABA and ABC copolymers containing both ECD and QCD ligands were synthesized with 96 monomer spacers as their B blocks. Although these polymers had lower densities of ECD and QCD peptides, their potencies correlated with the potencies of their corresponding homopolymers. In addition, no synergy between ECD and QCD mimics was observed. All the data suggest that QCD and ECD bind to the same complex of proteins that includes beta(1) integrin.

  11. Signal amplification strategies for DNA and protein detection based on polymeric nanocomposites and polymerization: A review.

    PubMed

    Zhou, Shaohong; Yuan, Liang; Hua, Xin; Xu, Lingling; Liu, Songqin

    2015-06-02

    Demand is increasing for ultrasensitive bioassays for disease diagnosis, environmental monitoring and other research areas. This requires novel signal amplification strategies to maximize the signal output. In this review, we focus on a series of significant signal amplification strategies based on polymeric nanocomposites and polymerization. Some common polymers are used as carriers to increase the local concentration of signal probes and/or biomolecules on their surfaces or in their interiors. Some polymers with special fluorescence and optical properties can efficiently transfer the excitation energy from a single site to the whole polymer backbone. This results in superior fluorescence signal amplification due to the resulting collective effort (integration of signal). Recent polymerization-based signal amplification strategies that employ atom transfer radical polymerization (ATRP) and photo-initiated polymerization are also summarized. Several distinctive applications of polymers in ultrasensitive bioanalysis are highlighted.

  12. In vitro polymerization of mussel polyphenolic proteins catalyzed by mushroom tyrosinase.

    PubMed

    Burzio, L A; Burzio, V A; Pardo, J; Burzio, L O

    2000-07-01

    The in vitro enzymatic polymerization of the polyphenolic protein purified from the mussels Aulacomya ater, Mytilus edulis chilensis and Choromytilus chorus was studied. Mushroom tyrosinase was used to oxidize the dopa residues present in these proteins, and polymerization was monitored by acid-urea polyacrylamide gel electrophoresis. The protein from A. ater polymerized at a faster rate than the other two. Amino acid analysis of the crosslinked protein showed a notable decrease in the content of dopa, but no significant change of other amino acids. This suggests that crosslink formation may be limited to the oxidized dopa derivatives of the protein molecules.

  13. The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA.

    PubMed

    Rossi, Matthew J; Mazina, Olga M; Bugreev, Dmitry V; Mazin, Alexander V

    2011-04-19

    The Holliday junction (HJ), a cross-shaped structure that physically links the two DNA helices, is a key intermediate in homologous recombination, DNA repair, and replication. Several helicase-like proteins are known to bind HJs and promote their branch migration (BM) by translocating along DNA at the expense of ATP hydrolysis. Surprisingly, the bacterial recombinase protein RecA and its eukaryotic homologue Rad51 also promote BM of HJs despite the fact they do not bind HJs preferentially and do not translocate along DNA. RecA/Rad51 plays a key role in DNA double-stranded break repair and homologous recombination. RecA/Rad51 binds to ssDNA and forms contiguous filaments that promote the search for homologous DNA sequences and DNA strand exchange. The mechanism of BM promoted by RecA/RAD51 is unknown. Here, we demonstrate that cycles of RecA/Rad51 polymerization and dissociation coupled with ATP hydrolysis drives the BM of HJs.

  14. Ascertaining effects of nanoscale polymeric interfaces on competitive protein adsorption at the individual protein level

    NASA Astrophysics Data System (ADS)

    Song, Sheng; Xie, Tian; Ravensbergen, Kristina; Hahm, Jong-In

    2016-02-01

    With the recent development of biomaterials and biodevices with reduced dimensionality, it is critical to comprehend protein adhesion processes to nanoscale solid surfaces, especially those occurring in a competitive adsorption environment. Complex sequences of adhesion events in competitive adsorption involving multicomponent protein systems have been extensively investigated, but our understanding is still limited primarily to macroscopic adhesion onto chemically simple surfaces. We examine the competitive adsorption behavior from a binary protein mixture containing bovine serum albumin and fibrinogen at the single protein level. We subsequently evaluate a series of adsorption and displacement processes occurring on both the macroscopic homopolymer and nanoscopic diblock copolymer surfaces, while systematically varying the protein concentration and incubation time. We identify the similarities and dissimilarities in competitive protein adsorption behavior between the two polymeric surfaces, the former presenting chemical uniformity at macroscale versus the latter exhibiting periodic nanointerfaces of chemically alternating polymeric segments. We then present our novel experimental finding of a large increase in the nanointerface-engaged residence time of the initially bound proteins and further explain the origin of this phenomenon manifested on nanoscale diblock copolymer surfaces. The outcomes of this study may provide timely insight into nanoscale competitive protein adsorption that is much needed in designing bioimplant and tissue engineering materials. In addition, the fundamental understanding gained from this study can be beneficial for the development of highly miniaturized biodevices and biomaterials fabricated by using nanoscale polymeric materials and interfaces.With the recent development of biomaterials and biodevices with reduced dimensionality, it is critical to comprehend protein adhesion processes to nanoscale solid surfaces, especially those

  15. Isolation and characterization of a polymerized prion protein.

    PubMed Central

    Lu, Bao-Yuan; Chang, Jui-Yoa

    2002-01-01

    A polymerized form of recombinant mouse prion protein (mPrP) domain 23-231 [mPrP-(23-231)], designated mPrP-z, was generated at acidic pH (pH 2-5) in the presence of selected concentrations of denaturant (2 M guanidinium chloride or 5 M urea). This isoform of mPrP is stable in acidic solution after removal of denaturant. It can be isolated and purified using reversed-phase HPLC or size-exclusion HPLC. mPrP-z bears structural properties that partially resemble those of scrapie prion. Unlike the native mPrP-(23-231) (mPrP-N), mPrP-z exhibits a high content of beta-sheet structure, as shown by CD spectroscopy, and exists as an oligomer with an approximate molecular mass of 340000 Da, as measured by light scattering. However, similarly to mPrP-N, mPrP-z contains the intact disulphide bond and is sensitive to digestion by proteinase K. PMID:11988079

  16. Reactivity of polymeric proanthocyanidins toward salivary proteins and their contribution to young red wine astringency.

    PubMed

    Sun, Baoshan; de Sá, Marta; Leandro, Conceição; Caldeira, Ilda; Duarte, Filomena L; Spranger, Isabel

    2013-01-30

    Recent studies have indicated the presence of significant amount of highly polymerized and soluble proanthocyanidins in red wine and such compounds interacted readily with proteins, suggesting that they might be particularly astringent. Thus, the objective of this work was to verify the astringency of polymeric proanthocyanidins and their contribution to red wine astringency. The precipitation reactions of the purified oligomeric procyanidins (degree of polymerization ranging from 2 to 12-15) and polymeric procyanidins (degree of polymerization ranging from 12-15 to 32-34) with human salivary proteins were studied; salivary proteins composition changes before and after the reaction was verified by SDS-PAGE and procyanidins composition changes by spectrometric, direct HPLC and thiolysis-HPLC methods. The astringency intensity of these two procyanidin fractions was evaluated by a sensory analysis panel. For verifying the correlation between polymeric proanthocyanidins and young red wine astringency, the levels of total oligomeric and total polymeric proanthocyanidins and other phenolic composition in various young red wines were quantified and the astringency intensities of these wines were evaluated by a sensory panel. The results showed that polymeric proanthocyanidins had much higher reactivity toward human salivary proteins and higher astringency intensity than the oligomeric ones. Furthermore, young red wine astringency intensities were highly correlated to levels of polymeric proanthocyanidins, particularly at low concentration range (correlation coefficient r = 0.9840) but not significant correlated to total polyphenols (r = 0.2343) or other individual phenolic compounds (generally r < 0.3). These results indicate the important contribution of polymeric proanthocyanidins to red wine astringency and the levels of polymeric polyphenols in red wines may be used as an indicator for its astringency.

  17. Structural and Functional Studies of H. seropedicae RecA Protein – Insights into the Polymerization of RecA Protein as Nucleoprotein Filament

    SciTech Connect

    Leite, Wellington C.; Galvão, Carolina W.; Saab, Sérgio C.; Iulek, Jorge; Etto, Rafael M.; Steffens, Maria B. R.; Chitteni-Pattu, Sindhu; Stanage, Tyler; Keck, James L.; Cox, Michael M.; Spies, Maria

    2016-07-22

    The bacterial RecA protein plays a role in the complex system of DNA damage repair. Here, we report the functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA). HsRecA protein is more efficient at displacing SSB protein from ssDNA than Escherichia coli RecA protein. HsRecA also promotes DNA strand exchange more efficiently. The three dimensional structure of HsRecA-ADP/ATP complex has been solved to 1.7 Å resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, that are similar to homologous bacterial RecA proteins. Comparative structural analysis showed that the N-terminal polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. Reconstruction of electrostatic potential from the hexameric structure of HsRecA-ADP/ATP revealed a high positive charge along the inner side, where ssDNA is bound inside the filament. The properties of this surface may explain the greater capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote DNA exchange relative to EcRecA. In conclusion, our functional and structural analyses provide insight into the molecular mechanisms of polymerization of bacterial RecA as a helical nucleoprotein filament.

  18. Structural and Functional Studies of H. seropedicae RecA Protein – Insights into the Polymerization of RecA Protein as Nucleoprotein Filament

    PubMed Central

    Galvão, Carolina W.; Saab, Sérgio C.; Iulek, Jorge; Etto, Rafael M.; Steffens, Maria B. R.; Chitteni-Pattu, Sindhu; Stanage, Tyler; Keck, James L.; Cox, Michael M.

    2016-01-01

    The bacterial RecA protein plays a role in the complex system of DNA damage repair. Here, we report the functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA). HsRecA protein is more efficient at displacing SSB protein from ssDNA than Escherichia coli RecA protein. HsRecA also promotes DNA strand exchange more efficiently. The three dimensional structure of HsRecA-ADP/ATP complex has been solved to 1.7 Å resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, that are similar to homologous bacterial RecA proteins. Comparative structural analysis showed that the N-terminal polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. Reconstruction of electrostatic potential from the hexameric structure of HsRecA-ADP/ATP revealed a high positive charge along the inner side, where ssDNA is bound inside the filament. The properties of this surface may explain the greater capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote DNA exchange relative to EcRecA. Our functional and structural analyses provide insight into the molecular mechanisms of polymerization of bacterial RecA as a helical nucleoprotein filament. PMID:27447485

  19. The putative pocket protein binding site of Autographa californica nucleopolyhedrovirus BV/ODV-C42 is required for virus-induced nuclear actin polymerization.

    PubMed

    Li, Kun; Wang, Yun; Bai, Huimin; Wang, Qian; Song, Jianhua; Zhou, Yuan; Wu, Chunchen; Chen, Xinwen

    2010-08-01

    Nuclear filamentous actin (F-actin) is essential for nucleocapsid morphogenesis of lepidopteran nucleopolyhedroviruses. Previously, we had demonstrated that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) BV/ODV-C42 (C42) is involved in nuclear actin polymerization by recruiting P78/83, an AcMNPV orf9-encoded N-WASP homology protein that is capable of activating an actin-related-protein 2/3 (Arp2/3) complex to initiate actin polymerization, to the nucleus. To further investigate the role of C42 in virus-induced actin polymerization, the recombinant bacmid vAc(p78/83nls-gfp), with a c42 knockout, p78/83 tagged with a nuclear localization signal coding sequence, and egfp as a reporter gene under the control of the Pp10 promoter, was constructed and transfected to Sf9 cells. In the nuclei of vAc(p78/83nls-gfp)-transfected cells, polymerized F-actin filaments were absent, whereas other actin polymerization elements (i.e., P78/83, G-actin, and Arp2/3 complex) were present. This in vivo evidence indicated that C42 actively participates in the nuclear actin polymerization process as a key element, besides its role in recruiting P78/83 to the nucleus. In order to collect in vitro evidence for the participation of C42 in actin polymerization, an anti-C42 antibody was used to neutralize the viral nucleocapsid, which is capable of initiating actin polymerization in vitro. Both the kinetics of pyrene-actin polymerization and F-actin-specific staining by phalloidin indicated that anti-C42 can significantly attenuate the efficiency of F-actin formation compared to that with control antibodies. Furthermore, we have identified the putative pocket protein binding sequence (PPBS) on C42 that is essential for C42 to exert its function in nuclear actin polymerization.

  20. An investigation on the analytical potential of polymerized liposomes bound to lanthanide ions for protein analysis.

    PubMed

    Santos, Marina; Roy, Bidhan C; Goicoechea, Héctor; Campiglia, Andres D; Mallik, Sanku

    2004-09-01

    We present a promising approach to protein sensing based on Eu3+ ions incorporated into polymerized liposomes. The sensitization of Eu3+ is accomplished with 5-aminosalicylic acid, which provides energy transfer for a stable reference signal and a wide wavelength excitation range free from protein interference. The lipophilic character of polymerized liposomes provides the appropriate platform for protein interaction with the lanthanide ion. Quantitative analysis is based on the linear relationship between the luminescence signal of Eu3+ and protein concentration. Because no spectral shift of the lanthanide luminescence is observed upon protein interaction, qualitative analysis is based on the luminescence lifetime of polymerized liposomes. This parameter, which changes significantly upon protein-liposome interaction, follows a well-behaved single-exponential decay that might be useful for protein identification. Copyright 2004 American Chemical Society

  1. Regulation of coat protein polymerization by the scaffolding protein of bacteriophage P22

    SciTech Connect

    Fuller, M.T.; King, J.

    1980-10-01

    In the morphogenesis of double stranded DNA phages, a precursor protein shell empty of DNA is first assembled and then filled with DNA. The assembly of the correctly dimensioned precursor shell (procapsid) of Salmonella bacteriophage P22 requires the interaction of some 420 coat protein subunits with approx. 200 scaffolding protein subunits to form a double shelled particle with the scaffolding protein on the inside. In the course of DNA packaging, all of the scaffolding protein subunits exit from the procapsid and participate in further rounds of procapsid assembly. To study the mechanism of shell assembly we have purified the coat and scaffolding protein subunits by selective dissociation of isolated procapsids. Both proteins can be obtained as soluble sununits in Tris buffer at near neutral pH. The coat protein sedimented in sucrose gradients as a roughly spherical monomer, while the scaffolding protein sedimented as if it were an elongated monomer. When the two proteins were mixed together in 1.5 M guanidine hydrochloride and dialyzed back to buffer at room temperature, procapsids formed which were very similar in morphology, sedimentation behavior, and protein composition to procapsids formed in vivo. Incubation of either protein alone under the same conditions did not yield any large structures. We interpret these results to mean that the assembly of the shell involves a switching of both proteins from their nonaggregating to their aggregating forms through their mutual interaction. The results are discussed in terms of the general problem of self-regulated assembly and the control of protein polymerization in morphogenesis.

  2. Epistructural Tension Promotes Protein Associations

    NASA Astrophysics Data System (ADS)

    Fernández, Ariel

    2012-05-01

    Epistructural tension is the reversible work per unit area required to span the aqueous interface of a soluble protein structure. The parameter accounts for the free-energy cost of imperfect hydration, involving water molecules with a shortage of hydrogen-bonding partnerships relative to bulk levels. The binding hot spots along protein-protein interfaces are identified with residues that contribute significantly to the epistructural tension in the free subunits. Upon association, such residues either displace or become deprived of low-coordination vicinal water molecules.

  3. HAM proteins promote organ indeterminacy

    PubMed Central

    2012-01-01

    HAIRY MERISTEM (HAM) proteins, members of the GRAS family of transcriptional regulators, are essential for maintenance of indeterminate growth in flowering plant shoots, loss-of-function ham mutants exhibiting a strikingly novel phenotype of shoot meristem arrest and differentiation. Specific cellular/molecular functions of HAM proteins underlying meristem maintenance are unknown. In this review, I highlight findings from recent analyses of Arabidopsis ham (Atham) loss-of-function phenotypes, including that HAM function limits the generation of clonally-derived meristem layers and that HAM function regulates CLAVATA3 expression. I consider how this new information both refines our understanding of the role of HAM proteins in regulating meristem structure and function, and may also suggest possible downstream HAM protein transcriptional targets. Finally, I note the significant phenotypic overlap between Atham phenotypes, and aintegumenta/anintegumenta-like6 double mutant phenotypes, suggesting meristem regulatory functions common to, and possible genetic interactions between, HAM and AINTEGUMENTA. PMID:22353859

  4. Novel polymeric scaffolds using protein microbubbles as porogen and growth factor carriers.

    PubMed

    Nair, Ashwin; Thevenot, Paul; Dey, Jagannath; Shen, Jinhui; Sun, Man-Wu; Yang, Jian; Tang, Liping

    2010-02-01

    Polymeric tissue engineering scaffolds prepared by conventional techniques like salt leaching and phase separation are greatly limited by their poor biomolecule-delivery abilities. Conventional methods of incorporation of various growth factors, proteins, and/or peptides on or in scaffold materials via different crosslinking and conjugation techniques are often tedious and may affect scaffold's physical, chemical, and mechanical properties. To overcome such deficiencies, a novel two-step porous scaffold fabrication procedure has been created in which bovine serum albumin microbubbles (henceforth MB) were used as porogen and growth factor carriers. Polymer solution mixed with MB was phase separated and then lyophilized to create porous scaffold. MB scaffold triggered substantially lesser inflammatory responses than salt-leached and conventional phase-separated scaffolds in vivo. Most importantly, the same technique was used to produce insulin-like growth factor-1 (IGF-1)-eluting porous scaffolds, simply by incorporating IGF-1-loaded MB (MB-IGF-1) with polymer solution before phase separation. In vitro such MB-IGF-1 scaffolds were able to promote cell growth to a much greater extent than scaffold soaked in IGF-1, confirming the bioactivity of the released IGF-1. Further, such MB-IGF-1 scaffolds elicited IGF-1-specific collagen production in the surrounding tissue in vivo. This novel growth factor-eluting scaffold fabrication procedure can be used to deliver a range of single or combination of bioactive biomolecules to substantially promote cell growth and function in degradable scaffold.

  5. Novel Polymeric Scaffolds Using Protein Microbubbles as Porogen and Growth Factor Carriers

    PubMed Central

    Nair, Ashwin; Thevenot, Paul; Dey, Jagannath; Shen, Jinhui; Sun, Man-Wu; Yang, Jian

    2010-01-01

    Polymeric tissue engineering scaffolds prepared by conventional techniques like salt leaching and phase separation are greatly limited by their poor biomolecule-delivery abilities. Conventional methods of incorporation of various growth factors, proteins, and/or peptides on or in scaffold materials via different crosslinking and conjugation techniques are often tedious and may affect scaffold's physical, chemical, and mechanical properties. To overcome such deficiencies, a novel two-step porous scaffold fabrication procedure has been created in which bovine serum albumin microbubbles (henceforth MB) were used as porogen and growth factor carriers. Polymer solution mixed with MB was phase separated and then lyophilized to create porous scaffold. MB scaffold triggered substantially lesser inflammatory responses than salt-leached and conventional phase-separated scaffolds in vivo. Most importantly, the same technique was used to produce insulin-like growth factor-1 (IGF-1)–eluting porous scaffolds, simply by incorporating IGF-1–loaded MB (MB-IGF-1) with polymer solution before phase separation. In vitro such MB-IGF-1 scaffolds were able to promote cell growth to a much greater extent than scaffold soaked in IGF-1, confirming the bioactivity of the released IGF-1. Further, such MB-IGF-1 scaffolds elicited IGF-1–specific collagen production in the surrounding tissue in vivo. This novel growth factor–eluting scaffold fabrication procedure can be used to deliver a range of single or combination of bioactive biomolecules to substantially promote cell growth and function in degradable scaffold. PMID:19327002

  6. Hydrophilic crosslinked-polymeric surface capable of effective suppression of protein adsorption

    NASA Astrophysics Data System (ADS)

    Kamon, Yuri; Inoue, Naoko; Mihara, Erika; Kitayama, Yukiya; Ooya, Tooru; Takeuchi, Toshifumi

    2016-08-01

    We investigated the nonspecific adsorption of proteins towards three hydrophilic crosslinked-polymeric thin layers prepared by surface-initiated atom transfer radical polymerization using N,N‧-methylenebisacrylamide, 2-(methacryloyloxy)ethyl-[N-(2-methacryloyloxy)ethyl]phosphorylcholine (MMPC), or 6,6‧-diacryloyl-trehalose crosslinkers. Protein binding experiments were performed by surface plasmon resonance with six proteins of different pI values including α-lactalbumin, bovine serum albumin (BSA), myoglobin, ribonuclease A, cytochrome C, and lysozyme in buffer solution at pH 7.4. All of the obtained crosslinked-polymeric thin layers showed low nonspecific adsorption of negatively charged proteins at pH 7.4 such as α-lactalbumin, BSA, and myoglobin. Nonspecific adsorption of positively charged proteins including ribonuclease A, cytochrome C, and lysozyme was the lowest for poly(MMPC). These results suggest poly(MMPC) can effectively reduce nonspecific adsorption of a wide range of proteins that are negatively or positively charged at pH 7.4. MMPC is a promising crosslinker for a wide range of polymeric materials requiring low nonspecific protein binding.

  7. Actin polymerization is stimulated by actin cross-linking protein palladin.

    PubMed

    Gurung, Ritu; Yadav, Rahul; Brungardt, Joseph G; Orlova, Albina; Egelman, Edward H; Beck, Moriah R

    2016-02-15

    The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the co-ordinated regulation of actin dynamics. However, the potential effect of palladin on actin dynamics has remained elusive. In the present study, we show that the actin-binding immunoglobulin-like domain of palladin, which is directly responsible for both actin binding and bundling, also stimulates actin polymerization in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation step of actin polymerization. Furthermore, palladin dramatically reduced depolymerization, slightly enhanced the elongation rate, and did not alter the critical concentration. Microscopy and in vitro cross-linking assays reveal differences in actin bundle architecture when palladin is incubated with actin before or after polymerization. These results suggest a model whereby palladin stimulates a polymerization-competent form of globular or monomeric actin (G-actin), akin to metal ions, either through charge neutralization or through conformational changes.

  8. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization.

    PubMed

    Lee, Wei Lin; Grimes, Jonathan M; Robinson, Robert C

    2015-03-01

    Pathogenic Yersinia species evade host immune systems through the injection of Yersinia outer proteins (Yops) into phagocytic cells. One Yop, YopO, also known as YpkA, induces actin-filament disruption, impairing phagocytosis. Here we describe the X-ray structure of Yersinia enterocolitica YopO in complex with actin, which reveals that YopO binds to an actin monomer in a manner that blocks polymerization yet allows the bound actin to interact with host actin-regulating proteins. SILAC-MS and biochemical analyses confirm that actin-polymerization regulators such as VASP, EVL, WASP, gelsolin and the formin diaphanous 1 are directly sequestered and phosphorylated by YopO through formation of ternary complexes with actin. This leads to a model in which YopO at the membrane sequesters actin from polymerization while using the bound actin as bait to recruit, phosphorylate and misregulate host actin-regulating proteins to disrupt phagocytosis.

  9. Yersinia effector YopO uses actin as bait to phosphorylate proteins that regulate actin polymerization

    PubMed Central

    Lee, Wei Lin; Grimes, Jonathan M; Robinson, Robert C

    2016-01-01

    Pathogenic Yersinia species evade host immune systems through the injection of Yersinia outer proteins (Yops) into phagocytic cells. One Yop, YopO, also known as YpkA, induces actin-filament disruption, impairing phagocytosis. Here we describe the X-ray structure of Yersinia enterocolitica YopO in complex with actin, which reveals that YopO binds to an actin monomer in a manner that blocks polymerization yet allows the bound actin to interact with host actin-regulating proteins. SILAC-MS and biochemical analyses confirm that actin-polymerization regulators such as VASP, EVL, WASP, gelsolin and the formin diaphanous 1 are directly sequestered and phosphorylated by YopO through formation of ternary complexes with actin. This leads to a model in which YopO at the membrane sequesters actin from polymerization while using the bound actin as bait to recruit, phosphorylate and misregulate host actin-regulating proteins to disrupt phagocytosis. PMID:25664724

  10. Actin polymerization-dependent activation of Cas-L promotes immunological synapse stability

    PubMed Central

    Santos, Luís C; Blair, David A; Kumari, Sudha; Cammer, Michael; Iskratsch, Thomas; Herbin, Olivier; Alexandropoulos, Konstantina; Dustin, Michael L; Sheetz, Michael P

    2016-01-01

    The immunological synapse formed between a T-cell and an antigen-presenting cell is important for cell–cell communication during T-cell-mediated immune responses. Immunological synapse formation begins with stimulation of the T-cell receptor (TCR). TCR microclusters are assembled and transported to the center of the immunological synapse in an actin polymerization-dependent process. However, the physical link between TCR and actin remains elusive. Here we show that lymphocyte-specific Crk-associated substrate (Cas-L), a member of a force sensing protein family, is required for transport of TCR microclusters and for establishing synapse stability. We found that Cas-L is phosphorylated at TCR microclusters in an actin polymerization-dependent fashion. Furthermore, Cas-L participates in a positive feedback loop leading to amplification of Ca2+ signaling, inside–out integrin activation, and actomyosin contraction. We propose a new role for Cas-L in T-cell activation as a mechanical transducer linking TCR microclusters to the underlying actin network and coordinating multiple actin-dependent structures in the immunological synapse. Our studies highlight the importance of mechanotransduction processes in T-cell-mediated immune responses. PMID:27359298

  11. Recent Advances in Protein and Peptide Drug Delivery: A Special Emphasis on Polymeric Nanoparticles

    PubMed Central

    Patel, Ashaben; Patel, Mitesh; Yang, Xiaoyan; Mitra, Ashim K.

    2015-01-01

    Proteins and peptides are widely indicated in many diseased states. Parenteral route is the most commonly employed method of administration for therapeutic proteins and peptides. However, requirement of frequent injections due to short in vivo half-life results in poor patient compliance. Non-invasive drug delivery routes such as nasal, transdermal, pulmonary, and oral offer several advantages over parenteral administration. Intrinsic physicochemical properties and low permeability across biological membrane limit protein delivery via non-invasive routes. One of the strategies to improve protein and peptide absorption is by delivering through nanostructured delivery carriers. Among nanocarriers, polymeric nanoparticles (NPs) have demonstrated significant advantages over other delivery systems. This article summarizes the application of polymeric NPs for protein and peptide drug delivery following oral, nasal, pulmonary, parenteral, transdermal, and ocular administrations. PMID:25106908

  12. Recent advances in protein and Peptide drug delivery: a special emphasis on polymeric nanoparticles.

    PubMed

    Patel, Ashaben; Patel, Mitesh; Yang, Xiaoyan; Mitra, Ashim K

    2014-01-01

    Proteins and peptides are widely indicated in many diseased states. Parenteral route is the most commonly em- ployed method of administration for therapeutic proteins and peptides. However, requirement of frequent injections due to short in vivo half-life results in poor patient compliance. Non-invasive drug delivery routes such as nasal, transdermal, pulmonary, and oral offer several advantages over parenteral administration. Intrinsic physicochemical properties and low permeability across biological membrane limit protein delivery via non-invasive routes. One of the strategies to improve protein and peptide absorption is by delivering through nanostructured delivery carriers. Among nanocarriers, polymeric nanoparticles (NPs) have demonstrated significant advantages over other delivery systems. This article summarizes the application of polymeric NPs for protein and peptide drug delivery following oral, nasal, pulmonary, parenteral, transder mal, and ocular administrations.

  13. Molecularly imprinted protein recognition thin films constructed by controlled/living radical polymerization.

    PubMed

    Sasaki, Shogo; Ooya, Tooru; Kitayama, Yukiya; Takeuchi, Toshifumi

    2015-02-01

    We demonstrated the synthesis of molecularly imprinted polymers (MIPs) with binding affinity toward a target protein, ribonuclease A (RNase) by atom transfer radical polymerization (ATRP) of acrylic acid, acrylamide, and N,N'-methylenebisacrylamide in the presence of RNase. The binding activity of the MIPs was evaluated by surface plasmon resonance (SPR) of the MIP thin layers prepared on the gold-coated sensor chips. The MIPs prepared by ATRP (MIP-ATRP) had a binding affinity toward RNase with larger binding amount compared to MIPs prepared by conventional free radical polymerization methods (MIP-RP). Moreover, protein selectivity was evaluated using reference proteins (cytochrome c, myoglobin, and α-lactalbumin) and was confirmed in MIP-ATRP of optimum film thickness determined experimentally to be 15-30 nm; however, protein selectivity was not achieved in all MIP-RP. We have shown that ATRP is powerful technique for preparing protein recognition materials by molecular imprinting.

  14. Prediction of heat-induced polymerization of different globular food proteins in mixtures with wheat gluten.

    PubMed

    Lambrecht, Marlies A; Rombouts, Ine; De Ketelaere, Bart; Delcour, Jan A

    2017-04-15

    Egg, soy or whey protein co-exists with wheat gluten in different food products. Different protein types impact each other during heat treatment. A positive co-protein effect occurs when heat-induced polymerization of a mixture of proteins is more intense than that of the isolated proteins. The intrinsic protein characteristics of globular proteins which enhance polymerization in mixtures with gluten are unknown. In this report, a model was developed to predict potential co-protein effects in mixtures of gluten and globular proteins during heating at 100°C. A negative co-protein effect with addition of lysozyme, no co-protein effect with soy glycinin or egg yolk and positive co-protein effects with bovine serum albumin, (S-)ovalbumin, egg white, whole egg, defatted egg yolk, wheat albumins and wheat globulins were detected. The level of accessible free sulfhydryl groups and the surface hydrophobicity of unfolded globular proteins were the main characteristics in determining the co-protein effects in gluten mixtures. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Effect of flour polymeric proteins on dough thermal properties and breadmaking characteristics for hard red spring wheat genotypes

    USDA-ARS?s Scientific Manuscript database

    The aim of this research was to investigate the effect of variation of flour polymeric proteins on rheological properties of dough under continuous mixing and thermal treatment for hard red spring (HRS) wheat genotypes grown in North Dakota, USA. Flour polymeric proteins were analyzed by size exclus...

  16. Advances in Polymer and Polymeric Nanostructures for Protein Conjugation

    PubMed Central

    González-Toro, Daniella C.; Thayumanavan, S.

    2013-01-01

    Linear polymers have been considered the best molecular structures for the formation of efficient protein conjugates due to their biological advantages, synthetic convenience and ease of functionalization. In recent years, much attention has been dedicated to develop synthetic strategies that produce the most control over protein conjugation utilizing linear polymers as scaffolds. As a result, different conjugate models, such as semitelechelic, homotelechelic, heterotelechelic and branched or star polymer conjugates, have been obtained that take advantage of these well-controlled synthetic strategies. Development of protein conjugates using nanostructures and the formation of said nanostructures from protein-polymer bioconjugates are other areas in the protein bioconjugation field. Although several polymer-protein technologies have been developed from these discoveries, few review articles have focused on the design and function of these polymers and nanostructures. This review will highlight some recent advances in protein-linear polymer technologies that employ protein covalent conjugation and successful protein-nanostructure bioconjugates (covalent conjugation as well) that have shown great potential for biological applications. PMID:24058205

  17. The effect of polymer surface modification on polymer-protein interaction via interfacial polymerization

    USDA-ARS?s Scientific Manuscript database

    Membrane separation is an important processing technology used for separating food ingredients and fractionating value-added components from food processing by-products. Long-term performance of polymeric membranes in food protein processing is impeded by formation of fouled layers on the membrane ...

  18. Polymerization and nucleic acid-binding properties of human L1 ORF1 protein

    PubMed Central

    Callahan, Kathryn E.; Hickman, Alison B.; Jones, Charles E.; Ghirlando, Rodolfo; Furano, Anthony V.

    2012-01-01

    The L1 (LINE 1) retrotransposable element encodes two proteins, ORF1p and ORF2p. ORF2p is the L1 replicase, but the role of ORF1p is unknown. Mouse ORF1p, a coiled-coil-mediated trimer of ∼42-kDa monomers, binds nucleic acids and has nucleic acid chaperone activity. We purified human L1 ORF1p expressed in insect cells and made two findings that significantly advance our knowledge of the protein. First, in the absence of nucleic acids, the protein polymerizes under the very conditions (0.05 M NaCl) that are optimal for high (∼1 nM)-affinity nucleic acid binding. The non-coiled-coil C-terminal half mediates formation of the polymer, an active conformer that is instantly resolved to trimers, or multimers thereof, by nucleic acid. Second, the protein has a biphasic effect on mismatched double-stranded DNA, a proxy chaperone substrate. It protects the duplex from dissociation at 37°C before eventually melting it when largely polymeric. Therefore, polymerization of ORF1p seemingly affects its interaction with nucleic acids. Additionally, polymerization of ORF1p at its translation site could explain the heretofore-inexplicable phenomenon of cis preference—the favored retrotransposition of the actively translated L1 transcript, which is essential for L1 survival. PMID:21937507

  19. Protein-surface interactions on stimuli-responsive polymeric biomaterials.

    PubMed

    Cross, Michael C; Toomey, Ryan G; Gallant, Nathan D

    2016-03-04

    Responsive surfaces: a review of the dependence of protein adsorption on the reversible volume phase transition in stimuli-responsive polymers. Specifically addressed are a widely studied subset: thermoresponsive polymers. Findings are also generalizable to other materials which undergo a similarly reversible volume phase transition. As of 2015, over 100,000 articles have been published on stimuli-responsive polymers and many more on protein-biomaterial interactions. Significantly, fewer than 100 of these have focused specifically on protein interactions with stimuli-responsive polymers. These report a clear trend of increased protein adsorption in the collapsed state compared to the swollen state. This control over protein interactions makes stimuli-responsive polymers highly useful in biomedical applications such as wound repair scaffolds, on-demand drug delivery, and antifouling surfaces. Outstanding questions are whether the protein adsorption is reversible with the volume phase transition and whether there is a time-dependence. A clear understanding of protein interactions with stimuli-responsive polymers will advance theoretical models, experimental results, and biomedical applications.

  20. Protein- and peptide-modified synthetic polymeric biomaterials.

    PubMed

    Krishna, Ohm D; Kiick, Kristi L

    2010-01-01

    This review presents an overview on bio-hybrid approaches of integrating the structural and functional features of proteins and peptides with synthetic polymers and the resulting unique properties in such hybrids, with a focus on bioresponsive/bioactive systems with biomaterials applications. The review is divided in two broad sections. First, we describe several examples of bio-hybrids produced by combining versatile synthetic polymers with proteins/enzymes and drugs that have resulted in (1) hybrid materials based on responsive polymers, (2) responsive hydrogels based on enzyme-catalyzed reactions, protein-protein interactions and protein-drug sensing, and (3) dynamic hydrogels based on conformational changes of a protein. Next, we present hybrids produced by combining synthetic polymers with peptides, classified based on the properties of the peptide domain: (1) peptides with different conformations, such as alpha-helical, coiled-coil, and beta-sheet; (2) peptides derived from structural protein domains such as silk, elastin, titin, and collagen; and (3) peptides with other biofunctional properties such as cell-binding domains and enzyme-recognized degradation domains. (c) 2010 Wiley Periodicals, Inc.

  1. Pearling instability of membrane tubes driven by curved proteins and actin polymerization

    NASA Astrophysics Data System (ADS)

    Jelerčič, U.; Gov, N. S.

    2015-12-01

    Membrane deformation inside living cells is crucial for the proper shaping of various intracellular organelles and is necessary during the fission/fusion processes that allow membrane recycling and transport (e.g. endocytosis). Proteins that induce membrane curvature play a key role in such processes, mostly by adsorbing to the membrane and forming a scaffold that deforms the membrane according to the curvature of the proteins. In this paper we explore the possibility of membrane tube destabilization through a pearling mechanism enabled by the combined effects of the adsorbed curved proteins and the actin polymerization that they recruit. The pearling instability can serve as the initiation for fission of the tube into vesicles. We find that adsorbed curved proteins are more likely to stabilize the tubes, while the actin polymerization can provide the additional constrictive force needed for the robust instability. We discuss the relevance of the theoretical results to in vivo and in vitro experiments.

  2. Polymerization of Quinone-Crosslinked Marine Bioadhesive Protein

    DTIC Science & Technology

    1988-10-05

    Rice-Ficht, A.C. (1987) Presclerotized eggshell protein from the liver fluke Fasciola hepatica . Biochem. 26, 7819-7825. Williams, T., Marumo, K...1988) A histidine-rich protein from the vitellarial cells of the liver fluke, Fasciola hepatica . To: J. Biol. Chem. Waite, J. H., Hansen, D. & Little, K...residues) separated from one another by a single glycine. In the trematode Fasciola , Dopas are commonly separated from one another by 2 amino acids (Glys

  3. Differential regulation of cortactin and N-WASP-mediated actin polymerization by missing in metastasis (MIM) protein.

    PubMed

    Lin, Jinxiu; Liu, Jiali; Wang, Ying; Zhu, Jianwei; Zhou, Kang; Smith, Nicole; Zhan, Xi

    2005-03-17

    Missing in metastasis (MIM) gene encodes an actin binding protein that is expressed at low levels in a subset of malignant cell lines. MIM protein tagged by green fluorescent protein (GFP) colocalizes with cortactin, an Arp2/3 complex activator, and interacts directly with the SH3 domain of cortactin. Recombinant full-length MIM promotes markedly cortactin and Arp2/3 complex-mediated actin polymerization in an SH3 dependent manner. In contrast, MIM-CT, a short splicing variant of MIM, binds poorly to cortactin in vitro and is unable to enhance actin polymerization. Full-length MIM binds to G-actin with a similar affinity as N-WASP-VCA, a constitutively active form of N-WASP, and inhibits N-WASP-VCA-mediated actin polymerization as analysed in vitro. The significance of the association of MIM with cortactin and G-actin was evaluated in NIH3T3 cells expressing several MIM constructs. Overexpression of full-length wild-type MIM-GFP inhibited markedly the motility of NIH3T3 cells induced by PDGF and that of human vein umbilical endothelial cells induced by sphingosine 1 phosphate. However, an MIM mutant with deletion of the WH2 domain, which is responsible for G-actin binding, enhanced cell motility. The motility inhibition imposed by MIM was compromised in the cells overexpressing N-WASP. In contrast, deletion of an MIM proline-rich domain, which is required for an optimal binding to cortactin, substantiated the MIM-mediated inhibition of cell motility. These data imply that MIM regulates cell motility by modulating different Arp2/3 activators in a distinguished manner.

  4. Protein- and Peptide-Modified Synthetic Polymeric Biomaterials

    PubMed Central

    Krishna, Ohm D.; Kiick, Kristi L.

    2015-01-01

    This review presents an overview on biohybrid approaches of integrating the structural and functional features of proteins and peptides with synthetic polymers and the resulting unique properties in such hybrids, with a focus on bioresponsive/bioactive systems with biomaterials applications. The review is divided in two broad sections. First, we describe several examples of biohybrids produced by combining versatile synthetic polymers with proteins/enzymes and drugs that have resulted in (1) hybrid materials based on responsive polymers, (2) responsive hydrogels based on enzyme-catalyzed reactions, protein–protein interactions and protein–drug sensing, and (3) dynamic hydrogels based on conformational changes of a protein. Next, we present hybrids produced by combining synthetic polymers with peptides, classified based on the properties of the peptide domain: (1) peptides with different conformations, such as α-helical, coiled-coil, and β-sheet; (2) peptides derived from structural protein domains such as silk, elastin, titin, and collagen; and (3) peptides with other biofunctional properties such as cell-binding domains and enzyme-recognized degradation domains. PMID:20091878

  5. Updates on smart polymeric carrier systems for protein delivery.

    PubMed

    El-Sherbiny, Ibrahim; Khalil, Islam; Ali, Isra; Yacoub, Magdi

    2017-10-01

    Smart materials are those materials that are responsive to chemical (organic molecules, chemical agents or specific agents), biochemical (protein, enzymes, growth factors, substrates or ligands), physical (electric field, magnetic field, temperature, pH, ionic strength or radiation) or mechanical (pressure or mechanical stress) signals. These responsive materials interact with the stimuli by changing their properties or conformational structures in a predictable manner. Recently, smart polymers have been utilized in various biomedical applications. Particularly, they have been used as a platform to synthesize stimuli-responsive systems that could deliver therapeutics to a specific site for a specific period with minimal adverse effects. For instance, stimuli-responsive polymers-based systems have been recently reported to deliver different bioactive molecules such as carbohydrates (heparin), chemotherapeutic agents (doxorubicin), small organic molecules (anti-coagulants), nucleic acids (siRNA), and proteins (growth factors and hormones). Protein therapeutics played a fundamental role in treatment of various chronic and some autoimmune diseases. For instance insulin has been used in treatment of diabetes. However, being a protein in nature, insulin delivery is limited by its instability, short half-life, and easy denaturation when administered orally. To overcome these challenges, and as highlighted in this review article, much research efforts have been recently devoted to design and develop convenient smart controlled nanosystems for protein therapeutics delivery.

  6. Bacterial DNA segregation dynamics mediated by the polymerizing protein ParF

    PubMed Central

    Barillà, Daniela; Rosenberg, Mark F; Nobbmann, Ulf; Hayes, Finbarr

    2005-01-01

    Prokaryotic DNA segregation most commonly involves members of the Walker-type ParA superfamily. Here we show that the ParF partition protein specified by the TP228 plasmid is a ParA ATPase that assembles into extensive filaments in vitro. Polymerization is potentiated by ATP binding and does not require nucleotide hydrolysis. Analysis of mutations in conserved residues of the Walker A motif established a functional coupling between filament dynamics and DNA partitioning. The partner partition protein ParG plays two separable roles in the ParF polymerization process. ParF is unrelated to prokaryotic polymerizing proteins of the actin or tubulin families, but is a homologue of the MinD cell division protein, which also assembles into filaments. The ultrastructures of the ParF and MinD polymers are remarkably similar. This points to an evolutionary parallel between DNA segregation and cytokinesis in prokaryotic cells, and reveals a potential molecular mechanism for plasmid and chromosome segregation mediated by the ubiquitous ParA-type proteins. PMID:15775965

  7. Caspase-11 promotes the fusion of phagosomes harboring pathogenic bacteria with lysosomes by modulating actin polymerization.

    PubMed

    Akhter, Anwari; Caution, Kyle; Abu Khweek, Arwa; Tazi, Mia; Abdulrahman, Basant A; Abdelaziz, Dalia H A; Voss, Oliver H; Doseff, Andrea I; Hassan, Hoda; Azad, Abul K; Schlesinger, Larry S; Wewers, Mark D; Gavrilin, Mikhail A; Amer, Amal O

    2012-07-27

    Inflammasomes are multiprotein complexes that include members of the NLR (nucleotide-binding domain leucine-rich repeat containing) family and caspase-1. Once bacterial molecules are sensed within the macrophage, the inflammasome is assembled, mediating the activation of caspase-1. Caspase-11 mediates caspase-1 activation in response to lipopolysaccharide and bacterial toxins, and yet its role during bacterial infection is unknown. Here, we demonstrated that caspase-11 was dispensable for caspase-1 activation in response to Legionella, Salmonella, Francisella, and Listeria. We also determined that active mouse caspase-11 was required for restriction of L. pneumophila infection. Similarly, human caspase-4 and caspase-5, homologs of mouse caspase-11, cooperated to restrict L. pneumophila infection in human macrophages. Caspase-11 promoted the fusion of the L. pneumophila vacuole with lysosomes by modulating actin polymerization through cofilin. However, caspase-11 was dispensable for the fusion of lysosomes with phagosomes containing nonpathogenic bacteria, uncovering a fundamental difference in the trafficking of phagosomes according to their cargo.

  8. Caspase-11 promotes the fusion of phagosomes harboring pathogenic bacteria with lysosomes by modulating actin polymerization

    PubMed Central

    Akhter, Anwari; Caution, Kyle; Khweek, Arwa Abu; Tazi, Mia; Abdulrahman, Basant A.; Abdelaziz, Dalia H.A.; Voss, Oliver H.; Doseff, Andrea I.; Hassan, Hoda; Azad, Abul K.; Schlesinger, Larry S.; Wewers, Mark D.; Gavrilin, Mikhail A.; Amer, Amal O.

    2012-01-01

    Summary Inflammasomes are multiprotein complexes that include members of the NLR (nucleotide-binding domain leucine-rich repeat containing) family and caspase-1. Once bacterial molecules are sensed within the macrophage, the inflammasome is assembled mediating the activation of caspase-1. Caspase-11 mediates caspase-1 activation in response to lipopolysaccharide and bacterial toxins. Yet, its role during bacterial infection is unknown. Here, we demonstrated that caspase-11 was dispensable for caspase-1 activation in response to Legionella, Salmonella, Francisella and Listeria. We also determined that active mouse caspase-11 was required for restriction of L. pneumophila infection. Similarly, human caspase-4 and 5, homologs of mouse caspase-11, cooperated to restrict L. pneumophila infection in human macrophages. Caspase-11 promoted the fusion of the L. pneumophila- vacuole with lysosomes by modulating actin polymerization through cofilin. However, caspase-11 was dispensable for the fusion of lysosomes with phagosomes containing non-pathogenic bacteria, uncovering a fundamental difference in the trafficking of phagosomes according to their cargo. PMID:22658523

  9. Practical considerations for preparing polymerized phospholipid bilayer capillary coatings for protein separations.

    PubMed

    Adem, Seid M; Mansfield, Elisabeth; Keogh, John P; Hall, Henry K; Aspinwall, Craig A

    2013-04-15

    Phosphorylcholine (PC) based phospholipid bilayers have proven useful as capillary coating materials due to their inherent resistance to non-specific protein adsorption. The primary limitation of this important class of capillary coatings remains the limited long-term chemical and physical stability of the coatings. Recently, a method for increasing phospholipid coating stability in fused silica capillaries via utilization of polymerized, synthetic phospholipids was reported. Here, we expand upon these studies by investigating polymerized lipid bilayer capillary coatings with respect to separation performance including run-to-run, day-to-day and column-to-column reproducibility and long-term stability. In addition, the effects of pH and capillary inner diameter on polymerized phospholipid coated capillaries were investigated to identify optimized coating conditions. The coatings are stabilized for protein separations across a wide range of pH values (4.0-9.3), a unique property for capillary coating materials. Additionally, smaller inner diameter capillaries (≤50 μm) were found to yield marked enhancements in coating stability and reproducibility compared to wider bore capillaries, demonstrating the importance of capillary size for separations employing polymerized phospholipid coatings. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. A polymeric bacterial protein activates dendritic cells via TLR4.

    PubMed

    Berguer, Paula M; Mundiñano, Juliana; Piazzon, Isabel; Goldbaum, Fernando A

    2006-02-15

    The enzyme lumazine synthase from Brucella spp. (BLS) is a highly immunogenic protein that folds as a stable dimer of pentamers. It is possible to insert foreign peptides and proteins at the 10 N terminus of BLS without disrupting its general folding, and these chimeras are very efficient to elicit systemic and oral immunity without adjuvants. In this study, we show that BLS stimulates bone marrow dendritic cells from mice in vitro to up-regulate the levels of costimulatory molecules (CD40, CD80, and CD86) and major histocompatibility class II Ag. Furthermore, the mRNA levels of several chemokines are increased, and proinflammatory cytokine secretion is induced upon exposure to BLS. In vivo, BLS increases the number of dendritic cells and their expression of CD62L in the draining lymph node. All of the observed effects are dependent on TLR4, and clearly independent of LPS contamination. The described characteristics of BLS make this protein an excellent candidate for vaccine development.

  11. Ethyl acetate extract from marine sponge Hyattella cribriformis exhibit potent anticancer activity by promoting tubulin polymerization as evidenced mitotic arrest and induction of apoptosis

    PubMed Central

    Annamalai, Pazhanimuthu; Thayman, Malini; Rajan, Sowmiya; Raman, Lakshmi Sundaram; Ramasubbu, Sankar; Perumal, Pachiappan

    2015-01-01

    Background: Marine sponges are important sources of bioactive compounds. Objective: This study investigated the anticancer properties of Hyattella cribriformis ethyl acetate (EA) fraction in various cancer and normal cell lines. Materials and Methods: anticancer assay was carried out in 15 cell lines to evaluate the anticancer potential of the EA fraction. Impact on cell cycle distribution was determined using flow cytometry. The fraction was investigated for interfering microtubules assembly in both in vitro and cellular assay. Further studies were conducted to determine the fraction induced cell death (apoptosis) using calcein/propidium iodide dual staining, activated caspase-3 and phosphorylation of Bcl-2 protein at Ser70. DNA fragmentation assay was performed to confirm the apoptosis. Results: EA fraction exhibited potent inhibition of cancer cell growth and resulted in 50% growth inhibition (GI50) of 0.27 μg/mL in A673 cell line. Sarcoma (MG-63, Saos-2) and ovarian (SK-OV-3 and OVCAR-3) cancer cell lines also showed superior anticancer activity GI50 of 1.0 μg/mL. Colon and breast cancer cell lines exhibited moderate GI compare other cancer cell lines and normal human lung fibroblast showed GI50 of 15.6 μg/mL. EA fraction showed potent G2/M phase arrest in A673 cell line and induced apoptosis at 48 h exposure. EA fraction promoted microtubule polymerization in tubulin polymerization assay and increased level of polymerized tubulin in the HeLa cells. Fraction induced the activation of caspase-3 and phosphorylation of Bcl-2 anti-apoptotic protein. Fraction induced DNA fragmentation in HeLa cells as evidence of apoptosis. Conclusion: Marine sponge H. cribriformis EA fraction exhibited potent anticancer activity through tubulin polymerization and induction of apoptosis. PMID:25829774

  12. Enhanced protein retention on poly(caprolactone) via surface initiated polymerization of acrylamide

    NASA Astrophysics Data System (ADS)

    Ma, Yuhao; Cai, Mengtan; He, Liu; Luo, Xianglin

    2016-01-01

    To enhance the biocompatibility or extend the biomedical application of poly(caprolactone) (PCL), protein retention on PCL surface is often required. In this study, poly(acrylamide) (PAAm) brushes were grown from PCL surface via surface-initiated atom transfer radical polymerization (SI-ATRP) and served as a protein-capturing platform. Grafted PAAm was densely packed on surface and exhibited superior protein retention ability. Captured protein was found to be resistant to washing under detergent environment. Furthermore, protein structure after being captured was investigated by circular dichroism (CD) spectroscopy, and the CD spectra verified that secondary structure of captured proteins was maintained, indicating no denaturation of protein happened for retention process.

  13. High throughput atmospheric pressure plasma-induced graft polymerization for identifying protein-resistant surfaces.

    PubMed

    Gu, Minghao; Kilduff, James E; Belfort, Georges

    2012-02-01

    Three critical aspects of searching for and understanding how to find highly resistant surfaces to protein adhesion are addressed here with specific application to synthetic membrane filtration. They include the (i) discovery of a series of previously unreported monomers from a large library of monomers with high protein resistance and subsequent low fouling characteristics for membrane ultrafiltration of protein-containing fluids, (ii) development of a new approach to investigate protein-resistant mechanisms from structure-property relationships, and (iii) adaptation of a new surface modification method, called atmospheric pressure plasma-induced graft polymerization (APP), together with a high throughput platform (HTP), for low cost vacuum-free synthesis of anti-fouling membranes. Several new high-performing chemistries comprising two polyethylene glycol (PEG), two amines and one zwitterionic monomers were identified from a library (44 commercial monomers) of five different classes of monomers as strong protein-resistant monomers. Combining our analysis here, using the Hansen solubility parameters (HSP) approach, and data from the literature, we conclude that strong interactions with water (hydrogen bonding) and surface flexibility are necessary for producing the highest protein resistance. Superior protein-resistant surfaces and subsequent anti-fouling performance was obtained with the HTP-APP as compared with our earlier HTP-photo graft-induced polymerization (PGP).

  14. Protein-Polymer Conjugates: Synthetic Approaches by Controlled Radical Polymerizations & Interesting Applications

    PubMed Central

    Grover, Gregory N.; Maynard, Heather D.

    2011-01-01

    Protein-polymer conjugates are of interest to researchers in diverse fields. Attachment of polymers to proteins results in improved pharmacokinetics, which is important in medicine. From an engineering standpoint, conjugates are exciting because they exhibit properties of both the biomolecules and synthetic polymers. This allows the activity of the protein to be altered or tuned, a key aspect in therapeutic design, anchoring conjugates to surfaces, and utilizing these materials for supramolecular self-assembly. Thus, there is broad interest in straightforward synthetic methods to make protein-polymer conjugates. Controlled radical polymerization (CRP) techniques have emerged as excellent strategies to make conjugates because the resulting polymers have narrow molecular weight distributions, targeted molecular weights, and attach to specific sites on proteins. Herein, recent advances in the synthesis and application of protein-polymer conjugates by CRP are highlighted. PMID:21071260

  15. Biomimetic Hydrogels Incorporating Polymeric Cell-Adhesive Peptide to Promote the 3D Assembly of Tumoroids

    PubMed Central

    Hao, Ying; Zerdoum, Aidan B.; Stuffer, Alexander J.; Rajasekaran, Ayyappan K.; Jia, Xinqiao

    2016-01-01

    Towards the goal of establishing physiologically relevant in vitro tumor models, we synthesized and characterized a biomimetic hydrogel using thiolated hyaluronic acid (HA-SH) and an acrylated copolymer carrying multiple copies of cell adhesive peptide (PolyRGD-AC). PolyRGD-AC was derived from a random copolymer of tert-butyl methacrylate (tBMA) and oligomeric (ethylene glycol) methacrylate (OEGMA), synthesized via atom transfer radical polymerization (ATRP). Acid hydrolysis of tert-butyl moieties revealed the carboxylates, through which acrylate groups were installed. Partial modification of the acrylate groups with a cysteine-containing RGD peptide generated PolyRGD-AC. When PolyRGD-AC was mixed with HA-SH under physiological conditions, a macroscopic hydrogel with an average elastic modulus of 630 Pa was produced. LNCaP prostate cancer cells encapsulated in HA-PolyRGD gels as dispersed single cells formed multicellular tumoroids by day 4 and reached an average diameter of ~95 μm by day 28. Cells in these structures were viable, formed cell-cell contacts through E-cadherin (E-CAD and displayed cortical organization of F-actin. Compared to the control gels prepared using PolyRDG, multivalent presentation of the RGD signal in the HA matrix increased cellular metabolism, promoted the development of larger tumoroids and enhanced the expression of E-CAD and integrins. Overall, hydrogels with multivalently immobilized RGD is a promising 3D culture platform for dissecting principles of tumorigenesis and for screening anticancer drugs. PMID:27723964

  16. The changes of proteins and polysaccharides in extracellular polymeric substance for Spirogyra fluviatilis under different salinity

    NASA Astrophysics Data System (ADS)

    Lee, Yichao; Chang, Shuiping

    2017-05-01

    Spirogyra is a genus of widely distributed, large green fresh water algae. This study discovered that changes in salinity can induce Spirogyra fluviatilis to produce amounts of extracellular polymeric substance (EPS) when controlling other environmental conditions. If culturing S. fluviatilis with salinity greater than a 3.0‰ medium for 4 hours, the secretion EPS will be changed. And the level of polysaccharides and proteins, the primary components of EPS, is slightly increased in accordance with the increase in the salinity. But the proteins to polysaccharides ratio changes are not significantly

  17. Chemical potential measurements of deoxyhemoglobin S polymerization. Determination of the phase diagram of an assembling protein.

    PubMed

    Prouty, M S; Schechter, A N; Parsegian, V A

    1985-08-05

    We have used the "osmotic stress" method to determine the phase diagram of deoxyhemoglobin S polymerization. This method involves equilibration, through a semipermeable membrane, of the protein with solutions of inert polymers of known osmotic pressure. With deoxyhemoglobin A and S solutions, in which we have demonstrated achievement of equilibrium, plots of osmotic pressure versus concentration initially agree closely with the results of other methods of measurement of colligative properties. However, once the known solubility value is exceeded for the deoxyhemoglobin S solutions at various temperatures, there is a rapid rise in hemoglobin concentration over a narrow osmotic pressure range and then a more gradual increase in concentration. We believe that these two regions correspond, respectively, to the onset of the polymerization process, and of subsequent continuing growth and compression or alignment of polymer. We derive the thermodynamic values for these processes and show that the behavior of the deoxyhemoglobin S system is analogous to the phase transition for a simple chemical system. These results are relevant to understanding the intracellular polymerization of deoxyhemoglobin S in sickle cell disease, and these concepts are applicable to other protein assembly systems.

  18. 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.

  19. A Steric Antagonism of Actin Polymerization by a Salmonella Virulence Protein

    SciTech Connect

    Margarit,S.; Davidson, W.; Frego, L.; Stebbins, F.

    2006-01-01

    Salmonella spp. require the ADP-ribosyltransferase activity of the SpvB protein for intracellular growth and systemic virulence. SpvB covalently modifies actin, causing cytoskeletal disruption and apoptosis. We report here the crystal structure of the catalytic domain of SpvB, and we show by mass spectrometric analysis that SpvB modifies actin at Arg177, inhibiting its ATPase activity. We also describe two crystal structures of SpvB-modified, polymerization-deficient actin. These structures reveal that ADP-ribosylation does not lead to dramatic conformational changes in actin, suggesting a model in which this large family of toxins inhibits actin polymerization primarily through steric disruption of intrafilament contacts.

  20. Engineering of the E. coli outer membrane protein FhuA to overcome the hydrophobic mismatch in thick polymeric membranes.

    PubMed

    Muhammad, Noor; Dworeck, Tamara; Fioroni, Marco; Schwaneberg, Ulrich

    2011-03-17

    Channel proteins like the engineered FhuA Δ1-159 often cannot insert into thick polymeric membranes due to a mismatch between the hydrophobic surface of the protein and the hydrophobic surface of the polymer membrane. To address this problem usually specific block copolymers are synthesized to facilitate protein insertion. Within this study in a reverse approach we match the protein to the polymer instead of matching the polymer to the protein. To increase the FhuA Δ1-159 hydrophobic surface by 1 nm, the last 5 amino acids of each of the 22 β-sheets, prior to the more regular periplasmatic β-turns, were doubled leading to an extended FhuA Δ1-159 (FhuA Δ1-159 Ext). The secondary structure prediction and CD spectroscopy indicate the β-barrel folding of FhuA Δ1-159 Ext. The FhuA Δ1-159 Ext insertion and functionality within a nanocontainer polymeric membrane based on the triblock copolymer PIB(1000)-PEG(6000)-PIB(1000) (PIB = polyisobutylene, PEG = polyethyleneglycol) has been proven by kinetic analysis using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine). Identical experiments with the unmodified FhuA Δ1-159 report no kinetics and presumably no insertion into the PIB(1000)-PEG(6000)-PIB(1000) membrane. Furthermore labeling of the Lys-NH(2) groups present in the FhuA Δ1-159 Ext channel, leads to controllability of in/out flux of substrates and products from the nanocontainer. Using a simple "semi rational" approach the protein's hydrophobic transmembrane region was increased by 1 nm, leading to a predicted lower hydrophobic mismatch between the protein and polymer membrane, minimizing the insertion energy penalty. The strategy of adding amino acids to the FhuA Δ1-159 Ext hydrophobic part can be further expanded to increase the protein's hydrophobicity, promoting the efficient embedding into thicker/more hydrophobic block copolymer membranes.

  1. A method to tune the shape of protein-encapsulated polymeric microspheres

    NASA Astrophysics Data System (ADS)

    Alteriis, Renato De; Vecchione, Raffaele; Attanasio, Chiara; Gregorio, Maria De; Porzio, Massimiliano; Battista, Edmondo; Netti, Paolo A.

    2015-07-01

    Protein encapsulation technologies of polymeric microspheres currently in use have been optimized to effectively protect their “protein cargo” from inactivation occurring in biological environments, preserving its bioactivity during release up to several weeks. The scenario of protein delivery would greatly benefit by strategies enabling the production of non-spherical particles. Herein we report an easy and effective stamp-based method to produce poly-lactic-glycolic-acid (PLGA) microparticles encapsulating Vascular Endothelial Growth Factor (VEGF) of different shapes. We demonstrate that PLGA microspheres can be deformed at room temperature exploiting solvent/non-solvent plasticization in order to preserve the properties of the starting microspheres. This gentle method allows the production of shaped particles that provide a prolonged release of VEGF in active form, as verified by an angiogenic assay. The retention of the biological activity of an extremely labile molecule, i.e. VEGF, lets us hypothesize that a wide variety of drug and protein encapsulated polymeric microspheres can be processed using this method.

  2. Automated frit inlet/frit outlet flow field-flow fractionation for protein characterization with emphasis on polymeric wheat proteins.

    PubMed

    Stevenson, S G; Ueno, T; Preston, K R

    1999-01-01

    A flow field-flow fractionation (FFF) unit fitted with a 254-nm spacer, frit inlet (FI), and frit outlet (FO) was automated for protein analysis by addition of a system controller, autosampler, and computer software to control pumps, sample loading, and data capture. Standard molecular size marker proteins and polymeric wheat storage protein extracts were used to assess the performance of the automated unit. Optimum resolution for these proteins was obtained with a sample inlet flow of 0.2 mL/min, a frit inlet flow (recirculating) of 1.4 mL/min, and a cross-flow (recirculating) of 5 mL/min using 0.05 M acetic acid containing 0.002% FL-70 as a carrier. Use of the FIFO FFF eliminates the requirement for stop-flow relaxation and pressure balancing, results in better reproducibility, and generates a 7-10-fold increase in sensitivity at the detector by concentrating fractions eluting from the channel. These improvements resulted in superior resolution of polymeric wheat protein fractions compared to those obtained previously using a standard channel with manual load and stop-flow relaxation, allowing accurate integration of peak or size range areas. Automation of this system allows unattended sample fractionation and hence markedly increases potential for sample throughput.

  3. Biophysical inhibition of pulmonary surfactant function by polymeric nanoparticles: role of surfactant protein B and C.

    PubMed

    Beck-Broichsitter, Moritz; Ruppert, Clemens; Schmehl, Thomas; Günther, Andreas; Seeger, Werner

    2014-11-01

    The current study investigated the mechanisms involved in the process of biophysical inhibition of pulmonary surfactant by polymeric nanoparticles (NP). The minimal surface tension of diverse synthetic surfactants was monitored in the presence of bare and surface-decorated (i.e. poloxamer 407) sub-100 nm poly(lactide) NP. Moreover, the influence of NP on surfactant composition (i.e. surfactant protein (SP) content) was studied. Dose-elevations of SP advanced the biophysical activity of the tested surfactant preparation. Surfactant-associated protein C supplemented phospholipid mixtures (PLM-C) were shown to be more susceptible to biophysical inactivation by bare NP than phospholipid mixture supplemented with surfactant protein B (PLM-B) and PLM-B/C. Surfactant function was hindered owing to a drastic depletion of the SP content upon contact with bare NP. By contrast, surface-modified NP were capable of circumventing unwanted surfactant inhibition. Surfactant constitution influences the extent of biophysical inhibition by polymeric NP. Steric shielding of the NP surface minimizes unwanted NP-surfactant interactions, which represents an option for the development of surfactant-compatible nanomedicines. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  4. Generation effects on the microstructure and product distribution in ethylene polymerization promoted by dendritic nickel catalysts.

    PubMed

    Benito, José M; de Jesús, Ernesto; de la Mata, F Javier; Flores, Juan C; Gómez, Rafael

    2005-11-07

    Carbosilane dendrimers Gn-[(ONNMe2)NiBr2]m, containing up to sixteen terminal pyridylimine nickel complexes, have been studied as catalysts for polymerization of ethylene; the microstructure and the oligomer/polymer distribution are significantly affected by the generation of the dendritic precursor.

  5. Bacillus anthracis SlaQ Promotes S-Layer Protein Assembly

    PubMed Central

    Nguyen-Mau, Sao-Mai; Oh, So-Young; Schneewind, Daphne I.; Missiakas, Dominique

    2015-01-01

    ABSTRACT Bacillus anthracis vegetative forms assemble an S-layer comprised of two S-layer proteins, Sap and EA1. A hallmark of S-layer proteins are their C-terminal crystallization domains, which assemble into a crystalline lattice once these polypeptides are deposited on the bacterial surface via association between their N-terminal S-layer homology domains and the secondary cell wall polysaccharide. Here we show that slaQ, encoding a small cytoplasmic protein conserved among pathogenic bacilli elaborating S-layers, is required for the efficient secretion and assembly of Sap and EA1. S-layer protein precursors cosediment with SlaQ, and SlaQ appears to facilitate Sap assembly. Purified SlaQ polymerizes and when mixed with purified Sap promotes the in vitro formation of tubular S-layer structures. A model is discussed whereby SlaQ, in conjunction with S-layer secretion factors SecA2 and SlaP, promotes localized secretion and S-layer assembly in B. anthracis. IMPORTANCE S-layer proteins are endowed with the propensity for self-assembly into crystalline arrays. Factors promoting S-layer protein assembly have heretofore not been reported. We identified Bacillus anthracis SlaQ, a small cytoplasmic protein that facilitates S-layer protein assembly in vivo and in vitro. PMID:26216847

  6. Bacillus anthracis SlaQ Promotes S-Layer Protein Assembly.

    PubMed

    Nguyen-Mau, Sao-Mai; Oh, So-Young; Schneewind, Daphne I; Missiakas, Dominique; Schneewind, Olaf

    2015-10-01

    Bacillus anthracis vegetative forms assemble an S-layer comprised of two S-layer proteins, Sap and EA1. A hallmark of S-layer proteins are their C-terminal crystallization domains, which assemble into a crystalline lattice once these polypeptides are deposited on the bacterial surface via association between their N-terminal S-layer homology domains and the secondary cell wall polysaccharide. Here we show that slaQ, encoding a small cytoplasmic protein conserved among pathogenic bacilli elaborating S-layers, is required for the efficient secretion and assembly of Sap and EA1. S-layer protein precursors cosediment with SlaQ, and SlaQ appears to facilitate Sap assembly. Purified SlaQ polymerizes and when mixed with purified Sap promotes the in vitro formation of tubular S-layer structures. A model is discussed whereby SlaQ, in conjunction with S-layer secretion factors SecA2 and SlaP, promotes localized secretion and S-layer assembly in B. anthracis. S-layer proteins are endowed with the propensity for self-assembly into crystalline arrays. Factors promoting S-layer protein assembly have heretofore not been reported. We identified Bacillus anthracis SlaQ, a small cytoplasmic protein that facilitates S-layer protein assembly in vivo and in vitro. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. A polymeric nanocoating on carbon nanotube arrays for developing imprinted protein sensor

    NASA Astrophysics Data System (ADS)

    Ren, L.; Zhao, H. Z.; Xu, C. J.; Yu, Y.; Wang, H. Z.; Lan, Y. C.; Wagner, D.; Naughton, M. J.; Ren, Z. F.; Chiles, T. C.; Cai, D.

    2010-03-01

    Polyphenol (PPn) was electrodeposited on carbon nanotubes (CNT) arrays at nanoscale thickness. PPn is a non-conductive polymer, so increase of sensor impedance was observed with high density CNT array, low density CNT array, and tip-polished CNT array (tCNTA), while tCNTA was determined to be the best nanosensor platform to incorporate the imprinted PPn coating due to the highest impedance increase. The PPn was characterized by transmission electron microscopy, electrochemical impedance spectroscopy and cyclic voltammetry for its thickness, uniformity, stability, resistivity and permittivity etc., as well as the protein entrapment and removal process. The density of the imprint was also evaluated by a PPn refilling experiment. Finally, ferritin was used as the template to develop a highly sensitive and selective protein nanosensor. Therefore, a novel strategy was demonstrated here to deposit and characterize polymeric nanocoating, also to evaluate imprints and detect proteins.

  8. Polymeric strong cation-exchange monolithic column for capillary liquid chromatography of peptides and proteins.

    PubMed

    Chen, Xin; Tolley, H Dennis; Lee, Milton L

    2009-08-01

    A strong cation-exchange (SCX) monolithic stationary phase was prepared in 75 microm id capillaries by direct in situ polymerization of sulfopropyl methacrylate and polyethylene glycol diacrylate in a ternary porogen system consisting of methanol, cyclohexanol, and water. The resulting monolith exhibited good dynamic binding capacity, fast kinetic adsorption of proteins, and high permeability. The monolith had a dynamic binding capacity of approximately 52 mg/mL of column volume for lysozyme and cytochrome C. The monolith was evaluated for SCX capillary LC of synthetic peptides, natural peptides, and protein standards. Fast separation of proteins was achieved in less than 4 min. The average peak capacity for peptides was 28 using a relatively steep gradient when hydrophobic interactions were suppressed with 40% acetonitrile.

  9. Competitive protein adsorption to soft polymeric layers: binary mixtures and comparison to theory.

    PubMed

    Oberle, Michael; Yigit, Cemil; Angioletti-Uberti, Stefano; Dzubiella, Joachim; Ballauff, Matthias

    2015-02-19

    Nanoparticles immersed in biological fluids readily adsorb proteins. The protein corona thus generated on the surface of the particles largely determines their biological fate. Since biological fluids, e.g., blood plasma, contain a large number of proteins, competitive adsorption must be considered. We study the competitive adsorption of lysozyme, cytochrome c, papain, and RNase A onto a soft charged polymeric layer. The experimental data of binary protein mixtures are compared to a theoretical model taking into account electrostatic and hydrophobic interactions between the proteins and the network. The interactions between bound proteins are modeled within a second virial approximation. The model possesses full generality and can be applied to the adsorption of an arbitrary number of protein types. The parameters describing the adsorption of a single protein type are obtained by isothermal titration calorimetry (ITC), while the competitive adsorption of a binary mixture is studied by fluorescence spectroscopy. The competitive adsorption can be predicted from the data related to the adsorption of the single types without adjustable parameters.

  10. Reversible Interactions of Proteins with Mixed Shell Polymeric Micelles: Tuning the Surface Hydrophobic/Hydrophilic Balance toward Efficient Artificial Chaperones.

    PubMed

    Wang, Jianzu; Song, Yiqing; Sun, Pingchuan; An, Yingli; Zhang, Zhenkun; Shi, Linqi

    2016-03-22

    Molecular chaperones can elegantly fine-tune its hydrophobic/hydrophilic balance to assist a broad spectrum of nascent polypeptide chains to fold properly. Such precious property is difficult to be achieved by chaperone mimicking materials due to limited control of their surface characteristics that dictate interactions with unfolded protein intermediates. Mixed shell polymeric micelles (MSPMs), which consist of two kinds of dissimilar polymeric chains in the micellar shell, offer a convenient way to fine-tune surface properties of polymeric nanoparticles. In the current work, we have fabricated ca. 30 kinds of MSPMs with finely tunable hydrophilic/hydrophobic surface properties. We investigated the respective roles of thermosensitive and hydrophilic polymeric chains in the thermodenaturation protection of proteins down to the molecular structure. Although the three kinds of thermosensitive polymers investigated herein can form collapsed hydrophobic domains on the micellar surface, we found distinct capability to capture and release unfolded protein intermediates, due to their respective affinity for proteins. Meanwhile, in terms of the hydrophilic polymeric chains in the micellar shell, poly(ethylene glycol) (PEG) excels in assisting unfolded protein intermediates to refold properly via interacting with the refolding intermediates, resulting in enhanced chaperone efficiency. However, another hydrophilic polymer-poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) severely deteriorates the chaperone efficiency of MSPMs, due to its protein-resistant properties. Judicious combination of thermosensitive and hydrophilic chains in the micellar shell lead to MSPM-based artificial chaperones with optimal efficacy.

  11. Polymeric adsorbent for removing toxic proteins from blood of patients with kidney failure.

    PubMed

    Davankov, V; Pavlova, L; Tsyurupa, M; Brady, J; Balsamo, M; Yousha, E

    2000-02-28

    A hypercrosslinked styrenic polymer with an enhanced proportion of mesopores in the range 2-20 nm has been developed. The principle of the synthesis consists of the suspension polymerization of divinylbenzene (or copolymerization of styrene with divinylbenzene) in the presence of a porogen that is a theta-solvent for polystyrene. On the scale of thermodynamic affinity, theta-solvents occupy a border position between good solvents and precipitating media for the growing polymer chains. In this case, microphase separation takes place during the final stages of the polymerization process. The polymer was shown to adsorb 93-98% of beta2-microglobulin from the blood or plasma of patients with chronic kidney failure. At the same time, large essential proteins, like albumin, are not removed to a significant extent, obviously, due to the size-exclusion effect and the difference in the hydrophobicity of the proteins. By replacing surface exposed pendant vinyl groups of the polymer with hydrophilic functional groups, the material was made hemocompatible, according to the standard battery of biocompatibility tests required by ISO 10993 guidelines. No adverse effects such as fever or hypotension were noted in dogs in direct hemoperfusion experiments with the polymer.

  12. A Visible Light Initiating System for Free Radical Promoted Cationic Polymerization

    DTIC Science & Technology

    1994-02-02

    bis-(4, 4’-dimethylaminoihenyl)methane as the main product and crystal violet, methyl violet, as well as the leuco rorms of these dyes as minor...cation in chain polymerization is reported. The system is based on electron transfer photoreduction of xanthene dye triplets by aromatic amines...Previously, Crivello and Lam 9 had reported attempts to use visible light absorbing dyes to sensitize the decomposition of iodonium salts to initiate

  13. Promotion of the halide effect in the formation of shaped metal nanocrystals via a hybrid cationic, polymeric stabilizer: Octahedra, cubes, and anisotropic growth

    NASA Astrophysics Data System (ADS)

    Sneed, Brian T.; Golden, Matthew C.; Liu, Yejing; Lee, Hiang K.; Andoni, Ilektra; Young, Allison P.; McMahon, Greg; Erdman, Natasha; Shibata, Masateru; Ling, Xing Yi; Tsung, Chia-Kuang

    2016-06-01

    To promote the effect of halide ions (Cl-, Br-, and I-) in facet-selective growth of {111} and {100} of shaped metal nanocrystals, we utilize PDADMAC, a hybrid cationic, polymeric stabilizer. SERS and synthesis experiments provide evidence supporting that the higher amount of PDADMA+ at surfaces promotes the local adsorption of halides, allowing the creation of Pd cubes, octahedra, and cuboctopods.

  14. 'Catalysts' for polyacrylamide gel polymerization and detection of proteins by silver staining.

    PubMed

    Hochstrasser, D F; Merril, C R

    1988-01-01

    The crosslinker diacrylyl-piperazine produces polyacrylamide gels which display improved electrophoretic separation of proteins and better physical strength. It also produces gels with improved detection of proteins by ammoniacal silver staining by reducing the background. This reduced background provided us with an opportunity to investigate residual background staining caused by the catalytic reagents utilized in the polymerization of acrylamide gels. The commonly used catalyst system, tetramethyl-ethylenediamine and ammonium persulfate was shown to be responsible for the yellow staining background found after a prolonged development time with silver staining. An alternate catalyst system has been designed to decrease further the formation of this background staining. Dimethyl-piperazine or tetramethylethylenediamine, potassium or ammonium persulfate, and sodium thiosulfate are shown to provide for gels which have excellent mechanical and staining characteristics. These catalytic systems produce little background staining despite prolonged development time with the ammoniacal silver stain, and they reduce background staining with the dichromate silver stain.

  15. Stimuli-Responsive Polymeric Systems for Controlled Protein and Peptide Delivery: Future Implications for Ocular Delivery.

    PubMed

    Mahlumba, Pakama; Choonara, Yahya E; Kumar, Pradeep; du Toit, Lisa C; Pillay, Viness

    2016-07-30

    Therapeutic proteins and peptides have become notable in the drug delivery arena for their compatibility with the human body as well as their high potency. However, their biocompatibility and high potency does not negate the existence of challenges resulting from physicochemical properties of proteins and peptides, including large size, short half-life, capability to provoke immune responses and susceptibility to degradation. Various delivery routes and delivery systems have been utilized to improve bioavailability, patient acceptability and reduce biodegradation. The ocular route remains of great interest, particularly for responsive delivery of macromolecules due to the anatomy and physiology of the eye that makes it a sensitive and complex environment. Research in this field is slowly gaining attention as this could be the breakthrough in ocular drug delivery of macromolecules. This work reviews stimuli-responsive polymeric delivery systems, their use in the delivery of therapeutic proteins and peptides as well as examples of proteins and peptides used in the treatment of ocular disorders. Stimuli reviewed include pH, temperature, enzymes, light, ultrasound and magnetic field. In addition, it discusses the current progress in responsive ocular drug delivery. Furthermore, it explores future prospects in the use of stimuli-responsive polymers for ocular delivery of proteins and peptides. Stimuli-responsive polymers offer great potential in improving the delivery of ocular therapeutics, therefore there is a need to consider them in order to guarantee a local, sustained and ideal delivery of ocular proteins and peptides, evading tissue invasion and systemic side-effects.

  16. Nucleocapsid protein structures from orthobunyaviruses reveal insight into ribonucleoprotein architecture and RNA polymerization.

    PubMed

    Ariza, Antonio; Tanner, Sian J; Walter, Cheryl T; Dent, Kyle C; Shepherd, Dale A; Wu, Weining; Matthews, Susan V; Hiscox, Julian A; Green, Todd J; Luo, Ming; Elliott, Richard M; Fooks, Anthony R; Ashcroft, Alison E; Stonehouse, Nicola J; Ranson, Neil A; Barr, John N; Edwards, Thomas A

    2013-06-01

    All orthobunyaviruses possess three genome segments of single-stranded negative sense RNA that are encapsidated with the virus-encoded nucleocapsid (N) protein to form a ribonucleoprotein (RNP) complex, which is uncharacterized at high resolution. We report the crystal structure of both the Bunyamwera virus (BUNV) N-RNA complex and the unbound Schmallenberg virus (SBV) N protein, at resolutions of 3.20 and 2.75 Å, respectively. Both N proteins crystallized as ring-like tetramers and exhibit a high degree of structural similarity despite classification into different orthobunyavirus serogroups. The structures represent a new RNA-binding protein fold. BUNV N possesses a positively charged groove into which RNA is deeply sequestered, with the bases facing away from the solvent. This location is highly inaccessible, implying that RNA polymerization and other critical base pairing events in the virus life cycle require RNP disassembly. Mutational analysis of N protein supports a correlation between structure and function. Comparison between these crystal structures and electron microscopy images of both soluble tetramers and authentic RNPs suggests the N protein does not bind RNA as a repeating monomer; thus, it represents a newly described architecture for bunyavirus RNP assembly, with implications for many other segmented negative-strand RNA viruses.

  17. Actin-binding Protein Drebrin Regulates HIV-1-triggered Actin Polymerization and Viral Infection*

    PubMed Central

    Gordón-Alonso, Mónica; Rocha-Perugini, Vera; Álvarez, Susana; Ursa, Ángeles; Izquierdo-Useros, Nuria; Martinez-Picado, Javier; Muñoz-Fernández, María A.; Sánchez-Madrid, Francisco

    2013-01-01

    HIV-1 contact with target cells triggers F-actin rearrangements that are essential for several steps of the viral cycle. Successful HIV entry into CD4+ T cells requires actin reorganization induced by the interaction of the cellular receptor/co-receptor complex CD4/CXCR4 with the viral envelope complex gp120/gp41 (Env). In this report, we analyze the role of the actin modulator drebrin in HIV-1 viral infection and cell to cell fusion. We show that drebrin associates with CXCR4 before and during HIV infection. Drebrin is actively recruited toward cell-virus and Env-driven cell to cell contacts. After viral internalization, drebrin clustering is retained in a fraction of the internalized particles. Through a combination of RNAi-based inhibition of endogenous drebrin and GFP-tagged expression of wild-type and mutant forms, we establish drebrin as a negative regulator of HIV entry and HIV-mediated cell fusion. Down-regulation of drebrin expression promotes HIV-1 entry, decreases F-actin polymerization, and enhances profilin local accumulation in response to HIV-1. These data underscore the negative role of drebrin in HIV infection by modulating viral entry, mainly through the control of actin cytoskeleton polymerization in response to HIV-1. PMID:23926103

  18. Polymeric biomaterials for nerve regeneration applications: From promoting cellular organization to the delivery of bioactive molecules

    NASA Astrophysics Data System (ADS)

    Delgado-Rivera, Roberto L.

    Thousands of new cases of injury to the central nervous system (CNS) occur each year in the USA and all over the world. However, despite recent advances, at present there is no cure for the resulting paraplegia or quadriplegia. This research is directed towards engineering biomaterial platforms to promote cellular organization at the surface of polymer scaffolds that will be conducive to proper regeneration of injured CNS. In addition, the formulation of a delivery system for neuroactive molecules using polymer-based materials will be evaluated to establish its potential to treat CNS disorders. Initial studies involved the chemical modification of an electrospun nonwoven matrix of nanofibers with fibroblast growth factor 2 (FGF-2). Nanofibers alone up-regulated FGF-2, albeit to a lesser extent than nanofibers covalently modified with FGF-2. These results underscore the importance of both surface topography and growth factor presentation on cellular function. Moreover, that FGF-2 modified nanofibrillar scaffolds may demonstrate utility in tissue engineering applications for replacement and regeneration of damaged tissue following CNS injury or disease. Subsequent research efforts focused on a novel micropatterning technique called microscale plasma-initiated patterning (microPIP). This patterning method uses a polydimethylsiloxane (PDMS) stamp to selectively protect regions of an underlying substrate from oxygen plasma treatment resulting in hydrophobic and hydrophilic regions. FGF-2 and laminin-1 were applied to an electrospun polyamide nanofibrillar matrix following plasma treatment. In this work it, was possible to demonstrate that textured surfaces, such as nanofibrillar scaffolds, can be micropatterned to provide external chemical cues for cellular organization. Finally, a microsphere system capable of encapsulating proteins while minimizing the mechanisms of protein degradation and providing a controlled release was investigated. Microspheres were comprised of

  19. Cyclase-associated proteins: CAPacity for linking signal transduction and actin polymerization.

    PubMed

    Hubberstey, Andrew V; Mottillo, Emilio P

    2002-04-01

    Many extracellular signals elicit changes in the actin cytoskeleton, which are mediated through an array of signaling proteins and pathways. One family of proteins that plays a role in regulating actin remodeling in response to cellular signals are the cyclase-associated proteins (CAPs). CAPs are highly conserved monomeric actin binding proteins present in a wide range of organisms including yeast, fly, plants, and mammals. The original CAP was isolated as a component of the Saccharomyces cerevisiae adenylyl cyclase complex that serves as an effector of Ras during nutritional signaling. CAPs are multifunctional molecules that contain domains involved in actin binding, adenylyl cyclase association in yeast, SH3 binding, and oligomerization. Genetic studies in yeast have implicated CAPs in vesicle trafficking and endocytosis. CAPs play a developmental role in multicellular organisms, and studies of Drosophila have illuminated the importance of the actin cytoskeleton during eye development and in establishing oocyte polarity. This review will highlight the critical structural and functional domains of CAPs, describe recent studies that have implied important roles for these proteins in linking cell signaling with actin polymerization, and highlight their roles in vesicle trafficking and development.

  20. The fabrication of superlow protein absorption zwitterionic coating by electrochemically mediated atom transfer radical polymerization and its application.

    PubMed

    Hu, Yichuan; Yang, Guang; Liang, Bo; Fang, Lu; Ma, Guanglong; Zhu, Qin; Chen, Shengfu; Ye, Xuesong

    2015-02-01

    A well-controllable electrochemically mediated surface-initiated atom transfer radical polymerization (e-siATRP) method for the fabrication of superlow protein absorption zwitterionic hydrogel coatings based on poly(sulbetaine methacrylate) (pSBMA) was developed in this work. The effects of the electric condition on polymerization as well as its antifouling performances both in vitro and in vivo were also investigated. Different potentials (-0.08 V, -0.15 V and -0.22 V) and polymerization times (from 8 to 48 h) were chosen to study the polymerization procedure. X-ray photoelectron spectroscopy, atomic force microscopy and ellipsometry measurements were used to characterize the properties of the polymer layers. Ellipsometry measurements showed that a higher potential provided faster polymerization and thicker polymer layers; however, the protein absorption experiments showed that the best polymerization condition was under a constant potential of -0.15 V and 32 h, under which the protein absorption was 0.8% in an enzyme-linked immunosorbent assay (compared to a bare gold electrode). The electrodes with a pSBMA coating effectively deduced the current sensitivity decay both in undiluted serum and in vivo. The usage of the commercially available polymerization monomer of SBMA, the simple convenient synthesis process regardless of the presence of oxygen and the excellent controllability of e-siATRP make it a very promising and universal technique in the preparation of zwitterionic polymer coatings, especially in the development of biocompatible material for implantable devices such as neural and biosensor electrodes.

  1. Promoters and proteins from Clostridium thermocellum and uses thereof

    DOEpatents

    Wu, J. H. David; Newcomb, Michael

    2012-11-13

    The present invention relates to an inducible and a high expression nucleic acid promoter isolated from Clostridium thermocellum. These promoters are useful for directing expression of a protein or polypeptide encoded by a nucleic acid molecule operably associated with the nucleic acid promoters. The present invention also relates to nucleic acid constructs including the C. thermocellum promoters, and expression vectors and hosts containing such nucleic acid constructs. The present invention also relates to protein isolated from Clostridium thermocellum, including a repressor protein. The present invention also provides methods of using the isolated promoters and proteins from Clostridium thermocellum, including methods for directing inducible in vitro and in vivo expression of a protein or polypeptide in a host, and methods of producing ethanol from a cellulosic biomass.

  2. Enhanced Degradation of Misfolded Proteins Promotes Tumorigenesis.

    PubMed

    Chen, Liang; Brewer, Michael D; Guo, Lili; Wang, Ruoxing; Jiang, Peng; Yang, Xiaolu

    2017-03-28

    An adequate cellular capacity to degrade misfolded proteins is critical for cell survival and organismal health. A diminished capacity is associated with aging and neurodegenerative diseases; however, the consequences of an enhanced capacity remain undefined. Here, we report that the ability to clear misfolded proteins is increased during oncogenic transformation and is reduced upon tumor cell differentiation. The augmented capacity mitigates oxidative stress associated with oncogenic growth and is required for both the initiation and maintenance of malignant phenotypes. We show that tripartite motif-containing (TRIM) proteins select misfolded proteins for proteasomal degradation. The higher degradation power in tumor cells is attributed to the upregulation of the proteasome and especially TRIM proteins, both mediated by the antioxidant transcription factor Nrf2. These findings establish a critical role of TRIMs in protein quality control, connect the clearance of misfolded proteins to antioxidant defense, and suggest an intrinsic characteristic of tumor cells.

  3. Characterization of poly(allylamine) as a polymeric ligand for ion-exchange protein chromatography.

    PubMed

    Li, Ming; Li, Yanying; Yu, Linling; Sun, Yan

    2017-02-24

    This work reports poly(allylamine) (PAA), as a polymeric ion-exchange ligand for protein chromatography. Sepharose FF was modified with PAA, and six anion exchangers with ionic capacities (ICs) from 165 to 618mmol/L were prepared. Inverse size exclusion chromatography, adsorption equilibrium, uptake kinetics and column elution were performed. It was found that both the adsorption capacity and effective diffusivity maintained low values in the IC range of 165-373mmol/L, but they started to increase beyond 373mmol/L, and increased by 80% and 23 times, respectively, when the IC reached 618mmol/L. Interestingly, a drastic decrease of pore size was observed around the IC of 373mmol/L. The results suggest that the PAA chains played an important role in protein adsorption by altering the inner pore structure of the gels. It is considered that, PAA chains turn from inextensible states with multipoint-grafting on the pore surface at low coupling densities (IC<373mmol/L) to closer, extended and flexible grafting states with less coupling points at higher coupling densities (IC>373mmol/L). These characters of the grafted chains at higher IC values benefit in protein adsorption by three-dimensional binding and encouraged the happening of "chain delivery" of bound proteins on the chains. Besides, the ion exchangers showed favorable adsorption and uptake properties in a wide ionic strength range, 0-500mmol/L NaCl, indicating much better salt tolerance feature than the so-far reported ion exchangers. Moreover, a mild condition of pH 5.0 offered effective recovery of bound proteins in elution chromatography. The results indicate that the PAA-based anion exchanger of a high IC value is promising for high-capacity protein chromatography dealing with feedstock of a wide range of ionic strengths.

  4. The β2 clamp in the Mycobacterium tuberculosis DNA polymerase III αβ2ε replicase promotes polymerization and reduces exonuclease activity

    PubMed Central

    Gu, Shoujin; Li, Wenjuan; Zhang, Hongtai; Fleming, Joy; Yang, Weiqiang; Wang, Shihua; Wei, Wenjing; Zhou, Jie; Zhu, Guofeng; Deng, Jiaoyu; Hou, Jian; Zhou, Ying; Lin, Shiqiang; Zhang, Xian-En; Bi, Lijun

    2016-01-01

    DNA polymerase III (DNA pol III) is a multi-subunit replication machine responsible for the accurate and rapid replication of bacterial genomes, however, how it functions in Mycobacterium tuberculosis (Mtb) requires further investigation. We have reconstituted the leading-strand replication process of the Mtb DNA pol III holoenzyme in vitro, and investigated the physical and functional relationships between its key components. We verify the presence of an αβ2ε polymerase-clamp-exonuclease replicase complex by biochemical methods and protein-protein interaction assays in vitro and in vivo and confirm that, in addition to the polymerase activity of its α subunit, Mtb DNA pol III has two potential proofreading subunits; the α and ε subunits. During DNA replication, the presence of the β2 clamp strongly promotes the polymerization of the αβ2ε replicase and reduces its exonuclease activity. Our work provides a foundation for further research on the mechanism by which the replication machinery switches between replication and proofreading and provides an experimental platform for the selection of antimicrobials targeting DNA replication in Mtb. PMID:26822057

  5. Annexin A1–containing extracellular vesicles and polymeric nanoparticles promote epithelial wound repair

    PubMed Central

    Leoni, Giovanna; Neumann, Philipp-Alexander; Kamaly, Nazila; Quiros, Miguel; Nishio, Hikaru; Jones, Hefin R.; Sumagin, Ronen; Hilgarth, Roland S.; Alam, Ashfaqul; Fredman, Gabrielle; Argyris, Ioannis; Rijcken, Emile; Kusters, Dennis; Reutelingsperger, Chris; Perretti, Mauro; Parkos, Charles A.; Farokhzad, Omid C.; Neish, Andrew S.; Nusrat, Asma

    2015-01-01

    Epithelial restitution is an essential process that is required to repair barrier function at mucosal surfaces following injury. Prolonged breaches in epithelial barrier function result in inflammation and further damage; therefore, a better understanding of the epithelial restitution process has potential for improving the development of therapeutics. In this work, we demonstrate that endogenous annexin A1 (ANXA1) is released as a component of extracellular vesicles (EVs) derived from intestinal epithelial cells, and these ANXA1-containing EVs activate wound repair circuits. Compared with healthy controls, patients with active inflammatory bowel disease had elevated levels of secreted ANXA1-containing EVs in sera, indicating that ANXA1-containing EVs are systemically distributed in response to the inflammatory process and could potentially serve as a biomarker of intestinal mucosal inflammation. Local intestinal delivery of an exogenous ANXA1 mimetic peptide (Ac2-26) encapsulated within targeted polymeric nanoparticles (Ac2-26 Col IV NPs) accelerated healing of murine colonic wounds after biopsy-induced injury. Moreover, one-time systemic administration of Ac2-26 Col IV NPs accelerated recovery following experimentally induced colitis. Together, our results suggest that local delivery of proresolving peptides encapsulated within nanoparticles may represent a potential therapeutic strategy for clinical situations characterized by chronic mucosal injury, such as is seen in patients with IBD. PMID:25664854

  6. Highly permeable polymeric membranes based on the incorporation of the functional water channel protein Aquaporin Z

    PubMed Central

    Kumar, Manish; Grzelakowski, Mariusz; Zilles, Julie; Clark, Mark; Meier, Wolfgang

    2007-01-01

    The permeability and solute transport characteristics of amphiphilic triblock-polymer vesicles containing the bacterial water-channel protein Aquaporin Z (AqpZ) were investigated. The vesicles were made of a block copolymer with symmetric poly-(2-methyloxazoline)-poly-(dimethylsiloxane)-poly-(2-methyloxazoline) (PMOXA15-PDMS110-PMOXA15) repeat units. Light-scattering measurements on pure polymer vesicles subject to an outwardly directed salt gradient in a stopped-flow apparatus indicated that the polymer vesicles were highly impermeable. However, a large enhancement in water productivity (permeability per unit driving force) of up to ≈800 times that of pure polymer was observed when AqpZ was incorporated. The activation energy (Ea) of water transport for the protein-polymer vesicles (3.4 kcal/mol) corresponded to that reported for water-channel-mediated water transport in lipid membranes. The solute reflection coefficients of glucose, glycerol, salt, and urea were also calculated, and indicated that these solutes are completely rejected. The productivity of AqpZ-incorporated polymer membranes was at least an order of magnitude larger than values for existing salt-rejecting polymeric membranes. The approach followed here may lead to more productive and sustainable water treatment membranes, whereas the variable levels of permeability obtained with different concentrations of AqpZ may provide a key property for drug delivery applications. PMID:18077364

  7. Photoinduced graft polymerization of 2-methacryloyloxyethyl phosphorylcholine on silicone hydrogels for reducing protein adsorption.

    PubMed

    Wang, Jing Jing; Liu, Fang

    2011-12-01

    The biomimetic synthetic methacrylate monomer containing a phosphorylcholine group, 2-methacryloyloxyethyl phosphorylcholine (MPC), has been widely used to improve the surface property of biomaterials. In the current report, both hydrophilic and antifouling surfaces were prepared on silicone hydrogels with MPC grafted by UV-induced free radical polymerization. The MPC-grafted silicone hydrogels were characterized by graft yield and static water contact angle (SCA) measurements. According to the results, the graft yield reached a maximum at 5 min of UV exposure time and 8 wt% MPC concentration. The modified silicone hydrogels possessed hydrophilic surfaces with the lowest water contact angle of 20º. The oxygen permeability of the MPC-grafted silicone hydrogels was as high as the unmodified silicone hydrogel. The mechanical property of silicone hydrogels was maintained at about 95% of the tensile strength and elastic modulus after the MPC grafting. The results of the in vitro single protein adsorption on the MPC-grafted silicone hydrogels were in agreement with the SCA measurements. The smaller the water contact angle, the greater was the protein repelling ability. The MPC-grafted silicone hydrogel is expected to be a novel biomaterial which possesses excellent surface hydrophilicity, antifouling property, oxygen permeability and mechanical property.

  8. Improving the binding capacities of protein A chromatographic materials by means of ligand polymerization.

    PubMed

    Freiherr von Roman, Matthias; Berensmeier, Sonja

    2014-06-20

    Protein A chromatography is one of the most important techniques used in the purification of monoclonal antibodies. Due to the low dynamic binding capacity of protein A chromatographic materials compared to other stationary phases, protein A chromatography is often discussed to be the bottleneck among current purification processes. Several approaches were tested within this study in order to maximize IgG binding capacities of current acrylamido-based based resins. Genetic engineering techniques were used in order to polymerize one of the IgG binding domains (B-domain) of protein A from Staphylococcus aureus (SpA) to achieve ligands with an increased length. The solution-binding ratio and the total size of ligand-antibody complexes were used to characterize the interaction potential of novel ligands, revealing a relatively linear dependency between the number of binding domains upon the amount of bound antibody molecules. This relationship was also valid up to a ligand which was comprised of 8 B-domains after attaching them onto acrylamido-based based stationary phases using epoxy coupling techniques. Equilibrium binding capacities of more than 80mghIgGmL(-1) were achieved using the B8 ligand. Furthermore, static binding capacities, especially for smaller ligands comprised of fewer B-domains, were improved up to 87mghIgGmL(-1) using site-specific coupling chemistry, which is an improvement of more than 20% compared to commercially available materials. In order to evaluate pore exclusion effects due to the use of prolonged affinity ligands, prepared materials were characterized regarding their effective intraparticle porosity and breakthrough capacity.

  9. Transport proteins promoting Escherichia coli pathogenesis.

    PubMed

    Tang, Fengyi; Saier, Milton H

    2014-01-01

    Escherichia coli is a genetically diverse species infecting hundreds of millions of people worldwide annually. We examined seven well-characterized E. coli pathogens causing urinary tract infections, gastroenteritis, pyelonephritis and haemorrhagic colitis. Their transport proteins were identified and compared with each other and a non-pathogenic E. coli K12 strain to identify transport proteins related to pathogenesis. Each pathogen possesses a unique set of protein secretion systems for export to the cell surface or for injecting effector proteins into host cells. Pathogens have increased numbers of iron siderophore receptors and ABC iron uptake transporters, but the numbers and types of low-affinity secondary iron carriers were uniform in all strains. The presence of outer membrane iron complex receptors and high-affinity ABC iron uptake systems correlated, suggesting co-evolution. Each pathovar encodes a different set of pore-forming toxins and virulence-related outer membrane proteins lacking in K12. Intracellular pathogens proved to have a characteristically distinctive set of nutrient uptake porters, different from those of extracellular pathogens. The results presented in this report provide information about transport systems relevant to various types of E. coli pathogenesis that can be exploited in future basic and applied studies.

  10. Transport proteins promoting Escherichia coli pathogenesis

    PubMed Central

    Tang, Fengyi; Saier, Milton H.

    2014-01-01

    Escherichia coli is a genetically diverse species infecting hundreds of millions of people worldwide annually. We examined seven well-characterized E. coli pathogens causing urinary tract infections, gastroenteritis, pyelonephritis and haemorrhagic colitis. Their transport proteins were identified and compared with each other and a non-pathogenic E. coli K12 strain to identify transport proteins related to pathogenesis. Each pathogen possesses a unique set of protein secretion systems for export to the cell surface or for injecting effector proteins into host cells. Pathogens have increased numbers of iron siderophore receptors and ABC iron uptake transporters, but the numbers and types of low-affinity secondary iron carriers were uniform in all strains. The presence of outer membrane iron complex receptors and high-affinity ABC iron uptake systems correlated, suggesting co-evolution. Each pathovar encodes a different set of pore-forming toxins and virulence-related outer membrane proteins lacking in K12. Intracellular pathogens proved to have a characteristically distinctive set of nutrient uptake porters, different from those of extracellular pathogens. The results presented in this report provide information about transport systems relevant to various types of E. coli pathogenesis that can be exploited in future basic and applied studies. PMID:24747185

  11. Polymerized ionic liquid-wrapped carbon nanotubes: the promising composites for direct electrochemistry and biosensing of redox protein.

    PubMed

    Xiao, Chunhui; Chu, Xiaochen; Wu, Bohua; Pang, Haili; Zhang, Xiaohua; Chen, Jinhua

    2010-03-15

    Polymerized ionic liquid-wrapped carbon nanotubes (PIL-CNTs) were firstly designed for direct electrochemistry and biosensing of redox proteins. The CNTs were coated successfully with polymerized ionic liquid (PIL) layer, as verified by transmission electron microscopy (TEM), thermogravimetric analysis (TGA) and Fourier transform infrared (FT-IR) spectroscopy. The PIL-CNTs were dispersed better in water and showed superior electrocatalysis toward O(2) and H(2)O(2) comparing to pristine CNTs and the mixture of IL monomer and CNTs. With glucose oxidase (GOD) as a protein model, the direct electrochemistry of the redox protein was investigated on the PIL-CNTs modified glassy carbon (GC) electrode and excellent direct electrochemical performance of GOD molecules was observed. The proposed biosensor (GOD/PIL-CNTs/GC electrode) displayed good analytical performance for glucose with linear response up to 6mM, response sensitivity of 0.853 microA mM(-1), good stability and selectivity.

  12. The Growth-Suppressive Function of the Polycomb Group Protein Polyhomeotic Is Mediated by Polymerization of Its Sterile Alpha Motif (SAM) Domain*

    PubMed Central

    Robinson, Angela K.; Leal, Belinda Z.; Chadwell, Linda V.; Wang, Renjing; Ilangovan, Udayar; Kaur, Yogeet; Junco, Sarah E.; Schirf, Virgil; Osmulski, Pawel A.; Gaczynska, Maria; Hinck, Andrew P.; Demeler, Borries; McEwen, Donald G.; Kim, Chongwoo A.

    2012-01-01

    Polyhomeotic (Ph), a member of the Polycomb Group (PcG), is a gene silencer critical for proper development. We present a previously unrecognized way of controlling Ph function through modulation of its sterile alpha motif (SAM) polymerization leading to the identification of a novel target for tuning the activities of proteins. SAM domain containing proteins have been shown to require SAM polymerization for proper function. However, the role of the Ph SAM polymer in PcG-mediated gene silencing was uncertain. Here, we first show that Ph SAM polymerization is indeed required for its gene silencing function. Interestingly, the unstructured linker sequence N-terminal to Ph SAM can shorten the length of polymers compared with when Ph SAM is individually isolated. Substituting the native linker with a random, unstructured sequence (RLink) can still limit polymerization, but not as well as the native linker. Consequently, the increased polymeric Ph RLink exhibits better gene silencing ability. In the Drosophila wing disc, Ph RLink expression suppresses growth compared with no effect for wild-type Ph, and opposite to the overgrowth phenotype observed for polymer-deficient Ph mutants. These data provide the first demonstration that the inherent activity of a protein containing a polymeric SAM can be enhanced by increasing SAM polymerization. Because the SAM linker had not been previously considered important for the function of SAM-containing proteins, our finding opens numerous opportunities to manipulate linker sequences of hundreds of polymeric SAM proteins to regulate a diverse array of intracellular functions. PMID:22275371

  13. The growth-suppressive function of the polycomb group protein polyhomeotic is mediated by polymerization of its sterile alpha motif (SAM) domain.

    PubMed

    Robinson, Angela K; Leal, Belinda Z; Chadwell, Linda V; Wang, Renjing; Ilangovan, Udayar; Kaur, Yogeet; Junco, Sarah E; Schirf, Virgil; Osmulski, Pawel A; Gaczynska, Maria; Hinck, Andrew P; Demeler, Borries; McEwen, Donald G; Kim, Chongwoo A

    2012-03-16

    Polyhomeotic (Ph), a member of the Polycomb Group (PcG), is a gene silencer critical for proper development. We present a previously unrecognized way of controlling Ph function through modulation of its sterile alpha motif (SAM) polymerization leading to the identification of a novel target for tuning the activities of proteins. SAM domain containing proteins have been shown to require SAM polymerization for proper function. However, the role of the Ph SAM polymer in PcG-mediated gene silencing was uncertain. Here, we first show that Ph SAM polymerization is indeed required for its gene silencing function. Interestingly, the unstructured linker sequence N-terminal to Ph SAM can shorten the length of polymers compared with when Ph SAM is individually isolated. Substituting the native linker with a random, unstructured sequence (RLink) can still limit polymerization, but not as well as the native linker. Consequently, the increased polymeric Ph RLink exhibits better gene silencing ability. In the Drosophila wing disc, Ph RLink expression suppresses growth compared with no effect for wild-type Ph, and opposite to the overgrowth phenotype observed for polymer-deficient Ph mutants. These data provide the first demonstration that the inherent activity of a protein containing a polymeric SAM can be enhanced by increasing SAM polymerization. Because the SAM linker had not been previously considered important for the function of SAM-containing proteins, our finding opens numerous opportunities to manipulate linker sequences of hundreds of polymeric SAM proteins to regulate a diverse array of intracellular functions.

  14. Incorporated glucosamine adversely affects the emulsifying properties of whey protein isolate polymerized by transglutaminase.

    PubMed

    Chen, Lin; Ullah, Niamat; Li, Chenyi; Hackman, Robert M; Li, Zhixi; Xu, Xinglian; Zhou, Guanghong; Feng, Xianchao

    2017-05-01

    Glucosamine (GlcN) and microbial transglutaminase (Tgase) are used separately or together to improve the emulsifying properties of whey protein isolate (WPI). However, little is known about how the emulsifying properties change when GlcN residues are incorporated into WPI cross-linked by Tgase. We used Tgase as a biocatalyst to cross-link WPI in the presence of GlcN in a liquid system for 12 h at a moderate temperature (25°C). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analyses indicated that protein polymerization and GlcN conjugation occurred simultaneously, phenomena also supported by the loss of free amines (9.4-20.5%). Addition of 5 U Tgase/g protein improved the emulsifying properties of moderately cross-linked WPI polymers. The Tgase-treated WPI polymers had a larger particle size (∼2.6-fold) than native WPI, which may have reduced coalescence and flocculation in an oil-in-water emulsion system. However, the incorporation of GlcN residues into WPI, predominantly via enzymatic glycation, partly inhibited the cross-links between the WPI molecules catalyzed by Tgase, reducing the size of the WPI polymers 0.81- to 0.86-fold). Consequently, WPI+GlcN conjugates provided less stability to the emulsion. Moreover, high NaCl concentration (0.2 M) decreased the emulsifying properties of the WPI+GlcN conjugates by neutralizing negative electric charges in the glycoconjugates. However, the improved emulsifying properties of WPI cross-linked by Tgase may be useful in food processing at higher NaCl concentrations due to the formation of the thicker steric barrier at the oil-water interface. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  15. Polymeric nanoparticles

    PubMed Central

    Bolhassani, Azam; Javanzad, Shabnam; Saleh, Tayebeh; Hashemi, Mehrdad; Aghasadeghi, Mohammad Reza; Sadat, Seyed Mehdi

    2014-01-01

    Nanocarriers with various compositions and biological properties have been extensively applied for in vitro/in vivo drug and gene delivery. The family of nanocarriers includes polymeric nanoparticles, lipid-based carriers (liposomes/micelles), dendrimers, carbon nanotubes, and gold nanoparticles (nanoshells/nanocages). Among different delivery systems, polymeric carriers have several properties such as: easy to synthesize, inexpensive, biocompatible, biodegradable, non-immunogenic, non-toxic, and water soluble. In addition, cationic polymers seem to produce more stable complexes led to a more protection during cellular trafficking than cationic lipids. Nanoparticles often show significant adjuvant effects in vaccine delivery since they may be easily taken up by antigen presenting cells (APCs). Natural polymers such as polysaccharides and synthetic polymers have demonstrated great potential to form vaccine nanoparticles. The development of new adjuvants or delivery systems for DNA and protein immunization is an expanding research field. This review describes polymeric carriers especially PLGA, chitosan, and PEI as vaccine delivery systems. PMID:24128651

  16. Protein nanocoatings on synthetic polymeric nanofibrous membranes designed as carriers for skin cells.

    PubMed

    Bacakova, Marketa; Pajorova, Julia; Stranska, Denisa; Hadraba, Daniel; Lopot, Frantisek; Riedel, Tomas; Brynda, Eduard; Zaloudkova, Margit; Bacakova, Lucie

    2017-01-01

    Protein-coated resorbable synthetic polymeric nanofibrous membranes are promising for the fabrication of advanced skin substitutes. We fabricated electrospun polylactic acid and poly(lactide-co-glycolic acid) nanofibrous membranes and coated them with fibrin or collagen I. Fibronectin was attached to a fibrin or collagen nanocoating, in order further to enhance the cell adhesion and spreading. Fibrin regularly formed a coating around individual nanofibers in the membranes, and also formed a thin noncontinuous nanofibrous mesh on top of the membranes. Collagen also coated most of the fibers of the membrane and randomly created a soft gel on the membrane surface. Fibronectin predominantly adsorbed onto a thin fibrin mesh or a collagen gel, and formed a thin nanofibrous structure. Fibrin nanocoating greatly improved the attachment, spreading, and proliferation of human dermal fibroblasts, whereas collagen nanocoating had a positive influence on the behavior of human HaCaT keratinocytes. In addition, fibrin stimulated the fibroblasts to synthesize fibronectin and to deposit it as an extracellular matrix. Fibrin coating also showed a tendency to improve the ultimate tensile strength of the nanofibrous membranes. Fibronectin attached to fibrin or to a collagen coating further enhanced the adhesion, spreading, and proliferation of both cell types.

  17. Protein-Nanocrystal Conjugates Support a Single Filament Polymerization Model in R1 Plasmid Segregation

    SciTech Connect

    Choi, Charina L.; Claridge, Shelley A.; Garner, Ethan C.; Alivisatos, A. Paul; Mullins, R. Dyche

    2008-07-15

    To ensure inheritance by daughter cells, many low-copy number bacterial plasmids, including the R1 drug-resistance plasmid, encode their own DNA segregation systems. The par operon of plasmid R1 directs construction of a simple spindle structure that converts free energy of polymerization of an actin-like protein, ParM, into work required to move sister plasmids to opposite poles of rod-shaped cells. The structures of individual components have been solved, but little is known about the ultrastructure of the R1 spindle. To determine the number of ParM filaments in a minimal R1 spindle, we used DNA-gold nanocrystal conjugates as mimics of the R1 plasmid. Wefound that each end of a single polar ParM filament binds to a single ParR/parC-gold complex, consistent with the idea that ParM filaments bind in the hollow core of the ParR/parC ring complex. Our results further suggest that multifilament spindles observed in vivo are associated with clusters of plasmidssegregating as a unit.

  18. Protein nanocoatings on synthetic polymeric nanofibrous membranes designed as carriers for skin cells

    PubMed Central

    Bacakova, Marketa; Pajorova, Julia; Stranska, Denisa; Hadraba, Daniel; Lopot, Frantisek; Riedel, Tomas; Brynda, Eduard; Zaloudkova, Margit; Bacakova, Lucie

    2017-01-01

    Protein-coated resorbable synthetic polymeric nanofibrous membranes are promising for the fabrication of advanced skin substitutes. We fabricated electrospun polylactic acid and poly(lactide-co-glycolic acid) nanofibrous membranes and coated them with fibrin or collagen I. Fibronectin was attached to a fibrin or collagen nanocoating, in order further to enhance the cell adhesion and spreading. Fibrin regularly formed a coating around individual nanofibers in the membranes, and also formed a thin noncontinuous nanofibrous mesh on top of the membranes. Collagen also coated most of the fibers of the membrane and randomly created a soft gel on the membrane surface. Fibronectin predominantly adsorbed onto a thin fibrin mesh or a collagen gel, and formed a thin nanofibrous structure. Fibrin nanocoating greatly improved the attachment, spreading, and proliferation of human dermal fibroblasts, whereas collagen nanocoating had a positive influence on the behavior of human HaCaT keratinocytes. In addition, fibrin stimulated the fibroblasts to synthesize fibronectin and to deposit it as an extracellular matrix. Fibrin coating also showed a tendency to improve the ultimate tensile strength of the nanofibrous membranes. Fibronectin attached to fibrin or to a collagen coating further enhanced the adhesion, spreading, and proliferation of both cell types. PMID:28223803

  19. Microencapsulation of Lactobacillus acidophilus NCFM using polymerized whey proteins as wall material.

    PubMed

    Jiang, Yujun; Zheng, Zhe; Zhang, Tiehua; Hendricks, Gregory; Guo, Mingruo

    2016-09-01

    Survivability of probiotics in foods is essential for developing functional food containing probiotics. We investigated polymerized whey protein (PWP)-based microencapsulation process which is developed for protecting probiotics like Lactobacillus acidophilus NCFM and compared with the method using sodium alginate (SA). The entrapment rate was 89.3 ± 4.8% using PWP, while it was 73.2 ± 1.4% for SA. The microencapsulated NCFM by PWP and SA were separately subjected to digestion juices and post-fermentation storage of fermented cows' and goats' milk using the encapsulated culture. The log viable count of NCFM in PWP-based microencapsulation was 4.56, compared with that of 4.26 in SA-based ones and 3.13 for free culture. Compared with using SA as wall material, PWP was more effective in protecting probiotic. Microencapsulation of L. acidophilus NCFM using PWP as wall material can be exploited in the development of fermented dairy products with better survivability of probiotic organism.

  20. Role of non-prolamin proteins and low molecular weight redox agents in protein folding and polymerization in wheat grains and influence on baking quality parameters.

    PubMed

    Osipova, Svetlana V; Permyakova, Marina D; Permyakov, Aleksey V

    2012-12-12

    The various enzyme systems and low molecular weight (LMW) redox agents are related to the folding and polymerization of prolamins in the ripening wheat grains and the formation of baking quality. Protein disulfide isomerases (PDIs) and cyclophylins accelerate "correct" folding of prolamins, which is most likely necessary for the subsequent formation of the macromolecular structure of the gluten protein matrix. PDIs are also involved in the polymerization of prolamins, catalyzing the oxidation of protein sulfhydryl groups. Molecular chaperone binding BiP protein facilitates folding of prolamins, with its role increasing in the stressful conditions. Reducing systems of thioredoxin and glutaredoxin, LMW redox pairs GSH/GSSG and Asc/DHAsc, thiol oxidases, and lipoxygenases (LOXs) regulate redox balance and the rate of polymerization of prolamins at the different stages of grain ripening. Additionally, LOX is probably involved in the protein-starch-lipid interactions between the starch granule and the protein matrix, mediated by puroindolines, determining the formation of grain texture. It is assumed that the high variability of baking quality in different environmental conditions is due to the interaction of labile enzyme systems with the storage proteins in the developing wheat caryopsis.

  1. Mussel-inspired new approach for polymerization and cross-linking of peptides and proteins containing tyrosines by Fremy's salt oxidation.

    PubMed

    Wilchek, Meir; Miron, Talia

    2015-03-18

    Our objective was to develop a method mimicking the natural process of coherence in marine mollusks, by direct chemical conversion of protein tyrosine residues to DOPA-o-quinones, which consequently generates polymerization and cross-linking. Fremy's salt, (ON(SO3K)2, was used to convert tyrosine residues in peptides and proteins to reactive o-quinones. The conversion of tyrosines to DOPA-o-quinones, and their ability to polymerize or cross-link, was tested on tyramine, peptides, and proteins. The peptides tested were as follows: biotin-PEG4-tyramine (PEG-BT), and two decapeptides (identical to the repeating units comprising the mussel's adhesive protein). The proteins tested were as follows: bovine pancreatic ribonuclease A (RNase), lysozyme, IgG, avidin, and streptavidin. The oxidized peptides and proteins were all shown to incorporate oxygen atoms and undergo polymerization and cross-linking, depending on the availability of nucleophiles, mostly lysine amino groups of proteins. All the peptides and the noninteracting proteins such as RNase and lysozyme underwent homopolymerization upon Fremy's salt oxidation. When Fremy's salt oxidaized PEG-BT was mixed with the above proteins, it did not react with any of these proteins because PEG-BT underwent fast self-polymerization. Conversely, streptavidin or avidin cross-linked with PEG-BT after preincubation, thus showing that biorecognition is a prerequisite for cross-linking. Polymerization and cross-linking also occurred, following Fremy's salt oxidation of interacting proteins such as avidin and strepavidin with biotinyilated lysozyme or biotinylated RNase. This indicates that only proteins in very close proximity readily cross-link and polymerize via tyrosine residues. Attempts to convert DOPA-quinone to DOPA by reduction with sodium dithionite (Na2S2O4), was successful as far as small peptides were used. Fremy's salt oxidation can serve as an easy and useful tool to polymerize and cross-link proteins, for

  2. Thermal analysis evaluation of mechanical properties changes promoted by gamma radiation on surgical polymeric textiles

    NASA Astrophysics Data System (ADS)

    Ferreira, L. M.; Casimiro, M. H.; Oliveira, C.; Cabeço Silva, M. E.; Marques Abreu, M. J.; Coelho, A.

    2002-05-01

    The large number of surgical operations with post-operative infection problems and the appearing of new infectious diseases, contribute to the development of new materials in order to answer the needs of health care services. This development must take into account the modifications promoted by sterilisation methods in materials, namely by gamma radiation. The differential scanning calorimetry (DSC) and thermogravimetry (TGA) techniques show that a nonwoven and a laminate textiles maintain a good molecular cohesion, do not showing high levels of degradation, for gamma radiation dose values lower than 100 kGy in nonwoven and 200 kGy in laminate materials. The tensile strength and the elongation decrease slowly for the nonwoven textile and decrease faster for the laminate textile for 25 and 80 kGy absorbed dose. This paper shows that the DSC and TGA techniques can be helpful for the prevision of mechanical changes occurred in the materials as a consequence of the gamma irradiation.

  3. Redox agents and N-ethylmaleimide affect protein polymerization during laboratory scale dry pasta production and cooking.

    PubMed

    Bruneel, Charlotte; Buggenhout, Joke; Lagrain, Bert; Brijs, Kristof; Delcour, Jan A

    2016-04-01

    Durum wheat (Triticum durum Desf.) semolina gluten proteins consist of monomeric gliadin and polymeric glutenin and determine the quality of pasta products made therefrom. During pasta drying, glutenin starts polymerizing already below 60 °C (65% relative humidity (RH)), whereas gliadin only is incorporated in the protein network at temperatures exceeding 68 °C (68% RH) through thiol (SH)/disulfide (SS) exchange reactions. Removal of free SH groups in glutenin by adding 2.3 μmol KBrO3 or KIO3 per g dry matter semolina protein (g protein) or 13.8 μmol N-ethylmaleimide/g protein reduces gliadin-glutenin cross-linking during pasta drying and/or cooking and yields cooked pasta of high quality. Introducing free SH groups by adding 13.8 μmol glutathione/g protein increases gliadin-glutenin cross-linking during pasta processing, resulting in cooked pasta of lower quality. We hypothesize that too much gliadin incorporation in the glutenin network during pasta processing tightens the protein network and results in lower cooking quality. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Cucurbit[6]uril-Promoted Click Chemistry for Protein Modification.

    PubMed

    Finbloom, Joel A; Han, Kenneth; Slack, Clancy C; Furst, Ariel L; Francis, Matthew B

    2017-07-19

    Azide-alkyne cycloaddition is a powerful reaction for the formation of bioconjugates. When catalyzed by Cu(I) or strain promotion, this cycloaddition is considered to be a "click" reaction with many applications in chemical biology and materials science. We report a new type of azide-alkyne click chemistry for the synthesis of protein conjugates using cucurbit[6]uril (CB6) supramolecular chemistry. CB6-promoted azide-alkyne cycloaddition has been previously used for the synthesis of rotaxanes but has not been applied to the development of complex bioconjugates. By developing new substrates for CB6 click that do not contain any cross-reactive functional groups and by optimizing reaction conditions, we converted CB6 click chemistry from a rotaxane synthesis tool into a useful bioconjugation technique. Using these new parameters, we synthesized a series of protein conjugates including protein-peptide, protein-DNA, protein-polymer, and protein-drug conjugates. We further demonstrated that CB6 click can be used in conjunction with strain-promoted azide-alkyne cycloaddition to generate distinct bioconjugates in protein mixtures. CB6 click is a promising new reaction for the development of protein conjugates and can be applied toward the synthesis of complex biomaterials for a wide range of applications.

  5. Phase transitions of multivalent proteins can promote clustering of membrane receptors

    PubMed Central

    Banjade, Sudeep; Rosen, Michael K

    2014-01-01

    Clustering of proteins into micrometer-sized structures at membranes is observed in many signaling pathways. Most models of clustering are specific to particular systems, and relationships between physical properties of the clusters and their molecular components are not well understood. We report biochemical reconstitution on supported lipid bilayers of protein clusters containing the adhesion receptor Nephrin and its cytoplasmic partners, Nck and N-WASP. With Nephrin attached to the bilayer, multivalent interactions enable these proteins to polymerize on the membrane surface and undergo two-dimensional phase separation, producing micrometer-sized clusters. Dynamics and thermodynamics of the clusters are modulated by the valencies and affinities of the interacting species. In the presence of the Arp2/3 complex, the clusters assemble actin filaments, suggesting that clustering of regulatory factors could promote local actin assembly at membranes. Interactions between multivalent proteins could be a general mechanism for cytoplasmic adaptor proteins to organize membrane receptors into micrometer-scale signaling zones. DOI: http://dx.doi.org/10.7554/eLife.04123.001 PMID:25321392

  6. Promoters, transcripts, and regulatory proteins of Mungbean yellow mosaic geminivirus.

    PubMed

    Shivaprasad, P V; Akbergenov, Rashid; Trinks, Daniela; Rajeswaran, R; Veluthambi, K; Hohn, Thomas; Pooggin, Mikhail M

    2005-07-01

    Geminiviruses package circular single-stranded DNA and replicate in the nucleus via a double-stranded intermediate. This intermediate also serves as a template for bidirectional transcription by polymerase II. Here, we map promoters and transcripts and characterize regulatory proteins of Mungbean yellow mosaic virus-Vigna (MYMV), a bipartite geminivirus in the genus Begomovirus. The following new features, which might also apply to other begomoviruses, were revealed in MYMV. The leftward and rightward promoters on DNA-B share the transcription activator AC2-responsive region, which does not overlap the common region that is nearly identical in the two DNA components. The transcription unit for BC1 (movement protein) includes a conserved, leader-based intron. Besides negative-feedback regulation of its own leftward promoter on DNA-A, the replication protein AC1, in cooperation with AC2, synergistically transactivates the rightward promoter, which drives a dicistronic transcription unit for the coat protein AV1. AC2 and the replication enhancer AC3 are expressed from one dicistronic transcript driven by a strong promoter mapped within the upstream AC1 gene. Early and constitutive expression of AC2 is consistent with its essential dual function as an activator of viral transcription and a suppressor of silencing.

  7. Promoters, Transcripts, and Regulatory Proteins of Mungbean Yellow Mosaic Geminivirus†

    PubMed Central

    Shivaprasad, P. V.; Akbergenov, Rashid; Trinks, Daniela; Rajeswaran, R.; Veluthambi, K.; Hohn, Thomas; Pooggin, Mikhail M.

    2005-01-01

    Geminiviruses package circular single-stranded DNA and replicate in the nucleus via a double-stranded intermediate. This intermediate also serves as a template for bidirectional transcription by polymerase II. Here, we map promoters and transcripts and characterize regulatory proteins of Mungbean yellow mosaic virus-Vigna (MYMV), a bipartite geminivirus in the genus Begomovirus. The following new features, which might also apply to other begomoviruses, were revealed in MYMV. The leftward and rightward promoters on DNA-B share the transcription activator AC2-responsive region, which does not overlap the common region that is nearly identical in the two DNA components. The transcription unit for BC1 (movement protein) includes a conserved, leader-based intron. Besides negative-feedback regulation of its own leftward promoter on DNA-A, the replication protein AC1, in cooperation with AC2, synergistically transactivates the rightward promoter, which drives a dicistronic transcription unit for the coat protein AV1. AC2 and the replication enhancer AC3 are expressed from one dicistronic transcript driven by a strong promoter mapped within the upstream AC1 gene. Early and constitutive expression of AC2 is consistent with its essential dual function as an activator of viral transcription and a suppressor of silencing. PMID:15956560

  8. Conjugation of Hyaluronic Acid onto Surfaces via the Interfacial Polymerization of Dopamine to Prevent Protein Adsorption.

    PubMed

    Huang, Renliang; Liu, Xia; Ye, Huijun; Su, Rongxin; Qi, Wei; Wang, Libing; He, Zhimin

    2015-11-10

    A versatile, convenient, and cost-effective method that can be used for grafting antifouling materials onto different surfaces is highly desirable in many applications. Here, we report the one-step fabrication of antifouling surfaces via the polymerization of dopamine and the simultaneous deposition of anionic hyaluronic acid (HA) on Au substrates. The water contact angle of the Au surfaces decreased from 84.9° to 24.8° after the attachment of a highly uniform polydopamine (PDA)/HA hybrid film. The results of surface plasmon resonance analysis showed that the Au-PDA/HA surfaces adsorbed proteins from solutions of bovine serum albumin, lysozyme, β-lactoglobulin, fibrinogen, and soybean milk in ultralow or low amounts (4.8-31.7 ng/cm(2)). The hydrophilicity and good antifouling performance of the PDA/HA surfaces is attributable to the HA chains that probably attached onto their upper surface via hydrogen bonding between PDA and HA. At the same time, the electrostatic repulsion between PDA and HA probably prevents the aggregation of PDA, resulting in the formation of a highly uniform PDA/HA hybrid film with the HA chains (with a stretched structure) on the upper surface. We also developed a simple method for removing this PDA/HA film and recycling the Au substrates by using an aqueous solution of NaOH as the hydrolyzing agent. The Au surface remained undamaged, and a PDA/HA film could be redeposited on the surface, with the surface exhibiting good antifouling performance even after 10 such cycles. Finally, it was found that this grafting method is applicable to other substrates, including epoxy resins, polystyrene, glass, and steel, owing to the strong adhesion of PDA with these substrates.

  9. Fractionation of proteins and carbohydrates of extracellular polymeric substances in a membrane bioreactor system.

    PubMed

    Malamis, Simos; Andreadakis, Andreas

    2009-07-01

    The major operational problem associated with membrane bioreactors (MBR) is membrane fouling, for which extracellular polymeric substances (EPS) are primarily responsible. In this work both the soluble and bound EPS (i.e. SMP and EPS) produced in an MBR system operating under sludge retention times (SRT) of 10, 15, 20 and 33 days were fractionized by means of membranes having variable molecular weight cutoffs (300 kDa, 100 kDa, 10 kDa & 1 kDa). The results show that increasing the SRT leads to a reduction of SMP and EPS and that these reductions are more pronounced for the SRTs in the range 10-20 days. This reduction is more significant for carbohydrates than for proteins. The decrease of SMP and EPS with increasing SRT from 10 to 20 days led to a significant decrease of the level of fouling. The further increase of SRT to 33 days did not significantly impact on the level of fouling as the SMP and EPS concentrations did not change much. Under the examined operating conditions, EPS were found to be composed mainly of large macromolecules having a size of 0.45 microm-300 kDa and to a lower extent of very small molecules (<1 kDa) that are not easily decomposed by the biomass activity. The majority of SMP is composed of very small molecules (<1 kDa), while some macromolecules in the range of 0.45 microm-300 kDa are present. Consequently, both EPS and SMP were found to have a bimodal character.

  10. Optimal Hydrophobicity in Ring-Opening Metathesis Polymerization-Based Protein Mimics Required for siRNA Internalization.

    PubMed

    deRonde, Brittany M; Posey, Nicholas D; Otter, Ronja; Caffrey, Leah M; Minter, Lisa M; Tew, Gregory N

    2016-06-13

    Exploring the role of polymer structure for the internalization of biologically relevant cargo, specifically siRNA, is of critical importance to the development of improved delivery reagents. Herein, we report guanidinium-rich protein transduction domain mimics (PTDMs) based on a ring-opening metathesis polymerization scaffold containing tunable hydrophobic moieties that promote siRNA internalization. Structure-activity relationships using Jurkat T cells and HeLa cells were explored to determine how the length of the hydrophobic block and the hydrophobic side chain compositions of these PTDMs impacted siRNA internalization. To explore the hydrophobic block length, two different series of diblock copolymers were synthesized: one series with symmetric block lengths and one with asymmetric block lengths. At similar cationic block lengths, asymmetric and symmetric PTDMs promoted siRNA internalization in the same percentages of the cell population regardless of the hydrophobic block length; however, with 20 repeat units of cationic charge, the asymmetric block length had greater siRNA internalization, highlighting the nontrivial relationships between hydrophobicity and overall cationic charge. To further probe how the hydrophobic side chains impacted siRNA internalization, an additional series of asymmetric PTDMs was synthesized that featured a fixed hydrophobic block length of five repeat units that contained either dimethyl (dMe), methyl phenyl (MePh), or diphenyl (dPh) side chains and varied cationic block lengths. This series was further expanded to incorporate hydrophobic blocks consisting of diethyl (dEt), diisobutyl (diBu), and dicyclohexyl (dCy) based repeat units to better define the hydrophobic window for which our PTDMs had optimal activity. High-performance liquid chromatography retention times quantified the relative hydrophobicities of the noncationic building blocks. PTDMs containing the MePh, diBu, and dPh hydrophobic blocks were shown to have superior

  11. Functional interactions between a glutamine synthetase promoter and MYB proteins.

    PubMed

    Gómez-Maldonado, Josefa; Avila, Concepción; Torre, Fernando; Cañas, Rafael; Cánovas, Francisco M; Campbell, Malcolm M

    2004-08-01

    In Scots pine (Pinus sylvestris), ammonium assimilation is catalysed by glutamine synthetase (GS) [EC 6.3.1.2], which is encoded by two genes, PsGS1a and PsGS1b. PsGS1b is expressed in the vascular tissue throughout the plant body, where it is believed to play a role in recycling ammonium released by various facets of metabolism. The mechanisms that may underpin the transcriptional regulation of PsGS1b were explored. The PsGS1b promoter contains a region that is enriched in previously characterized cis-acting elements, known as AC elements. Pine nuclear proteins bound these AC element-rich regions in a tissue-specific manner. As previous experiments had shown that R2R3-MYB transcription factors could interact with AC elements, the capacity of the AC elements in the PsGS1b promoter to interact with MYB proteins was examined. Two MYB proteins from loblolly pine (Pinus taeda), PtMYB1 and PtMYB4, bound to the PsGS1b promoter were able to activate transcription from this promoter in yeast, arabidopsis and pine cells. Immunolocalization experiments revealed that the two MYB proteins were most abundant in cells previously shown to accumulate PsGS1b transcripts. Immunoprecipitation analysis and supershift electrophoretic mobility shift assays implicated these same two proteins in the formation of complexes between pine nuclear extracts and the PsGS1b promoter. Given that these MYB proteins were previously shown to have the capacity to activate gene expression related to lignin biosynthesis, we hypothesize that they may function to co-regulate lignification, a process that places significant demands on nitrogen recycling, and GS, the major enzyme involved in the nitrogen recycling pathway.

  12. In vivo human Cartilage oligomeric matrix protein (COMP) promoter activity.

    PubMed

    Posey, Karen L; Davies, Sherri; Bales, Elise S; Haynes, Richard; Sandell, Linda J; Hecht, Jacqueline T

    2005-12-01

    Cartilage oligomeric matrix protein (COMP) is a large extracellular matrix protein whose function is unknown. Mutations in COMP cause pseudoachondroplasia and multiple epiphyseal dysplasia, two skeletal dysplasias which are associated with intracellular retention of COMP in chondrocytes. In contrast, COMP null mice are normal suggesting gene redundancy or that the detrimental effect is associated with mutant COMP rather than the absence of functional COMP. To define the elements that regulate COMP transcription and tissue-specificity, we have evaluated the human COMP promoter driving fusion gene expression in vitro and in vivo. COMP promoter activity is higher in rat chondrosarcoma cells (RCS) than in a fibroblast cell line. In RCS cells, expression of a reporter gene containing 1.7 kb of the human COMP promoter was three-fold higher than all shorter COMP promoter constructs. In transgenic mice, 1.7 kb of the human COMP promoter is active early in development in the limbs, spine, and eye. As development progresses, promoter activity diminishes in the eye and migrates from the center to the ends of the long bones. On the other hand, while 375 bp of the human COMP promoter is sufficient for proper tissue-specific expression, levels are less than those found with the 1.7-COMP promoter. The expression pattern of both promoters recapitulates endogenous cartilage COMP expression in mice. Our findings indicate that the elements required for chondrocyte-specific expression lie within 375 bp of the translational start site, while DNA enhancer elements are located between 1.0 to 1.7 kb.

  13. Promoting protein crystallization using a plate with simple geometry.

    PubMed

    Chen, Rui-Qing; Yin, Da-Chuan; Liu, Yong-Ming; Lu, Qin-Qin; He, Jin; Liu, Yue

    2014-03-01

    Increasing the probability of obtaining protein crystals in crystallization screening is always an important goal for protein crystallography. In this paper, a new method called the cross-diffusion microbatch (CDM) method is presented, which aims to efficiently promote protein crystallization and increase the chance of obtaining protein crystals. In this method, a very simple crystallization plate was designed in which all crystallization droplets are in one sealed space, so that a variety of volatile components from one droplet can diffuse into any other droplet via vapour diffusion. Crystallization screening and reproducibility tests indicate that this method could be a potentially powerful technique in practical protein crystallization screening. It can help to obtain crystals with higher probability and at a lower cost, while using a simple and easy procedure.

  14. An Artificial Reaction Promoter Modulates Mitochondrial Functions via Chemically Promoting Protein Acetylation

    PubMed Central

    Shindo, Yutaka; Komatsu, Hirokazu; Hotta, Kohji; Ariga, Katsuhiko; Oka, Kotaro

    2016-01-01

    Acetylation, which modulates protein function, is an important process in intracellular signalling. In mitochondria, protein acetylation regulates a number of enzymatic activities and, therefore, modulates mitochondrial functions. Our previous report showed that tributylphosphine (PBu3), an artificial reaction promoter that promotes acetylransfer reactions in vitro, also promotes the reaction between acetyl-CoA and an exogenously introduced fluorescent probe in mitochondria. In this study, we demonstrate that PBu3 induces the acetylation of mitochondrial proteins and a decrease in acetyl-CoA concentration in PBu3-treated HeLa cells. This indicates that PBu3 can promote the acetyltransfer reaction between acetyl-CoA and mitochondrial proteins in living cells. PBu3-induced acetylation gradually reduced mitochondrial ATP concentrations in HeLa cells without changing the cytoplasmic ATP concentration, suggesting that PBu3 mainly affects mitochondrial functions. In addition, pyruvate, which is converted into acetyl-CoA in mitochondria and transiently increases ATP concentrations in the absence of PBu3, elicited a further decrease in mitochondrial ATP concentrations in the presence of PBu3. Moreover, the application and removal of PBu3 reversibly alternated mitochondrial fragmentation and elongation. These results indicate that PBu3 enhances acetyltransfer reactions in mitochondria and modulates mitochondrial functions in living cells. PMID:27374857

  15. Ampakines promote spine actin polymerization, long-term potentiation, and learning in a mouse model of Angelman syndrome.

    PubMed

    Baudry, Michel; Kramar, Eniko; Xu, Xiaobo; Zadran, Homera; Moreno, Stephanie; Lynch, Gary; Gall, Christine; Bi, Xiaoning

    2012-08-01

    Angelman syndrome (AS) is a neurodevelopmental disorder largely due to abnormal maternal expression of the UBE3A gene leading to the deletion of E6-associated protein. AS subjects have severe cognitive impairments for which there are no therapeutic interventions. Mouse models (knockouts of the maternal Ube3a gene: 'AS mice') of the disorder have substantial deficits in long-term potentiation (LTP) and learning. Here we report a clinically plausible pharmacological treatment that ameliorates both deficits. AS mice were injected ip twice daily for 5 days with vehicle or the ampakine CX929; drugs of this type enhance fast EPSCs by positively modulating AMPA receptors. Theta burst stimulation (TBS) produced a normal enhancement of field EPSPs in hippocampal slices prepared from vehicle-treated AS mice but LTP decreased steadily to baseline; however, LTP in slices from ampakine-treated AS mice stabilized at levels found in wild-type controls. TBS-induced actin polymerization within dendritic spines, an essential event for stabilizing LTP, was severely impaired in slices from vehicle-treated AS mice but not in those from ampakine-treated AS mice. Long-term memory scores in a fear conditioning paradigm were reduced by 50% in vehicle-treated AS mice but were comparable to values for littermate controls in the ampakine-treated AS mice. We propose that AS is associated with a profound defect in activity-driven spine cytoskeletal reorganization, resulting in a loss of the synaptic plasticity required for the encoding of long-term memory. Notably, the spine abnormality along with the LTP and learning impairments can be reduced by a minimally invasive drug treatment.

  16. Improved blood compatibility of segmented polyurethane by polymeric additives having phospholipid polar group. II. Dispersion state of the polymeric additive and protein adsorption on the surface.

    PubMed

    Ishihara, K; Shibata, N; Tanaka, S; Iwasaki, Y; Kurosaki, T; Nakabayashi, N

    1996-11-01

    To improve the blood compatibility of a segmented polyurethane (SPU), phospholipid polymer, i.e., 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymerized with cyclohexyl methacrylate or 2-ethylhexyl methacrylate, was blended into SPU as a polymeric additive. The blending was achieved by a solvent-evaporation technique from a homogeneous solution containing both the SPU and the MPC polymer. Surface analysis of the SPU membrane blended with the MPC polymer (SPU/MPC polymer membrane) revealed that the MPC polymer was concentrated at the surface of the SPU membrane which contacted the substrate, Teflon, compared with that which contacted air during the membrane-formation period. The dispersion state of the MPC polymer in the SPU membrane was evaluated in detail by staining the MPC unit with osmium tetraoxide. When sonication was applied during preparation of the mixed solution containing SPU and the MPC polymer, the dispersion of the MPC polymer in the SPU membrane was different from that without sonication. That is, the size of the domains of the MPC polymer became smaller but the number of the domains increased. The amount of the MPC polymer mixed with SPU affected the dispersion state. Plasma proteins adsorbed on the SPU/MPC polymer membrane surface after contact with human plasma were detected by gold-colloid-labeled immunoassay. Both albumin and fibrinogen were observed on the SPU membrane; however, the amount of these proteins was reduced on the SPU/MPC polymer membrane. Thus it was concluded that the blood compatibility of the SPU was effectively improved by the blending of the MPC polymer.

  17. ATP hydrolysis by a domain related to translation factor GTPases drives polymerization of a static bacterial morphogenetic protein.

    PubMed

    Castaing, Jean-Philippe; Nagy, Attila; Anantharaman, Vivek; Aravind, L; Ramamurthi, Kumaran S

    2013-01-08

    The assembly of static supramolecular structures is a culminating event of developmental programs. One such structure, the proteinaceous shell (called the coat) that surrounds spores of the bacterium Bacillus subtilis, is composed of about 70 different proteins and represents one of the most durable biological structures known. The coat is built atop a basement layer that contains an ATPase (SpoIVA) that forms a platform required for coat assembly. Here, we show that SpoIVA belongs to the translation factors class of P-loop GTPases and has evolutionarily lost the ability to bind GTP; instead, it uses ATP hydrolysis to drive its self-assembly into static filaments. We demonstrate that ATP hydrolysis is required by every subunit for incorporation into the growing polymer by inducing a conformational change that drives polymerization of a nucleotide-free filament. SpoIVA therefore differs from other self-organizing polymers (dynamic cytoskeletal structures and static intermediate filaments) in that it uses ATP hydrolysis to self-assemble, not disassemble, into a static polymer. We further show that polymerization requires a critical concentration that we propose is only achieved once SpoIVA is recruited to the surface of the developing spore, thereby ensuring that SpoIVA polymerization only occurs at the correct subcellular location during spore morphogenesis.

  18. Functional analysis of bipartite begomovirus coat protein promoter sequences

    SciTech Connect

    Lacatus, Gabriela; Sunter, Garry

    2008-06-20

    We demonstrate that the AL2 gene of Cabbage leaf curl virus (CaLCuV) activates the CP promoter in mesophyll and acts to derepress the promoter in vascular tissue, similar to that observed for Tomato golden mosaic virus (TGMV). Binding studies indicate that sequences mediating repression and activation of the TGMV and CaLCuV CP promoter specifically bind different nuclear factors common to Nicotiana benthamiana, spinach and tomato. However, chromatin immunoprecipitation demonstrates that TGMV AL2 can interact with both sequences independently. Binding of nuclear protein(s) from different crop species to viral sequences conserved in both bipartite and monopartite begomoviruses, including TGMV, CaLCuV, Pepper golden mosaic virus and Tomato yellow leaf curl virus suggests that bipartite begomoviruses bind common host factors to regulate the CP promoter. This is consistent with a model in which AL2 interacts with different components of the cellular transcription machinery that bind viral sequences important for repression and activation of begomovirus CP promoters.

  19. Tumor promotion by depleting cells of protein kinase C delta.

    PubMed Central

    Lu, Z; Hornia, A; Jiang, Y W; Zang, Q; Ohno, S; Foster, D A

    1997-01-01

    Tumor-promoting phorbol esters activate, but then deplete cells of, protein kinase C (PKC) with prolonged treatment. It is not known whether phorbol ester-induced tumor promotion is due to activation or depletion of PKC. In rat fibroblasts overexpressing the c-Src proto-oncogene, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced anchorage-independent growth and other transformation-related phenotypes. The appearance of transformed phenotypes induced by TPA in these cells correlated not with activation but rather with depletion of expressed PKC isoforms. Consistent with this observation, PKC inhibitors also induced transformed phenotypes in c-Src-overexpressing cells. Bryostatin 1, which inhibited the TPA-induced down-regulation of the PKCdelta isoform specifically, blocked the tumor-promoting effects of TPA, implicating PKCdelta as the target of the tumor-promoting phorbol esters. Consistent with this hypothesis, expression of a dominant negative PKCdelta mutant in cells expressing c-Src caused transformation of these cells, and rottlerin, a protein kinase inhibitor with specificity for PKCdelta, like TPA, caused transformation of c-Src-overexpressing cells. These data suggest that the tumor-promoting effect of phorbol esters is due to depletion of PKCdelta, which has an apparent tumor suppressor function. PMID:9154841

  20. Rad54 protein promotes branch migration of Holliday junctions.

    PubMed

    Bugreev, Dmitry V; Mazina, Olga M; Mazin, Alexander V

    2006-08-03

    Homologous recombination has a crucial function in the repair of DNA double-strand breaks and in faithful chromosome segregation. The mechanism of homologous recombination involves the search for homology and invasion of the ends of a broken DNA molecule into homologous duplex DNA to form a cross-stranded structure, a Holliday junction (HJ). A HJ is able to undergo branch migration along DNA, generating increasing or decreasing lengths of heteroduplex. In both prokaryotes and eukaryotes, the physical evidence for HJs, the key intermediate in homologous recombination, was provided by electron microscopy. In bacteria there are specialized enzymes that promote branch migration of HJs. However, in eukaryotes the identity of homologous recombination branch-migration protein(s) has remained elusive. Here we show that Rad54, a Swi2/Snf2 protein, binds HJ-like structures with high specificity and promotes their bidirectional branch migration in an ATPase-dependent manner. The activity seemed to be conserved in human and yeast Rad54 orthologues. In vitro, Rad54 has been shown to stimulate DNA pairing of Rad51, a key homologous recombination protein. However, genetic data indicate that Rad54 protein might also act at later stages of homologous recombination, after Rad51 (ref. 13). Novel DNA branch-migration activity is fully consistent with this late homologous recombination function of Rad54 protein.

  1. Efficiency of serum protein removal from skim milk with ceramic and polymeric membranes at 50 degrees C.

    PubMed

    Zulewska, J; Newbold, M; Barbano, D M

    2009-04-01

    Raw milk (2,710 kg) was separated at 4 degrees C, the skim milk was pasteurized (72 degrees C, 16 s), split into 3 batches, and microfiltered using pilot-scale ceramic uniform transmembrane pressure (UTP; Membralox model EP1940GL0.1microA, 0.1 microm alumina, Pall Corp., East Hills, NY), ceramic graded permeability (GP; Membralox model EP1940GL0.1microAGP1020, 0.1 microm alumina, Pall Corp.), and polymeric spiral-wound (SW; model FG7838-OS0x-S, 0.3 microm polyvinylidene fluoride, Parker-Hannifin, Process Advanced Filtration Division, Tell City, IN) membranes. There were differences in flux among ceramic UTP, ceramic GP, and polymeric SW microfiltration membranes (54.08, 71.79, and 16.21 kg/m2 per hour, respectively) when processing skim milk at 50 degrees C in a continuous bleed-and-feed 3x process. These differences in flux among the membranes would influence the amount of membrane surface area required to process a given volume of milk in a given time. Further work is needed to determine if these differences in flux are maintained over longer processing times. The true protein contents of the microfiltration permeates from UTP and GP membranes were higher than from SW membranes (0.57, 0.56, and 0.38%, respectively). Sodium-dodecyl-sulfate-PAGE gels for permeates revealed a higher casein proportion in GP and SW permeate than in UTP permeate, with the highest passage of casein through the GP membrane under the operational conditions used in this study. The slight cloudiness of the permeates produced using the GP and SW systems may have been due to the presence of a small amount of casein, which may present an obstacle in their use in applications when clarity is an important functional characteristic. More beta-lactoglobulin passed through the ceramic membranes than through the polymeric membrane. The efficiency of removal of serum proteins in a continuous bleed-and-feed 3x process at 50 degrees C was 64.40% for UTP, 61.04% for GP, and 38.62% for SW microfiltration

  2. Growth promoting hormonal implant pellets coated with a polymeric, porous film promote weight gain by grazing beef heifers and steers for up to 200 days.

    PubMed

    Cleale, R M; Edmonds, J D; Edmonds, M; Hunsaker, B D; Kraft, L A; Smith, L L; Yazwinski, T A

    2015-04-01

    Two studies evaluated growth promoting effects of implant pellets (IP), each containing 3.5 mg estradiol benzoate (EB) and 25 mg trenbolone acetate (TBA), to which a polymeric, porous coating was applied. Trial 1 evaluated performance of heifers (n = 70/treatment, initial BW = 188 ± 2.2 kg) and steers (n = 70/treatment, initial BW = 194 ± 2.2 kg) implanted subcutaneously in the ear with 0 (SC), 2 (2IP), 4 (4IP), or 6 (6IP) pellets that delivered EB/TBA (mg/mg) doses of 0/0, 7/50, 14/100, and 21/150, respectively, over grazing periods of 202 d (heifers) or 203 d (steers). Animals received experimental treatments on d 0 and over the grazing period were managed as single groups by sex in a rotational grazing system. When pasture forage availability became limited, cattle were supplemented with preserved forage but not concentrate supplements. Weight gains by heifers treated with 2IP, 4IP, and 6IP were greater (P < 0.05) than SC heifers but not different from each other. Weight gains by steers treated with 2IP, 4IP, and 6IP were greater than SC steers (P < 0.05), and ADG by steers treated with 6IP was greater (P < 0.05) than steers given 2IP or 4IP. Trial 2 was a multisite grazing study performed with heifers and steers to compare ADG after treatment with one 6-pellet, coated implant delivering 21 mg EB and 150 mg TBA (6IP) to sham treated negative controls (SC) over a grazing period of at least 200 d. A completely random design was used at each site, with the goal to treat 70 cattle per site, treatment, and sex; data were pooled across sites. Heifers (n = 558, initial BW = 229 ± 16 kg) and steers (n = 555, initial BW = 235 ± 20 kg) grazed in rotational programs consistent with regional practices for an average of 202 d. When necessary, cattle were supplemented with preserved forage, but no concentrate supplements were fed. Over 202 d, ADG by heifers treated with 6IP was 11.3% greater (P = 0.0035) than SC heifers (0.64 ± 0.06 kg/d), and ADG by steers treated with

  3. Titanium Dioxide Photocatalytic Polymerization of Acrylamide for Gel Electrophoresis (TIPPAGE) of Proteins and Structural Identification by Mass Spectrometry

    PubMed Central

    Zhang, Wenyang; Yuan, Zhiwei; Huang, Lulu; Kang, Jie; Jiang, Ruowei; Zhong, Hongying

    2016-01-01

    Polyacrylamide gel electrophoresis (PAGE) coupled with mass spectrometry has been well established for separating, identifying and quantifying protein mixtures from cell lines, tissues or other biological samples. The copolymerization process of acrylamide and bis-acrylamide is the key to mastering this powerful technique. In general, this is a vinyl addition reaction initiated by free radical-generating reagents such as ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) under basic pH and degassing experimental condition. We report herein a photocatalytic polymerization approach that is based on photo-generated hydroxyl radicals with nanoparticles of titanium dioxide. It was shown that the polymerization process is greatly accelerated in acidic condition when ultraviolet light shots on the gel solution containing TiO2 nanoparticles without degassing. This feature makes it very useful in preparing Triton X-100 acid urea (TAU) gel that has been developed for separating basic proteins such as histones and variants in acidic experimental condition. Additionally, the presence of titanium dioxide in the gel not only improves mechanistic property of gels but also changes the migration pattern of different proteins that have different affinities to titanium dioxide. PMID:26865351

  4. Titanium Dioxide Photocatalytic Polymerization of Acrylamide for Gel Electrophoresis (TIPPAGE) of Proteins and Structural Identification by Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Zhang, Wenyang; Yuan, Zhiwei; Huang, Lulu; Kang, Jie; Jiang, Ruowei; Zhong, Hongying

    2016-02-01

    Polyacrylamide gel electrophoresis (PAGE) coupled with mass spectrometry has been well established for separating, identifying and quantifying protein mixtures from cell lines, tissues or other biological samples. The copolymerization process of acrylamide and bis-acrylamide is the key to mastering this powerful technique. In general, this is a vinyl addition reaction initiated by free radical-generating reagents such as ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) under basic pH and degassing experimental condition. We report herein a photocatalytic polymerization approach that is based on photo-generated hydroxyl radicals with nanoparticles of titanium dioxide. It was shown that the polymerization process is greatly accelerated in acidic condition when ultraviolet light shots on the gel solution containing TiO2 nanoparticles without degassing. This feature makes it very useful in preparing Triton X-100 acid urea (TAU) gel that has been developed for separating basic proteins such as histones and variants in acidic experimental condition. Additionally, the presence of titanium dioxide in the gel not only improves mechanistic property of gels but also changes the migration pattern of different proteins that have different affinities to titanium dioxide.

  5. Tubulin binding protein, CacyBP/SIP, induces actin polymerization and may link actin and tubulin cytoskeletons.

    PubMed

    Schneider, Gabriela; Nieznanski, Krzysztof; Jozwiak, Jolanta; Slomnicki, Lukasz P; Redowicz, Maria J; Filipek, Anna

    2010-11-01

    CacyBP/SIP, originally identified as a S100A6 target, was shown to interact with some other S100 proteins as well as with Siah-1, Skp1, tubulin and ERK1/2 kinases (reviewed in Schneider and Filipek, Amino Acids, 2010). Here, we show that CacyBP/SIP interacts and co-localizes with actin in NB2a cells. Using a zero-length cross-linker we found that both proteins bound directly to each other. Co-sedimentation assays revealed that CacyBP/SIP induced G-actin polymerization and formation of unique circular actin filament bundles. The N-terminal fragment of CacyBP/SIP (residues 1-179) had similar effect on actin polymerization as the entire CacyBP/SIP protein, while the C-terminal one (residues 178-229) had not. To check the influence of CacyBP/SIP on cell morphology as well as on cell adhesion and migration, a stable NIH 3T3 cell line with an increased level of CacyBP/SIP was generated. We found that the adhesion and migration rates of the modified cells were changed in comparison with the control ones. Interestingly, the co-sedimentation and proximity ligation assays indicated that CacyBP/SIP could simultaneously interact with tubulin and actin, suggesting that CacyBP/SIP might link actin and tubulin cytoskeletons.

  6. Highly protein-resistant coatings and suspension cell culture thereon from amphiphilic block copolymers prepared by RAFT polymerization.

    PubMed

    Haraguchi, Kazutoshi; Kubota, Kazuomi; Takada, Tetsuo; Mahara, Saori

    2014-06-09

    Novel amphiphilic block copolymers composed of hydrophobic (poly(2-methoxyethyl acrylate): M) and hydrophilic (poly(N,N-dimethylacrylamide): D) segments were synthesized by living radical polymerization: a reversible addition-fragmentation chain-transfer polymerization. Two types of amphiphilic block copolymers, triblock (MDM) and 4-arm block ((MD)4) copolymers with specific compositions (D/M = (750-1500)/250), were prepared by a versatile one-pot synthesis. These copolymers show good adhesion to various types of substrates (e.g., polystyrene, polycarbonate, polypropylene, Ti, and glass), and the surface coating showed high protein repellency and a low contact angle for water, regardless of the substrate. The two opposing characteristics of high protein repellency and good substrate adhesion were achieved by the combined effects of the molecular architecture of the block copolymers, the high molecular weight, and the characteristics of each segment, that is, low protein adsorption capability of both segments and low glass transition temperature of the hydrophobic segment. Further, a polystyrene dish coated with the MDM block copolymer could be sterilized by γ-ray irradiation and used as a good substrate for a suspension cell culture that exhibits low cell adhesion and good cell growth.

  7. Titanium Dioxide Photocatalytic Polymerization of Acrylamide for Gel Electrophoresis (TIPPAGE) of Proteins and Structural Identification by Mass Spectrometry.

    PubMed

    Zhang, Wenyang; Yuan, Zhiwei; Huang, Lulu; Kang, Jie; Jiang, Ruowei; Zhong, Hongying

    2016-02-11

    Polyacrylamide gel electrophoresis (PAGE) coupled with mass spectrometry has been well established for separating, identifying and quantifying protein mixtures from cell lines, tissues or other biological samples. The copolymerization process of acrylamide and bis-acrylamide is the key to mastering this powerful technique. In general, this is a vinyl addition reaction initiated by free radical-generating reagents such as ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) under basic pH and degassing experimental condition. We report herein a photocatalytic polymerization approach that is based on photo-generated hydroxyl radicals with nanoparticles of titanium dioxide. It was shown that the polymerization process is greatly accelerated in acidic condition when ultraviolet light shots on the gel solution containing TiO2 nanoparticles without degassing. This feature makes it very useful in preparing Triton X-100 acid urea (TAU) gel that has been developed for separating basic proteins such as histones and variants in acidic experimental condition. Additionally, the presence of titanium dioxide in the gel not only improves mechanistic property of gels but also changes the migration pattern of different proteins that have different affinities to titanium dioxide.

  8. Integration of RAFT polymerization and click chemistry to fabricate PAMPS modified macroporous polypropylene membrane for protein fouling mitigation.

    PubMed

    Wang, Yun; Wang, Li-Li; He, Xiao-Chun; Zhang, Zi-Jun; Yu, Hai-Yin; Gu, Jia-Shan

    2014-12-01

    A copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) grafting-to method was used to tether alkyne-terminated poly(2-acrylamido-2-methyl propane sulfonic acid) (alkyne-PAMPS) to the azide functionalized macroporous polypropylene membrane (MPPM-N3). Alkyne-PAMPS was synthesized by the reversible addition-fragmentation chain transfer polymerization (RAFT) of AMPS with an alkyne-terminated trithiocarbonate served as a chain transfer agent. The combination of RAFT polymerization with click chemistry to graft polymer to the surface of polypropylene membrane produced relatively high grafting density and controllable grafting chain length. The structure and composition of the modified and unmodified MPPM surfaces were analyzed by attenuated total reflection-Fourier transform infrared spectroscopy (ATR/FT-IR), X-ray photoelectron spectroscopy (XPS); field emission scanning electron microscopy (FE-SEM) was employed to observe the morphological changes on the membrane surface. The permeation performances were tested by the filtration of protein dispersion. The experimental results show that with the grafting degree going up, the relative flux reduction decreases, while the relative flux recovery ratio increases, and the protein fouling is obviously mitigated by tethering PAMPS to the membrane surface. The modified membranes can be potentially applied for fouling reduction during the filtration of proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Comparison of two chemical extraction methods for proteins and polysaccharides of Spirogyra fluviatilis in extracellular polymeric substances

    NASA Astrophysics Data System (ADS)

    Chang, Shuiping; Lee, Yichao

    2017-05-01

    Under natural circumstances, Spirogyra fluviatilis excretes large amounts of extracellular polymeric substances (EPS) and forms a mucilage layer. This study used two algal EPS extraction methods such as NH4OH and formaldehyde-NaOH to compare the extraction yields of EPS and the levels of its primary components of polysaccharides and proteins. The results indicated that the extraction yields of EPS using formaldehyde-NaOH and NH4OH are 8.05 mg EPS/g-fresh algae and 5.26 mg EPS/g-fresh algae, respectively. The ratio of proteins to polysaccharides in EPS is around 0.24 to 0.34, which is not a significant difference between the two extraction methods. In other words, the level of polysaccharides is always higher than the level of proteins in EPS extraction with both two methods

  10. Regulatory elements of the Staphylococcus aureus protein A (Spa) promoter.

    PubMed

    Gao, Jinxin; Stewart, George C

    2004-06-01

    Staphylococcal protein A (Spa) is an important virulence factor of Staphylococcus aureus. Transcription of the spa determinant occurs during the exponential growth phase and is repressed when the cells enter the postexponential growth phase. Regulation of spa expression has been found to be complicated, with regulation involving multiple factors, including Agr, SarA, SarS, SarT, Rot, and MgrA. Our understanding of how these factors work on the spa promoter to regulate spa expression is incomplete. To identify regulatory sites within the spa promoter, analysis of deletion derivatives of the promoter in host strains deficient in one or more of the regulatory factors was undertaken, and several critical features of spa regulation were revealed. The transcriptional start sites of spa were determined by primer extension. The spa promoter sequences were subcloned in front of a promoterless chloramphenicol acetyltransferase reporter gene. Various lengths of spa truncations with the same 3' end were constructed, and the resultant plasmids were transduced into strains with different regulatory genetic backgrounds. Our results identified upstream promoter sequences necessary for Agr system regulation of spa expression. The cis elements for SarS activity, an activator of spa expression, and for SarA activity, a repressor of spa expression, were identified. The well-characterized SarA consensus sequence on the spa promoter was found to be insufficient for SarA repression of the spa promoter. Full repression required the presence of a second consensus site adjacent to the SarS binding site. Sequences directly upstream of the core promoter sequence were found to stimulate transcription.

  11. Evolution of Drosophila ribosomal protein gene core promoters

    PubMed Central

    Ma, Xiaotu; Zhang, Kangyu; Li, Xiaoman

    2011-01-01

    The coordinated expression of ribosomal protein genes (RPGs) has been well documented in many species. Previous analyses of RPG promoters focus only on Fungi and mammals. Recognizing this gap and using a comparative genomics approach, we utilize a motif-finding algorithm that incorporates cross-species conservation to identify several significant motifs in Drosophila RPG promoters. As a result, significant differences of the enriched motifs in RPG promoter are found among Drosophila, Fungi, and mammals, demonstrating the evolutionary dynamics of the ribosomal gene regulatory network. We also report a motif present in similar numbers of RPGs among Drosophila species which does not appear to be conserved at the individual RPG gene level. A module-wise stabilizing selection theory is proposed to explain this observation. Overall, our results provide significant insight into the fast-evolving nature of transcriptional regulation in the RPG module. PMID:19059316

  12. Evolution of Drosophila ribosomal protein gene core promoters.

    PubMed

    Ma, Xiaotu; Zhang, Kangyu; Li, Xiaoman

    2009-03-01

    The coordinated expression of ribosomal protein genes (RPGs) has been well documented in many species. Previous analyses of RPG promoters focus only on Fungi and mammals. Recognizing this gap and using a comparative genomics approach, we utilize a motif-finding algorithm that incorporates cross-species conservation to identify several significant motifs in Drosophila RPG promoters. As a result, significant differences of the enriched motifs in RPG promoter are found among Drosophila, Fungi, and mammals, demonstrating the evolutionary dynamics of the ribosomal gene regulatory network. We also report a motif present in similar numbers of RPGs among Drosophila species which does not appear to be conserved at the individual RPG gene level. A module-wise stabilizing selection theory is proposed to explain this observation. Overall, our results provide significant insight into the fast-evolving nature of transcriptional regulation in the RPG module.

  13. Protein binding elements in the human beta-polymerase promoter.

    PubMed Central

    Englander, E W; Wilson, S H

    1990-01-01

    The core promoter for human DNA polymerase beta contains discrete binding sites for mammalian nuclear proteins, as revealed by DNasel footprinting and gel mobility shift assays. Two sites correspond to sequences identical with the Sp1 factor binding element, and a third site includes an eight residue palindromic sequence, TGACGTCA, known as the CRE element of several cAMP responsive promoters; the 5 to 10 residues flanking this palindrome on each side have no apparent sequence homology with known elements in other promoters. Nuclear extract from a variety of tissues and cells were examined; these included rat liver and testes and cultured cells of human and hamster origin. The DNasel footprint is strong over and around the palindromic element for each of the extracts and is equivalent in size (approximately 22 residues); footprinting over the Sp1 binding sites is seen also. Two potential tissue-specific binding sites, present in liver but not in testes, were found corresponding to residues -13 to -10 and +33 to +48, respectively. Protein binding to the palindromic element was confirmed by an electrophoretic mobility shift assay with the core promoter as probe. Binding specificity of the 22 residue palindromic element, as revealed by oligonucleotide competition, is different from that of AP-1 binding element. Controlled proteolysis with trypsin was used to study structural properties of proteins forming the mobility shift bands. Following digestion with trypsin, most of the palindrome binding activity of each extract corresponded to a sharp, faster migrating band, potentially representing a DNA binding domain of the palindrome binding protein. Images PMID:2315044

  14. Protein-resistant polyurethane via surface-initiated atom transfer radical polymerization of oligo(ethylene glycol) methacrylate.

    PubMed

    Jin, Zhilin; Feng, Wei; Zhu, Shiping; Sheardown, Heather; Brash, John L

    2009-12-15

    Protein-resistant polyurethane (PU) surfaces were prepared by surface-initiated simultaneous normal and reverse atom transfer radical polymerization (s-ATRP) of poly(oligo(ethylene glycol) methacrylate) (poly (OEGMA)). Oxygen plasma treatment was employed for initial activation of the PU surface. The grafted polymer chain length was adjusted by varying the molar ratio of monomer to sacrificial initiator in solution from 5:1 to 200:1. The modified PU surfaces were characterized by water contact angle, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). Protein adsorption experiments from tris-buffered saline (TBS) and plasma were carried out to evaluate the protein-resistance of the surfaces. Adsorption from single and binary protein solutions as well as from plasma was significantly reduced after modification. Adsorption decreased with increasing poly(OEGMA) chain length. Fibrinogen (Fg) adsorption on the 200:1 monomer/initiator surface was in the range of 3-33 ng/cm(2) representing 96-99% reduction compared with the unmodified PU. Fg adsorption from 0.01-10% plasma was as low as 1-5 ng/cm(2). Moreover, binary protein adsorption experiments using Fg and lysozyme (Lys) showed that protein size is a factor in the protein resistance of these surfaces.

  15. Interplay between Penicillin-binding proteins and SEDS proteins promotes bacterial cell wall synthesis.

    PubMed

    Leclercq, Sophie; Derouaux, Adeline; Olatunji, Samir; Fraipont, Claudine; Egan, Alexander J F; Vollmer, Waldemar; Breukink, Eefjan; Terrak, Mohammed

    2017-02-24

    Bacteria utilize specialized multi-protein machineries to synthesize the essential peptidoglycan (PG) cell wall during growth and division. The divisome controls septal PG synthesis and separation of daughter cells. In E. coli, the lipid II transporter candidate FtsW is thought to work in concert with the PG synthases penicillin-binding proteins PBP3 and PBP1b. Yet, the exact molecular mechanisms of their function in complexes are largely unknown. We show that FtsW interacts with PBP1b and lipid II and that PBP1b, FtsW and PBP3 co-purify suggesting that they form a trimeric complex. We also show that the large loop between transmembrane helices 7 and 8 of FtsW is important for the interaction with PBP3. Moreover, we found that FtsW, but not the other flippase candidate MurJ, impairs lipid II polymerization and peptide cross-linking activities of PBP1b, and that PBP3 relieves these inhibitory effects. All together the results suggest that FtsW interacts with lipid II preventing its polymerization by PBP1b unless PBP3 is also present, indicating that PBP3 facilitates lipid II release and/or its transfer to PBP1b after transport across the cytoplasmic membrane. This tight regulatory mechanism is consistent with the cell's need to ensure appropriate use of the limited pool of lipid II.

  16. Interplay between Penicillin-binding proteins and SEDS proteins promotes bacterial cell wall synthesis

    PubMed Central

    Leclercq, Sophie; Derouaux, Adeline; Olatunji, Samir; Fraipont, Claudine; Egan, Alexander J. F.; Vollmer, Waldemar; Breukink, Eefjan; Terrak, Mohammed

    2017-01-01

    Bacteria utilize specialized multi-protein machineries to synthesize the essential peptidoglycan (PG) cell wall during growth and division. The divisome controls septal PG synthesis and separation of daughter cells. In E. coli, the lipid II transporter candidate FtsW is thought to work in concert with the PG synthases penicillin-binding proteins PBP3 and PBP1b. Yet, the exact molecular mechanisms of their function in complexes are largely unknown. We show that FtsW interacts with PBP1b and lipid II and that PBP1b, FtsW and PBP3 co-purify suggesting that they form a trimeric complex. We also show that the large loop between transmembrane helices 7 and 8 of FtsW is important for the interaction with PBP3. Moreover, we found that FtsW, but not the other flippase candidate MurJ, impairs lipid II polymerization and peptide cross-linking activities of PBP1b, and that PBP3 relieves these inhibitory effects. All together the results suggest that FtsW interacts with lipid II preventing its polymerization by PBP1b unless PBP3 is also present, indicating that PBP3 facilitates lipid II release and/or its transfer to PBP1b after transport across the cytoplasmic membrane. This tight regulatory mechanism is consistent with the cell’s need to ensure appropriate use of the limited pool of lipid II. PMID:28233869

  17. Basic Equations in Statics and Kinetics of Protein Polymerization and the Mechanism of the Formation and Dissociation of Amyloid Fibrils Revealed by Pressure Perturbation.

    PubMed

    Tachibana, Hideki

    2015-01-01

    Studies of the pressure-dissociation of several amyloid or amyloid-like fibrils have shown that the fibril state is considerably voluminous. Quantitative characterization of the protein fibrillation reaction with respect to volumetric parameters is necessary to elucidate mechanisms of amyloid fibrillation in molecular terms such as protein cavity and hydration. Here we discuss, firstly, basic equations in statics and kinetics of protein polymerization as employed to obtain thermodynamic, volumetric, and kinetic parameters. Equilibrium treatment of the reactions with the scheme such as one-step polymerization, linear-association polymerization, or nucleation-dependent polymerization, and kinetic treatment of seeded linear-polymerization or spontaneous nucleation-elongation polymerization are described. In particular we will detail kinetics of the dissociation of fibrils which have been produced under the linear-association mechanism and therefore the length-distribution of which conforms to a geometric sequence in the degree of polymerization with a common ratio r, which is less than, and usually very close to, unity. In this case, an observed macroscopic rate of dissociation is shown to be a product of the microscopic elementary dissociation rate constant and a factor (1-r), extremely reduced compared with the intrinsic elementary rate. Secondly, we discuss protein conformational states in fibrillogenesis with molecular and volumetric observations reported, such as the unfolded state responsible for the association with seeds and the extension of amyloid fibrils, the transition state in which protein cavity formation and dehydration occur to intermediate levels, and the fibril state in which they occur to final respective levels which, in some cases, depend on the maturity of the fibril.

  18. Identification of protein pheromones that promote aggressive behaviour.

    PubMed

    Chamero, Pablo; Marton, Tobias F; Logan, Darren W; Flanagan, Kelly; Cruz, Jason R; Saghatelian, Alan; Cravatt, Benjamin F; Stowers, Lisa

    2007-12-06

    Mice use pheromones, compounds emitted and detected by members of the same species, as cues to regulate social behaviours such as pup suckling, aggression and mating. Neurons that detect pheromones are thought to reside in at least two separate organs within the nasal cavity: the vomeronasal organ (VNO) and the main olfactory epithelium (MOE). Each pheromone ligand is thought to activate a dedicated subset of these sensory neurons. However, the nature of the pheromone cues and the identity of the responding neurons that regulate specific social behaviours are largely unknown. Here we show, by direct activation of sensory neurons and analysis of behaviour, that at least two chemically distinct ligands are sufficient to promote male-male aggression and stimulate VNO neurons. We have purified and analysed one of these classes of ligand and found its specific aggression-promoting activity to be dependent on the presence of the protein component of the major urinary protein (MUP) complex, which is known to comprise specialized lipocalin proteins bound to small organic molecules. Using calcium imaging of dissociated vomeronasal neurons (VNs), we have determined that the MUP protein activates a sensory neuron subfamily characterized by the expression of the G-protein Galpha(o) subunit (also known as Gnao) and Vmn2r putative pheromone receptors (V2Rs). Genomic analysis indicates species-specific co-expansions of MUPs and V2Rs, as would be expected among pheromone-signalling components. Finally, we show that the aggressive behaviour induced by the MUPs occurs exclusively through VNO neuronal circuits. Our results substantiate the idea of MUP proteins as pheromone ligands that mediate male-male aggression through the accessory olfactory neural pathway.

  19. Strand invasion promoted by recombination protein of coliphage

    NASA Astrophysics Data System (ADS)

    Rybalchenko, Nataliya; Golub, Efim I.; Bi, Baoyuan; Radding, Charles M.

    2004-12-01

    Studies of phage in vivo have indicated that its own recombination enzymes, protein and exonuclease, are capable of catalyzing two dissimilar pathways of homologous recombination that are widely distributed in nature: single-strand annealing and strand invasion. The former is an enzymatic splicing of overlapping ends of broken homologous DNA molecules, whereas the latter is characterized by the formation of a three-stranded synaptic intermediate and subsequent strand exchange. Previous studies in vitro have shown that protein has annealing activity, and that exonuclease, acting on branched substrates, can produce a perfect splice that requires only ligation for completion. The present study shows that protein can initiate strand invasion in vitro, as evidenced both by the formation of displacement loops (D-loops) in superhelical DNA and by strand exchange between colinear single-stranded and double-stranded molecules. Thus, protein can catalyze steps that are central to both strand annealing and strand invasion pathways of recombination. These observations add protein to a set of diverse proteins that appear to promote recognition of homology by a unitary mechanism governed by the intrinsic dynamic properties of base pairs in DNA. genetic recombination | phage λ

  20. Monocyte/macrophage and protein interactions with non-fouling plasma polymerized tetraglyme and chemically modified polystyrene surfaces: In vitro and in vivo studies

    NASA Astrophysics Data System (ADS)

    Shen, Mingchao

    2001-07-01

    Biomaterials become encapsulated by fibrous tissues after implantation in soft tissues. Monocytes and macrophages are believed to play important roles in this response. The hypothesis tested in this dissertation is that material surface chemistry determines the amount of adsorbed proteins, which mediate monocyte adhesion, activation, and the foreign body response. On chemically modified polystyrene surfaces, monocyte adhesion in vitro was promoted by preadsorbed fibrinogen, fibronectin, and IgG, and increased with increasing amount of adsorbed fibrinogen. Adsorbed proteins and material surface chemistry mediated monocyte activation. TNFalpha release, procoagulant activity, and multinucleated foreign body giant cell (FBGC) formation was at least two-fold higher on IgG than other protein adsorbed surfaces. Adsorbed IgG and fibrinogen triggered monocyte intracellular calcium changes. FBGC formation was the highest on the hydrophobic polystyrene surface. Materials that greatly reduce non-specific protein adsorption may reduce the foreign body response to implanted materials. Radio-frequency plasma polymerized tetraglyme (CH3O(CH2CH2O)4CH 3) surfaces contained PEO-like chemical species and reduced fibrinogen adsorption to less than 10 ng/cm2. Monocyte adhesion to tetraglyme in vitro was also greatly reduced. Monocyte adhesion correlated linearly to the amount of adsorbed fibrinogen on a series of tetraglyme surfaces deposited at different plasma powers. Multivariate analysis using partial least squares regression identified the key surface spectra variables from electron spectroscopy for chemical analysis (ESCA) and time of flight secondary ion mass spectrometry (ToF-SIMS) that contributed to the non-fouling properties of tetraglyme. However, leukocyte adhesion to surfaces implanted subcutaneously in mice for 1 or 28 days did not correlate with protein adsorption and was higher on tetraglyme than the FEP control. Fibrous encapsulation to tetraglyme implanted for 28 days

  1. Ezrin regulates microvillus morphogenesis by promoting distinct activities of Eps8 proteins

    PubMed Central

    Zwaenepoel, Ingrid; Naba, Alexandra; Menezes Lyra Da Cunha, Marcel; Del Maestro, Laurence; Formstecher, Etienne; Louvard, Daniel; Arpin, Monique

    2012-01-01

    The mechanisms that regulate actin filament polymerization resulting in the morphogenesis of the brush border microvilli in epithelial cells remain unknown. Eps8, the prototype of a family of proteins capable of capping and bundling actin filaments, has been shown to bundle the microvillar actin filaments. We report that Eps8L1a, a member of the Eps8 family and a novel ezrin-interacting partner, controls microvillus length through its capping activity. Depletion of Eps8L1a leads to the formation of long microvilli, whereas its overexpression has the opposite effect. We demonstrate that ezrin differentially modulates the actin-capping and -bundling activities of Eps8 and Eps8L1a during microvillus assembly. Coexpression of ezrin with Eps8 promotes the formation of membrane ruffles and tufts of microvilli, whereas expression of ezrin and Eps8L1a induces the clustering of actin-containing structures at the cell surface. These distinct morphological changes are neither observed when a mutant of ezrin defective in its binding to Eps8/Eps8L1a is coexpressed with Eps8 or Eps8L1a nor observed when ezrin is expressed with mutants of Eps8 or Eps8L1a defective in the actin-bundling or -capping activities, respectively. Our data show a synergistic effect of ezrin and Eps8 proteins in the assembly and organization of actin microvillar filaments. PMID:22262457

  2. Step-by-step strategy for protein enrichment and proteome characterisation of extracellular polymeric substances in wastewater treatment systems.

    PubMed

    Silva, Ana F; Carvalho, Gilda; Soares, Renata; Coelho, Ana V; Barreto Crespo, M Teresa

    2012-08-01

    Extracellular polymeric substances (EPS) are keys in biomass aggregation and settleability in wastewater treatment systems. In membrane bioreactors (MBR), EPS are an important factor as they are considered to be largely responsible for membrane fouling. Proteins were shown to be the major component of EPS produced by activated sludge and to be correlated with the properties of the sludge, like settling, hydrophobicity and cell aggregation. Previous EPS proteomic studies of activated sludge revealed several problems, like the interference of other EPS molecules in protein analysis. In this study, a successful strategy was outlined to identify the proteins from soluble and bound EPS extracted from activated sludge of a lab-scale MBR. EPS samples were first subjected to pre-concentration through lyophilisation, centrifugal ultrafiltration or concentration with a dialysis membrane coated by a highly absorbent powder of polyacrylate-polyalcohol, preceded or not by a dialysis step. The highest protein concentration factors were achieved with the highly absorbent powder method without previous dialysis step. Four protein precipitation methods were then tested: acetone, trichloroacetic acid (TCA), perchloric acid and a commercial kit. Protein profiles were compared in 4-12 % sodium dodecyl sulphate polyacrylamide gel electrophoresis gels. Both acetone and TCA should be applied for the highest coverage for soluble EPS proteins, whereas TCA was the best method for bound EPS proteins. All visible bands of selected profiles were subjected to mass spectrometry analysis. A high number of proteins (25-32 for soluble EPS and 17 for bound EPS) were identified. As a conclusion of this study, a workflow is proposed for the successful proteome characterisation of soluble and bound EPS from activated sludge samples.

  3. The LIM Protein Zyxin Binds CARP-1 and Promotes Apoptosis

    PubMed Central

    Hervy, Martial; Hoffman, Laura M.; Jensen, Christopher C.; Smith, Mark; Beckerle, Mary C.

    2010-01-01

    Zyxin is a dual-function LIM domain protein that regulates actin dynamics in response to mechanical stress and shuttles between focal adhesions and the cell nucleus. Here we show that zyxin contributes to UV-induced apoptosis. Exposure of wild-type fibroblasts to UV-C irradiation results in apoptotic cell death, whereas cells harboring a homozygous disruption of the zyxin gene display a statistically significant survival advantage. To gain insight into the molecular mechanism by which zyxin promotes apoptotic signaling, we expressed an affinity-tagged zyxin variant in zyxin-null cells and isolated zyxin-associated proteins from cell lysates under physiological conditions. A 130-kDa protein that was co-isolated with zyxin was identified by microsequence analysis as the Cell Cycle and Apoptosis Regulator Protein-1 (CARP-1). CARP-1 associates with the LIM region of zyxin. Zyxin lacking the CARP-1 binding region shows reduced proapoptotic activity in response to UV-C irradiation. We demonstrate that CARP-1 is a nuclear protein. Zyxin is modified by phosphorylation in cells exposed to UV-C irradiation, and nuclear accumulation of zyxin is induced by UV-C exposure. These findings highlight a novel mechanism for modulating the apoptotic response to UV irradiation. PMID:20852740

  4. Optimization of Landscape Phage Fusion Protein-Modified Polymeric PEG-PE Micelles for Improved Breast Cancer Cell Targeting

    PubMed Central

    Wang, Tao; Petrenko, Valery A.; Torchilin, Vladimir P.

    2015-01-01

    Amphiphilic landscape phage fusion proteins with high affinity and selectivity towards breast cancer MCF-7 (Michigan Cancer Foundation-7) cells self-assemble with polymeric PEG-PE conjugates to form mixed micelles (phage-micelles) capable of cancer cell-targeted delivery of poorly-soluble drugs. While the PEG corona provides the stability and longevity to the micelles, its presence is a potential steric difficulties for the interaction of phage fusion protein with cell surface targets. We attempted to address this problem by controlling the length of the PEG block and the phage fusion protein quantity, selecting the optimal ones to produce a reasonable retention of the targeting affinity and selectivity of the MCF-7-specific phage fusion protein. Three PEG-PE conjugates with different PEG lengths were used to construct phage- and plain-micelles, followed by FACS analysis of the effect of the PEG length on their binding affinity and selectivity towards target MCF-7 cells using either a MCF-7 cell monoculture or a cell co-culture model composed of target cancer MCF-7 cells and non-target, non-cancer C166 cells expressing GFP (Green Fluorescent Protein). Both, the length of PEG and quantity of phage fusion protein had a profound impact on the targetability of the phage-micelles. Phage-micelles prepared with PEG2k-PE achieved a desirable binding affinity and selectivity. Incorporation of a minimal concentration of phage protein, up to 0.5%, produced maximal targeting efficiency towards MCF-7 cells. Overall, phage-micelles with PEG2k-PE and 0.5% of phage protein represent the optimal formulation for targeting towards breast cancer cells. PMID:26451274

  5. Mitogen Activated Protein Kinase Activated Protein Kinase 2 Regulates Actin Polymerization and Vascular Leak in Ventilator Associated Lung Injury

    PubMed Central

    Damarla, Mahendra; Hasan, Emile; Boueiz, Adel; Le, Anne; Pae, Hyun Hae; Montouchet, Calypso; Kolb, Todd; Simms, Tiffany; Myers, Allen; Kayyali, Usamah S.; Gaestel, Matthias; Peng, Xinqi; Reddy, Sekhar P.; Damico, Rachel; Hassoun, Paul M.

    2009-01-01

    Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2−/− mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2−/− mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HVT MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling. PMID:19240800

  6. The enamel protein amelotin is a promoter of hydroxyapatite mineralization.

    PubMed

    Abbarin, Nastaran; San Miguel, Symone; Holcroft, James; Iwasaki, Kengo; Ganss, Bernhard

    2015-05-01

    Amelotin (AMTN) is a recently discovered protein that is specifically expressed during the maturation stage of dental enamel formation. It is localized at the interface between the enamel surface and the apical surface of ameloblasts. AMTN knock-out mice have hypomineralized enamel, whereas transgenic mice overexpressing AMTN have a compact but disorganized enamel hydroxyapatite (HA) microstructure, indicating a possible involvement of AMTN in regulating HA mineralization directly. In this study, we demonstrated that recombinant human (rh) AMTN dissolved in a metastable buffer system, based on light scattering measurements, promotes HA precipitation. The mineral precipitates were characterized by scanning and transmission electron microscopy and electron diffraction. Colloidal gold immunolabeling of AMTN in the mineral deposits showed that protein molecules were associated with HA crystals. The binding affinity of rh-AMTN to HA was found to be comparable to that of amelogenin, the major protein of the forming enamel matrix. Overexpression of AMTN in mouse calvaria cells also increased the formation of calcium deposits in the culture medium. Overexpression of AMTN during the secretory stage of enamel formation in vivo resulted in rapid and uncontrolled enamel mineralization. Site-specific mutagenesis of the potential serine phosphorylation motif SSEEL reduced the in vitro mineral precipitation to less than 25%, revealing that this motif is important for the HA mineralizing function of the protein. A synthetic short peptide containing the SSEEL motif was only able to facilitate mineralization in its phosphorylated form ((P)S(P) SEEL), indicating that this motif is necessary but not sufficient for the mineralizing properties of AMTN. These findings demonstrate that AMTN has a direct influence on biomineralization by promoting HA mineralization and suggest a critical role for AMTN in the formation of the compact aprismatic enamel surface layer during the maturation

  7. Structured Functional Domains of Myelin Basic Protein: Cross Talk between Actin Polymerization and Ca2+-Dependent Calmodulin Interaction

    PubMed Central

    Bamm, Vladimir V.; De Avila, Miguel; Smith, Graham S.T.; Ahmed, Mumdooh A.M.; Harauz, George

    2011-01-01

    The 18.5-kDa myelin basic protein (MBP), the most abundant isoform in human adult myelin, is a multifunctional, intrinsically disordered protein that maintains compact assembly of the sheath. Solution NMR spectroscopy and a hydrophobic moment analysis of MBP's amino-acid sequence have previously revealed three regions with high propensity to form strongly amphipathic α-helices. These regions, located in the central, N- and C-terminal parts of the protein, have been shown to play a role in the interactions of MBP with cytoskeletal proteins, Src homology 3-domain-containing proteins, Ca2+-activated calmodulin (Ca2+-CaM), and myelin-mimetic membrane bilayers. Here, we have further characterized the structure-function relationship of these three domains. We constructed three recombinant peptides derived from the 18.5-kDa murine MBP: (A22–K56), (S72–S107), and (S133–S159) (which are denoted α1, α2, and α3, respectively). We used a variety of biophysical methods (circular dichroism spectroscopy, isothermal titration calorimetry, transmission electron microscopy, fluorimetry, and solution NMR spectroscopy and chemical shift index analysis) to characterize the interactions of these peptides with actin and Ca2+-CaM. Our results show that all three peptides can adopt α-helical structure inherently even in aqueous solution. Both α1- and α3-peptides showed strong binding with Ca2+-CaM, and both adopted an α-helical conformation upon interaction, but the binding of the α3-peptide appeared to be more dynamic. Only the α1-peptide exhibited actin polymerization and bundling activity, and the addition of Ca2+-CaM resulted in depolymerization of actin that had been polymerized by α1. The results of this study proved that there is an N-terminal binding domain in MBP for Ca2+-CaM (in addition to the primary site located in the C-terminus), and that it is sufficient for CaM-induced actin depolymerization. These three domains of MBP represent molecular recognition

  8. A 25-kD inhibitor of actin polymerization is a low molecular mass heat shock protein

    PubMed Central

    1991-01-01

    The 25-kD inhibitor of actin polymerization (25-kD IAP), isolated from turkey smooth muscle (Miron, T., M. Wilchek, and B. Geiger, 1988. Eur. J. Biochem. 178:543-553), is shown here to be a low molecular mass heat shock protein (HSP). Direct sequence analysis of the purified protein, as well as cloning and sequencing of the respective cDNA, disclosed a high degree of homology (67% identity, 80% similarity) to the human 27- kD HSP. Southern blot of chicken genomic DNA disclosed one band, suggesting the presence of a single gene, and Northern blot analysis revealed abundant transcript of approximately 1 kb in gizzard and heart tissues and lower amounts in total 18-d chick embryo RNA and in cultured fibroblasts. Exposure of the latter cells to 45 degrees C resulted in over 15-fold increase in the apparent level of the 25-kD IAP protein, confirming that its expression is regulated by heat shock. Immunofluorescent microscopic localization indicated that after heat treatment, the levels of the 25-kD IAP were markedly increased and the protein was apparently associated with cytoplasmic granules. Heat shock also had a transient, yet prominent, effect on the microfilament system in cultured fibroblasts: stress fibers disintegrated within 10-15 min after incubation at 45 degrees C, yet upon further incubation at the elevated temperature, conspicuous actin bundles were apparently reformed. PMID:2071672

  9. Multi-protein Delivery by Nanodiamonds Promotes Bone Formation

    PubMed Central

    Moore, L.; Gatica, M.; Kim, H.; Osawa, E.; Ho, D.

    2013-01-01

    Bone morphogenetic proteins (BMPs) are well-studied regulators of cartilage and bone development that have been Food and Drug Administration (FDA)-approved for the promotion of bone formation in certain procedures. BMPs are seeing more use in oral and maxillofacial surgeries because of recent FDA approval of InFUSE® for sinus augmentation and localized alveolar ridge augmentation. However, the utility of BMPs in medical and dental applications is limited by the delivery method. Currently, BMPs are delivered to the surgical site by the implantation of bulky collagen sponges. Here we evaluate the potential of detonation nanodiamonds (NDs) as a delivery vehicle for BMP-2 and basic fibroblast growth factor (bFGF). Nanodiamonds are biocompatible, 4- to 5-nm carbon nanoparticles that have previously been used to deliver a wide variety of molecules, including proteins and peptides. We find that both BMP-2 and bFGF are readily loaded onto NDs by physisorption, forming a stable colloidal solution, and are triggered to release in slightly acidic conditions. Simultaneous delivery of BMP-2 and bFGF by ND induces differentiation and proliferation in osteoblast progenitor cells. Overall, we find that NDs provide an effective injectable alternative for the delivery of BMP-2 and bFGF to promote bone formation. PMID:24045646

  10. Multi-protein delivery by nanodiamonds promotes bone formation.

    PubMed

    Moore, L; Gatica, M; Kim, H; Osawa, E; Ho, D

    2013-11-01

    Bone morphogenetic proteins (BMPs) are well-studied regulators of cartilage and bone development that have been Food and Drug Administration (FDA)-approved for the promotion of bone formation in certain procedures. BMPs are seeing more use in oral and maxillofacial surgeries because of recent FDA approval of InFUSE(®) for sinus augmentation and localized alveolar ridge augmentation. However, the utility of BMPs in medical and dental applications is limited by the delivery method. Currently, BMPs are delivered to the surgical site by the implantation of bulky collagen sponges. Here we evaluate the potential of detonation nanodiamonds (NDs) as a delivery vehicle for BMP-2 and basic fibroblast growth factor (bFGF). Nanodiamonds are biocompatible, 4- to 5-nm carbon nanoparticles that have previously been used to deliver a wide variety of molecules, including proteins and peptides. We find that both BMP-2 and bFGF are readily loaded onto NDs by physisorption, forming a stable colloidal solution, and are triggered to release in slightly acidic conditions. Simultaneous delivery of BMP-2 and bFGF by ND induces differentiation and proliferation in osteoblast progenitor cells. Overall, we find that NDs provide an effective injectable alternative for the delivery of BMP-2 and bFGF to promote bone formation.

  11. A small subunit processome protein promotes cancer by altering translation.

    PubMed

    Yang, H W; Kim, T-M; Song, S S; Menon, L; Jiang, X; Huang, W; Black, P M; Park, P J; Carroll, R S; Johnson, M D

    2015-08-20

    Dysregulation of ribosome biogenesis or translation can promote cancer, but the underlying mechanisms remain unclear. UTP18 is a component of the small subunit processome, a nucleolar multi-protein complex whose only known function is to cleave pre-ribosomal RNA to yield the 18S ribosomal RNA component of 40S ribosomal subunits. Here, we show that UTP18 also alters translation to promote stress resistance and growth, and that UTP18 is frequently gained and overexpressed in cancer. We observed that UTP18 localizes to the cytoplasm in a subset of cells, and that serum withdrawal increases cytoplasmic UTP18 localization. Cytoplasmic UTP18 associates with the translation complex and Hsp90 to upregulate the translation of IRES-containing transcripts such as HIF1a, Myc and VEGF, thereby inducing stress resistance. Hsp90 inhibition decreases cytoplasmic UTP18 and UTP18-induced increases in translation. Importantly, elevated UTP18 expression correlates with increased aggressiveness and decreased survival in numerous cancers. Enforced UTP18 overexpression promotes transformation and tumorigenesis, whereas UTP18 knockdown inhibits these processes. This stress adaptation mechanism is thus co-opted for growth by cancers, and its inhibition may represent a promising new therapeutic target.

  12. Multilayered films fabricated from an oligoarginine-conjugated protein promote efficient surface-mediated protein transduction.

    PubMed

    Jewell, Christopher M; Fuchs, Stephen M; Flessner, Ryan M; Raines, Ronald T; Lynn, David M

    2007-03-01

    The conjugation of cationic protein transduction domains to proteins results in an increase in the extent to which proteins are internalized by cells. This investigation sought to determine whether the conjugation of a protein transduction domain to a functional protein could be used to facilitate the incorporation of the protein into multilayered polyelectrolyte films and, subsequently, whether these films could be used to promote surface-mediated protein transduction. We demonstrate that it is possible to fabricate multilayered assemblies 80 nm thick using sodium polystyrene sulfonate (SPS) and bovine pancreatic ribonuclease (RNase A) conjugated to the cationic protein transduction domain nonaarginine (R(9)) using an entirely aqueous layer-by-layer process. We demonstrate further that the conjugation of R(9) to RNase A permits the assembly of multilayered films under conditions that do not allow for the incorporation of the unmodified protein. This result suggests that R(9) functions as a cationic anchor and serves to increase the strength of electrostatic interactions with SPS and facilitate layer-by-layer assembly. We also demonstrate that RNase A-R(9)/SPS films dissolve rapidly in physiologically relevant media and that macroscopic objects coated with these materials can be used to mediate high levels of protein transduction in mammalian cells. These results suggest the basis of general methods that could contribute to the design of materials that permit spatial and temporal control over the delivery of therapeutic proteins to cells and tissues.

  13. Monitoring Protein Fouling on Polymeric Membranes Using Ultrasonic Frequency-Domain Reflectometry

    PubMed Central

    Kujundzic, Elmira; Greenberg, Alan R.; Fong, Robin; Hernandez, Mark

    2011-01-01

    Novel signal-processing protocols were used to extend the in situ sensitivity of ultrasonic frequency-domain reflectometry (UFDR) for real-time monitoring of microfiltration (MF) membrane fouling during protein purification. Different commercial membrane materials, with a nominal pore size of 0.2 μm, were challenged using bovine serum albumin (BSA) and amylase as model proteins. Fouling induced by these proteins was observed in flat-sheet membrane filtration cells operating in a laminar cross-flow regime. The detection of membrane-associated proteins using UFDR was determined by applying rigorous statistical methodology to reflection spectra of ultrasonic signals obtained during membrane fouling. Data suggest that the total power reflected from membrane surfaces changes in response to protein fouling at concentrations as low as 14 μg/cm2, and results indicate that ultrasonic spectra can be leveraged to detect and monitor protein fouling on commercial MF membranes. PMID:24957732

  14. Polymerization degrees, molecular weights and protein-binding affinities of condensed tannin fractions from a Leucaena leucocephala hybrid.

    PubMed

    Saminathan, Mookiah; Tan, Hui Yin; Sieo, Chin Chin; Abdullah, Norhani; Wong, Clemente Michael Vui Ling; Abdulmalek, Emilia; Ho, Yin Wan

    2014-06-12

    Condensed tannins (CTs) form insoluble complexes with proteins and are able to protect them from degradation, which could lead to rumen bypass proteins. Depending on their degrees of polymerization (DP) and molecular weights, CT fractions vary in their capability to bind proteins. In this study, purified condensed tannins (CTs) from a Leucaena leucocephala hybrid were fractionated into five different molecular weight fractions. The structures of the CT fractions were investigated using 13C-NMR. The DP of the CT fractions were determined using a modified vanillin assay and their molecular weights were determined using Q-TOF LC-MS. The protein-binding affinities of the respective CT fractions were determined using a protein precipitation assay. The DP of the five CT fractions (fractions F1-F5) measured by the vanillin assay in acetic acid ranged from 4.86 to 1.56. The 13C-NMR results showed that the CT fractions possessed monomer unit structural heterogeneity. The number-average molecular weights (Mn) of the different fractions were 1265.8, 1028.6, 652.2, 562.2, and 469.6 for fractions F1, F2, F3, F4, and F5, respectively. The b values representing the CT quantities needed to bind half of the maximum precipitable bovine serum albumin increased with decreasing molecular weight--from fraction F1 to fraction F5 with values of 0.216, 0.295, 0.359, 0.425, and 0.460, respectively. This indicated that higher molecular weight fractions of CTs from L. leucocephala have higher protein-binding affinities than those with lower molecular weights.

  15. CHIP Regulates Osteoclast Formation through Promoting TRAF6 Protein Degradation

    PubMed Central

    Li, Shan; Shu, Bing; Zhang, Yanquan; Li, Jia; Guo, Junwei; Wang, Yinyin; Ren, Fangli; Xiao, Guozhi; Chang, Zhijie; Chen, Di

    2014-01-01

    Objective Carboxyl terminus of Hsp70-interacting protein (CHIP or STUB1) is an E3 ligase and regulates the stability of several proteins which are involved in tumor growth and metastasis. However, the role of CHIP in bone growth and bone remodeling in vivo has not been reported. The objective of this study is to investigate the role and mechanism of CHIP in regulation of bone mass and bone remodeling. Methods The bone phenotype of Chip−/− mice was examined by histology, histomorphometry and micro-CT analyses. The regulatory mechanism of CHIP on the degradation of TRAF6 and the inhibition of NF-κB signaling was examined by immunoprecipitation (IP), western blotting and luciferase reporter assays. Results In this study, we found that deletion of the Chip gene leads to osteopenic phenotype and increased osteoclast formation. We further found that TRAF6, as a novel substrate of CHIP, is up-regulated in Chip−/− osteoclasts. TRAF6 is critical for RANKL-induced osteoclastogenesis. TRAF6 is an adaptor protein which functions as an E3 ligase to regulate the activation of TAK1 and the I-κB kinase (IKK) and is a key regulator of NF-κB signaling. CHIP interacts with TRAF6 to promote TRAF6 ubiquitination and proteasome degradation. CHIP inhibits p65 nuclear translocation, leading to the repression of the TRAF6-mediated NF-κB transcription. Conclusion CHIP inhibits NF-κB signaling via promoting TRAF6 degradation and plays an important role in osteoclastogenesis and bone remodeling, suggesting that it may be a novel therapeutic target for the treatment of bone loss associated diseases. PMID:24578159

  16. Host SAMHD1 Protein Promotes HIV-1 Recombination in Macrophages*

    PubMed Central

    Nguyen, Laura A.; Kim, Dong-Hyun; Daly, Michele B.; Allan, Kevin C.; Kim, Baek

    2014-01-01

    Template switching can occur during the reverse transcription of HIV-1. Deoxynucleotide triphosphate (dNTP) concentrations have been biochemically shown to impact HIV-1 reverse transcriptase (RT)-mediated strand transfer. Lowering the dNTP concentrations promotes RT pausing and RNA template degradation by RNase H activity of the RT, subsequently leading to strand transfer. Terminally differentiated/nondividing macrophages, which serve as a key HIV-1 reservoir, contain extremely low dNTP concentrations (20–50 nm), which results from the cellular dNTP hydrolyzing sterile α motif and histidine aspartic domain containing protein 1 (SAMHD1) protein, when compared with activated CD4+ T cells (2–5 μm). In this study, we first observed that HIV-1 template switching efficiency was nearly doubled in human primary macrophages when compared with activated CD4+ T cells. Second, SAMHD1 degradation by viral protein X (Vpx), which elevates cellular dNTP concentrations, decreased HIV-1 template switching efficiency in macrophages to the levels comparable with CD4+ T cells. Third, differentiated SAMHD1 shRNA THP-1 cells have a 2-fold increase in HIV-1 template switching efficiency. Fourth, SAMHD1 degradation by Vpx did not alter HIV-1 template switching efficiency in activated CD4+ T cells. Finally, the HIV-1 V148I RT mutant that is defective in dNTP binding and has DNA synthesis delay promoted RT stand transfer when compared with wild type RT, particularly at low dNTP concentrations. Here, we report that SAMHD1 regulation of the dNTP concentrations influences HIV-1 template switching efficiency, particularly in macrophages. PMID:24352659

  17. A Lumry-Eyring Nucleated-Polymerization (LENP) Model of Protein Aggregation Kinetics 2. Competing Growth via Condensation- and Chain-Polymerization

    PubMed Central

    Li, Yi; Roberts, Christopher J.

    2009-01-01

    The Lumry-Eyring with nucleated-polymerization (LENP) model from part 1 (Andrews and Roberts, J. Phys. Chem. B 2007, 111, 7897 7913) is expanded to explicitly account for kinetic contributions from aggregate-aggregate condensation polymerization. Experimentally accessible quantities described by the resulting model include monomer mass fraction (m), weight-average molecular weight (Mw), and ratio of Mw to number-average molecular weight (Mn) as a function of time (t). Analysis of global model behavior illustrates ways to identify which steps in the overall aggregation process are kinetically important, based on the qualitative behavior of m, Mw, and Mw/Mn vs. t, and based on whether bulk phase separation or precipitation occurs. For cases in which all aggregates remain soluble, moment equations are provided that permit straightforward numerical regression of experimental data to give separate time scales or inverse rate coefficients for nucleation and for growth by chain and condensation polymerization. Analysis of simulated data indicates that it may be possible to neglect condensation reactions if only early-time data are considered, and also highlights difficulties in conclusively distinguishing between alternative mechanisms of condensation even when kinetics are monitored with both m and wM. PMID:19368365

  18. Protein promoting vibrations: Subpicosecond enzyme dynamics and catalysis

    NASA Astrophysics Data System (ADS)

    Mincer, Joshua S.

    The coupling of protein dynamics to enzymatic reactions is explored, specifically with reference to subpicosecond quasioscillatory motions, protein promoting vibrations (PPVS), that dynamically modulate the reaction potential energy surface on the same timescale as the reaction barrier passage itself. Computational algorithms to study these motions are presented. The first identifies a PPV in a specific enzymatic reaction, if it exists. The second identifies protein residues, the motions of which drive the PPV. Application to the enzyme horse liver alcohol dehydrogenase (HLADH) explains experimental mutagenesis studies and makes predictions for the many residues as yet not studied. PPV contribution to rate enhancement in HLADH is considered in light of these studies. The reaction rate theory developed by Antoniou and Schwartz is generalized to include an electron coordinate, allowing for the modeling of enzymes that catalyze proton-coupled electron transfer (PCET) reactions. Model system calculations yield kinetic parameters and primary HID kinetic isotope effects (KIEs) in qualitative agreement with experiment and allow for insights into KIE temperature dependence as well as KIE masking when these reactions are coupled to a PPV. Finally, PPV coupling to bond cleavage reactions in general is investigated in the specific context of PPV coupling to reaction center electron density in the enzyme purine nucleoside phosphorylase.

  19. Polymeric microspheres

    DOEpatents

    Walt, David R.; Mandal, Tarun K.; Fleming, Michael S.

    2004-04-13

    The invention features core-shell microsphere compositions, hollow polymeric microspheres, and methods for making the microspheres. The microspheres are characterized as having a polymeric shell with consistent shell thickness.

  20. Succination of thiol groups in adipose tissue proteins in diabetes: succination inhibits polymerization and secretion of adiponectin.

    PubMed

    Frizzell, Norma; Rajesh, Mathur; Jepson, Matthew J; Nagai, Ryoji; Carson, James A; Thorpe, Suzanne R; Baynes, John W

    2009-09-18

    S-(2-Succinyl)cysteine (2SC) is formed by reaction of the Krebs cycle intermediate fumarate with cysteine residues in protein, a process termed succination of protein. Both fumarate and succination of proteins are increased in adipocytes cultured in high glucose medium (Nagai, R., Brock, J. W., Blatnik, M., Baatz, J. E., Bethard, J., Walla, M. D., Thorpe, S. R., Baynes, J. W., and Frizzell, N. (2007) J. Biol. Chem. 282, 34219-34228). We show here that succination of protein is also increased in epididymal, mesenteric, and subcutaneous adipose tissue of diabetic (db/db) mice and that adiponectin is a major target for succination in both adipocytes and adipose tissue. Cys-39, which is involved in cross-linking of adiponectin monomers to form trimers, was identified as a key site of succination of adiponectin in adipocytes. 2SC was detected on two of seven monomeric forms of adiponectin immunoprecipitated from adipocytes and epididymal adipose tissue. Based on densitometry, 2SC-adiponectin accounted for approximately 7 and 8% of total intracellular adiponectin in cells and tissue, respectively. 2SC was found only in the intracellular, monomeric forms of adiponectin and was not detectable in polymeric forms of adiponectin in cell culture medium or plasma. We conclude that succination of adiponectin blocks its incorporation into trimeric and higher molecular weight, secreted forms of adiponectin. We propose that succination of proteins is a biomarker of mitochondrial stress and accumulation of Krebs cycle intermediates in adipose tissue in diabetes and that succination of adiponectin may contribute to the decrease in plasma adiponectin in diabetes.

  1. Lytic Promoters Express Protein during Herpes Simplex Virus Latency

    PubMed Central

    Russell, Tiffany A.; Tscharke, David C.

    2016-01-01

    Herpes simplex virus (HSV) has provided the prototype for viral latency with previously well-defined acute or lytic and latent phases. More recently, the deep quiescence of HSV latency has been questioned with evidence that lytic genes can be transcribed in this state. However, to date the only evidence that these transcripts might be translated has come from immunological studies that show activated T cells persist in the nervous system during latency. Here we use a highly sensitive Cre-marking model to show that lytic and latent phases are less clearly defined in two significant ways. First, around half of the HSV spread leading to latently infected sites occurred beyond the initial acute infection and second, we show direct evidence that lytic promoters can drive protein expression during latency. PMID:27348812

  2. Self-assembling polymeric nanoparticles for enhanced intra-articular anti-inflammatory protein delivery

    NASA Astrophysics Data System (ADS)

    Whitmire, Rachel Elisabeth

    Osteoarthritis (OA) affects 26 million Americans, or approximately 14% of the adult population. The incidence of OA is predicted to dramatically increase in the next 20 years as the US grows older and the rate of obesity continues to increase. There are currently no clinical interventions that cure OA. Current biomaterial delivery systems exhibit several limitations. First, most drug-delivery particles are hydrophobic, which is not optimal for hydrophilic protein encapsulation. Second, hydrophobic particles, such as PLGA, could cause wear damage to the already-fragile OA cartilage structure. Additionally, these particles usually suffer from non-specific protein adsorption, which causes increased phagocytosis and can lead to increased inflammation. New therapies that increase the effectiveness of OA treatments or reverse OA disease progression will greatly decrease the economic costs and individual pain associated with this disease. The goal of this thesis was to develop a new drug-delivering material to deliver anti-inflammatory protein for treating OA. Our central hypothesis for this work is that a controlled release/presentation system will more effectively deliver anti-inflammatory protein therapies to the OA joint. The primary goal of this work was to synthesize a block copolymer that could self-assemble into injectable, sub-micron-scale particles and would allow an anti-inflammatory protein, IL-1ra, to be tethered to its surface for efficient protein delivery. The block copolymer incorporated an oligo-ethylene monomer for tissue compatibility and non-fouling behavior, a 4-nitrophenol group for efficient protein tethering, and cyclohexyl methacrylate, a hydrophobic monomer, for particle stability. We engineered the copolymer and tested it in both in vitro culture experiments and an in vivo model to evaluate protein retention in the knee joint. The rationale for this project was that the rational design and synthesis of a new drug- and protein

  3. Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins.

    PubMed

    Courtemanche, Naomi; Pollard, Thomas D; Chen, Qian

    2016-06-01

    When tagged with a fluorescent protein, actin is not fully functional, so the LifeAct peptide fused to a fluorescent protein is widely used to localize actin filaments in live cells. However, we find that these fusion proteins have many concentration-dependent effects on actin assembly in vitro and in fission yeast cells. mEGFP-LifeAct inhibits actin assembly during endocytosis as well as assembly and constriction of the cytokinetic contractile ring. Purified mEGFP-LifeAct and LifeAct-mCherry bind actin filaments with Kd values of ∼10 μM. LifeAct-mCherry can promote actin filament nucleation and either promote or inhibit filament elongation. Both separately and together, profilin and formins suppress these effects. LifeAct-mCherry can also promote or inhibit actin filament severing by cofilin. These concentration-dependent effects mean that caution is necessary when overexpressing LifeAct fusion proteins to label actin filaments in cells. Therefore, we used low micromolar concentrations of tagged LifeAct to follow assembly and disassembly of actin filaments in cells. Careful titrations also gave an estimate of a peak of ∼190,000 actin molecules (∼500 μm) in the fission yeast contractile ring. These filaments shorten from ∼500 to ∼100 subunits as the ring constricts.

  4. Plasma-induced graft-polymerization of polyethylene glycol acrylate on polypropylene films: chemical characterization and evaluation of the protein adsorption.

    PubMed

    Zanini, Stefano; Riccardi, Claudia; Grimoldi, Elisa; Colombo, Claudia; Villa, Anna Maria; Natalello, Antonino; Gatti-Lafranconi, Pietro; Lotti, Marina; Doglia, Silvia Maria

    2010-01-01

    This work deals with the optimization of argon plasma-induced graft-polymerization of polyethylene glycol acrylate (PEGA) on polypropylene (PP) films in order to obtain surfaces with a reduced protein adsorption for possible biomedical applications. To this end, we examined the protein adsorption on the treated and untreated surfaces. The graft-polymerization process consisted of four steps: (a) plasma pre-activation of the PP substrates; (b) immersion in a PEGA solution; (c) argon plasma-induced graft-polymerization; (d) washing and drying of the samples. The efficiency of these processes was evaluated in terms of the amount of grafted polymer, coverage uniformity and substrates wettability. The process was monitored by contact angle measurements, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), X-ray Photoelectron Spectroscopy (XPS) and atomic force microscopy (AFM) analyses. The stability of the obtained thin films was evaluated in water and in Phosphate Buffer Saline (PBS) at 37 degrees C. The adsorption of fibrinogen and green fluorescent protein (GFP)--taken as model proteins--on the differently prepared surfaces was evaluated through a fluorescence approach using laser scanning confocal microscopy with photon counting detection. After plasma treatments of short duration, the protein adsorption decreases by about 60-70% with respect to that of the untreated film, while long plasma exposure resulted in a higher protein adsorption, due to damaging of the grafted polymer.

  5. Artificial chaperones based on mixed shell polymeric micelles: insight into the mechanism of the interaction of the chaperone with substrate proteins using Förster resonance energy transfer.

    PubMed

    Wang, Jianzu; Yin, Tao; Huang, Fan; Song, Yiqing; An, Yingli; Zhang, Zhenkun; Shi, Linqi

    2015-05-20

    Controlled and reversible interactions between polymeric nanoparticles and proteins have gained more and more attention with the hope to address many biological issues such as prevention of protein denaturation, interference of the fibrillation of disease relative proteins, removing of toxic biomolecules as well as targeting delivery of proteins, etc. In such cases, proper analytic techniques are needed to reveal the underlying mechanism of the particle-protein interactions. In the current work, Förster Resonance Energy Transfer (FRET) was used to investigate the interaction of our tailor designed artificial chaperone based on mixed shell polymeric micelles (MSPMs) with their substrate proteins. We designed a new kind of MSPMs with fluorescent acceptors precisely placed at the desired locations as well as hydrophobic domains which can adsorb unfolded proteins with a propensity to aggregate. Interactions of such model micelles with a donor-labeled protein-FITC-lysozyme, was monitored by FRET. The fabrication strategy of MSPMs makes it possible to control the accurate location of the acceptor, which is critical to reveal some unexpected insights of the micelle-protein interactions upon heating and cooling. Preadsorption of native proteins onto the hydrophobic domains of the MSPMs is a key step to prevent thermo-denaturation by diminishing interprotein aggregations. Reversible protein adsorption during heating and releasing during cooling have been confirmed. Conclusions from the FRET effect are in line with the measurement of residual enzymatic activity.

  6. Harmine promotes osteoblast differentiation through bone morphogenetic protein signaling

    SciTech Connect

    Yonezawa, Takayuki; Lee, Ji-Won; Hibino, Ayaka; Asai, Midori; Hojo, Hironori; Cha, Byung-Yoon; Teruya, Toshiaki; Nagai, Kazuo; Chung, Ung-Il; Yagasaki, Kazumi; and others

    2011-06-03

    Highlights: {yields} Harmine promotes the activity and mRNA expression of ALP. {yields} Harmine enhances the expressions of osteocalcin mRNA and protein. {yields} Harmine induces osteoblastic mineralization. {yields} Harmine upregulates the mRNA expressions of BMPs, Runx2 and Osterix. {yields} BMP signaling pathways are involved in the actions of harmine. -- Abstract: Bone mass is regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. We previously reported that harmine, a {beta}-carboline alkaloid, inhibits osteoclast differentiation and bone resorption in vitro and in vivo. In this study, we investigated the effects of harmine on osteoblast proliferation, differentiation and mineralization. Harmine promoted alkaline phosphatase (ALP) activity in MC3T3-E1 cells without affecting their proliferation. Harmine also increased the mRNA expressions of the osteoblast marker genes ALP and Osteocalcin. Furthermore, the mineralization of MC3T3-E1 cells was enhanced by treatment with harmine. Harmine also induced osteoblast differentiation in primary calvarial osteoblasts and mesenchymal stem cell line C3H10T1/2 cells. Structure-activity relationship studies using harmine-related {beta}-carboline alkaloids revealed that the C3-C4 double bond and 7-hydroxy or 7-methoxy group of harmine were important for its osteogenic activity. The bone morphogenetic protein (BMP) antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated harmine-promoted ALP activity. In addition, harmine increased the mRNA expressions of Bmp-2, Bmp-4, Bmp-6, Bmp-7 and its target gene Id1. Harmine also enhanced the mRNA expressions of Runx2 and Osterix, which are key transcription factors in osteoblast differentiation. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by harmine treatment. Taken together, these results indicate that harmine enhances osteoblast differentiation probably by inducing the expressions of

  7. Superparamagnetic lysozyme surface-imprinted polymer prepared by atom transfer radical polymerization and its application for protein separation.

    PubMed

    Gai, Qing-Qing; Qu, Feng; Liu, Zong-Jian; Dai, Rong-Ji; Zhang, Yu-Kui

    2010-07-30

    Molecular imprinting as a promising and facile separation technique has received much attention because of their high selectivity for target molecules. In this study, the superparamagnetic lysozyme surface-imprinted polymer was prepared by a novel fabricating protocol, the grafting of the imprinted polymer on magnetic particles in aqueous media was done by atom transfer radical polymerization (ATRP), and the properties of the imprinted polymer were characterized in detail. Its high selective adsorption and recognition to lysozyme demonstrated the separation ability of the magnetic imprinted material to template molecule, and it has been used for quick and direct separation of lysozyme from the mixture of standard proteins and real egg white samples under an external magnetic field. Furthermore, the elution of lysozyme from the imprinted material was achieved by PEG/sulphate aqueous two-phase system, which caused lysozyme not only desorption from the imprinted materials but also redistribution in the top and bottom phase of aqueous two-phase system. The aqueous two-phase system exhibited some of the extraction and enrichment effect to desorbed lysozyme. Our results showed that ATRP is a promising method for the protein molecularly imprinted polymer preparation.

  8. Protein purification with polymeric affinity membranes containing functionalized poly(acid) brushes.

    PubMed

    Jain, Parul; Vyas, Mukesh Kumar; Geiger, James H; Baker, Gregory L; Bruening, Merlin L

    2010-04-12

    Porous nylon membranes modified with poly(acid) brushes and their derivatives can rapidly purify proteins via ion-exchange and metal-ion affinity interactions. Membranes containing poly(2-(methacryloyloxy)ethyl succinate) (poly(MES)) brushes bind 118 +/- 8 mg of lysozyme per cm(3) of membrane and facilitate purification of lysozyme from chicken egg white. Moreover, functionalization of the poly(MES) brushes with nitrilotriacetate (NTA)-Ni(2+) complexes yields membranes that bind poly(histidine)-tagged (His-tagged) ubiquitin with a capacity of 85 +/- 2 mg of protein per cm(3) of membrane. Most importantly, the membranes modified with poly(MES)-NTA-Ni(2+) allow isolation of His-tagged cellular retinaldehyde-binding protein directly from a cell extract in <10 min, and the protein purity is comparable to that achieved with commercial affinity columns. Therefore, porous nylon membranes containing functionalized poly(MES) brushes are attractive candidates for rapid, high-capacity purification of His-tagged proteins from cell extracts.

  9. Generation of an Artificial Double Promoter for Protein Expression in Bacillus subtilis through a Promoter Trap System

    PubMed Central

    Yang, Mingming; Zhang, Weiwei; Ji, Shengyue; Cao, Pinghua; Chen, Yulin; Zhao, Xin

    2013-01-01

    Bacillus subtilis is an attractive host for production of recombinant proteins. Promoters and expression plasmid backbones have direct impacts on the efficiency of gene expression. To screen and isolate strong promoters, a promoter trap vector pShuttleF was developed in this study. Using the vector, approximately 1000 colonies containing likely promoters from Bacillus licheniformis genomic DNA were obtained. Amongst them, pShuttle-09 exhibited the highest β-Gal activities in both Escherichia coli and B. subtilis. The activity of pShuttle-09 in B. subtilis was eight times of that of the P43 promoter, a commonly used strong promoter for B. subtilis. A sequence analysis showed that pShuttle-09 contained PluxS and truncated luxS in-frame fused with the reporter gene as well as another fragment upstream of PluxS containing a putative promoter. This putative promoter was a hybrid promoter and its β-Gal activity was higher than PluxS. Reconstructing the hybrid promoter from pShuttle-09 to PlapS further improved the β-Gal production by 60%. The usefulness of our promoter trap system is likely due to random shuffling and recombination of DNA fragments and adoption of a rapid and high-throughput screening. Thus, our data provide additional evidence to support the concept of using a promoter trap system to create new promoters. PMID:23409173

  10. Transient Biocompatible Polymeric Platforms for Long-Term Controlled Release of Therapeutic Proteins and Vaccines

    PubMed Central

    Acar, Handan; Banerjee, Saikat; Shi, Heliang; Jamshidi, Reihaneh; Hashemi, Nastaran; Cho, Michael W.; Montazami, Reza

    2016-01-01

    Polymer-based interpenetrating networks (IPNs) with controllable and programmable degradation and release kinetics enable unique opportunities for physisorption and controlled release of therapeutic proteins or vaccines while their chemical and structural integrities are conserved. This paper presents materials, a simple preparation method, and release kinetics of a series of long-term programmable, biocompatible, and biodegradable polymer-based IPN controlled release platforms. Release kinetics of the gp41 protein was controlled over a 30-day period via tuning and altering the chemical structure of the IPN platforms. Post-release analysis confirmed structural conservation of the gp41 protein throughout the process. Cell viability assay confirmed biocompatibility and non-cytotoxicity of the IPNs. PMID:28546855

  11. The Postsynaptic Density Proteins Homer and Shank Form a Polymeric Network Structure

    SciTech Connect

    Hayashi, M.; Tang, C; Verpelli, C; Narayanan, R; Stearns, M; Xu, R; Li, H; Sala, C; Hayashi, Y

    2009-01-01

    The postsynaptic density (PSD) is crucial for synaptic functions, but the molecular architecture retaining its structure and components remains elusive. Homer and Shank are among the most abundant scaffolding proteins in the PSD, working synergistically for maturation of dendritic spines. Here, we demonstrate that Homer and Shank, together, form a mesh-like matrix structure. Crystallographic analysis of this region revealed a pair of parallel dimeric coiled coils intercalated in a tail-to-tail fashion to form a tetramer, giving rise to the unique configuration of a pair of N-terminal EVH1 domains at each end of the coiled coil. In neurons, the tetramerization is required for structural integrity of the dendritic spines and recruitment of proteins to synapses. We propose that the Homer-Shank complex serves as a structural framework and as an assembly platform for other PSD proteins.

  12. Localized surface plasmon resonance nanosensing of C-reactive protein with poly(2-methacryloyloxyethyl phosphorylcholine)-grafted gold nanoparticles prepared by surface-initiated atom transfer radical polymerization.

    PubMed

    Kitayama, Yukiya; Takeuchi, Toshifumi

    2014-06-03

    Highly sensitive and selective protein nanosensing based on localized surface plasmon resonance (LSPR) of gold nanoparticles (AuNPs) on which polymerized specific ligands were grafted as an artificial protein recognition layer for the target protein were demonstrated. As a model, optical nanosensing for C-reactive protein (CRP), a known biomarker for chronic inflammation that predicts the risk of arteriosclerosis or heart attacks, was achieved by measuring the shift of LSPR spectra derived from the change of permittivity of poly(2-methacryloyloxyethyl phosphorylcholine)-grafted AuNPs (PMPC-g-AuNPs) upon interacting with CRP, in which the PMPC-g-AuNPs layer were grafted on AuNPs by surface-initiated atom transfer radical polymerization (ATRP). This nanosensing system was effective even for detecting CRP concentrations in a human serum solution diluted to 1% (w/w), at which point a limit of detection was ~50 ng/mL and nonspecific adsorption of other proteins was negligible. The nanosensing system using specific ligand-grafted AuNPs has several strengths, such as low preparation cost, avoiding the need for expensive instruments, no necessary complex pretreatments, and high stability, because it does not contain biobased molecules. We believe this novel synthetic route for protein nanosensors, composed of AuNPs and a polymerized specific ligand utilizing surface-initiated controlled/living radical polymerization, will provide a foundation for the design and synthesis of nanosensors targeting various other biomarker proteins, paving the way for future advances in the field of biosensing.

  13. Energetics of Polymerization: A Contribution to an Understanding of Protein Synthesis.

    ERIC Educational Resources Information Center

    Friedmann, Herbert C.

    1986-01-01

    Discusses the various ways that textbooks treat the energetics of protein synthesis. Offers an approach to explaining the phenomenon by emphasizing the ordering aspects of the process. Describes the participation of compounds such as ATP and GTP in the ordering process. (TW)

  14. Pennycress protein isolate: Pilot plant production and application in films polymeric composites

    USDA-ARS?s Scientific Manuscript database

    This work scaled up the process of producing pennycress protein isolates (PPI) using 5 kg starting material (previously 100 g in bench-scale research). Defatted press cake, produced by prepressing and hexane extraction, was mixed with preheated 50 L of aqueous NaOH (pH 10) for 90 min in a jacketed k...

  15. Impact by condensed tannins with different mean degrees of polymerization on protein precipitation

    USDA-ARS?s Scientific Manuscript database

    Condensed tannins (CTs) isolated from white clover (Trifolium repens) flowers and big trefoil (Lotus pedunculatus) leaves were evaluated in precipitation studies with bovine serum albumin (BSA), lysozyme (LYS), and alfalfa leaf protein (ALF). The CTs were of similar compositions, but differed in the...

  16. A FRET-based method for monitoring septin polymerization and binding of septin-associated proteins

    PubMed Central

    Booth, E.A.; Thorner, J.

    2016-01-01

    Much about septin function has been inferred from in vivo studies using mainly genetic methods, and much of what we know about septin organization has been obtained through examination of static structures in vitro primarily by electron microscopy. Deeper mechanistic insight requires real-time analysis of the dynamics of the assembly of septin-based structures and how other proteins associate with them. We describe here a Förster resonance energy transfer (FRET)-based approach for measuring in vitro the rate and extent of filament formation from septin complexes, binding of other proteins to septin structures, and the apparent affinities of these interactions. FRET is particularly well suited for interrogating protein–protein interactions, especially on a rapid timescale; the spectral change provides an unambiguous indication of whether two elements within the system under study are associating and serves as a molecular-level “ruler” because it is very sensitive to the separation between the donor and acceptor fluorophores over biologically relevant distances (≤ 10 nm). The necessary procedures involve generation of appropriate cysteine-less and single cysteine-containing septin variants, expression and purification of the heterooctameric complexes containing them, efficient labeling of the purified complexes with desired fluorophores, fluorimetric measurement of FRET, and appropriate safeguards and controls in data acquisition and analysis. Our methods can be used to interrogate the effects of buffer conditions, small molecules, and septin-binding proteins on septin filament assembly or stability; determine the effect of alternative septin subunits, mutational alterations, or posttranslational modifications on assembly; and, delineate the location of septin-binding proteins. PMID:27473902

  17. The synthesis of magnetic lysozyme-imprinted polymers by means of distillation-precipitation polymerization for selective protein enrichment.

    PubMed

    Cao, Jiali; Zhang, Xihao; He, Xiwen; Chen, Langxing; Zhang, Yukui

    2014-02-01

    A protein imprinting approach for the synthesis of core-shell structure nanoparticles with a magnetic core and molecularly imprinted polymer (MIP) shell was developed using a simple distillation-precipitation polymerization method. In this work, Fe3O4 magnetic nanoparticles were first synthesized through a solvothermal method and then were conveniently surface-modified with 3-(methacryloyloxy)propyltrimethoxylsilane as anchor molecules to donate vinyl groups. Next a high-density MIP shell was coated onto the surface of the magnetic nanoparticles by the copolymerization of functional monomer acrylamide (AAm), cross-linking agent N,N'-methylenebisacrylamide (MBA), the initiator azodiisobutyronitrile (AIBN), and protein in acetonitrile heated at reflux. The morphology, adsorption, and recognition properties of the magnetic molecularly imprinted nanoparticles were investigated by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), vibrating sample magnetometer (VSM), and rebinding experiments. The resulting MIP showed a high adsorption capacity (104.8 mg g(-1)) and specific recognition (imprinting factor=7.6) to lysozyme (Lyz). The as-prepared Fe3O4@Lyz-MIP nanoparticles with a mean diameter of 320 nm were coated with an MIP shell that was 20 nm thick, which enabled Fe3O4@Lyz-MIP to easily reach adsorption equilibrium. The high magnetization saturation (40.35 emu g(-1)) endows the materials with the convenience of magnetic separation under an external magnetic field and allows them to be subsequently reused. Furthermore, Fe3O4@Lyz-MIP could selectively extract a target protein from real egg-white samples under an external magnetic field.

  18. Effects of proteins on absorption by the rat of iron from polymeric and low-molecular-weight iron species

    SciTech Connect

    Berner, L.; Miller, D.

    1986-03-05

    To examine effects of proteins on Fe absorption from polymeric ferric hydroxides (polys) or low-molecular-weight complexes (LMW Fe), 2 studies were conducted. First, anemic rats were given /sup 59/Fe-labeled polys or LMW Fe in the presence and absence of pepsin-digested soy protein isolate, casein, and BSA. The doses were introduced into ligated duodenal segments for 1 hr. Uptake into the carcass of /sup 59/Fe from polys was doubled in the presence of BSA (7.8 vs 16.1%, p < .05) while casein and soy had no effect (5.5 and 6.5%). Absorption of /sup 59/Fe from LMW Fe was 7X greater than from polys; BSA and casein had no effect but soy depressed Fe uptake by almost 50% (57.4 vs 35.5%, p < .05). The second experiment repeated the first except that the proteins were not pepsin-digested and the doses were given by gastric intubation. All Fe, whether from polys or LMW Fe, was highly available (although in vitro digestions reveal that polys are not depolymerized to a large degree under simulated stomach conditions). Soy depressed Fe uptake from both sources (92.9 vs. 81.6%, LMW Fe and 85.4 vs 73.7%, polys) while casein and BSA had no effect. These results show: (1) BSA can depolymerize polys in the rat duodenum, thus enhancing absorption; (2) soy isolate generally depressed Fe uptake; and (3) the rat stomach appears to have an exceptional capacity for equalizing Fe sources.

  19. Microfluidics-assisted engineering of polymeric microcapsules with high encapsulation efficiency for protein drug delivery.

    PubMed

    Pessi, Jenni; Santos, Hélder A; Miroshnyk, Inna; JoukoYliruusi; Weitz, David A; Mirza, Sabiruddin

    2014-09-10

    In this study, microfluidic technology was employed to develop protein formulations. The microcapsules were produced with a biphasic flow to create water-oil-water (W/O/W) double emulsion droplets with ultrathin shells. Optimized microcapsule formulations containing 1% (w/w) bovine serum albumin (BSA) in the inner phase were prepared with poly(vinyl alcohol), polycaprolactone and polyethylene glycol. All the particles were found to be intact and with a particle size of 23-47 μm. Furthermore, the particles were monodisperse, non-porous and stable up to 4 weeks. The encapsulation efficiency of BSA in the microcapsules was 84%. The microcapsules released 30% of their content within 168 h. This study demonstrates that microfluidics is a powerful technique for engineering formulations for therapeutic proteins.

  20. Monomeric, Oligomeric and Polymeric Proteins in Huntington Disease and Other Diseases of Polyglutamine Expansion

    PubMed Central

    Hoffner, Guylaine; Djian, Philippe

    2014-01-01

    Huntington disease and other diseases of polyglutamine expansion are each caused by a different protein bearing an excessively long polyglutamine sequence and are associated with neuronal death. Although these diseases affect largely different brain regions, they all share a number of characteristics, and, therefore, are likely to possess a common mechanism. In all of the diseases, the causative protein is proteolyzed, becomes abnormally folded and accumulates in oligomers and larger aggregates. The aggregated and possibly the monomeric expanded polyglutamine are likely to play a critical role in the pathogenesis and there is increasing evidence that the secondary structure of the protein influences its toxicity. We describe here, with special attention to huntingtin, the mechanisms of polyglutamine aggregation and the modulation of aggregation by the sequences flanking the polyglutamine. We give a comprehensive picture of the characteristics of monomeric and aggregated polyglutamine, including morphology, composition, seeding ability, secondary structure, and toxicity. The structural heterogeneity of aggregated polyglutamine may explain why polyglutamine-containing aggregates could paradoxically be either toxic or neuroprotective. PMID:24961702

  1. MinCD cell division proteins form alternating co-polymeric cytomotive filaments

    PubMed Central

    Ghosal, Debnath; Trambaiolo, Daniel; Amos, Linda A.; Löwe, Jan

    2014-01-01

    Summary During bacterial cell division, filaments of the tubulin-like protein FtsZ assemble at midcell to form the cytokinetic Z-ring. Its positioning is regulated by the oscillation of MinCDE proteins. MinC is activated by MinD through an unknown mechanism and prevents Z-ring assembly anywhere but midcell. Here, using X-ray crystallography, electron microscopy and in vivo analyses we show that MinD activates MinC by forming a new class of alternating copolymeric filaments that show similarity to eukaryotic septin filaments A non-polymerising mutation in MinD causes aberrant cell division in E. coli. MinCD copolymers bind to membrane, interact with FtsZ, and are disassembled by MinE. Imaging a functional msfGFP-MinC fusion protein in MinE deleted cells reveals filamentous structures. EM imaging of our reconstitution of the MinCD-FtsZ interaction on liposome surfaces reveals a plausible mechanism for regulation of FtsZ ring assembly by MinCD copolymers. PMID:25500731

  2. Facile trypsin immobilization in polymeric membranes for rapid, efficient protein digestion.

    PubMed

    Xu, Fei; Wang, Wei-Han; Tan, Yu-Jing; Bruening, Merlin L

    2010-12-15

    Sequential adsorption of poly(styrene sulfonate) and trypsin in nylon membranes provides a simple, inexpensive method to create stable, microporous reactors for fast protein digestion. The high local trypsin concentration and short radial diffusion distances in membrane pores facilitate proteolysis in residence times of a few seconds, and the minimal pressure drop across the thin membranes allows their use in syringe filters. Membrane digestion and subsequent MS analysis of bovine serum albumin provide 84% sequence coverage, which is higher than the 71% coverage obtained with in-solution digestion for 16 h or the <50% sequence coverages of other methods that employ immobilized trypsin. Moreover, trypsin-modified membranes digest protein in the presence of 0.05 wt % sodium dodecyl sulfate (SDS), whereas in-solution digestion under similar conditions yields no peptide signals in mass spectra even after removal of SDS. These membrane reactors, which can be easily prepared in any laboratory, have a shelf life of several months and continuously digest protein for at least 33 h without significant loss of activity.

  3. Use of polymeric membranes for purification of an E. coli expressed biotherapeutic protein.

    PubMed

    Muthukumar, S; Rathore, Anurag S

    2016-01-01

    Polymers have had a significant impact on the field of bioseparations in the past few decades. Most recently, membrane chromatography has emerged as an efficient alternative to the conventional packed-bed chromatography by eliminating the diffusion-related limitations associated with the traditional resin beads. In this article, we examine six membrane adsorbers for purification of granulocyte colony-stimulating factor (GCSF), an Escherichia coli-based biotherapeutic. These adsorbers differ either in their base matrix or in the surface chemistry. The role of interactions between the filter surfaces and the protein molecules in effecting these separations is the focus of the article.

  4. Protein microarrays on hybrid polymeric thin films prepared by self-assembly of polyelectrolytes for multiple-protein immunoassays.

    PubMed

    Zhou, Xichun; Zhou, Jizhong

    2006-03-01

    We report here the development and characterization of protein microarrays fabricated on nanoengineered 3-D polyelectrolyte thin films (PET) deposited on glass slide by consecutive adsorption of polyelectrolytes via self-assembly technique. Antibodies or antigens were immobilized in the PET-coated glass slides by electrostatic adsorption and entrapment of porous structure of the 3-D polymer film and thus establishing a platform for parallel analysis. Both antigen and antibody microarrays were fabricated on the PET-coated slides, and direct and indirect immunoassays on protein microarrays for multiple-analyte detection were demonstrated. Microarrays produced on these PET-coated slides have consistent spot morphology and provide performance features needed for proteomic analysis. The protein microarrays on the PET films provide LOD as low as 6 pg/mL and dynamic ranges up to three orders of magnitude, which are wider than the protein microarrays fabricated on aldehyde and poly-L-lysine functionalized slides. The PET films constructed by self-assembly technique in aqueous solution is green chemistry based, cost-effective method to generate 3-D thin film coatings on glass surface, and the coated slide is well suited for immobilizing many types of biological molecules so that a wide variety of microarray formats can be developed on this type of slide.

  5. Group B Streptococcus pilus sortase regulation: a single mutation in the lid region induces pilin protein polymerization in vitro.

    PubMed

    Cozzi, Roberta; Zerbini, Francesca; Assfalg, Michael; D'Onofrio, Mariapina; Biagini, Massimiliano; Martinelli, Manuele; Nuccitelli, Annalisa; Norais, Nathalie; Telford, John L; Maione, Domenico; Rinaudo, C Daniela

    2013-08-01

    Gram-positive bacteria build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates. Despite the availability of several crystal structures, pilus-related C sortases remain poorly characterized to date, and their mechanisms of transpeptidation and regulation need to be further investigated. The available 3-dimensional structures of these enzymes reveal a typical sortase fold, except for the presence of a unique feature represented by an N-terminal highly flexible loop known as the "lid." This region interacts with the residues composing the catalytic triad and covers the active site, thus maintaining the enzyme in an autoinhibited state and preventing the accessibility to the substrate. It is believed that enzyme activation may occur only after lid displacement from the catalytic domain. In this work, we provide the first direct evidence of the regulatory role of the lid, demonstrating that it is possible to obtain in vitro an efficient polymerization of pilin subunits using an active C sortase lid mutant carrying a single residue mutation in the lid region. Moreover, biochemical analyses of this recombinant mutant reveal that the lid confers thermodynamic and proteolytic stability to the enzyme.

  6. A Balance of Capping Protein and Profilin Functions Is Required to Regulate Actin Polymerization in Drosophila Bristle

    PubMed Central

    Hopmann, Roberta; Miller, Kathryn G.

    2003-01-01

    Profilin is a well-characterized protein known to be important for regulating actin filament assembly. Relatively few studies have addressed how profilin interacts with other actin-binding proteins in vivo to regulate assembly of complex actin structures. To investigate the function of profilin in the context of a differentiating cell, we have studied an instructive genetic interaction between mutations in profilin (chickadee) and capping protein (cpb). Capping protein is the principal protein in cells that caps actin filament barbed ends. When its function is reduced in the Drosophila bristle, F-actin levels increase and the actin cytoskeleton becomes disorganized, causing abnormal bristle morphology. chickadee mutations suppress the abnormal bristle phenotype and associated abnormalities of the actin cytoskeleton seen in cpb mutants. Furthermore, overexpression of profilin in the bristle mimics many features of the cpb loss-of-function phenotype. The interaction between cpb and chickadee suggests that profilin promotes actin assembly in the bristle and that a balance between capping protein and profilin activities is important for the proper regulation of F-actin levels. Furthermore, this balance of activities affects the association of actin structures with the membrane, suggesting a link between actin filament dynamics and localization of actin structures within the cell. PMID:12529431

  7. Differential polymerization of the two main protein components of dragline silk during fibre spinning

    NASA Astrophysics Data System (ADS)

    Sponner, Alexander; Unger, Eberhard; Grosse, Frank; Weisshart, Klaus

    2005-10-01

    Spider silks are some of the strongest materials found in nature. Achieving the high tensile strength and elasticity of the dragline of orb-weaving spiders, such as Nephila clavipes, is a principal goal in biomimetics research. The dragline has a composite nature and is predominantly made up by two proteins, the major ampullate spidroins 1 and 2 (refs 3,6,7), which can be considered natural block copolymers. On the basis of their molecular structures both spidroins are thought to contribute, in different ways, to the mechanical properties of dragline silk. The spinning process itself is also considered important for determining the observed features by shaping the hierarchical structure of the fibre. Here we study the heterogeneous distribution of proteins along the radial axis of the fibre. This heterogeneity is generated during the conversion of the liquid spinning dope into solid fibre. Whereas spidroin 1 is distributed almost uniformly within the fibre core, spidroin 2 is missing in the periphery and is tightly packed in certain core areas. Our findings suggest that the role of spidroin 2 in the spinning process could be to facilitate the formation of fibrils and contribute directly to the elasticity of the silk.

  8. Development of a Gastric Absorptive, Immediate Responsive, Oral Protein-Loaded Versatile Polymeric Delivery System.

    PubMed

    Kondiah, Pierre P D; Choonara, Yahya E; Tomar, Lomas K; Tyagi, Charu; Kumar, Pradeep; du Toit, Lisa C; Marimuthu, Thashree; Modi, Girish; Pillay, Viness

    2017-02-15

    A multifunctional platform to deliver three diverse proteins of insulin, interferon beta (INF-β) and erythropoietin (EPO), using a novel copolymeric microparticulate system of TMC-PEGDMA-MAA, was synthesised as an intelligent pH-responsive 2-fold gastric and intestinal absorptive system. Physiochemical and physicomechanical studies proved the degree of crystallinity that supported the controlled protein delivery of the microparticulate system. The copolymer was tableted before undertaking in vitro and in vivo analysis. After 2.5 h in simulated gastric fluid (SGF), insulin showed a fractional release of 3.2% in comparison to simulated intestinal fluid (SIF), in which a maximum of 83% of insulin was released. Similarly, INF-β and EPO released 3 and 9.7% in SGF and a maximum of 74 and 81.3% in SIF, respectively. In vivo studies demonstrated a significant decrease in blood glucose by 54.19% within 4 h post-dosing, and the comparator formulation provided 74.6% decrease in blood glucose within the same time period. INF-β peak bioavailable dose in serum was calculated to be 1.3% in comparison to an SC formulation having a peak concentration of 0.9%, demonstrating steady-state release for 24 h. EPO-loaded copolymeric microparticles had a 1.6% peak bioavailable concentration, in comparison to the 6.34% peak concentration after 8 h from the SC comparator formulation.

  9. Differential polymerization of the two main protein components of dragline silk during fibre spinning.

    PubMed

    Sponner, Alexander; Unger, Eberhard; Grosse, Frank; Weisshart, Klaus

    2005-10-01

    Spider silks are some of the strongest materials found in nature. Achieving the high tensile strength and elasticity of the dragline of orb-weaving spiders, such as Nephila clavipes, is a principal goal in biomimetics research. The dragline has a composite nature and is predominantly made up by two proteins, the major ampullate spidroins 1 and 2 (refs 3, 6, 7), which can be considered natural block copolymers. On the basis of their molecular structures both spidroins are thought to contribute, in different ways, to the mechanical properties of dragline silk. The spinning process itself is also considered important for determining the observed features by shaping the hierarchical structure of the fibre. Here we study the heterogeneous distribution of proteins along the radial axis of the fibre. This heterogeneity is generated during the conversion of the liquid spinning dope into solid fibre. Whereas spidroin 1 is distributed almost uniformly within the fibre core, spidroin 2 is missing in the periphery and is tightly packed in certain core areas. Our findings suggest that the role of spidroin 2 in the spinning process could be to facilitate the formation of fibrils and contribute directly to the elasticity of the silk.

  10. Integrin β4 regulates SPARC protein to promote invasion.

    PubMed

    Gerson, Kristin D; Shearstone, Jeffrey R; Maddula, V S R Krishna; Seligmann, Bruce E; Mercurio, Arthur M

    2012-03-23

    The α6β4 integrin (referred to as "β4" integrin) is a receptor for laminins that promotes carcinoma invasion through its ability to regulate key signaling pathways and cytoskeletal dynamics. An analysis of published Affymetrix GeneChip data to detect downstream effectors involved in β4-mediated invasion of breast carcinoma cells identified SPARC, or secreted protein acidic and rich in cysteine. This glycoprotein has been shown to play an important role in matrix remodeling and invasion. Our analysis revealed that manipulation of β4 integrin expression and signaling impacted SPARC expression and that SPARC facilitates β4-mediated invasion. Expression of β4 in β4-deficient cells reduced the expression of a specific microRNA (miR-29a) that targets SPARC and impedes invasion. In cells that express endogenous β4, miR-29a expression is low and β4 ligation facilitates the translation of SPARC through a TOR-dependent mechanism. The results obtained in this study demonstrate that β4 can regulate SPARC expression and that SPARC is an effector of β4-mediated invasion. They also highlight a potential role for specific miRNAs in executing the functions of integrins.

  11. Colonic protein fermentation and promotion of colon carcinogenesis by thermolyzed casein

    PubMed Central

    Corpet, Denis E.; Yin, Y.; Zhang, X. M.; Rémésy, C.; Stamp, D.; Medline, A.; Thompson, L.U.; Bruce, W. R.; Archer, M. C.

    1995-01-01

    Thermolyzed casein is known to promote the growth of aberrant crypt foci (ACF) and colon cancer when it is fed to rats that have been initiated with azoxymethane. We speculated that the promotion was a consequence of increased colonic protein fermentation (i.e., that the thermolysis of the casein decreases its digestibility, increases the amount of protein reaching the colon, and increases colonic protein fermentation and that the potentially toxic products of this fermentation promote colon carcinogenesis). We found that the thermolysis of casein reduces its digestibility and increases colonic protein fermentation, as assessed by fecal ammonium and urinary phenol, cresol, and indol-3-ol. Thermolysis of two other proteins, soy and egg white protein, also increases colonic protein fermentation with increased fecal ammonia and urinary phenols, and thermolysis of all three proteins increases the levels of ammonia and butyric, valeric, and i-valeric acids in the cecal contents. We found, however, that the increased protein fermentation observed with thermolysis is not associated with promotion of colon carcinogenesis. With casein, the kinetics of protein fermentation with increasing thermolysis time are clearly different from the kinetics of promotion of ACF growth. The formation of the fermentation products was highest when the protein was thermolyzed for one hour, whereas promotion was highest for protein that had been thermolyzed for two or more hours. With soy and egg white, thermolysis increased colonic protein fermentation but did not promote colon carcinogenesis. Thus, although thermolysis of dietary casein increases colonic protein fermentation, products of this fermentation do not appear to be responsible for the promotion of colon carcinogenesis. Indeed, the results suggest that protein fermentation products do not play an important role in colon cancer promotion. PMID:7603887

  12. Use of amino acid-based polymeric material for isolation of a protein from poison

    NASA Astrophysics Data System (ADS)

    Erol, Kadir; Köse, Kazım; Güngüneş, Hakan; Köse, Dursun Ali

    2017-02-01

    Melittin is a small protein with 126 amino acid residues which exists in the bee and snake venom. In this study, the Fe(II)-Ni(II) double-salt incorporated poly(2-hydroxyethyl methacrylate-N-methacryloyl-L-aspartic acid), poly(HEMA-MAsp), magnetic microparticles were synthesized for the separation of melittin. Fourier transform infrared spectroscopy, scanning electron microscopy, vibrating sample magnetometer and Mossbauer spectroscopy techniques were used for the characterization. The melittin adsorption capacity of magnetic microparticles in aqueous solution were identified as 69.60 mg/g, whereas that in real be venom solution was 46.80 mg/g microparticle. The adsorption-desorption cycle was repeated 5 times and no significant decrease were observed in the adsorption capacity.

  13. The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

    PubMed

    Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

    2016-05-17

    Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  14. Glutamate promotes SSB protein-protein Interactions via intrinsically disordered regions.

    PubMed

    Kozlov, Alexander G; Shinn, Min Kyung; Weiland, Elizabeth A; Lohman, Timothy M

    2017-09-01

    E. coli single strand (ss) DNA binding protein (SSB) is an essential protein that binds to ssDNA intermediates formed during genome maintenance. SSB homotetramers bind ssDNA in several modes that differ in occluded site size and cooperativity. High "unlimited" cooperativity is associated with the 35 site size ((SSB)35) mode at low [NaCl], whereas the 65 site size ((SSB)65) mode formed at higher [NaCl] (> 200mM), where ssDNA wraps completely around the tetramer, displays "limited" cooperativity forming dimers of tetramers. It was previously thought that high cooperativity was associated only with the (SSB)35 binding mode. However, we show here that highly cooperative binding also occurs in the (SSB)65/(SSB)56 binding modes at physiological salt concentrations containing either glutamate or acetate. Highly cooperative binding requires the 56 amino acid intrinsically disordered C-terminal linker (IDL) that connects the DNA binding domain with the 9 amino acid C-terminal acidic tip that is involved in SSB binding to other proteins involved in genome maintenance. These results suggest that high cooperativity involves interactions between IDL regions from different SSB tetramers. Glutamate, which is preferentially excluded from protein surfaces, may generally promote interactions between intrinsically disordered regions of proteins. Since glutamate is the major monovalent anion in E. coli, these results suggest that SSB likely binds to ssDNA with high cooperativity in vivo. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Inaccurate measurement of a polymeric IgA myeloma protein by nephelometric and fluorometric instrumentation.

    PubMed

    Levinson, S S; Goldman, J O; Markyvech, L; Nathan, L E

    1986-11-01

    The concentration of IgA in a serum was 5.99 g/L as assayed nephelometrically with reagent from one company, but varied between 5 and 3 g/L (for sixfold and 36-fold dilutions, respectively) without giving a definitive answer when assayed with reagent from another source. Immunofixation electrophoresis indicated an IgA lambda monoclonal protein of 45 g/L. Radial immunodiffusion showed two components, having a total concentration of 41 g/L. By fluorometry the IgA was 3.1 g/L. Increasing the dilution caused the (dilution-corrected) lower values to increase. Although the most frequent cause of such discrepant findings is an IgA2 myeloma, which occurs in about one of every 100 myeloma cases, Ouchterlony double diffusion indicated the major component to be IgA1. A polymer, Mr 670,000, was identified by column chromatography. Contrary to the usual behavior of polymers assayed with radial immunodiffusion, which underestimates their concentration, this polymer reached equivalency in agreement with its true concentration as assayed by the Mancini-Heremans technique.

  16. Single polysaccharide assembly protein that integrates polymerization, termination, and chain-length quality control

    PubMed Central

    Williams, Danielle M.; Ovchinnikova, Olga G.; Koizumi, Akihiko; Mainprize, Iain L.; Kimber, Matthew S.; Lowary, Todd L.

    2017-01-01

    Lipopolysaccharides (LPS) are essential outer membrane glycolipids in most gram-negative bacteria. Biosynthesis of the O-antigenic polysaccharide (OPS) component of LPS follows one of three widely distributed strategies, and similar processes are used to assemble other bacterial surface glycoconjugates. This study focuses on the ATP-binding cassette (ABC) transporter-dependent pathway, where glycans are completed on undecaprenyl diphosphate carriers at the cytosol:membrane interface, before export by the ABC transporter. We describe Raoultella terrigena WbbB, a prototype for a family of proteins that, remarkably, integrates several key activities in polysaccharide biosynthesis into a single polypeptide. WbbB contains three glycosyltransferase (GT) modules. Each of the GT102 and GT103 modules characterized here represents a previously unrecognized GT family. They form a polymerase, generating a polysaccharide of [4)-α-Rhap-(1→3)-β-GlcpNAc-(1→] repeat units. The polymer chain is terminated by a β-linked Kdo (3-deoxy-d-manno-oct-2-ulosonic acid) residue added by a third GT module belonging to the recently discovered GT99 family. The polymerase GT modules are separated from the GT99 chain terminator by a coiled-coil structure that forms a molecular ruler to determine product length. Different GT modules in the polymerase domains of other family members produce diversified OPS structures. These findings offer insight into glycan assembly mechanisms and the generation of antigenic diversity as well as potential tools for glycoengineering. PMID:28137848

  17. Regulation of WASH-Dependent Actin Polymerization and Protein Trafficking by Ubiquitination

    PubMed Central

    Hao, Yi-Heng; Doyle, Jennifer M.; Ramanathan, Saumya; Gomez, Timothy S.; Jia, Da; Xu, Ming; Chen, Zhijian J.; Billadeau, Daniel D.; Rosen, Michael K.; Potts, Patrick Ryan

    2013-01-01

    SUMMARY Endosomal protein trafficking is an essential cellular process that is deregulated in several diseases and targeted by pathogens. Here, we describe a novel role for ubiquitination in this process. We find that the novel E3 RING ubiquitin ligase, MAGE-L2-TRIM27, localizes to endosomes through interactions with the Retromer complex. Knockdown of MAGE-L2-TRIM27 or the Ube2O E2 ubiquitin-conjugating enzyme significantly impaired Retromer-mediated transport. We further demonstrate that MAGE-L2-TRIM27 ubiquitin ligase activity is required for nucleation of endosomal F-actin by the WASH regulatory complex, a known regulator of Retromer-mediated transport. Mechanistic studies showed that MAGE-L2-TRIM27 facilitates K63-linked ubiquitination of WASH K220. Significantly, disruption of WASH ubiquitination impaired endosomal F-actin nucleation and Retromer-dependent transport. These findings provide a cellular and molecular function for MAGE-L2-TRIM27 and reveal novel aspects of retrograde transport, including an unappreciated role of K63-linked ubiquitination and identification of an activating signal of the WASH regulatory complex. PMID:23452853

  18. Insights into the variability of nucleated amyloid polymerization by a minimalistic model of stochastic protein assembly

    NASA Astrophysics Data System (ADS)

    Eugène, Sarah; Xue, Wei-Feng; Robert, Philippe; Doumic, Marie

    2016-05-01

    Self-assembly of proteins into amyloid aggregates is an important biological phenomenon associated with human diseases such as Alzheimer's disease. Amyloid fibrils also have potential applications in nano-engineering of biomaterials. The kinetics of amyloid assembly show an exponential growth phase preceded by a lag phase, variable in duration as seen in bulk experiments and experiments that mimic the small volumes of cells. Here, to investigate the origins and the properties of the observed variability in the lag phase of amyloid assembly currently not accounted for by deterministic nucleation dependent mechanisms, we formulate a new stochastic minimal model that is capable of describing the characteristics of amyloid growth curves despite its simplicity. We then solve the stochastic differential equations of our model and give mathematical proof of a central limit theorem for the sample growth trajectories of the nucleated aggregation process. These results give an asymptotic description for our simple model, from which closed form analytical results capable of describing and predicting the variability of nucleated amyloid assembly were derived. We also demonstrate the application of our results to inform experiments in a conceptually friendly and clear fashion. Our model offers a new perspective and paves the way for a new and efficient approach on extracting vital information regarding the key initial events of amyloid formation.

  19. Paclitaxel-Loaded Polymeric Micelles Modified with MCF-7 Cell-Specific Phage Protein: Enhanced Binding to Target Cancer Cells and Increased Cytotoxicity

    PubMed Central

    Wang, Tao; Petrenko, Valery A.; Torchilin, Vladimir P.

    2010-01-01

    Polymeric micelles are used as pharmaceutical carriers to increase solubility and bioavailability of poorly water-soluble drugs. Different ligands are used to prepare targeted polymeric micelles. Earlier, we developed the method for use of specific landscape phage fusion coat proteins as targeted delivery ligands and demonstrated the efficiency of this approach with doxorubicin-loaded PEGylated liposomes. Here, we describe a MCF-7 cell-specific micellar formulation self-assembled from the mixture of the micelle-forming amphiphilic polyethylene glycol-phosphatidylethanolamine (PEG-PE) conjugate, MCF-7-specific landscape phage fusion coat protein, and the hydrophobic drug paclitaxel. These micelles demonstrated a very low CMC value and specific binding to target cells. Using an in vitro co-culture model, FACS analysis, and fluorescence microscopy we showed that MCF-7 targeted phage micelles preferential bound to target cells compared to non-target cells. As a result, targeted paclitaxel-loaded phage micelles demonstrated a significantly higher cytotoxicity towards target MCF-7 cells than free drug or non-targeted micelle formulations, but failed to show such a differential toxicity towards non-target C166 cells. Overall, cancer cell-specific phage proteins identified from phage display peptide libraries can serve as targeting ligands (“substitute antibody”) for polymeric micelle-based pharmaceutical preparations. PMID:20518562

  20. Promotion of Bone Morphogenetic Protein Signaling by Tetraspanins and Glycosphingolipids

    PubMed Central

    Szymczak, Lindsey C.; Aydin, Taner; Yun, Sijung; Constas, Katharine; Schaeffer, Arielle; Ranjan, Sinthu; Kubba, Saad; Alam, Emad; McMahon, Devin E.; He, Jingpeng; Shwartz, Neta; Tian, Chenxi; Plavskin, Yevgeniy; Lindy, Amanda; Dad, Nimra Amir; Sheth, Sunny; Amin, Nirav M.; Zimmerman, Stephanie; Liu, Dennis; Schwarz, Erich M.; Smith, Harold; Krause, Michael W.; Liu, Jun

    2015-01-01

    Bone morphogenetic proteins (BMPs) belong to the transforming growth factor β (TGFβ) superfamily of secreted molecules. BMPs play essential roles in multiple developmental and homeostatic processes in metazoans. Malfunction of the BMP pathway can cause a variety of diseases in humans, including cancer, skeletal disorders and cardiovascular diseases. Identification of factors that ensure proper spatiotemporal control of BMP signaling is critical for understanding how this pathway is regulated. We have used a unique and sensitive genetic screen to identify the plasma membrane-localized tetraspanin TSP-21 as a key new factor in the C. elegans BMP-like “Sma/Mab” signaling pathway that controls body size and postembryonic M lineage development. We showed that TSP-21 acts in the signal-receiving cells and genetically functions at the ligand-receptor level. We further showed that TSP-21 can associate with itself and with two additional tetraspanins, TSP-12 and TSP-14, which also promote Sma/Mab signaling. TSP-12 and TSP-14 can also associate with SMA-6, the type I receptor of the Sma/Mab pathway. Finally, we found that glycosphingolipids, major components of the tetraspanin-enriched microdomains, are required for Sma/Mab signaling. Our findings suggest that the tetraspanin-enriched membrane microdomains are important for proper BMP signaling. As tetraspanins have emerged as diagnostic and prognostic markers for tumor progression, and TSP-21, TSP-12 and TSP-14 are all conserved in humans, we speculate that abnormal BMP signaling due to altered expression or function of certain tetraspanins may be a contributing factor to cancer development. PMID:25978409

  1. Promotion of tribological and hydrophobic properties of a coating on TPE substrates by atmospheric plasma-polymerization

    NASA Astrophysics Data System (ADS)

    Sainz-García, Elisa; Alba-Elías, Fernando; Múgica-Vidal, Rodolfo; Pantoja-Ruiz, Mariola

    2016-05-01

    Thermoplastic elastomers (TPE) are used in the automotive sealing industry with the objective of producing anti-friction and hydrophobic components. At present, the anti-friction property is achieved by the electrostatic flocking, which sometimes produces an irregular coating. Therefore, this paper's objective is the promotion of adhesion of an anti-friction (based on the silane aminopropyltriethoxysilane-APTES-) and hydrophobic (based on the fluorinated precursor 1-perfluorohexene-PFH-) coating by the adhesion promoter, APTES. Different mixtures of APTES and PFH have been applied to a TPE substrate by an Atmospheric Pressure Plasma Jet (APPJ) system with Dielectric Barrier Discharge (DBD) in order to determine the optimal mixture of precursors. The main difficulty in this work lies in the hydrophilic character of APTES and the low adhesion of the fluorinated coatings. The sample coated with a mixture of 50% APTES and 50% PFH (A50P50) was found to be the best one to satisfy both properties at the same time, despite not having the highest dynamic water contact angle (WCA) or the lowest friction coefficient.

  2. A novel bi-directional promoter system allows tunable recombinant protein production in Pichia pastoris.

    PubMed

    Rajamanickam, Vignesh; Metzger, Karl; Schmid, Christian; Spadiut, Oliver

    2017-09-13

    The methylotrophic yeast Pichia pastoris is a well-studied host organism for recombinant protein production, which is usually regulated either by a constitutive promoter (e.g. promoter of glyceraldehyde-3-phosphate dehydrogenase; PGAP) or an inducible promoter (e.g. promoter of alcohol oxidase 1; PAOX1). Both promoter systems have several advantages and disadvantages; with one of the main disadvantages being their lack of tunability. Various novel promoter systems, which are either inducible or de-repressed, allowing higher degrees of freedom, have been reported. Recently, bi-directional promoter systems in P. pastoris with two promoter systems regulating recombinant expression of one or more genes were developed. In this study, we introduce a novel bi-directional promoter system combining a modified catalase promoter system (PDC; derepressible and inducible) and the traditional PAOX1, allowing tunable recombinant protein production. We characterized a recombinant P. pastoris strain, carrying the novel bi-directional promoter system, during growth and production in three dynamic bioreactor cultivations. We cloned the model enzyme cellobiohydralase downstream of either promoter and applied different feeding strategies to determine the physiological boundaries of the strain. We succeeded in demonstrating tunability of recombinant protein production solely in response to the different feeding strategies and identified a mixed feed regime allowing highest productivity. In this feasibility study, we present the first controlled bioreactor experiments with a recombinant P. pastoris strain carrying a novel bi-directional promotor combination of a catalase promoter variant (PDC) and the traditional PAOX1. We demonstrated that this bi-directional promoter system allows tunable recombinant protein expression only in response to the available C-sources. This bi-directional promoter system offers a high degree of freedom for bioprocess design and development, making bi

  3. Ubiquitin promoter-terminator cassette promotes genetically stable expression of the taste-modifying protein miraculin in transgenic lettuce.

    PubMed

    Hirai, Tadayoshi; Shohael, Abdullah Mohammad; Kim, You-Wang; Yano, Megumu; Ezura, Hiroshi

    2011-12-01

    Lettuce is a commercially important leafy vegetable that is cultivated worldwide, and it is also a target crop for plant factories. In this study, lettuce was selected as an alternative platform for recombinant miraculin production because of its fast growth, agronomic value, and wide availability. The taste-modifying protein miraculin is a glycoprotein extracted from the red berries of the West African native shrub Richadella dulcifica. Because of its limited natural availability, many attempts have been made to produce this protein in suitable alternative hosts. We produced transgenic lettuce with miraculin gene driven either by the ubiquitin promoter/terminator cassette from lettuce or a 35S promoter/nos terminator cassette. Miraculin gene expression and miraculin accumulation in both cassettes were compared by quantitative real-time PCR analysis, Western blotting, and enzyme-linked immunosorbent assay. The expression level of the miraculin gene and protein in transgenic lettuce was higher and more genetically stable in the ubiquitin promoter/terminator cassette than in the 35S promoter/nos terminator cassette. These results demonstrated that the ubiquitin promoter/terminator cassette is an efficient platform for the genetically stable expression of the miraculin protein in lettuce and hence this platform is of benefit for recombinant miraculin production on a commercial scale.

  4. Determination of paclitaxel in hyaluronic acid polymeric micelles in rat blood by protein precipitation-micelle breaking method: application to a pharmacokinetic study.

    PubMed

    Liu, Yanhua; Sun, Jin; Lian, He; Li, Xin; Cao, Wen; Bai, Liming; Wang, Yongjun; He, Zhonggui

    2013-09-15

    An efficient dissociation of paclitaxel (PTX) from the home-made hyaluronic acid-octadecyl (HA-C18) polymeric micelles formulation in rat blood could not be achieved using previously published PTX analytical methods. So, we intended to develop the micelle-breaking method to determine paclitaxel encapsulated in the HA-C18 polymeric micelles in blood. The pretreatment method of blood samples adopted a simple one-step protein precipitation-micelle breaking process with methanol as micelle-breaking and protein precipitant solvents for complete extraction of PTX from HA-C18 micelles in blood. The micelle breaking efficiency of methanol was as high as 97.7%. Separation was carried out by gradient elution on an Acquity UPLC BEH C18 column with a mobile phase consisting of water (containing 0.1% formic acid) and acetonitrile. A total single run time was as short as 3.0min. Detection was performed by triple-quadrupole mass spectrometry with positive electrospray ionization as source ionization in multiple-reaction monitoring mode at m/z 854.3→286.2 for PTX and m/z 808.5→527.3 for the internal standard, docetaxel. The method demonstrated good linearity at the concentrations ranging from 20 to 10,000ng/mL. The intra- and inter-day relative standard deviations were less than 9.9%. The mean extraction recoveries of PTX and IS were 94.7% and 87.5%, respectively. In summary, the methanol protein precipitation-micelle breaking method could extract PTX completely from the polymeric micelles. Finally, the method was successfully applied to a pharmacokinetic study of the home-made PTX-loaded HA-C18 polymeric micelles and Taxol solution after intravenous administration in rats. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Seminal Plasma Proteins as Androgen Receptor Corregulators Promote Prostate Cancer Growth

    DTIC Science & Technology

    2016-12-01

    Award Number: W81XWH-13-1-0412 TITLE: Seminal Plasma Proteins as Androgen Receptor Corregulators Promote Prostate Cancer Growth PRINCIPAL...SUBTITLE Seminal Plasma Proteins as Androgen Receptor Corregulators Promote Prostate Cancer Growth 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-13...semenogelin I (SgI) in the presence of zinc, promote prostate cancer growth via functioning as androgen receptor (AR) co-activators. Using cell lines

  6. Combination of electrografting and atom-transfer radical polymerization for making the stainless steel surface antibacterial and protein antiadhesive.

    PubMed

    Ignatova, Milena; Voccia, Samuel; Gilbert, Bernard; Markova, Nadya; Cossement, Damien; Gouttebaron, Rachel; Jérôme, Robert; Jérôme, Christine

    2006-01-03

    A two-step "grafting from" method has been successfully carried out, which is based on the electrografting of polyacrylate chains containing an initiator for the atom transfer radical polymerization (ATRP) of 2-(tert-butylamino)ethyl methacrylate (TBAEMA) or copolymerization of TBAEMA with either monomethyl ether of poly(ethylene oxide) methacrylate (PEOMA) or acrylic acid (AA) or styrene. The chemisorption of this type of polymer brushes onto stainless steel surfaces has potential in orthopaedic surgery. These films have been characterized by ATR-FTIR, Raman spectroscopy, atomic force microscopy (AFM), and measurement of contact angles of water. The polymer formed in solution by ATRP and that one detached on purpose from the surface have been analyzed by size exclusion chromathography (SEC) and (1)H NMR spectroscopy. The strong adherence of the films onto stainless steel has been assessed by peeling tests. AFM analysis has shown that addition of hydrophilic comonomers to the grafted chains decreases the surface roughness. According to dynamic quartz crystal microbalance experiments, proteins (e.g., fibrinogen) are more effectively repelled whenever copolymer brushes contain neutral hydrophilic (PEOMA) co-units rather than negatively charged groups (PAA salt). Moreover, a 2- to 3-fold decrease in the fibrinogen adsorption is observed when TBAEMA is copolymerized with either PEOMA or AA rather than homopolymerized or copolymerized with styrene. Compared to the bare stainless steel surface, brushes of polyTBAEMA, poly(TBAEMA-co-PEOMA) and poly(TBAEMA-co-AA) decrease the bacteria adhesion by 3 to 4 orders of magnitude as revealed by Gram-positive bacteria S. aureus adhesion tests.

  7. Hybrid polymeric-protein nano-carriers (HPPNC) for targeted delivery of TGFβ inhibitors to hepatocellular carcinoma cells.

    PubMed

    Hanafy, Nemany A N; Quarta, Alessandra; Di Corato, Riccardo; Dini, Luciana; Nobile, Concetta; Tasco, Vittorianna; Carallo, Sonia; Cascione, Mariafrancesca; Malfettone, Andrea; Soukupova, Jitka; Rinaldi, Rosaria; Fabregat, Isabel; Leporatti, Stefano

    2017-08-01

    TGFβ1 pathway antagonists have been considered promising therapies to attenuate TGFβ downstream signals in cancer cells. Inhibiting peptides, as P-17 in this study, are bound to either TGFβ1 or its receptors, blocking signal transduction. However, for efficient use of these TGFβ1antagonist as target therapeutic tools, improvement in their delivery is required. Here, a plasmid carrying specific shDNA (SHT-DNA), small interfering RNA (siRNA), and the peptide (P-17) were loaded separately into folic acid (FA)-functionalized nano-carriers made of Bovine Serum Albumin (BSA). The two building blocks of the carrier, (BSA and FA) were used because of the high affinity of albumin for liver and for the overexpression of folate receptors on the membrane of hepatocellular carcinoma cells. The empty and the encapsulated carriers were thoroughly investigated to characterize their structure, to evaluate the colloidal stability and the surface functionalization. The entrapment of SHT-DNA, siRNA and P-17, respectively, was demonstrated by morphological and quantitative analysis. Finally, cellular studies were performed to assess the targeting efficiency of the hybrid carriers. These vectors were used because of the high affinity of albumin for liver and for the overexpression of folate receptors on the membrane hepatocellular carcinoma cells. The empty and the encapsulated carriers were thoroughly investigated to characterize their structure, to evaluate the colloidal stability and the surface functionalization. The entrapment of SHT-DNA, siRNA and P-17, respectively, was demonstrated by morphological and quantitative analysis. A novel fabrication of Hybrid Polymeric-Protein Nano-Carriers (HPPNC) for delivering TGF β1 inhibitors to HCC cells has been developed. SHT-DNA, siRNA and P-17 have been successfully encapsulated. TGF β1 inhibitors-loaded HPPNC were efficiently uptaken by HLF cells.

  8. Double promoter expression systems for recombinant protein production by industrial microorganisms.

    PubMed

    Öztürk, Sibel; Ergün, Burcu Gündüz; Çalık, Pınar

    2017-09-12

    Using double promoter expression systems is a promising approach to increase heterologous protein production. In this review, current double promoter expression systems for the production of recombinant proteins (r-proteins) by industrially important bacteria, Bacillus subtilis and Escherichia coli; and yeasts, Saccharomyces cerevisiae and Pichia pastoris, are discussed by assessing their potentials and drawbacks. Double promoter expression systems need to be designed to maintain a higher specific product formation rate within the production domain. While bacterial double promoter systems have been constructed as chimeric tandem promoters, yeast dual promoter systems have been developed as separate expression cassettes. To increase production and productivity, the optimal transcriptional activity should be justified either by simultaneously satisfying the requirements of both promoters, or by consecutively stimulating the changeover from one to another in a biphasic process or via successive-iterations. Thus, considering the dynamics of a fermentation process, double promoters can be classified according to their operational mechanisms, as: i) consecutively operating double promoter systems, and ii) simultaneously operating double promoter systems. Among these metabolic design strategies, extending the expression period with two promoters activated under different conditions, or enhancing the transcriptional activity with two promoters activated under similar conditions within the production domain, can be applied independently from the host. Novel studies with new insights, which aim a rational systematic design and construction of dual promoter expression vectors with tailored transcriptional activity, will empower r-protein production with enhanced production and productivity. Finally, the current state-of-the-art review emphasizes the advantages of double promoter systems along with the necessity for discovering new promoters for the development of more

  9. Identification of procollagen promoter DNA-binding proteins: effects of dexamethasone

    SciTech Connect

    Sweeney, C.; Cutroneo, K.R.

    1987-05-01

    Glucocorticoids selectively decrease procollagen synthesis by decreasing procollagen mRNA transcription. Dexamethasone coordinately decreased total cellular type I and type III procollagen mRNAs in mouse embryonic skin fibroblasts. Since sequence specific DNA-binding proteins are known to modulate eukaryotic gene expression the authors identified in mouse fibroblasts nuclear proteins which bind to types I and III procollagen promoter DNAs. Nuclear proteins were electrophoresed, blotted onto nitrocellulose and probed with /sup 32/P-end-labeled type I and type III procollagen promoter DNAs in the presence of equimolar amounts of /sup 32/P-end-labeled vector DNA. Differences in total DNA binding were noted by the densitometric scans of the nuclear proteins. Dexamethasone treatment enhanced total DNA binding. Increasing the NaCl concentration decreased the number of promoter DNA-binding proteins without altering the relative specificity for the promoter DNAs. Promoter DNA binding to nuclear proteins was also inhibited by increasing concentrations of E. coli DNA. The number of DNA-binding proteins was greater for type III procollagen promoter DNA. The effect of dexamethasone treatment on promoter DNA binding to nuclear proteins was determined.

  10. Phylum-wide analysis of genes/proteins related to the last steps of assembly and export of extracellular polymeric substances (EPS) in cyanobacteria

    NASA Astrophysics Data System (ADS)

    Pereira, Sara B.; Mota, Rita; Vieira, Cristina P.; Vieira, Jorge; Tamagnini, Paula

    2015-10-01

    Many cyanobacteria produce extracellular polymeric substances (EPS) with particular characteristics (e.g. anionic nature and presence of sulfate) that make them suitable for industrial processes such as bioremediation of heavy metals or thickening, suspending or emulsifying agents. Nevertheless, their biosynthetic pathway(s) are still largely unknown, limiting their utilization. In this work, a phylum-wide analysis of genes/proteins putatively involved in the assembly and export of EPS in cyanobacteria was performed. Our results demonstrated that most strains harbor genes encoding proteins related to the three main pathways: Wzy-, ABC transporter-, and Synthase-dependent, but often not the complete set defining one pathway. Multiple gene copies are mainly correlated to larger genomes, and the strains with reduced genomes (e.g. the clade of marine unicellular Synechococcus and Prochlorococcus), seem to have lost most of the EPS-related genes. Overall, the distribution of the different genes/proteins within the cyanobacteria phylum raises the hypothesis that cyanobacterial EPS production may not strictly follow one of the pathways previously characterized. Moreover, for the proteins involved in EPS polymerization, amino acid patterns were defined and validated constituting a novel and robust tool to identify proteins with similar functions and giving a first insight to which polymer biosynthesis they are related to.

  11. Atom transfer radical polymerization to fabricate monodisperse poly[glycidyl methacrylate-co-poly (ethylene glycol) methacrylate] microspheres and its application for protein affinity purification.

    PubMed

    Yu, Ling; Shi, Zhuan Zhuan; Li, Chang Ming

    2015-09-01

    Poly[glycidyl methacrylate-co-poly (ethylene glycol) methacrylate] microspheres for the first time were successfully synthesized by atom transfer radical polymerization (ATRP) method at room temperature. The co-polymerization approach was investigated to delicately control the microsphere morphology and size-distribution by reaction conditions including solvent percentage, monomer loading and rotation speed. The results show that the average size of the microspheres is ∼5.7 μm with coexistence of epoxy, hydroxyl and ether groups, which provide plentiful functional sites for protein anchoring. The mechanism of the microsphere formation is proposed. The microsphere successfully demonstrates its unique application for affinity purification of proteins, in which the functional epoxy group facilitates a simple and efficient protein covalent immobilization to purify immunoglobulin G on the microspheres, while the hydrophilic poly (ethylene glycol) motif can repulse nonspecific protein adsorption for good specificity. This microspheres can be used in broad protein biosensors due to their abundant functional groups and high surface to volume ratio.

  12. Phylum-wide analysis of genes/proteins related to the last steps of assembly and export of extracellular polymeric substances (EPS) in cyanobacteria.

    PubMed

    Pereira, Sara B; Mota, Rita; Vieira, Cristina P; Vieira, Jorge; Tamagnini, Paula

    2015-10-06

    Many cyanobacteria produce extracellular polymeric substances (EPS) with particular characteristics (e.g. anionic nature and presence of sulfate) that make them suitable for industrial processes such as bioremediation of heavy metals or thickening, suspending or emulsifying agents. Nevertheless, their biosynthetic pathway(s) are still largely unknown, limiting their utilization. In this work, a phylum-wide analysis of genes/proteins putatively involved in the assembly and export of EPS in cyanobacteria was performed. Our results demonstrated that most strains harbor genes encoding proteins related to the three main pathways: Wzy-, ABC transporter-, and Synthase-dependent, but often not the complete set defining one pathway. Multiple gene copies are mainly correlated to larger genomes, and the strains with reduced genomes (e.g. the clade of marine unicellular Synechococcus and Prochlorococcus), seem to have lost most of the EPS-related genes. Overall, the distribution of the different genes/proteins within the cyanobacteria phylum raises the hypothesis that cyanobacterial EPS production may not strictly follow one of the pathways previously characterized. Moreover, for the proteins involved in EPS polymerization, amino acid patterns were defined and validated constituting a novel and robust tool to identify proteins with similar functions and giving a first insight to which polymer biosynthesis they are related to.

  13. Effect of microfiltration concentration factor on serum protein removal from skim milk using spiral-wound polymeric membranes.

    PubMed

    Beckman, S L; Barbano, D M

    2013-10-01

    Our objective was to determine the effect of concentration factor (CF) on the removal of serum protein (SP) from skim milk during microfiltration (MF) at 50 °C using a 0.3-μm-pore-size spiral-wound (SW) polymeric polyvinylidene fluoride (PVDF) membrane. Pasteurized (72°C for 16 s) skim milk was MF (50 °C) at 3 CF (1.50, 2.25, and 3.00×), each on a separate day of processing starting with skim milk. Two phases of MF were used at each CF, with an initial startup-stabilization phase (40 min in full recycle mode) to achieve the desired CF, followed by a steady-state phase (90-min feed-and-bleed with recycle) where data was collected. The experiment was replicated 3 times, and SP removal from skim milk was quantified at each CF. System pressures, flow rates, CF, and fluxes were monitored during the 90-min run. Permeate flux increased (12.8, 15.3, and 19.0 kg/m(2) per hour) with decreasing CF from 3.00 to 1.50×, whereas fouled water flux did not differ among CF, indicating that the effect of membrane fouling on hydraulic resistance of the membrane was similar at all CF. However, the CF used when microfiltering skim milk (50°C) with a 0.3-μm polymeric SW PVDF membrane did affect the percentage of SP removed. As CF increased from 1.50 to 3.00×, the percentage of SP removed from skim milk increased from 10.56 to 35.57%, in a single stage bleed-and-feed MF system. Percentage SP removal from skim milk was lower than the theoretical value. Rejection of SP during MF of skim milk with SW PVDF membranes was caused by fouling of the membrane, not by the membrane itself and differences in the foulant characteristic among CF influenced SP rejection more than it influenced hydraulic resistance. We hypothesize that differences in the conditions near the surface of the membrane and within the pores during the first few minutes of processing, when casein micelles pass through the membrane, influenced the rejection of SP because more pore size narrowing and plugging occurred at

  14. Fibronectin matrix polymerization regulates smooth muscle cell phenotype through a Rac1 dependent mechanism.

    PubMed

    Shi, Feng; Long, Xiaochun; Hendershot, Allison; Miano, Joseph M; Sottile, Jane

    2014-01-01

    Smooth muscle cells are maintained in a differentiated state in the vessel wall, but can be modulated to a synthetic phenotype following injury. Smooth muscle phenotypic modulation is thought to play an important role in the pathology of vascular occlusive diseases. Phenotypically modulated smooth muscle cells exhibit increased proliferative and migratory properties that accompany the downregulation of smooth muscle cell marker proteins. Extracellular matrix proteins, including fibronectin, can regulate the smooth muscle phenotype when used as adhesive substrates. However, cells produce and organize a 3-dimensional fibrillar extracellular matrix, which can affect cell behavior in distinct ways from the protomeric 2-dimensional matrix proteins that are used as adhesive substrates. We previously showed that the deposition/polymerization of fibronectin into the extracellular matrix can regulate the deposition and organization of other extracellular matrix molecules in vitro. Further, our published data show that the presence of a fibronectin polymerization inhibitor results in increased expression of smooth muscle cell differentiation proteins and inhibits vascular remodeling in vivo. In this manuscript, we used an in vitro cell culture system to determine the mechanism by which fibronectin polymerization affects smooth muscle phenotypic modulation. Our data show that fibronectin polymerization decreases the mRNA levels of multiple smooth muscle differentiation genes, and downregulates the levels of smooth muscle α-actin and calponin proteins by a Rac1-dependent mechanism. The expression of smooth muscle genes is transcriptionally regulated by fibronectin polymerization, as evidenced by the increased activity of luciferase reporter constructs in the presence of a fibronectin polymerization inhibitor. Fibronectin polymerization also promotes smooth muscle cell growth, and decreases the levels of actin stress fibers. These data define a Rac1-dependent pathway wherein

  15. Ligand-Promoted Protein Folding by Biased Kinetic Partitioning

    PubMed Central

    Hingorani, Karan S.; Metcalf, Matthew C.; Deming, Derrick T.; Garman, Scott C.; Powers, Evan T.; Gierasch, Lila M.

    2017-01-01

    Protein folding in cells occurs in the presence of high concentrations of endogenous binding partners, and exogenous binding partners have been exploited as pharmacological chaperones. A combined mathematical modeling and experimental approach shows that a ligand improves the folding of a destabilized protein by biasing the kinetic partitioning between folding and alternative fates (aggregation or degradation). Computationally predicted inhibition of test protein aggregation and degradation as a function of ligand concentration are validated by experiments in two disparate cellular systems. PMID:28218913

  16. Protein Kinase Cϵ (PKCϵ) Promotes Synaptogenesis through Membrane Accumulation of the Postsynaptic Density Protein PSD-95.

    PubMed

    Sen, Abhik; Hongpaisan, Jarin; Wang, Desheng; Nelson, Thomas J; Alkon, Daniel L

    2016-08-05

    Protein kinase Cϵ (PKCϵ) promotes synaptic maturation and synaptogenesis via activation of synaptic growth factors such as BDNF, NGF, and IGF. However, many of the detailed mechanisms by which PKCϵ induces synaptogenesis are not fully understood. Accumulation of PSD-95 to the postsynaptic density (PSD) is known to lead to synaptic maturation and strengthening of excitatory synapses. Here we investigated the relationship between PKCϵ and PSD-95. We show that the PKCϵ activators dicyclopropanated linoleic acid methyl ester and bryostatin 1 induce phosphorylation of PSD-95 at the serine 295 residue, increase the levels of PSD-95, and enhance its membrane localization. Elimination of the serine 295 residue in PSD-95 abolished PKCϵ-induced membrane accumulation. Knockdown of either PKCϵ or JNK1 prevented PKCϵ activator-mediated membrane accumulation of PSD-95. PKCϵ directly phosphorylated PSD-95 and JNK1 in vitro Inhibiting PKCϵ, JNK, or calcium/calmodulin-dependent kinase II activity prevented the effects of PKCϵ activators on PSD-95 phosphorylation. Increase in membrane accumulation of PKCϵ and phosphorylated PSD-95 (p-PSD-95(S295)) coincided with an increased number of synapses and increased amplitudes of excitatory post-synaptic potentials (EPSPs) in adult rat hippocampal slices. Knockdown of PKCϵ also reduced the synthesis of PSD-95 and the presynaptic protein synaptophysin by 30 and 44%, respectively. Prolonged activation of PKCϵ increased synapse number by 2-fold, increased presynaptic vesicle density, and greatly increased PSD-95 clustering. These results indicate that PKCϵ promotes synaptogenesis by activating PSD-95 phosphorylation directly through JNK1 and calcium/calmodulin-dependent kinase II and also by inducing expression of PSD-95 and synaptophysin. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Protein Kinase Cϵ (PKCϵ) Promotes Synaptogenesis through Membrane Accumulation of the Postsynaptic Density Protein PSD-95*

    PubMed Central

    Sen, Abhik; Hongpaisan, Jarin; Wang, Desheng; Nelson, Thomas J.; Alkon, Daniel L.

    2016-01-01

    Protein kinase Cϵ (PKCϵ) promotes synaptic maturation and synaptogenesis via activation of synaptic growth factors such as BDNF, NGF, and IGF. However, many of the detailed mechanisms by which PKCϵ induces synaptogenesis are not fully understood. Accumulation of PSD-95 to the postsynaptic density (PSD) is known to lead to synaptic maturation and strengthening of excitatory synapses. Here we investigated the relationship between PKCϵ and PSD-95. We show that the PKCϵ activators dicyclopropanated linoleic acid methyl ester and bryostatin 1 induce phosphorylation of PSD-95 at the serine 295 residue, increase the levels of PSD-95, and enhance its membrane localization. Elimination of the serine 295 residue in PSD-95 abolished PKCϵ-induced membrane accumulation. Knockdown of either PKCϵ or JNK1 prevented PKCϵ activator-mediated membrane accumulation of PSD-95. PKCϵ directly phosphorylated PSD-95 and JNK1 in vitro. Inhibiting PKCϵ, JNK, or calcium/calmodulin-dependent kinase II activity prevented the effects of PKCϵ activators on PSD-95 phosphorylation. Increase in membrane accumulation of PKCϵ and phosphorylated PSD-95 (p-PSD-95S295) coincided with an increased number of synapses and increased amplitudes of excitatory post-synaptic potentials (EPSPs) in adult rat hippocampal slices. Knockdown of PKCϵ also reduced the synthesis of PSD-95 and the presynaptic protein synaptophysin by 30 and 44%, respectively. Prolonged activation of PKCϵ increased synapse number by 2-fold, increased presynaptic vesicle density, and greatly increased PSD-95 clustering. These results indicate that PKCϵ promotes synaptogenesis by activating PSD-95 phosphorylation directly through JNK1 and calcium/calmodulin-dependent kinase II and also by inducing expression of PSD-95 and synaptophysin. PMID:27330081

  18. Evaluation of an injectable polymeric delivery system for controlled and localized release of biological factors to promote therapeutic angiogenesis

    NASA Astrophysics Data System (ADS)

    Rocker, Adam John

    Cardiovascular disease remains as the leading cause of death worldwide and is frequently associated with partial or full occlusion of coronary arteries. Currently, angioplasty and bypass surgery are the standard approaches for treating patients with these ischemic heart conditions. However, a large number of patients cannot undergo these procedures. Therapeutic angiogenesis provides a minimally invasive tool for treating cardiovascular diseases by inducing new blood vessel growth from the existing vasculature. Angiogenic growth factors can be delivered locally through gene, cell, and protein therapy. Natural and synthetic polymer growth factor delivery systems are under extensive investigation due their widespread applications and promising therapeutic potential. Although biocompatible, natural polymers often suffer from batch-to-batch variability which can cause unpredictable growth factor release rates. Synthetic polymers offer advantages for growth factor delivery as they can be easily modified to control release kinetics. During the angiogenesis process, vascular endothelial growth factor (VEGF) is necessary to initiate neovessel formation while platelet-derived growth factor (PDGF) is needed later to help stabilize and mature new vessels. In the setting of myocardial infarction, additional anti-inflammatory cytokines like IL-10 are needed to help optimize cardiac repair and limit the damaging effects of inflammation following infarction. To meet these angiogenic and anti-inflammatory needs, an injectable polymer delivery system created from a sulfonated reverse thermal gel encapsulating micelle nanoparticles was designed and evaluated. The sulfonate groups on the thermal gel electrostatically bind to VEGF which controls its release rate, while the micelles are loaded with PDGF and are slowly released as the gel degrades. IL-10 was loaded into the system as well and diffused from the gel over time. An in vitro release study was performed which demonstrated the

  19. JNK inhibitor SP600125 promotes the formation of polymerized tubulin, leading to G2/M phase arrest, endoreduplication, and delayed apoptosis.

    PubMed

    Moon, Dong Oh; Kim, Mun Ock; Kang, Chang Hee; Lee, Jae Dong; Choi, Yung Hyun; Kim, Gi Young

    2009-09-30

    The JNK inhibitor SP600125 strongly inhibits cell proliferation in many human cancer cells by blocking cell-cycle progression and inducing apoptosis. Despite extensive study, the mechanism by which SP600125 inhibits mitosis-related effects in human leukemia cells remains unclear. We investigated the effects of SP600125 on the inhibition of cell proliferation and the cell cycle, and on microtubule dynamics in vivo and in vitro. Treatment of synchronized leukemia cells with varying concentrations of SP600125 results in significant G2/M cell cycle arrest with elevated p21 levels, phosphorylation of histone H3 within 24 h, and endoreduplication with elevated Cdk2 protein levels after 48 h. SP600125 also induces significant abnormal microtubule dynamics in vivo. High concentrations of SP600125 (200 microM) were required to disorganize microtubule polymerization in vitro. Additionally, SP600125- induced delayed apoptosis and cell death was accompanied by significant poly ADP-ribose polymerase (PARP) cleavage and caspase-3 activity in the late phase (at 72 h). Endoreduplication showed a greater increase in ectopic Bcl-2-expressing U937 cells at 72 h than in wild-type U937 cells without delayed apoptosis. These results indicate that Bcl-2 suppresses apoptosis and SP600125-induced G2/M arrest and endoreduplication. Therefore, we suggest that SP600125 induces mitotic arrest by inducing abnormal spindle microtubule dynamics.

  20. Development of high-productivity, strong cation-exchange adsorbers for protein capture by graft polymerization from membranes with different pore sizes

    PubMed Central

    Chenette, Heather C.S.; Robinson, Julie R.; Hobley, Eboni; Husson, Scott M.

    2012-01-01

    This paper describes the surface modification of macroporous membranes using ATRP (atom transfer radical polymerization) to create cation-exchange adsorbers with high protein binding capacity at high product throughput. The work is motivated by the need for a more economical and rapid capture step in downstream processing of protein therapeutics. Membranes with three reported nominal pore sizes (0.2, 0.45, 1.0 μm) were modified with poly(3-sulfopropyl methacrylate, potassium salt) tentacles, to create a high density of protein binding sites. A special formulation was used in which the monomer was protected by a crown ether to enable surface-initiated ATRP of this cationic polyelectrolyte. Success with modification was supported by chemical analysis using Fourier-transform infrared spectroscopy and indirectly by measurement of pure water flux as a function of polymerization time. Uniformity of modification within the membranes was visualized with confocal laser scanning microscopy. Static and dynamic binding capacities were measured using lysozyme protein to allow comparisons with reported performance data for commercial cation-exchange materials. Dynamic binding capacities were measured for flow rates ranging from 13 to 109 column volumes (CV)/min. Results show that this unique ATRP formulation can be used to fabricate cation-exchange membrane adsorbers with dynamic binding capacities as high as 70 mg/mL at a throughput of 100 CV/min and unprecedented productivity of 300 mg/mL/min. PMID:23175597

  1. Myofilament Protein Alterations Promote Physical Disability in Aging and Disease

    PubMed Central

    Miller, Mark S.; Toth, Michael J.

    2014-01-01

    Skeletal muscle contractile function declines with age and age-associated diseases. Although muscle atrophy undoubtedly contributes to this decrease, recent findings suggest that reduced myofilament protein content and function also may participate. Based on these data, we propose that age- and disease-related alterations in myofilament proteins represent one molecular mechanism contributing to the development of physical disability. PMID:23392279

  2. Promoter-specific trans activation and repression by human cytomegalovirus immediate-early proteins involves common and unique protein domains.

    PubMed Central

    Stenberg, R M; Fortney, J; Barlow, S W; Magrane, B P; Nelson, J A; Ghazal, P

    1990-01-01

    trans activation of promoters by viral regulatory proteins provides a useful tool to study coordinate control of gene expression. Immediate-early (IE) regions 1 and 2 of human cytomegalovirus (CMV) code for a series of proteins that originate from differentially spliced mRNAs. These IE proteins are proposed to regulate the temporal expression of the viral genome. To examine the structure and function of the IE proteins, we used linker insertion mutagenesis of the IE gene region as well as cDNA expression vector cloning of the abundant IE mRNAs. We showed that IE1 and IE2 proteins of CMV exhibit promoter-specific differences in their modes of action by either trans activating early and IE promoters or repressing the major IE promoter (MIEP). Transient cotransfection experiments with permissive human cells revealed a synergistic interaction between the 72- and the 86-kilodalton (kDa) IE proteins in trans activating an early promoter. In addition, transfection studies revealed that the 72-kDa protein was capable of trans activating the MIEP. In contrast, the 86-kDa protein specifically repressed the MIEP and this repression was suppressed by the 72-kDa protein. Furthermore, observations based on the primary sequence structure revealed a modular arrangement of putative regulatory motifs that could either potentiate or repress gene expression. These modular domains are either shared or unique among the IE proteins. From these data, we propose a model for IE protein function in the coordinate control of CMV gene expression. Images PMID:2157043

  3. Response gene to complement 32 protein promotes macrophage phagocytosis via activation of protein kinase C pathway.

    PubMed

    Tang, Rui; Zhang, Gui; Chen, Shi-You

    2014-08-15

    Macrophage phagocytosis plays an important role in host defense. The molecular mechanism, especially factors regulating the phagocytosis, however, is not completely understood. In the present study, we found that response gene to complement 32 (RGC-32) is an important regulator of phagocytosis. Although RGC-32 is induced and abundantly expressed in macrophage during monocyte-macrophage differentiation, RGC-32 appears not to be important for this process because RGC-32-deficient bone marrow progenitor can normally differentiate to macrophage. However, both peritoneal macrophages and bone marrow-derived macrophages with RGC-32 deficiency exhibit significant defects in phagocytosis, whereas RGC-32-overexpressed macrophages show increased phagocytosis. Mechanistically, RGC-32 is recruited to macrophage membrane where it promotes F-actin assembly and the formation of phagocytic cups. RGC-32 knock-out impairs F-actin assembly. RGC-32 appears to interact with PKC to regulate PKC-induced phosphorylation of F-actin cross-linking protein myristoylated alanine-rich protein kinase C substrate. Taken together, our results demonstrate for the first time that RGC-32 is a novel membrane regulator for macrophage phagocytosis.

  4. Characterization and protein-adsorption behavior of deposited organic thin film onto titanium by plasma polymerization with hexamethyldisiloxane.

    PubMed

    Hayakawa, Tohru; Yoshinari, Masao; Nemoto, Kimiya

    2004-01-01

    Plasma polymerized hexamethyldisiloxane (HMDSO) thin film was deposited onto titanium using a radio-frequency apparatus for the surface modification of titanium. A titanium disk was first polished using colloidal silica at pH=9.8. Plasma-polymerized HMDSO films were firmly attached to the titanium by heating the titanium to a temperature of approximately 250 degrees C. The thickness of the deposited film was 0.07-0.35mum after 10-60min of plasma polymerization. The contact angle with respect to double distilled water significantly increased after HMDSO coating. X-ray photoelectron spectroscopy revealed that the deposited thin film consisted of Si, C, and O atoms. No Ti peaks were observed on the deposited surface. The deposited HMDSO film was stable during 2-weeks immersion in phosphate buffer saline solution. Fourier transform reflection-absorption spectroscopy showed the formation of Si-H, Si-C, C-H, and Cz.dbnd6;O bonds in addition to Si-O-Si bonds. Quartz crystal microbalance-dissipation measurement demonstrated that the deposition of HMDSO thin films on titanium has a benefit for fibronectin adsorption at the early stage. In conclusion, plasma polymerization is a promising technique for the surface modification of titanium. HMDSO-coated titanium has potential application as a dental implant material.

  5. Effect of heparin on protein aggregation: inhibition versus promotion.

    PubMed

    Xu, Yisheng; Seeman, Daniel; Yan, Yunfeng; Sun, Lianhong; Post, Jared; Dubin, Paul L

    2012-05-14

    The effect of heparin on both native and denatured protein aggregation was investigated by turbidimetry and dynamic light scattering (DLS). Turbidimetric data show that heparin is capable of inhibiting and reversing the native aggregation of bovine serum albumin (BSA), β-lactoglobulin (BLG), and Zn-insulin at a pH near pI and at low ionic strength I; however, the results vary with regard to the range of pH, I, and protein-heparin stoichiometry required to achieve these effects. The kinetics of this process were studied to determine the mechanism by which interaction with heparin could result in inhibition or reversal of native protein aggregates. For each protein, the binding of heparin to distinctive intermediate aggregates formed at different times in the aggregation process dictates the outcome of complexation. This differential binding was explained by changes in the affinity of a given protein for heparin, partly due to the effects of protein charge anisotropy as visualized by electrostatic modeling. The heparin effect can be further extended to include inhibition of denaturing protein aggregation, as seen from the kinetics of BLG aggregation under conditions of thermally induced unfolding with and without heparin.

  6. pH-sensitive polymeric nanoparticles to improve oral bioavailability of peptide/protein drugs and poorly water-soluble drugs.

    PubMed

    Wang, Xue-Qing; Zhang, Qiang

    2012-10-01

    pH-sensitive polymeric nanoparticles are promising for oral drug delivery, especially for peptide/protein drugs and poorly water-soluble medicines. This review describes current status of pH-sensitive polymeric nanoparticles for oral drug delivery and introduces the mechanisms of drug release from them as well as possible reasons for absorption improvement, with emphasis on our contribution to this field. pH-sensitive polymeric nanoparticles are prepared mainly with polyanions, polycations, their mixtures or cross-linked polymers. The mechanisms of drug release are the result of carriers' dissolution, swelling or both of them at specific pH. The possible reasons for improvement of oral bioavailability include the following: improve drug stability, enhance mucoadhesion, prolong resident time in GI tract, ameliorate intestinal permeability and increase saturation solubility and dissolution rate for poorly water-soluble drugs. As for the advantages of pH-sensitive nanoparticles over conventional nanoparticles, we conclude that (1) most carriers used are enteric-coating materials and their safety has been approved. (2) The rapid dissolution or swelling of carriers at specific pH results in quick drug release and high drug concentration gradient, which is helpful for absorption. (3) At the specific pH carriers dissolve or swell, and the bioadhesion of carriers to mucosa becomes high because nanoparticles turn from solid to gel, which can facilitate drug absorption.

  7. Multistage skin tumor promotion: involvement of a protein kinase

    SciTech Connect

    Mamrack, M.; Slaga, T. J.

    1980-01-01

    Current information suggests that chemical carcinogenesis is a multistep process with one of the best studied models in this regard being the two-stage carcinogenesis system using mouse skin. The effects of several carcinogens and tumor promoters in various sequences of application were studied to examine the nature of the process. The actions of several tumor inhibitors were compared. (ACR)

  8. AmpA protein functions by different mechanisms to influence early cell type specification and to modulate cell adhesion and actin polymerization in Dictyostelium discoideum.

    PubMed

    Cost, Hoa N; Noratel, Elizabeth F; Blumberg, Daphne D

    2013-01-01

    The Dictyostelium discoideum ampA gene encodes a multifunctional regulator protein that modulates cell-cell and cell-substrate adhesions and actin polymerization during growth and is necessary for correct cell type specification and patterning during development. Insertional inactivation of the ampA gene results in defects that define two distinct roles for the ampA gene during development. AmpA is necessary in a non-cell autonomous manner to prevent premature expression of a prespore gene marker. It is also necessary in a cell autonomous manner for the anterior like cells, which express the ampA gene, to migrate to the upper cup during culmination. It is also necessary to prevent excessive cell-cell agglutination when cells are developed in a submerged suspension culture. Here, we demonstrate that a supernatant source of AmpA protein, added extracellularly, can prevent the premature mis-expression of the prespore marker. Synthetic oligopeptides are used to identify the domain of the AmpA protein that is important for preventing cells from mis-expressing the prespore gene. We further demonstrate that a factor capable of inducing additional cells to express the prespore gene marker accumulates extracellularly in the absence of AmpA protein. While the secreted AmpA acts extracellularly to suppress prespore gene expression, the effects of AmpA on cell agglutination and on actin polymerization in growing cells are not due to an extracellular role of secreted AmpA protein. Rather, these effects appear to reflect a distinct cell autonomous role of the ampA gene. Finally, we show that secretion of AmpA protein is brought about by elevating the levels of expression of ampA so that the protein accumulates to an excessive level.

  9. Zwitterionic sulfobetaine-grafted poly(vinylidene fluoride) membrane surface with stably anti-protein-fouling performance via a two-step surface polymerization

    NASA Astrophysics Data System (ADS)

    Li, Qian; Bi, Qiu-Yan; Zhou, Bo; Wang, Xiao-Lin

    2012-03-01

    A zwitterionic polymer, poly(3-(methacryloylamino) propyl-dimethyl-(3-sulfopropyl) ammonium hydroxide) (poly(MPDSAH)) was successfully grafted in high density from the surface of poly(vinylidene fluoride) (PVDF) hollow fiber membrane via a two-step polymerization. Poly(2-hydroxyethyl methacrylate) (poly(HEMA)) chains were firstly grafted from outside surface of PVDF membrane through atom transfer radical polymerization (ATRP) to provide the initiation sites for subsequent cerium (Ce (IV))-induced graft copolymerization of polyMPDSAH in the presence of N,N'-ethylene bisacrylamide (EBAA) as a cross-linking agent. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS) confirmed that the EBAA could stimulate zwitterionic polymers grafting onto the membrane surface. The dense poly(MPDSAH) layers on the PVDF membrane surface were revealed by the scanning electron microscope (SEM). The mechanical property of PVDF membrane was improved by the zwitterionic surface layers. The gravimetry results indicated the grafting amount increased to 520 μg/cm2 for a copolymerization time of more than 3 h. Static and dynamic water contact angle measurements showed that the surface hydrophilicity of the PVDF membranes was significantly enhanced. As the grafting amount reached 513 μg cm-2, the value of contact angle dropped to 22.1° and the amount of protein adsorption decreased to zero. The cyclic experiments for BSA solution filtration demonstrated that the extent of protein fouling was significantly reduced and most of the fouling was reversible. The grafted polymer layer on the PVDF membrane showed a good stability during the membrane cleaning process. The experimental results concluded a good prospect in obtaining the sulfobetaine-modified PVDF membranes with high mechanical strength, good anti-protein-fouling performance, and long-term stability via the two-step polymerization.

  10. Methods for promoting wound healing and muscle regeneration with the cell signaling protein Nell1

    DOEpatents

    Culiat, Cymbeline T

    2014-11-04

    The present invention provides methods for promoting wound healing and treating muscle atrophy in a mammal in need. The method comprises administering to the mammal a Nell1 protein or a Nell1 nucleic acid molecule.

  11. Methods for promoting wound healing and muscle regeneration with the cell signaling protein Nell1

    DOEpatents

    Culiat, Cymbeline T [Oak Ridge, TN

    2011-03-22

    The present invention provides methods for promoting wound healing and treating muscle atrophy in a mammal in need. The method comprises administering to the mammal a Nell1 protein or a Nell1 nucleic acid molecule.

  12. Validated LC-MS/MS method for simultaneous determination of doxorubicin and curcumin in polymeric micelles in subcellular compartments of MCF-7/Adr cells by protein precipitation-ultrasonic breaking method.

    PubMed

    Wang, Jinling; Li, Ying; Ma, Wenzhuan; Wang, Xiaohui; Tu, Pengfei

    2017-06-01

    A specific and sensitive LC-MS/MS with protein precipitation- ultrasonic breaking method has been developed and validated for simultaneous determination of doxorubicin (DOX) and curcumin (Cur) in DOX and Cur co-loaded hyaluronic acid-vitamin E succinatemicelles [(DOX + Cur)-polymeric micelles (PMs)] in subcellular compartments of resistant MCF-7/Adr cells. Sequential extraction of four subcellular protein fractions (cytosolic, membrane/organelle, nucleic and cytoskeleton) was performed directly from MCF-7/Adr cells after incubation with (DOX + Cur)-PMs. An ultrasonic breaking-methanol precipitation method was used for extraction of the fractions, and the micelle breaking efficiency with methanol was 98.1 and 97.6% for DOX and Cur, respectively. The analytes were analyzed using positive electrospray ionization coupled with multiple reaction monitoring. The calibration curves were linear over a concentration range of 0.5-400 ng/mL for DOX and 2-2000 ng/mL for Cur, and the recovery for the two analytes were >85% with negligible matrix effect. The intra-day and inter-day precision was <10.80% and relative error was within ±7.70%. The developed method was successfully applied for subcellular determination of DOX and Cur in MCF-7/Adr cells. Moreover, Cur and (DOX + Cur)-PMs had a marked promoting effect on the distribution of DOX in the nucleic protein fraction. Copyright © 2016 John Wiley & Sons, Ltd.

  13. Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility

    PubMed Central

    Young, Barry P.; Loewen, Christopher J.; Mayor, Thibault

    2016-01-01

    Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations. PMID:27448207

  14. Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility.

    PubMed

    Comyn, Sophie A; Young, Barry P; Loewen, Christopher J; Mayor, Thibault

    2016-07-01

    Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations.

  15. Facile preparation of organic-inorganic hybrid polymeric ionic liquid monolithic column with a one-pot process for protein separation in capillary electrochromatography.

    PubMed

    Liu, Cuicui; Deng, Qiliang; Fang, Guozhen; Feng, Xue; Qian, Hailong; Wang, Shuo

    2014-11-01

    An organic-inorganic hybrid monolithic column based on 1-vinyl-3-dodecylimidazolium bromide (VC12Im(+)Br(-)) has been prepared in a single step by combining radical copolymerization with a non-hydrolytic sol-gel (NHSG) process. The NHSG process was significantly shortened to 6 h by using formic acid as catalyst. For comparison, we also prepared polymeric ionic liquid (PIL) monolithic columns by hydrolytic sol-gel and organic polymeric process, respectively. The resulting monolithic columns were characterized by Fourier transform infrared spectra, scanning electron microscopy, and Brunauer-Emmett-Teller. Under the capillary electrochromatography mode, these columns were applied to separate alkylbenzenes, anilines, and proteins, respectively. The results indicated that the NHSG-based hybrid PIL monolithic column exhibited the highest column efficiency among the three types of columns; organic solvent, commonly required by the traditional columns to achieve satisfactory separation efficiency for proteins, was absent in the NHSG-based hybrid PIL monolithic column because of the biocompatibility of the VC12Im(+)Br(-), which was beneficial to analysis of protein containing samples. In order to demonstrate its application potential, the developed NHSG-based hybrid PIL monolithic column was also employed to separate egg white sample.

  16. Study of protein modifications induced by phorbol ester tumor promoters in mouse skin

    SciTech Connect

    Nelson, K.G.

    1981-08-01

    The purpose of this study was to determine if the phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) induced any specific changes in mouse epidermal proteins using the high resolution technique of two-dimensional electrophoresis. To accomplish this goal of determining the specificity and possibly the stage in promotion with which these protein changes were associated, epidermal proteins were analyzed (1) after treatment of adult mouse epidermis with several weakly promoting hyperplasiogenic agents, (2) following treatment with TPA in combination with various inhibitors of tumor promotion, (3) in basal kerotinocytes isolated from adult epidermis following treatment with TPA or several weakly promoting agents, and (4) during an initiation-promotion experiment. Evidence was found which indicated that the potent tumor promoter TPA as well as the weakly promoting hyperplasiogenic agents, mezerein, ethylphenylpropiolate (EPP), and mechanical abrasion, induced similar modifications of epidermal proteins, particularly among the keratins. These keratin modifications progressed with time following treatment resulting in a keratin pattern which resembled that of newborn epidermis.

  17. High levels of protein expression using different mammalian CMV promoters in several cell lines.

    PubMed

    Xia, Wei; Bringmann, Peter; McClary, John; Jones, Patrick P; Manzana, Warren; Zhu, Ying; Wang, Soujuan; Liu, Yi; Harvey, Susan; Madlansacay, Mary Rose; McLean, Kirk; Rosser, Mary P; MacRobbie, Jean; Olsen, Catherine L; Cobb, Ronald R

    2006-01-01

    With the recent completion of the human genome sequencing project, scientists are faced with the daunting challenge of deciphering the function of these newly found genes quickly and efficiently. Equally as important is to produce milligram quantities of the therapeutically relevant gene products as quickly as possible. Mammalian expression systems provide many advantages to aid in this task. Mammalian cell lines have the capacity for proper post-translational modifications including proper protein folding and glycosylation. In response to the needs described above, we investigated the protein expression levels driven by the human CMV in the presence or absence of intron A, the mouse and rat CMV promoters with intron A, and the MPSV promoter in plasmid expression vectors. We evaluated the different promoters using an in-house plasmid vector backbone. The protein expression levels of four genes of interest driven by these promoters were evaluated in HEK293EBNA and CHO-K1 cells. Stable and transient transfected cells were utilized. In general, the full-length human CMV, in the presence of intron A, gave the highest levels of protein expression in transient transfections in both cell lines. However, the MPSV promoter resulted in the highest levels of stable protein expression in CHO-K1 cells. Using the CMV driven constitutive promoters in the presence of intron A, we have been able to generate >10 microg/ml of recombinant protein using transient transfections.

  18. Soy protein isolate inhibits hepatic tumor promotion in mice fed a high-fat liquid diet.

    PubMed

    Mercer, Kelly E; Pulliam, Casey F; Pedersen, Kim B; Hennings, Leah; Ronis, Martin Jj

    2017-03-01

    Alcoholic and nonalcoholic fatty liver diseases are risk factors for development of hepatocellular carcinoma, but the underlying mechanisms are poorly understood. On the other hand, ingestion of soy-containing diets may oppose the development of certain cancers. We previously reported that replacing casein with a soy protein isolate reduced tumor promotion in the livers of mice with alcoholic liver disease after feeding a high fat ethanol liquid diet following initiation with diethylnitrosamine. Feeding soy protein isolate inhibited processes that may contribute to tumor promotion including inflammation, sphingolipid signaling, and Wnt/β-catenin signaling. We have extended these studies to characterize liver tumor promotion in a model of nonalcoholic fatty liver disease produced by chronic feeding of high-fat liquid diets in the absence of ethanol. Mice treated with diethylnitrosamine on postnatal day 14 were fed a high-fat liquid diet made with casein or SPI as the sole protein source for 16 weeks in adulthood. Relative to mice fed normal chow, a high fat/casein diet led to increased tumor promotion, hepatocyte proliferation, steatosis, and inflammation. Replacing casein with soy protein isolate counteracted these effects. The high fat diets also resulted in a general increase in transcripts for Wnt/β-catenin pathway components, which may be an important mechanism, whereby hepatic tumorigenesis is promoted. However, soy protein isolate did not block Wnt signaling in this nonalcoholic fatty liver disease model. We conclude that replacing casein with soy protein isolate blocks development of steatosis, inflammation, and tumor promotion in diethylnitrosamine-treated mice fed high fat diets. Impact statement The impact of dietary components on cancer is a topic of great interest for both the general public and the scientific community. Liver cancer is currently the second leading form of cancer deaths worldwide. Our study has addressed the effect of the protein

  19. Poly(glutamic acid) poly(ethylene glycol) hydrogels prepared by photoinduced polymerization: Synthesis, characterization, and preliminary release studies of protein drugs.

    PubMed

    Yang, Zhiqiang; Zhang, Yuehua; Markland, Peter; Yang, Victor C

    2002-10-01

    A class of new biodegradable hydrogels based on poly(ethylene glycol) methacrylate-graft-poly(glutamic acid) and poly(ethylene glycol) dimethacrylate was synthesized by photoinduced polymerization. Because all the polymeric constituents were highly hydrophilic, crosslinking could be performed in aqueous solutions. This type of crosslinked hydrogel was prepared by modifying a select number of acidic side-groups on poly(glutamic acid) with poly(ethylene glycol) methacrylate. These modified chains were then crosslinked in the presence of poly(ethylene glycol) dimethacrylate under a photoinduced polymerization at a wavelength of 365 nm. Swelling experiments were conducted to study the crosslinking density, pH-responsive behavior, and degradation of the hydrogel. Results showed that the degree of swelling of this type of hydrogels increased as the crosslinker concentration (or density) was reduced. Because of the presence of acidic side chains on poly(glutamic acid), swelling behavior was found to be pH-responsive, increasing at high pH in response to the increase in the amount of ionized acidic side chains. The degradation rate of these hydrogels also varied with pH. More rapid degradation was observed under stronger alkaline conditions because of the hydrolysis of the ester bonds between the crosslinker and the polymer backbone. Practically useful degradation rates could be achieved for such hydrogels under physiological conditions. Drug release rates from these hydrogels were found to be proportional to the protein molecular weight and the crosslinker density; increasing at lower protein molecular weight or crosslinker density. The preliminary findings presented in this article suggest that this class of biodegradable hydrogels could be an attractive avenue for drug delivery applications. The specific photoinduced crosslinking chemistry used would permit hydrogels to be synthesized in existence of the entrapped macromolecular drugs including peptides, proteins, and

  20. Influence of casein on flux and passage of serum proteins during microfiltration using polymeric spiral-wound membranes at 50°C.

    PubMed

    Zulewska, Justyna; Barbano, David M

    2013-04-01

    Raw milk (approximately 1,800 kg) was separated at 4°C, pasteurized (at 72°C for 16s), and split into 2 batches. One batch (620 kg) was microfiltered (MF) using pilot-scale ceramic uniform transmembrane pressure Membralox membranes (model EP1940GL0.1 μA, 0.1-μm alumina; Pall Corp., East Hills, NY) to produce retentate and permeate. The permeate from the MF uniform transmembrane pressure was casein-free skim milk (CFSM). The CFSM was MF using polymeric spiral-wound (SW) membranes (model FG7838-OS0x-S, 0.3 μm; Parker-Hannifin Corp., Process Advanced Filtration Division, Tell City, IN) at a concentration factor of 3× and temperature of 50°C. Following the processing of CFSM, the second batch of skim milk (1,105 kg) was processed using the same polymeric membranes to determine how casein content in the feed material for MF with polymeric membranes affects the performance of the system. There was little resistance to passage of milk serum proteins (SP) through a 0.3-μm polyvinylidene fluoride (PVDF) SW membrane at 50°C and no detectable increase in hydraulic resistance of the membrane when processing CFSM. Therefore, milk SP contributed little, if any, to fouling of the PVDF membrane. In contrast, when processing skim milk containing a normal concentration of casein, the flux was much lower than when processing CFSM (17.2 vs. 80.2 kg/m(2) per hour, respectively) and the removal of SP from skim milk with a single-pass 3× bleed-and-feed MF system was also much lower than from CFSM (35.2 vs. 59.5% removal, respectively). Thus, when processing skim milk with a PVDF SW membrane, casein was the major protein foulant that increased hydraulic resistance and reduced passage of SP through the membrane.

  1. Preparation of a thick polymer brush layer composed of poly(2-methacryloyloxyethyl phosphorylcholine) by surface-initiated atom transfer radical polymerization and analysis of protein adsorption resistance.

    PubMed

    Inoue, Yuuki; Onodera, Yuya; Ishihara, Kazuhiko

    2016-05-01

    The purpose of this study was to prepare a thick polymer brush layer composed of poly(2-methacryloyloxyethyl phosphorylcholine (MPC)) and assess its resistance to protein adsorption from the dissolved state of poly(MPC) chains in an aqueous condition. The thick poly(MPC) brush layer was prepared through the surface-initiated atom transfer radical polymerization (SI-ATRP) of MPC with a free initiator from an initiator-immobilized substrate at given [Monomer]/[Free initiator] ratios. The ellipsometric thickness of the poly(MPC) brush layers could be controlled by the polymerization degree of the poly(MPC) chains. The thickness of the poly(MPC) brush layer in an aqueous medium was larger than that in air, and this tendency became clearer when the polymerization degree of the poly(MPC) increased. The maximum thickness of the poly(MPC) brush layer in an aqueous medium was around 110 nm. The static air contact angle of the poly(MPC) brush layer in water indicated a reasonably hydrophilic nature, which was independent of the thickness of the poly(MPC) brush layer at the surface. This result occurred because the hydrated state of the poly(MPC) chains is not influenced by the environment surrounding them. Finally, as measured with a quartz crystal microbalance, the amount of protein adsorbed from a fetal bovine serum solution (10% in phosphate-buffered saline) on the original substrate was 420 ng/cm(2). However, the poly(MPC) brush layer reduced this value dramatically to less than 50 ng/cm(2). This effect was independent of the thickness of the poly(MPC) brush layer for thicknesses between 20 nm and about 110 nm. These results indicated that the surface covered with a poly(MPC) brush layer is a promising platform to avoid biofouling and could also be applied to analyze the reactions of biological molecules with a high signal/noise ratio.

  2. Kunststoffe (Polymere)

    NASA Astrophysics Data System (ADS)

    Weißbach, Wolfgang

    Polymere bestehen aus Riesen- oder Makromolekülen, die durch chemische Reaktionen aus einfachen, niedermolekularen Verbindungen entstehen, den Monomeren. Ausgangsstoffe sind überwiegend Kohlenwasserstoffe (KW), die größte Gruppe der C-Verbindungen. Sie müssen reaktionsfähige Stellen besitzen, das sind OH-Gruppen oder Dopppelbindungen.

  3. Iron promotes protein insolubility and aging in C. elegans

    PubMed Central

    Klang, Ida M.; Schilling, Birgit; Sorensen, Dylan J.; Sahu, Alexandria K.; Kapahi, Pankaj; Andersen, Julie K.; Swoboda, Peter; Killilea, David W.; Gibson, Bradford W.; Lithgow, Gordon J.

    2014-01-01

    Many late-onset proteotoxic diseases are accompanied by a disruption in homeostasis of metals (metallostasis) including iron, copper and zinc. Although aging is the most prominent risk factor for these disorders, the impact of aging on metallostasis and its role in proteotoxic disease remain poorly understood. Moreover, it is not clear whether a loss of metallostasis influences normal aging. We have investigated the role of metallostasis in longevity of Caenorhabditis elegans. We found that calcium, copper, iron, and manganese levels increase as a function of age, while potassium and phosphorus levels tend to decrease. Increased dietary iron significantly accelerated the age-related accumulation of insoluble protein, a molecular pathology of aging. Proteomic analysis revealed widespread effects of dietary iron in multiple organelles and tissues. Pharmacological interventions to block accumulation of specific metals attenuated many models of proteotoxicity and extended normal lifespan. Collectively, these results suggest that a loss of metallostasis with aging contributes to age-related protein aggregation. PMID:25554795

  4. scyllo-Inositol promotes robust mutant Huntingtin protein degradation.

    PubMed

    Lai, Aaron Y; Lan, Cynthia P; Hasan, Salwa; Brown, Mary E; McLaurin, Joanne

    2014-02-07

    Huntington disease is characterized by neuronal aggregates and inclusions containing polyglutamine-expanded huntingtin protein and peptide fragments (polyQ-Htt). We have used an established cell-based assay employing a PC12 cell line overexpressing truncated exon 1 of Htt with a 103-residue polyQ expansion that yields polyQ-Htt aggregates to investigate the fate of polyQ-Htt-drug complexes. scyllo-Inositol is an endogenous inositol stereoisomer known to inhibit accumulation and toxicity of the amyloid-β peptide and α-synuclein. In light of these properties, we investigated the effect of scyllo-inositol on polyQ-Htt accumulation. We show that scyllo-inositol lowered the number of visible polyQ-Htt aggregates and robustly decreased polyQ-Htt protein abundance without concomitant cellular toxicity. We found that scyllo-inositol-induced polyQ-Htt reduction was by rescue of degradation pathways mediated by the lysosome and by the proteasome but not autophagosomes. The rescue of degradation pathways was not a direct result of scyllo-inositol on the lysosome or proteasome but due to scyllo-inositol-induced reduction in mutant polyQ-Htt protein levels.

  5. A secreted protein promotes cleavage furrow maturation during cytokinesis.

    PubMed

    Xu, Xuehong; Vogel, Bruce E

    2011-01-25

    Developmental modifications in cell shape depend on dynamic interactions between the extracellular matrix and cytoskeleton. In contrast, existing models of cytokinesis describe substantial cell surface remodeling that involves many intracellular regulatory and structural proteins but includes no contribution from the extracellular matrix [1-3]. Here, we show that extracellular hemicentins assemble at the cleavage furrow of dividing cells in the C. elegans germline and in preimplantation mouse embryos. In the absence of hemicentin, cleavage furrows form but retract prior to completion, resulting in multinucleate cells. In addition to their role in tissue organization, the data indicate that hemicentins are the first secreted proteins required during mammalian development and the only known secreted proteins required for cytokinesis, with an evolutionarily conserved role in stabilizing and preventing retraction of nascent cleavage furrows. Together with studies showing that extracellular polysaccharides are required for cytokinesis in diverse species [4-9], our data suggest that assembly of a cell type-specific extracellular matrix may be a general requirement for cleavage furrow maturation and contractile ring function during cytokinesis. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. A Secreted Protein Promotes Cleavage Furrow Maturation during Cytokinesis

    PubMed Central

    Xu, Xuehong; Vogel, Bruce E.

    2010-01-01

    Summary Developmental modifications in cell shape depend on dynamic interactions between the extracellular matrix and cytoskeleton. In contrast, existing models of cytokinesis describe substantial cell surface remodeling that involves many intracellular regulatory and structural proteins but includes no contribution from the extracellular matrix [1–3]. Here, we show that extracellular hemicentins assemble at the cleavage furrow of dividing cells in the C. elegans germline and in preimplantation mouse embryos. In the absence of hemicentin, cleavage furrows form but retract prior to completion, resulting in multinucleate cells. In addition to their role in tissue organization, the data indicate that hemicentins are the first secreted proteins required during mammalian development and the only known secreted proteins required for cytokinesis, with an evolutionarily conserved role in stabilizing and preventing retraction of nascent cleavage furrows. Together with studies showing that extracellular polysaccharides are required for cytokinesis in diverse species [4–9], our data suggest that assembly of a cell type-specific extracellular matrix may be a general requirement for cleavage furrow maturation and contractile ring function during cytokinesis. PMID:21215633

  7. The mitochondrial ARTS protein promotes apoptosis through targeting XIAP

    PubMed Central

    Gottfried, Yossi; Rotem, Asaf; Lotan, Rona; Steller, Hermann; Larisch, Sarit

    2004-01-01

    ARTS is an unusual septin-like mitochondrial protein that was originally shown to mediate TGF-beta-induced apoptosis. Recently, we found that ARTS is also important for cell killing by other pro-apoptotic factors, such as arabinoside, etoposide, staurosporine and Fas. In Drosophila, the IAP antagonists Reaper, Hid and Grim are essential for the induction of virtually all apoptotic cell death. We found that mutations in peanut, which encodes a Drosophila homologue of ARTS, can dominantly suppress cell killing by Reaper, Hid and Grim, indicating that peanut acts downstream or in parallel to these. In mammalian cells, ARTS is released from mitochondria upon pro-apoptotic stimuli and then binds to XIAP. Binding of ARTS to XIAP is direct, as recombinant ARTS and XIAP proteins can bind to each other in vitro. ARTS binding to XIAP is specific and related to its pro-apoptotic function, as mutant forms of ARTS (or related septins) that fail to bind XIAP failed to induce apoptosis. ARTS leads to decreased XIAP protein levels and caspase activation. Our data suggest that ARTS induces apoptosis by antagonizing IAPs. PMID:15029247

  8. Identifying solubility-promoting buffers for intrinsically disordered proteins prior to purification.

    PubMed

    Churion, Kelly A; Bondos, Sarah E

    2012-01-01

    Intrinsically disordered proteins are anticipated to be more prone to aggregation than folded, stable proteins. Chemical additives included in the buffer can help maintain proteins in a soluble, monomeric state. However, the array of chemicals that impact protein solubility is staggering, precluding iterative testing of chemical conditions during purification. Herein, we describe a filter-based aggregation assay to rapidly identify chemical additives that maintain solubility for a protein of interest. A hierarchical approach to buffer selection is provided, in which the type of chemical which best improves solubility is first determined, followed by identifying the optimal chemical and its most effective concentration. Finally, combinations of chemical additives can be assessed if necessary. Although this assay can be applied to purified protein, partially purified protein, or aggregated protein, this protocol specifically details the use of this assay for crude cell lysate. This approach allows identification of solubility-promoting buffers prior to the initial protein purification.

  9. Several different upstream promoter elements can potentiate transactivation by the BPV-1 E2 protein.

    PubMed Central

    Ham, J; Dostatni, N; Arnos, F; Yaniv, M

    1991-01-01

    The enhancer and upstream promoter regions of RNA polymerase II transcribed genes modulate the rate of transcription initiation and establish specific patterns of gene expression. Both types of region consist of clusters of DNA binding sites for nuclear proteins. To determine how efficiently the same factor can activate transcription when acting as an enhancer or promoter factor, we have studied transactivation by the BPV-1 E2 protein, a papillomavirus transcriptional regulator. By cotransfecting a BPV-1 E2 expression vector and a series of reporter plasmids containing well-defined chimeric promoters we have found that whilst E2 can strongly stimulate complex promoters such as that of the HSV tk gene, it does not efficiently activate constructions containing only a TATA box and initiation site. We show that insertion of upstream promoter elements, but not of spacer DNA, between E2 binding sites and the TATA box greatly increases E2 activation. This effect was observed with more than one type of upstream promoter element, is not related to the strength of the promoter and is unlikely to result from co-operative DNA binding by E2 and the transcription factors tested. These results would suggest that E2 has the properties of an enhancer rather than promoter factor and that in certain cases promoter and enhancer factors may affect different steps in the process of transcriptional activation. Images PMID:1655407

  10. Pseudomonas aeruginosa and tumor necrosis factor-alpha attenuate Clara cell secretory protein promoter function.

    PubMed

    Harrod, Kevin S; Jaramillo, Richard J

    2002-02-01

    The Clara cell secretory protein (CCSP, also CC-10/uterglobin) is a 16-kD homodimeric protein abundantly expressed in the airways of mammals. Although the molecular function is unknown, gene-targeting studies indicate CCSP as a regulator of lung inflammation following acute respiratory infection or injury. CCSP is decreased in the lungs of mice following acute Pseudomonas aeruginosa (P.a.) infection. In the present study, the role of decreased promoter function in the regulation of CCSP by P.a. was assessed using an in vitro co-culture system and in vivo studies of transgenic mice. CCSP promoter activity in lung epithelial cells was markedly decreased by P.a. or tumor necrosis factor-alpha (TNF-alpha) in a dose-dependent manner. Regulation of CCSP promoter function by either P.a. or TNF-alpha was localized to the proximal 166 bp flanking region of the CCSP promoter activity. Decreased regulation of the CCSP promoter by P.a. or TNF-alpha was specific to CCSP, as human surfactant protein D (SP-D) promoter activity was unaffected or increased by P.a. or TNF-alpha, respectively. A neutralizing antibody against human TNF-alpha was able to reverse both the TNF-alpha- mediated as well as P.a.-mediated decrease in CCSP promoter function in lung epithelial cells. TNF-alpha secretion by lung epithelial cells coincided with the decrease in CCSP promoter function following P.a. administration. Using a transgenic mouse model, P.a. administration to the lung markedly attenuated CCSP promoter-conferred gene expression in vivo. The attenuation of CCSP promoter activity in lung epithelial cells by P.a. involves, in part, autocrine/paracrine secretion of TNF-alpha, which in turn regulates CCSP transcription through cis-active elements in the proximal promoter region.

  11. The molecular chaperone Hsp70 promotes the proteolytic removal of oxidatively damaged proteins by the proteasome

    PubMed Central

    Reeg, Sandra; Jung, Tobias; Castro, José P.; Davies, Kelvin J.A.; Henze, Andrea; Grune, Tilman

    2016-01-01

    One hallmark of aging is the accumulation of protein aggregates, promoted by the unfolding of oxidized proteins. Unraveling the mechanism by which oxidized proteins are degraded may provide a basis to delay the early onset of features, such as protein aggregate formation, that contribute to the aging phenotype. In order to prevent aggregation of oxidized proteins, cells recur to the 20S proteasome, an efficient turnover proteolysis complex. It has previously been shown that upon oxidative stress the 26S proteasome, another form, dissociates into the 20S form. A critical player implicated in its dissociation is the Heat Shock Protein 70 (Hsp70), which promotes an increase in free 20S proteasome and, therefore, an increased capability to degrade oxidized proteins. The aim of this study was to test whether or not Hsp70 is involved in cooperating with the 20S proteasome for a selective degradation of oxidatively damaged proteins. Our results demonstrate that Hsp70 expression is induced in HT22 cells as a result of mild oxidative stress conditions. Furthermore, Hsp70 prevents the accumulation of oxidized proteins and directly promotes their degradation by the 20S proteasome. In contrast the expression of the Heat shock cognate protein 70 (Hsc70) was not changed in recovery after oxidative stress and Hsc70 has no influence on the removal of oxidatively damaged proteins. We were able to demonstrate in HT22 cells, in brain homogenates from 129/SV mice and in vitro, that there is an increased interaction of Hsp70 with oxidized proteins, but also with the 20S proteasome, indicating a role of Hsp70 in mediating the interaction of oxidized proteins with the 20S proteasome. Thus, our data clearly implicate an involvement of Hsp70 oxidatively damaged protein degradation by the 20S proteasome. PMID:27498116

  12. Platelet interaction with polymerizing fibrin.

    PubMed

    Niewiarowski, S; Regoeczi, E; Stewart, G J; Senyl, A F; Mustard, J F

    1972-03-01

    Interaction of washed pig, rabbit, or human platelets with fibrinogen was studied during its transition to fibrin using photometric, isotopic, and electron microscopic techniques. Untreated fibrinogen and fully polymerized fibrin had no detectable effect on platelets. Fibrinogen, incubated with low concentrations of reptilase or thrombin, formed intermediate products which readily became associated with platelets and caused their aggregation. Neutralization of the thrombin did not prevent this interaction. In the absence of fibrinogen, reptilase did not affect platelets. The interaction of polymerizing fibrin with platelets was accompanied by small losses of platelet constituents (serotonin, adenine nucleotides, platelet factor 4, and lactic dehydrogenase). This loss did not appear to be the result of the platelet release reaction. Inhibitors of the release reaction or of adenosine diphosphate (ADP)-induced aggregation did not prevent the interaction of platelets with polymerizing fibrin. Apyrase or prostaglandin E(1) (PGE(1)) reduced the extent of platelet aggregation by polymerizing fibrin, but the amount of protein associated with platelets was slightly increased. The interaction of polymerizing fibrin with platelets was completely inhibited by ethylenediaminetetraacetate (EDTA) or ethylene glycol bis (beta-aminoethyl ether) N, N,N',N'-tetraacetic acid (EGTA).Fibers formed in solutions of polymerizing fibrin were larger in the presence than in the absence of washed platelets, suggesting that platelets affect fibrin polymerization. The adherence of platelets to polymerizing fibrin may be responsible for the establishment of links between platelets and fibrin in hemostatic plugs and thrombi.

  13. Drosophila Golgi membrane protein Ema promotes autophagosomal growth and function.

    PubMed

    Kim, Sungsu; Naylor, Sarah A; DiAntonio, Aaron

    2012-05-01

    Autophagy is a self-degradative process in which cellular material is enclosed within autophagosomes and trafficked to lysosomes for degradation. Autophagosomal biogenesis is well described; however mechanisms controlling the growth and ultimate size of autophagosomes are unclear. Here we demonstrate that the Drosophila membrane protein Ema is required for the growth of autophagosomes. In an ema mutant, autophagosomes form in response to starvation and developmental cues, and these autophagosomes can mature into autolysosomes; however the autophagosomes are very small, and autophagy is impaired. In fat body cells, Ema localizes to the Golgi complex and is recruited to the membrane of autophagosomes in response to starvation. The Drosophila Golgi protein Lva also is recruited to the periphery of autophagosomes in response to starvation, and this recruitment requires ema. Therefore, we propose that Golgi is a membrane source for autophagosomal growth and that Ema facilitates this process. Clec16A, the human ortholog of Ema, is a candidate autoimmune susceptibility locus. Expression of Clec16A can rescue the autophagosome size defect in the ema mutant, suggesting that regulation of autophagosome morphogenesis may be a fundamental function of this gene family.

  14. Macrophage Stimulating Protein May Promote Tubular Regeneration after Acute Injury

    PubMed Central

    Cantaluppi, Vincenzo; Biancone, Luigi; Romanazzi, Giuseppe Mauriello; Figliolini, Federico; Beltramo, Silvia; Galimi, Francesco; Camboni, Maria Gavina; Deriu, Elisa; Conaldi, Piergiulio; Bottelli, Antonella; Orlandi, Viviana; Herrera, Maria Beatriz; Pacitti, Alfonso; Segoloni, Giuseppe Paolo; Camussi, Giovanni

    2008-01-01

    Macrophage-stimulating protein (MSP) exerts proliferative and antiapoptotic effects, suggesting that it may play a role in tubular regeneration after acute kidney injury. In this study, elevated plasma levels of MSP were found both in critically ill patients with acute renal failure and in recipients of renal allografts during the first week after transplantation. In addition, MSP and its receptor, RON, were markedly upregulated in the regenerative phase after glycerol-induced tubular injury in mice. In vitro, MSP stimulated tubular epithelial cell proliferation and conferred resistance to cisplatin-induced apoptosis by inhibiting caspase activation and modulating Fas, mitochondrial proteins, Akt, and extracellular signal–regulated kinase. MSP also enhanced migration, scattering, branching morphogenesis, tubulogenesis, and mesenchymal de-differentiation of surviving tubular cells. In addition, MSP induced an embryonic phenotype characterized by Pax-2 expression. In conclusion, MSP is upregulated during the regeneration of injured tubular cells, and it exerts multiple biologic effects that may aid recovery from acute kidney injury. PMID:18614774

  15. Mesoscopic model and free energy landscape for protein-DNA binding sites: analysis of cyanobacterial promoters.

    PubMed

    Tapia-Rojo, Rafael; Mazo, Juan José; Hernández, José Ángel; Peleato, María Luisa; Fillat, María F; Falo, Fernando

    2014-10-01

    The identification of protein binding sites in promoter sequences is a key problem to understand and control regulation in biochemistry and biotechnological processes. We use a computational method to analyze promoters from a given genome. Our approach is based on a physical model at the mesoscopic level of protein-DNA interaction based on the influence of DNA local conformation on the dynamics of a general particle along the chain. Following the proposed model, the joined dynamics of the protein particle and the DNA portion of interest, only characterized by its base pair sequence, is simulated. The simulation output is analyzed by generating and analyzing the Free Energy Landscape of the system. In order to prove the capacity of prediction of our computational method we have analyzed nine promoters of Anabaena PCC 7120. We are able to identify the transcription starting site of each of the promoters as the most populated macrostate in the dynamics. The developed procedure allows also to characterize promoter macrostates in terms of thermo-statistical magnitudes (free energy and entropy), with valuable biological implications. Our results agree with independent previous experimental results. Thus, our methods appear as a powerful complementary tool for identifying protein binding sites in promoter sequences.

  16. Transactivation of the parathyroid hormone promoter by specificity proteins and the nuclear factor Y complex.

    PubMed

    Alimov, Alexander P; Park-Sarge, Ok-Kyong; Sarge, Kevin D; Malluche, Hartmut H; Koszewski, Nicholas J

    2005-08-01

    We previously identified a highly conserved specificity protein 1 (Sp1) DNA element in mammalian PTH promoters that acted as an enhancer of gene transcription and bound Sp1 and Sp3 proteins present in parathyroid gland nuclear extracts. More recently, a nuclear factor (NF)-Y element (NF-Y(prox)) was also described by our group, which was located approximately 30 bp downstream from the Sp1 site in the human PTH (hPTH) promoter and by itself acted as a weak enhancer of gene transcription. We now report that Sp proteins and NF-Y can synergistically enhance transcription of a minimal hPTH promoter construct. Positioning of the Sp1 DNA element appears to be critical for this synergism because deviations of one half of a helical turn caused an approximate 60% decrease in transactivation. Finally, examination of the bovine PTH (bPTH) promoter also revealed Sp1/NF-Y synergism, in conjunction with the identification of an analogous NF-Y binding site similarly positioned downstream from the bPTH Sp1 element. In summary, synergistic transactivation of the hPTH and bPTH promoters is observed by Sp proteins and the NF-Y complex. The conservation of this transactivation in the human and bovine promoters suggests that this may be a principle means of enhancing PTH gene transcription.

  17. Correlation of MGMT promoter methylation status with gene and protein expression levels in glioblastoma

    PubMed Central

    Uno, Miyuki; Oba-Shinjo, Sueli Mieko; Camargo, Anamaria Aranha; Moura, Ricardo Pereira; de Aguiar, Paulo Henrique; Cabrera, Hector Navarro; Begnami, Marcos; Rosemberg, Sérgio; Teixeira, Manoel Jacobsen; Marie, Suely Kazue Nagahashi

    2011-01-01

    OBJECTIVES: 1) To correlate the methylation status of the O6-methylguanine-DNA-methyltransferase (MGMT) promoter to its gene and protein expression levels in glioblastoma and 2) to determine the most reliable method for using MGMT to predict the response to adjuvant therapy in patients with glioblastoma. BACKGROUND: The MGMT gene is epigenetically silenced by promoter hypermethylation in gliomas, and this modification has emerged as a relevant predictor of therapeutic response. METHODS: Fifty-one cases of glioblastoma were analyzed for MGMT promoter methylation by methylation-specific PCR and pyrosequencing, gene expression by real time polymerase chain reaction, and protein expression by immunohistochemistry. RESULTS: MGMT promoter methylation was found in 43.1% of glioblastoma by methylation-specific PCR and 38.8% by pyrosequencing. A low level of MGMT gene expression was correlated with positive MGMT promoter methylation (p = 0.001). However, no correlation was found between promoter methylation and MGMT protein expression (p = 0.297). The mean survival time of glioblastoma patients submitted to adjuvant therapy was significantly higher among patients with MGMT promoter methylation (log rank = 0.025 by methylation-specific PCR and 0.004 by pyrosequencing), and methylation was an independent predictive factor that was associated with improved prognosis by multivariate analysis. DISCUSSION AND CONCLUSION: MGMT promoter methylation status was a more reliable predictor of susceptibility to adjuvant therapy and prognosis of glioblastoma than were MGMT protein or gene expression levels. Methylation-specific polymerase chain reaction and pyrosequencing methods were both sensitive methods for determining MGMT promoter methylation status using DNA extracted from frozen tissue. PMID:22012047

  18. Cytomegalovirus immediate early proteins promote stemness properties in glioblastoma

    PubMed Central

    Soroceanu, Liliana; Matlaf, Lisa; Khan, Sabeena; Akhavan, Armin; Singer, Eric; Bezrookove, Vladimir; Decker, Stacy; Ghanny, Saleena; Hadaczek, Piotr; Bengtsson, Henrik; Ohlfest, John; Luciani-Torres, Maria-Gloria; Harkins, Lualhati; Perry, Arie; Guo, Hong; Soteropoulos, Patricia; Cobbs, Charles S

    2015-01-01

    Glioblastoma (GBM) is the most common and aggressive human brain tumor. Human cytomegalovirus (HCMV) immediate early (IE) proteins that are endogenously expressed in GBM cells are strong viral transactivators with onconcogenic properties. Here, we show how HCMV IE are preferentially expressed in glioma stem-like cells (GSC), where they co-localize with the other GBM stemness markers, CD133, Nestin, and Sox2. In patient-derived GSC that are endogenously infected with HCMV, attenuating IE expression by an RNA-i-based strategy, was sufficient to inhibit tumorsphere formation, Sox2 expression, cell cycle progression, and cell survival. Conversely, HCMV infection of HMCV-negative GSC elicited robust self-renewal and proliferation of cells that could be partially reversed by IE attenuation. In HCMV-positive GSC, IE attenuation induced a molecular program characterized by enhanced expression of mesenchymal markers and pro-inflammatory cytokines, resembling the therapeutically-resistant GBM phenotype. Mechanistically, HCMV/IE regulation of Sox2 occurred via inhibition of miRNA-145, a negative regulator of Sox2 protein expression. In a spontaneous mouse model of glioma, ectopic expression of the IE1 gene (UL123) specifically increased Sox2 and Nestin levels in the IE1-positive tumors, upregulating stemness and proliferation markers in vivo. Similarly, human GSC infected with the HCMV strain Towne but not the IE1-deficient strain CR208 showed enhanced growth as tumorspheres and intracranial tumor xenografts, compared to mock-infected human GSC. Overall, our findings offer new mechanistic insights into how HCMV/IE control stemness properties in glioblastoma cells. PMID:26239477

  19. Strong seed-specific protein expression from the Vigna radiata storage protein 8SGα promoter in transgenic Arabidopsis seeds.

    PubMed

    Chen, Mo-Xian; Zheng, Shu-Xiao; Yang, Yue-Ning; Xu, Chao; Liu, Jie-Sheng; Yang, Wei-Dong; Chye, Mee-Len; Li, Hong-Ye

    2014-03-20

    Vigna radiata (mung bean) is an important crop plant and is a major protein source in developing countries. Mung bean 8S globulins constitute nearly 90% of total seed storage protein and consist of three subunits designated as 8SGα, 8SGα' and 8SGβ. The 5'-flanking sequences of 8SGα' has been reported to confer high expression in transgenic Arabidopsis seeds. In this study, a 472-bp 5'-flanking sequence of 8SGα was identified by genome walking. Computational analysis subsequently revealed the presence of numerous putative seed-specific cis-elements within. The 8SGα promoter was then fused to the gene encoding β-glucuronidase (GUS) to create a reporter construct for Arabidopsis thaliana transformation. The spatial and temporal expression of 8SGα∷GUS, as investigated using GUS histochemical assays, showed GUS expression exclusively in transgenic Arabidopsis seeds. Quantitative GUS assays revealed that the 8SGα promoter showed 2- to 4-fold higher activity than the Cauliflower Mosaic Virus (CaMV) 35S promoter. This study has identified a seed-specific promoter of high promoter strength, which is potentially useful for directing foreign protein expression in seed bioreactors. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Promotion

    PubMed Central

    Alam, Hasan B.

    2013-01-01

    This article gives an overview of the promotion process in an academic medical center. A description of different promotional tracks, tenure and endowed chairs, and the process of submitting an application is provided. Finally, some practical advice about developing skills and attributes that can help with academic growth and promotion is dispensed. PMID:24436683

  1. Centriolar satellites assemble centrosomal microcephaly proteins to recruit CDK2 and promote centriole duplication.

    PubMed

    Kodani, Andrew; Yu, Timothy W; Johnson, Jeffrey R; Jayaraman, Divya; Johnson, Tasha L; Al-Gazali, Lihadh; Sztriha, Lāszló; Partlow, Jennifer N; Kim, Hanjun; Krup, Alexis L; Dammermann, Alexander; Krogan, Nevan J; Walsh, Christopher A; Reiter, Jeremy F

    2015-08-22

    Primary microcephaly (MCPH) associated proteins CDK5RAP2, CEP152, WDR62 and CEP63 colocalize at the centrosome. We found that they interact to promote centriole duplication and form a hierarchy in which each is required to localize another to the centrosome, with CDK5RAP2 at the apex, and CEP152, WDR62 and CEP63 at sequentially lower positions. MCPH proteins interact with distinct centriolar satellite proteins; CDK5RAP2 interacts with SPAG5 and CEP72, CEP152 with CEP131, WDR62 with MOONRAKER, and CEP63 with CEP90 and CCDC14. These satellite proteins localize their cognate MCPH interactors to centrosomes and also promote centriole duplication. Consistent with a role for satellites in microcephaly, homozygous mutations in one satellite gene, CEP90, may cause MCPH. The satellite proteins, with the exception of CCDC14, and MCPH proteins promote centriole duplication by recruiting CDK2 to the centrosome. Thus, centriolar satellites build a MCPH complex critical for human neurodevelopment that promotes CDK2 centrosomal localization and centriole duplication.

  2. Centriolar satellites assemble centrosomal microcephaly proteins to recruit CDK2 and promote centriole duplication

    PubMed Central

    Kodani, Andrew; Yu, Timothy W; Johnson, Jeffrey R; Jayaraman, Divya; Johnson, Tasha L; Al-Gazali, Lihadh; Sztriha, Lāszló; Partlow, Jennifer N; Kim, Hanjun; Krup, Alexis L; Dammermann, Alexander; Krogan, Nevan J; Walsh, Christopher A; Reiter, Jeremy F

    2015-01-01

    Primary microcephaly (MCPH) associated proteins CDK5RAP2, CEP152, WDR62 and CEP63 colocalize at the centrosome. We found that they interact to promote centriole duplication and form a hierarchy in which each is required to localize another to the centrosome, with CDK5RAP2 at the apex, and CEP152, WDR62 and CEP63 at sequentially lower positions. MCPH proteins interact with distinct centriolar satellite proteins; CDK5RAP2 interacts with SPAG5 and CEP72, CEP152 with CEP131, WDR62 with MOONRAKER, and CEP63 with CEP90 and CCDC14. These satellite proteins localize their cognate MCPH interactors to centrosomes and also promote centriole duplication. Consistent with a role for satellites in microcephaly, homozygous mutations in one satellite gene, CEP90, may cause MCPH. The satellite proteins, with the exception of CCDC14, and MCPH proteins promote centriole duplication by recruiting CDK2 to the centrosome. Thus, centriolar satellites build a MCPH complex critical for human neurodevelopment that promotes CDK2 centrosomal localization and centriole duplication. DOI: http://dx.doi.org/10.7554/eLife.07519.001 PMID:26297806

  3. Poly(oligo(ethylene glycol)acrylamide) brushes by surface initiated polymerization: effect of macromonomer chain length on brush growth and protein adsorption from blood plasma.

    PubMed

    Kizhakkedathu, Jayachandran N; Janzen, Johan; Le, Yevgeniya; Kainthan, Rajesh K; Brooks, Donald E

    2009-04-09

    Three hydrolytically stable polyethyleneglycol (PEG)-based N-substituted acrylamide macromonomers, methoxypolyethyleneglycol (350) acrylamide (MPEG350Am) methoxypolyethyleneglycol (750) acrylamide(MPEG750Am) and methoxypolyethyleneglycol (2000)acrylamide (MPEG2000Am) with increasing PEG chain length were synthesized. Surface-initiated aqueous atom transfer radical polymerization (ATRP) using CuCl/1,1,4,7,10,10-hexamethyl triethylene tetramine (HMTETA) catalyst was utilized to generate dense polymer brushes from these monomers via an ester linker group on the surface of model polystyrene (PS) particles. The molecular weight, hydrodynamic thickness, and graft densities of the grafted polymer layers were controlled by changing the reaction parameters of monomer concentration, addition of Cu(II)Cl2, and sodium chloride. The graft densities of surface-grafted brushes decreased with increasing PEG macromonomer chain length, 350 > 750 > 2000, under similar experimental conditions. The molecular weight of grafts increased with increase in monomer concentration, and only selected conditions produced narrow distributed polymer chains. The molecular weight of grafted polymer chains differs significantly to those formed in solution. The hydrodynamic thicknesses of the grafted polymer layers were fitted to the Daoud and Cotton model (DCM) for brush height on spherical surfaces. The results show that the size of the pendent groups on the polymer chains has a profound effect on the hydrodynamic thickness of the brush for a given degree of polymerization. The new PEG-based surfaces show good protection against nonspecific protein adsorption from blood plasma compared to the bare surface. Protein adsorption decreased with increasing surface density of grafted polymer chains. Poly(MPEG750Am) brushes were more effective in preventing protein adsorption than poly(MPEG350Am) even at low graft densities, presumably due to the increase in PEG content in the grafted layer.

  4. Differential expression and ligand binding indicate alternative functions for zebrafish polymeric immunoglobulin receptor (pIgR) and a family of pIgR-like (PIGRL) proteins

    PubMed Central

    Kortum, Amanda N.; Rodriguez-Nunez, Ivan; Yang, Jibing; Shim, Juyoung; Runft, Donna; O’Driscoll, Marci L; Haire, Robert N.; Cannon, John P.; Turner, Poem M.; Litman, Ronda T.; Kim, Carol H.; Neely, Melody N.; Litman, Gary W.; Yoder, Jeffrey A.

    2014-01-01

    The polymeric immunoglobulin (Ig) receptor (pIgR) is an integral transmembrane glycoprotein that plays an important role in the mammalian immune response by transporting soluble polymeric Igs across mucosal epithelial cells. Single pIgR genes, which are expressed in lymphoid organs including mucosal tissues, have been identified in several teleost species. A single pigr gene has been identified on zebrafish chromosome 2 along with a large multigene family consisting of 29 pigr-like (PIGRL) genes. Full length transcripts from 10 different PIGRL genes that encode secreted and putative inhibitory membrane bound receptors have been characterized. Although PIGRL and pigr transcripts are detected in immune tissues, only PIGRL transcripts can be detected in lymphoid and myeloid cells. In contrast to pIgR which binds Igs, certain PIGRL proteins bind phospholipids. PIGRL transcript levels are increased after infection with Streptococcus iniae, suggesting a role for PIGRL genes during bacterial challenge. Transcript levels of PIGRL genes are decreased after infection with Snakehead rhabdovirus, suggesting that viral infection may suppress PIGRL function. PMID:24469064

  5. P5-type sulfhydryl oxidoreductase promotes the sorting of proteins to protein body I in rice endosperm cells

    PubMed Central

    Onda, Yayoi; Kawagoe, Yasushi

    2013-01-01

    In rice (Oryza sativa) endosperm cells, oxidative protein folding is necessary for the sorting of storage proteins to protein bodies, PB-I and PB-II. Here we examined the role of sulfhydryl oxidoreductase PDIL2;3 (a human P5 ortholog) in the endoplasmic reticulum (ER), using GFP-AB, a PB-I marker in which the N-terminal region (AB) of α-globulin is fused to green fluorescent protein (GFP). RNAi knockdown of PDIL2;3 inhibited the accumulation of GFP-AB in PB-I and promoted its exit from the ER. We discuss the role of PDIL2;3 in retaining proteins within the ER and specifying their localization to PB-I through disulfide bond formation. PMID:23299424

  6. Compensation for differences in gene copy number among yeast ribosomal proteins is encoded within their promoters

    PubMed Central

    Zeevi, Danny; Sharon, Eilon; Lotan-Pompan, Maya; Lubling, Yaniv; Shipony, Zohar; Raveh-Sadka, Tali; Keren, Leeat; Levo, Michal; Weinberger, Adina; Segal, Eran

    2011-01-01

    Coordinate regulation of ribosomal protein (RP) genes is key for controlling cell growth. In yeast, it is unclear how this regulation achieves the required equimolar amounts of the different RP components, given that some RP genes exist in duplicate copies, while others have only one copy. Here, we tested whether the solution to this challenge is partly encoded within the DNA sequence of the RP promoters, by fusing 110 different RP promoters to a fluorescent gene reporter, allowing us to robustly detect differences in their promoter activities that are as small as ∼10%. We found that single-copy RP promoters have significantly higher activities, suggesting that proper RP stoichiometry is indeed partly encoded within the RP promoters. Notably, we also partially uncovered how this regulation is encoded by finding that RP promoters with higher activity have more nucleosome-disfavoring sequences and characteristic spatial organizations of these sequences and of binding sites for key RP regulators. Mutations in these elements result in a significant decrease of RP promoter activity. Thus, our results suggest that intrinsic (DNA-dependent) nucleosome organization may be a key mechanism by which genomes encode biologically meaningful promoter activities. Our approach can readily be applied to uncover how transcriptional programs of other promoters are encoded. PMID:22009988

  7. Use of the arabinose p(bad) promoter for tightly regulated display of proteins on bacteriophage.

    PubMed

    Huang, W; McKevitt, M; Palzkill, T

    2000-06-27

    Phage display is a widely used method to optimize the binding characteristics of protein-ligand interactions. In addition, it has been used to clone genes from genomic and cDNA libraries based on their ligand-binding characteristics. One difficulty often encountered when expressing heterologous proteins by phage display is the toxicity of the protein on the Escherichia coli host. Previous studies have shown that heterologous protein expression can be tightly controlled using plasmids with the P(BAD) promoter of the arabinose operon of E. coli, and the araC gene, which is both a positive and negative regulator of the promoter. We constructed a set of phage display vectors that utilize the P(BAD) promoter to control the expression of proteins on the surface of the M13 bacteriophage. These vectors exhibit tightly controlled expression of proteins on the surface of the phage. In addition, the amount of protein displayed on the phage is modulated by the amount of arabinose present in the growth medium during phage propagation. This may be useful for altering the stringency of binding enrichment during phage display.

  8. The human cut homeodomain protein represses transcription from the c-myc promoter.

    PubMed Central

    Dufort, D; Nepveu, A

    1994-01-01

    Studies of the c-myc promoter have shown that efficient transcription initiation at the P2 start site as well as the block to elongation of transcription require the presence of the ME1a1 protein binding site upstream of the P2 TATA box. Following fractionation by size exclusion chromatography, three protein-ME1a1 DNA complexes, a, b, and c, were detected by electrophoretic mobility shift assay. A cDNA encoding a protein present in complex c was isolated by screening of an expression library with an ME1a1 DNA probe. This cDNA was found to encode the human homolog of the Drosophila Cut homeodomain protein. The bacterially expressed human Cut (hu-Cut) protein bound to the ME1a1 site, and antibodies against hu-Cut inhibited the ME1a1 binding activity c in nuclear extracts. In cotransfection experiments, the hu-Cut protein repressed transcription from the c-myc promoter, and this repression was shown to be dependent on the presence of the ME1a1 site. Using a reporter construct with a heterologous promoter, we found that c-myc exon 1 sequences were also necessary, in addition to the ME1a1 site, for repression by Cut. Taken together, these results suggest that the human homolog of the Drosophila Cut homeodomain protein is involved in regulation of the c-myc gene. Images PMID:8196661

  9. The apoptotic protein tBid promotes leakage by altering membrane curvature.

    PubMed

    Epand, Raquel F; Martinou, Jean-Claude; Fornallaz-Mulhauser, Monique; Hughes, Donald W; Epand, Richard M

    2002-09-06

    The apoptotic protein tBid is effective in promoting both leakage and lipid mixing in liposomes composed of cardiolipin and phosphatidylcholine at a molar ratio of 1:2 in the presence of calcium. When half of the phosphatidylcholine component of these liposomes is replaced with phosphatidylethanolamine, a lipid that promotes negative membrane curvature, the rates of both leakage and lipid mixing caused by tBid are substantially increased. Replacement of cardiolipin with phosphatidylglycerol, a lipid that is structurally similar to cardiolipin but does not promote negative membrane curvature in the presence of calcium, prevents the tBid from promoting leakage. The promotion of leakage by tBid is also inhibited by several substances that promote positive membrane curvature, including lysophosphatidylcholine, tritrpticin, a potent antimicrobial peptide, and cyclosporin A, a known inhibitor of cytochrome c release from mitochondria. We directly measured the effect of tBid on membrane curvature by (31)P NMR. We found that tBid promotes the formation of highly curved non-lamellar phases. All of these data are consistent with the hypothesis that tBid promotes negative curvature, and as a result it destabilizes bilayer membranes. Bcl-X(L) inhibits leakage and lipid mixing induced by tBid. Bcl-X(L) is anti-apoptotic. It reduces the promotion of non-bilayer phases by tBid, although by itself Bcl-X(L) is capable of promoting their formation. Bcl-X(L) has little effect on liposomal integrity. Our results suggest that the anti-apoptotic activity of Bcl-X(L) is not a consequence of its interaction with membranes, but rather with other proteins, such as tBid.

  10. Seed storage protein gene promoters contain conserved DNA motifs in Brassicaceae, Fabaceae and Poaceae

    PubMed Central

    Fauteux, François; Strömvik, Martina V

    2009-01-01

    Background Accurate computational identification of cis-regulatory motifs is difficult, particularly in eukaryotic promoters, which typically contain multiple short and degenerate DNA sequences bound by several interacting factors. Enrichment in combinations of rare motifs in the promoter sequence of functionally or evolutionarily related genes among several species is an indicator of conserved transcriptional regulatory mechanisms. This provides a basis for the computational identification of cis-regulatory motifs. Results We have used a discriminative seeding DNA motif discovery algorithm for an in-depth analysis of 54 seed storage protein (SSP) gene promoters from three plant families, namely Brassicaceae (mustards), Fabaceae (legumes) and Poaceae (grasses) using backgrounds based on complete sets of promoters from a representative species in each family, namely Arabidopsis (Arabidopsis thaliana (L.) Heynh.), soybean (Glycine max (L.) Merr.) and rice (Oryza sativa L.) respectively. We have identified three conserved motifs (two RY-like and one ACGT-like) in Brassicaceae and Fabaceae SSP gene promoters that are similar to experimentally characterized seed-specific cis-regulatory elements. Fabaceae SSP gene promoter sequences are also enriched in a novel, seed-specific E2Fb-like motif. Conserved motifs identified in Poaceae SSP gene promoters include a GCN4-like motif, two prolamin-box-like motifs and an Skn-1-like motif. Evidence of the presence of a variant of the TATA-box is found in the SSP gene promoters from the three plant families. Motifs discovered in SSP gene promoters were used to score whole-genome sets of promoters from Arabidopsis, soybean and rice. The highest-scoring promoters are associated with genes coding for different subunits or precursors of seed storage proteins. Conclusion Seed storage protein gene promoter motifs are conserved in diverse species, and different plant families are characterized by a distinct combination of conserved motifs

  11. Surface Density of the Hendra G Protein Modulates Hendra F Protein-Promoted Membrane Fusion: Role for Hendra G Protein Trafficking and Degradation

    PubMed Central

    Whitman, Shannon D.; Dutch, Rebecca Ellis

    2007-01-01

    Hendra virus, like most paramyxoviruses, requires both a fusion (F) and attachment (G) protein for promotion of cell-cell fusion. Recent studies determined that Hendra F is proteolytically processed by the cellular protease cathepsin L after endocytosis. This unique cathepsin L processing results in a small percentage of Hendra F on the cell surface. To determine how the surface densities of the two Hendra glycoproteins affect fusion promotion, we performed experiments that varied the levels of glycoproteins expressed in transfected cells. Using two different fusion assays, we found a marked increase in fusion when expression of the Hendra G protein was increased, with a 1:1 molar ratio of Hendra F:G on the cell surface resulting in optimal membrane fusion. Our results also showed that Hendra G protein levels are modulated by both more rapid protein turnover and slower protein trafficking than is seen for Hendra F. PMID:17328935

  12. Surface density of the Hendra G protein modulates Hendra F protein-promoted membrane fusion: Role for Hendra G protein trafficking and degradation

    SciTech Connect

    Whitman, Shannon D.; Dutch, Rebecca Ellis . E-mail: rdutc2@uky.edu

    2007-07-05

    Hendra virus, like most paramyxoviruses, requires both a fusion (F) and attachment (G) protein for promotion of cell-cell fusion. Recent studies determined that Hendra F is proteolytically processed by the cellular protease cathepsin L after endocytosis. This unique cathepsin L processing results in a small percentage of Hendra F on the cell surface. To determine how the surface densities of the two Hendra glycoproteins affect fusion promotion, we performed experiments that varied the levels of glycoproteins expressed in transfected cells. Using two different fusion assays, we found a marked increase in fusion when expression of the Hendra G protein was increased, with a 1:1 molar ratio of Hendra F:G on the cell surface resulting in optimal membrane fusion. Our results also showed that Hendra G protein levels are modulated by both more rapid protein turnover and slower protein trafficking than is seen for Hendra F.

  13. Water promotes the sealing of nanoscale packing defects in folding proteins.

    PubMed

    Fernández, Ariel

    2014-05-21

    A net dipole moment is shown to arise from a non-Debye component of water polarization created by nanoscale packing defects on the protein surface. Accordingly, the protein electrostatic field exerts a torque on the induced dipole, locally impeding the nucleation of ice at the protein-water interface. We evaluate the solvent orientation steering (SOS) as the reversible work needed to align the induced dipoles with the Debye electrostatic field and computed the SOS for the variable interface of a folding protein. The minimization of the SOS is shown to drive protein folding as evidenced by the entrainment of the total free energy by the SOS energy along trajectories that approach a Debye limit state where no torque arises. This result suggests that the minimization of anomalous water polarization at the interface promotes the sealing of packing defects, thereby maintaining structural integrity and committing the protein chain to fold.

  14. Chain Reaction Polymerization.

    ERIC Educational Resources Information Center

    McGrath, James E.

    1981-01-01

    The salient features and importance of chain-reaction polymerization are discussed, including such topics as the thermodynamics of polymerization, free-radical polymerization kinetics, radical polymerization processes, copolymers, and free-radical chain, anionic, cationic, coordination, and ring-opening polymerizations. (JN)

  15. Chain Reaction Polymerization.

    ERIC Educational Resources Information Center

    McGrath, James E.

    1981-01-01

    The salient features and importance of chain-reaction polymerization are discussed, including such topics as the thermodynamics of polymerization, free-radical polymerization kinetics, radical polymerization processes, copolymers, and free-radical chain, anionic, cationic, coordination, and ring-opening polymerizations. (JN)

  16. Zinc-Induced Polymerization of Killer-Cell Ig-like Receptor into Filaments Promotes Its Inhibitory Function at Cytotoxic Immunological Synapses.

    PubMed

    Kumar, Santosh; Rajagopalan, Sumati; Sarkar, Pabak; Dorward, David W; Peterson, Mary E; Liao, Hsien-Shun; Guillermier, Christelle; Steinhauser, Matthew L; Vogel, Steven S; Long, Eric O

    2016-04-07

    The inhibitory function of killer cell immunoglobulin-like receptors (KIR) that bind HLA-C and block activation of human natural killer (NK) cells is dependent on zinc. We report that zinc induced the assembly of soluble KIR into filamentous polymers, as detected by electron microscopy, which depolymerized after zinc chelation. Similar KIR filaments were isolated from lysates of cells treated with zinc, and membrane protrusions enriched in zinc were detected on whole cells by scanning electron microscopy and imaging mass spectrometry. Two independent mutations in the extracellular domain of KIR, away from the HLA-C binding site, impaired zinc-driven polymerization and inhibitory function. KIR filaments formed spontaneously, without the addition of zinc, at functional inhibitory immunological synapses of NK cells with HLA-C(+) cells. Adding to the recent paradigm of signal transduction through higher order molecular assemblies, zinc-induced polymerization of inhibitory KIR represents an unusual mode of signaling by a receptor at the cell surface.

  17. Promoter methylation of yes-associated protein (YAP1) gene in polycystic ovary syndrome.

    PubMed

    Jiang, Li-Le; Xie, Juan-Ke; Cui, Jin-Quan; Wei, Duo; Yin, Bao-Li; Zhang, Ya-Nan; Chen, Yuan-Hui; Han, Xiao; Wang, Qian; Zhang, Cui-Lian

    2017-01-01

    DNA methylation modification has been proved to influence the phenotype of polycystic ovary syndrome (PCOS). Genome-wide association studies (GWAS) demonstrate that yes-associated protein (YAP1) genetic sites are associated with PCOS. The study aims to detect the methylation status of YAP1 promoter in ovary granulosa cells (GCs) of PCOS patients and explore novel therapeutic targets for PCOS. Randomized controlled trial was applied and a total of 72 women were included in the study, including 36 cases of PCOS patients and 36 cases of health controls. Ovary GCs were extracted from in vitro fertilization embryo transfer. Methylation status of YAP1 promoter was detected by bisulfite sequencing PCR (BSP). Protein and mRNA expression of YAP1 were measured by western blotting and real-time quantitate PCR. Overall methylation level of YAP1 promoter region from PCOS group was significantly lower than that from control group. CpG sites analysis revealed that 12 sites (-443, -431, -403, -371, -331, -120, -49, -5, +1, +9, +15, +22) were significantly hypomethylated in women with PCOS (P < 0.05). A significant upregulation of YAP1 mRNA and protein expression levels was observed. Testosterone concentration could alleviate the methylation status and demonstrate obvious dose-dependent relation. Our research achievements manifest that hypomethylation of YAP1 promoter promotes the YAP1 expression, which plays a key role in the pathogenesis and accelerate PCOS.

  18. Promoter methylation of yes-associated protein (YAP1) gene in polycystic ovary syndrome

    PubMed Central

    Jiang, Li-Le; Xie, Juan-Ke; Cui, Jin-Quan; Wei, Duo; Yin, Bao-Li; Zhang, Ya-Nan; Chen, Yuan-Hui; Han, Xiao; Wang, Qian; Zhang, Cui-Lian

    2017-01-01

    Abstract Background: DNA methylation modification has been proved to influence the phenotype of polycystic ovary syndrome (PCOS). Genome-wide association studies (GWAS) demonstrate that yes-associated protein (YAP1) genetic sites are associated with PCOS. The study aims to detect the methylation status of YAP1 promoter in ovary granulosa cells (GCs) of PCOS patients and explore novel therapeutic targets for PCOS. Methods: Randomized controlled trial was applied and a total of 72 women were included in the study, including 36 cases of PCOS patients and 36 cases of health controls. Ovary GCs were extracted from in vitro fertilization embryo transfer. Methylation status of YAP1 promoter was detected by bisulfite sequencing PCR (BSP). Protein and mRNA expression of YAP1 were measured by western blotting and real-time quantitate PCR. Results: Overall methylation level of YAP1 promoter region from PCOS group was significantly lower than that from control group. CpG sites analysis revealed that 12 sites (−443, −431, −403, −371, −331, −120, −49, −5, +1, +9, +15, +22) were significantly hypomethylated in women with PCOS (P < 0.05). A significant upregulation of YAP1 mRNA and protein expression levels was observed. Testosterone concentration could alleviate the methylation status and demonstrate obvious dose–dependent relation. Conclusion: Our research achievements manifest that hypomethylation of YAP1 promoter promotes the YAP1 expression, which plays a key role in the pathogenesis and accelerate PCOS. PMID:28079802

  19. Biokompatible Polymere

    NASA Astrophysics Data System (ADS)

    Ha, Suk-Woo; Wintermantel, Erich; Maier, Gerhard

    Der klinische Einsatz von synthetischen Polymeren begann in den 60-er Jahren in Form von Einwegartikeln, wie beispielsweise Spritzen und Kathetern, vor allem aufgrund der Tatsache, dass Infektionen infolge nicht ausreichender Sterilität der wiederverwendbaren Artikel aus Glas und metallischen Werkstoffen durch den Einsatz von sterilen Einwegartikeln signifikant reduziert werden konnten [1]. Die Einführung der medizinischen Einwegartikel aus Polymeren erfolgte somit nicht nur aus ökonomischen, sondern auch aus hygienischen Gründen. Wegen der steigenden Anzahl synthetischer Polymere und dem zunehmenden Bedarf an ärztlicher Versorgung reicht die Anwendung von Polymeren in der Medizin von preisgünstigen Einwegartikeln, die nur kurzzeitig intrakorporal eingesetzt werden, bis hin zu Implantaten, welche über eine längere Zeit grossen Beanspruchungen im menschlichen Körper ausgesetzt sind. Die steigende Verbreitung von klinisch eingesetzten Polymeren ist auf ihre einfache und preisgünstige Verarbeitbarkeit in eine Vielzahl von Formen und Geometrien sowie auf ihr breites Eigenschaftsspektrum zurückzuführen. Polymere werden daher in fast allen medizinischen Bereichen eingesetzt.

  20. Polymerized supramolecular assemblies and biocompatibility

    NASA Astrophysics Data System (ADS)

    O'Brien, David F.

    2001-03-01

    The creation of durable, biomembrane-mimetic coatings for inorganic and polymeric surfaces that are biocompatible, i.e. resistant to nonspecific protein adsorption, remains an important goal that is expected to impact numerous fields. It has already been shown that the physical stability of lipid bilayer vesicles can be dramatically enhanced by cross-linking polymerization of reactive lipids, such as phosphatidylcholines. Bilayers of these same lipids on clean silicon dioxide surfaces can be formed by fusion of small bilayer vesicles with the surface. Radical initiated polymerization of these supported bilayers yields a stable poly(lipid) film that is not perturbed upon exposure to surfactant. Moreover, the cross-linked bilayer film can be removed from water into air with retention of the poly(lipid) bilayer structure. These polymerized bilayer films could be repeatedly transferred from water to air to water with no obvious change in their biocompatibility. The supported bilayer films were equally resistant to non-specific protein adsorption before and after polymerization. This indicates that biocompatible nature of the phosphorylcholine head group of the lipids was not compromised by polymerization of the lipids. The ability to maintain surface biocompatibility of membranes while substantially increasing their stability would appear to extend the technological uses of supramolecular assemblies of lipids.

  1. NSs protein of rift valley fever virus promotes posttranslational downregulation of the TFIIH subunit p62.

    PubMed

    Kalveram, Birte; Lihoradova, Olga; Ikegami, Tetsuro

    2011-07-01

    Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) is an important emerging pathogen of humans and ruminants. Its NSs protein has previously been identified as a major virulence factor that suppresses host defense through three distinct mechanisms: it directly inhibits beta interferon (IFN-β) promoter activity, it promotes the degradation of double-stranded RNA-dependent protein kinase (PKR), and it suppresses host transcription by disrupting the assembly of the basal transcription factor TFIIH through sequestration of its p44 subunit. Here, we report that in addition to PKR, NSs also promotes the degradation of the TFIIH subunit p62. Infection of cells with the RVFV MP-12 vaccine strain reduced p62 protein levels to below the detection limit early in the course of infection. This NSs-mediated downregulation of p62 was posttranslational, as it was unaffected by pharmacological inhibition of transcription or translation and MP-12 infection had no effect on p62 mRNA levels. Treatment of cells with proteasome inhibitors but not inhibition of lysosomal acidification or nuclear export resulted in a stabilization of p62 in the presence of NSs. Furthermore, p62 could be coprecipitated with NSs from lysates of infected cells. These data suggest that the RVFV NSs protein is able to interact with the TFIIH subunit p62 inside infected cells and promotes its degradation, which can occur directly in the nucleus.

  2. Discovery of a super-strong promoter enables efficient production of heterologous proteins in cyanobacteria.

    PubMed

    Zhou, Jie; Zhang, Haifeng; Meng, Hengkai; Zhu, Yan; Bao, Guanhui; Zhang, Yanping; Li, Yin; Ma, Yanhe

    2014-03-28

    Cyanobacteria are oxygenic photosynthetic prokaryotes that play important roles in the global carbon cycle. Recently, engineered cyanobacteria capable of producing various small molecules from CO2 have been developed. However, cyanobacteria are seldom considered as factories for producing proteins, mainly because of the lack of efficient strong promoters. Here, we report the discovery and verification of a super-strong promoter P(cpc560), which contains two predicted promoters and 14 predicted transcription factor binding sites (TFBSs). Using P(cpc560), functional proteins were produced at a level of up to 15% of total soluble protein in the cyanobacterium Synechocystis sp. 6803, a level comparable to that produced in Escherichia coli. We demonstrated that the presence of multiple TFBSs in P(cpc560) is crucial for its promoter strength. Genetically transformable cyanobacteria neither have endotoxins nor form inclusion bodies; therefore, P(cpc560) opens the possibility to use cyanobacteria as alternative hosts for producing heterogeneous proteins from CO2 and inorganic nutrients.

  3. Microfilament regulatory protein MENA increases activity of RhoA and promotes metastasis of hepatocellular carcinoma.

    PubMed

    Lin, Ling; Yang, Xiao-Mei; Li, Jun; Zhang, Yan-Li; Qin, Wenxin; Zhang, Zhi-Gang

    2014-09-10

    Mammalian enabled (MENA), usually known as a direct regulator of microfilament polymerization and bundling, promotes metastasis in various cancers. Here we focus on the role of MENA in hepatocellular carcinoma (HCC) metastasis and the relevant mechanism from the view of RhoA activity regulation. By HCC tissue microarray analysis, we found that MENA expression was positively associated with satellite lesions (P<0.01) and vascular invasion (P<0.01). Cases with membrane reinforcement of MENA staining in HCC tissues had significantly higher rates of early recurrence in the intermediate MENA expression group. Knockdown of MENA significantly suppressed HCC cell migration and invasion in vitro, as well as their intrahepatic and distant metastasis in vivo. Knockdown of MENA also decreased filopodia and stress fibers in SMMC-7721 cells. Furthermore, a decrease of RhoA activity was detected by a pull-down assay in SMMC-7721-shMENA cells. The ROCK inhibitor, Y-27632, suppressed migration of both MENA knockdown SMMC-7721 cells and control cells, but diminished their difference. Thus, our findings suggest that MENA promotes HCC cell motility by activating RhoA.

  4. An OmpA-Like Protein from Acinetobacter spp. Stimulates Gastrin and Interleukin-8 Promoters

    PubMed Central

    Ofori-Darko, Ernest; Zavros, Yana; Rieder, Gabriele; Tarlé, Susan A.; Van Antwerp, Mary; Merchant, Juanita L.

    2000-01-01

    Bacterial overgrowth in the stomach may occur under conditions of diminished or absent acid secretion. Under these conditions, secretion of the hormone gastrin is elevated. Alternatively, bacterial factors may directly stimulate gastrin. Consistent with this hypothesis, we found that mice colonized for 2 months with a mixed bacterial culture of opportunistic pathogens showed an increase in serum gastrin. To examine regulation of gene expression by bacterial proteins, stable transformants of AGS cells expressing gastrin or interleukin-8 (IL-8) promoters were cocultured with live organisms. Both whole-cell sonicates and a heat-stable fraction were also coincubated with the cells. A level of 108 organisms per ml stimulated both the gastrin and IL-8 promoters. Heat-stable proteins prepared from these bacterial sonicates stimulated the promoter significantly more than the live organism or unheated sonicates. A 38-kDa heat-stable protein stimulating the gastrin and IL-8 promoters was cloned and found to be an OmpA-related protein. Immunoblotting using antibody to the OmpA-like protein identified an Acinetobacter sp. as the bacterial species that expressed this protein and colonized the mouse stomach. Moreover, reintubation of mice with a pure culture of the Acinetobacter sp. caused gastritis. We conclude that bacterial colonization of the stomach may increase serum gastrin levels in part through the ability of the bacteria to produce OmpA-like proteins that directly stimulate gastrin and IL-8 gene expression. These results implicate OmpA-secreting bacteria in the activation of gastrin gene expression and raise the possibility that a variety of organisms may contribute to the increase in serum gastrin and subsequent epithelial cell proliferation in the hypochlorhydric stomach. PMID:10816525

  5. Enhanced green fluorescent protein expression in Pleurotus ostreatus for in vivo analysis of fungal laccase promoters.

    PubMed

    Amore, Antonella; Honda, Yoichi; Faraco, Vincenza

    2012-10-01

    The laccase family of Pleurotus ostreatus has been widely characterized, and studies of the genes coding for laccase isoenzymes in P. ostreatus have so far led to the identification of four different genes and the corresponding cDNAs, poxc, pox1, poxa1b and poxa3. Analyses of P. ostreatus laccase promoters poxc, pox1, poxa1b and poxa3 have allowed identification of several putative response elements, and sequences of metal-responsive elements involved in the formation of complexes with fungal proteins have been identified in poxc and poxa1b promoters. In this work, development of a system for in vivo analysis of P. ostreatus laccase promoter poxc by enhanced green fluorescent protein expression is performed, based on a poly ethylene glycol-mediated procedure for fungal transformation. A quantitative measurement of fluorescence expressed in P. ostreatus transformants is hereby reported for the first time for this fungus.

  6. G-Protein Signaling Protein-17 (RGS17) is Upregulated and Promotes Tumor Growth and Migration in Human Colorectal Carcinoma.

    PubMed

    Li, Ling; Luo, He-Sheng

    2017-03-23

    Colorectal carcinoma is one of the leading causes of cancer-related deaths and has a high tendency for metastasis, which makes it a priority to find novel methods to diagnose and treat colorectal carcinoma in the very early stage. Herein, we studied the role of regulators of G-protein signaling (RGS) family protein RGS17 in colorectal carcinoma growth and metastasis. We found that RGS17 was upregulated in both clinical colorectal carcinoma tissues and cultured colorectal carcinoma cells. Knockdown of RGS17 by specific siRNA decreased, whereas overexpression of RGS17 with expression plasmid increased cell proliferation rate in cultured cells. Consistently, a mouse model of colorectal carcinoma also showed that depletion of RGS17 significantly inhibited tumor growth in vivo. Moreover, transwell assay showed that RGS17 promoted colorectal carcinoma cell migration and invasion abilities. These data suggest that RGS17 is overexpressed in colorectal carcinoma and promotes cell proliferation, migration and invasion.

  7. Identification of a novel DNA-binding protein to osmotin promoter.

    PubMed

    Xu, P; Ling, J; Li, D; Hasegawa, P M; Bressan, R A

    1998-12-01

    One novel osmotin promoter, binding-protein (OPBP1) gene, was isolated from salt-adapted tobacco suspension cells using yeast one-hybrid system. The OPBP1 interacted specifically in vivo with FA, a DNA sequence from the 5 upstream region of osmotin gene, which was essential for osmotin responsiveness. The deduced amino acid sequence of OPBP1 contained a conserved motif of a new gene family, AP2 family. This protein did not contain the typical motif found in the most known DNA-binding proteins and transcription factors.

  8. Hepatitis B viral core protein disrupts human host gene expression by binding to promoter regions

    PubMed Central

    2012-01-01

    Background The core protein (HBc) of hepatitis B virus (HBV) has been implicated in the malignant transformation of chronically-infected hepatocytes and displays pleiotropic functions, including RNA- and DNA-binding activities. However, the mechanism by which HBc interacts with the human genome to exert effects on hepatocyte function remains unknown. This study investigated the distribution of HBc binding to promoters in the human genome and evaluated its effects on the related genes’ expression. Results Whole-genome chromatin immunoprecipitation microarray (ChIP-on-chip) analysis was used to identify HBc-bound human gene promoters. Gene Ontology and pathway analyses were performed on related genes. The quantitative polymerase chain reaction assay was used to verify ChIP-on-chip results. Five novel genes were selected for luciferase reporter assay evaluation to assess the influence of HBc promoter binding. The HBc antibody immunoprecipitated approximately 3100 human gene promoters. Among these, 1993 are associated with known biological processes, and 2208 regulate genes with defined molecular functions. In total, 1286 of the related genes mediate primary metabolic processes, and 1398 encode proteins with binding activity. Sixty-four of the promoters regulate genes related to the mitogen-activated protein kinase (MAPK) pathways, and 41 regulate Wnt/beta-catenin pathway genes. The reporter gene assay indicated that HBc binding up-regulates proto-oncogene tyrosine-protein kinase (SRC), type 1 insulin-like growth factor receptor (IGF1R), and neurotrophic tyrosine kinase receptor 2 (NTRK2), and down-regulates v-Ha-ras Harvey rat sarcoma viral oncogene (HRAS). Conclusion HBc has the ability to bind a large number of human gene promoters, and can disrupt normal host gene expression. Manipulation of the transcriptional profile in HBV-infected hepatocytes may represent a key pathogenic mechanism of HBV infection. PMID:23088787

  9. Stimulation of basal transcription from the mouse mammary tumor virus promoter by Oct proteins.

    PubMed Central

    Kim, M H; Peterson, D O

    1995-01-01

    The steroid hormone-inducible promoter of mouse mammary tumor virus (MMTV) contains three overlapping sequences related to the consensus octamer motif ATGCAAAT. Basal promoter activity in the absence of hormone induction from a template in which all three octamer elements were mutated was decreased by two-to threefold in in vitro transcription assays. Oct-1 protein purified from HeLa cell nuclear extracts, as well as recombinant Oct-1 expressed in bacteria, recognized MMTV octamer-related sequences, as shown by DNase I footprinting. Furthermore, rabbit polyclonal antiserum directed against recombinant Oct-1 completely inhibited the formation of specific complexes between MMTV octamer-related sequences and proteins present in nuclear extracts of HeLa cells, indicating that Oct-1 is the major protein in HeLa nuclear extracts that recognizes octamer-related sequences in the MMTV promoter. In addition, depletion of Oct-1 from the nuclear extract by using Oct-1-specific antiserum or a sequence-specific DNA affinity resin decreased in vitro transcription from the wild-type MMTV promoter to a level identical to that obtained from a promoter in which all three octamer-related sequences were mutated. Addition of purified HeLa Oct-1 or recombinant Oct-1 to the depleted extract selectively increased transcription from the wild-type relative to the mutated promoter, demonstrating that Oct-1 transcription factor stimulates basal transcription from the MMTV promoter. A similar effect was observed when purified recombinant Oct-2 was added to the Oct-1-depleted extract, suggesting that Oct-2 may play an important role in MMTV transcription in B cells. PMID:7609037

  10. The effect of polymer surface modification on polymer-protein interaction via interfacial polymerization and hydrophilic polymer grafting

    USDA-ARS?s Scientific Manuscript database

    Protein membrane separation is prone to fouling on the membrane surface resulting from protein adsorption onto the surface. Surface modification of synthetic membranes is one way to reduce fouling. We investigated surface modification of polyethersulfone (PES) as a way of improving hydrophilicity ...

  11. Isolation and characterization of oil palm constitutive promoter derived from ubiquitin extension protein (uep1) gene.

    PubMed

    Masura, Subhi Siti; Parveez, Ghulam Kadir Ahmad; Ismail, Ismanizan

    2010-09-30

    The ubiquitin extension protein (uep1) gene was identified as a constitutively expressed gene in oil palm. We have isolated and characterized the 5' region of the oil palm uep1 gene, which contains an 828 bp sequence upstream of the uep1 translational start site. Construction of a pUEP1 transformation vector, which contains gusA reporter gene under the control of uep1 promoter, was carried out for functional analysis of the promoter through transient expression studies. It was found that the 5' region of uep1 functions as a constitutive promoter in oil palm and could drive GUS expression in all tissues tested, including embryogenic calli, embryoid, immature embryo, young leaflet from mature palm, green leaf, mesocarp and meristematic tissues (shoot tip). This promoter could also be used in dicot systems as it was demonstrated to be capable of driving gusA gene expression in tobacco.

  12. Uniform accumulation of recombinant miraculin protein in transgenic tomato fruit using a fruit-ripening-specific E8 promoter.

    PubMed

    Hirai, Tadayoshi; Kim, You-Wang; Kato, Kazuhisa; Hiwasa-Tanase, Kyoko; Ezura, Hiroshi

    2011-12-01

    The E8 promoter, a tomato fruit-ripening-specific promoter, and the CaMV 35S promoter, a constitutive promoter, were used to express the miraculin gene encoding the taste-modifying protein in tomato. The accumulation of miraculin protein and mRNA was compared among transgenic tomatoes expressing the miraculin gene driven by these promoters. Recombinant miraculin protein predominantly accumulated in transgenic tomato lines using the E8 promoter (E8-MIR) only at the red fruit stage. The accumulations were almost uniform among all fruit tissues. When the 35S promoter (35S-MIR) was used, miraculin accumulation in the exocarp was much higher than in other tissues, indicating that the miraculin accumulation pattern can be regulated by using different types of promoters. We also discuss the potential of the E8-MIR lines for practical use.

  13. Pineapple translation factor SUI1 and ribosomal protein L36 promoters drive constitutive transgene expression patterns in Arabidopsis thaliana.

    PubMed

    Koia, Jonni; Moyle, Richard; Hendry, Caroline; Lim, Lionel; Botella, José Ramón

    2013-03-01

    The availability of a variety of promoter sequences is necessary for the genetic engineering of plants, in basic research studies and for the development of transgenic crops. In this study, the promoter and 5' untranslated regions of the evolutionally conserved protein translation factor SUI1 gene and ribosomal protein L36 gene were isolated from pineapple and sequenced. Each promoter was translationally fused to the GUS reporter gene and transformed into the heterologous plant system Arabidopsis thaliana. Both the pineapple SUI1 and L36 promoters drove GUS expression in all tissues of Arabidopsis at levels comparable to the CaMV35S promoter. Transient assays determined that the pineapple SUI1 promoter also drove GUS expression in a variety of climacteric and non-climacteric fruit species. Thus the pineapple SUI1 and L36 promoters demonstrate the potential for using translation factor and ribosomal protein genes as a source of promoter sequences that can drive constitutive transgene expression patterns.

  14. TIM-family proteins promote infection of multiple enveloped viruses through virion-associated phosphatidylserine.

    PubMed

    Jemielity, Stephanie; Wang, Jinyize J; Chan, Ying Kai; Ahmed, Asim A; Li, Wenhui; Monahan, Sheena; Bu, Xia; Farzan, Michael; Freeman, Gordon J; Umetsu, Dale T; Dekruyff, Rosemarie H; Choe, Hyeryun

    2013-03-01

    Human T-cell Immunoglobulin and Mucin-domain containing proteins (TIM1, 3, and 4) specifically bind phosphatidylserine (PS). TIM1 has been proposed to serve as a cellular receptor for hepatitis A virus and Ebola virus and as an entry factor for dengue virus. Here we show that TIM1 promotes infection of retroviruses and virus-like particles (VLPs) pseudotyped with a range of viral entry proteins, in particular those from the filovirus, flavivirus, New World arenavirus and alphavirus families. TIM1 also robustly enhanced the infection of replication-competent viruses from the same families, including dengue, Tacaribe, Sindbis and Ross River viruses. All interactions between TIM1 and pseudoviruses or VLPs were PS-mediated, as demonstrated with liposome blocking and TIM1 mutagenesis experiments. In addition, other PS-binding proteins, such as Axl and TIM4, promoted infection similarly to TIM1. Finally, the blocking of PS receptors on macrophages inhibited the entry of Ebola VLPs, suggesting that PS receptors can contribute to infection in physiologically relevant cells. Notably, infection mediated by the entry proteins of Lassa fever virus, influenza A virus and SARS coronavirus was largely unaffected by TIM1 expression. Taken together our data show that TIM1 and related PS-binding proteins promote infection of diverse families of enveloped viruses, and may therefore be useful targets for broad-spectrum antiviral therapies.

  15. Neural regeneration protein is a novel chemoattractive and neuronal survival-promoting factor

    SciTech Connect

    Gorba, Thorsten; Bradoo, Privahini; Antonic, Ana; Marvin, Keith; Liu, Dong-Xu; Lobie, Peter E.; Reymann, Klaus G.; Gluckman, Peter D.; Sieg, Frank . E-mail: fsieg@neurenpharma.com

    2006-10-01

    Neurogenesis and neuronal migration are the prerequisites for the development of the central nervous system. We have identified a novel rodent gene encoding for a neural regeneration protein (NRP) with an activity spectrum similar to the chemokine stromal-derived factor (SDF)-1, but with much greater potency. The Nrp gene is encoded as a forward frameshift to the hypothetical alkylated DNA repair protein AlkB. The predicted protein sequence of NRP contains domains with homology to survival-promoting peptide (SPP) and the trefoil protein TFF-1. The Nrp gene is first expressed in neural stem cells and expression continues in glial lineages. Recombinant NRP and NRP-derived peptides possess biological activities including induction of neural migration and proliferation, promotion of neuronal survival, enhancement of neurite outgrowth and promotion of neuronal differentiation from neural stem cells. NRP exerts its effect on neuronal survival by phosphorylation of the ERK1/2 and Akt kinases, whereas NRP stimulation of neural migration depends solely on p44/42 MAP kinase activity. Taken together, the expression profile of Nrp, the existence in its predicted protein structure of domains with similarities to known neuroprotective and migration-inducing factors and the high potency of NRP-derived synthetic peptides acting in femtomolar concentrations suggest it to be a novel gene of relevance in cellular and developmental neurobiology.

  16. Tobacco arabinogalactan protein NtEPc can promote banana (Musa AAA) somatic embryogenesis.

    PubMed

    Shu, H; Xu, L; Li, Z; Li, J; Jin, Z; Chang, S

    2014-12-01

    Banana is an important tropical fruit worldwide. Parthenocarpy and female sterility made it impossible to improve banana varieties through common hybridization. Genetic transformation for banana improvement is imperative. But the low rate that banana embryogenic callus was induced made the transformation cannot be performed in many laboratories. Finding ways to promote banana somatic embryogenesis is critical for banana genetic transformation. After tobacco arabinogalactan protein gene NtEPc was transformed into Escherichia coli (DE3), the recombinant protein was purified and filter-sterilized. A series of the sterilized protein was added into tissue culture medium. It was found that the number of banana immature male flowers developing embryogenic calli increased significantly in the presence of NtEPc protein compared with the effect of the control medium. Among the treatments, explants cultured on medium containing 10 mg/l of NtEPc protein had the highest chance to develop embryogenic calli. The percentage of lines that developed embryogenic calli on this medium was about 12.5 %. These demonstrated that NtEPc protein can be used to promote banana embryogenesis. This is the first paper that reported that foreign arabinogalactan protein (AGP) could be used to improve banana somatic embryogenesis.

  17. Activation of Hsp70 reduces neurotoxicity by promoting polyglutamine protein degradation.

    PubMed

    Wang, Adrienne M; Miyata, Yoshinari; Klinedinst, Susan; Peng, Hwei-Ming; Chua, Jason P; Komiyama, Tomoko; Li, Xiaokai; Morishima, Yoshihiro; Merry, Diane E; Pratt, William B; Osawa, Yoichi; Collins, Catherine A; Gestwicki, Jason E; Lieberman, Andrew P

    2013-02-01

    We sought new strategies to reduce amounts of the polyglutamine androgen receptor (polyQ AR) and achieve benefits in models of spinobulbar muscular atrophy, a protein aggregation neurodegenerative disorder. Proteostasis of the polyQ AR is controlled by the heat shock protein 90 (Hsp90)- and Hsp70-based chaperone machinery, but mechanisms regulating the protein's turnover are incompletely understood. We demonstrate that overexpression of Hsp70 interacting protein (Hip), a co-chaperone that enhances binding of Hsp70 to its substrates, promotes client protein ubiquitination and polyQ AR clearance. Furthermore, we identify a small molecule that acts similarly to Hip by allosterically promoting Hsp70 binding to unfolded substrates. Like Hip, this synthetic co-chaperone enhances client protein ubiquitination and polyQ AR degradation. Both genetic and pharmacologic approaches targeting Hsp70 alleviate toxicity in a Drosophila model of spinobulbar muscular atrophy. These findings highlight the therapeutic potential of allosteric regulators of Hsp70 and provide new insights into the role of the chaperone machinery in protein quality control.

  18. Melatonin enhances thermotolerance by promoting cellular protein protection in tomato plants.

    PubMed

    Xu, Wen; Cai, Shu-Yu; Zhang, Yun; Wang, Yu; Ahammed, Golam Jalal; Xia, Xiao-Jian; Shi, Kai; Zhou, Yan-Hong; Yu, Jing-Quan; Reiter, Russel J; Zhou, Jie

    2016-11-01

    Melatonin is a pleiotropic signaling molecule that provides physiological protection against diverse environmental stresses in plants. Nonetheless, the mechanisms for melatonin-mediated thermotolerance remain largely unknown. Here, we report that endogenous melatonin levels increased with a rise in ambient temperature and that peaked at 40°C. Foliar pretreatment with an optimal dose of melatonin (10 μmol/L) or the overexpression of N-acetylserotonin methyltransferase (ASMT) gene effectively ameliorated heat-induced photoinhibition and electrolyte leakage in tomato plants. Both exogenous melatonin treatment and endogenous melatonin manipulation by overexpression of ASMT decreased the levels of insoluble and ubiquitinated proteins, but enhanced the expression of heat-shock proteins (HSPs) to refold denatured and unfolded proteins under heat stress. Meanwhile, melatonin also induced expression of several ATG genes and formation of autophagosomes to degrade aggregated proteins under the same stress. Proteomic profile analyses revealed that protein aggregates for a large number of biological processes accumulated in wild-type plants. However, exogenous melatonin treatment or overexpression of ASMT reduced the accumulation of aggregated proteins. Aggregation responsive proteins such as HSP70 and Rubisco activase were preferentially accumulated and ubiquitinated in wild-type plants under heat stress, while melatonin mitigated heat stress-induced accumulation and ubiquitination of aggregated proteins. These results suggest that melatonin promotes cellular protein protection through induction of HSPs and autophagy to refold or degrade denatured proteins under heat stress in tomato plants. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. Evidence Supports the Use of Soy Protein to Promote Cardiometabolic Health and Muscle Development.

    PubMed

    Paul, Greg; Mendelson, Garry J

    2015-01-01

    Consumption of adequate amounts of dietary protein can help individuals maintain a healthy body composition, especially when combined with resistance exercise and during weight loss. It is well established that dietary protein intake supports muscle development and helps reduce loss of lean body mass during weight loss. Numerous studies have demonstrated the efficacy of soy protein intake for promoting fat loss while preserving muscle mass and supporting lean body mass gains. In fact, soy protein and animal-based proteins both support weight loss and weight maintenance equally as part of an energy-restricted diet; however, soy protein offers additional cardiometabolic advantages. Key teaching points: Soy protein is a high-quality, plant-based protein that can be consumed throughout the life span. More human clinical studies have been conducted to assess the cholesterol-lowering effects of soy protein than any other cholesterol-lowering food ingredient. Ingestion of proteins with unique and complementary characteristics like soy, whey, and casein helps resistance-trained individuals achieve significant muscle growth. Recent research supports the efficacy of consuming a combination of soy, whey, and casein after resistance exercise to extend the time period that muscle building occurs.

  20. The innate immune protein calprotectin promotes Pseudomonas aeruginosa and Staphylococcus aureus interaction

    PubMed Central

    Wakeman, Catherine A.; Moore, Jessica L.; Noto, Michael J.; Zhang, Yaofang; Singleton, Marc D.; Prentice, Boone M.; Gilston, Benjamin A.; Doster, Ryan S.; Gaddy, Jennifer A.; Chazin, Walter J.; Caprioli, Richard M.; Skaar, Eric P.

    2016-01-01

    Microorganisms form biofilms containing differentiated cell populations. To determine factors driving differentiation, we herein visualize protein and metal distributions within Pseudomonas aeruginosa biofilms using imaging mass spectrometry. These in vitro experiments reveal correlations between differential protein distribution and metal abundance. Notably, zinc- and manganese-depleted portions of the biofilm repress the production of anti-staphylococcal molecules. Exposure to calprotectin (a host protein known to sequester metal ions at infectious foci) recapitulates responses occurring within metal-deplete portions of the biofilm and promotes interaction between P. aeruginosa and Staphylococcus aureus. Consistent with these results, the presence of calprotectin promotes co-colonization of the murine lung, and polymicrobial communities are found to co-exist in calprotectin-enriched airspaces of a cystic fibrosis lung explant. These findings, which demonstrate that metal fluctuations are a driving force of microbial community structure, have clinical implications because of the frequent occurrence of P. aeruginosa and S. aureus co-infections. PMID:27301800

  1. Label-free detection and identification of protein ligands captured by receptors in a polymerized planar lipid bilayer using MALDI-TOF MS

    PubMed Central

    Liang, Boying; Ju, Yue; Joubert, James R.; Kaleta, Erin J.; Lopez, Rodrigo; Jones, Ian W.; Hall, Henry K.; Ratnayaka, Saliya N.; Wysocki, Vicki H.; Saavedra, S. Scott

    2015-01-01

    Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) coupled with affinity capture is a well-established method to extract biological analytes from complex samples followed by label-free detection and identification. Many bioanalytes of interest bind to membrane-associated receptors, however, the matrices and high vacuum conditions inherent to MALDI-TOF MS make it largely incompatible with the use of artificial lipid membranes with incorporated receptors as platforms for detection of captured proteins and peptides. Here we show that cross-linking polymerization of a planar supported lipid bilayer (PSLB) provides the stability needed for MALDI-TOF MS analysis of proteins captured by receptors embedded in the membrane. PSLBs composed of poly(bis-SorbPC) and doped with the ganglioside receptors GM1 and GD1a were used for affinity capture of the B-subunits of cholera toxin, heat-labile enterotoxin, and pertussis toxin. The three toxins were captured simultaneously, then detected and identified by MS based on differences in their molecular weights. Poly(bis-SorbPC) PSLBs are inherently resistant to nonspecific protein adsorption, which allowed selective toxin detection to be achieved in complex matrices (bovine serum and shrimp extract). Using GM1-cholera toxin B as a model receptor-ligand pair, the minimal detectable concentration of toxin was estimated to be 4 nM. On-plate trypsin digestion of bound cholera toxin B followed by MS/MS analysis of digested peptides was performed successfully, demonstrating the feasibility of using the PSLB-based affinity capture platform for identification of unknown, membrane-associated proteins. Overall, this work demonstrates that combining a poly(lipid) affinity capture platform with MALDI-TOF MS detection is a viable approach for capture and proteomic characterization of membrane-associated proteins in a label-free manner. PMID:25694144

  2. Label-free detection and identification of protein ligands captured by receptors in a polymerized planar lipid bilayer using MALDI-TOF MS.

    PubMed

    Liang, Boying; Ju, Yue; Joubert, James R; Kaleta, Erin J; Lopez, Rodrigo; Jones, Ian W; Hall, Henry K; Ratnayaka, Saliya N; Wysocki, Vicki H; Saavedra, S Scott

    2015-04-01

    Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) coupled with affinity capture is a well-established method to extract biological analytes from complex samples followed by label-free detection and identification. Many bioanalytes of interest bind to membrane-associated receptors; however, the matrices and high-vacuum conditions inherent to MALDI-TOF MS make it largely incompatible with the use of artificial lipid membranes with incorporated receptors as platforms for detection of captured proteins and peptides. Here we show that cross-linking polymerization of a planar supported lipid bilayer (PSLB) provides the stability needed for MALDI-TOF MS analysis of proteins captured by receptors embedded in the membrane. PSLBs composed of poly(bis-sorbylphosphatidylcholine) (poly(bis-SorbPC)) and doped with the ganglioside receptors GM1 and GD1a were used for affinity capture of the B subunits of cholera toxin, heat-labile enterotoxin, and pertussis toxin. The three toxins were captured simultaneously, then detected and identified by MS on the basis of differences in their molecular weights. Poly(bis-SorbPC) PSLBs are inherently resistant to nonspecific protein adsorption, which allowed selective toxin detection to be achieved in complex matrices (bovine serum and shrimp extract). Using GM1-cholera toxin subunit B as a model receptor-ligand pair, we estimated the minimal detectable concentration of toxin to be 4 nM. On-plate tryptic digestion of bound cholera toxin subunit B followed by MS/MS analysis of digested peptides was performed successfully, demonstrating the feasibility of using the PSLB-based affinity capture platform for identification of unknown, membrane-associated proteins. Overall, this work demonstrates that combining a poly(lipid) affinity capture platform with MALDI-TOF MS detection is a viable approach for capture and proteomic characterization of membrane-associated proteins in a label-free manner.

  3. Evaluation of the Saccharomyces cerevisiae ADH2 promoter for protein synthesis.

    PubMed

    Lee, K Michael; DaSilva, Nancy A

    2005-04-30

    The Saccharomyces cerevisiae ADH2 promoter (P(ADH2)) is repressed several hundred-fold in the presence of glucose; transcription is initiated once the glucose in the medium is exhausted. The promoter can thus be utilized for effective regulation of recombinant gene expression in S. cerevisiae without the addition of an inducer. To evaluate this promoter in the absence of plasmid copy number and stability variations, the P(ADH2)-lacZ cassette was integrated into the yeast chromosomes. The effects of medium composition, glucose concentration and cultivation time on promoter derepression and expression level were investigated. Maximum protein activity was obtained after 48 h of growth in complex YPD medium containing 1% glucose. The widely used S. cerevisiae GAL1 and CUP1 promoters both require the addition of an inducer [galactose and copper(II) ion, respectively] before regulated genes will be expressed. The strengths of these three different promoters were compared for cells containing one copy of an integrated lacZ gene under their control. The ADH2 promoter was superior for all induction strategies investigated.

  4. Role of Ingested Amino Acids and Protein in the Promotion of Resistance Exercise–Induced Muscle Protein Anabolism123

    PubMed Central

    Rasmussen, Blake B

    2016-01-01

    The goal of this critical review is to comprehensively assess the evidence for the molecular, physiologic, and phenotypic skeletal muscle responses to resistance exercise (RE) combined with the nutritional intervention of protein and/or amino acid (AA) ingestion in young adults. We gathered the literature regarding the translational response in human skeletal muscle to acute exposure to RE and protein/AA supplements and the literature describing the phenotypic skeletal muscle adaptation to RE and nutritional interventions. Supplementation of protein/AAs with RE exhibited clear protein dose–dependent effects on translational regulation (protein synthesis) through mammalian target of rapamycin complex 1 (mTORC1) signaling, which was most apparent through increases in p70 ribosomal protein S6 kinase 1 (S6K1) phosphorylation, compared with postexercise recovery in the fasted or carbohydrate-fed state. These acute findings were critically tested via long-term exposure to RE training (RET) and protein/AA supplementation, and it was determined that a diminishing protein/AA supplement effect occurs over a prolonged exposure stimulus after exercise training. Furthermore, we found that protein/AA supplements, combined with RET, produced a positive, albeit minor, effect on the promotion of lean mass growth (when assessed in >20 participants/treatment); a negligible effect on muscle mass; and a negligible to no additional effect on strength. A potential concern we discovered was that the majority of the exercise training studies were underpowered in their ability to discern effects of protein/AA supplementation. Regardless, even when using optimal methodology and large sample sizes, it is clear that the effect size for protein/AA supplementation is low and likely limited to a subset of individuals because the individual variability is high. With regard to nutritional intakes, total protein intake per day, rather than protein timing or quality, appears to be more of a factor

  5. X-ray and cryo-EM structures of monomeric and filamentous actin-like protein MamK reveal changes associated with polymerization.

    PubMed

    Löwe, Jan; He, Shaoda; Scheres, Sjors H W; Savva, Christos G

    2016-11-22

    Magnetotactic bacteria produce iron-rich magnetic nanoparticles that are enclosed by membrane invaginations to form magnetosomes so they are able to sense and act upon Earth's magnetic field. In Magnetospirillum and other magnetotactic bacteria, to combine their magnetic moments, magnetosomes align along filaments formed by a bacterial actin homolog, MamK. Here, we present the crystal structure of a nonpolymerizing mutant of MamK from Magnetospirillum magneticum AMB-1 at 1.8-Å resolution, revealing its close similarity to actin and MreB. The crystals contain AMPPNP-bound monomeric MamK in two different conformations. To investigate conformational changes associated with polymerization, we used unmodified MamK protein and cryo-EM with helical 3D reconstruction in RELION to obtain a density map and a fully refined atomic model of MamK in filamentous form at 3.6-Å resolution. The filament is parallel (polar) double-helical, with a rise of 52.2 Å and a twist of 23.8°. As shown previously and unusually for actin-like filaments, the MamK subunits from each of the two strands are juxtaposed, creating an additional twofold axis along the filament. Compared with monomeric MamK, ADP-bound MamK in the filament undergoes a conformational change, rotating domains I and II against each other to further close the interdomain cleft between subdomains IB and IIB. The domain movement causes several loops to close around the nucleotide-binding pocket. Glu-143, a key residue for catalysis coordinating the magnesium ion, moves closer, presumably switching nucleotide hydrolysis upon polymerization-one of the hallmarks of cytomotive filaments of the actin type.

  6. Are the interactions between recombinant prion proteins and polymeric surfaces related to the hydrophilic/hydrophobic balance?

    PubMed

    Vrlinic, Tjasa; Debarnot, Dominique; Legeay, Gilbert; Coudreuse, Arnaud; El Moualij, Benaissa; Zorzi, Willy; Perret-Liaudet, Armand; Quadrio, Isabelle; Mozetic, Miran; Poncin-Epaillard, Fabienne

    2012-06-01

    New non-fouling tubes are developed and their influence on the adhesion of neuroproteins is studied. Recombinant prion proteins are considered as a single component representative of hydrophobic proteins. Samples are stored for 24 h at 4 °C in tubes coated with two different coatings: poly(N-isopropylacrylamide) as a hydrophilic surface and a plasma-fluorinated coating as a hydrophobic one. The protein adhesion is monitored by ELISA tests, XPS and confocal microscopy. It appears that the highest recovery of recombinant prion protein in the liquid phase is obtained with the hydrophilic surface while the hydrophobic character of the storage tube induces an important amount of biological loss. However, the recovery is not complete even for tubes coated with poly(N-isopropylacrylamide).

  7. Radiation-induced graft polymerization is the key to develop high-performance functional materials for protein purification

    NASA Astrophysics Data System (ADS)

    Saito, Kyoichi; Tsuneda, Satoshi; Kim, Min; Kubota, Noboru; Sugita, Kazuyuki; Sugo, Takanobu

    1999-05-01

    We have described a preparation scheme for immobilizing polymer chains at a uniformly high density onto a microfiltration membrane. Highly efficient protein recovery was demonstrated by the results of the determination of breakthrough and elution curves. The three requirements of high rate, high capacity, and repeated use for the protein recovery were satisfied by ensuring the occurrence of convection, multilayer binding, and hydrophilization, respectively. In addition, easy scale-up to fabrication of a membrane module was verified on a small scale.

  8. 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.

  9. Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription.

    PubMed

    Knight, Britta; Kubik, Slawomir; Ghosh, Bhaswar; Bruzzone, Maria Jessica; Geertz, Marcel; Martin, Victoria; Dénervaud, Nicolas; Jacquet, Philippe; Ozkan, Burak; Rougemont, Jacques; Maerkl, Sebastian J; Naef, Félix; Shore, David

    2014-08-01

    In yeast, ribosome production is controlled transcriptionally by tight coregulation of the 138 ribosomal protein genes (RPGs). RPG promoters display limited sequence homology, and the molecular basis for their coregulation remains largely unknown. Here we identify two prevalent RPG promoter types, both characterized by upstream binding of the general transcription factor (TF) Rap1 followed by the RPG-specific Fhl1/Ifh1 pair, with one type also binding the HMG-B protein Hmo1. We show that the regulatory properties of the two promoter types are remarkably similar, suggesting that they are determined to a large extent by Rap1 and the Fhl1/Ifh1 pair. Rapid depletion experiments allowed us to define a hierarchy of TF binding in which Rap1 acts as a pioneer factor required for binding of all other TFs. We also uncovered unexpected features underlying recruitment of Fhl1, whose forkhead DNA-binding domain is not required for binding at most promoters, and Hmo1, whose binding is supported by repeated motifs. Finally, we describe unusually micrococcal nuclease (MNase)-sensitive nucleosomes at all RPG promoters, located between the canonical +1 and -1 nucleosomes, which coincide with sites of Fhl1/Ifh1 and Hmo1 binding. We speculate that these "fragile" nucleosomes play an important role in regulating RPG transcriptional output. © 2014 Knight et al.; Published by Cold Spring Harbor Laboratory Press.

  10. Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription

    PubMed Central

    Knight, Britta; Kubik, Slawomir; Ghosh, Bhaswar; Bruzzone, Maria Jessica; Geertz, Marcel; Martin, Victoria; Dénervaud, Nicolas; Jacquet, Philippe; Ozkan, Burak; Rougemont, Jacques; Maerkl, Sebastian J.; Naef, Félix

    2014-01-01

    In yeast, ribosome production is controlled transcriptionally by tight coregulation of the 138 ribosomal protein genes (RPGs). RPG promoters display limited sequence homology, and the molecular basis for their coregulation remains largely unknown. Here we identify two prevalent RPG promoter types, both characterized by upstream binding of the general transcription factor (TF) Rap1 followed by the RPG-specific Fhl1/Ifh1 pair, with one type also binding the HMG-B protein Hmo1. We show that the regulatory properties of the two promoter types are remarkably similar, suggesting that they are determined to a large extent by Rap1 and the Fhl1/Ifh1 pair. Rapid depletion experiments allowed us to define a hierarchy of TF binding in which Rap1 acts as a pioneer factor required for binding of all other TFs. We also uncovered unexpected features underlying recruitment of Fhl1, whose forkhead DNA-binding domain is not required for binding at most promoters, and Hmo1, whose binding is supported by repeated motifs. Finally, we describe unusually micrococcal nuclease (MNase)-sensitive nucleosomes at all RPG promoters, located between the canonical +1 and −1 nucleosomes, which coincide with sites of Fhl1/Ifh1 and Hmo1 binding. We speculate that these “fragile” nucleosomes play an important role in regulating RPG transcriptional output. PMID:25085421

  11. Fast Protein Translation Can Promote Co- and Posttranslational Folding of Misfolding-Prone Proteins.

    PubMed

    Trovato, Fabio; O'Brien, Edward P

    2017-05-09

    Chemical kinetic modeling has previously been used to predict that fast-translating codons can enhance cotranslational protein folding by helping to avoid misfolded intermediates. Consistent with this prediction, protein aggregation in yeast and worms was observed to increase when translation was globally slowed down, possibly due to increased cotranslational misfolding. Observation of similar behavior in molecular simulations would confirm predictions from the simpler chemical kinetic model and provide a molecular perspective on cotranslational folding, misfolding, and the impact of translation speed on these processes. All-atom simulations cannot reach the timescales relevant to protein synthesis, and most conventional structure-based coarse-grained models do not allow for nonnative structure formation. Here, we introduce a protocol to incorporate misfolding using the functional forms of publicly available force fields. With this model we create two artificial proteins that are capable of undergoing structural transitions between a native and a misfolded conformation and simulate their synthesis by the ribosome. Consistent with the chemical kinetic predictions, we find that rapid synthesis of misfolding-prone nascent-chain segments increases the fraction of folded proteins by kinetically partitioning more molecules through on-pathway intermediates, decreasing the likelihood of sampling misfolded conformations. Novel to this study, to our knowledge, we observe that differences in protein dynamics, arising from different translation-elongation schedules, can persist long after the nascent protein has been released from the ribosome, and that a sufficient level of energetic frustration is needed for fast-translating codons to be beneficial for folding. These results provide further evidence that fast-translating codons can be as biologically important as pause sites in coordinating cotranslational folding. Copyright © 2017 Biophysical Society. Published by Elsevier

  12. Protein-DNA Interactions at the Opossum Npt2a Promoter are Dependent upon NHERF-1.

    PubMed

    Clark, Barbara J; Murray, Rebecca D; Salyer, Sarah A; Tyagi, Samuel C; Arumugam, Cibi; Khundmiri, Syed J; Lederer, Eleanor D

    2016-01-01

    Phosphate homeostasis is controlled by the renal reabsorption of Pi by the type IIa sodium phosphate cotransporter, Npt2a, which is localized in the proximal tubule brush border membrane. Regulation of Npt2a expression is a key control point to maintain phosphate homeostasis with most studies focused on regulating protein levels in the brush border membrane. Molecular mechanisms that control Npt2a mRNA, however, remain to be defined. We have reported that Npt2a mRNA and protein levels correlate directly with the expression of the Na+/H+ exchanger regulatory factor 1 (NHERF-1) using opossum kidney (OK) cells and the NHERF-1-deficient OK-H cells. The goal of this study was to determine whether NHERF-1 contributes to transcriptional and/or post-transcriptional mechanisms controlling Npt2a mRNA levels. Npt2a mRNA half-life was compared between OK and NHERF-1 deficient OK-H cell lines. oNpt2a promoter-reporter gene assays and electrophoretic mobility shift assays (EMSA) were used identify a NHERF-1 responsive region within the oNpt2a proximal promoter. Npt2a mRNA half-life is the same in OK and OK-H cells. The NHERF-1 responsive region lies within the proximal promoter in a region that contains a highly conserved CAATT box and G-rich element. Specific protein-DNA complex formation with the CAATT element is altered by the absence of NHERF-1 (OK v OK-H EMSA) although NHERF-1 does not directly contribute to complex formation. NHERF-1 helps maintain steady-state Npt2a mRNA levels in OK cells through indirect mechanisms that help promote protein-DNA interactions at the Npt2a proximal promoter. © 2016 The Author(s) Published by S. Karger AG, Basel.

  13. KLHL40 deficiency destabilizes thin filament proteins and promotes nemaline myopathy.

    PubMed

    Garg, Ankit; O'Rourke, Jason; Long, Chengzu; Doering, Jonathan; Ravenscroft, Gianina; Bezprozvannaya, Svetlana; Nelson, Benjamin R; Beetz, Nadine; Li, Lin; Chen, She; Laing, Nigel G; Grange, Robert W; Bassel-Duby, Rhonda; Olson, Eric N

    2014-08-01

    Nemaline myopathy (NM) is a congenital myopathy that can result in lethal muscle dysfunction and is thought to be a disease of the sarcomere thin filament. Recently, several proteins of unknown function have been implicated in NM, but the mechanistic basis of their contribution to disease remains unresolved. Here, we demonstrated that loss of a muscle-specific protein, kelch-like family member 40 (KLHL40), results in a nemaline-like myopathy in mice that closely phenocopies muscle abnormalities observed in KLHL40-deficient patients. We determined that KLHL40 localizes to the sarcomere I band and A band and binds to nebulin (NEB), a protein frequently implicated in NM, as well as a putative thin filament protein, leiomodin 3 (LMOD3). KLHL40 belongs to the BTB-BACK-kelch (BBK) family of proteins, some of which have been shown to promote degradation of their substrates. In contrast, we found that KLHL40 promotes stability of NEB and LMOD3 and blocks LMOD3 ubiquitination. Accordingly, NEB and LMOD3 were reduced in skeletal muscle of both Klhl40-/- mice and KLHL40-deficient patients. Loss of sarcomere thin filament proteins is a frequent cause of NM; therefore, our data that KLHL40 stabilizes NEB and LMOD3 provide a potential basis for the development of NM in KLHL40-deficient patients.

  14. KLHL40 deficiency destabilizes thin filament proteins and promotes nemaline myopathy

    PubMed Central

    Garg, Ankit; O’Rourke, Jason; Long, Chengzu; Doering, Jonathan; Ravenscroft, Gianina; Bezprozvannaya, Svetlana; Nelson, Benjamin R.; Beetz, Nadine; Li, Lin; Chen, She; Laing, Nigel G.; Grange, Robert W.; Bassel-Duby, Rhonda; Olson, Eric N.

    2014-01-01

    Nemaline myopathy (NM) is a congenital myopathy that can result in lethal muscle dysfunction and is thought to be a disease of the sarcomere thin filament. Recently, several proteins of unknown function have been implicated in NM, but the mechanistic basis of their contribution to disease remains unresolved. Here, we demonstrated that loss of a muscle-specific protein, kelch-like family member 40 (KLHL40), results in a nemaline-like myopathy in mice that closely phenocopies muscle abnormalities observed in KLHL40-deficient patients. We determined that KLHL40 localizes to the sarcomere I band and A band and binds to nebulin (NEB), a protein frequently implicated in NM, as well as a putative thin filament protein, leiomodin 3 (LMOD3). KLHL40 belongs to the BTB-BACK-kelch (BBK) family of proteins, some of which have been shown to promote degradation of their substrates. In contrast, we found that KLHL40 promotes stability of NEB and LMOD3 and blocks LMOD3 ubiquitination. Accordingly, NEB and LMOD3 were reduced in skeletal muscle of both Klhl40–/– mice and KLHL40-deficient patients. Loss of sarcomere thin filament proteins is a frequent cause of NM; therefore, our data that KLHL40 stabilizes NEB and LMOD3 provide a potential basis for the development of NM in KLHL40-deficient patients. PMID:24960163

  15. Cdc37 Promotes the Stability of Protein Kinases Cdc28 and Cak1

    PubMed Central

    Farrell, Alison; Morgan, David O.

    2000-01-01

    In the budding yeast Saccharomyces cerevisiae, Cdc37 is required for the productive formation of Cdc28-cyclin complexes. The cdc37-1 mutant arrests at Start with low levels of Cdc28 protein, which is predominantly unphosphorylated at Thr169, fails to bind cyclin, and has little protein kinase activity. We show here that Cdc28 and not cyclin is specifically defective in the cdc37-1 mutant and that Cdc37 likely does not act as an assembly factor for Cdc28-cyclin complex formation. We have also found that the levels and activity of the protein kinase Cak1 are significantly reduced in the cdc37-1 mutant. Pulse-chase analysis indicates that Cdc28 and Cak1 proteins are both destabilized when Cdc37 function is absent during but not after translation. In addition, Cdc37 promotes the production of Cak1, but not that of Cdc28, when coexpressed in insect cells. We conclude that budding yeast Cdc37, like its higher eukaryotic homologs, promotes the physical integrity of multiple protein kinases, perhaps by virtue of a cotranslational role in protein folding. PMID:10629030

  16. Promoter architecture and transcriptional regulation of Abf1-dependent ribosomal protein genes in Saccharomyces cerevisiae.

    PubMed

    Fermi, Beatrice; Bosio, Maria Cristina; Dieci, Giorgio

    2016-07-27

    In Saccharomyces cerevisiae, ribosomal protein gene (RPG) promoters display binding sites for either Rap1 or Abf1 transcription factors. Unlike Rap1-associated promoters, the small cohort of Abf1-dependent RPGs (Abf1-RPGs) has not been extensively investigated. We show that RPL3, RPL4B, RPP1A, RPS22B and RPS28A/B share a common promoter architecture, with an Abf1 site upstream of a conserved element matching the sequence recognized by Fhl1, a transcription factor which together with Ifh1 orchestrates Rap1-associated RPG regulation. Abf1 and Fhl1 promoter association was confirmed by ChIP and/or gel retardation assays. Mutational analysis revealed a more severe requirement of Abf1 than Fhl1 binding sites for RPG transcription. In the case of RPS22B an unusual Tbf1 binding site promoted both RPS22B and intron-hosted SNR44 expression. Abf1-RPG down-regulation upon TOR pathway inhibition was much attenuated at defective mutant promoters unable to bind Abf1. TORC1 inactivation caused the expected reduction of Ifh1 occupancy at RPS22B and RPL3 promoters, but unexpectedly it entailed largely increased Abf1 association with Abf1-RPG promoters. We present evidence that Abf1 recruitment upon nutritional stress, also observed for representative ribosome biogenesis genes, favours RPG transcriptional rescue upon nutrient replenishment, thus pointing to nutrient-regulated Abf1 dynamics at promoters as a novel mechanism in ribosome biogenesis control. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  17. Promoter architecture and transcriptional regulation of Abf1-dependent ribosomal protein genes in Saccharomyces cerevisiae

    PubMed Central

    Fermi, Beatrice; Bosio, Maria Cristina; Dieci, Giorgio

    2016-01-01

    In Saccharomyces cerevisiae, ribosomal protein gene (RPG) promoters display binding sites for either Rap1 or Abf1 transcription factors. Unlike Rap1-associated promoters, the small cohort of Abf1-dependent RPGs (Abf1-RPGs) has not been extensively investigated. We show that RPL3, RPL4B, RPP1A, RPS22B and RPS28A/B share a common promoter architecture, with an Abf1 site upstream of a conserved element matching the sequence recognized by Fhl1, a transcription factor which together with Ifh1 orchestrates Rap1-associated RPG regulation. Abf1 and Fhl1 promoter association was confirmed by ChIP and/or gel retardation assays. Mutational analysis revealed a more severe requirement of Abf1 than Fhl1 binding sites for RPG transcription. In the case of RPS22B an unusual Tbf1 binding site promoted both RPS22B and intron-hosted SNR44 expression. Abf1-RPG down-regulation upon TOR pathway inhibition was much attenuated at defective mutant promoters unable to bind Abf1. TORC1 inactivation caused the expected reduction of Ifh1 occupancy at RPS22B and RPL3 promoters, but unexpectedly it entailed largely increased Abf1 association with Abf1-RPG promoters. We present evidence that Abf1 recruitment upon nutritional stress, also observed for representative ribosome biogenesis genes, favours RPG transcriptional rescue upon nutrient replenishment, thus pointing to nutrient-regulated Abf1 dynamics at promoters as a novel mechanism in ribosome biogenesis control. PMID:27016735

  18. Mdm36 is a mitochondrial fission-promoting protein in Saccharomyces cerevisiae.

    PubMed

    Hammermeister, Miriam; Schödel, Kerstin; Westermann, Benedikt

    2010-07-15

    The division of mitochondrial membranes is a complex process mediated by the dynamin-related protein Dnm1 in yeast, acting in concert with several cofactors. We have identified Mdm36 as a mitochondria-associated protein required for efficient mitochondrial division. Deltamdm36 mutants contain highly interconnected mitochondrial networks that strikingly resemble known fission mutants. Furthermore, mitochondrial fission induced by depolymerization of the actin cytoskeleton is blocked in Deltamdm36 mutants, and the number of Dnm1 clusters on mitochondrial tips is reduced. Double mutant analyses indicate that Mdm36 acts antagonistically to fusion-promoting components, such as Fzo1 and Mdm30. The cell cortex-associated protein Num1 was shown previously to interact with Dnm1 and promote mitochondrial fission. We observed that mitochondria are highly motile and that their localization is not restricted to the cell periphery in Deltamdm36 and Deltanum1 mutants. Intriguingly, colocalization of Num1 and Dnm1 is abolished in the absence of Mdm36. These data suggest that Mdm36 is required for mitochondrial division by facilitating the formation of protein complexes containing Dnm1 and Num1 at the cell cortex. We propose a model that Mdm36-dependent formation of cell cortex anchors is required for the generation of tension on mitochondrial membranes to promote mitochondrial fission by Dnm1.

  19. Lamellipodin promotes actin assembly by clustering Ena/VASP proteins and tethering them to actin filaments

    PubMed Central

    Hansen, Scott D; Mullins, R Dyche

    2015-01-01

    Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly. DOI: http://dx.doi.org/10.7554/eLife.06585.001 PMID:26295568

  20. Identification of regions within the Legionella pneumophila VipA effector protein involved in actin binding and polymerization and in interference with eukaryotic organelle trafficking.

    PubMed

    Bugalhão, Joana N; Mota, Luís Jaime; Franco, Irina S

    2016-02-01

    The Legionella pneumophila effector protein VipA is an actin nucleator that co-localizes with actin filaments and early endosomes in infected macrophages and which interferes with organelle trafficking when expressed in yeast. To identify the regions of VipA involved in its subcellular localization and functions, we ectopically expressed specific VipA mutant proteins in eukaryotic cells. This indicated that the characteristic punctate distribution of VipA depends on its NH2 -terminal (amino acid residues 1-133) and central coiled-coil (amino acid residues 133-206) regions, and suggested a role for the COOH-terminal (amino acid residues 206-339) region in association with actin filaments and for the NH2 -terminal in co-localization with early endosomes. Co-immunoprecipitation and in vitro assays showed that the COOH-terminal region of VipA is necessary and sufficient to mediate actin binding, and is essential but insufficient to induce microfilament formation. Assays in yeast revealed that the NH2 and the COOH-terminal regions, and possibly an NPY motif within the NH2 region of VipA, are necessary for interference with organelle trafficking. Overall, this suggests that subversion of eukaryotic vesicular trafficking by VipA involves both its ability to associate with early endosomes via its NH2 -terminal region and its capacity to bind and polymerize actin through its COOH-terminal region.

  1. TATA-Binding Protein Mutants That Increase Transcription from Enhancerless and Repressed Promoters In Vivo

    PubMed Central

    Geisberg, Joseph V.; Struhl, Kevin

    2000-01-01

    Using a genetic screen, we isolated three TATA-binding protein (TBP) mutants that increase transcription from promoters that are repressed by the Cyc8-Tup1 or Sin3-Rpd3 corepressors or that lack an enhancer element, but not from an equivalently weak promoter with a mutated TATA element. Increased transcription is observed when the TBP mutants are expressed at low levels in the presence of wild-type TBP. These TBP mutants are unable to support cell viability, and they are toxic in strains lacking Rpd3 histone deacetylase or when expressed at higher levels. Although these mutants do not detectably bind TATA elements in vitro, genetic and chromatin immunoprecipitation experiments indicate that they act directly at promoters and do not increase transcription by titration of a negative regulatory factor(s). The TBP mutants are mildly defective for associating with promoters responding to moderate or strong activators; in addition, they are severely defective for RNA polymerase (Pol) III but not Pol I transcription. These results suggest that, with respect to Pol II transcription, the TBP mutants specifically increase expression from core promoters. Biochemical analysis indicates that the TBP mutants are unaffected for TFIID complex formation, dimerization, and interactions with either the general negative regulator NC2 or the N-terminal inhibitory domain of TAF130. We speculate that these TBP mutants have an unusual structure that allows them to preferentially access TATA elements in chromatin templates. These TBP mutants define a criterion by which promoters repressed by Cyc8-Tup1 or Sin3-Rpd3 resemble enhancerless, but not TATA-defective, promoters; hence, they support the idea that these corepressors inhibit the function of activator proteins rather than the Pol II machinery. PMID:10669725

  2. Phosphoinositides and membrane curvature switch the mode of actin polymerization via selective recruitment of toca-1 and Snx9.

    PubMed

    Gallop, Jennifer L; Walrant, Astrid; Cantley, Lewis C; Kirschner, Marc W

    2013-04-30

    The membrane-cytosol interface is the major locus of control of actin polymerization. At this interface, phosphoinositides act as second messengers to recruit membrane-binding proteins. We show that curved membranes, but not flat ones, can use phosphatidylinositol 3-phosphate [PI(3)P] along with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to stimulate actin polymerization. In this case, actin polymerization requires the small GTPase cell cycle division 42 (Cdc42), the nucleation-promoting factor neural Wiskott-Aldrich syndrome protein (N-WASP) and the actin nucleator the actin-related protein (Arp) 2/3 complex. In liposomes containing PI(4,5)P2 as the sole phosphoinositide, actin polymerization requires transducer of Cdc42 activation-1 (toca-1). In the presence of phosphatidylinositol 3-phosphate, polymerization is both more efficient and independent of toca-1. Under these conditions, sorting nexin 9 (Snx9) can be implicated as a specific adaptor that replaces toca-1 to mobilize neural Wiskott-Aldrich syndrome protein and the Arp2/3 complex. This switch in phosphoinositide and adaptor specificity for actin polymerization from membranes has implications for how different types of actin structures are generated at precise times and locations in the cell.

  3. PIP3-binding proteins promote age-dependent protein aggregation and limit survival in C. elegans.

    PubMed

    Ayyadevara, Srinivas; Balasubramaniam, Meenakshisundaram; Johnson, Jay; Alla, Ramani; Mackintosh, Samuel G; Shmookler Reis, Robert J

    2016-08-02

    Class-I phosphatidylinositol 3-kinase (PI3KI) converts phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3). PIP3 comprises two fatty-acid chains that embed in lipid-bilayer membranes, joined by glycerol to inositol triphosphate. Proteins with domains that specifically bind that head-group (e.g. pleckstrin-homology [PH] domains) are thus tethered to the inner plasma-membrane surface where they have an enhanced likelihood of interaction with other PIP3-bound proteins, in particular other components of their signaling pathways. Null alleles of the C. elegans age-1 gene, encoding the catalytic subunit of PI3KI, lack any detectable class-I PI3K activity and so cannot form PIP3. These mutant worms survive almost 10-fold longer than the longest-lived normal control, and are highly resistant to a variety of stresses including oxidative and electrophilic challenges. Traits associated with age-1 mutation are widely believed to be mediated through AKT-1, which requires PIP3 for both tethering and activation. Active AKT complex phosphorylates and thereby inactivates the DAF-16/FOXO transcription factor. However, extensive evidence indicates that pleiotropic effects of age-1-null mutations, including extreme longevity, cannot be explained by insulin like-receptor/AKT/FOXO signaling alone, suggesting involvement of other PIP3-binding proteins. We used ligand-affinity capture to identify membrane-bound proteins downstream of PI3KI that preferentially bind PIP3. Computer modeling supports a subset of candidate proteins predicted to directly bind PIP3 in preference to PIP2, and functional testing by RNAi knockdown confirmed candidates that partially mediate the stress-survival, aggregation-reducing and longevity benefits of PI3KI disruption. PIP3-specific candidate sets are highly enriched for proteins previously reported to affect translation, stress responses, lifespan, proteostasis, and lipid transport.

  4. Identification of a KRAB-zinc finger protein binding to the Rpe65 gene promoter.

    PubMed

    Lu, Zhongjian; Poliakov, Eugenia; Redmond, T Michael

    2006-05-01

    We wish to identify transcriptional factors involved in regulation binding to the proximal promoter region of the RPE65 gene that confers RPE-specific expression. We incubated human D407 RPE cell nuclear extract with double-stranded (sense 5-prime biotinylated) oligonucleotides, based on the RPE65 proximal gene promoter, bound to streptavidin-Dynabeads. Bound nuclear proteins were eluted, separated on SDS-PAGE, and analyzed by mass spectrometry. Peptide sequence was used to identify cDNA clones that were subcloned into pCDNA3.1 for expression and co-transfection into D407 cells to assess transcriptional activation of mouse Rpe65 gene promoter/reporter constructs. SiRNA interference was used to suppress ZNF492 expression. We identified a D407 nuclear protein binding to biotinylated-DNA/streptavidin beads as the product of clone KIAA1473 encoding a protein named ZNF492. ZNF492 has an open reading frame of 531 amino acids with a truncated N-terminus and lacks the usual Krüppel-associated box-A (KRAB-A) while KRAB-B remains intact and has 12 C2H2 zinc-fingers in tandem arrangement. Co-expression in D407 cells of ZNF492 protein did not activate TR1, a mouse Rpe65 gene promoter/reporter construct with 49-bp 5-prime flanking sequence, but did activate construct TR2, containing 188-bp 5-prime flanking sequence, by 2.5-fold, and the longer constructs TR4, containing 655-bp 5-prime flanking sequence, and TR5, containing 1240-bp 5-prime flanking sequence, by about 2-fold. SiRNA-mediated suppression of ZNF492 in D407 resulted in decreased Rpe65 promoter activity. We have identified ZNF492, a KRAB-zinc finger protein, by its interaction with immobilized RPE65 promoter DNA sequence. This KRAB-zinc finger protein serves as a moderate transcriptional factor for Rpe65 gene upregulation. In ZNF492, absence of KRAB-A might reduce or prevent co-repressor binding to account for the modest upregulation of Rpe65 gene expression.

  5. Platelet adhesion and protein adsorption on silicone rubber surface by ozone-induced grafted polymerization with carboxybetaine monomer.

    PubMed

    Zhou, Jun; Yuan, Jiang; Zang, Xiaopeng; Shen, Jian; Lin, Sicong

    2005-03-10

    Platelet adhesion and protein adsorption on the silicone rubber film grafted with N,N'-dimethyl-N-methacryloyloxyethyl-N-(2-carboxyethyl) ammonium (DMMCA) was studied. The grafting was carried out by means of ozone-induced method and was confirmed by ATR-FTIR and XPS investigations. The grafted films possessed relatively hydrophilic surface revealed by contact angle measurement. The blood compatibility of the grafted film was evaluated in vitro by platelet adhesion in platelet-rich plasma (PRP) and protein absorption in bovine fibrinogen (BFG) using silicone film as the reference. No substantial platelet adhesion was observed for the grafted films incubated in PRP for 60 and 180 min. The protein absorption was also significantly reduced after incubated in bovine fibrinogen for 60 min. Both the results indicated that the blood compatibility of silicone rubber was greatly improved by ozone-induced grafting of carboxybetaine zwitterionic polymer onto its surface.

  6. Promoter Identification and Transcription Analysis of Penicillin-Binding Protein Genes in Streptococcus pneumoniae R6

    PubMed Central

    Peters, Katharina; Pipo, Julia; Schweizer, Inga; Hakenbeck, Regine

    2016-01-01

    Penicillin-binding proteins (PBPs) are membrane-associated enzymes, which are involved in the last two steps of peptidoglycan biosynthesis, and some of them are key players in cell division. Furthermore, they are targets of β-lactams, the most widely used antibiotics. Nevertheless, very little is known about the expression and regulation of PBP genes. Using transcriptional mapping, we now determined the promoter regions of PBP genes from the laboratory strain Streptococcus pneumoniae R6 and examined the expression profile of these six promoters. The extended −10 region is highly conserved and complies with a σA-type promoter consensus sequence. In contrast, the −35 region is poorly conserved, indicating the possibility for differential PBP regulation. All PBP promoters were constitutively expressed and highly active during the exponential and early stationary growth phase. However, the individual expression of PBP promoters varied approximately fourfold, with pbp1a being the highest and pbp3 the lowest. Furthermore, the deletion of one nucleotide in the spacer region of the PBP3 promoter reduced pbp3 expression ∼10-fold. The addition of cefotaxime above the minimal inhibitory concentration (MIC) did not affect PBP expression in the penicillin-sensitive R6 strain. No evidence for regulation of S. pneumoniae PBP genes was obtained. PMID:27409661

  7. Promoter Identification and Transcription Analysis of Penicillin-Binding Protein Genes in Streptococcus pneumoniae R6.

    PubMed

    Peters, Katharina; Pipo, Julia; Schweizer, Inga; Hakenbeck, Regine; Denapaite, Dalia

    2016-09-01

    Penicillin-binding proteins (PBPs) are membrane-associated enzymes, which are involved in the last two steps of peptidoglycan biosynthesis, and some of them are key players in cell division. Furthermore, they are targets of β-lactams, the most widely used antibiotics. Nevertheless, very little is known about the expression and regulation of PBP genes. Using transcriptional mapping, we now determined the promoter regions of PBP genes from the laboratory strain Streptococcus pneumoniae R6 and examined the expression profile of these six promoters. The extended -10 region is highly conserved and complies with a σ(A)-type promoter consensus sequence. In contrast, the -35 region is poorly conserved, indicating the possibility for differential PBP regulation. All PBP promoters were constitutively expressed and highly active during the exponential and early stationary growth phase. However, the individual expression of PBP promoters varied approximately fourfold, with pbp1a being the highest and pbp3 the lowest. Furthermore, the deletion of one nucleotide in the spacer region of the PBP3 promoter reduced pbp3 expression ∼10-fold. The addition of cefotaxime above the minimal inhibitory concentration (MIC) did not affect PBP expression in the penicillin-sensitive R6 strain. No evidence for regulation of S. pneumoniae PBP genes was obtained.

  8. Thermal Conductive and Mechanical Properties of Polymeric Composites Based on Solution-Exfoliated Boron Nitride and Graphene Nanosheets: A Morphology-Promoted Synergistic Effect.

    PubMed

    Cui, Xieliang; Ding, Peng; Zhuang, Nan; Shi, Liyi; Song, Na; Tang, Shengfu

    2015-09-02

    In this work, we reported a synergistic effect of boron nitride (BN) with graphene nanosheets on the enhancement of thermal conductive and mechanical properties of polymeric composites. Here, few layered BN (s-BN) and graphene (s-GH) were used and obtained by liquid exfoliation method. The polystyrene (PS) and polyamide 6 (PA) composites were obtained via solution blending method and subsequently hot-pressing. The experimental results suggested that the thermal conductivity (TC) of the PS and PA composites increases with additional introduction of s-BN. For example, compared with the composites containing 20 wt % s-GH, additional introduction of only 1.5 wt % s-BN could increase the TC up to 38 and 34% in polystyrene (PS) and polyamide 6 (PA) matrix, respectively. Meanwhile, the mechanical properties of the composites were synchronously enhanced. It was found that s-BN filled in the interspaces of s-GH sheets and formed s-BN/s-GH stacked structure, which were helpful for the synchronously improving TC and mechanical properties of the polymeric materials.

  9. Effects of protein molecular weight on the intrinsic material properties and release kinetics of wet spun polymeric microfiber delivery systems.

    PubMed

    Lavin, Danya M; Zhang, Linda; Furtado, Stacia; Hopkins, Richard A; Mathiowitz, Edith

    2013-01-01

    Wet spun microfibers have great potential for the design of multifunctional controlled release scaffolds. Understanding aspects of drug delivery and mechanical strength, specific to protein molecular weight, may aid in the optimization and development of wet spun fiber platforms. This study investigated the intrinsic material properties and release kinetics of poly(l-lactic acid) (PLLA) and poly(lactic-co-glycolic acid) (PLGA) wet spun microfibers encapsulating proteins with varying molecular weights. A cryogenic emulsion technique developed in our laboratory was used to encapsulate insulin (5.8 kDa), lysozyme (14.3 kDa) and bovine serum albumin (BSA, 66.0 kDa) within wet spun microfibers (~100 μm). Protein loading was found to significantly influence mechanical strength and drug release kinetics of PLGA and PLLA microfibers in a molecular-weight-dependent manner. BSA encapsulation resulted in the most significant decrease in strength and ductility for both PLGA and PLLA microfibers. Interestingly, BSA-loaded PLGA microfibers had a twofold increase (8±2 MPa to 16±1 MPa) in tensile strength and a fourfold increase (3±1% to 12±6%) in elongation until failure in comparison to PLLA microfibers. PLGA and PLLA microfibers exhibited prolonged protein release up to 63 days in vitro. Further analysis with the Korsmeyer-Peppas kinetic model determined that the mechanism of protein release was dependent on Fickian diffusion. These results emphasize the critical role protein molecular weight has on the properties of wet spun filaments, highlighting the importance of designing small molecular analogues to replace growth factors with large molecular weights.

  10. Invadopodia proteins, cortactin, N-WASP and WIP differentially promote local invasiveness in ameloblastoma.

    PubMed

    Siar, Chong Huat; Rahman, Zainal Ariff Bin Abdul; Tsujigiwa, Hidetsugu; Mohamed Om Alblazi, Kamila; Nagatsuka, Hitoshi; Ng, Kok Han

    2016-09-01

    Cell migration and invasion through interstitial tissues are dependent upon several specialized characteristics of the migratory cell notably generation of proteolytic membranous protrusions or invadopodia. Ameloblastoma is a benign odontogenic epithelial neoplasm with a locally infiltrative behaviour. Cortactin and MMT1-MMP are two invadopodia proteins implicated in its local invasiveness. Other invadopodia regulators, namely N-WASP, WIP and Src kinase remain unclarified. This study addresses their roles in ameloblastoma. Eighty-seven paraffin-embedded ameloblastoma cases (20 unicystic, 47 solid/multicystic, 3 desmoplastic and 17 recurrent) were subjected to immunohistochemistry for expression of cortactin, N-WASP, WIP, Src kinase and F-actin, and findings correlated with clinicopathological parameters. Invadopodia proteins (except Src kinase) and F-actin were widely detected in ameloblastoma (cortactin: n = 73/87, 83.9%; N-WASP: n = 59/87; 67.8%; WIP: n = 77/87; 88.5%; and F-actin: n = 87/87, 100%). Protein localization was mainly cytoplasmic and/or membranous, and occasionally nuclear for F-actin. Cortactin, which functions as an actin-scaffolding protein, demonstrated significantly higher expression levels within ameloblastoma tumoral epithelium than in stroma (P < 0.05). N-WASP, which coordinates actin polymerization and invadopodia-mediated extracellular matrix degradation, was overexpressed in the solid/multicystic subtype (P < 0.05). WIP, an upstream regulator of N-WASP, and F-actin were significantly upregulated along the tumour invasive front compared to tumour centres (P < 0.05). Except for males with cortactin overexpression, other clinical parameters (age, ethnicity and anatomical site) showed no significant correlations. Present results suggest that local invasiveness of ameloblastoma is dependent upon the migratory potential of its tumour cells as defined by their distribution of cortactin, N-WASP and WIP in correlation with F-actin cytoskeletal

  11. Application of RGS box proteins to evaluate G-protein selectivity in receptor-promoted signaling.

    PubMed

    Hains, Melinda D; Siderovski, David P; Harden, T Kendall

    2004-01-01

    Regulator of G-protein signaling (RGS) domains bind directly to GTP-bound Galpha subunits and accelerate their intrinsic GTPase activity by up to several thousandfold. The selectivity of RGS proteins for individual Galpha subunits has been illustrated. Thus, the expression of RGS proteins can be used to inhibit signaling pathways activated by specific G protein-coupled receptors (GPCRs). This article describes the use of specific RGS domain constructs to discriminate among G(i/o), Gq-and G(12/13)-mediated activation of phospholipase C (PLC) isozymes in COS-7 cells. Overexpression of the N terminus of GRK2 (amino acids 45-178) or p115 RhoGEF (amino acids 1-240) elicited selective inhibition of Galphaq- or Galpha(12/13)-mediated signaling to PLC activation, respectively. In contrast, RGS2 overexpression was found to inhibit PLC activation by both G(i/o)- and Gq-coupled GPCRs. RGS4 exhibited dramatic receptor selectivity in its inhibitory actions; of the G(i/o)- and Gq-coupled GPCRs tested (LPA1, LPA2, P2Y1, S1P3), only the Gq-coupled lysophosphatidic acid-activated LPA2 receptor was found to be inhibited by RGS4 overexpression.

  12. Opioid peptides derived from food proteins suppress aggregation and promote reactivation of partly unfolded stressed proteins.

    PubMed

    Artemova, N V; Bumagina, Z M; Kasakov, A S; Shubin, V V; Gurvits, B Ya

    2010-02-01

    A new view of the opioid peptides is presented. The potential of small peptides derived from precursor food proteins, to bind to partly unfolded stressed proteins to prevent their irreversible aggregation and inactivation has been demonstrated in various in vitro test systems: dithiothreitol-induced aggregation of alpha-lactalbumin (LA), heat-induced aggregation of alcohol dehydrogenase (ADH), and aggregation and inactivation of bovine erythrocyte carbonic anhydrase (CA) in the process of its refolding after removal of stress conditions. Using dynamic light scattering (DLS), turbidimetry, fluorescence, and circular dichroism measurements protective effects of the synthetic opioid peptides: exorphin C from wheat gluten (Tyr-Pro-Ile-Ser-Leu), rubiscolin-5 from spinach ribulose-bisphosphate-carboxylase/oxygenase (Rubisco) (Tyr-Pro-Leu-Asp-Leu), and hemorphin-6 from bovine hemoglobin (Tyr-Pro-Trp-Thr-Gln-Arg) have been revealed. We have demonstrated the concentration-dependent suppression of light scattering intensity of aggregates of LA and ADH in the presence of the peptides, the population of nanoparticles with higher hydrodynamic radii being shifted to the lower ones, accompanied by an increase in the lag period of aggregation. The presence of the peptides in the refolding solution was shown to assist reactivation of CA and enhance the yield of the CA soluble protein. The results suggest that bioactive food protein fragments may be regarded as exogenous supplements to the endogenous defense mechanisms of the human organism under stress conditions.

  13. F-box protein 7 mutations promote protein aggregation in mitochondria and inhibit mitophagy.

    PubMed

    Zhou, Zhi Dong; Xie, Shao Ping; Sathiyamoorthy, Sushmitha; Saw, Wuan Ting; Sing, Tan Ye; Ng, Shin Hui; Chua, Heidi Pek Hup; Tang, Alyssa Mei Yan; Shaffra, Fathima; Li, Zeng; Wang, Hongyan; Ho, Patrick Ghim Hoe; Lai, Mitchell Kim Peng; Angeles, Dario C; Lim, Tit Meng; Tan, Eng-King

    2015-11-15

    The mutations of F-box protein 7 (FBXO7) gene (T22M, R378G and R498X) are associated with a severe form of autosomal recessive juvenile-onset Parkinson's disease (PD) (PARK 15). Here we demonstrated that wild-type (WT) FBXO7 is a stress response protein and it can play both cytoprotective and neurotoxic roles. The WT FBXO7 protein is vital to cell mitophagy and can facilitate mitophagy to protect cells, whereas mutant FBXO7 inhibits mitophagy. Upon stress, the endogenous WT FBXO7 gets up-regulated, concentrates into mitochondria and forms FBXO7 aggregates in mitochondria. However, FBXO7 mutations aggravate deleterious FBXO7 aggregation in mitochondria. The FBXO7 aggregation and toxicity can be alleviated by Proline, glutathione (GSH) and coenzyme Q10, whereas deleterious FBXO7 aggregation in mitochondria can be aggravated by prohibitin 1 (PHB1), a mitochondrial protease inhibitor. The overexpression of WT FBXO7 could lead to FBXO7 protein aggregation and dopamine neuron degeneration in transgenic Drosophila heads. The elevated FBXO7 expression and aggregation were identified in human fibroblast cells from PD patients. FBXO7 can also form aggregates in brains of PD and Alzheimer's disease. Our study provides novel pathophysiologic insights and suggests that FBXO7 may be a potential therapeutic target in FBXO7-linked neuron degeneration in PD.

  14. TACC3 is a microtubule plus end–tracking protein that promotes axon elongation and also regulates microtubule plus end dynamics in multiple embryonic cell types

    PubMed Central

    Nwagbara, Belinda U.; Faris, Anna E.; Bearce, Elizabeth A.; Erdogan, Burcu; Ebbert, Patrick T.; Evans, Matthew F.; Rutherford, Erin L.; Enzenbacher, Tiffany B.; Lowery, Laura Anne

    2014-01-01

    Microtubule plus end dynamics are regulated by a conserved family of proteins called plus end–tracking proteins (+TIPs). It is unclear how various +TIPs interact with each other and with plus ends to control microtubule behavior. The centrosome-associated protein TACC3, a member of the transforming acidic coiled-coil (TACC) domain family, has been implicated in regulating several aspects of microtubule dynamics. However, TACC3 has not been shown to function as a +TIP in vertebrates. Here we show that TACC3 promotes axon outgrowth and regulates microtubule dynamics by increasing microtubule plus end velocities in vivo. We also demonstrate that TACC3 acts as a +TIP in multiple embryonic cell types and that this requires the conserved C-terminal TACC domain. Using high-resolution live-imaging data on tagged +TIPs, we show that TACC3 localizes to the extreme microtubule plus end, where it lies distal to the microtubule polymerization marker EB1 and directly overlaps with the microtubule polymerase XMAP215. TACC3 also plays a role in regulating XMAP215 stability and localizing XMAP215 to microtubule plus ends. Taken together, our results implicate TACC3 as a +TIP that functions with XMAP215 to regulate microtubule plus end dynamics. PMID:25187649

  15. Phage φ29 Regulatory Protein p4 Stabilizes the Binding of the RNA Polymerase to the Late Promoter in a Process Involving Direct Protein-Protein Contacts

    NASA Astrophysics Data System (ADS)

    Nuez, Beatriz; Rojo, Fernando; Salas, Margarita

    1992-12-01

    Transcription from the late promoter, PA3, of Bacillus subtilis phage φ29 is activated by the viral regulatory protein p4. A kinetic analysis of the activation process has revealed that the role of protein p4 is to stabilize the binding of RNA polymerase to the promoter as a closed complex without significantly affecting further steps of the initiation process. Electrophoretic band-shift assays performed with a DNA fragment spanning only the protein p4 binding site showed that RNA polymerase could efficiently retard the complex formed by protein p4 bound to the DNA. Similarly, when a DNA fragment containing only the RNA polymerase-binding region of PA3 was used, p4 greatly stimulated the binding of RNA polymerase to the DNA. These results strongly suggest that p4 and RNA polymerase contact each other at the PA3 promoter. In the light of current knowledge of the p4 activation mechanism, we propose that direct contacts between the two proteins participate in the activation process.

  16. Phage phi 29 regulatory protein p4 stabilizes the binding of the RNA polymerase to the late promoter in a process involving direct protein-protein contacts.

    PubMed

    Nuez, B; Rojo, F; Salas, M

    1992-12-01

    Transcription from the late promoter, PA3, of Bacillus subtilis phage phi 29 is activated by the viral regulatory protein p4. A kinetic analysis of the activation process has revealed that the role of protein p4 is to stabilize the binding of RNA polymerase to the promoter as a closed complex without significantly affecting further steps of the initiation process. Electrophoretic band-shift assays performed with a DNA fragment spanning only the protein p4 binding site showed that RNA polymerase could efficiently retard the complex formed by protein p4 bound to the DNA. Similarly, when a DNA fragment containing only the RNA polymerase-binding region of PA3 was used, p4 greatly stimulated the binding of RNA polymerase to the DNA. These results strongly suggest that p4 and RNA polymerase contact each other at the PA3 promoter. In the light of current knowledge of the p4 activation mechanism, we propose that direct contacts between the two proteins participate in the activation process.

  17. Promotion of protein crystal growth by actively switching crystal growth mode via femtosecond laser ablation

    NASA Astrophysics Data System (ADS)

    Tominaga, Yusuke; Maruyama, Mihoko; Yoshimura, Masashi; Koizumi, Haruhiko; Tachibana, Masaru; Sugiyama, Shigeru; Adachi, Hiroaki; Tsukamoto, Katsuo; Matsumura, Hiroyoshi; Takano, Kazufumi; Murakami, Satoshi; Inoue, Tsuyoshi; Yoshikawa, Hiroshi Y.; Mori, Yusuke

    2016-11-01

    Large single crystals with desirable shapes are essential for various scientific and industrial fields, such as X-ray/neutron crystallography and crystalline devices. However, in the case of proteins the production of such crystals is particularly challenging, despite the efforts devoted to optimization of the environmental, chemical and physical parameters. Here we report an innovative approach for promoting the growth of protein crystals by directly modifying the local crystal structure via femtosecond laser ablation. We demonstrate that protein crystals with surfaces that are locally etched (several micrometers in diameter) by femtosecond laser ablation show enhanced growth rates without losing crystal quality. Optical phase-sensitive microscopy and X-ray topography imaging techniques reveal that the local etching induces spiral growth, which is energetically advantageous compared with the spontaneous two-dimensional nucleation growth mode. These findings prove that femtosecond laser ablation can actively switch the crystal growth mode, offering flexible control over the size and shape of protein crystals.

  18. Promoting Tag Removal of a MBP-Fused Integral Membrane Protein by TEV Protease.

    PubMed

    Chen, Yanke; Li, Qichang; Yang, Jun; Xie, Hao

    2017-03-01

    Tag removal is a prerequisite issue for structural and functional analysis of affinity-purified membrane proteins. The present study took a MBP-fused membrane protein, MrpF, as a model to investigate the tag removal by TEV protease. Influences of the linking sequence between TEV cleavage site and MrpF on protein expression and predicted secondary structure were investigated. The steric accessibility of TEV protease to cleavage site of MBP-fused MrpF was explored. It was found that reducing the size of hydrophilic group of detergents and/or extending the linking sequence between cleavage site and target protein can significantly improve the accessibility of the cleavage site and promote tag removal by TEV protease.

  19. A new method for the production of gelatin microparticles for controlled protein release from porous polymeric scaffolds.

    PubMed

    Ozkizilcik, Asya; Tuzlakoglu, Kadriye

    2014-03-01

    Tissue engineering using scaffolds and growth factors is a crucial approach in bone regeneration and repair. The combination of bioactive agents carrying microparticles with porous scaffolds can be an efficient solution when controlled release of bio-signalling molecules is required. The present study was based on a recent approach using a biodegradable scaffold and protein-loaded microparticles produced in an innovative manner in which protein loss is minimized during the loading process. Bovine serum albumin (BSA)-loaded gelatin microparticles were obtained by grinding freeze-dried membranes of gelatin and BSA. Porous scaffolds (250-355 µm pore size) produced from a polyactide (PLLA) and polycaprolactone (PCL) blend by salt leaching/supercritical CO₂ methods were used for the experiments. Gelatin microparticles containing three different BSA amounts were incorporated into the porous scaffolds by using a surfactant. In vitro release profiles showed up to 90% protein loading efficiency. This novel method appears to be an effective approach for producing particles that can minimize protein loss during the loading process.

  20. Protein precipitation behavior of condensed tannins from Lotus pedunculatus and Trifolium repens with different mean degrees of polymerization

    USDA-ARS?s Scientific Manuscript database

    The precipitation of bovine serum albumin (BSA), lysozyme (LYS), and alfalfa leaf protein (ALF) by two large- and two medium-sized condensed tannin (CT) fractions of similar flavan-3-ol subunit composition is described. CT fractions isolated from white clover flowers and big trefoil leaves exhibited...

  1. TPA-inducible proteins may account for sensitivity to promotion of transformation

    SciTech Connect

    Hirano, K.; Smith, B.; Colburn, N.H.

    1986-05-01

    The preneoplastic JB6 mouse epidermal cell system includes cell lines sensitive (P/sup +/) or resistant (P/sup -/) to tumor promoter induced neoplastic transformation. The authors investigated whether a difference in TPA-inducible proteins may explain this differential sensitivity. The synthesis of a 39 Kd cytoplasmic protein (Major Excreted Protein) was TPA-inducible, but to a similar extent in both P/sup +/ and P/sup -/ cells. TPA stimulated phosphorylation but not synthesis of the previously described stress protein pp80 in both P/sup +/ and P/sup -/ cells from 1 to 5 hr after treatment. Pulse labelling of P/sup +/ and P/sup -/ cells with /sup 35/S-methionine revealed a TPA dependent P/sup +/ specific transient increase in the synthesis of 58Kd protein. Induction was observed at 1 hr, and returned to basal levels by 4 hr and 20 hr, in nuclear and cytoplasmic fractions, respectively. This protein is not phosphorylated in response to TPA treatment. P/sup +/ cells differ from P/sup -/ cells in one or more genes that specify sensitivity to promotion of transformation, designated pro genes. Antibodies to three peptides representing the pro-1 open reading frame were used in immunoprecipitation and Western blotting to isolate the pro-1 gene product. A 43 Kd protein was immunologically responsive to the pro-1 peptide antibodies, and showed an increased signal 40 min after TPA treatment. Since the predicted molecular weight of a pro-1 gene product is only 7 Kd, the possibility of a modification of the protein by poly(ADP-ribosylation) or glycosylation is being investigated.

  2. The heat shock protein 60 promotes progesterone synthesis in mitochondria of JEG-3 cells.

    PubMed

    Monreal-Flores, Jessica; Espinosa-García, María Teresa; García-Regalado, Alejandro; Arechavaleta-Velasco, Fabian; Martínez, Federico

    2017-06-01

    Progesterone synthesis in human placenta is essential to maintain pregnancy. The limiting step in placental progesterone synthesis is cholesterol transport from the cytoplasm to the inner mitochondrial membrane. Multiple proteins located in mitochondrial contact sites seem to play a key role in this process. Previously, our group identified the heat shock protein 60 (HSP60) as part of mitochondrial contact sites in human placenta, suggesting its participation in progesterone synthesis. Here, we examined the role of HSP60 in progesterone synthesis. Our results show that over-expression of HSP60 in human placental choriocarcinoma cells (JEG-3) and human embryonic kidney 293 cells (HEK293) promotes progesterone synthesis. Furthermore, incubation of the HSP60 recombinant protein with intact isolated mitochondria from JEG-3 cells also promotes progesterone synthesis in a dose-related fashion. We also show that HSP60 interacts with STARD3 and P450scc proteins from mitochondrial membrane contact sites. Finally, we show that the HSP60 recombinant protein binds cholesterol. Ours results demonstrate that HSP60 participates in mitochondrial progesterone synthesis. These findings provide novel insights into progesterone synthesis in the human placenta and its role in maintaining pregnancy. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  3. Ets-1 facilitates nuclear entry of NFAT proteins and their recruitment to the IL-2 promoter

    PubMed Central

    Tsao, Hsiao-Wei; Tai, Tzong-Shyuan; Tseng, William; Chang, Hui-Hsin; Grenningloh, Roland; Miaw, Shi-Chuen; Ho, I-Cheng

    2013-01-01

    E26 transformation-specific sequence 1 (Ets-1), the prototype of the ETS family of transcription factors, is critical for the expression of IL-2 by murine Th cells; however, its mechanism of action is still unclear. Here we show that Ets-1 is also essential for optimal production of IL-2 by primary human Th cells. Although Ets-1 negatively regulates the expression of Blimp1, a known suppressor of IL-2 expression, ablation of B lymphocyte-induced maturation protein 1 (Blimp1) does not rescue the expression of IL-2 by Ets-1-deficient Th cells. Instead, Ets-1 physically and functionally interacts with the nuclear factor of activated T-cells (NFAT) and is required for the recruitment of NFAT to the IL-2 promoter. In addition, Ets-1 is located in both the nucleus and cytoplasm of resting Th cells. Nuclear Ets-1 quickly exits the nucleus in response to calcium-dependent signals and competes with NFAT proteins for binding to protein components of noncoding RNA repressor of NFAT complex (NRON), which serves as a cytoplasmic trap for phosphorylated NFAT proteins. This nuclear exit of Ets-1 precedes rapid nuclear entry of NFAT and Ets-1 deficiency results in impaired nuclear entry, but not dephosphorylation, of NFAT proteins. Thus, Ets-1 promotes the expression of IL-2 by modulating the activity of NFAT. PMID:24019486

  4. Ets-1 facilitates nuclear entry of NFAT proteins and their recruitment to the IL-2 promoter.

    PubMed

    Tsao, Hsiao-Wei; Tai, Tzong-Shyuan; Tseng, William; Chang, Hui-Hsin; Grenningloh, Roland; Miaw, Shi-Chuen; Ho, I-Cheng

    2013-09-24

    E26 transformation-specific sequence 1 (Ets-1), the prototype of the ETS family of transcription factors, is critical for the expression of IL-2 by murine Th cells; however, its mechanism of action is still unclear. Here we show that Ets-1 is also essential for optimal production of IL-2 by primary human Th cells. Although Ets-1 negatively regulates the expression of Blimp1, a known suppressor of IL-2 expression, ablation of B lymphocyte-induced maturation protein 1 (Blimp1) does not rescue the expression of IL-2 by Ets-1-deficient Th cells. Instead, Ets-1 physically and functionally interacts with the nuclear factor of activated T-cells (NFAT) and is required for the recruitment of NFAT to the IL-2 promoter. In addition, Ets-1 is located in both the nucleus and cytoplasm of resting Th cells. Nuclear Ets-1 quickly exits the nucleus in response to calcium-dependent signals and competes with NFAT proteins for binding to protein components of noncoding RNA repressor of NFAT complex (NRON), which serves as a cytoplasmic trap for phosphorylated NFAT proteins. This nuclear exit of Ets-1 precedes rapid nuclear entry of NFAT and Ets-1 deficiency results in impaired nuclear entry, but not dephosphorylation, of NFAT proteins. Thus, Ets-1 promotes the expression of IL-2 by modulating the activity of NFAT.

  5. The Potyviral P3 Protein Targets Eukaryotic Elongation Factor 1A to Promote the Unfolded Protein Response and Viral Pathogenesis.

    PubMed

    Luan, Hexiang; Shine, M B; Cui, Xiaoyan; Chen, Xin; Ma, Na; Kachroo, Pradeep; Zhi, Haijan; Kachroo, Aardra

    2016-09-01

    The biochemical function of the potyviral P3 protein is not known, although it is known to regulate virus replication, movement, and pathogenesis. We show that P3, the putative virulence determinant of soybean mosaic virus (SMV), targets a component of the translation elongation complex in soybean. Eukaryotic elongation factor 1A (eEF1A), a well-known host factor in viral pathogenesis, is essential for SMV virulence and the associated unfolded protein response (UPR). Silencing GmEF1A inhibits accumulation of SMV and another ER-associated virus in soybean. Conversely, endoplasmic reticulum (ER) stress-inducing chemicals promote SMV accumulation in wild-type, but not GmEF1A-knockdown, plants. Knockdown of genes encoding the eEF1B isoform, which is important for eEF1A function in translation elongation, has similar effects on UPR and SMV resistance, suggesting a link to translation elongation. P3 and GmEF1A promote each other's nuclear localization, similar to the nuclear-cytoplasmic transport of eEF1A by the Human immunodeficiency virus 1 Nef protein. Our results suggest that P3 targets host elongation factors resulting in UPR, which in turn facilitates SMV replication and place eEF1A upstream of BiP in the ER stress response during pathogen infection.

  6. The transmembrane LRR protein DMA-1 promotes dendrite branching and growth in C. elegans.

    PubMed

    Liu, Oliver W; Shen, Kang

    2011-12-04

    Dendrites often adopt complex branched structures. The development and organization of these arbors fundamentally determine the potential input and connectivity of a given neuron. The cell-surface receptors that control dendritic branching remain poorly understood. We found that, in Caenorhabditis elegans, a previously uncharacterized transmembrane protein containing extracellular leucine-rich repeat (LRR) domains, which we named DMA-1 (dendrite-morphogenesis-abnormal), promotes dendrite branching and growth. Sustained expression of dma-1 was found only in the elaborately branched sensory neurons PVD and FLP. Genetic analysis revealed that the loss of dma-1 resulted in much reduced dendritic arbors, whereas overexpression of dma-1 resulted in excessive branching. Forced expression of dma-1 in neurons with simple dendrites was sufficient to promote ectopic branching. Worms lacking dma-1 were defective in sensing harsh touch. DMA-1 is the first transmembrane LRR protein to be implicated in dendritic branching and expands the breadth of roles of LRR receptors in nervous system development.

  7. Relating Promoter Sequences to the Proteins that Bind to Them: A Comparison Study.

    NASA Astrophysics Data System (ADS)

    Glass, Kimberly

    2007-03-01

    Chromatin Immunoprecipitation (ChIP-on-ChIP) microarray data reveals that the proteins H3K9dimethyl and RNA-Polymerase II are exclusive regarding their binding to the promoter region of genes. When comparing the base pair sequences of the promoters that bind to Pol2 versus H3K9, striking differences appear. The mononucleotides have fundamentally different behaviors in each group. In addition, motifs that cluster before the transcriptional start site also generally have a strong enrichment in one group compared to the other. Using this knowledge a model can be developed that allows one to calculate a probability that a promoter will bind to either H3K9 or Pol2 based on its base pair sequence.

  8. A biodegradable polymeric system for peptide–protein delivery assembled with porous microspheres and nanoparticles, using an adsorption/infiltration process

    PubMed Central

    Alcalá-Alcalá, Sergio; Urbán-Morlán, Zaida; Aguilar-Rosas, Irene; Quintanar-Guerrero, David

    2013-01-01

    A biodegradable polymeric system is proposed for formulating peptides and proteins. The systems were assembled through the adsorption of biodegradable polymeric nanoparticles onto porous, biodegradable microspheres by an adsorption/infiltration process with the use of an immersion method. The peptide drug is not involved in the manufacturing of the nanoparticles or in obtaining the microspheres; thus, contact with the organic solvent, interfaces, and shear forces required for the process are prevented during drug loading. Leuprolide acetate was used as the model peptide, and poly(d,l-lactide-co-glycolide) (PLGA) was used as the biodegradable polymer. Leuprolide was adsorbed onto different amounts of PLGA nanoparticles (25 mg/mL, 50 mg/mL, 75 mg/mL, and 100 mg/mL) in a first stage; then, these were infiltrated into porous PLGA microspheres (100 mg) by dipping the structures into a microsphere suspension. In this way, the leuprolide was adsorbed onto both surfaces (ie, nanoparticles and microspheres). Scanning electron microscopy studies revealed the formation of a nanoparticle film on the porous microsphere surface that becomes more continuous as the amount of infiltrated nanoparticles increases. The adsorption efficiency and release rate are dependent on the amount of adsorbed nanoparticles. As expected, a greater adsorption efficiency (~95%) and a slower release rate were seen (~20% of released leuprolide in 12 hours) when a larger amount of nanoparticles was adsorbed (100 mg/mL of nanoparticles). Leuprolide acetate begins to be released immediately when there are no infiltrated nanoparticles, and 90% of the peptide is released in the first 12 hours. In contrast, the systems assembled in this study released less than 44% of the loaded drug during the same period of time. The observed release profiles denoted a Fickian diffusion that fit Higuchi’s model (t1/2). The manufacturing process presented here may be useful as a potential alternative for formulating

  9. Protein phosphatase 5 promotes hepatocarcinogenesis through interaction with AMP-activated protein kinase.

    PubMed

    Chen, Yao-Li; Hung, Man-Hsin; Chu, Pei-Yi; Chao, Tzu-I; Tsai, Ming-Hsien; Chen, Li-Ju; Hsiao, Yung-Jen; Shih, Chih-Ting; Hsieh, Feng-Shu; Chen, Kuen-Feng

    2017-08-15

    The serine-threonine protein phosphatase family members are known as critical regulators of various cellular functions, such as survival and transformation. Growing evidence suggests that pharmacological manipulation of phosphatase activity exhibits therapeutic benefits. Ser/Thr protein phosphatase 5 (PP5) is known to participate in glucocorticoid receptor (GR) and stress-induced signaling cascades that regulate cell growth and apoptosis, and has been shown to be overexpressed in various human malignant diseases. However, the role of PP5 in hepatocellular carcinoma (HCC) and whether PP5 may be a viable therapeutic target for HCC treatment are unknown. Here, by analyzing HCC clinical samples obtained from 215 patients, we found that overexpression of PP5 is tumor specific and associated with worse clinical outcomes. We further characterized the oncogenic properties of PP5 in HCC cells. Importantly, both silencing of PP5 with lentiviral-mediated short hairpin RNA (shRNA) and chemical inhibition of PP5 phosphatase activity using the natural compound cantharidin/norcantharidin markedly suppressed the growth of HCC cells and tumors in vitro and in vivo. Moreover, we identified AMP-activated protein kinase (AMPK) as a novel downstream target of oncogenic PP5 and demonstrated that the antitumor mechanisms underlying PP5 inhibition involve activation of AMPK signaling. Overall, our results establish a pathological function of PP5 in hepatocarcinogenesis via affecting AMPK signaling and suggest that PP5 inhibition is an attractive therapeutic approach for HCC. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Regulation of viral and cellular promoter activity by polyomavirus early proteins.

    PubMed Central

    Pannuti, A; La Mantia, G; Lania, L

    1987-01-01

    The chloramphenicol-acetyl-transferase (CAT) expression system has been utilized to study the ability of the polyomavirus (Py) early proteins, the 100K large T, the 55K middle T and 22K small T-antigens, to activate a variety of eukaryotic promoters (the SV40 early, the alpha 2(1) collagen, the rabbit beta-globin, the polyomavirus early and the H-2 class I) in both transient and stable expression assays. We have found that either the complete polyomavirus early region or a plasmid expressing only the 55K middle T-antigen are capable of stimulating the expression of all the promoter-CAT plasmids in transient co-transfection experiments in both NIH-3T3 and Rat-2 cells. Conversely, the Py early proteins do not stimulate the transcription of most of the promoter-CAT genes stably introduced in the cell chromosomes, with the exception of H-2 class I promoter, when stimulation of transcription has been observed upon infection with recombinant retrovirus encoding the Py middle T-antigen. Images PMID:3029721

  11. Human leucine zipper protein promotes hepatic steatosis via induction of apolipoprotein A-IV.

    PubMed

    Kang, Minsoo; Kim, Jeonghan; An, Hyoung-Tae; Ko, Jesang

    2017-02-28

    The molecular mechanism of stress-induced hepatic steatosis is not well known. Human leucine zipper protein (LZIP) regulates the expression of genes involved in inflammation, cell migration, and stress response. The aim of this study was to determine the regulatory role of LZIP in stress-induced hepatic steatosis. We used a microarray analysis to identify LZIP-induced genes involved in hepatic lipid metabolism. LZIP increased the expression of apolipoprotein A-IV (APOA4) mRNA. In the presence of stress inducer, APOA4 promoter analysis was performed and LZIP-induced lipid accumulation was monitored in mouse primary cells and human tissues. Under Golgi stress conditions, LZIP underwent proteolytic cleavage and was phosphorylated by AKT to protect against proteasome degradation. The stabilized N-terminal LZIP was translocated to the nucleus, where it directly bound to the APOA4 promoter, leading to APOA4 induction. LZIP-induced APOA4 expression resulted in increased absorption of surrounding free fatty acids. LZIP also promoted hepatic steatosis in mouse liver. Both LZIP and APOA4 were highly expressed in human steatosis samples. Our findings indicate that LZIP is a novel modulator of APOA4 expression and hepatic lipid metabolism. LZIP might be a therapeutic target for developing treatment strategies for hepatic steatosis and related metabolic diseases.-Kang, M., Kim, J., An, H.-T., Ko, J. Human leucine zipper protein promotes hepatic steatosis via induction of apolipoprotein A-IV.

  12. A-kinase anchoring protein-Lbc promotes pro-fibrotic signaling in cardiac fibroblasts.

    PubMed

    Cavin, Sabrina; Maric, Darko; Diviani, Dario

    2014-02-01

    In response to stress or injury the heart undergoes an adverse remodeling process associated with cardiomyocyte hypertrophy and fibrosis. Transformation of cardiac fibroblasts to myofibroblasts is a crucial event initiating the fibrotic process. Cardiac myofibroblasts invade the myocardium and secrete excess amounts of extracellular matrix proteins, which cause myocardial stiffening, cardiac dysfunctions and progression to heart failure. While several studies indicate that the small GTPase RhoA can promote profibrotic responses, the exchange factors that modulate its activity in cardiac fibroblasts are yet to be identified. In the present study, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor (GEF) activity, is critical for activating RhoA and transducing profibrotic signals downstream of type I angiotensin II receptors (AT1Rs) in cardiac fibroblasts. In particular, our results indicate that suppression of AKAP-Lbc expression by infecting adult rat ventricular fibroblasts with lentiviruses encoding AKAP-Lbc specific short hairpin (sh) RNAs strongly reduces the ability of angiotensin II to promote RhoA activation, differentiation of cardiac fibroblasts to myofibroblasts, collagen deposition as well as myofibroblast migration. Interestingly, AT1Rs promote AKAP-Lbc activation via a pathway that requires the α subunit of the heterotrimeric G protein G12. These findings identify AKAP-Lbc as a key Rho-guanine nucleotide exchange factor modulating profibrotic responses in cardiac fibroblasts.

  13. Activator protein 1 promotes the transcriptional activation of IRAK-M.

    PubMed

    Jin, Peipei; Bo, Lulong; Liu, Yongjian; Lu, Wenbin; Lin, Shengwei; Bian, Jinjun; Deng, Xiaoming

    2016-10-01

    Interleukin-1 receptor-associated kinase M (IRAK-M) is a well-known negative regulator for Toll-like receptor signaling, which can regulate immune homeostasis and tolerance in a number of pathological settings. However, the mechanism for IRAK-M regulation at transcriptional level remains largely unknown. In this study, a 1.4kb upstream sequence starting from the major IRAK-M transcriptional start site was cloned into luciferase reporter vector pGL3-basic to construct the full-length IRAK-M promoter. Luciferase reporter plasmids harboring the full-length and the deletion mutants of IRAK-M were transfected into 293T and A549 cells, and their relative luciferase activity was measured. The results demonstrated that activator protein 1(AP-1) cis-element plays a crucial role in IRAK-M constitutive gene transcription. Silencing of c-Fos and/or c-Jun expression suppressed the IRAK-M promoter activity as well as its mRNA and protein expressions. As a specific inhibitor for AP-1 activation, SP600125 also significantly suppressed the basal transcriptional activity of IRAK-M, the binding activity of c-Fos/c-Jun with IRAK-M promoter, and IRAK-M protein expression. Taken together, the result of this study highlights the importance of AP-1 in IRAK-M transcription, which offers more information on the role of IRAK-M in infectious and non-infectious diseases.

  14. Genomic structure, gene expression, and promoter analysis of human multidrug resistance-associated protein 7

    SciTech Connect

    Kao, Hsin-Hsin; Chang, Ming-Shi; Cheng, Jan-Fang; Huang, Jin-Ding

    2002-03-15

    The multidrug resistance-associated protein (MRP) subfamily transporters associated with anticancer drug efflux are attributed to the multidrug-resistance of cancer cells. The genomic organization of human multidrug resistance-associated protein 7 (MRP7) was identified. The human MRP7 gene, consisting of 22 exons and 21 introns, greatly differs from other members of the human MRP subfamily. A splicing variant of human MRP7, MRP7A, expressed in most human tissues, was also characterized. The 1.93-kb promoter region of MRP7 was isolated and shown to support luciferase activity at a level 4- to 5-fold greater than that of the SV40 promoter. Basal MRP7 gene expression was regulated by 2 regions in the 5-flanking region at 1,780 1,287 bp, and at 611 to 208 bp. In Madin-Darby canine kidney (MDCK) cells, MRP7 promoter activity was increased by 226 percent by genotoxic 2-acetylaminofluorene and 347 percent by the histone deacetylase inhibitor, trichostatin A. The protein was expressed in the membrane fraction of transfected MDCK cells.

  15. Binding and Release between Polymeric Carrier and Protein Drug: pH Mediated Interplay of Coulomb forces, Hydrogen Bonding, van der Waals Interactions and Entropy.

    PubMed

    De Luca, Sergio; Chen, Fan; Seal, Prasenjit; Stenzel, Martina H; Smith, Sean C

    2017-09-07

    The accelerating search for new types of drugs and delivery strategies poses the challenge to understand the mechanism of delivery. To this end, a detailed atomistic picture of binding between the drug and the carrier is quintessential. While many studies focus on the electrostatics of drug-vector interactions, it has also been pointed out that entropic factors relating to water and counter ions can play an important role. By carrying out extensive molecular dynamics simulations and subsequently validating with experiment, we shed light herein on the binding in aqueous solution between a protein drug and a polymeric carrier. We examined the complexation between the polymer, poly (ethylene glycol) methyl ether acrylate-b-poly(carboxyethyl acrylate (PEGMEA-b-PCEA) and the protein, egg white lysozyme, a system that acts as a model for polymer-vector / protein-drug delivery systems. The complexation has been visualized and characterized using contact maps and hydrogen bonding analyses for five independent simulations of the complex, each running over 100 ns. Binding at physiological pH is, as expected, mediated by coulombic attraction between the positively charged protein and negatively charged carboxylate groups on the polymer. However, we find that consideration of electrostatics alone is insufficient to explain the complexation behaviour at low pH. Intra-complex hydrogen bonds, van der Waals interactions also water-water interactions dictate that the polymer does not release the protein at pH 4.8 or indeed at pH 3.2, even though the Coulombic attractions are largely removed as carboxylate groups on the polymer become titrated. Experiments in aqueous solution carried out at pH = 7.0, 4.5, and 3.0 confirm the veracity of the computed binding behaviour. Overall, these combined simulation and experimental results illustrate that coulomb interactions need to be complemented with consideration of other entropic forces, mediated by van der Waals interactions and hydrogen

  16. Dopamine polymerization promoted by a catecholase biomimetic Cu(II)(μ-OH)Cu(II) complex containing a triazine-based ligand.

    PubMed

    de Oliveira, José A F; da Silva, Marcos P; de Souza, Bernardo; Camargo, Tiago P; Szpoganicz, Bruno; Neves, Ademir; Bortoluzzi, Adailton J

    2016-10-04

    We describe herein the catecholase-like catalytic activity and dopamine polymerization by using a dinuclear [LCu(II)(μ-OH)2Cu(II)](ClO4)2 (1) complex where L is the dinucleating triazine-based ligand 6-chloro-N(2),N(2),N(4),N(4)-tetrakis(pyridin-2-ylmethyl)-1,3,5-triazine-2,4-diamine. The kinetic parameters (kcat = 0.318 s(-1), KM = 1.6 × 10(-3) mol L(-1), and kcat/KM = 198.8 L s(-1) mol(-1)), mechanistic insights into the oxidation of 3,5-di-tert-butyl catechol and early characterization of poly(dopamine) are presented.

  17. Insight in the role of bovine serum albumin for promoting the in situ surface growth of polyhydroxybutyrate (PHB) on patterned surfaces via enzymatic surface-initiated polymerization.

    PubMed

    Niamsiri, Nuttawee; Bergkvist, Magnus; Delamarre, Soazig C; Cady, Nathan C; Coates, Geoffrey W; Ober, Christopher K; Batt, Carl A

    2007-10-15

    Polyhydroxyalkanoates (PHAs) are a family of aliphatic polyesters produced by a variety of microorganisms as a reserve of carbon and energy. Enzymes involved in the synthesis of PHAs can be utilized to produce polymers in vitro, both in bulk and on solid surfaces. Here, site-specific attachment of the key catalytic enzyme, PHA synthase, on lithographically patterned surfaces and subsequent addition of (R)-3-hydroxybutyryl-CoA substrate allowed us to fabricate spatially ordered polyhydroxybutyrate (PHB) polymeric structures via an in situ enzymatic surface-initiated polymerization (ESIP). By varying the reaction conditions, we enhanced the growth of PHB on solid surfaces and analyzed the resulting structures by fluorescence microscopy, atomic force microscopy (AFM), attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, and gel permeation chromatography (GPC). We found that stabilization of smaller PHB granule structures by an addition of bovine serum albumin (BSA) was the most important factor for a successful synthesis of a PHB layer up to 1mum in thickness, consisting mainly of larger cluster assemblies of PHB granules that cover the entire patterned area. Immunofluorescence detection and surface contact angle analysis revealed that BSA was physically bound to the PHB polymer all through the cluster, and reduced the overall hydrophobicity of the polymer surface. Based on information obtained from AFM, kinetic measurements and various polymer characterization methods, a plausible model for roles of BSA in the enhancement of PHB formation on surfaces is discussed. Furthermore, by using biotinylated BSA conjugates, we were able to incorporate biotin groups into the PHB polymer matrix, thus generating a bioactive surface that can be used for displaying other functional biomolecules through streptavidin-biotin interaction on the PHB structures. Because of its versatility, our fabrication strategy is expected to be a useful surface

  18. Myb and Ets proteins cooperate in transcriptional activation of the mim-1 promoter.

    PubMed Central

    Dudek, H; Tantravahi, R V; Rao, V N; Reddy, E S; Reddy, E P

    1992-01-01

    In the generation of the acutely transforming avian retrovirus E26, both myb and ets genes have been transduced, leading to the production of a Gag-Myb-Ets fusion protein. This co-occurrence of v-myb and v-ets oncogenes suggests that the two might have a functional relationship. To look for such a relationship, we tested the transcriptional activation activity of Myb alone or with coexpressed Ets-1 or Ets-2. Using the promoter of the v-Myb-inducible mim-1 gene as a target, we found that full-length c-Myb gene products were poor activators of transcription, while an oncogenic (truncated) form of this protein was a strong trans-activator. However, coexpression of Ets-2 with full-length or truncated forms of Myb greatly increased trans-activation. Coexpression of Ets-1, Fos, Jun, or Myc with Myb did not increase trans-activation of the mim-1 promoter. The ability of Myb and Ets-2 to transactivate was cooperative, since Ets-2 alone gave little or no activation. Bacterially synthesized Ets-2 protein was found to bind specifically to the mim-1 promoter, suggesting that it may be a target for both Myb and Ets proteins. Thus, Myb and Ets proteins can cooperate in transcriptional activation, and their co-occurrence in the E26 virus may reflect a functional relationship between these two oncoproteins. Truncated forms of Myb may have a reduced need for cooperating factors such as Ets-2, and this might constitute an important mechanism associated with oncogenic activation. Images PMID:1741383

  19. Metastasis-associated protein 1 promotes tumor invasion by downregulation of E-cadherin.

    PubMed

    Weng, Wenhao; Yin, Jiayi; Zhang, Yue; Qiu, Jin; Wang, Xinghe

    2014-03-01

    Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors. Upregulation of metastasis-associated protein 1 (MTA1) has been reported to contribute to the development of esophageal squamous cell carcinoma. Therefore, the objective of our study was to identify the molecular mechanisms of MTA1 underlying the invasion and metastasis of ESCC. We overexpressed MTA1 in ESCC cells to examine the role of MTA1 in the regulation of the cell invasion. In addition, using luciferase reporter assay and electrophoretic mobility shift assays, we evaluated the binding of MTA1 to the promoter of E-cadherin. We found that MTA1 overexpression promotes invasiveness of the human esophageal carcinoma cell line EC-9706. This effect was accompanied by downregulation of the epithelial cell marker E-cadherin and upregulation of vimentin and MMP-9 luciferase reporter assays showed that MTA1 inhibited the promoter activity of E-cadherin and that this was dependent on Snail, Slug and HDAC1. We also found that Snail and Slug bound the E-boxes in the promoter of E-cadherin and recruited MTA1 and HDAC1 to suppress E-cadherin expression, as confirmed by electrophoretic mobility shift and chromatin immunoprecipitation assays. MTA1 promotes tumor invasion by downregulation of E-cadherin. These results demonstrate a novel role for MTA1 in the regulation of esophageal squamous cell carcinoma invasion and provide insight into the mechanisms involved in this process.

  20. The Lipid Droplet Protein Hypoxia-inducible Gene 2 Promotes Hepatic Triglyceride Deposition by Inhibiting Lipolysis*

    PubMed Central

    DiStefano, Marina T.; Danai, Laura V.; Roth Flach, Rachel J.; Chawla, Anil; Pedersen, David J.; Guilherme, Adilson; Czech, Michael P.

    2015-01-01

    The liver is a major site of glucose, fatty acid, and triglyceride (TG) synthesis and serves as a major regulator of whole body nutrient homeostasis. Chronic exposure of humans or rodents to high-calorie diets promotes non-alcoholic fatty liver disease, characterized by neutral lipid accumulation in lipid droplets (LD) of hepatocytes. Here we show that the LD protein hypoxia-inducible gene 2 (Hig2/Hilpda) functions to enhance lipid accumulation in hepatocytes by attenuating TG hydrolysis. Hig2 expression increased in livers of mice on a high-fat diet and during fasting, two states associated with enhanced hepatic TG content. Hig2 expressed in primary mouse hepatocytes localized to LDs and promoted LD TG deposition in the presence of oleate. Conversely, tamoxifen-inducible Hig2 deletion reduced both TG content and LD size in primary hepatocytes from mice harboring floxed alleles of Hig2 and a cre/ERT2 transgene controlled by the ubiquitin C promoter. Hepatic TG was also decreased by liver-specific deletion of Hig2 in mice with floxed Hig2 expressing cre controlled by the albumin promoter. Importantly, we demonstrate that Hig2-deficient hepatocytes exhibit increased TG lipolysis, TG turnover, and fatty acid oxidation as compared with controls. Interestingly, mice with liver-specific Hig2 deletion also display improved glucose tolerance. Taken together, these data indicate that Hig2 plays a major role in promoting lipid sequestration within LDs in mouse hepatocytes through a mechanism that impairs TG degradation. PMID:25922078

  1. The Lipid Droplet Protein Hypoxia-inducible Gene 2 Promotes Hepatic Triglyceride Deposition by Inhibiting Lipolysis.

    PubMed

    DiStefano, Marina T; Danai, Laura V; Roth Flach, Rachel J; Chawla, Anil; Pedersen, David J; Guilherme, Adilson; Czech, Michael P

    2015-06-12

    The liver is a major site of glucose, fatty acid, and triglyceride (TG) synthesis and serves as a major regulator of whole body nutrient homeostasis. Chronic exposure of humans or rodents to high-calorie diets promotes non-alcoholic fatty liver disease, characterized by neutral lipid accumulation in lipid droplets (LD) of hepatocytes. Here we show that the LD protein hypoxia-inducible gene 2 (Hig2/Hilpda) functions to enhance lipid accumulation in hepatocytes by attenuating TG hydrolysis. Hig2 expression increased in livers of mice on a high-fat diet and during fasting, two states associated with enhanced hepatic TG content. Hig2 expressed in primary mouse hepatocytes localized to LDs and promoted LD TG deposition in the presence of oleate. Conversely, tamoxifen-inducible Hig2 deletion reduced both TG content and LD size in primary hepatocytes from mice harboring floxed alleles of Hig2 and a cre/ERT2 transgene controlled by the ubiquitin C promoter. Hepatic TG was also decreased by liver-specific deletion of Hig2 in mice with floxed Hig2 expressing cre controlled by the albumin promoter. Importantly, we demonstrate that Hig2-deficient hepatocytes exhibit increased TG lipolysis, TG turnover, and fatty acid oxidation as compared with controls. Interestingly, mice with liver-specific Hig2 deletion also display improved glucose tolerance. Taken together, these data indicate that Hig2 plays a major role in promoting lipid sequestration within LDs in mouse hepatocytes through a mechanism that impairs TG degradation.

  2. Mineralization-inhibiting effects of transglutaminase-crosslinked polymeric osteopontin.

    PubMed

    Hoac, Betty; Nelea, Valentin; Jiang, Wenge; Kaartinen, Mari T; McKee, Marc D

    2017-08-01

    Osteopontin (OPN) belongs to the SIBLING family (Small, Integrin-Binding LIgand N-linked Glycoproteins) of mineral-binding matrix proteins found in bones and teeth. OPN is a well-known inhibitor of matrix mineralization, and enzymatic modification of OPN can affect this inhibitory function. In bone, OPN exists both as a monomer and as a high-molecular-weight polymer - the latter is formed by transglutaminase-mediated crosslinking of glutamine and lysine residues in OPN to create homotypic protein assemblies. OPN can be covalently crosslinked by transglutaminase 2 (TG2) and Factor XIII-A. Polymeric OPN has increased binding to collagen and promotes osteoblast adhesion, but despite these initial observations, its role in mineralization is not clear. In this study, we investigated the effect of polymerized OPN on mineralization using a hydroxyapatite crystal growth assay and mineralizing MC3T3-E1 osteoblast cultures. In the cultures, endogenous polymeric OPN was detected after mineralization occurred. In cell-free conditions, TG2 was used to crosslink bovine OPN into its polymeric form, and atomic force microscopy and dynamic light scattering revealed variably-sized, large branched aggregates ranging across hundreds of nanometers. These OPN polymers inhibited the growth of hydroxyapatite crystals in solution at concentrations similar to monomeric OPN, although the crosslinking slightly reduced its inhibitory potency. When added to MC3T3-E1 osteoblast cultures, this exogenous polymeric OPN essentially did not inhibit mineralization when given during the later mineralization stages of culture; however, cultures treated early and then continuously with polymeric OPN throughout both the matrix assembly and mineral deposition stages showed reduced mineralization. Immunoblotting of protein extracts from these continuously treated cultures revealed exogenous OPN polymers incorporated into mature matrix that had not yet mineralized. These results suggest that in bone, the

  3. cAMP and cAMP-dependent protein kinase regulate the human heat shock protein 70 gene promoter activity.

    PubMed

    Choi, H S; Li, B; Lin, Z; Huang, E; Liu, A Y

    1991-06-25

    The theme of this study is an evaluation of the involvement of cAMP and cAMP-dependent protein kinase (PKA) in the regulation of the human heat shock protein (hsp) 70 gene promoter. Expression of a highly specific protein inhibitor of PKA (pRSVPKI) inhibited the basal as well as heat- and cadmium-induced expression of the cotransfected pHBCAT, a human hsp 70 promoter-driven reporter gene; this inhibition was dependent on the amount of pRSVPKI used. The effect of an expression vector of the RI regulatory subunit of PKA, pMTREV, was similar to that of pRSVPKI; pMTREV inhibited both the basal as well as the heat-induced expression of pHBCAT. The specificity of effects of these expression vectors was demonstrated by the lack of effect of a mutant PKI gene and by the unaffected expression of a reference gene (pRSV beta gal) under these conditions. Analysis of the effects of dibutyryl cAMP (1 mM), forskolin (10 microM), and 8-Br-cAMP (1 mM) on the transient expression of pHBCAT showed that these cAMP-elevating agents stimulated the hsp 70 promoter activity, whereas cAMP (1 mM) was without effect. Chloramphenicol acetyltransferase gene constructs with truncated or mutated hsp 70 promoter were used to define the cis-acting DNA element(s) that confer this cAMP stimulation; the heat induced (42 degrees C) expression was used as a control. Mutation of the adenovirus transcription factor element (pLSN-40/-26) greatly reduced the basal level of expression; forskolin had little or no effect on this adenovirus transcription factor-minus promoter, although the promoter activity was very heat inducible. The absence of a functional heat shock consensus element (HSE) in the construct pLSPNWT rendered the promoter heat insensitive; this construct was forskolin responsive although the magnitude of this stimulation was reduced when compared with that of a control construct with HSE. These results were corroborated by studies using consensus sequence of ATF (ATFE) and HSE as competitors

  4. Achieving efficient protein expression in Trichoderma reesei by using strong constitutive promoters

    PubMed Central

    2012-01-01

    Backgrounds The fungus Trichoderma reesei is an important workhorse for expression of homologous or heterologous genes, and the inducible cbh1 promoter is generally used. However, constitutive expression is more preferable in some cases than inducible expression that leads to production of unwanted cellulase components. In this work, constitutive promoters of T. reesei were screened and successfully used for high level homologous expression of xylanase II. Results The transcriptional profiles of 13 key genes that participate in glucose metabolism in T. reesei were analyzed by quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR). The results indicated that the mRNA levels of pdc (encoding pyruvate decarboxylase) and eno (encoding enolase) genes were much higher than other genes under high glucose conditions. Recombinant T. reesei strains that homologously expressed xylanase II were constructed by using the promoters of the pdc and eno genes, and they respectively produced 9266 IU/ml and 8866 IU/ml of xylanase activities in the cultivation supernatant in a medium with high glucose concentration. The productivities of xylanase II were 1.61 g/L (with the pdc promoter) and 1.52 g/L (with the eno promoter), approximately accounted for 83% and 82% of the total protein secreted by T. reesei, respectively. Conclusions This work demonstrates the screening of constitutive promoters by using RT-qPCR in T. reesei, and has obtained the highest expression of recombinant xylanase II to date by using these promoters. PMID:22709462

  5. Sal-like protein 2 upregulates p16 expression through a proximal promoter element

    PubMed Central

    Wu, Zhenghua; Cheng, Kebin; Shi, Lidan; Li, Zheqi; Negi, Hema; Gao, Guangwei; Kamle, Suchitra; Li, Dawei

    2015-01-01

    Sal-like protein 2 (Sall2), a homeotic transcription factor, is a putative tumor suppressor. We have previously shown that Sall2 activates the transcription of tumor suppressor gene p21 and suppresses tumorigenesis through cell cycle inhibition and induction of apoptosis. To investigate additional Sall2-regulated downstream genes, we analyzed the differences in mRNA expression profiles with and without exogenously expressed Sall2. We identified 1616 Sall2-responsive genes through gene expression arrays. Promoter-reporter assays of p16INK4A and several other tumor-related genes indicated that the Sall2 regulation of these promoters was not significantly different between the two major forms of Sall2 with alternative exon 1 or exon 1A. Additional analysis showed that Sall2-induced p16 promoter activation was Sall2 dose-dependent. Deletion and site-directed mutagenesis of the p16 promoter identified a consensus Sall2 binding site (GGGTGGG) proximal to the p16 transcription start site and was critical for p16 promoter activation. Finally, to confirm the significance of Sall2-activated p16 expression in cell cycle regulation, we co-transfected the SKOV3 cells with a Sall2 expression construct and a p16 minigene and also co-transfected the ES-2 cells with a Sall2 expression construct and the siRNA against p16 for flow cytometry analysis. Our results showed that Sall2 enhanced the p16 minigene blocking of cell cycle progression and p16 knockdown with siRNA abolished most of the Sall2 inhibition of cell cycle progression. These findings indicate that Sall2 targets multiple cell cycle regulators, including p16, through their promoters, adding knowledge to the understanding of Sall2 and p16 gene regulation, and how Sall2 deregulation may promote cancer formation. PMID:25580951

  6. Promoter Recognition by Extracytoplasmic Function σ Factors: Analyzing DNA and Protein Interaction Motifs

    PubMed Central

    Guzina, Jelena

    2016-01-01

    ABSTRACT Extracytoplasmic function (ECF) σ factors are the largest and the most diverse group of alternative σ factors, but their mechanisms of transcription are poorly studied. This subfamily is considered to exhibit a rigid promoter structure and an absence of mixing and matching; both −35 and −10 elements are considered necessary for initiating transcription. This paradigm, however, is based on very limited data, which bias the analysis of diverse ECF σ subgroups. Here we investigate DNA and protein recognition motifs involved in ECF σ factor transcription by a computational analysis of canonical ECF subfamily members, much less studied ECF σ subgroups, and the group outliers, obtained from recently sequenced bacteriophages. The analysis identifies an extended −10 element in promoters for phage ECF σ factors; a comparison with bacterial σ factors points to a putative 6-amino-acid motif just C-terminal of domain σ2, which is responsible for the interaction with the identified extension of the −10 element. Interestingly, a similar protein motif is found C-terminal of domain σ2 in canonical ECF σ factors, at a position where it is expected to interact with a conserved motif further upstream of the −10 element. Moreover, the phiEco32 ECF σ factor lacks a recognizable −35 element and σ4 domain, which we identify in a homologous phage, 7-11, indicating that the extended −10 element can compensate for the lack of −35 element interactions. Overall, the results reveal greater flexibility in promoter recognition by ECF σ factors than previously recognized and raise the possibility that mixing and matching also apply to this group, a notion that remains to be biochemically tested. IMPORTANCE ECF σ factors are the most numerous group of alternative σ factors but have been little studied. Their promoter recognition mechanisms are obscured by the large diversity within the ECF σ factor group and the limited similarity with the well

  7. A novel lumazine synthase molecule from Brucella significantly promotes the immune-stimulation effects of antigenic protein.

    PubMed

    Du, Z Q; Wang, J Y

    2015-10-27

    Brucella, an intracellular parasite that infects some livestock and humans, can damage or destroy the reproductive system of livestock. The syndrome is referred to as brucellosis and often occurs in pastoral areas; it is contagious from livestock to humans. In this study, the intact Brucella suis outer membrane protein 31 (omp31) gene was cloned, recombinantly expressed, and examined as a subunit vaccine candidate. The intact Brucella lumazine synthase (bls) gene was cloned and recombinantly expressed to study polymerization function in vitro. Non-reducing gel electrophoresis showed that rBs-BLS existed in different forms in vitro, including as a dimer and a pentamer. An enzyme-linked immunosorbent assay result showed that rOmp31 protein could induce production of an antibody in rabbits. However, the rOmp31-BLS fusion protein could elicit a much higher antibody titer in rabbits; this construct involved fusion of the Omp31 molecule with the BLS molecule. Our results indicate that Omp31 is involved in immune stimulation, while BLS has a polymerizing function based on rOmp31-BLS fusion protein immunogenicity. These data suggest that Omp31 is an ideal subunit vaccine candidate and that the BLS molecule is a favorable transport vector for antigenic proteins.

  8. Heterologous protein expression in Trichoderma reesei using the cbhII promoter.

    PubMed

    Meng, Fanju; Wei, Dongzhi; Wang, Wei

    2013-09-01

    To express homologous or heterologous proteins in fungi, a protein expression system using the promoter of cellobiohydrolase II gene (cbhII) was constructed by generating an expression vector called pWEIIF00. The obtained vector possesses the left and right borders, a hygromycin phosphotransferase B selective marker and a strong promoter and terminator of cbhII from Trichoderma reesei. It can easily undergo random recombination. The applicability of the vector was tested by red fluorescent protein gene (DsRed2) expression detection in T. reesei Rut C30. Using this system, a recombinant Cel5A variant, N342R (Qin et al., 2008), was then selected to express in Rut-C30. Compared to that of the parent strain, integration of the N342R gene resulted in 31.09% increased carboxymethyl-cellulose-degrading (CMCase) activity at pH 5.0 and 56.06% increased activity at pH 6.0. The increased CMCase activity of the recombinant strains would be beneficial for its application uses in multiple industries. The vector constructed in this study can used in fungi to produce industrial proteins.

  9. The Arabidopsis PLAT domain protein1 promotes abiotic stress tolerance and growth in tobacco.

    PubMed

    Hyun, Tae Kyung; Albacete, Alfonso; van der Graaff, Eric; Eom, Seung Hee; Großkinsky, Dominik K; Böhm, Hannah; Janschek, Ursula; Rim, Yeonggil; Ali, Walid Wahid; Kim, Soo Young; Roitsch, Thomas

    2015-08-01

    Plant growth and consequently crop yield can be severely compromised by abiotic and biotic stress conditions. Transgenic approaches that resulted in increased tolerance against abiotic stresses often were typically accompanied by adverse effects on plant growth and fitness under optimal growing conditions. Proteins that belong to the PLAT-plant-stress protein family harbour a single PLAT (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase) domain and are ubiquitously present in monocot and dicot plant species. Until now, only limited data is available for PLAT-plant-stress family members, which suggested that these proteins in general could promote tolerance towards stress responses. We studied the function of the Arabidopsis PLAT-plant-stress protein AtPLAT1 employing heterologous gain-of-function analysis in tobacco. AtPLAT1 conferred increased abiotic stress tolerance in tobacco, evident by improved tolerance towards cold, drought and salt stresses, and promoted growth, reflected by a faster development under non-stressed conditions. However, the overexpression of AtPLAT1 in tobacco reduced the tolerance towards biotic stress conditions and, therefore, could be involved in regulating the crosstalk between abiotic and biotic stress responses. Thus, we showed that heterologously expressed AtPLAT1 functions as positive regulator of abiotic stress tolerance and plant growth, which could be an important new asset for strategies to develop plants with improved abiotic stress tolerance, without growth and subsequent yield penalties under optimal growth conditions.

  10. A liver stress-endocrine nexus promotes metabolic integrity during dietary protein dilution.

    PubMed

    Maida, Adriano; Zota, Annika; Sjøberg, Kim A; Schumacher, Jonas; Sijmonsma, Tjeerd P; Pfenninger, Anja; Christensen, Marie M; Gantert, Thomas; Fuhrmeister, Jessica; Rothermel, Ulrike; Schmoll, Dieter; Heikenwälder, Mathias; Iovanna, Juan L; Stemmer, Kerstin; Kiens, Bente; Herzig, Stephan; Rose, Adam J

    2016-09-01

    Dietary protein intake is linked to an increased incidence of type 2 diabetes (T2D). Although dietary protein dilution (DPD) can slow the progression of some aging-related disorders, whether this strategy affects the development and risk for obesity-associated metabolic disease such as T2D is unclear. Here, we determined that DPD in mice and humans increases serum markers of metabolic health. In lean mice, DPD promoted metabolic inefficiency by increasing carbohydrate and fat oxidation. In nutritional and polygenic murine models of obesity, DPD prevented and curtailed the development of impaired glucose homeostasis independently of obesity and food intake. DPD-mediated metabolic inefficiency and improvement of glucose homeostasis were independent of uncoupling protein 1 (UCP1), but required expression of liver-derived fibroblast growth factor 21 (FGF21) in both lean and obese mice. FGF21 expression and secretion as well as the associated metabolic remodeling induced by DPD also required induction of liver-integrated stress response-driven nuclear protein 1 (NUPR1). Insufficiency of select nonessential amino acids (NEAAs) was necessary and adequate for NUPR1 and subsequent FGF21 induction and secretion in hepatocytes in vitro and in vivo. Taken together, these data indicate that DPD promotes improved glucose homeostasis through an NEAA insufficiency-induced liver NUPR1/FGF21 axis.

  11. Type II integral membrane protein, TM of J paramyxovirus promotes cell-to-cell fusion.

    PubMed

    Li, Zhuo; Hung, Cher; Paterson, Reay G; Michel, Frank; Fuentes, Sandra; Place, Ryan; Lin, Yuan; Hogan, Robert J; Lamb, Robert A; He, Biao

    2015-10-06

    Paramyxoviruses include many important animal and human pathogens. Most paramyxoviruses have two integral membrane proteins: fusion protein (F) and attachment proteins hemagglutinin, hemagglutinin-neuraminidase, or glycoprotein (G), which are critical for viral entry into cells. J paramyxovirus (JPV) encodes four integral membrane proteins: F, G, SH, and transmembrane (TM). The function of TM is not known. In this work, we have generated a viable JPV lacking TM (JPV∆TM). JPV∆TM formed opaque plaques compared with JPV. Quantitative syncytia assays showed that JPV∆TM was defective in promoting cell-to-cell fusion (i.e., syncytia formation) compared with JPV. Furthermore, cells separately expressing F, G, TM, or F plus G did not form syncytia whereas cells expressing F plus TM formed some syncytia. However, syncytia formation was much greater with coexpression of F, G, and TM. Biochemical analysis indicates that F, G, and TM interact with each other. A small hydrophobic region in the TM ectodomain from amino acid residues 118 to 132, the hydrophobic loop (HL), was important for syncytial promotion, suggesting that the TM HL region plays a critical role in cell-to-cell fusion.

  12. A liver stress-endocrine nexus promotes metabolic integrity during dietary protein dilution

    PubMed Central

    Maida, Adriano; Zota, Annika; Sjøberg, Kim A.; Sijmonsma, Tjeerd P.; Pfenninger, Anja; Christensen, Marie M.; Gantert, Thomas; Fuhrmeister, Jessica; Rothermel, Ulrike; Schmoll, Dieter; Heikenwälder, Mathias; Iovanna, Juan L.; Stemmer, Kerstin; Herzig, Stephan; Rose, Adam J.

    2016-01-01

    Dietary protein intake is linked to an increased incidence of type 2 diabetes (T2D). Although dietary protein dilution (DPD) can slow the progression of some aging-related disorders, whether this strategy affects the development and risk for obesity-associated metabolic disease such as T2D is unclear. Here, we determined that DPD in mice and humans increases serum markers of metabolic health. In lean mice, DPD promoted metabolic inefficiency by increasing carbohydrate and fat oxidation. In nutritional and polygenic murine models of obesity, DPD prevented and curtailed the development of impaired glucose homeostasis independently of obesity and food intake. DPD-mediated metabolic inefficiency and improvement of glucose homeostasis were independent of uncoupling protein 1 (UCP1), but required expression of liver-derived fibroblast growth factor 21 (FGF21) in both lean and obese mice. FGF21 expression and secretion as well as the associated metabolic remodeling induced by DPD also required induction of liver-integrated stress response–driven nuclear protein 1 (NUPR1). Insufficiency of select nonessential amino acids (NEAAs) was necessary and adequate for NUPR1 and subsequent FGF21 induction and secretion in hepatocytes in vitro and in vivo. Taken together, these data indicate that DPD promotes improved glucose homeostasis through an NEAA insufficiency–induced liver NUPR1/FGF21 axis. PMID:27548521

  13. Ubiquitin-like protein UBL5 promotes the functional integrity of the Fanconi anemia pathway.

    PubMed

    Oka, Yasuyoshi; Bekker-Jensen, Simon; Mailand, Niels

    2015-05-12

    Ubiquitin and ubiquitin-like proteins (UBLs) function in a wide array of cellular processes. UBL5 is an atypical UBL that does not form covalent conjugates with cellular proteins and which has a known role in modulating pre-mRNA splicing. Here, we report an unexpected involvement of human UBL5 in promoting the function of the Fanconi anemia (FA) pathway for repair of DNA interstrand crosslinks (ICLs), mediated by a specific interaction with the central FA pathway component FANCI. UBL5-deficient cells display spliceosome-independent reduction of FANCI protein stability, defective FANCI function in response to DNA damage and hypersensitivity to ICLs. By mapping the sequence determinants underlying UBL5-FANCI binding, we generated separation-of-function mutants to demonstrate that key aspects of FA pathway function, including FANCI-FANCD2 heterodimerization, FANCD2 and FANCI monoubiquitylation and maintenance of chromosome stability after ICLs, are compromised when the UBL5-FANCI interaction is selectively inhibited by mutations in either protein. Together, our findings establish UBL5 as a factor that promotes the functionality of the FA DNA repair pathway.

  14. Tumor suppressor protein C53 antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation.

    PubMed

    Jiang, Hai; Wu, Jianchun; He, Chen; Yang, Wending; Li, Honglin

    2009-04-01

    Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint. More recently, Wang et al. (2007) found that C53/LZAP may function as a tumor suppressor by way of inhibiting NF-kappaB signaling. We report here the identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexpression. Intriguingly, we found that C53 interacts with Chk1 and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell-cycle progression and DNA damage response.

  15. DMA Modulus as a Screening Parameter for Compatibility of Polymeric Containment Materials with Various Solutions for use in Space Shuttle Microgravity Protein Crystal Growth (PCG) Experiments

    NASA Technical Reports Server (NTRS)

    Wingard, Charles Doug; Munafo, Paul M. (Technical Monitor)

    2002-01-01

    Protein crystals are grown in microgravity experiments inside the Space Shuttle during orbit. Such crystals are basically grown in a five-component system containing a salt, buffer, polymer, organic and water. During these experiments, a number of different polymeric containment materials must be compatible with up to hundreds of different PCG solutions in various concentrations for durations up to 180 days. When such compatibility experiments are performed at NASA/MSFC (Marshall Space Flight Center) simultaneously on containment material samples immersed in various solutions in vials, the samples are rather small out of necessity. DMA4 modulus was often used as the primary screening parameter for such small samples as a pass/fail criterion for incompatibility issues. In particular, the TA Instruments DMA 2980 film tension clamp was used to test rubber O-rings as small in I.D. as 0.091 in. by cutting through the cross-section at one place, then clamping the stretched linear cord stock at each end. The film tension clamp was also used to successfully test short length samples of medical/surgical grade tubing with an O.D. of 0.125 in.

  16. Method of preparing water purification membranes. [polymerization of allyl amine as thin films in plasma discharge

    NASA Technical Reports Server (NTRS)

    Hollahan, J. R.; Wydeven, T. J., Jr. (Inventor)

    1974-01-01

    Allyl amine and chemically related compounds are polymerized as thin films in the presence of a plasma discharge. The monomer compound can be polymerized by itself or in the presence of an additive gas to promote polymerization and act as a carrier. The polymerized films thus produced show outstanding advantages when used as reverse osmosis membranes.

  17. Gadd45a Protein Promotes Skeletal Muscle Atrophy by Forming a Complex with the Protein Kinase MEKK4*♦

    PubMed Central

    Bullard, Steven A.; Seo, Seongjin; Schilling, Birgit; Dyle, Michael C.; Dierdorff, Jason M.; Ebert, Scott M.; DeLau, Austin D.; Gibson, Bradford W.; Adams, Christopher M.

    2016-01-01

    Skeletal muscle atrophy is a serious and highly prevalent condition that remains poorly understood at the molecular level. Previous work found that skeletal muscle atrophy involves an increase in skeletal muscle Gadd45a expression, which is necessary and sufficient for skeletal muscle fiber atrophy. However, the direct mechanism by which Gadd45a promotes skeletal muscle atrophy was unknown. To address this question, we biochemically isolated skeletal muscle proteins that associate with Gadd45a as it induces atrophy in mouse skeletal muscle fibers in vivo. We found that Gadd45a interacts with multiple proteins in skeletal muscle fibers, including, most prominently, MEKK4, a mitogen-activated protein kinase kinase kinase that was not previously known to play a role in skeletal muscle atrophy. Furthermore, we found that, by forming a complex with MEKK4 in skeletal muscle fibers, Gadd45a increases MEKK4 protein kinase activity, which is both sufficient to induce skeletal muscle fiber atrophy and required for Gadd45a-mediated skeletal muscle fiber atrophy. Together, these results identify a direct biochemical mechanism by which Gadd45a induces skeletal muscle atrophy and provide new insight into the way that skeletal muscle atrophy occurs at the molecular level. PMID:27358404

  18. Gadd45a Protein Promotes Skeletal Muscle Atrophy by Forming a Complex with the Protein Kinase MEKK4.

    PubMed

    Bullard, Steven A; Seo, Seongjin; Schilling, Birgit; Dyle, Michael C; Dierdorff, Jason M; Ebert, Scott M; DeLau, Austin D; Gibson, Bradford W; Adams, Christopher M

    2016-08-19

    Skeletal muscle atrophy is a serious and highly prevalent condition that remains poorly understood at the molecular level. Previous work found that skeletal muscle atrophy involves an increase in skeletal muscle Gadd45a expression, which is necessary and sufficient for skeletal muscle fiber atrophy. However, the direct mechanism by which Gadd45a promotes skeletal muscle atrophy was unknown. To address this question, we biochemically isolated skeletal muscle proteins that associate with Gadd45a as it induces atrophy in mouse skeletal muscle fibers in vivo We found that Gadd45a interacts with multiple proteins in skeletal muscle fibers, including, most prominently, MEKK4, a mitogen-activated protein kinase kinase kinase that was not previously known to play a role in skeletal muscle atrophy. Furthermore, we found that, by forming a complex with MEKK4 in skeletal muscle fibers, Gadd45a increases MEKK4 protein kinase activity, which is both sufficient to induce skeletal muscle fiber atrophy and required for Gadd45a-mediated skeletal muscle fiber atrophy. Together, these results identify a direct biochemical mechanism by which Gadd45a induces skeletal muscle atrophy and provide new insight into the way that skeletal muscle atrophy occurs at the molecular level. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Human Herpesvirus 8 Interleukin-6 Interacts with Calnexin Cycle Components and Promotes Protein Folding.

    PubMed

    Chen, Daming; Xiang, Qiwang; Nicholas, John

    2017-09-06

    Viral interleukin-6 (vIL-6) encoded by human herpesvirus 8 (HHV-8) is believed to contribute via mitogenic, survival, and angiogenic activities to HHV-8-associated Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease through autocrine or paracrine mechanisms during latency or productive replication. There is direct evidence that vIL-6 promotes latently infected PEL cell viability and proliferation and also viral productive replication in PEL and endothelial cells. These activities are mediated largely through endoplasmic reticulum (ER)-localized vIL-6, which can induce signal transduction via the gp130 signaling receptor, activating mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription (STAT) signaling, and interactions of vIL-6 with the ER membrane protein vitamin K epoxide reductase complex subunit 1 variant 2 (VKORC1v2). The latter functional axis involves suppression of pro-apoptotic lysosomal protein cathepsin D by promotion of the ER-associated degradation of ER-transiting, pre-proteolytically processed pro-cathepsin D. Other interactions of VKORC1v2 and activities of vIL-6 via the receptor have not been reported. Here, we show that both vIL-6 and VKORC1v2 interact with calnexin cycle proteins UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1), catalyzing monoglucosylation of N-glycans, and oppositely-acting glucosidase II (GlucII) and that vIL-6 can promote protein folding. This activity was found to require VKORC1v2 and UGGT1, to involve vIL-6 associations with VKORC1v2, UGGT1 and GlucII, and to operate in the context of productively infected cells. These findings document new VKORC1v2-associated interactions and activities of vIL-6, revealing novel mechanisms of vIL-6 function within the ER compartment.IMPORTANCE HHV-8 vIL-6 pro-survival (latent) and pro-replication functions are mediated from the ER compartment through both gp130 receptor-mediated signal transduction and

  20. Antibodies against a Surface Protein of Streptococcus pyogenes Promote a Pathological Inflammatory Response

    PubMed Central

    Kahn, Fredrik; Mörgelin, Matthias; Shannon, Oonagh; Norrby-Teglund, Anna; Herwald, Heiko; Olin, Anders I.; Björck, Lars

    2008-01-01

    Streptococcal toxic shock syndrome (STSS) caused by Streptococcus pyogenes is a clinical condition with a high mortality rate despite modern intensive care. A key feature of STSS is excessive plasma leakage leading to hypovolemic hypotension, disturbed microcirculation and multiorgan failure. Previous work has identified a virulence mechanism in STSS where M1 protein of S. pyogenes forms complexes with fibrinogen that activate neutrophils to release heparin-binding protein (HBP), an inducer of vascular leakage. Here, we report a marked inter-individual difference in the response to M1 protein–induced HBP release, a difference found to be related to IgG antibodies directed against the central region of the M1 protein. To elicit massive HBP release, such antibodies need to be part of the M1 protein–fibrinogen complexes. The data add a novel aspect to bacterial pathogenesis where antibodies contribute to the severity of disease by promoting a pathologic inflammatory response. PMID:18787689

  1. CDK6 binds and promotes the degradation of the EYA2 protein

    PubMed Central

    Kohrt, Dawn; Crary, Jennifer; Zimmer, Marc; Patrick, Aaron N; Ford, Heide L; Hinds, Philip W; Grossel, Martha J

    2014-01-01

    Cyclin-dependent kinase 6 (Cdk6) is a D-Cyclin-activated kinase that is directly involved in driving the cell cycle through inactivation of pRB in G1 phase. Increasingly, evidence suggests that CDK6, while directly driving the cell cycle, may only be essential for proliferation of specialized cell types, agreeing with the notion that CDK6 also plays an important role in differentiation. Here, evidence is presented that CDK6 binds to and promotes degradation of the EYA2 protein. The EYA proteins are a family of proteins that activate genes essential for the development of multiple organs, regulate cell proliferation, and are misregulated in several types of cancer. This interaction suggests that CDK6 regulates EYA2 activity, a mechanism that could be important in development and in cancer. PMID:24196439

  2. Effects of polymeric formula vs elemental formula in neonatal piglets with short bowel syndrome.

    PubMed

    Hua, Zheng; Turner, Justine M; Mager, Diana R; Sigalet, David L; Wizzard, Pamela R; Nation, Patrick N; Ball, Ron O; Pencharz, Paul B; Wales, Paul W

    2014-05-01

    Intestinal adaptation is important for recovery in short bowel syndrome (SBS). This process is dependent on the presence of enteral nutrition (EN) and trophic factors, such as glucagon-like peptide-2 (GLP-2). In clinical practice, elemental formula is often used to feed neonates with SBS, whereas animal studies suggest polymeric formula promotes better intestinal adaptation. In neonatal piglet models of SBS, with or without ileum, we compared the elemental with the polymeric formula, including the effect on endogenous GLP-2. Forty-eight piglets underwent 75% mid-intestinal resection with jejunoileal anastomosis, 75% distal-intestinal resection with jejunocolic anastomosis (JC), or sham without resection. Parenteral nutrition (PN) started postoperatively, tapering as EN was increased, according to clinical criteria, based on diarrhea and weight. Within groups, piglets were randomized to an isocaloric/isonitrogenous elemental (amino acid) or polymeric (intact protein) diet. Plasma GLP-2 and histology for adaptation were measured at 14 days. Within both SBS and control groups, no difference in adaptation was observed according to diet. A difference was observed only within the JC piglet group with regard to clinical outcomes. In these piglets, compared with elemental formula, the polymeric formula was associated with more diarrhea ( P = .023) and longer duration of PN support (P = .047). An overall benefit of the polymeric formula over the elemental formula on gut adaptation was not observed. Furthermore, SBS piglets without ileum had less ability to tolerate polymeric formula, contributing to more days of PN support.

  3. Promotion of beta-glucan synthase activity in corn microsomal membranes by calcium and protein phosphorylation

    NASA Technical Reports Server (NTRS)

    Paliyath, G.; Poovaiah, B. W.

    1988-01-01

    Regulation of the activity of beta-glucan synthase was studied using microsomal preparations from corn coleoptiles. The specific activity as measured by the incorporation of glucose from uridine diphospho-D-[U-14C]glucose varied between 5 to 15 pmol (mg protein)-1 min-1. Calcium promoted beta-glucan synthase activity and the promotion was observed at free calcium concentrations as low as 1 micromole. Kinetic analysis of substrate-velocity curve showed an apparent Km of 1.92 x 10(-4) M for UDPG. Calcium increased the Vmax from 5.88 x 10(-7) mol liter-1 min-1 in the absence of calcium to 9.52 x 10(-7) mol liter-1 min-1 and 1.66 x 10(-6) mol liter-1 min-1 in the presence of 0.5 mM and 1 mM calcium, respectively. The Km values remained the same under these conditions. Addition of ATP further increased the activity above the calcium-promoted level. Sodium fluoride, a phosphoprotein phosphatase inhibitor, promoted glucan synthase activity indicating that phosphorylation and dephosphorylation are involved in the regulation of the enzyme activity. Increasing the concentration of sodium fluoride from 0.25 mM to 10 mM increased glucan synthase activity five-fold over the + calcium + ATP control. Phosphorylation of membrane proteins also showed a similar increase under these conditions. Calmodulin, in the presence of calcium and ATP stimulated glucan synthase activity substantially, indicating that calmodulin could be involved in the calcium-dependent phosphorylation and promotion of beta-glucan synthase activity. The role of calcium in mediating auxin action is discussed.

  4. A phagemid vector using the E. coli phage shock promoter facilitates phage display of toxic proteins.

    PubMed

    Beekwilder, J; Rakonjac, J; Jongsma, M; Bosch, D

    1999-03-04

    Phage display is a powerful tool with which to adapt the specificity of protease inhibitors. To this end, a library of variants of the potato protease inhibitor PI2 was introduced in a canonical phagemid vector. Although PI2 is a natural trypsin inhibitor, we were unable to select trypsin-binding variants from the library. Instead, only mutants carrying deletions or amber stop codons were found. Bacteria carrying these mutations had a much faster growth rate than those carrying the wt PI2-encoding gene, even when the promoter was repressed. To overcome these problems, two new phagemid vectors for g3-mediated phage display were constructed. The first vector has a lower plasmid copy number, as compared to the canonical vector. Bacteria harboring this new vector are much less affected by the presence of the PI2-g3 fusion gene, which appears from a markedly reduced growth retardation. A second vector was equipped with the promoter of the Escherichia coli psp operon, instead of the lac promoter, to control the PI2-g3 gene fusion expression. The psp promoter is induced upon helper phage infection. A phagemid vector with this promoter controlling a PI2-g3 gene fusion did not affect the viability of the host. Furthermore, both new vectors were shown to produce phage particles that display the inhibitor protein and were therefore considered suitable for phage display. The inhibitor library was introduced in both new vectors. Trypsin-binding phages with inhibitory sequences were selected, instead of sequences with stop codons or deletions. This demonstrates the usefulness of these new vectors for phage display of proteins that affect the viability of E. coli.

  5. Promotion of beta-glucan synthase activity in corn microsomal membranes by calcium and protein phosphorylation

    NASA Technical Reports Server (NTRS)

    Paliyath, G.; Poovaiah, B. W.

    1988-01-01

    Regulation of the activity of beta-glucan synthase was studied using microsomal preparations from corn coleoptiles. The specific activity as measured by the incorporation of glucose from uridine diphospho-D-[U-14C]glucose varied between 5 to 15 pmol (mg protein)-1 min-1. Calcium promoted beta-glucan synthase activity and the promotion was observed at free calcium concentrations as low as 1 micromole. Kinetic analysis of substrate-velocity curve showed an apparent Km of 1.92 x 10(-4) M for UDPG. Calcium increased the Vmax from 5.88 x 10(-7) mol liter-1 min-1 in the absence of calcium to 9.52 x 10(-7) mol liter-1 min-1 and 1.66 x 10(-6) mol liter-1 min-1 in the presence of 0.5 mM and 1 mM calcium, respectively. The Km values remained the same under these conditions. Addition of ATP further increased the activity above the calcium-promoted level. Sodium fluoride, a phosphoprotein phosphatase inhibitor, promoted glucan synthase activity indicating that phosphorylation and dephosphorylation are involved in the regulation of the enzyme activity. Increasing the concentration of sodium fluoride from 0.25 mM to 10 mM increased glucan synthase activity five-fold over the + calcium + ATP control. Phosphorylation of membrane proteins also showed a similar increase under these conditions. Calmodulin, in the presence of calcium and ATP stimulated glucan synthase activity substantially, indicating that calmodulin could be involved in the calcium-dependent phosphorylation and promotion of beta-glucan synthase activity. The role of calcium in mediating auxin action is discussed.

  6. Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Ion Channel Activity Promotes Virus Fitness and Pathogenesis

    PubMed Central

    Nieto-Torres, Jose L.; DeDiego, Marta L.; Verdiá-Báguena, Carmina; Jimenez-Guardeño, Jose M.; Regla-Nava, Jose A.; Fernandez-Delgado, Raul; Castaño-Rodriguez, Carlos; Alcaraz, Antonio; Torres, Jaume; Aguilella, Vicente M.; Enjuanes, Luis

    2014-01-01

    Deletion of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) envelope (E) gene attenuates the virus. E gene encodes a small multifunctional protein that possesses ion channel (IC) activity, an important function in virus-host interaction. To test the contribution of E protein IC activity in virus pathogenesis, two recombinant mouse-adapted SARS-CoVs, each containing one single amino acid mutation that suppressed ion conductivity, were engineered. After serial infections, mutant viruses, in general, incorporated compensatory mutations within E gene that rendered active ion channels. Furthermore, IC activity conferred better fitness in competition assays, suggesting that ion conductivity represents an advantage for the virus. Interestingly, mice infected with viruses displaying E protein IC activity, either with the wild-type E protein sequence or with the revertants that restored ion transport, rapidly lost weight and died. In contrast, mice infected with mutants lacking IC activity, which did not incorporate mutations within E gene during the experiment, recovered from disease and most survived. Knocking down E protein IC activity did not significantly affect virus growth in infected mice but decreased edema accumulation, the major determinant of acute respiratory distress syndrome (ARDS) leading to death. Reduced edema correlated with lung epithelia integrity and proper localization of Na+/K+ ATPase, which participates in edema resolution. Levels of inflammasome-activated IL-1β were reduced in the lung airways of the animals infected with viruses lacking E protein IC activity, indicating that E protein IC function is required for inflammasome activation. Reduction of IL-1β was accompanied by diminished amounts of TNF and IL-6 in the absence of E protein ion conductivity. All these key cytokines promote the progression of lung damage and ARDS pathology. In conclusion, E protein IC activity represents a new determinant for SARS-CoV virulence. PMID:24788150

  7. Pbx homeodomain proteins direct Myod activity to promote fast-muscle differentiation.

    PubMed

    Maves, Lisa; Waskiewicz, Andrew Jan; Paul, Biswajit; Cao, Yi; Tyler, Ashlee; Moens, Cecilia B; Tapscott, Stephen J

    2007-09-01

    The basic helix-loop-helix (bHLH) transcription factor Myod directly regulates gene expression throughout the program of skeletal muscle differentiation. It is not known how a Myod-driven myogenic program is modulated to achieve muscle fiber-type-specific gene expression. Pbx homeodomain proteins mark promoters of a subset of Myod target genes, including myogenin (Myog); thus, Pbx proteins might modulate the program of myogenesis driven by Myod. By inhibiting Pbx function in zebrafish embryos, we show that Pbx proteins are required in order for Myod to induce the expression of a subset of muscle genes in the somites. In the absence of Pbx function, expression of myog and of fast-muscle genes is inhibited, whereas slow-muscle gene expression appears normal. By knocking down Pbx or Myod function in combination with another bHLH myogenic factor, Myf5, we show that Pbx is required for Myod to regulate fast-muscle, but not slow-muscle, development. Furthermore, we show that Sonic hedgehog requires Myod in order to induce both fast- and slow-muscle markers but requires Pbx only to induce fast-muscle markers. Our results reveal that Pbx proteins modulate Myod activity to drive fast-muscle gene expression, thus showing that homeodomain proteins can direct bHLH proteins to establish a specific cell-type identity.

  8. HyCCAPP as a tool to characterize promoter DNA-protein interactions in Saccharomyces cerevisiae.

    PubMed

    Guillen-Ahlers, Hector; Rao, Prahlad K; Levenstein, Mark E; Kennedy-Darling, Julia; Perumalla, Danu S; Jadhav, Avinash Y L; Glenn, Jeremy P; Ludwig-Kubinski, Amy; Drigalenko, Eugene; Montoya, Maria J; Göring, Harald H; Anderson, Corianna D; Scalf, Mark; Gildersleeve, Heidi I S; Cole, Regina; Greene, Alexandra M; Oduro, Akua K; Lazarova, Katarina; Cesnik, Anthony J; Barfknecht, Jared; Cirillo, Lisa A; Gasch, Audrey P; Shortreed, Michael R; Smith, Lloyd M; Olivier, Michael

    2016-06-01

    Currently available methods for interrogating DNA-protein interactions at individual genomic loci have significant limitations, and make it difficult to work with unmodified cells or examine single-copy regions without specific antibodies. In this study, we describe a physiological application of the Hybridization Capture of Chromatin-Associated Proteins for Proteomics (HyCCAPP) methodology we have developed. Both novel and known locus-specific DNA-protein interactions were identified at the ENO2 and GAL1 promoter regions of Saccharomyces cerevisiae, and revealed subgroups of proteins present in significantly different levels at the loci in cells grown on glucose versus galactose as the carbon source. Results were validated using chromatin immunoprecipitation. Overall, our analysis demonstrates that HyCCAPP is an effective and flexible technology that does not require specific antibodies nor prior knowledge of locally occurring DNA-protein interactions and can now be used to identify changes in protein interactions at target regions in the genome in response to physiological challenges. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Proteins of the Rpf (resuscitation promoting factor) family are peptidoglycan hydrolases.

    PubMed

    Telkov, M V; Demina, G R; Voloshin, S A; Salina, E G; Dudik, T V; Stekhanova, T N; Mukamolova, G V; Kazaryan, K A; Goncharenko, A V; Young, M; Kaprelyants, A S

    2006-04-01

    The secreted Micrococcus luteus protein, Rpf, is required for successful resuscitation of dormant "non-culturable" M. luteus cells and for growth stimulation in poor media. The biochemical mechanism of Rpf action remained unknown. Theoretical predictions of Rpf domain architecture and organization, together with a recent NMR analysis of the protein structure, indicate that the conserved Rpf domain has a lysozyme-like fold. In the present study, we found that both the secreted native protein and the recombinant protein lyse crude preparations of M. luteus cell walls. They also hydrolyze 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside, a synthetic substrate for peptidoglycan muramidases, with optimum activity at pH 6. The Rpf protein also has weak proteolytic activity against N-CBZ-Gly-Gly-Arg-beta-naphthylamide, a substrate for trypsin-like enzymes. Rpf activity towards 4-methylumbelliferyl-beta-D-N,N',N''-triacetylchitotrioside was reduced when the glutamate residue at position 54, invariant for all Rpf family proteins and presumably involved in catalysis, was altered. The same amino acid substitution resulted in impaired resuscitation activity of Rpf. The data indicate that Rpf is a peptidoglycan-hydrolyzing enzyme, and strongly suggest that this specific activity is responsible for its growth promotion and resuscitation activity. A possible mechanism of Rpf-mediated resuscitation is discussed.

  10. Proteins dominate in the surface layers formed on materials exposed to extracellular polymeric substances from bacterial cultures.

    PubMed

    Yang, Yi; Wikieł, Agata J; Dall'Agnol, Leonardo T; Eloy, Pierre; Genet, Michel J; Moura, José J G; Sand, Wolfgang; Dupont-Gillain, Christine C; Rouxhet, Paul G

    2016-01-01

    The chemical compositions of the surface conditioning layers formed by different types of solutions (from isolated EPS to whole culture media), involving different bacterial strains relevant for biocorrosion were compared, as they may influence the initial step in biofilm formation. Different substrata (polystyrene, glass, steel) were conditioned and analyzed by X-ray photoelectron spectroscopy. Peak decomposition and assignment were validated by correlations between independent spectral data and the ubiquitous presence of organic contaminants on inorganic substrata was taken into account. Proteins or peptides were found to be a major constituent of all conditioning layers and polysaccharides were not present in appreciable concentrations; the proportion of nitrogen which may be due to DNA was lower than 15%. There was no significant difference between the compositions of the adlayers formed from different conditioning solutions, except for the adlayers produced with tightly bound EPS extracted from D. alaskensis.

  11. PSEUDO RESPONSE REGULATORs stabilize CONSTANS protein to promote flowering in response to day length.

    PubMed

    Hayama, Ryosuke; Sarid-Krebs, Liron; Richter, René; Fernández, Virginia; Jang, Seonghoe; Coupland, George

    2017-04-03

    Seasonal reproduction in many organisms requires detection of day length. This is achieved by integrating information on the light environment with an internal photoperiodic time-keeping mechanism. Arabidopsis thaliana promotes flowering in response to long days (LDs), and CONSTANS (CO) transcription factor represents a photoperiodic timer whose stability is higher when plants are exposed to light under LDs. Here, we show that PSEUDO RESPONSE REGULATOR (PRR) proteins directly mediate this stabilization. PRRs interact with and stabilize CO at specific times during the day, thereby mediating its accumulation under LDs. PRR-mediated stabilization increases binding of CO to the promoter of FLOWERING LOCUS T (FT), leading to enhanced FT transcription and early flowering under these conditions. PRRs were previously reported to contribute to timekeeping by regulating CO transcription through their roles in the circadian clock. We propose an additional role for PRRs in which they act upon CO protein to promote flowering, directly coupling information on light exposure to the timekeeper and allowing recognition of LDs.

  12. Ixodes scapularis dystroglycan-like protein promotes Borrelia burgdorferi migration from the gut.

    PubMed

    Coumou, Jeroen; Narasimhan, Sukanya; Trentelman, Jos J; Wagemakers, Alex; Koetsveld, Joris; Ersoz, Jasmin I; Oei, Anneke; Fikrig, Erol; Hovius, Joppe W

    2016-03-01

    The causative agent of Lyme borreliosis, Borrelia burgdorferi, is transmitted by Ixodes ticks. During tick feeding, B. burgdorferi migrates from the tick gut to the salivary glands from where transmission to the host occurs. B. burgdorferi-interacting tick proteins might serve as vaccine targets to thwart B. burgdorferi transmission. A previous screening for B. burgdorferi-interacting Ixodes scapularis gut proteins identified an I. scapularis putative dystroglycan protein (ISCW015049). Here, we describe the ISCW015049's protein structure and its cellular location in the tick gut in relation to B. burgdorferi migration. Secondly, in vivo B. burgdorferi-tick attachment murine models were performed to study the role of ISCW015049 during B. burgdorferi migration and transmission. In silico analysis confirmed that ISCW015049 is similar to dystroglycan and was named I. scapularis dystroglycan-like protein (ISDLP). Confocal microscopy of gut tissue showed that ISDLP is expressed on the surface of gut cells, is upregulated during tick feeding, and is expressed significantly higher in infected ticks compared to uninfected ticks. Inhibition of ISDLP by RNA interference (RNAi) resulted in lower B. burgdorferi transmission to mice. In conclusion, we have identified a dystroglycan-like protein in I. scapularis gut that can bind to B. burgdorferi and promotes B. burgdorferi migration from the tick gut. Key messages: B. burgdorferi exploits tick proteins to orchestrate its transmission to the host. B. burgdorferi is able bind to an I. scapularis dystroglycan-like protein (ISDLP). Inhibition of ISDLP in ticks results in lower B. burgdorferi transmission to mice. ISDLP is a potential target to prevent Lyme borreliosis.

  13. Dynamic Loading and Unloading of Proteins in Polymeric Stomatocytes: Formation of an Enzyme-Loaded Supramolecular Nanomotor.

    PubMed

    Abdelmohsen, Loai K E A; Nijemeisland, Marlies; Pawar, Gajanan M; Janssen, Geert-Jan A; Nolte, Roeland J M; van Hest, Jan C M; Wilson, Daniela A

    2016-02-23

    Self-powered artificial nanomotors are currently attracting increased interest as mimics of biological motors but also as potential components of nanomachinery, robotics, and sensing devices. We have recently described the controlled shape transformation of polymersomes into bowl-shaped stomatocytes and the assembly of platinum-driven nanomotors. However, the platinum encapsulation inside the structures was low; only 50% of the structures contained the catalyst and required both high fuel concentrations for the propelling of the nanomotors and harsh conditions for the shape transformation. Application of the nanomotors in a biological setting requires the nanomotors to be efficiently propelled by a naturally available energy source and at biological relevant concentrations. Here we report a strategy for enzyme entrapment and nanomotor assembly via controlled and reversible folding of polymersomes into stomatocytes under mild conditions, allowing the encapsulation of the proteins inside the stomach with almost 100% efficiency and retention of activity. The resulting enzyme-driven nanomotors are capable of propelling these structures at low fuel concentrations (hydrogen peroxide or glucose) via a one-enzyme or two-enzyme system. The confinement of the enzymes inside the stomach does not hinder their activity and in fact facilitates the transfer of the substrates, while protecting them from the deactivating influences of the media. This is particularly important for future applications of nanomotors in biological settings especially for systems where fast autonomous movement occurs at physiological concentrations of fuel.

  14. Sumoylation of Rap1 mediates the recruitment of TFIID to promote transcription of ribosomal protein genes.

    PubMed

    Chymkowitch, Pierre; Nguéa, Aurélie P; Aanes, Håvard; Koehler, Christian J; Thiede, Bernd; Lorenz, Susanne; Meza-Zepeda, Leonardo A; Klungland, Arne; Enserink, Jorrit M

    2015-06-01

    Transcription factors are abundant Sumo targets, yet the global distribution of Sumo along the chromatin and its physiological relevance in transcription are poorly understood. Using Saccharomyces cerevisiae, we determined the genome-wide localization of Sumo along the chromatin. We discovered that Sumo-enriched genes are almost exclusively involved in translation, such as tRNA genes and ribosomal protein genes (RPGs). Genome-wide expression analysis showed that Sumo positively regulates their transcription. We also discovered that the Sumo consensus motif at RPG promoters is identical to the DNA binding motif of the transcription factor Rap1. We demonstrate that Rap1 is a molecular target of Sumo and that sumoylation of Rap1 is important for cell viability. Furthermore, Rap1 sumoylation promotes recruitment of the basal transcription machinery, and sumoylation of Rap1 cooperates with the target of rapamycin kinase complex 1 (TORC1) pathway to promote RPG transcription. Strikingly, our data reveal that sumoylation of Rap1 functions in a homeostatic feedback loop that sustains RPG transcription during translational stress. Taken together, Sumo regulates the cellular translational capacity by promoting transcription of tRNA genes and RPGs. © 2015 Chymkowitch et al.; Published by Cold Spring Harbor Laboratory Press.

  15. Sumoylation of Rap1 mediates the recruitment of TFIID to promote transcription of ribosomal protein genes

    PubMed Central

    Chymkowitch, Pierre; Nguéa P, Aurélie; Aanes, Håvard; Koehler, Christian J.; Thiede, Bernd; Lorenz, Susanne; Meza-Zepeda, Leonardo A.; Klungland, Arne; Enserink, Jorrit M.

    2015-01-01

    Transcription factors are abundant Sumo targets, yet the global distribution of Sumo along the chromatin and its physiological relevance in transcription are poorly understood. Using Saccharomyces cerevisiae, we determined the genome-wide localization of Sumo along the chromatin. We discovered that Sumo-enriched genes are almost exclusively involved in translation, such as tRNA genes and ribosomal protein genes (RPGs). Genome-wide expression analysis showed that Sumo positively regulates their transcription. We also discovered that the Sumo consensus motif at RPG promoters is identical to the DNA binding motif of the transcription factor Rap1. We demonstrate that Rap1 is a molecular target of Sumo and that sumoylation of Rap1 is important for cell viability. Furthermore, Rap1 sumoylation promotes recruitment of the basal transcription machinery, and sumoylation of Rap1 cooperates with the target of rapamycin kinase complex 1 (TORC1) pathway to promote RPG transcription. Strikingly, our data reveal that sumoylation of Rap1 functions in a homeostatic feedback loop that sustains RPG transcription during translational stress. Taken together, Sumo regulates the cellular translational capacity by promoting transcription of tRNA genes and RPGs. PMID:25800674

  16. Pax3 and regulation of the melanocyte-specific tyrosinase-related protein-1 promoter.

    PubMed

    Galibert, M D; Yavuzer, U; Dexter, T J; Goding, C R

    1999-09-17

    Previous work has established that the melanocyte-specific tyrosinase-related protein-1 (TRP-1) promoter is regulated positively by the microphthalmia-associated transcription factor Mitf, acting through the conserved M box and negatively by the T-box factor Tbx2, which can bind two "melanocyte-specific elements" termed the MSEu and MSEi. Both the MSEu and MSEi, which share a 6-base pair GTGTGA consensus, are also recognized by a previously unidentified melanocyte-specific factor, MSF. Here we show using a combination of DNA binding assays, proteolytic clipping, and anti-Pax3 antibodies that MSF is indistinguishable from Pax3, a paired homeodomain transcription factor implicated genetically in melanocyte development and the regulation of the Mitf promoter. Consistent with Pax3 being able to bind the TRP-1 promoter, Pax3 is expressed in melanocytes and melanomas, and TRP-1 promoter activity is up-regulated by Pax3. The results identify a novel role for Pax3 in the expression of TRP-1, and the potential role of Pax3 in the melanocyte lineage is discussed.

  17. The Effect of Salts in Promoting Specific and Competitive Interactions between Zinc Finger Proteins and Metals

    NASA Astrophysics Data System (ADS)

    Li, Gongyu; Yuan, Siming; Zheng, Shihui; Chen, Yuting; Zheng, Zhen; Liu, Yangzhong; Huang, Guangming

    2017-09-01

    Specific protein-metal interactions (PMIs) fulfill essential functions in cells and organic bodies, and activation of these functions in vivo are mostly modulated by the complex environmental factors, including pH value, small biomolecules, and salts. Specifically, the role of salts in promoting specific PMIs and their competition among various metals has remained untapped mainly due to the difficulty to distinguish nonspecific PMIs from specific PMIs by classic spectroscopic techniques. Herein, we report Hofmeister salts differentially promote the specific PMIs by combining nanoelectrospray ionization mass spectrometry and spectroscopic techniques (fluorescence measurement and circular dichroism). Furthermore, to explore the influence of salts in competitive binding between metalloproteins and various metals, we designed a series of competitive experiments and applied to a well-defined model system, the competitive binding of zinc (II) and arsenic (III) to holo-promyelocytic leukemia protein (PML). These experiments not only provided new insights at the molecular scale as complementary to previous NMR and spectroscopic results, but also deduced the relative binding ability between zinc finger proteins and metals at the molecular scale, which avoids the mass spectrometric titration-based determination of binding constants that is frequently affected and often degraded by variable solution conditions including salt contents. [Figure not available: see fulltext.

  18. Introduction of the carbohydrate-activated promoter P(malK) for recombinant protein production.

    PubMed

    Boström, M; Larsson, G

    2002-07-01

    A production protocol for the use of the malK promoter was established. The protocol includes two phases: an initial fed-batch phase on glucose to reach a high cell density and a fed-batch phase on maltose for production of the desired recombinant protein. It is suggested that this cultivation scheme could be used for all promoters that are catabolite repressed by glucose and where growth and production need to be separated. The specific feature of this system is shown by its ability to control the rate of synthesis of the product protein, ss-galactosidase. In the production phase with a constant feed or an exponential feeding of 0.1 h(-1) it took 4 h longer to reach the maximum specific production rate than with the higher dilution rates of 0.25 h(-1) and 0.4 h(-1), respectively. In the above experiments a dilution rate of 0.3 h(-1) in the growth phase was used. The volumetric production of this system could furthermore be extended to 40 h. All protocol procedures so far tested resulted in the same maximum production rate, but reached in different lengths of time. It is argued that this system is particularly well suited for the production of proteins that have a complex structure and/or need to be produced in a soluble form or to be exported to the periplasm.

  19. Loss of Par3 promotes lung adenocarcinoma metastasis through 14-3-3ζ protein

    PubMed Central

    Tong, Song; Xia, Tian; Fan, Kai; Jiang, Ke; Zhai, Wei; Li, Jing-Song; Wang, Si-Hua; Wang, Jian-Jun

    2016-01-01

    Partitioning defective protein 3 (Par3) can activate the Tiam1/Rac pathway to inhibit invasion and metastasis in many cancers; however, the role of Par3 in lung adenocarcinoma remains unknown. Here we show that Par3 is downregulated in lung adenocarcinoma tissues and is associated with higher rates of lymph node metastasis and recurrence. Our functional study demonstrated that knock-down of Par3 promoted lung adenocarcinoma cell growth, cell migration, tumor formation, and metastasis, all of which were effectively inhibited when 14-3-3ζ was silenced. We found that Par3 binded with 14-3-3ζ protein and also showed that Par3 abrogated the binding of 14-3-3ζ to Tiam1, which was responsible for Rac1 activation. Knock-down of 14-3-3ζ inhibited Tiam1/Rac-GTP activation and blocked the invasive behavior of cells lacking Par3. These data suggest that loss of Par3 promotes metastatic behavior in lung adenocarcinoma cells through 14-3-3ζ protein. PMID:27588399

  20. Activation of a calcium-dependent protein kinase involved in the Azospirillum growth promotion in rice.

    PubMed

    Ribaudo, Claudia M; Curá, José A; Cantore, María L

    2017-02-01

    Rice seedlings (Oryza sativa) inoculated with the plant growth-promoting rhizobacteria Azospirillum brasilense FT326 showed an enhanced development of the root system 3 days after inoculation. Later on, a remarkable enlargement of shoots was also evident. An increase in the Ca(2+)-dependent histone kinase activity was also detected as a result of inoculation. The biochemical characterization and Western-blot analysis of the kinase strongly supports the hypothesis that it belongs to a member of the rice CDPK family. The fact that the amount of the protein did not change upon inoculation seems to indicate that a posttranslational activation is responsible for the change in the enzymatic activity. An in-gel kinase experiment identified a 46 kDa CDPK like protein kinase as a putative component of the signal transduction pathway triggered by Azospirillum inoculation. To our knowledge, this is the first report on the possible involvement of a Ca(2+)-dependent protein kinase in promotion of rice plants growth by A. brasilense.

  1. A novel DNA replication origin identified in the human heat shock protein 70 gene promoter.

    PubMed Central

    Taira, T; Iguchi-Ariga, S M; Ariga, H

    1994-01-01

    A general and sensitive method for the mapping of initiation sites of DNA replication in vivo, developed by Vassilev and Johnson, has revealed replication origins in the region of simian virus 40 ori, in the regions upstream from the human c-myc gene and downstream from the Chinese hamster dihydrofolate reductase gene, and in the enhancer region of the mouse immunoglobulin heavy-chain gene. Here we report that the region containing the promoter of the human heat shock protein 70 (hsp70) gene was identified as a DNA replication origin in HeLa cells by this method. Several segments of the region were cloned into pUC19 and examined for autonomously replicating sequence (ARS) activity. The plasmids carrying the segments replicated episomally and semiconservatively when transfected into HeLa cells. The segments of ARS activity contained the sequences previously identified as binding sequences for a c-myc protein complex (T. Taira, Y. Negishi, F. Kihara, S. M. M. Iguchi-Ariga, and H. Ariga, Biochem. Biophys. Acta 1130:166-174, 1992). Mutations introduced within the c-myc protein complex binding sequences abolished the ARS activity. Moreover, the ARS plasmids stably replicated at episomal state for a long time in established cell lines. The results suggest that the promoter region of the human hsp70 gene plays a role in DNA replication as well as in transcription. Images PMID:8065368

  2. Loss of Par3 promotes lung adenocarcinoma metastasis through 14-3-3ζ protein.

    PubMed

    Song, Tong; Tian, Xia; Kai, Fan; Ke, Jiang; Wei, Zhai; Jing-Song, Li; Si-Hua, Wang; Jian-Jun, Wang

    2016-09-27

    Partitioning defective protein 3 (Par3) can activate the Tiam1/Rac pathway to inhibit invasion and metastasis in many cancers; however, the role of Par3 in lung adenocarcinoma remains unknown. Here we show that Par3 is downregulated in lung adenocarcinoma tissues and is associated with higher rates of lymph node metastasis and recurrence. Our functional study demonstrated that knock-down of Par3 promoted lung adenocarcinoma cell growth, cell migration, tumor formation, and metastasis, all of which were effectively inhibited when 14-3-3ζ was silenced. We found that Par3 binded with 14-3-3ζ protein and also showed that Par3 abrogated the binding of 14-3-3ζ to Tiam1, which was responsible for Rac1 activation. Knock-down of 14-3-3ζ inhibited Tiam1/Rac-GTP activation and blocked the invasive behavior of cells lacking Par3. These data suggest that loss of Par3 promotes metastatic behavior in lung adenocarcinoma cells through 14-3-3ζ protein.

  3. Microsomal triglyceride transfer protein promotes the secretion of Xenopus laevis vitellogenin A1.

    PubMed

    Sellers, Jeremy A; Hou, Li; Schoenberg, Daniel R; Batistuzzo de Medeiros, Silvia R; Wahli, Walter; Shelness, Gregory S

    2005-04-08

    Vitellogenins (Vtg) are ancient lipid transport and storage proteins and members of the large lipid transfer protein (LLTP) gene family, which includes insect apolipophorin II/I, apolipoprotein B (apoB), and the microsomal triglyceride transfer protein (MTP). Lipidation of Vtg occurs at its site of synthesis in vertebrate liver, insect fat body, and nematode intestine; however, the mechanism of Vtg lipid acquisition is unknown. To explore whether Vtg biogenesis requires the apoB cofactor and LLTP family member, MTP, Vtg was expressed in COS cells with and without coexpression of the 97-kDa subunit of human MTP. Expression of Vtg alone gave rise to a approximately 220-kDa apoprotein, which was predominantly confined to an intracellular location. Coexpression of Vtg with human MTP enhanced Vtg secretion by 5-fold, without dramatically affecting its intracellular stability. A comparison of wild type and a triglyceride transfer-defective form of MTP revealed that both were capable of promoting Vtg secretion, whereas only wild type MTP could promote the secretion of apoB41 (amino-terminal 41% of apoB). These studies demonstrate that the biogenesis of Vtg is MTP-dependent and that MTP is the likely ancestral member of the LLTP gene family.

  4. Human hedgehog interacting protein expression and promoter methylation in medulloblastoma cell lines and primary tumor samples

    PubMed Central

    Shahi, Mehdi H.; Afzal, Mohammad; Sinha, Subrata; Eberhart, Charles G.; Rey, Juan A.; Fan, Xing

    2015-01-01

    Medulloblastoma is the most common pediatric brain tumor and its development is affected by genetic and epigenetic factors. In this study we found there is low or no expression of the hedgehog interacting protein (HHIP), a negative regulator of the sonic hedgehog pathway, in most medulloblastoma cell lines and primary samples explored. We proceeded to promoter methylation assays of this gene by MCA-Meth, and found that HHIP was hypermethylated in all medulloblastoma cell lines, but only in 2 out of 14 (14%) primary tumor samples. Methylation correlated with low or unexpressed HHIP in cell lines but not in primary tumor samples. These results suggest the possibility of epigenetic regulation of HHIP in medulloblastoma, similarly to gastric, hepatic and pancreatic cancer. However, HHIP seems to be not only under regulation of promoter methylation, but under other factors involved in the control of its low levels of expression in medulloblastoma. PMID:20853133

  5. Telomere binding protein TRB1 is associated with promoters of translation machinery genes in vivo.

    PubMed

    Schrumpfová, Petra Procházková; Vychodilová, Ivona; Hapala, Jan; Schořová, Šárka; Dvořáček, Vojtěch; Fajkus, Jiří

    2016-01-01

    Recently we characterised TRB1, a protein from a single-myb-histone family, as a structural and functional component of telomeres in Arabidopsis thaliana. TRB proteins, besides their ability to bind specifically to telomeric DNA using their N-terminally positioned myb-like domain of the same type as in human shelterin proteins TRF1 or TRF2, also possess a histone-like domain which is involved in protein-protein interactions e.g., with POT1b. Here we set out to investigate the genome-wide localization pattern of TRB1 to reveal its preferential sites of binding to chromatin in vivo and its potential functional roles in the genome-wide context. Our results demonstrate that TRB1 is preferentially associated with promoter regions of genes involved in ribosome biogenesis, in addition to its roles at telomeres. This preference coincides with the frequent occurrence of telobox motifs in the upstream regions of genes in this category, but it is not restricted to the presence of a telobox. We conclude that TRB1 shows a specific genome-wide distribution pattern which suggests its role in regulation of genes involved in biogenesis of the translational machinery, in addition to its preferential telomeric localization.

  6. Myocardial Reloading after Extracorporeal Membrane Oxygenation Alters Substrate Metabolism While Promoting Protein Synthesis

    SciTech Connect

    Kajimoto, Masaki; Priddy, Colleen M.; Ledee, Dolena; Xu, Chun; Isern, Nancy G.; Olson, Aaron; Des Rosiers, Christine; Portman, Michael A.

    2013-08-19

    Extracorporeal membrane oxygenation (ECMO) unloads the heart providing a bridge to recovery in children after myocardial stunning. Mortality after ECMO remains high.Cardiac substrate and amino acid requirements upon weaning are unknown and may impact recovery. We assessed the hypothesis that ventricular reloading modulates both substrate entry into the citric acid cycle (CAC) and myocardial protein synthesis. Fourteen immature piglets (7.8-15.6 kg) were separated into 2 groups based on ventricular loading status: 8 hour-ECMO (UNLOAD) and post-wean from ECMO (RELOAD). We infused [2-13C]-pyruvate as an oxidative substrate and [13C6]-L-leucine, as a tracer of amino acid oxidation and protein synthesis into the coronary artery. RELOAD showed marked elevations in myocardial oxygen consumption above baseline and UNLOAD. Pyruvate uptake was markedly increased though RELOAD decreased pyruvate contribution to oxidative CAC metabolism.RELOAD also increased absolute concentrations of all CAC intermediates, while maintaining or increasing 13C-molar percent enrichment. RELOAD also significantly increased cardiac fractional protein synthesis rates by >70% over UNLOAD. Conclusions: RELOAD produced high energy metabolic requirement and rebound protein synthesis. Relative pyruvate decarboxylation decreased with RELOAD while promoting anaplerotic pyruvate carboxylation and amino acid incorporation into protein rather than to the CAC for oxidation. These perturbations may serve as therapeutic targets to improve contractile function after ECMO.

  7. G-protein coupled receptor BAI3 promotes myoblast fusion in vertebrates

    PubMed Central

    Hamoud, Noumeira; Tran, Viviane; Croteau, Louis-Philippe; Kania, Artur; Côté, Jean-François

    2014-01-01

    Muscle fibers form as a result of myoblast fusion, yet the cell surface receptors regulating this process are unknown in vertebrates. In Drosophila, myoblast fusion involves the activation of the Rac pathway by the guanine nucleotide exchange factor Myoblast City and its scaffolding protein ELMO, downstream of cell-surface cell-adhesion receptors. We previously showed that the mammalian ortholog of Myoblast City, DOCK1, functions in an evolutionarily conserved manner to promote myoblast fusion in mice. In search for regulators of myoblast fusion, we identified the G-protein coupled receptor brain-specific angiogenesis inhibitor (BAI3) as a cell surface protein that interacts with ELMO. In cultured cells, BAI3 or ELMO1/2 loss of function severely impaired myoblast fusion without affecting differentiation and cannot be rescued by reexpression of BAI3 mutants deficient in ELMO binding. The related BAI protein family member, BAI1, is functionally distinct from BAI3, because it cannot rescue the myoblast fusion defects caused by the loss of BAI3 function. Finally, embryonic muscle precursor expression of a BAI3 mutant unable to bind ELMO was sufficient to block myoblast fusion in vivo. Collectively, our findings provide a role for BAI3 in the relay of extracellular fusion signals to their intracellular effectors, identifying it as an essential transmembrane protein for embryonic vertebrate myoblast fusion. PMID:24567399

  8. Neuronal receptor activity-modifying protein 1 promotes energy expenditure in mice.

    PubMed

    Zhang, Zhongming; Liu, Xuebo; Morgan, Donald A; Kuburas, Adisa; Thedens, Daniel R; Russo, Andrew F; Rahmouni, Kamal

    2011-04-01

    Receptor activity-modifying proteins (RAMPs) 1, 2, and 3 are unusual accessory proteins that dictate the binding specificity of two G protein-coupled receptors involved in energy homeostasis: calcitonin gene-related peptide (CGRP) and amylin receptors. These proteins are expressed throughout the central nervous system (CNS), including in the brain regions involved in the regulation of energy homeostasis, but the significance of CNS RAMPs in the control of energy balance remains unknown. To examine the functional significance of modulating neuronal RAMP1, we assessed the effect of overexpressing human RAMP1 (hRAMP1) in the CNS on body energy balance. Nestin/hRAMP1 transgenic mice have a remarkably decreased body weight associated with reduced fat mass and circulating leptin levels. The transgenic mice exhibited higher energy expenditure as indicated by increased oxygen consumption, body temperature, and sympathetic tone subserving brown adipose tissue (BAT). Consistent with this, the nestin/hRAMP1 transgenic mice had elevated BAT mRNA levels of peroxisome proliferator-activated receptor γ coactivator 1α and uncoupling protein 1 and 3, and these changes can be reversed by chronic blockade of sympathetic nervous system signaling. Furthermore, metabolic response to amylin was enhanced in the nestin/hRAMP1 mice whereas the response to CGRP was blunted, possibly the result of higher expression of CGRP in the CNS. These data demonstrate that CNS RAMP1 plays a pivotal role in the regulation of energy homeostasis by promoting energy expenditure.

  9. AlkB homolog 3-mediated tRNA demethylation promotes protein synthesis in cancer cells

    PubMed Central

    Ueda, Yuko; Ooshio, Ikumi; Fusamae, Yasuyuki; Kitae, Kaori; Kawaguchi, Megumi; Jingushi, Kentaro; Hase, Hiroaki; Harada, Kazuo; Hirata, Kazumasa; Tsujikawa, Kazutake

    2017-01-01

    The mammalian AlkB homolog (ALKBH) family of proteins possess a 2-oxoglutarate- and Fe(II)-dependent oxygenase domain. A similar domain in the Escherichia coli AlkB protein catalyzes the oxidative demethylation of 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) in both DNA and RNA. AlkB homolog 3 (ALKBH3) was also shown to demethylate 1-meA and 3-meC (induced in single-stranded DNA and RNA by a methylating agent) to reverse the methylation damage and retain the integrity of the DNA/RNA. We previously reported the high expression of ALKBH3 in clinical tumor specimens and its involvement in tumor progression. In this study, we found that ALKBH3 effectively demethylated 1-meA and 3-meC within endogenously methylated RNA. Moreover, using highly purified recombinant ALKBH3, we identified N6-methyladenine (N6-meA) in mammalian transfer RNA (tRNA) as a novel ALKBH3 substrate. An in vitro translation assay showed that ALKBH3-demethylated tRNA significantly enhanced protein translation efficiency. In addition, ALKBH3 knockdown in human cancer cells impaired cellular proliferation and suppressed the nascent protein synthesis that is usually accompanied by accumulation of the methylated RNAs. Thus, our data highlight a novel role for ALKBH3 in tumor progression via RNA demethylation and subsequent protein synthesis promotion. PMID:28205560

  10. Crystal structure of the tumor-promoter okadaic acid bound to protein phosphatase-1.

    PubMed

    Maynes, J T; Bateman, K S; Cherney, M M; Das, A K; Luu, H A; Holmes, C F; James, M N

    2001-11-23

    Protein phosphatase-1 (PP1) plays a key role in dephosphorylation in numerous biological processes such as glycogen metabolism, cell cycle regulation, smooth muscle contraction, and protein synthesis. Microorganisms produce a variety of inhibitors of PP1, which include the microcystin class of inhibitors and okadaic acid, the latter being the major cause of diarrhetic shellfish poisoning and a powerful tumor promoter. We have determined the crystal structure of the molecular complex of okadaic acid bound to PP1 to a resolution of 1.9 A. This structure reveals that the acid binds in a hydrophobic groove adjacent to the active site of the protein and interacts with basic residues within the active site. Okadaic acid exhibits a cyclic structure, which is maintained via an intramolecular hydrogen bond. This is reminiscent of other macrocyclic protein phosphatase inhibitors. The inhibitor-bound enzyme shows very little conformational change when compared with two other PP1 structures, except in the inhibitor-sensitive beta12-beta13 loop region. The selectivity of okadaic acid for protein phosphatases-1 and -2A but not PP-2B (calcineurin) may be reassessed in light of this study.

  11. RNF20 promotes the polyubiquitination and proteasome-dependent degradation of AP-2α protein.

    PubMed

    Ren, Peng; Sheng, Zhifeng; Wang, Yijun; Yi, Xin; Zhou, Qiuzhi; Zhou, Jianlin; Xiang, Shuanglin; Hu, Xiang; Zhang, Jian

    2014-02-01

    Transcription factor activator protein 2α (AP-2α) is a negative regulator of adipogenesis by repressing the transcription of CCAAT/enhancer binding protein (C/EBPα) gene. During adipogenesis, AP-2α is degraded, leading to transcriptional up-regulation of C/EBPα. However, the mechanism for AP-2α degradation is not clear. Here, using immunoprecipitation assay and mass spectrometry, we identified ring finger protein 20 (RNF20) as an AP-2α-interacting protein in 3T3-L1 preadipocytes. RNF20 has been proved to be an E3 ubiquitin ligase for both histone H2B and tumor suppressor ErbB3-binding protein 1 (Ebp1). In this study, we demonstrated that RNF20 co-localized and interacted with AP-2α, and promoted its polyubiquitination and proteasome-dependent degradation. Over-expression of RNF20 inhibited the activity of AP-2α and rescued the C/EBPα expression which was inhibited by AP-2α. These results suggested that RNF20 may play roles in adipocyte differentiation by stimulating ubiquitin-proteasome-dependent degradation of AP-2α.

  12. A human TATA binding protein-related protein with altered DNA binding specificity inhibits transcription from multiple promoters and activators.

    PubMed

    Moore, P A; Ozer, J; Salunek, M; Jan, G; Zerby, D; Campbell, S; Lieberman, P M

    1999-11-01

    The TATA binding protein (TBP) plays a central role in eukaryotic and archael transcription initiation. We describe the isolation of a novel 23-kDa human protein that displays 41% identity to TBP and is expressed in most human tissue. Recombinant TBP-related protein (TRP) displayed barely detectable binding to consensus TATA box sequences but bound with slightly higher affinities to nonconsensus TATA sequences. TRP did not substitute for TBP in transcription reactions in vitro. However, addition of TRP potently inhibited basal and activated transcription from multiple promoters in vitro and in vivo. General transcription factors TFIIA and TFIIB bound glutathione S-transferase-TRP in solution but failed to stimulate TRP binding to DNA. Preincubation of TRP with TFIIA inhibited TBP-TFIIA-DNA complex formation and addition of TFIIA overcame TRP-mediated transcription repression. TRP transcriptional repression activity was specifically reduced by mutations in TRP that disrupt the TFIIA binding surface but not by mutations that disrupt the TFIIB or DNA binding surface of TRP. These results suggest that TFIIA is a primary target of TRP transcription inhibition and that TRP may modulate transcription by a novel mechanism involving the partial mimicry of TBP functions.

  13. MGMT promoter methylation and correlation with protein expression in primary central nervous system lymphoma.

    PubMed

    Toffolatti, L; Scquizzato, E; Cavallin, S; Canal, F; Scarpa, M; Stefani, P M; Gherlinzoni, F; Dei Tos, A P

    2014-11-01

    The O (6)-methylguanine-DNA-methyltransferase (MGMT) gene encodes for a DNA repairing enzyme of which silencing by promoter methylation is involved in brain tumorigenesis. MGMT promoter methylation represents a favorable prognostic factor and has been associated with a better response to alkylating agents in glioma and systemic lymphoma. Primary central nervous system lymphoma (PCNSL) is a rare and aggressive extranodal malignant lymphoma. The current standard of care, based on high-dose methotrexate chemotherapy, has improved prognosis but outcome remains poor for a majority of patients. Therapeutic progress in this field is conditioned by limited biological and molecular knowledge about the disease. Temozolomide has recently emerged as an alternative option for PCNSL treatment. We aimed to analyze the MGMT gene methylation status in a series of 24 PCNSLs, to investigate the relationship between methylation status of the gene and immunohistochemical expression of MGMT protein and to evaluate the possible prognostic significance of these biomarkers. Our results confirm that methylation of the MGMT gene and loss of MGMT protein are frequent events in these lymphomas (54 % of our cases) and suggest that they are gender and age related. MGMT methylation showed high correlation with loss of protein expression (concordance correlation coefficient = -0.49; Fisher exact test: p < 0.01), different from what has been observed in other brain tumors. In the subgroup of ten patients who received high dose chemotherapy, the presence of methylated MGMT promoter (n = 4), seems to be associated with a prolonged overall survival (>60 months in three of four patients). The prognostic significance of these molecular markers in PCNSL needs to be further studied in groups of patients treated in a homogeneous way.

  14. Method for forming polymerized microfluidic devices

    DOEpatents

    Sommer, Gregory J.; Hatch, Anson V.; Wang, Ying-Chih; Singh, Anup K.; Renzi, Ronald F.; Claudnic, Mark R.

    2013-03-12

    Methods for making a microfluidic device according to embodiments of the present invention include defining.about.cavity. Polymer precursor solution is positioned in the cavity, and exposed to light to begin the polymerization process and define a microchannel. In some embodiments, after the polymerization process is partially complete, a solvent rinse is performed, or fresh polymer precursor introduced into the microchannel. This may promote removal of unpolymerized material from the microchannel and enable smaller feature sizes. The polymer precursor solution may contain an iniferter. Polymerized features therefore may be capped with the iniferter, which is photoactive. The iniferter may aid later binding of a polyacrylamide gel to the microchannel surface.

  15. Method for forming polymerized microfluidic devices

    DOEpatents

    Sommer, Gregory J [Livermore, CA; Hatch, Anson V [Tracy, CA; Wang, Ying-Chih [Pleasanton, CA; Singh, Anup K [Danville, CA; Renzi, Ronald F [Tracy, CA; Claudnic, Mark R [Livermore, CA

    2011-11-01

    Methods for making a micofluidic device according to embodiments of the present invention include defining a cavity. Polymer precursor solution is positioned in the cavity, and exposed to light to begin the polymerization process and define a microchannel. In some embodiments, after the polymerization process is partially complete, a solvent rinse is performed, or fresh polymer precursor introduced into the microchannel. This may promote removal of unpolymerized material from the microchannel and enable smaller feature sizes. The polymer precursor solution may contain an iniferter. Polymerized features therefore may be capped with the iniferter, which is photoactive. The iniferter may aid later binding of a polyacrylamide gel to the microchannel surface.

  16. Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling

    PubMed Central

    2009-01-01

    Background Plant Growth Promoting Rhizobacteria (PGPR), Pseudomonas fluorescens strain KH-1 was found to exhibit plant growth promotional activity in rice under both in-vitro and in-vivo conditions. But the mechanism underlying such promotional activity of P. fluorescens is not yet understood clearly. In this study, efforts were made to elucidate the molecular responses of rice plants to P. fluorescens treatment through protein profiling. Two-dimensional polyacrylamide gel electrophoresis strategy was adopted to identify the PGPR responsive proteins and the differentially expressed proteins were analyzed by mass spectrometry. Results Priming of P. fluorescens, 23 different proteins found to be differentially expressed in rice leaf sheaths and MS analysis revealed the differential expression of some important proteins namely putative p23 co-chaperone, Thioredoxin h- rice, Ribulose-bisphosphate carboxylase large chain precursor, Nucleotide diPhosphate kinase, Proteosome sub unit protein and putative glutathione S-transferase protein. Conclusion Functional analyses of the differential proteins were reported to be directly or indirectly involved in growth promotion in plants. Thus, this study confirms the primary role of PGPR strain KH-1 in rice plant growth promotion. PMID:20034395

  17. Mitogen-associated protein kinase- and protein kinase A-dependent regulation of rhodopsin promoter expression in zebrafish rod photoreceptor cells.

    PubMed

    Yu, Chuan-Jiang; Gao, Yan; Willis, Callie L; Li, Ping; Tiano, Joseph P; Nakamura, Paul A; Hyde, David R; Li, Lei

    2007-02-15

    Mitogen-associated protein kinase (MAPK)- and protein kinase A (PKA)-dependent signal transductions play important roles in the regulation of gene expression. Both MAPK and PKA pathways can be activated by light exposure. In this study, we investigated the effect of light on MAPK and PKA signal transduction and their roles in the regulation of rhodopsin promoter expression by using transgenic zebrafish [Tg(rhod::GFP)]. The Tg(rhod::GFP) fish express short half-life GFP that is under the transcriptional control of the zebrafish rhodopsin promoter and can therefore be used for in vivo studies of rhodopsin gene transcription in live cells. Blue light plays a role in the regulation of rhodopsin promoter expression via an MAPK-mediated signal transduction cascade. Blue light excites cryptochromes (CRY), which activate the downstream PKC-dependent MAPK signal pathway. White light, on the other hand, regulates rhodopsin promoter expression via a G-protein-coupled cAMP-dependent PKA pathway. White light promotes dopamine release in the retina, which activates dopamine receptors and the downstream PKA pathway. Blocking MAPK signaling diminishes the blue light-induced increases in rhodopsin promoter expression, but this treatment has no effect on white light-mediated rhodopsin promoter expression. Conversely, blocking the PKA pathway diminishes the white light-induced rhodopsin promoter expression but does not affect rhodopsin promoter expression regulated by blue light. Together, the data suggest that MAPK and PKA regulate rhodopsin transcription through parallel signal transduction pathways.

  18. Common Functional Genetic Variants in Catecholamine Storage Vesicle Protein Promoter Motifs Interact to Trigger Systemic Hypertension

    PubMed Central

    Zhang, Kuixing; Rao, Fangwen; Wang, Lei; Rana, Brinda K.; Ghosh, Sajalendu; Mahata, Manjula; Salem, Rany M.; Rodriguez-Flores, Juan L.; Fung, Maple M.; Waalen, Jill; Tayo, Bamidele; Taupenot, Laurent; Mahata, Sushil K.; O'Connor, Daniel T.

    2010-01-01

    Objectives The purpose of this study was to explore transcriptional mechanisms whereby genetic variation in the CHGB promoter influence BP and hypertension. Background Hypertension is a complex trait in which deranged autonomic control of the circulation may be an etiological culprit. Chromogranin B (CHGB) is a major soluble protein in the core of catecholamine storage vesicles, playing a necessary (catalytic) role in the biogenesis of secretory vesicles. Previously we found that genetic variation at CHGB influenced plasma CHGB expression as well as autonomic function, and that BP association was maximal towards the 5′ end of the gene. Methods After polymorphism discovery, we functionally characterized the 2 common variants in the proximal CHGB promoter, A-296C and A-261T, which lay within the same haplotype block in black and white populations. CHGB promoter activity was studied by haplotype/luciferase reporter transfection. Transcriptional mechanisms were probed by EMSA and ChIP. Results The A-296C variant disrupted a c-FOS motif, and exhibited differential mobility shifting to chromaffin cell nuclear proteins during EMSA, differential binding of endogenous c-FOS on ChIP, and differential transcriptional response to exogenous c-FOS. A-261T disrupted motifs for SRY and YY1, with similar consequences for gel mobility during EMSA, endogenous factor binding during ChIP, and transcriptional responses to the exogenous factors. 2-SNP haplotype analyses demonstrated a profound (p∼3×10-20) effect of CHGB promoter variation on BP in the European ancestry population, with a rank order of CTpromoter activity in cella. Site-by-site interactions at A-296C and A-261T yielded highly non-additive effects on SBP and DBP. CHGB haplotype effects on BP were also noted in an independent (African ancestry) sample. In a

  19. TP53 Promoter Methylation in Primary Glioblastoma: Relationship with TP53 mRNA and Protein Expression and Mutation Status

    PubMed Central

    Szybka, Malgorzata; Malachowska, Beata; Fendler, Wojciech; Potemski, Piotr; Piaskowski, Sylwester; Jaskolski, Dariusz; Papierz, Wielislaw; Skowronski, Wieslaw; Och, Waldemar; Kordek, Radzislaw

    2014-01-01

    Reduced expression of TP53 by promoter methylation has been reported in several neoplasms. It remains unclear whether TP53 promoter methylation is associated with reduced transcriptional and protein expression in glioblastoma (GB). The aim of our work was to study the impact of TP53 methylation and mutations on TP53 mRNA level and protein expression in 42 molecularly characterized primary GB tumors. We also evaluate the impact of all molecular alterations on the overall patient survival. The frequency of TP53 promoter methylation was found in 21.4%. To the best of our knowledge, this is the first report showing such high frequency of TP53 promoter methylation in primary GB. There was no relation between TP53 promoter methylation and TP53 mRNA level (p=0.5722) and between TP53 promoter methylation and TP53 protein expression (p=0.2045). No significant associations were found between TP53 mRNA expression and mutation of TP53 gene (p=0.9076). However, significant association between TP53 mutation and TP53 protein expression was found (p=0.0016). Our data suggest that in primary GB TP53 promoter methylation does not play a role in silencing of TP53 transcriptional and protein expression and is probably regulated by other genetic and epigenetic mechanisms associated with genes involved in the TP53 pathway. PMID:24506545

  20. Construction and validation of a mCherry protein vector for promoter analysis in Lactobacillus acidophilus.

    PubMed

    Mohedano, M Luz; García-Cayuela, Tomás; Pérez-Ramos, Adrián; Gaiser, Rogier A; Requena, Teresa; López, Paloma

    2015-02-01

    Lactobacilli are widespread in natural environments and are increasingly being investigated as potential health modulators. In this study, we have adapted the broad-host-range vector pNZ8048 to express the mCherry protein (pRCR) to expand the usage of the mCherry protein for analysis of gene expression in Lactobacillus. This vector is also able to replicate in Streptococcus pneumoniae and Escherichia coli. The usage of pRCR as a promoter probe was validated in Lactobacillus acidophilus by characterizing the regulation of lactacin B expression. The results show that the regulation is exerted at the transcriptional level, with lbaB gene expression being specifically induced by co-culture of the L. acidophilus bacteriocin producer and the S. thermophilus STY-31 inducer bacterium.

  1. TIPE3 Is The Transfer Protein Of Lipid Second Messengers That Promote Cancer

    PubMed Central

    Fayngerts, Svetlana A.; Wu, Jianping; Oxley, Camilla L.; Liu, Xianglan; Vourekas, Anastassios; Cathopoulis, Terry; Wang, Zhaojun; Cui, Jian; Liu, Suxia; Sun, Honghong; Lemmon, Mark A.; Zhang, Lining

    2014-01-01

    Summary More than half of human cancers have aberrantly upregulated phosphoinositide signals; yet how phospholipid signals are controlled during tumorigenesis is not fully understood. We report here that TIPE3 (TNFAIP8L3) is the transfer protein of phosphoinositide second messengers that promote cancer. High-resolution crystal structure of TIPE3 shows a large hydrophobic cavity that is occupied by a phospholipid-like molecule. TIPE3 preferentially captures and shuttles two lipid second messengers, i.e., phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, and increases their levels in the plasma membrane. Importantly, human cancers have markedly upregulated TIPE3 expression. Knocking out TIPE3 diminishes tumorigenesis whereas enforced TIPE3 expression enhances it in vivo. Thus, the function and metabolism of phosphoinositide second messengers are controlled by a specific transfer protein during tumorigenesis. PMID:25242044

  2. Nuclear envelope morphology constrains diffusion and promotes asymmetric protein segregation in closed mitosis.

    PubMed

    Boettcher, Barbara; Marquez-Lago, Tatiana T; Bayer, Mathias; Weiss, Eric L; Barral, Yves

    2012-06-25

    During vegetative growth, Saccharomyces cerevisiae cells divide asymmetrically: the mother cell buds to produce a smaller daughter cell. This daughter asymmetrically inherits the transcription factor Ace2, which activates daughter-specific transcriptional programs. In this paper, we investigate when and how this asymmetry is established and maintained. We show that Ace2 asymmetry is initiated in the elongated, but undivided, anaphase nucleus. At this stage, the nucleoplasm was highly compartmentalized; little exchange was observed for nucleoplasmic proteins between mother and bud. Using photobleaching and in silico modeling, we show that diffusion barriers compartmentalize the nuclear membranes. In contrast, the behavior of proteins in the nucleoplasm is well explained by the dumbbell shape of the anaphase nucleus. This compartmentalization of the nucleoplasm promoted Ace2 asymmetry in anaphase nuclei. Thus, our data indicate that yeast cells use the process of closed mitosis and the morphological constraints associated with it to asymmetrically segregate nucleoplasmic components.

  3. The degradation of mixed lineage kinase domain-like protein promotes neuroprotection after ischemic brain injury

    PubMed Central

    Zhou, Yanlong; Zhou, Beiqun; Tu, Hui; Tang, Yan; Xu, Chen; Chen, Yanbo; Zhao, Zhong; Miao, Zhigang

    2017-01-01

    Mixed lineage kinase domain-like (MLKL) protein was recently found to play a critical role in necrotic cell death. To explore its role in neurological diseases, we measured MLKL protein expression after ischemia injury in a mouse model. We found that MLKL expression significantly increased 12 h after ischemia/reperfusion (I/R) injury with peak levels at 48 h. Inhibition of MLKL by intraperitoneal administration of NSA significantly reduced infarct volume and improved neurological deficits after 75 min of ischemia and 24 h of reperfusion. Further, we found NSA reduced MLKL levels via the ubiquitination proteasome pathway, but not by inhibiting RNA transcription. Interestingly, NSA administration increased cleaved PARP-1 levels, indicating the protective effects of MLKL inhibition is not related to apoptosis. These findings suggest MLKL is a new therapeutic target for neurological pathologies like stroke. Therefore, promoting degradation of MLKL may be a novel avenue to reduce necrotic cell death after ischemic brain injury. PMID:28978125

  4. The degradation of mixed lineage kinase domain-like protein promotes neuroprotection after ischemic brain injury.

    PubMed

    Zhou, Yanlong; Zhou, Beiqun; Tu, Hui; Tang, Yan; Xu, Chen; Chen, Yanbo; Zhao, Zhong; Miao, Zhigang

    2017-09-15

    Mixed lineage kinase domain-like (MLKL) protein was recently found to play a critical role in necrotic cell death. To explore its role in neurological diseases, we measured MLKL protein expression after ischemia injury in a mouse model. We found that MLKL expression significantly increased 12 h after ischemia/reperfusion (I/R) injury with peak levels at 48 h. Inhibition of MLKL by intraperitoneal administration of NSA significantly reduced infarct volume and improved neurological deficits after 75 min of ischemia and 24 h of reperfusion. Further, we found NSA reduced MLKL levels via the ubiquitination proteasome pathway, but not by inhibiting RNA transcription. Interestingly, NSA administration increased cleaved PARP-1 levels, indicating the protective effects of MLKL inhibition is not related to apoptosis. These findings suggest MLKL is a new therapeutic target for neurological pathologies like stroke. Therefore, promoting degradation of MLKL may be a novel avenue to reduce necrotic cell death after ischemic brain injury.

  5. Activation of the beta interferon promoter by paramyxoviruses in the absence of virus protein synthesis.

    PubMed

    Killip, M J; Young, D F; Precious, B L; Goodbourn, S; Randall, R E

    2012-02-01

    Conflicting reports exist regarding the requirement for virus replication in interferon (IFN) induction by paramyxoviruses. Our previous work has demonstrated that pathogen-associated molecular patterns capable of activating the IFN-induction cascade are not normally generated during virus replication, but are associated instead with the presence of defective interfering (DI) viruses. We demonstrate here that DIs of paramyxoviruses, including parainfluenza virus 5, mumps virus and Sendai virus, can activate the IFN-induction cascade and the IFN-β promoter in the absence of virus protein synthesis. As virus protein synthesis is an absolute requirement for paramyxovirus genome replication, our results indicate that these DI viruses do not require replication to activate the IFN-induction cascade.

  6. The tumour suppressor APC promotes HIV-1 assembly via interaction with Gag precursor protein

    PubMed Central

    Miyakawa, Kei; Nishi, Mayuko; Matsunaga, Satoko; Okayama, Akiko; Anraku, Masaki; Kudoh, Ayumi; Hirano, Hisashi; Kimura, Hirokazu; Morikawa, Yuko; Yamamoto, Naoki; Ono, Akira; Ryo, Akihide

    2017-01-01

    Diverse cellular proteins and RNAs are tightly regulated in their subcellular localization to exert their local function. Here we report that the tumour suppressor adenomatous polyposis coli protein (APC) directs the localization and assembly of human immunodeficiency virus (HIV)-1 Gag polyprotein at distinct membrane components to enable the efficient production and spread of infectious viral particles. A proteomic analysis and subsequent biomolecular interaction assay reveals that the carboxyl terminus of APC interacts with the matrix region of Gag. Ectopic expression of APC, but not its familial adenomatous polyposis-related truncation mutant, prominently enhances HIV-1 production. Conversely, the depletion of APC leads to a significant decrease in membrane targeting of viral components, resulting in the severe loss of production of infectious virions. Furthermore, APC promotes the directional assembly of viral components at virological synapses, thereby facilitating cell-to-cell viral transmission. These findings reveal an unexpected role of APC in the directional spread of HIV-1. PMID:28134256

  7. Polymerization of perfluorobutadiene

    NASA Technical Reports Server (NTRS)

    Newman, J.; Toy, M. S.

    1970-01-01

    Diisopropyl peroxydicarbonate dissolved in liquid perfluorobutadiene is conducted in a sealed vessel at the autogenous pressure of polymerization. Reaction temperature, ratio of catalyst to monomer, and amount of agitation determine degree of polymerization and product yield.

  8. Hypusine-containing protein eIF5A promotes translation elongation.

    PubMed

    Saini, Preeti; Eyler, Daniel E; Green, Rachel; Dever, Thomas E

    2009-05-07

    Translation elongation factors facilitate protein synthesis by the ribosome. Previous studies identified two universally conserved translation elongation factors, EF-Tu in bacteria (known as eEF1A in eukaryotes) and EF-G (eEF2), which deliver aminoacyl-tRNAs to the ribosome and promote ribosomal translocation, respectively. The factor eIF5A (encoded by HYP2 and ANB1 in Saccharomyces cerevisiae), the sole protein in eukaryotes and archaea to contain the unusual amino acid hypusine (N(epsilon)-(4-amino-2-hydroxybutyl)lysine), was originally identified based on its ability to stimulate the yield (endpoint) of methionyl-puromycin synthesis-a model assay for first peptide bond synthesis thought to report on certain aspects of translation initiation. Hypusine is required for eIF5A to associate with ribosomes and to stimulate methionyl-puromycin synthesis. Because eIF5A did not stimulate earlier steps of translation initiation, and depletion of eIF5A in yeast only modestly impaired protein synthesis, it was proposed that eIF5A function was limited to stimulating synthesis of the first peptide bond or that eIF5A functioned on only a subset of cellular messenger RNAs. However, the precise cellular role of eIF5A is unknown, and the protein has also been linked to mRNA decay, including the nonsense-mediated mRNA decay pathway, and to nucleocytoplasmic transport. Here we use molecular genetic and biochemical studies to show that eIF5A promotes translation elongation. Depletion or inactivation of eIF5A in the yeast S. cerevisiae resulted in the accumulation of polysomes and an increase in ribosomal transit times. Addition of recombinant eIF5A from yeast, but not a derivative lacking hypusine, enhanced the rate of tripeptide synthesis in vitro. Moreover, inactivation of eIF5A mimicked the effects of the eEF2 inhibitor sordarin, indicating that eIF5A might function together with eEF2 to promote ribosomal translocation. Because eIF5A is a structural homologue of the bacterial

  9. Effects of polymerization and nucleotide identity on the conformational dynamics of the bacterial actin homolog MreB.

    PubMed

    Colavin, Alexandre; Hsin, Jen; Huang, Kerwyn Casey

    2014-03-04

    The assembly of protein filaments drives many cellular processes, from nucleoid segregation, growth, and division in single cells to muscle contraction in animals. In eukaryotes, shape and motility are regulated through cycles of polymerization and depolymerization of actin cytoskeletal networks. In bacteria, the actin homolog MreB forms filaments that coordinate the cell-wall synthesis machinery to regulate rod-shaped growth and contribute to cellular stiffness through unknown mechanisms. Like actin, MreB is an ATPase and requires ATP to polymerize, and polymerization promotes nucleotide hydrolysis. However, it is unclear whether other similarities exist between MreB and actin because the two proteins share low sequence identity and have distinct cellular roles. Here, we use all-atom molecular dynamics simulations to reveal surprising parallels between MreB and actin structural dynamics. We observe that MreB exhibits actin-like polymerization-dependent structural changes, wherein polymerization induces flattening of MreB subunits, which restructures the nucleotide-binding pocket to favor hydrolysis. MreB filaments exhibited nucleotide-dependent intersubunit bending, with hydrolyzed polymers favoring a straighter conformation. We use steered simulations to demonstrate a coupling between intersubunit bending and the degree of flattening of each subunit, suggesting cooperative bending along a filament. Taken together, our results provide molecular-scale insight into the diversity of structural states of MreB and the relationships among polymerization, hydrolysis, and filament properties, which may be applicable to other members of the broad actin family.

  10. Vegan proteins may reduce risk of cancer, obesity, and cardiovascular disease by promoting increased glucagon activity.

    PubMed

    McCarty, M F

    1999-12-01

    Amino acids modulate the secretion of both insulin and glucagon; the composition of dietary protein therefore has the potential to influence the balance of glucagon and insulin activity. Soy protein, as well as many other vegan proteins, are higher in non-essential amino acids than most animal-derived food proteins, and as a result should preferentially favor glucagon production. Acting on hepatocytes, glucagon promotes (and insulin inhibits) cAMP-dependent mechanisms that down-regulate lipogenic enzymes and cholesterol synthesis, while up-regulating hepatic LDL receptors and production of the IGF-I antagonist IGFBP-1. The insulin-sensitizing properties of many vegan diets--high in fiber, low in saturated fat--should amplify these effects by down-regulating insulin secretion. Additionally, the relatively low essential amino acid content of some vegan diets may decrease hepatic IGF-I synthesis. Thus, diets featuring vegan proteins can be expected to lower elevated serum lipid levels, promote weight loss, and decrease circulating IGF-I activity. The latter effect should impede cancer induction (as is seen in animal studies with soy protein), lessen neutrophil-mediated inflammatory damage, and slow growth and maturation in children. In fact, vegans tend to have low serum lipids, lean physiques, shorter stature, later puberty, and decreased risk for certain prominent 'Western' cancers; a vegan diet has documented clinical efficacy in rheumatoid arthritis. Low-fat vegan diets may be especially protective in regard to cancers linked to insulin resistance--namely, breast and colon cancer--as well as prostate cancer; conversely, the high IGF-I activity associated with heavy ingestion of animal products may be largely responsible for the epidemic of 'Western' cancers in wealthy societies. Increased phytochemical intake is also likely to contribute to the reduction of cancer risk in vegans. Regression of coronary stenoses has been documented during low-fat vegan diets