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Sample records for protein vitronectin increases

  1. Formation of soluble amyloid oligomers and amyloid fibrils by the multifunctional protein vitronectin

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    Langen Ralf

    2008-10-01

    Full Text Available Abstract Background The multifunctional protein vitronectin is present within the deposits associated with Alzheimer disease (AD, age-related macular degeneration (AMD, atherosclerosis, systemic amyloidoses, and glomerulonephritis. The extent to which vitronectin contributes to amyloid formation within these plaques, which contain misfolded, amyloidogenic proteins, and the role of vitronectin in the pathophysiology of the aforementioned diseases is currently unknown. The investigation of vitronectin aggregation is significant since the formation of oligomeric and fibrillar structures are common features of amyloid proteins. Results We observed vitronectin immunoreactivity in senile plaques of AD brain, which exhibited overlap with the amyloid fibril-specific OC antibody, suggesting that vitronectin is deposited at sites of amyloid formation. Of particular interest is the growing body of evidence indicating that soluble nonfibrillar oligomers may be responsible for the development and progression of amyloid diseases. In this study we demonstrate that both plasma-purified and recombinant human vitronectin readily form spherical oligomers and typical amyloid fibrils. Vitronectin oligomers are toxic to cultured neuroblastoma and retinal pigment epithelium (RPE cells, possibly via a membrane-dependent mechanism, as they cause leakage of synthetic vesicles. Oligomer toxicity was attenuated in RPE cells by the anti-oligomer A11 antibody. Vitronectin fibrils contain a C-terminal protease-resistant fragment, which may approximate the core region of residues essential to amyloid formation. Conclusion These data reveal the propensity of vitronectin to behave as an amyloid protein and put forth the possibilities that accumulation of misfolded vitronectin may contribute to aggregate formation seen in age-related amyloid diseases.

  2. Bovine S protein (vitronectin increases phagocytosis of Streptococcus dysgalactiae Aumento na fagocitose de Streptococcus dysgalactiae pela ação da proteína S bovina (vitronectina

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    Laerte Francisco Filippsen

    1999-01-01

    Full Text Available The effects of bovine S protein (vitronectin on phagocytosis of Streptococcus dysgalactiae strains isolated from cattle with mastitis were investigated. Phagocytized streptococci were determined by a fluorometric microassay using glass adherent polymorphonuclear neutrophils (PMN. Preincubation of S. dysgalactiae with bovine S protein significantly increased their phagocytosis by PMN. Bovine S protein had no effect on phagocytic killing of non-S protein binding S. pyogenes cultures. Enzymatic digestion of the bovine S protein binding sites on S. dysgalactiae with pronase resulted in a significative reduction of the effects of S protein on phagocytosis. It could thus be concluded that in addition to its role as a promoter of cellular adhesion and complement inhibitor, bovine S protein may also influence the phagocytosis of S. dysgalactiae during inflammatory processes.Foram investigados os efeitos da proteína S bovina (vitronectina na fagocitose de amostras de Streptococcus dysgalactiae isoladas de bovinos com mastite. A determinação do número de estreptococos fagocitados foi realizada pelo método fluorométrico utilizando neutrófilos polimorfonucleares (NPM aderidos em lâminas de vidro. A pré-incubação do S. dysgalactiae com a proteína S bovina aumentou significativamente a sua fagocitose por NPM. A proteína S bovina não causou efeito na fagocitose de culturas de S. pyogenes, já que não apresentam sítios de ligação para esta proteína. A digestão enzimática com pronase dos sítios de ligação S. dysgalactiae para a proteína S bovina resultou numa significativa redução do efeito da proteína S na fagocitose. Pode-se concluir que além do papel como promotor da adesão celular e inibidor do complemento, a proteína S bovina pode também influir na fagocitose do S. dysgalactiae durante os processos inflamatórios.

  3. Pathogenic Leptospira species acquire factor H and vitronectin via the surface protein LcpA.

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    da Silva, Ludmila Bezerra; Miragaia, Lidia Dos Santos; Breda, Leandro Carvalho Dantas; Abe, Cecilia Mari; Schmidt, Mariana Costa Braga; Moro, Ana Maria; Monaris, Denize; Conde, Jonas Nascimento; Józsi, Mihály; Isaac, Lourdes; Abreu, Patrícia Antônia Estima; Barbosa, Angela Silva

    2015-03-01

    Upon infection, pathogenic Leptospira species bind several complement regulators in order to overcome host innate immunity. We previously characterized a 20-kDa leptospiral surface protein which interacts with C4b binding protein (C4BP): leptospiral complement regulator-acquiring protein A (LcpA). Here we show that LcpA also interacts with human factor H (FH), which remains functionally active once bound to the protein. Antibodies directed against short consensus repeat 20 (SCR20) inhibited binding of FH to LcpA by approximately 90%, thus confirming that this particular domain is involved in the interaction. We have also shown for the first time that leptospires bind human vitronectin and that the interaction is mediated by LcpA. Coincubation with heparin blocked LcpA-vitronectin interaction in a dose-dependent manner, strongly suggesting that binding may occur through the heparin binding domains of vitronectin. LcpA also bound to the terminal pathway component C9 and inhibited Zn(2+)-induced polymerization and membrane attack complex (MAC) formation. Competitive binding assays indicated that LcpA interacts with C4BP, FH, and vitronectin through distinct sites. Taken together, our findings indicate that LcpA may play a role in leptospiral immune evasion. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  4. Serum osteopontin and vitronectin levels in systemic sclerosis.

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    Gundogdu, Baris; Yolbas, Servet; Yilmaz, Musa; Aydin, Suleyman; Koca, Sulayman Serdar

    2017-11-01

    Osteopontin a matricellular protein has pro-fibrotic effects and binds integrin such as αvβ1 and αvβ3. Vitronectin is one of the integrin αvβ3 ligands and is a multifunctional glycoprotein. The aim of the present study was to evaluate serum osteopontin and vitronectin levels in a cohort of patients with systemic sclerosis (SSc). Eighty-six patients with SSc, 46 patients with systemic lupus erythematosus (SLE), and 38 healthy controls (HC) were enrolled in the study. Serum osteopontin, vitronectin, IL-6, and TGF-β levels were analyzed. Serum osteopontin levels were higher in the SSc and SLE groups compared to the HC group (p < 0.01 and p < 0.001, respectively). However, it was not correlated with disease activity and severity scores in the SSc group. On the other hand, serum vitronectin levels were lower in the SSc group than in the SLE and HC groups (p < 0.001 for both). These results may suggest that osteopontin levels may be increased due to the inflammatory process and osteopontin has not a specific role on fibrosis in SSc. On the other hand, serum vitronectin levels decrease in SSc in contrast to SLE. It may be concluded that the one cause of decreased serum vitronectin levels in SSc may be its accumulation in fibrotic area.

  5. Design of a Vitronectin-Based Recombinant Protein as a Defined Substrate for Differentiation of Human Pluripotent Stem Cells into Hepatocyte-Like Cells.

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    Masato Nagaoka

    Full Text Available Maintenance and differentiation of human pluripotent stem cells (hPSCs usually requires culture on a substrate for cell adhesion. A commonly used substratum is Matrigel purified from Engelbreth-Holm-Swarm sarcoma cells, and consists of a complex mixture of extracellular matrix proteins, proteoglycans, and growth factors. Several studies have successfully induced differentiation of hepatocyte-like cells from hPSCs. However, most of these studies have used Matrigel as a cell adhesion substrate, which is not a defined culture condition. In an attempt to generate a substratum that supports undifferentiated properties and differentiation into hepatic lineage cells, we designed novel substrates consisting of vitronectin fragments fused to the IgG Fc domain. hPSCs adhered to these substrates via interactions between integrins and the RGD (Arg-Gly-Asp motif, and the cells maintained their undifferentiated phenotypes. Using a previously established differentiation protocol, hPSCs were efficiently differentiated into mesendodermal and hepatic lineage cells on a vitronectin fragment-containing substrate. We found that full-length vitronectin did not support stable cell adhesion during the specification stage. Furthermore, the vitronectin fragment with the minimal RGD-containing domain was sufficient for differentiation of human induced pluripotent stem cells into hepatic lineage cells under completely defined conditions that facilitate the clinical application of cells differentiated from hPSCs.

  6. Incorporation of vitronectin into fibrin clots. Evidence for a binding interaction between vitronectin and gamma A/gamma' fibrinogen.

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    Podor, Thomas J; Campbell, Stephanie; Chindemi, Paul; Foulon, Denise M; Farrell, David H; Walton, Philip D; Weitz, Jeffrey I; Peterson, Cynthia B

    2002-03-01

    Vitronectin is an abundant plasma protein that regulates coagulation, fibrinolysis, complement activation, and cell adhesion. Recently, we demonstrated that plasma vitronectin inhibits fibrinolysis by mediating the interaction of type 1 plasminogen activator inhibitor with fibrin (Podor, T. J., Peterson, C. B., Lawrence, D. A., Stefansson, S., Shaughnessy, S. G., Foulon, D. M., Butcher, M., and Weitz, J. I. (2000) J. Biol. Chem. 275, 19788-19794). The current studies were undertaken to further examine the interactions between vitronectin and fibrin(ogen). Comparison of vitronectin levels in plasma with those in serum indicates that approximately 20% of plasma vitronectin is incorporated into the clot. When the time course of biotinylated-vitronectin incorporation into clots formed from (125)I-fibrinogen is monitored, vitronectin incorporation into the clot parallels that of fibrinogen in the absence or presence of activated factor XIII. Vitronectin binds specifically to fibrin matrices with an estimated K(d) of approximately 0.6 microm. Additional vitronectin subunits are assembled on fibrin-bound vitronectin multimers through self-association. Confocal microscopy of fibrin clots reveals the globular vitronectin aggregates anchored at intervals along the fibrin fibrils. This periodicity raised the possibility that vitronectin interacts with the gamma A/gamma' variant of fibrin(ogen) that represents about 10% of total fibrinogen. In support of this concept, the vitronectin which contaminates fibrinogen preparations co-purifies with the gamma A/gamma' fibrinogen fraction, and clots formed from gamma A/gamma' fibrinogen preferentially bind vitronectin. These studies reveal that vitronectin associates with fibrin during coagulation, and may thereby modulate hemostasis and inflammation.

  7. Identification of outer membrane Porin D as a vitronectin-binding factor in cystic fibrosis clinical isolates of Pseudomonas aeruginosa

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    Paulsson, Magnus; Singh, Birendra; Al-Jubair, Tamim

    2015-01-01

    BACKGROUND: Pseudomonas aeruginosa is a pathogen that frequently colonizes patients with cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD). Several pathogens are known to bind vitronectin to increase their virulence. Vitronectin has been shown to enhance P. aeruginosa adhesion...

  8. Vitronectin in human breast carcinomas

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    Aaboe, Mads; Offersen, Birgitte Vrou; Christensen, Anni

    2003-01-01

    We have analysed the occurrence of the extracellular glycoprotein vitronectin in carcinomas and normal tissue of human breast. Immunohistochemical analysis of carcinomas revealed a strong vitronectin accumulation in extracellular matrix (ECM) around some cancer cell clusters and in the subendothe......We have analysed the occurrence of the extracellular glycoprotein vitronectin in carcinomas and normal tissue of human breast. Immunohistochemical analysis of carcinomas revealed a strong vitronectin accumulation in extracellular matrix (ECM) around some cancer cell clusters...... and in the subendothelial area of some blood vessels. In normal tissue, vitronectin had a homogeneous periductal occurrence, with local accumulation much lower than that in the carcinomas. Using a new solid phase radioligand assay, the vitronectin concentrations of extracts of carcinomas and normal breast tissue were...... is not synthesised locally in breast tissue but derived by leakage from vessels, followed by extracellular accumulation in patterns distinctly different in carcinomas and normal tissue. The observation of a high vitronectin content in the carcinomas and its localisation in the tissue contributes to the clarification...

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  10. Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin.

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    De Lorenzi, Valentina; Sarra Ferraris, Gian Maria; Madsen, Jeppe B; Lupia, Michela; Andreasen, Peter A; Sidenius, Nicolai

    2016-07-01

    Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI-1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR Moreover, we show that PAI-1 counteracts the negative feedback and behaves as a proteolysis-triggered stabilizer of uPAR-mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N-terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process. © 2016 The Authors.

  11. Deposition of C3, the terminal complement complex and vitronectin in primary biliary cirrhosis and primary sclerosing cholangitis

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    Garred, P; Lyon, H; Christoffersen, P

    1993-01-01

    -dependent cytotoxic mechanisms in the pathogenesis. Therefore, we investigated liver biopsy specimens from 21 patients with PBC, six patients with PSC and six controls for complement deposits by immunohistochemistry using polyclonal and monoclonal antibodies against C3d, the terminal complement complex (TCC......) and vitronectin (S-protein). We found C3d, TCC and vitronectin deposits only in the portal tracts. C3d and TCC were present in the walls of the hepatic arteries and in the connective tissue stroma but never around the bile ducts. We found vitronectin deposits throughout the connective tissue, often independent...... of the TCC deposits. When vitronectin and TCC were co-localized, the staining patterns were inverse; that is, intense staining for TCC accompanied weak staining for vitronectin and vice versa. Occasionally complete dissociation between TCC and vitronectin staining was observed. Deposits of TCC...

  12. Vitronectin--master controller or micromanager?

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    Leavesley, David I; Kashyap, Abhishek S; Croll, Tristan; Sivaramakrishnan, Manaswini; Shokoohmand, Ali; Hollier, Brett G; Upton, Zee

    2013-10-01

    The concept that the mammalian glycoprotein vitronectin acts as a biological 'glue' and key controller of mammalian tissue repair and remodelling activity is emerging from nearly 50 years of experimental in vitro and in vivo data. Unexpectedly, the vitronectin-knockout (VN-KO) mouse was found to be viable and to have largely normal phenotype. However, diligent observation revealed that the VN-KO animal exhibits delayed coagulation and poor wound healing. This is interpreted to indicate that VN occupies a role in the earliest events of thrombogenesis and tissue repair. VN is the foundation upon which the thrombus grows in an organised structure. In addition to sealing the wound, the thrombus also serves to protect the underlying tissue from oxidation, is a reservoir of mitogens and tissue repair mediators, and provides a provisional scaffold for the repairing tissue. In the absence of VN (e.g., VN-KO animal), this cascade is disrupted before it begins. A wide variety of biologically active species associate with VN. Although initial studies were focused on mitogens, other classes of bioactives (e.g., glycosaminoglycans and metalloproteinases) are now also known to specifically interact with VN. Although some interactions are transient, others are long-lived and often result in multi-protein complexes. Multi-protein complexes provide several advantages: prolonging molecular interactions, sustaining local concentrations, facilitating co-stimulation of cell surface receptors and thereby enhancing cellular/biological responses. We contend that these, or equivalent, multi-protein complexes facilitate VN polyfunctionality in vivo. It is also likely that many of the species demonstrated to associate with VN in vitro, also associate with VN in vivo in similar multi-protein complexes. Thus, the predominant biological function of VN is that of a master controller of the extracellular environment; informing, and possibly instructing cells 'where' to behave, 'when' to behave

  13. Human Vitronectin-Derived Peptide Covalently Grafted onto Titanium Surface Improves Osteogenic Activity: A Pilot In Vivo Study on Rabbits.

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    Cacchioli, Antonio; Ravanetti, Francesca; Bagno, Andrea; Dettin, Monica; Gabbi, Carlo

    2009-10-01

    Peptide and protein exploitation for the biochemical functionalization of biomaterial surfaces allowed fabricating biomimetic devices able to evoke and promote specific and advantageous cell functions in vitro and in vivo. In particular, cell adhesion improvement to support the osseointegration of implantable devices has been thoroughly investigated. This study was aimed at checking the biological activity of the (351-359) human vitronectin precursor (HVP) sequence, mapped on the human vitronectin protein; the peptide was covalently linked to the surface of titanium cylinders, surgically inserted in the femurs of New Zealand white rabbits and analyzed at short experimental time points (4, 9, and 16 days after surgery). To assess the osteogenic activity of the peptide, three vital fluorochromic bone markers were used (calcein green, xylenol orange, and calcein blue) to stain the areas of newly grown bone. Static and dynamic histomorphometric parameters were measured at the bone-implant interface and at different distances from the surface. The biological role of the (351-359)HVP sequence was checked by comparing peptide-grafted samples and controls, analyzing how and how much its effects change with time across the bone regions surrounding the implant surface. The results obtained reveal a major activity of the investigated peptide 4 days after surgery, within the bone region closest to the implant surface, and larger bone to implant contact 9 and 16 days after surgery. Thus, improved primary fixation of endosseous devices can be foreseen, resulting in an increased osteointegration.

  14. Conserved Patterns of Microbial Immune Escape: Pathogenic Microbes of Diverse Origin Target the Human Terminal Complement Inhibitor Vitronectin via a Single Common Motif.

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    Teresia Hallström

    Full Text Available Pathogenicity of many microbes relies on their capacity to resist innate immunity, and to survive and persist in an immunocompetent human host microbes have developed highly efficient and sophisticated complement evasion strategies. Here we show that different human pathogens including Gram-negative and Gram-positive bacteria, as well as the fungal pathogen Candida albicans, acquire the human terminal complement regulator vitronectin to their surface. By using truncated vitronectin fragments we found that all analyzed microbial pathogens (n = 13 bound human vitronectin via the same C-terminal heparin-binding domain (amino acids 352-374. This specific interaction leaves the terminal complement complex (TCC regulatory region of vitronectin accessible, allowing inhibition of C5b-7 membrane insertion and C9 polymerization. Vitronectin complexed with the various microbes and corresponding proteins was thus functionally active and inhibited complement-mediated C5b-9 deposition. Taken together, diverse microbial pathogens expressing different structurally unrelated vitronectin-binding molecules interact with host vitronectin via the same conserved region to allow versatile control of the host innate immune response.

  15. Identification and therapeutic potential of a vitronectin binding region of meningococcal msf.

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    Darryl J Hill

    Full Text Available The human pathogen Neisseria meningitides (Nm attains serum resistance via a number of mechanisms, one of which involves binding to the host complement regulator protein vitronectin. We have shown previously that the Meningococcal surface fibril (Msf, a trimeric autotransporter, binds to the activated form of vitronectin (aVn to increase Nm survival in human serum. In this study, we aimed to identify the aVn-binding region of Msf to assess its potential as an antigen which can elicit antibodies that block aVn binding and/or possess bactericidal properties. Using several recombinant Msf fragments spanning its surface-exposed region, the smallest aVn-binding recombinants were found to span residues 1-86 and 39-124. The use of further deletion constructs and overlapping recombinant Msf fragments suggested that a region of Msf comprising residues 39-82 may be primarily important for aVn binding and that other regions may also be involved but to a lesser extent. Molecular modelling implicated K66 and K68, conserved in all available Msf sequences, to be involved in the interaction. Recombinant fragments which bound to aVn were able to reduce the survival advantage conveyed by aVn-interaction in serum bactericidal assays. Antibodies raised against one such fragment inhibited aVn binding to Msf. In addition, the antibodies enhanced specific killing of Msf-expressing Nm in a dose-dependent manner. Overall, this study identifies an aVn-binding region of Msf, an adhesin known to impart serum resistance properties to the pathogen; and shows that this region of Msf can elicit antibodies with dual properties which reduce pathogen survival within the host and thus has potential as a vaccine antigen.

  16. Identification and therapeutic potential of a vitronectin binding region of meningococcal msf.

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    Hill, Darryl J; Griffiths, Natalie J; Borodina, Elena; Andreae, Clio A; Sessions, Richard B; Virji, Mumtaz

    2015-01-01

    The human pathogen Neisseria meningitides (Nm) attains serum resistance via a number of mechanisms, one of which involves binding to the host complement regulator protein vitronectin. We have shown previously that the Meningococcal surface fibril (Msf), a trimeric autotransporter, binds to the activated form of vitronectin (aVn) to increase Nm survival in human serum. In this study, we aimed to identify the aVn-binding region of Msf to assess its potential as an antigen which can elicit antibodies that block aVn binding and/or possess bactericidal properties. Using several recombinant Msf fragments spanning its surface-exposed region, the smallest aVn-binding recombinants were found to span residues 1-86 and 39-124. The use of further deletion constructs and overlapping recombinant Msf fragments suggested that a region of Msf comprising residues 39-82 may be primarily important for aVn binding and that other regions may also be involved but to a lesser extent. Molecular modelling implicated K66 and K68, conserved in all available Msf sequences, to be involved in the interaction. Recombinant fragments which bound to aVn were able to reduce the survival advantage conveyed by aVn-interaction in serum bactericidal assays. Antibodies raised against one such fragment inhibited aVn binding to Msf. In addition, the antibodies enhanced specific killing of Msf-expressing Nm in a dose-dependent manner. Overall, this study identifies an aVn-binding region of Msf, an adhesin known to impart serum resistance properties to the pathogen; and shows that this region of Msf can elicit antibodies with dual properties which reduce pathogen survival within the host and thus has potential as a vaccine antigen.

  17. Is vitronectin the velcro that binds the gametes together?

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    Fusi, F M; Bernocchi, N; Ferrari, A; Bronson, R A

    1996-11-01

    Evidence has been presented that the adhesion of human spermatozoa to the oolemma is mediated by integrins recognizing the Arg-Gly-Asp sequence (RGD). Fibronectin and vitronectin, glycoproteins that contain functional RGD sequences, are both present on human spermatozoa, and integrins that recognize these ligands have been detected on spermatozoa and eggs. In this work, we studied the effects of oligopeptides specifically designed to block fibronectin or vitronectin receptors on the interaction of human spermatozoa with zona-free hamster oocytes. GRGDdSP, a peptide blocking cell attachment to fibronectin, was without effect, while GdRGDSP, which blocks both fibronectin and vitronectin receptors, significantly inhibited the binding of human spermatozoa to the oolemma of zona-free hamster eggs, in a concentration-dependent manner, over a range 1-100 microM. As these experiments suggested that a vitronectin receptor plays a role in sperm-oolemmal adhesion, we performed a series of experiments studying the effects of exogenous vitronectin, when added to spermatozoa and oocytes, on gamete interactions. Sperm-oolemmal adherence, as well as sperm aggregation, was promoted by vitronectin, over range of 2.2 nM to 1 microM, but only in the presence of calcium ions. We propose that vitronectin released during the sperm acrosome reaction is recognized by both gametes and plays a role in their adhesion.

  18. Interactions of plasminogen activator inhibitor-1 with vitronectin involve an extensive binding surface and induce mutual conformational rearrangements

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    Blouse, Grant E; Dupont, Daniel Miotto; Schar, Christine R

    2009-01-01

    In order to explore early events during the association of plasminogen activator inhibitor-1 (PAI-1) with its cofactor vitronectin, we have applied a robust strategy that combines protein engineering, fluorescence spectroscopy, and rapid reaction kinetics. Fluorescence stopped-flow experiments de...

  19. Selection of a Novel Aptamer Against Vitronectin Using Capillary Electrophoresis and Next Generation Sequencing

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    Christopher H Stuart

    2016-01-01

    Full Text Available Breast cancer (BC results in ≃40,000 deaths each year in the United States and even among survivors treatment of the disease may have devastating consequences, including increased risk for heart disease and cognitive impairment resulting from the toxic effects of chemotherapy. Aptamer-mediated drug delivery can contribute to improved treatment outcomes through the selective delivery of chemotherapy to BC cells, provided suitable cancer-specific antigens can be identified. We report here the use of capillary electrophoresis in conjunction with next generation sequencing to develop the first vitronectin (VN binding aptamer (VBA-01; Kd 405 nmol/l, the first aptamer to vitronectin (VN; Kd = 405 nmol/l, a protein that plays an important role in wound healing and that is present at elevated levels in BC tissue and in the blood of BC patients relative to the corresponding nonmalignant tissues. We used VBA-01 to develop DVBA-01, a dimeric aptamer complex, and conjugated doxorubicin (Dox to DVBA-01 (7:1 ratio using pH-sensitive, covalent linkages. Dox conjugation enhanced the thermal stability of the complex (60.2 versus 46.5°C and did not decrease affinity for the VN target. The resulting DVBA-01-Dox complex displayed increased cytotoxicity to MDA-MB-231 BC cells that were cultured on plasticware coated with VN (1.8 × 10−6mol/l relative to uncoated plates (2.4 × 10−6 mol/l, or plates coated with the related protein fibronectin (2.1 × 10−6 mol/l. The VBA-01 aptamer was evaluated for binding to human BC tissue using immunohistochemistry and displayed tissue specific binding and apparent association with BC cells. In contrast, a monoclonal antibody that preferentially binds to multimeric VN primarily stained extracellular matrix and vessel walls of BC tissue. Our results indicate a strong potential for using VN-targeting aptamers to improve drug delivery to treat BC.

  20. A composite role of vitronectin and urokinase in the modulation of cell morphology upon expression of the urokinase receptor

    DEFF Research Database (Denmark)

    Hillig, Thore; Engelholm, Lars H; Ingvarsen, Signe

    2008-01-01

    The urokinase receptor, urokinase receptor (uPAR), is a glycosylphosphatidylinositol-anchored membrane protein engaged in pericellular proteolysis and cellular adhesion, migration, and modulation of cell morphology. A direct matrix adhesion is mediated through the binding of uPAR to vitronectin......, and this event is followed by downstream effects including changes in the cytoskeletal organization. However, it remains unclear whether the adhesion through uPAR-vitronectin is the only event capable of initiating these morphological rearrangements or whether lateral interactions between uPAR and integrins can...... a different single-site mutant, uPAR(Y57A), in the presence of a synthetic uPAR-binding peptide, as well as with wild-type uPAR, which underwent cytoskeletal rearrangements even when cultivated in uPA-deficient serum. Blocking of integrins with an Arg-Gly-Asp-containing peptide counteracted the matrix...

  1. Integrin-mediated adhesion of human mesenchymal stem cells to extracellular matrix proteins adsorbed to polymer surfaces

    International Nuclear Information System (INIS)

    Dånmark, S; Mustafa, K; Finne-Wistrand, A; Albertsson, A-C; Patarroyo, M

    2012-01-01

    In vitro, degradable aliphatic polyesters are widely used as cell carriers for bone tissue engineering, despite their lack of biological cues. Their biological active surface is rather determined by an adsorbed layer of proteins from the surrounding media. Initial cell fate, including adhesion and proliferation, which are key properties for efficient cell carriers, is determined by the adsorbed layer of proteins. Herein we have investigated the ability of human bone marrow derived stem cells (hBMSC) to adhere to extracellular matrix (ECM) proteins, including fibronectin and vitronectin which are present in plasma and serum. hBMSC expressed integrins for collagens, laminins, fibronectin and vitronectin. Accordingly, hBMSC strongly adhered to these purified ECM proteins by using the corresponding integrins. Although purified fibronectin and vitronectin adsorbed to aliphatic polyesters to a lower extent than to cell culture polystyrene, these low levels were sufficient to mediate adhesion of hBMSC. It was found that plasma- and serum-coated polystyrene adsorbed significant levels of both fibronectin and vitronectin, and fibronectin was identified as the major adhesive component of plasma for hBMSC; however, aliphatic polyesters adsorbed minimal levels of fibronectin under similar conditions resulting in impaired cell adhesion. Altogether, the results suggest that the efficiency of aliphatic polyesters cell carriers could be improved by increasing their ability to adsorb fibronectin. (paper)

  2. Increasing Household Protein Consumption Through Minilivestock ...

    African Journals Online (AJOL)

    Mini-livestock production can be a major contributor of a more balanced diet for both rural and urban settlements. The attributes of mini-livestock gives it the potential of increasing household protein consumption as well as being a source of income. Mini-livestock production can be practiced in rural and urban settlements ...

  3. A composite role of vitronectin and urokinase in the modulation of cell morphology upon expression of the urokinase receptor

    DEFF Research Database (Denmark)

    Engelholm, L.H.; Ingvarsen, S.; Madsen, D.H.

    2008-01-01

    , and this event is followed by downstream effects including changes in the cytoskeletal organization. However, it remains unclear whether the adhesion through uPAR-vitronectin is the only event capable of initiating these morphological rearrangements or whether lateral interactions between uPAR and integrins can...... adhesion and cytoskeletal rearrangements, whereas a mutant uPAR, uPAR(W32A) with defective vitronectin binding, failed to induce both phenomena. However, upon saturation of uPAR(W32A) with the protease ligand, pro-uPA, or its receptor-binding domain, the ability to induce cytoskeletal rearrangements...... was restored, although this did not rescue the uPAR-vitronectin binding and adhesion capability. On the other hand, using other uPAR variants, we could show that uPAR-vitronectin adhesion is indeed capable and sufficient to induce the same morphological rearrangements. This was shown with cells expressing...

  4. Cyr61/CCN1 displays high-affinity binding to the somatomedin B(1-44 domain of vitronectin.

    Directory of Open Access Journals (Sweden)

    Ivo M B Francischetti

    2010-02-01

    Full Text Available Cyr61 is a member of the CCN (Cyr61, connective tissue growth, NOV family of extracellular-associated (matricellular proteins that present four distinct functional modules, namely insulin-like growth factor binding protein (IGFBP, von Willebrand factor type C (vWF, thrombospondin type 1 (TSP, and C-terminal growth factor cysteine knot (CT domain. While heparin sulphate proteoglycans reportedly mediate the interaction of Cyr61 with the matrix and cell surface, the role of other extracellular associated proteins has not been revealed.In this report, surface plasmon resonance (SPR experiments and solid-phase binding assays demonstrate that recombinant Cyr61 interacts with immobilized monomeric or multimeric vitronectin (VTNC with K(D in the nanomolar range. Notably, the binding site for Cyr61 was identified as the somatomedin B domain (SMTB(1-44 of VTNC, which mediates its interaction with PAI-1, uPAR, and integrin alphav beta3. Accordingly, PAI-1 outcompetes Cyr61 for binding to immobilized SMTB(1-44, and Cyr61 attenuates uPAR-mediated U937 adhesion to VTNC. In contrast, isothermal titration calorimetry shows that Cyr61 does not display high-affinity binding for SMTB(1-44 in solution. Nevertheless, competitive ELISA revealed that multimeric VTNC, heat-modified monomeric VTNC, or SMTB(1-44 at high concentrations attenuate Cyr61 binding to immobilized VTNC, while monomeric VTNC was ineffective. Therefore, immobilization of VTNC exposes cryptic epitopes that recognize Cyr61 with high affinity, as reported for a number of antibodies, beta-endorphin, and other molecules.The finding that Cyr61 interacts with the SMTB(1-44 domain suggests that VTNC represent a point of anchorage for CCN family members to the matrix. Results are discussed in the context of the role of CCN and VTNC in matrix biology and angiogenesis.

  5. The 82-plex plasma protein signature that predicts increasing inflammation

    DEFF Research Database (Denmark)

    Tepel, Martin; Beck, Hans C; Tan, Qihua

    2015-01-01

    The objective of the study was to define the specific plasma protein signature that predicts the increase of the inflammation marker C-reactive protein from index day to next-day using proteome analysis and novel bioinformatics tools. We performed a prospective study of 91 incident kidney....... The prediction model selected and validated 82 plasma proteins which determined increased next-day C-reactive protein (area under receiver-operator-characteristics curve, 0.772; 95% confidence interval, 0.669 to 0.876; P signature (P ....001) was associated with observed increased next-day C-reactive protein. The 82-plex protein signature outperformed routine clinical procedures. The category-free net reclassification index improved with 82-plex plasma protein signature (total net reclassification index, 88.3%). Using the 82-plex plasma protein...

  6. Protein intake does not increase vastus lateralis muscle protein synthesis during cycling

    DEFF Research Database (Denmark)

    Hulston, CJ; Wolsk, Emil; Grøndahl, Thomas Sahl

    2011-01-01

    PURPOSE: This study aimed to investigate the effect of protein ingestion on leg protein turnover and vastus lateralis muscle protein synthesis during bicycle exercise and recovery. METHODS: Eight healthy males participated in two experiments in which they ingested either a carbohydrate solution...... sampling, and blood flow measurements. Muscle protein synthesis was calculated from the incorporation of l-[ring-C6]phenylalanine into protein. RESULTS: Consuming protein during exercise increased leg protein synthesis and decreased net leg protein breakdown; however, protein ingestion did not increase...... protein synthesis within the highly active vastus lateralis muscle (0.029%·h(-1), ± 0.004%·h(-1), and 0.030%·h(-1), ± 0.003%·h(-1), in CHO and CHO + P, respectively; P = 0.88). In contrast, consuming protein, during exercise and recovery, increased postexercise vastus lateralis muscle protein synthesis...

  7. Evaluation of Vitronectin Expression in Prostate Cancer and the Clinical Significance of the Association of Vitronectin Expression with Prostate Specific Antigen in Detecting Prostate Cancer.

    Science.gov (United States)

    Niu, Yue; Zhang, Ling; Bi, Xing; Yuan, Shuai; Chen, Peng

    2016-03-05

    To detect the expression of vitronectin (VTN) in the tissues and blood serum of prostate cancer (PCa) patients, and evaluate its clinical significance and to evaluate the significance of the combined assay of VTN and prostate specific antigens (PSA) in PCa diagnosis. To detect the expression of VTN as a potential marker for PCa diagnosis and prognosis, immunohistochemistry was performed on the tissues of 32 patients with metastatic PCa (PCaM), 34 patients with PCa without metastasis (PCa), and 41 patients with benign prostatic hyperplasia (BPH). The sera were then subjected to Western blot analysis. All cases were subsequently examined to determine the concentrations of PSA and VTN in the sera. The collected data were collated and analyzed. The positive expression rates of VTN in the tissues of the BPH and PCa groups (including PCa and PCaM groups) were 75.61% and 45.45%, respectively (P = .005). VTN was more highly expressed in the sera of the BPH patients (0.83 ± 0.07) than in the sera of the PCa patients (0.65 ± 0.06) (P < .05). It was also more highly expressed in the sera of the PCa patients than in the sera of the PCaM patients (0.35 ± 0.08) (P < .05). In the diagnosis of BPH and PCa, the Youden indexes of PSA detection, VTN detection, and combined detection were 0.2620, 0.3468, and 0.5635; the kappa values were 0.338, 0.304, and 0.448, respectively, and the areas under the receiver operating characteristic curve were 0.625, 0.673, and 0.703 (P < .05), respectively. VTN levels in sera may be used as a potential marker of PCa for the diagnosis and assessment of disease progression and metastasis. The combined detection of VTN and PSA in sera can be clinically applied in PCa diagnosis. .

  8. Increasing protein stability by improving beta-turns.

    Science.gov (United States)

    Fu, Hailong; Grimsley, Gerald R; Razvi, Abbas; Scholtz, J Martin; Pace, C Nick

    2009-11-15

    Our goal was to gain a better understanding of how protein stability can be increased by improving beta-turns. We studied 22 beta-turns in nine proteins with 66-370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some beta-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein, Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase alpha-subunit, and Maltose binding protein. Of the 15 single proline mutations, 11 increased stability (Average = 0.8 +/- 0.3; Range = 0.3-1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. On the basis of this and our previous work, we conclude that proteins can generally be stabilized by replacing nonproline residues with proline residues at the i + 1 position of Type I and II beta-turns and at the i position in Type II beta-turns. Other turn positions can sometimes be used if the phi angle is near -60 degrees for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in beta-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in beta-turns that could be replaced by Gly to increase protein stability. Improving beta-turns by substituting Pro residues is a generally useful way of increasing protein stability. 2009 Wiley-Liss, Inc.

  9. The relationship between vitronectin and hepatic insulin resistance in type 2 diabetes mellitus.

    Science.gov (United States)

    Cao, Yan; Li, Xinyu; Lu, Chong; Zhan, Xiaorong

    2018-05-18

    The World Health Organization (WHO) estimates that approximately 300 million people will suffer from diabetes mellitus by 2025. Type 2 diabetes mellitus (T2DM) is much more prevalent. T2DM comprises approximately 90% of diabetes mellitus cases, and it is caused by a combination of insulin resistance and inadequate compensatory insulin secretory response. In this study, we aimed to compare the plasma vitronectin (VN) levels between patients with T2DM and insulin resistance (IR) and healthy controls. Seventy patients with IR and 70 age- and body mass index (BMI)-matched healthy controls were included in the study. The insulin, Waist-to-Hip Ratio (WHR), C-peptide (CP) and VN levels of all participants were examined. The homeostasis model of assessment for insulin resistence index (HOMA-IR (CP)) formula was used to calculate insulin resistance. The levels of BMI, fasting plasma gluose (FPG), 2-hour postprandial glucose (2hPG), glycated hemoglobins (HbA1c), and HOMA-IR (CP) were significantly elevated in case group compared with controls. VN was found to be significantly decreased in case group. (VN Mean (Std): 8.55 (2.92) versus 12.88 (1.26) ng/mL p insulin resistance in patients with T2DM.

  10. Protein energy malnutrition increases arginase activity in monocytes and macrophages.

    Science.gov (United States)

    Corware, Karina; Yardley, Vanessa; Mack, Christopher; Schuster, Steffen; Al-Hassi, Hafid; Herath, Shanthi; Bergin, Philip; Modolell, Manuel; Munder, Markus; Müller, Ingrid; Kropf, Pascale

    2014-01-01

    Protein energy malnutrition is commonly associated with immune dysfunctions and is a major factor in susceptibility to infectious diseases. In this study, we evaluated the impact of protein energy malnutrition on the capacity of monocytes and macrophages to upregulate arginase, an enzyme associated with immunosuppression and increased pathogen replication. Our results show that monocytes and macrophages are significantly increased in the bone marrow and blood of mice fed on a protein low diet. No alteration in the capacity of bone marrow derived macrophages isolated from malnourished mice to phagocytose particles, to produce the microbicidal molecule nitric oxide and to kill intracellular Leishmania parasites was detected. However, macrophages and monocytes from malnourished mice express significantly more arginase both in vitro and in vivo. Using an experimental model of visceral leishmaniasis, we show that following protein energy malnutrition, the increased parasite burden measured in the spleen of these mice coincided with increased arginase activity and that macrophages provide a more permissive environment for parasite growth. Taken together, these results identify a novel mechanism in protein energy malnutrition that might contributes to increased susceptibility to infectious diseases by upregulating arginase activity in myeloid cells.

  11. Shortening a loop can increase protein native state entropy.

    Science.gov (United States)

    Gavrilov, Yulian; Dagan, Shlomi; Levy, Yaakov

    2015-12-01

    Protein loops are essential structural elements that influence not only function but also protein stability and folding rates. It was recently reported that shortening a loop in the AcP protein may increase its native state conformational entropy. This effect on the entropy of the folded state can be much larger than the lower entropic penalty of ordering a shorter loop upon folding, and can therefore result in a more pronounced stabilization than predicted by polymer model for loop closure entropy. In this study, which aims at generalizing the effect of loop length shortening on native state dynamics, we use all-atom molecular dynamics simulations to study how gradual shortening a very long or solvent-exposed loop region in four different proteins can affect their stability. For two proteins, AcP and Ubc7, we show an increase in native state entropy in addition to the known effect of the loop length on the unfolded state entropy. However, for two permutants of SH3 domain, shortening a loop results only with the expected change in the entropy of the unfolded state, which nicely reproduces the observed experimental stabilization. Here, we show that an increase in the native state entropy following loop shortening is not unique to the AcP protein, yet nor is it a general rule that applies to all proteins following the truncation of any loop. This modification of the loop length on the folded state and on the unfolded state may result with a greater effect on protein stability. © 2015 Wiley Periodicals, Inc.

  12. Mechanisms underlying the attachment and spreading of human osteoblasts: from transient interactions to focal adhesions on vitronectin-grafted bioactive surfaces.

    Science.gov (United States)

    Brun, Paola; Scorzeto, Michele; Vassanelli, Stefano; Castagliuolo, Ignazio; Palù, Giorgio; Ghezzo, Francesca; Messina, Grazia M L; Iucci, Giovanna; Battaglia, Valentina; Sivolella, Stefano; Bagno, Andrea; Polzonetti, Giovanni; Marletta, Giovanni; Dettin, Monica

    2013-04-01

    The features of implant devices and the reactions of bone-derived cells to foreign surfaces determine implant success during osseointegration. In an attempt to better understand the mechanisms underlying osteoblasts attachment and spreading, in this study adhesive peptides containing the fibronectin sequence motif for integrin binding (Arg-Gly-Asp, RGD) or mapping the human vitronectin protein (HVP) were grafted on glass and titanium surfaces with or without chemically induced controlled immobilization. As shown by total internal reflection fluorescence microscopy, human osteoblasts develop adhesion patches only on specifically immobilized peptides. Indeed, cells quickly develop focal adhesions on RGD-grafted surfaces, while HVP peptide promotes filopodia, structures involved in cellular spreading. As indicated by immunocytochemistry and quantitative polymerase chain reaction, focal adhesions kinase activation is delayed on HVP peptides with respect to RGD while an osteogenic phenotypic response appears within 24h on osteoblasts cultured on both peptides. Cellular pathways underlying osteoblasts attachment are, however, different. As demonstrated by adhesion blocking assays, integrins are mainly involved in osteoblast adhesion to RGD peptide, while HVP selects osteoblasts for attachment through proteoglycan-mediated interactions. Thus an interfacial layer of an endosseous device grafted with specifically immobilized HVP peptide not only selects the attachment and supports differentiation of osteoblasts but also promotes cellular migration. Copyright © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  13. Animal Agriculture as Panacea for Increased Protein Intake in Nigeria

    African Journals Online (AJOL)

    The world's population is expected to increase from 6 billion to more than 7.5 billion by 2020. This burgeoning population may require a doubling of animal protein and a corresponding doubling of feed grains demand. Nigeria is currently the most populous black nation in the world with the threat of nutritional deficiency ...

  14. Consumption of Milk Protein or Whey Protein Results in a Similar Increase in Muscle Protein Synthesis in Middle Aged Men.

    Science.gov (United States)

    Mitchell, Cameron J; McGregor, Robin A; D'Souza, Randall F; Thorstensen, Eric B; Markworth, James F; Fanning, Aaron C; Poppitt, Sally D; Cameron-Smith, David

    2015-10-21

    The differential ability of various milk protein fractions to stimulate muscle protein synthesis (MPS) has been previously described, with whey protein generally considered to be superior to other fractions. However, the relative ability of a whole milk protein to stimulate MPS has not been compared to whey. Sixteen healthy middle-aged males ingested either 20 g of milk protein (n = 8) or whey protein (n = 8) while undergoing a primed constant infusion of ring (13)C₆ phenylalanine. Muscle biopsies were obtained 120 min prior to consumption of the protein and 90 and 210 min afterwards. Resting myofibrillar fractional synthetic rates (FSR) were 0.019% ± 0.009% and 0.021% ± 0.018% h(-1) in the milk and whey groups respectively. For the first 90 min after protein ingestion the FSR increased (p whey groups respectively with no difference between groups (p = 0.810). FSR returned to baseline in both groups between 90 and 210 min after protein ingestion. Despite evidence of increased rate of digestion and leucine availability following the ingestion of whey protein, there was similar activation of MPS in middle-aged men with either 20 g of milk protein or whey protein.

  15. Protein enrichment of familiar foods as an innovative strategy to increase protein intake in institutionalized elderly

    NARCIS (Netherlands)

    Beelen, J.; Roos, de N.M.; Groot, de C.P.G.M.

    2017-01-01

    Objective
    To increase the protein intake of older adults, protein enrichment of familiar foods and drinks might be an effective and attractive alternative for oral nutritional supplements (ONS). We performed a pilot study to test whether these products could help institutionalized elderly to

  16. SGLT2 Protein Expression Is Increased in Human Diabetic Nephropathy

    Science.gov (United States)

    Wang, Xiaoxin X.; Levi, Jonathan; Luo, Yuhuan; Myakala, Komuraiah; Herman-Edelstein, Michal; Qiu, Liru; Wang, Dong; Peng, Yingqiong; Grenz, Almut; Lucia, Scott; Dobrinskikh, Evgenia; D'Agati, Vivette D.; Koepsell, Hermann; Kopp, Jeffrey B.; Rosenberg, Avi Z.; Levi, Moshe

    2017-01-01

    There is very limited human renal sodium gradient-dependent glucose transporter protein (SGLT2) mRNA and protein expression data reported in the literature. The first aim of this study was to determine SGLT2 mRNA and protein levels in human and animal models of diabetic nephropathy. We have found that the expression of SGLT2 mRNA and protein is increased in renal biopsies from human subjects with diabetic nephropathy. This is in contrast to db-db mice that had no changes in renal SGLT2 protein expression. Furthermore, the effect of SGLT2 inhibition on renal lipid content and inflammation is not known. The second aim of this study was to determine the potential mechanisms of beneficial effects of SGLT2 inhibition in the progression of diabetic renal disease. We treated db/db mice with a selective SGLT2 inhibitor JNJ 39933673. We found that SGLT2 inhibition caused marked decreases in systolic blood pressure, kidney weight/body weight ratio, urinary albumin, and urinary thiobarbituric acid-reacting substances. SGLT2 inhibition prevented renal lipid accumulation via inhibition of carbohydrate-responsive element-binding protein-β, pyruvate kinase L, SCD-1, and DGAT1, key transcriptional factors and enzymes that mediate fatty acid and triglyceride synthesis. SGLT2 inhibition also prevented inflammation via inhibition of CD68 macrophage accumulation and expression of p65, TLR4, MCP-1, and osteopontin. These effects were associated with reduced mesangial expansion, accumulation of the extracellular matrix proteins fibronectin and type IV collagen, and loss of podocyte markers WT1 and synaptopodin, as determined by immunofluorescence microscopy. In summary, our study showed that SGLT2 inhibition modulates renal lipid metabolism and inflammation and prevents the development of nephropathy in db/db mice. PMID:28196866

  17. Protein Enrichment of Familiar Foods as an Innovative Strategy to Increase Protein Intake in Institutionalized Elderly.

    Science.gov (United States)

    Beelen, J; de Roos, N M; de Groot, L C P G M

    2017-01-01

    To increase the protein intake of older adults, protein enrichment of familiar foods and drinks might be an effective and attractive alternative for oral nutritional supplements (ONS). We performed a pilot study to test whether these products could help institutionalized elderly to reach a protein intake of 1.2 gram per kg body weight per day (g/kg/d). Intervention study with one treatment group (no control group). Dietary assessment was done before and at the end of a 10-day intervention. Two care facilities in Gelderland, the Netherlands: a residential care home and a rehabilitation center. 22 elderly subjects (13 women, 9 men; mean age 83.0±9.4 years). We used a variety of newly developed protein enriched regular foods and drinks, including bread, soups, fruit juices, and instant mashed potatoes. Dietary intake was assessed on two consecutive days before and at the end of the intervention, using food records filled out by research assistants. Energy and macronutrient intake was calculated using the 2013 Dutch food composition database. Changes in protein intake were evaluated using paired t-tests. Protein intake increased by 11.8 g/d (P=0.003); from 0.96 to 1.14 g/kg/d (P=0.002). This increase is comparable to protein provided by one standard portion of ONS. The intake of energy and other macronutrients did not change significantly. At the end of the intervention more elderly reached a protein intake level of 1.2 g/kg/d than before (9 vs 4). Protein intake significantly increased during breakfast (+3.7 g) and during the evening (+2.2 g). Including familiar protein enriched foods and drinks in the menu helped to meet protein recommendations in institutionalized elderly.

  18. Increasing Protein Charge State When Using Laser Electrospray Mass Spectrometry

    Science.gov (United States)

    Karki, Santosh; Flanigan, Paul M.; Perez, Johnny J.; Archer, Jieutonne J.; Levis, Robert J.

    2015-05-01

    Femtosecond (fs) laser vaporization is used to transfer cytochrome c, myoglobin, lysozyme, and ubiquitin from the condensed phase into an electrospray (ES) plume consisting of a mixture of a supercharging reagent, m-nitrobenzyl alcohol ( m-NBA), and trifluoroacetic acid (TFA), acetic acid (AA), or formic acid (FA). Interaction of acid-sensitive proteins like cytochrome c and myoglobin with the highly charged ES droplets resulted in a shift to higher charge states in comparison with acid-stable proteins like lysozyme and ubiquitin. Laser electrospray mass spectrometry (LEMS) measurements showed an increase in both the average charge states (Zavg) and the charge state with maximum intensity (Zmode) for acid-sensitive proteins compared with conventional electrospray ionization mass spectrometry (ESI-MS) under equivalent solvent conditions. A marked increase in ion abundance of higher charge states was observed for LEMS in comparison with conventional electrospray for cytochrome c (ranging from 19+ to 21+ versus 13+ to 16+) and myoglobin (ranging from 19+ to 26+ versus 18+ to 21+) using an ES solution containing m-NBA and TFA. LEMS measurements as a function of electrospray flow rate yielded increasing charge states with decreasing flow rates for cytochrome c and myoglobin.

  19. Mechanical stimulation increases proliferation, differentiation and protein expression in culture

    DEFF Research Database (Denmark)

    Grossi, Alberto; Yadav, Kavita; Lawson, Moira Ann

    2007-01-01

    Myogenesis is a complex sequence of events, including the irreversible transition from the proliferation-competent myoblast stage into fused, multinucleated myotubes. Myogenic differentiation is regulated by positive and negative signals from surrounding tissues. Stimulation due to stretch- or load...... to elucidate also the signaling pathway by which this mechanical stimulation can causes an increase in protein expression. When mechanically stimulated via laminin receptors on cell surface, C(2)C(12) cells showed an increase in cell proliferation and differentiation. Populations undergoing mechanical...... stimulation through laminin receptors show an increase in expression of Myo-D, myogenin and an increase in ERK1/2 phosphorylation. Cells stimulated via fibronectin receptors show no significant increases in fusion competence. We conclude that load induced signalling through integrin containing laminin...

  20. Bactericidal Permeability-Increasing Proteins Shape Host-Microbe Interactions

    Directory of Open Access Journals (Sweden)

    Fangmin Chen

    2017-04-01

    Full Text Available We characterized bactericidal permeability-increasing proteins (BPIs of the squid Euprymna scolopes, EsBPI2 and EsBPI4. They have molecular characteristics typical of other animal BPIs, are closely related to one another, and nest phylogenetically among invertebrate BPIs. Purified EsBPIs had antimicrobial activity against the squid’s symbiont, Vibrio fischeri, which colonizes light organ crypt epithelia. Activity of both proteins was abrogated by heat treatment and coincubation with specific antibodies. Pretreatment under acidic conditions similar to those during symbiosis initiation rendered V. fischeri more resistant to the antimicrobial activity of the proteins. Immunocytochemistry localized EsBPIs to the symbiotic organ and other epithelial surfaces interacting with ambient seawater. The proteins differed in intracellular distribution. Further, whereas EsBPI4 was restricted to epithelia, EsBPI2 also occurred in blood and in a transient juvenile organ that mediates hatching. The data provide evidence that these BPIs play different defensive roles early in the life of E. scolopes, modulating interactions with the symbiont.

  1. Discovery of an imidazopyridine-containing 1,4-benzodiazepine nonpeptide vitronectin receptor (alpha v beta 3) antagonist with efficacy in a restenosis model.

    Science.gov (United States)

    Keenan, R M; Lago, M A; Miller, W H; Ali, F E; Cousins, R D; Hall, L B; Hwang, S M; Jakas, D R; Kwon, C; Louden, C; Nguyen, T T; Ohlstein, E H; Rieman, D J; Ross, S T; Samanen, J M; Smith, B R; Stadel, J; Takata, D T; Vickery, L; Yuan, C C; Yue, T L

    1998-11-17

    In the 3-oxo-1,4-benzodiazepine-2-acetic acid series of vitronectin receptor (alpha v beta 3) antagonists, a compound containing an imidazopyridine arginine mimetic was discovered which had sufficient potency and i.v. pharmacokinetics for demonstration of efficacy in a rat restenosis model.

  2. Combining modularity, conservation, and interactions of proteins significantly increases precision and coverage of protein function prediction

    Directory of Open Access Journals (Sweden)

    Sers Christine T

    2010-12-01

    Full Text Available Abstract Background While the number of newly sequenced genomes and genes is constantly increasing, elucidation of their function still is a laborious and time-consuming task. This has led to the development of a wide range of methods for predicting protein functions in silico. We report on a new method that predicts function based on a combination of information about protein interactions, orthology, and the conservation of protein networks in different species. Results We show that aggregation of these independent sources of evidence leads to a drastic increase in number and quality of predictions when compared to baselines and other methods reported in the literature. For instance, our method generates more than 12,000 novel protein functions for human with an estimated precision of ~76%, among which are 7,500 new functional annotations for 1,973 human proteins that previously had zero or only one function annotated. We also verified our predictions on a set of genes that play an important role in colorectal cancer (MLH1, PMS2, EPHB4 and could confirm more than 73% of them based on evidence in the literature. Conclusions The combination of different methods into a single, comprehensive prediction method infers thousands of protein functions for every species included in the analysis at varying, yet always high levels of precision and very good coverage.

  3. Increased Baseline C-Reactive Protein Concentrations Are Associated with Increased Risk of Infections

    DEFF Research Database (Denmark)

    Zacho, Jeppe; Benfield, Thomas; Tybjærg-Hansen, Anne

    2016-01-01

    BACKGROUND: The acute-phase reactant C-reactive protein (CRP) increases rapidly during an infection. We tested the hypothesis that chronic low-level increases in CRP are associated with an increased risk of infectious disease. METHODS: We studied 9660 individuals from a prospective general...... population cohort, including 3592 in whom infectious disease developed, and another 60 896 individuals from a cross-sectional general population study, of whom 13 332 developed infectious disease; 55% were women, and the mean age was 57 years. Hospital diagnoses of infections in 1977-2010 were based....... RESULTS: Individuals with CRP >3 mg/L had 1.2 and 1.7 times increased risk of infectious disease, in the prospective general population cohort and the cross-sectional general population study, respectively, compared with individuals with CRP

  4. A low protein diet increases the hypoxic tolerance in Drosophila.

    Directory of Open Access Journals (Sweden)

    Paul Vigne

    2006-12-01

    Full Text Available Dietary restriction is well known to increase the life span of a variety of organisms from yeast to mammals, but the relationships between nutrition and the hypoxic tolerance have not yet been considered. Hypoxia is a major cause of cell death in myocardial infarction and stroke. Here we forced hypoxia-related death by exposing one-day-old male Drosophila to chronic hypoxia (5% O(2 and analysed their survival. Chronic hypoxia reduced the average life span from 33.6 days to 6.3 days when flies were fed on a rich diet. A demographic analysis indicated that chronic hypoxia increased the slope of the mortality trajectory and not the short-term risk of death. Dietary restriction produced by food dilution, by yeast restriction, or by amino acid restriction partially reversed the deleterious action of hypoxia. It increased the life span of hypoxic flies up to seven days, which represented about 25% of the life time of an hypoxic fly. Maximum survival of hypoxic flies required only dietary sucrose, and it was insensitive to drugs such as rapamycin and resveratrol, which increase longevity of normoxic animals. The results thus uncover a new link between protein nutrition, nutrient signalling, and resistance to hypoxic stresses.

  5. ACUTE PHASE PROTEIN INCREASE IN HIGH ALTITUDE MOUNTAINEERS

    Directory of Open Access Journals (Sweden)

    Tolga Saka

    Full Text Available ABSTRACT Introduction: Many middle-aged Turks go hiking in mountains to breathe some fresh air or to maintain fitness. Objective: This study investigated the effects of regular high altitude mountain climbing on the metabolic and hematological responses of mountaineers. Methods: Hematological and biochemical parameters were studied, as well as some hormonal values of 21 mountaineers and 16 healthy age-matched sedentary volunteers. Results: The neutrophil to lymphocyte ratio (NLR was significantly lower (p<0.04 in mountaineers compared with the sedentary group. Total protein (p<0.001 and albumin (p<0.001 were lower, while the levels of ferritin (p<0.04, creatine (p<0.03 and creatine phosphokinase (p<0.01 were higher in mountaineers. Other hematological and biochemical parameters, i.e., erythrocytes, leukocytes, hemoglobin and hematocrit, did not change significantly. Conclusion: Our results show that regular exposure to high altitude increased the serum levels of some acute phase proteins with anti-inflammatory properties.

  6. Ribosome reinitiation at leader peptides increases translation of bacterial proteins.

    Science.gov (United States)

    Korolev, Semen A; Zverkov, Oleg A; Seliverstov, Alexandr V; Lyubetsky, Vassily A

    2016-04-16

    Short leader genes usually do not encode stable proteins, although their importance in expression control of bacterial genomes is widely accepted. Such genes are often involved in the control of attenuation regulation. However, the abundance of leader genes suggests that their role in bacteria is not limited to regulation. Specifically, we hypothesize that leader genes increase the expression of protein-coding (structural) genes via ribosome reinitiation at the leader peptide in the case of a short distance between the stop codon of the leader gene and the start codon of the structural gene. For instance, in Actinobacteria, the frequency of leader genes at a distance of 10-11 bp is about 70 % higher than the mean frequency within the 1 to 65 bp range; and it gradually decreases as the range grows longer. A pronounced peak of this frequency-distance relationship is also observed in Proteobacteria, Bacteroidetes, Spirochaetales, Acidobacteria, the Deinococcus-Thermus group, and Planctomycetes. In contrast, this peak falls to the distance of 15-16 bp and is not very pronounced in Firmicutes; and no such peak is observed in cyanobacteria and tenericutes. Generally, this peak is typical for many bacteria. Some leader genes located close to a structural gene probably play a regulatory role as well.

  7. Effects of increasing dietary protein levels on growth, feed utilization ...

    African Journals Online (AJOL)

    Yomi

    2012-01-05

    Jan 5, 2012 ... The effect of different dietary protein levels on growth performance and on feed utilization of catfish. (Heterobranchus ... (Legendre, 1991) because of its taste, fast growth rate ..... diet containing 40% protein had high growth with low food intake and feed ... protein rate (45%) combined with a bad utilization of.

  8. Increased renal alpha-epithelial sodium channel (ENAC) protein and increased ENAC activity in normal pregnancy.

    Science.gov (United States)

    West, Crystal; Zhang, Zheng; Ecker, Geoffrey; Masilamani, Shyama M E

    2010-11-01

    Pregnancy-mediated sodium (Na) retention is required to provide an increase in plasma volume for the growing fetus. The mechanisms responsible for this Na retention are not clear. We first used a targeted proteomics approach and found that there were no changes in the protein abundance compared with virgin rats of the β or γ ENaC, type 3 Na(+)/H(+) exchanger (NHE3), bumetanide-sensitive cotransporter (NKCC2), or NaCl cotransporter (NCC) in mid- or late pregnancy. In contrast, we observed marked increases in the abundance of the α-ENaC subunit. The plasma volume increased progressively during pregnancy with the greatest plasma volume being evident in late pregnancy. ENaC inhibition abolished the difference in plasma volume status between virgin and pregnant rats. To determine the in vivo activity of ENaC, we conducted in vivo studies of rats in late pregnancy (days 18-20) and virgin rats to measure the natriuretic response to ENaC blockade (with benzamil). The in vivo activity of ENaC (U(Na)V postbenzamil-U(Na)V postvehicle) was markedly increased in late pregnancy, and this difference was abolished by pretreatment with the mineralocorticoid receptor antagonist, eplerenone. These findings demonstrate that the increased α-ENaC subunit of pregnancy is associated with an mineralocorticoid-dependent increase in ENaC activity. Further, we show that ENaC activity is a major contributor of plasma volume status in late pregnancy. These changes are likely to contribute to the renal sodium retention and plasma volume expansion required for an optimal pregnancy.

  9. Increased Prevalence of Activated Protein C Resistance During ...

    African Journals Online (AJOL)

    Background: Acquired resistance to protein C in pregnancy has been established as one of the factors associated with ..... diabetes, sickle cell disease, smoking, anti-phospholipid syndrome inherited thrombophilia, and previous history of.

  10. Distribution of cytoskeletal proteins, integrins, leukocyte adhesion molecules and extracellular matrix proteins in plastic-embedded human and rat kidneys

    NARCIS (Netherlands)

    van Goor, H; Coers, W; van der Horst, MLC; Suurmeijer, AJH

    2001-01-01

    OBJECTIVE: To study the distribution of cytoskeletal proteins (actin, alpha -actinin, vinculin, beta -tubulin, keratin, vimentin, desmin), adhesion molecules for cell-matrix interations (very later antigens [VLA1-6], beta1, beta2 [CD18], vitronectin receptor [alphav beta3], CD 11b), leukocyte

  11. Ingestion of Wheat Protein Increases In Vivo Muscle Protein Synthesis Rates in Healthy Older Men in a Randomized Trial.

    Science.gov (United States)

    Gorissen, Stefan Hm; Horstman, Astrid Mh; Franssen, Rinske; Crombag, Julie Jr; Langer, Henning; Bierau, Jörgen; Respondek, Frederique; van Loon, Luc Jc

    2016-09-01

    Muscle mass maintenance is largely regulated by basal muscle protein synthesis and the capacity to stimulate muscle protein synthesis after food intake. The postprandial muscle protein synthetic response is modulated by the amount, source, and type of protein consumed. It has been suggested that plant-based proteins are less potent in stimulating postprandial muscle protein synthesis than animal-derived proteins. However, few data support this contention. We aimed to assess postprandial plasma amino acid concentrations and muscle protein synthesis rates after the ingestion of a substantial 35-g bolus of wheat protein hydrolysate compared with casein and whey protein. Sixty healthy older men [mean ± SEM age: 71 ± 1 y; body mass index (in kg/m(2)): 25.3 ± 0.3] received a primed continuous infusion of l-[ring-(13)C6]-phenylalanine and ingested 35 g wheat protein (n = 12), 35 g wheat protein hydrolysate (WPH-35; n = 12), 35 g micellar casein (MCas-35; n = 12), 35 g whey protein (Whey-35; n = 12), or 60 g wheat protein hydrolysate (WPH-60; n = 12). Plasma and muscle samples were collected at regular intervals. The postprandial increase in plasma essential amino acid concentrations was greater after ingesting Whey-35 (2.23 ± 0.07 mM) than after MCas-35 (1.53 ± 0.08 mM) and WPH-35 (1.50 ± 0.04 mM) (P protein synthesis rates increased after ingesting MCas-35 (P protein synthesis rates above basal rates (0.049% ± 0.007%/h; P = 0.02). The myofibrillar protein synthetic response to the ingestion of 35 g casein is greater than after an equal amount of wheat protein. Ingesting a larger amount of wheat protein (i.e., 60 g) substantially increases myofibrillar protein synthesis rates in healthy older men. This trial was registered at clinicaltrials.gov as NCT01952639. © 2016 American Society for Nutrition.

  12. Zinc finger protein 521 overexpression increased transcript levels of ...

    Indian Academy of Sciences (India)

    2016-02-12

    Feb 12, 2016 ... Zinc finger protein 521 is highly expressed in brain, neural stem cells and early progenitors of the human .... Membranes were blocked for 1 h with 10% skim milk and ..... fat-like development of white fat and thermogenesis.

  13. Enhanced Stability of a Protein with Increasing Temperature

    DEFF Research Database (Denmark)

    Vinther, Joachim Møllesøe; Kristensen, Søren M; Led, Jens J

    2010-01-01

    The unusual stability of a structured but locally flexible protein, human growth hormone (hGH) at pH 2.7, was investigated using the temperature dependence of the nanosecond-picosecond dynamics of the backbone amide groups obtained from (15)N NMR relaxation data. It is found that the flexibility ...

  14. Sex differences in snack food reinforcement in response to increasing dietary protein

    Science.gov (United States)

    BRACKGROUND: Protein is posited to play a dynamic role in energy balance and reward-driven eating behavior. However, little is known about the effect of increasing protein intake on snack food reinforcement. OBJECTIVE: We sought to determine the extent to which increasing dietary protein changes th...

  15. The stability and formation of native proteins from unfolded monomers is increased through interactions with unrelated proteins.

    Directory of Open Access Journals (Sweden)

    Claudia Rodríguez-Almazán

    Full Text Available The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins.

  16. Foods with increased protein content: A qualitative study on European consumer preferences and perceptions.

    Science.gov (United States)

    Banovic, Marija; Arvola, Anne; Pennanen, Kyösti; Duta, Denisa E; Brückner-Gühmann, Monika; Lähteenmäki, Liisa; Grunert, Klaus G

    2018-06-01

    Foods with increased protein content have rapidly become one of the fastest-growing product categories targeting image- and health-focused consumers. However, it is not clear whether consumers really understand the difference between 'inherently rich in protein' and 'artificially increased protein'. This study used a qualitative focus group approach to investigate the consumer preferences and perceptions of foods with increased protein content among mixed-age and older population in four European countries. In total fifty-two participants were involved in the study. Understanding of the concept of foods with 'increased protein' content was limited. Both older and mixed-age participants could not differentiate between natural sources of protein and foods with increased protein content, no matter whether foods with animal or plant proteins were mentioned. Older participants expressed more scepticism towards foods with increased protein content than mixed-age participants. The combination of protein type and food carrier closer to conventional foods received more acceptance among both older and mixed-age participants. Future use and acceptance of foods with increased protein content will depend on the extent to which consumer concerns about incorporating additional protein into a diet can be responded. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. A Protein Disulfide Isomerase Gene Fusion Expression System That Increases the Extracellular Productivity of Bacillus brevis

    Science.gov (United States)

    Kajino, Tsutomu; Ohto, Chikara; Muramatsu, Masayoshi; Obata, Shusei; Udaka, Shigezo; Yamada, Yukio; Takahashi, Haruo

    2000-01-01

    We have developed a versatile Bacillus brevis expression and secretion system based on the use of fungal protein disulfide isomerase (PDI) as a gene fusion partner. Fusion with PDI increased the extracellular production of heterologous proteins (light chain of immunoglobulin G, 8-fold; geranylgeranyl pyrophosphate synthase, 12-fold). Linkage to PDI prevented the aggregation of the secreted proteins, resulting in high-level accumulation of fusion proteins in soluble and biologically active forms. We also show that the disulfide isomerase activity of PDI in a fusion protein is responsible for the suppression of the aggregation of the protein with intradisulfide, whereas aggregation of the protein without intradisulfide was prevented even when the protein was fused to a mutant PDI whose two active sites were disrupted, suggesting that another PDI function, such as chaperone-like activity, synergistically prevented the aggregation of heterologous proteins in the PDI fusion expression system. PMID:10653729

  18. Increased nitration and carbonylation of proteins in MRL +/+ mice exposed to trichloroethene: Potential role of protein oxidation in autoimmunity

    International Nuclear Information System (INIS)

    Wang Gangduo; Wang Jianling; Ma Huaxian; Khan, M. Firoze

    2009-01-01

    Even though reactive oxygen and nitrogen species (RONS) are implicated as mediators of autoimmune diseases (ADs), little is known about contribution of protein oxidation (carbonylation and nitration) in the pathogenesis of such diseases. The focus of this study was, therefore, to establish a link between protein oxidation and induction and/or exacerbation of autoimmunity. To achieve this, female MRL +/+ mice were treated with trichloroethene (TCE), an environmental contaminant known to induce autoimmune response, for 6 or 12 weeks (10 mmol/kg, i.p., every 4 th day). TCE treatment resulted in significantly increased formation of nitrotyrosine (NT) and induction of iNOS in the serum at both 6 and 12 weeks of treatment, but the response was greater at 12 weeks. Likewise, TCE treatment led to greater NT formation, and iNOS protein and mRNA expression in the livers and kidneys. Moreover, TCE treatment also caused significant increases (∼3 fold) in serum protein carbonyls (a marker of protein oxidation) at both 6 and 12 weeks. Significantly increased protein carbonyls were also observed in the livers and kidneys (2.1 and 1.3 fold, respectively) at 6 weeks, and to a greater extent at 12 weeks (3.5 and 2.1 fold, respectively) following TCE treatment. The increases in TCE-induced protein oxidation (carbonylation and nitration) were associated with significant increases in Th1 specific cytokine (IL-2, IFN-γ) release into splenocyte cultures. These results suggest an association between protein oxidation and induction/exacerbation of autoimmune response. The results present a potential mechanism by which oxidatively modified proteins could contribute to TCE-induced autoimmune response and necessitates further investigations for clearly establishing the role of protein oxidation in the pathogenesis of ADs.

  19. Muscarinic agonists and phorbol esters increase tyrosine phosphorylation of a 40-kilodalton protein in hippocampal slices

    International Nuclear Information System (INIS)

    Stratton, K.R.; Worley, P.F.; Huganir, R.L.; Baraban, J.M.

    1989-01-01

    The authors have used the hippocampal slice preparation to investigate the regulation of protein tyrosine phosphorylation in brain. After pharmacological treatment of intact slices, proteins were separated by electrophoresis, and levels of protein tyrosine phosphorylation were assessed by immunoblotting with specific anti-phosphotyrosine antibodies. Phorbol esters, activators of the serine- and threonine-phosphorylating enzyme protein kinase C, selectively increase tyrosine phosphorylation of a soluble protein with an apparent molecular mass of approximately 40 kilodaltons. Muscarinic agonists such as carbachol and oxotremorine M that strongly activate the inositol phospholipid system also increase tyrosine phosphorylation of this protein. Neurotransmitter activation of the inositol phospholipid system and protein kinase C appears to trigger a cascade leading to increased tyrosine phosphorylation

  20. Mild electrical stimulation with heat stimulation increase heat shock protein 70 in articular chondrocyte.

    Science.gov (United States)

    Hiraoka, Nobuyuki; Arai, Yuji; Takahashi, Kenji A; Mazda, Osam; Kishida, Tsunao; Honjo, Kuniaki; Tsuchida, Shinji; Inoue, Hiroaki; Morino, Saori; Suico, Mary Ann; Kai, Hirofumi; Kubo, Toshikazu

    2013-06-01

    The objective of this study is to investigate the effects of mild electrical stimulation (MES) and heat stress (HS) on heat shock protein 70 (HSP70), that protects chondrocytes and enhances cartilage matrix metabolism, in chondrocyte and articular cartilage. Rabbit articular chondrocytes were treated with MES and/or HS. The safeness was assessed by LDH assay and morphology. HSP70 protein, ubiquitinated proteins and HSP70 mRNA were examined by Western blotting and real-time PCR. Rat knee joints were treated with MES and/or HS. HSP70 protein, ubiquitinated proteins, HSP70 mRNA and proteoglycan core protein (PG) mRNA in articular cartilage were investigated. In vitro, HS increased HSP70 mRNA and HSP70 protein. MES augmented ubiquitinated protein and HSP70 protein, but not HSP70 mRNA. MES + HS raised HSP70 mRNA and ubiquitinated protein, and significantly increased HSP70 protein. In vivo, HS and MES + HS treatment augmented HSP70 mRNA. HS modestly augmented HSP70 protein. MES + HS significantly increased HSP70 protein and ubiquitinated proteins. PG mRNA was markedly raised by MES + HS. This study demonstrated that MES, in combination with HS, increases HSP70 protein in chondrocytes and articular cartilage, and promotes cartilage matrix metabolism in articular cartilage. MES in combination with HS can be a novel physical therapy for osteoarthritis by inducing HSP70 in articular cartilage. Copyright © 2013 Orthopaedic Research Society.

  1. Post-Weaning Protein Malnutrition Increases Blood Pressure and Induces Endothelial Dysfunctions in Rats

    Science.gov (United States)

    Siman, Fabiana D. M.; Silveira, Edna A.; Meira, Eduardo F.; da Costa, Carlos P.; Vassallo, Dalton V.; Padilha, Alessandra S.

    2012-01-01

    Malnutrition during critical periods in early life may increase the subsequent risk of hypertension and metabolic diseases in adulthood, but the underlying mechanisms are still unclear. We aimed to evaluate the effects of post-weaning protein malnutrition on blood pressure and vascular reactivity in aortic rings (conductance artery) and isolated-perfused tail arteries (resistance artery) from control (fed with Labina®) and post-weaning protein malnutrition rats (offspring that received a diet with low protein content for three months). Systolic and diastolic blood pressure and heart rate increased in the post-weaning protein malnutrition rats. In the aortic rings, reactivity to phenylephrine (10−10–3.10−4 M) was similar in both groups. Endothelium removal or L-NAME (10−4 M) incubation increased the response to phenylephrine, but the L-NAME effect was greater in the aortic rings from the post-weaning protein malnutrition rats. The protein expression of the endothelial nitric oxide isoform increased in the aortic rings from the post-weaning protein malnutrition rats. Incubation with apocynin (0.3 mM) reduced the response to phenylephrine in both groups, but this effect was higher in the post-weaning protein malnutrition rats, suggesting an increase of superoxide anion release. In the tail artery of the post-weaning protein malnutrition rats, the vascular reactivity to phenylephrine (0.001–300 µg) and the relaxation to acetylcholine (10−10–10−3 M) were increased. Post-weaning protein malnutrition increases blood pressure and induces vascular dysfunction. Although the vascular reactivity in the aortic rings did not change, an increase in superoxide anion and nitric oxide was observed in the post-weaning protein malnutrition rats. However, in the resistance arteries, the increased vascular reactivity may be a potential mechanism underlying the increased blood pressure observed in this model. PMID:22529948

  2. Vitellogenin-RNAi and ovariectomy each increase lifespan, increase protein storage, and decrease feeding, but are not additive in grasshoppers.

    Science.gov (United States)

    Tetlak, Alicia G; Burnett, Jacob B; Hahn, Daniel A; Hatle, John D

    2015-12-01

    Reduced reproduction has been shown to increase lifespan in many animals, yet the mechanisms behind this trade-off are unclear. We addressed this question by combining two distinct, direct means of life-extension via reduced reproduction, to test whether they were additive. In the lubber grasshopper, Romalea microptera, ovariectomized (OVX) individuals had a ~20% increase in lifespan and a doubling of storage relative to controls (Sham operated). Similarly, young female grasshoppers treated with RNAi against vitellogenin (the precursor to egg yolk protein) had increased fat body mass and halted ovarian growth. In this study, we compared VgRNAi to two control groups that do not reduce reproduction, namely buffer injection (Buffer) and injection with RNAi against a hexameric storage protein (Hex90RNAi). Each injection treatment was tested with and without ovariectomy. Hence, we tested feeding, storage, and lifespans in six groups: OVX and Buffer, OVX and Hex90RNAi, OVX and VgRNAi, Sham and Buffer, Sham and Hex90RNAi, and Sham and VgRNAi. Ovariectomized grasshoppers and VgRNAi grasshoppers each had similar reductions in feeding (~40%), increases in protein storage in the hemolymph (150-300%), and extensions in lifespan (13-21%). Ovariectomized grasshoppers had higher vitellogenin protein levels than did VgRNAi grasshoppers. Last but not least, when ovariectomy and VgRNAi were applied together, there was no greater effect on feeding, protein storage, or longevity. Hence, feeding regulation, and protein storage in insects, may be conserved components of life-extension via reduced reproduction.

  3. Altering adsorbed proteins or cellular gene expression in bone-metastatic cancer cells affects PTHrP and Gli2 without altering cell growth

    Directory of Open Access Journals (Sweden)

    Jonathan M. Page

    2015-09-01

    Full Text Available The contents of this data in brief are related to the article titled “Matrix Rigidity Regulates the Transition of Tumor Cells to a Bone-Destructive Phenotype through Integrin β3 and TGF-β Receptor Type II”. In this DIB we will present our supplemental data investigating Integrin expression, attachment of cells to various adhesion molecules, and changes in gene expression in multiple cancer cell lines. Since the interactions of Integrins with adsorbed matrix proteins are thought to affect the ability of cancer cells to interact with their underlying substrates, we examined the expression of Integrin β1, β3, and β5 in response to matrix rigidity. We found that only Iβ3 increased with increasing substrate modulus. While it was shown that fibronectin greatly affects the expression of tumor-produced factors associated with bone destruction (parathyroid hormone-related protein, PTHrP, and Gli2, poly-l-lysine, vitronectin and type I collagen were also analyzed as potential matrix proteins. Each of the proteins was independently adsorbed on both rigid and compliant polyurethane films which were subsequently used to culture cancer cells. Poly-l-lysine, vitronectin and type I collagen all had negligible effects on PTHrP or Gli2 expression, but fibronectin was shown to have a dose dependent effect. Finally, altering the expression of Iβ3 demonstrated that it is required for tumor cells to respond to the rigidity of the matrix, but does not affect other cell growth or viability. Together these data support the data presented in our manuscript to show that the rigidity of bone drives Integrinβ3/TGF-β crosstalk, leading to increased expression of Gli2 and PTHrP.

  4. Increased Protein Stability and Decreased Protein Turnover in the Caenorhabditis elegans Ins/IGF-1 daf-2 Mutant.

    Science.gov (United States)

    Depuydt, Geert; Shanmugam, Nilesh; Rasulova, Madina; Dhondt, Ineke; Braeckman, Bart P

    2016-12-01

    In Caenorhabditis elegans, cellular proteostasis is likely essential for longevity. Autophagy has been shown to be essential for lifespan extension of daf-2 insulin/IGF mutants. Therefore, it can be hypothesized that daf-2 mutants achieve this phenotype by increasing protein turnover. However, such a mechanism would exert a substantial energy cost. By using classical 35 S pulse-chase labeling, we observed that protein synthesis and degradation rates are decreased in young adults of the daf-2 insulin/IGF mutants. Although reduction of protein turnover may be energetically favorable, it may lead to accumulation and aggregation of damaged proteins. As this has been shown not to be the case in daf-2 mutants, another mechanism must exist to maintain proteostasis in this strain. We observed that proteins isolated from daf-2 mutants are more soluble in acidic conditions due to increased levels of trehalose. This suggests that trehalose may decrease the potential for protein aggregation and increases proteostasis in the daf-2 mutants. We postulate that daf-2 mutants save energy by decreasing protein turnover rates and instead stabilize their proteome by trehalose. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America.

  5. Increase in local protein concentration by field-inversion gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Paulus Aran

    2007-09-01

    Full Text Available Abstract Background Proteins that migrate through cross-linked polyacrylamide gels (PAGs under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE. Results Separation of model protein species and large protein complexes was compared between FIGE and constant field electrophoresis (CFE in different percentages of PAGs. Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times. These results revealed an increase in band intensity per defined gel volume. A biphasic protein relative mobility shift was observed in percentages of PAGs up to 14%. However, the effect of FIGE on protein separation was stochastic at higher PAG percentage. Rat liver lysates subjected to FIGE in the second-dimension separation of two-dimensional polyarcylamide gel electrophoresis (2D PAGE showed a 20% increase in the number of discernible spots compared with CFE. Nine common spots from both FIGE and CFE were selected for peptide sequencing by mass spectrometry (MS, which revealed higher final ion scores of all nine protein spots from FIGE. Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE. Conclusion The present investigation suggests that FIGE under appropriate conditions improves protein separation efficiency during PAGE as a result of increased local protein concentration. FIGE can be implemented with minimal additional instrumentation in any laboratory setting. Despite the tradeoff of longer running times, FIGE can be a powerful protein

  6. Increase in local protein concentration by field-inversion gel electrophoresis.

    Science.gov (United States)

    Tsai, Henghang; Low, Teck Yew; Freeby, Steve; Paulus, Aran; Ramnarayanan, Kalpana; Cheng, Chung-Pui Paul; Leung, Hon-Chiu Eastwood

    2007-09-26

    Proteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE). Separation of model protein species and large protein complexes was compared between FIGE and constant field electrophoresis (CFE) in different percentages of PAGs. Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times. These results revealed an increase in band intensity per defined gel volume. A biphasic protein relative mobility shift was observed in percentages of PAGs up to 14%. However, the effect of FIGE on protein separation was stochastic at higher PAG percentage. Rat liver lysates subjected to FIGE in the second-dimension separation of two-dimensional polyarcylamide gel electrophoresis (2D PAGE) showed a 20% increase in the number of discernible spots compared with CFE. Nine common spots from both FIGE and CFE were selected for peptide sequencing by mass spectrometry (MS), which revealed higher final ion scores of all nine protein spots from FIGE. Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE. The present investigation suggests that FIGE under appropriate conditions improves protein separation efficiency during PAGE as a result of increased local protein concentration. FIGE can be implemented with minimal additional instrumentation in any laboratory setting. Despite the tradeoff of longer running times, FIGE can be a powerful protein separation tool.

  7. The rate of synthesis and decomposition of tissue proteins in hypokinesia and increased muscular activity

    Science.gov (United States)

    Fedorov, I. V.; Chernyy, A. V.; Fedorov, A. I.

    1978-01-01

    During hypokinesia and physical loading (swimming) of rats, the radioactivity of skeletal muscle, liver, kidney, heart, and blood proteins was determined after administration of radioactive amino acids. Tissue protein synthesis decreased during hypokinesia, and decomposition increased. Both synthesis and decomposition increased during physical loading, but anabolic processes predominated in the total tissue balance. The weights of the animals decreased in hypokinesia and increased during increased muscle activity.

  8. COPS5 (Jab1) protein increases β site processing of amyloid precursor protein and amyloid β peptide generation by stabilizing RanBP9 protein levels.

    Science.gov (United States)

    Wang, Hongjie; Dey, Debleena; Carrera, Ivan; Minond, Dmitriy; Bianchi, Elisabetta; Xu, Shaohua; Lakshmana, Madepalli K

    2013-09-13

    Increased processing of amyloid precursor protein (APP) and accumulation of neurotoxic amyloid β peptide (Aβ) in the brain is central to the pathogenesis of Alzheimer's disease (AD). Therefore, the identification of molecules that regulate Aβ generation is crucial for future therapeutic approaches for AD. We demonstrated previously that RanBP9 regulates Aβ generation in a number of cell lines and primary neuronal cultures by forming tripartite protein complexes with APP, low-density lipoprotein-related protein, and BACE1, consequently leading to increased amyloid plaque burden in the brain. RanBP9 is a scaffold protein that exists and functions in multiprotein complexes. To identify other proteins that may bind RanBP9 and regulate Aβ levels, we used a two-hybrid analysis against a human brain cDNA library and identified COPS5 as a novel RanBP9-interacting protein. This interaction was confirmed by coimmunoprecipitation experiments in both neuronal and non-neuronal cells and mouse brain. Colocalization of COPS5 and RanBP9 in the same subcellular compartments further supported the interaction of both proteins. Furthermore, like RanBP9, COPS5 robustly increased Aβ generation, followed by increased soluble APP-β (sAPP-β) and decreased soluble-APP-α (sAPP-α) levels. Most importantly, down-regulation of COPS5 by siRNAs reduced Aβ generation, implying that endogenous COPS5 regulates Aβ generation. Finally, COPS5 levels were increased significantly in AD brains and APΔE9 transgenic mice, and overexpression of COPS5 strongly increased RanBP9 protein levels by increasing its half-life. Taken together, these results suggest that COPS5 increases Aβ generation by increasing RanBP9 levels. Thus, COPS5 is a novel RanBP9-binding protein that increases APP processing and Aβ generation by stabilizing RanBP9 protein levels.

  9. Relating the effects of protein type and content in increased-protein cheese pies to consumers' perception of satiating capacity.

    Science.gov (United States)

    Marcano, J; Varela, P; Fiszman, S

    2015-02-01

    Since proteins have been shown to have the highest satiation-inducing effects of all the macronutrients, increasing the protein level is one of the main strategies for designing foods with enhanced satiating capacity. However, few studies analyze the effect that protein addition has on the texture and flavor characteristics of the target food item to relate it to the expected satiating capacity it elicits. The present work studied cheese pies with three levels of soy and whey proteins. Since the protein level altered the rheological behavior of the batters before baking and the texture of the baked pies, the feasibility of adding several protein levels for obtaining a range of final products was investigated. A check-all-that-apply questionnaire containing 32 sensory and non-sensory characteristics of the samples was given to consumers (n = 131) who also scored the perceived samples' satiating capacity. The results showed that the type and content of protein contributed distinctive sensory characteristics to the samples that could be related to their satiating capacity perception. Harder and drier samples (high protein levels) were perceived as more satiating with less perceptible sweet and milky cheese pie characteristic flavors. Soy contributed an off-flavour. These results will contribute to a better understanding of the interrelation of all these factors, aiding the development of highly palatable solid foods with enhanced satiating capacities.

  10. Metabolisable protein supply to lactating dairy cows increased with increasing dry matter concentration in grass-clover silage

    DEFF Research Database (Denmark)

    Johansen, Marianne; Hellwing, Anne Louise Frydendahl; Lund, Peter

    2017-01-01

    The aim of this experiment was to study the effect of increased dry matter (DM) concentration in grass-clover silage, obtained by extending the pre-wilting period before ensiling, on the amount of metabolisable protein (MP) supplied to lactating dairy cows. Spring growth and first regrowth of grass...... and faeces, respectively, were collected over 94 h to cover the diurnal variation, pooled, and subsequently analysed. Rumen fluid was collected in same sampling procedure. To estimate the duodenal flow of microbial protein, microbes were isolated from the rumen and analysed for amino acids (AA) and purines...... flow of AA. The higher duodenal flow of AA derived from a lower rumen degradation of feed protein and a tendency towards a higher microbial synthesis in the rumen. Fibre digestibility and CH4 production were not affected by silage DM concentration. In conclusion, MP concentration in grass-clover silage...

  11. A positive feedback-based gene circuit to increase the production of a membrane protein

    Directory of Open Access Journals (Sweden)

    Gennis Robert B

    2010-05-01

    Full Text Available Abstract Background Membrane proteins are an important class of proteins, playing a key role in many biological processes, and are a promising target in pharmaceutical development. However, membrane proteins are often difficult to produce in large quantities for the purpose of crystallographic or biochemical analyses. Results In this paper, we demonstrate that synthetic gene circuits designed specifically to overexpress certain genes can be applied to manipulate the expression kinetics of a model membrane protein, cytochrome bd quinol oxidase in E. coli, resulting in increased expression rates. The synthetic circuit involved is an engineered, autoinducer-independent variant of the lux operon activator LuxR from V. fischeri in an autoregulatory, positive feedback configuration. Conclusions Our proof-of-concept experiments indicate a statistically significant increase in the rate of production of the bd oxidase membrane protein. Synthetic gene networks provide a feasible solution for the problem of membrane protein production.

  12. Exogenous insulin does not increase muscle protein synthesis rate when administered systemically: a systematic review

    NARCIS (Netherlands)

    Trommelen, J.; Groen, B.; Hamer, H.M.; Groot, de C.P.G.M.; Loon, van L.J.C.

    2015-01-01

    Background Though it is well appreciated that insulin plays an important role in the regulation of muscle protein metabolism, there is much discrepancy in the literature on the capacity of exogenous insulin administration to increase muscle protein synthesis rates in vivo in humans. Objective To

  13. Clofibrate-induced increases in peroxisomal proteins: effect on synthesis, degradation, and mRNA activity

    International Nuclear Information System (INIS)

    Mortensen, R.M.

    1983-01-01

    The effect of clofibrate on the polypeptide composition of peroxisomes was determined. A simple method was developed for the isolation of peroxisomes with a purity of 90-95% using sedimentation in a metrizamide gradient. The specific activities of HD did not change with clofibrate treatment so that the increases in enzyme activities are solely due to increases in protein amounts. The hepatic concentration of HD increased 63 times. The HD synthesis rate, as measured by the incorporation of [ 3 H]leucine, increased 74 times, so that the increase in the synthesis was sufficient to account for the increase in protein. Clofibrate caused no discernible change in the degradation rate of HD labeled with [ 14 C]bicarbonate. The half-life of HD was approximately 2 days. The translatable mRBA coding for HD increased 55 times. This value is not significantly different from the increase in HD protein or in HD synthesis. This observation was also true for several other peroxisomal proteins. Therefore, clofibrate causes an increase in the mRNA activity, which increases the synthesis of HD leading to an accumulation of protein and enzyme activity. The kinetics of the clofibrate-induced changes in HD synthesis rate, protein level, and enzymatic activity was analyzed using a simple model which included the half-lives of the drug, mRNA, and protein. The best fit of the model to the data gave an mRNA half-life of 10 hours and a protein half-life of 1.8 days, with no significant change by clofibrate

  14. Increased precision for analysis of protein-ligand dissociation constants determined from chemical shift titrations

    Energy Technology Data Exchange (ETDEWEB)

    Markin, Craig J.; Spyracopoulos, Leo, E-mail: leo.spyracopoulos@ualberta.ca [University of Alberta, Department of Biochemistry (Canada)

    2012-06-15

    NMR is ideally suited for the analysis of protein-protein and protein ligand interactions with dissociation constants ranging from {approx}2 {mu}M to {approx}1 mM, and with kinetics in the fast exchange regime on the NMR timescale. For the determination of dissociation constants (K{sub D}) of 1:1 protein-protein or protein-ligand interactions using NMR, the protein and ligand concentrations must necessarily be similar in magnitude to the K{sub D}, and nonlinear least squares analysis of chemical shift changes as a function of ligand concentration is employed to determine estimates for the parameters K{sub D} and the maximum chemical shift change ({Delta}{delta}{sub max}). During a typical NMR titration, the initial protein concentration, [P{sub 0}], is held nearly constant. For this condition, to determine the most accurate parameters for K{sub D} and {Delta}{delta}{sub max} from nonlinear least squares analyses requires initial protein concentrations that are {approx}0.5 Multiplication-Sign K{sub D}, and a maximum concentration for the ligand, or titrant, of {approx}10 Multiplication-Sign [P{sub 0}]. From a practical standpoint, these requirements are often difficult to achieve. Using Monte Carlo simulations, we demonstrate that co-variation of the ligand and protein concentrations during a titration leads to an increase in the precision of the fitted K{sub D} and {Delta}{delta}{sub max} values when [P{sub 0}] > K{sub D}. Importantly, judicious choice of protein and ligand concentrations for a given NMR titration, combined with nonlinear least squares analyses using two independent variables (ligand and protein concentrations) and two parameters (K{sub D} and {Delta}{delta}{sub max}) is a straightforward approach to increasing the accuracy of measured dissociation constants for 1:1 protein-ligand interactions.

  15. Increasing the production yield of recombinant protein in transgenic seeds by expanding the deposition space within the intracellular compartment

    OpenAIRE

    Takaiwa, Fumio

    2013-01-01

    Seeds must maintain a constant level of nitrogen in order to germinate. When recombinant proteins are produced while endogenous seed protein expression is suppressed, the production levels of the foreign proteins increase to compensate for the decreased synthesis of endogenous proteins. Thus, exchanging the production of endogenous seed proteins for that of foreign proteins is a promising approach to increase the yield of foreign recombinant proteins. Providing a space for the deposition of r...

  16. Quantitative analysis of core fucosylation of serum proteins in liver diseases by LC-MS-MRM.

    Science.gov (United States)

    Ma, Junfeng; Sanda, Miloslav; Wei, Renhuizi; Zhang, Lihua; Goldman, Radoslav

    2018-02-07

    Aberrant core fucosylation of proteins has been linked to liver diseases. In this study, we carried out multiple reaction monitoring (MRM) quantification of core fucosylated N-glycopeptides of serum proteins partially deglycosylated by a combination of endoglycosidases (endoF1, endoF2, and endoF3). To minimize variability associated with the preparatory steps, the analysis was performed without enrichment of glycopeptides or fractionation of serum besides the nanoRP chromatography. Specifically, we quantified core fucosylation of 22 N-glycopeptides derived from 17 proteins together with protein abundance of these glycoproteins in a cohort of 45 participants (15 disease-free control, 15 fibrosis and 15 cirrhosis patients) using a multiplex nanoUPLC-MS-MRM workflow. We find increased core fucosylation of 5 glycopeptides at the stage of liver fibrosis (i.e., N630 of serotransferrin, N107 of alpha-1-antitrypsin, N253 of plasma protease C1 inhibitor, N397 of ceruloplasmin, and N86 of vitronectin), increase of additional 6 glycopeptides at the stage of cirrhosis (i.e., N138 and N762 of ceruloplasmin, N354 of clusterin, N187 of hemopexin, N71 of immunoglobulin J chain, and N127 of lumican), while the degree of core fucosylation of 10 glycopeptides did not change. Interestingly, although we observe an increase in the core fucosylation at N86 of vitronectin in liver fibrosis, core fucosylation decreases on the N169 glycopeptide of the same protein. Our results demonstrate that the changes in core fucosylation are protein and site specific during the progression of fibrotic liver disease and independent of the changes in the quantity of N-glycoproteins. It is expected that the fully optimized multiplex LC-MS-MRM assay of core fucosylated glycopeptides will be useful for the serologic assessment of the fibrosis of liver. We have quantified the difference in core fucosylation among three comparison groups (healthy control, fibrosis and cirrhosis patients) using a sensitive and

  17. Effect of experimentally increased protein supply to postpartum dairy cows on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis.

    Science.gov (United States)

    Larsen, M; Røntved, C M; Theil, P K; Khatun, M; Lauridsen, C; Kristensen, N B

    2017-05-01

    The effect of experimentally increasing the postpartum protein supply on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis was studied using 8 periparturient Holstein cows in a complete randomized design. At calving, cows were assigned to abomasal infusion of water (CTRL) or casein (CAS) in addition to a lactation diet. Casein infusion was gradually decreased from 696 ± 1 g/d at +2 d relative to calving (DRTC) to 212 ± 10 g/d at +29 DRTC to avoid excessive supply. Synthesis rate of plasma proteins was measured at -14, +4, +15, and +29 DRTC by measuring [C]Phe isotopic enrichment in arterial plasma free Phe, total plasma proteins, and albumin after 3, 5, and 7 h of jugular ring[C]Phe infusion. Plasma volume was determined at +4 and +29 DRTC by dilution of a [I]BSA dose. Synthesis rate of tissue protein in biopsied rumen papillae was determined by measuring [C]Phe isotopic enrichment, and mRNA expression of selected genes was measured by real-time qPCR. Total and differential leukocyte counts were performed and immune responsiveness of monocytes was evaluated by tumor necrosis factor ɑ (TNFɑ) concentration on ex vivo whole blood stimulation with Escherichia coli lipopolysaccharide (LPS) and responsiveness of T-lymphocytes by interferon γ (IFNγ) concentration on stimulation with Staphylococcus aureus enterotoxin β (SEB). Further, ELISA plasma concentrations of IgM, IgA, and IgG were determined. The DRTC affected the majority of investigated parameters as expected. The CAS treatment increased milk protein yield (P = 0.04), and tended to lower TNFɑ (P = 0.06), and lowered IFNγ (P = 0.03) responsiveness per monocyte and lymphocyte, respectively, compared with CTRL. Further, fractional synthesis rate of albumin was greater at +4 DRTC for CAS compared with CTRL but did not differ by +29 DRTC (interaction: P = 0.01). In rumen papillae, synthesis rate of tissue protein was greater for CAS compared with CTRL (P protein supply seem to

  18. Binding proteins enhance specific uptake rate by increasing the substrate-transporter encounter rate.

    Science.gov (United States)

    Bosdriesz, Evert; Magnúsdóttir, Stefanía; Bruggeman, Frank J; Teusink, Bas; Molenaar, Douwe

    2015-06-01

    Microorganisms rely on binding-protein assisted, active transport systems to scavenge for scarce nutrients. Several advantages of using binding proteins in such uptake systems have been proposed. However, a systematic, rigorous and quantitative analysis of the function of binding proteins is lacking. By combining knowledge of selection pressure and physiochemical constraints, we derive kinetic, thermodynamic, and stoichiometric properties of binding-protein dependent transport systems that enable a maximal import activity per amount of transporter. Under the hypothesis that this maximal specific activity of the transport complex is the selection objective, binding protein concentrations should exceed the concentration of both the scarce nutrient and the transporter. This increases the encounter rate of transporter with loaded binding protein at low substrate concentrations, thereby enhancing the affinity and specific uptake rate. These predictions are experimentally testable, and a number of observations confirm them. © 2015 FEBS.

  19. Balanced trafficking between the ER and the Golgi apparatus increases protein secretion in yeast

    DEFF Research Database (Denmark)

    Bao, Jichen; Huang, Mingtao; Petranovic, Dina

    2018-01-01

    of ADP-ribosylation factor GTP activating proteins, Gcs1p and Glo3p, which are involved in the process of COPI-coated vesicle formation. Engineering the retrograde trafficking increased the secretion of alpha-amylase but did not induce production of reactive oxygen species. An expanded ER membrane......The yeast Saccharomyces cerevisiae is widely used as a cell factory to produce recombinant proteins. However, S. cerevisiae naturally secretes only a few proteins, such as invertase and the mating alpha factor, and its secretory capacity is limited. It has been reported that engineering protein...... recombinant proteins, endoglucanase I from Trichoderma reesei and glucan-1,4-alpha-glucosidase from Rhizopus oryzae, indicating overexpression of GLO3 in a SEC16 moderate overexpression strain might be a general strategy for improving production of secreted proteins by yeast....

  20. Protein Carbamylation: A Marker Reflecting Increased Age-Related Cell Oxidation

    Directory of Open Access Journals (Sweden)

    Julia Carracedo

    2018-05-01

    Full Text Available Carbamylation is a post-translational modification of proteins that may partake in the oxidative stress-associated cell damage, and its increment has been recently proposed as a “hallmark of aging”. The molecular mechanisms associated with aging are related to an increased release of free radicals. We have studied whether carbamylated proteins from the peripheral blood of healthy subjects are related to oxidative damage and aging, taking into account the gender and the immune profile of the subjects. The study was performed in healthy human volunteers. The detection of protein carbamylation and malondialdehyde (MDA levels was evaluated using commercial kits. The immune profile was calculated using parameters of immune cell function. The results show that the individuals from the elderly group (60–79 years old have increased carbamylated protein and MDA levels. When considered by gender, only men between 60 and 79 years old showed significantly increased carbamylated proteins and MDA levels. When those subjects were classified by their immune profile, the carbamylated protein levels were higher in those with an older immune profile. In conclusion, the carbamylation of proteins in peripheral blood is related to age-associated oxidative damage and to an aging functional immunological signature. Our results suggest that carbamylated proteins may play an important role at the cellular level in the aging process.

  1. Mutation of exposed hydrophobic amino acids to arginine to increase protein stability

    Directory of Open Access Journals (Sweden)

    Czaplicki Jerzy

    2004-07-01

    Full Text Available Abstract Background One strategy to increase the stability of proteins is to reduce the area of water-accessible hydrophobic surface. Results In order to test it, we replaced 14 solvent-exposed hydrophobic residues of acetylcholinesterase by arginine. The stabilities of the resulting proteins were tested using denaturation by high temperature, organic solvents, urea and by proteolytic digestion. Conclusion Altough the mutational effects were rather small, this strategy proved to be successful since half of the mutants showed an increased stability. This stability may originate from the suppression of unfavorable interactions of nonpolar residues with water or from addition of new hydrogen bonds with the solvent. Other mechanisms may also contribute to the increased stability observed with some mutants. For example, introduction of a charge at the surface of the protein may provide a new coulombic interaction on the protein surface.

  2. High dietary protein intake is associated with an increased body weight and total death risk.

    Science.gov (United States)

    Hernández-Alonso, Pablo; Salas-Salvadó, Jordi; Ruiz-Canela, Miguel; Corella, Dolores; Estruch, Ramón; Fitó, Montserrat; Arós, Fernando; Gómez-Gracia, Enrique; Fiol, Miquel; Lapetra, José; Basora, Josep; Serra-Majem, Lluis; Muñoz, Miguel Ángel; Buil-Cosiales, Pilar; Saiz, Carmen; Bulló, Mònica

    2016-04-01

    High dietary protein diets are widely used to manage overweight and obesity. However, there is a lack of consensus about their long-term efficacy and safety. Therefore, the aim of this study was to assess the effect of long-term high-protein consumption on body weight changes and death outcomes in subjects at high cardiovascular risk. A secondary analysis of the PREDIMED trial was conducted. Dietary protein was assessed using a food-frequency questionnaire during the follow-up. Cox proportional hazard models were used to estimate the multivariate-adjusted hazard ratio (HR) and 95% confidence intervals (95%CI) for protein intake in relation to the risk of body weight and waist circumference changes, cardiovascular disease, cardiovascular death, cancer death and total death. Higher total protein intake, expressed as percentage of energy, was significantly associated with a greater risk of weight gain when protein replaced carbohydrates (HR: 1.90; 95%CI: 1.05, 3.46) but not when replaced fat (HR: 1.69; 95%CI: 0.94, 3.03). However, no association was found between protein intake and waist circumference. Contrary, higher total protein intake was associated with a greater risk of all-cause death in both carbohydrate and fat substitution models (HR: 1.59; 95%CI: 1.08, 2.35; and HR: 1.66; 95%CI: 1.13, 2.43, respectively). A higher consumption of animal protein was associated with an increased risk of fatal and non-fatal outcomes when protein substituted carbohydrates or fat. Higher dietary protein intake is associated with long-term increased risk of body weight gain and overall death in a Mediterranean population at high cardiovascular risk. Copyright © 2015 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

  3. Specific Increase of Protein Levels by Enhancing Translation Using Antisense Oligonucleotides Targeting Upstream Open Frames.

    Science.gov (United States)

    Liang, Xue-Hai; Shen, Wen; Crooke, Stanley T

    2017-01-01

    A number of diseases are caused by low levels of key proteins; therefore, increasing the amount of specific proteins in human bodies is of therapeutic interest. Protein expression is downregulated by some structural or sequence elements present in the 5' UTR of mRNAs, such as upstream open reading frames (uORF). Translation initiation from uORF(s) reduces translation from the downstream primary ORF encoding the main protein product in the same mRNA, leading to a less efficient protein expression. Therefore, it is possible to use antisense oligonucleotides (ASOs) to specifically inhibit translation of the uORF by base-pairing with the uAUG region of the mRNA, redirecting translation machinery to initiate from the primary AUG site. Here we review the recent findings that translation of specific mRNAs can be enhanced using ASOs targeting uORF regions. Appropriately designed and optimized ASOs are highly specific, and they act in a sequence- and position-dependent manner, with very minor off-target effects. Protein levels can be increased using this approach in different types of human and mouse cells, and, importantly, also in mice. Since uORFs are present in around half of human mRNAs, the uORF-targeting ASOs may thus have valuable potential as research tools and as therapeutics to increase the levels of proteins for a variety of genes.

  4. Hydrophobic interaction chromatography in dual salt system increases protein binding capacity.

    Science.gov (United States)

    Senczuk, Anna M; Klinke, Ralph; Arakawa, Tsutomu; Vedantham, Ganesh; Yigzaw, Yinges

    2009-08-01

    Hydrophobic interaction chromatography (HIC) uses weakly hydrophobic resins and requires a salting-out salt to promote protein-resin interaction. The salting-out effects increase with protein and salt concentration. Dynamic binding capacity (DBC) is dependent on the binding constant, as well as on the flow characteristics during sample loading. DBC increases with the salt concentration but decreases with increasing flow rate. Dynamic and operational binding capacity have a major raw material cost/processing time impact on commercial scale production of monoclonal antibodies. In order to maximize DBC the highest salt concentration without causing precipitation is used. We report here a novel method to maintain protein solubility while increasing the DBC by using a combination of two salting-out salts (referred to as dual salt). In a series of experiments, we explored the dynamic capacity of a HIC resin (TosoBioscience Butyl 650M) with combinations of salts. Using a model antibody, we developed a system allowing us to increase the dynamic capacity up to twofold using the dual salt system over traditional, single salt system. We also investigated the application of this novel approach to several other proteins and salt combinations, and noted a similar protein solubility and DBC increase. The observed increase in DBC in the dual salt system was maintained at different linear flow rates and did not impact selectivity.

  5. Increased adsorption of histidine-tagged proteins onto tissue culture polystyrene

    DEFF Research Database (Denmark)

    Holmberg, Maria; Hansen, Thomas Steen; Lind, Johan Ulrik

    2012-01-01

    and ethylenediaminetetraacetic acid (EDTA), as well as adsorption performed at different pH and ionic strength indicates that the high adsorption is caused by electrostatic interaction between negatively charged carboxylate groups on the TCPS surface and positively charged histidine residues in the proteins. Pre......In this study we compare histidine-tagged and native proteins with regards to adsorption properties. We observe significantly increased adsorption of proteins with an incorporated polyhistidine amino acid motif (HIS-tag) onto tissue culture polystyrene (TCPS) compared to similar proteins without...... a HIS-tag. The effect is not observed on polystyrene (PS). Adsorption experiments have been performed at physiological pH (7.4) and the effect was only observed for the investigated proteins that have pI values below or around 7.4. Competitive adsorption experiments with imidazole...

  6. Lower growth temperature increases alternative pathway capacity and alternative oxidase protein in tobacco.

    Science.gov (United States)

    Vanlerberghe, G C; McIntosh, L

    1992-09-01

    Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow) have been used to study the effect of growth temperature on the CN-resistant, salicylhydroxamic acid-sensitive alternative pathway of respiration. Mitochondria isolated from cells maintained at 30 degrees C had a low capacity to oxidize succinate via the alternative pathway, whereas mitochondria isolated from cells 24 h after transfer to 18 degrees C displayed, on average, a 5-fold increase in this capacity (from 7 to 32 nanoatoms oxygen per milligram protein per minute). This represented an increase in alternative pathway capacity from 18 to 45% of the total capacity of electron transport. This increased capacity was lost upon transfer of cells back to 30 degrees C. A monoclonal antibody to the terminal oxidase of the alternative pathway (the alternative oxidase) from Sauromatum guttatum (T.E. Elthon, R.L. Nickels, L. McIntosh [1989] Plant Physiology 89: 1311-1317) recognized a 35-kilodalton mitochondrial protein in tobacco. There was an excellent correlation between the capacity of the alternative path in isolated tobacco mitochondria and the levels of this 35-kilodalton alternative oxidase protein. Cycloheximide could inhibit both the increased level of the 35-kilodalton alternative oxidase protein and the increased alternative pathway capacity normally seen upon transfer to 18 degrees C. We conclude that transfer of tobacco cells to the lower temperature increases the capacity of the alternative pathway due, at least in part, to de novo synthesis of the 35-kilodalton alternative oxidase protein.

  7. Effects of experimentally increased protein supply to postpartum dairy cows on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis

    DEFF Research Database (Denmark)

    Larsen, Mogens; Røntved, Christine Maria; Theil, Peter Kappel

    2017-01-01

    The effect of experimentally increasing the postpartum protein supply on plasma protein synthesis, rumen tissue proliferation, and immune homeostasis was studied using 8 periparturient Holstein cows in a complete randomized design. At calving, cows were assigned to abomasal infusion of water (CTRL......) or casein (CAS) in addition to a lactation diet. Casein infusion was gradually decreased from 696 ± 1 g/d at +2 d relative to calving (DRTC) to 212 ± 10 g/d at +29 DRTC to avoid excessive supply. Synthesis rate of plasma proteins was measured at –14, +4, +15, and +29 DRTC by measuring [13C]Phe isotopic...... enrichment in arterial plasma free Phe, total plasma proteins, and albumin after 3, 5, and 7 h of jugular ring[13C]Phe infusion. Plasma volume was determined at +4 and +29 DRTC by dilution of a [125I]BSA dose. Synthesis rate of tissue protein in biopsied rumen papillae was determined by measuring [13C...

  8. Protein synthesis levels are increased in a subset of individuals with Fragile X syndrome

    DEFF Research Database (Denmark)

    Jacquemont, Sébastien; Pacini, Laura; Jønch, Aia E

    2018-01-01

    architecture and plasticity. Preclinical studies revealed that pharmacological interventions restore those deficits, which are thought to mediate the FXS cognitive and behavioral symptoms. Here we characterized the de novo rate of protein synthesis in patients with FXS and their relationship with clinical...... severity. We measured the rate of protein synthesis in fibroblasts derived from 32 individuals with FXS and from 17 controls as well as in fibroblasts and primary neurons of 27 Fmr1 KO mice and 20 controls. Here we show that levels of protein synthesis are increased in fibroblasts of individuals with FXS...... and Fmr1 KO mice. However, this cellular phenotype displays a broad distribution and a proportion of fragile X individuals and Fmr1 KO mice do not show increased levels of protein synthesis, having measures in the normal range. Because the same Fmr1 KO animal measures in fibroblasts predict those...

  9. Poly(zwitterionic)protein conjugates offer increased stability without sacrificing binding affinity or bioactivity

    Science.gov (United States)

    Keefe, Andrew J.; Jiang, Shaoyi

    2012-01-01

    Treatment with therapeutic proteins is an attractive approach to targeting a number of challenging diseases. Unfortunately, the native proteins themselves are often unstable in physiological conditions, reducing bioavailability and therefore increasing the dose that is required. Conjugation with poly(ethylene glycol) (PEG) is often used to increase stability, but this has a detrimental effect on bioactivity. Here, we introduce conjugation with zwitterionic polymers such as poly(carboxybetaine). We show that poly(carboxybetaine) conjugation improves stability in a manner similar to PEGylation, but that the new conjugates retain or even improve the binding affinity as a result of enhanced protein-substrate hydrophobic interactions. This chemistry opens a new avenue for the development of protein therapeutics by avoiding the need to compromise between stability and affinity.

  10. Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces.

    Directory of Open Access Journals (Sweden)

    Yingying Zhu

    Full Text Available Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish or non-meat proteins (casein or soy for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota.

  11. Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces.

    Science.gov (United States)

    Zhu, Yingying; Lin, Xisha; Li, He; Li, Yingqiu; Shi, Xuebin; Zhao, Fan; Xu, Xinglian; Li, Chunbao; Zhou, Guanghong

    2016-01-01

    Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish) or non-meat proteins (casein or soy) for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs) were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota.

  12. Intake of Meat Proteins Substantially Increased the Relative Abundance of Genus Lactobacillus in Rat Feces

    Science.gov (United States)

    Zhu, Yingying; Lin, Xisha; Li, He; Li, Yingqiu; Shi, Xuebin; Zhao, Fan; Xu, Xinglian; Li, Chunbao; Zhou, Guanghong

    2016-01-01

    Diet has been shown to have a critical influence on gut bacteria and host health, and high levels of red meat in diet have been shown to increase colonic DNA damage and thus be harmful to gut health. However, previous studies focused more on the effects of meat than of meat proteins. In order to investigate whether intake of meat proteins affects the composition and metabolic activities of gut microbiota, feces were collected from growing rats that were fed with either meat proteins (from beef, pork or fish) or non-meat proteins (casein or soy) for 14 days. The resulting composition of gut microbiota was profiled by sequencing the V4-V5 region of the 16S ribosomal RNA genes and the short chain fatty acids (SCFAs) were analyzed using gas chromatography. The composition of gut microbiota and SCFA levels were significantly different between the five diet groups. At a recommended dose of 20% protein in the diet, meat protein-fed rats had a higher relative abundance of the beneficial genus Lactobacillus, but lower levels of SCFAs and SCFA-producing bacteria including Fusobacterium, Bacteroides and Prevotella, compared with the soy protein-fed group. Further work is needed on the regulatory pathways linking dietary protein intake to gut microbiota. PMID:27042829

  13. Mutation of exposed hydrophobic amino acids to arginine to increase protein stability

    OpenAIRE

    Strub, Caroline; Alies, Carole; Lougarre, Andrée; Ladurantie, Caroline; Czaplicki, Jerzy; Fournier, Didier

    2004-01-01

    Abstract Background One strategy to increase the stability of proteins is to reduce the area of water-accessible hydrophobic surface. Results In order to test it, we replaced 14 solvent-exposed hydrophobic residues of acetylcholinesterase by arginine. The stabilities of the resulting proteins were tested using denaturation by high temperature, organic solvents, urea and by proteolytic digestion. Conclusion Altough the mutational effects were rather small, this strategy proved to be successful...

  14. Whole body and forearm substrate metabolism in hyperthyroidism: evidence of increased basal muscle protein breakdown.

    Science.gov (United States)

    Riis, Anne Lene Dalkjaer; Jørgensen, Jens Otto Lunde; Gjedde, Signe; Nørrelund, Helene; Jurik, Anne Grethe; Nair, K S; Ivarsen, Per; Weeke, Jørgen; Møller, Niels

    2005-06-01

    Thyroid hormones have significant metabolic effects, and muscle wasting and weakness are prominent clinical features of chronic hyperthyroidism. To assess the underlying mechanisms, we examined seven hyperthyroid women with Graves' disease before (Ht) and after (Eut) medical treatment and seven control subjects (Ctr). All subjects underwent a 3-h study in the postabsorptive state. After regional catheterization, protein dynamics of the whole body and of the forearm muscles were measured by amino acid tracer dilution technique using [15N]phenylalanine and [2H4]tyrosine. Before treatment, triiodothyronine was elevated (6.6 nmol/l) and whole body protein breakdown was increased 40%. The net forearm release of phenylalanine was increased in hyperthyroidism (microg.100 ml(-1).min(-1)): -7.0 +/- 1.2 Ht vs. -3.8 +/- 0.8 Eut (P = 0.04), -4.2 +/- 0.3 Ctr (P = 0.048). Muscle protein breakdown, assessed by phenylalanine rate of appearance, was increased (microg.100 ml(-1).min(-1)): 15.5 +/- 2.0 Ht vs. 9.6 +/- 1.4 Eut (P = 0.03), 9.9 +/- 0.6 Ctr (P = 0.02). Muscle protein synthesis rate did not differ significantly. Muscle mass and muscle function were decreased 10-20% before treatment. All abnormalities were normalized after therapy. In conclusion, our results show that hyperthyroidism is associated with increased muscle amino acid release resulting from increased muscle protein breakdown. These abnormalities can explain the clinical manifestations of sarcopenia and myopathy.

  15. Dystropathology Increases Energy Expenditure and Protein Turnover in the Mdx Mouse Model of Duchenne Muscular Dystrophy

    Science.gov (United States)

    Radley-Crabb, Hannah G.; Marini, Juan C.; Sosa, Horacio A.; Castillo, Liliana I.; Grounds, Miranda D.; Fiorotto, Marta L.

    2014-01-01

    The skeletal muscles in Duchenne muscular dystrophy and the mdx mouse model lack functional dystrophin and undergo repeated bouts of necrosis, regeneration, and growth. These processes have a high metabolic cost. However, the consequences for whole body energy and protein metabolism, and on the dietary requirements for these macronutrients at different stages of the disease, are not well-understood. This study used juvenile (4- to 5- wk-old) and adult (12- to 14-wk-old) male dystrophic C57BL/10ScSn-mdx/J and age-matched C57BL/10ScSn/J control male mice to measure total and resting energy expenditure, food intake, spontaneous activity, body composition, whole body protein turnover, and muscle protein synthesis rates. In juvenile mdx mice that have extensive muscle damage, energy expenditure, muscle protein synthesis, and whole body protein turnover rates were higher than in age-matched controls. Adaptations in food intake and decreased activity were insufficient to meet the increased energy and protein needs of juvenile mdx mice and resulted in stunted growth. In (non-growing) adult mdx mice with less severe dystropathology, energy expenditure, muscle protein synthesis, and whole body protein turnover rates were also higher than in age-matched controls. Food intake was sufficient to meet their protein and energy needs, but insufficient to result in fat deposition. These data show that dystropathology impacts the protein and energy needs of mdx mice and that tailored dietary interventions are necessary to redress this imbalance. If not met, the resultant imbalance blunts growth, and may limit the benefits of therapies designed to protect and repair dystrophic muscles. PMID:24586653

  16. A Minimalistic Resource Allocation Model to Explain Ubiquitous Increase in Protein Expression with Growth Rate.

    Directory of Open Access Journals (Sweden)

    Uri Barenholz

    Full Text Available Most proteins show changes in level across growth conditions. Many of these changes seem to be coordinated with the specific growth rate rather than the growth environment or the protein function. Although cellular growth rates, gene expression levels and gene regulation have been at the center of biological research for decades, there are only a few models giving a base line prediction of the dependence of the proteome fraction occupied by a gene with the specific growth rate. We present a simple model that predicts a widely coordinated increase in the fraction of many proteins out of the proteome, proportionally with the growth rate. The model reveals how passive redistribution of resources, due to active regulation of only a few proteins, can have proteome wide effects that are quantitatively predictable. Our model provides a potential explanation for why and how such a coordinated response of a large fraction of the proteome to the specific growth rate arises under different environmental conditions. The simplicity of our model can also be useful by serving as a baseline null hypothesis in the search for active regulation. We exemplify the usage of the model by analyzing the relationship between growth rate and proteome composition for the model microorganism E.coli as reflected in recent proteomics data sets spanning various growth conditions. We find that the fraction out of the proteome of a large number of proteins, and from different cellular processes, increases proportionally with the growth rate. Notably, ribosomal proteins, which have been previously reported to increase in fraction with growth rate, are only a small part of this group of proteins. We suggest that, although the fractions of many proteins change with the growth rate, such changes may be partially driven by a global effect, not necessarily requiring specific cellular control mechanisms.

  17. Strong Selection Significantly Increases Epistatic Interactions in the Long-Term Evolution of a Protein.

    Directory of Open Access Journals (Sweden)

    Aditi Gupta

    2016-03-01

    Full Text Available Epistatic interactions between residues determine a protein's adaptability and shape its evolutionary trajectory. When a protein experiences a changed environment, it is under strong selection to find a peak in the new fitness landscape. It has been shown that strong selection increases epistatic interactions as well as the ruggedness of the fitness landscape, but little is known about how the epistatic interactions change under selection in the long-term evolution of a protein. Here we analyze the evolution of epistasis in the protease of the human immunodeficiency virus type 1 (HIV-1 using protease sequences collected for almost a decade from both treated and untreated patients, to understand how epistasis changes and how those changes impact the long-term evolvability of a protein. We use an information-theoretic proxy for epistasis that quantifies the co-variation between sites, and show that positive information is a necessary (but not sufficient condition that detects epistasis in most cases. We analyze the "fossils" of the evolutionary trajectories of the protein contained in the sequence data, and show that epistasis continues to enrich under strong selection, but not for proteins whose environment is unchanged. The increase in epistasis compensates for the information loss due to sequence variability brought about by treatment, and facilitates adaptation in the increasingly rugged fitness landscape of treatment. While epistasis is thought to enhance evolvability via valley-crossing early-on in adaptation, it can hinder adaptation later when the landscape has turned rugged. However, we find no evidence that the HIV-1 protease has reached its potential for evolution after 9 years of adapting to a drug environment that itself is constantly changing. We suggest that the mechanism of encoding new information into pairwise interactions is central to protein evolution not just in HIV-1 protease, but for any protein adapting to a changing

  18. Antisense oligonucleotides targeting translation inhibitory elements in 5' UTRs can selectively increase protein levels.

    Science.gov (United States)

    Liang, Xue-Hai; Sun, Hong; Shen, Wen; Wang, Shiyu; Yao, Joyee; Migawa, Michael T; Bui, Huynh-Hoa; Damle, Sagar S; Riney, Stan; Graham, Mark J; Crooke, Rosanne M; Crooke, Stanley T

    2017-09-19

    A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5' UTRs of some mRNAs. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5' UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5' UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Increased Protein Structural Resolution from Diethylpyrocarbonate-based Covalent Labeling and Mass Spectrometric Detection

    Science.gov (United States)

    Zhou, Yuping; Vachet, Richard W.

    2012-04-01

    Covalent labeling and mass spectrometry are seeing increased use together as a way to obtain insight into the 3-dimensional structure of proteins and protein complexes. Several amino acid specific (e.g., diethylpyrocarbonate) and non-specific (e.g., hydroxyl radicals) labeling reagents are available for this purpose. Diethylpyrocarbonate (DEPC) is a promising labeling reagent because it can potentially probe up to 30% of the residues in the average protein and gives only one reaction product, thereby facilitating mass spectrometric analysis. It was recently reported, though, that DEPC modifications are labile for some amino acids. Here, we show that label loss is more significant and widespread than previously thought, especially for Ser, Thr, Tyr, and His residues, when relatively long protein digestion times are used. Such label loss ultimately decreases the amount of protein structural information that is obtainable with this reagent. We find, however, that the number of DEPC modified residues and, thus, protein structural information, can be significantly increased by decreasing the time between the covalent labeling reaction and the mass spectrometric analysis. This is most effectively accomplished using short (e.g., 2 h) proteolytic digestions with enzymes such as immobilized chymotrypsin or Glu-C rather than using methods (e.g., microwave or ultrasonic irradiation) that accelerate proteolysis in other ways. Using short digestion times, we show that the percentage of solvent accessible residues that can be modified by DEPC increases from 44% to 67% for cytochrome c, 35% to 81% for myoglobin, and 76% to 95% for β-2-microglobulin. In effect, these increased numbers of modified residues improve the protein structural resolution available from this covalent labeling method. Compared with typical overnight digestion conditions, the short digestion times decrease the average distance between modified residues from 11 to 7 Å for myoglobin, 13 to 10 Å for

  20. The transient nature of Bunyamwera orthobunyavirus NSs protein expression: effects of increased stability of NSs protein on virus replication.

    Science.gov (United States)

    van Knippenberg, Ingeborg; Fragkoudis, Rennos; Elliott, Richard M

    2013-01-01

    The NSs proteins of bunyaviruses are the viral interferon antagonists, counteracting the host's antiviral response to infection. During high-multiplicity infection of cultured mammalian cells with Bunyamwera orthobunyavirus (BUNV), NSs is rapidly degraded after reaching peak levels of expression at 12hpi. Through the use of inhibitors this was shown to be the result of proteasomal degradation. A recombinant virus (rBUN4KR), in which all four lysine residues in NSs were replaced by arginine residues, expresses an NSs protein (NSs4KR) that is resistant to degradation, confirming that degradation is lysine-dependent. However, despite repeated attempts, no direct ubiquitylation of NSs in infected cells could be demonstrated. This suggests that degradation of NSs, although lysine-dependent, may be achieved through an indirect mechanism. Infection of cultured mammalian cells or mice indicated no disadvantage for the virus in having a non-degradable NSs protein: in fact rBUN4KR had a slight growth advantage over wtBUNV in interferon-competent cells, presumably due to the increased and prolonged presence of NSs. In cultured mosquito cells there was no difference in growth between wild-type BUNV and rBUN4KR, but surprisingly NSs4KR was not stabilised compared to the wild-type NSs protein.

  1. TIMP-1 increases expression and phosphorylation of proteins associated with drug resistance in breast cancer cells

    DEFF Research Database (Denmark)

    Hekmat, Omid; Munk, Stephanie; Fogh, Louise

    2013-01-01

    may explain the resistance phenotype to topoisomerase inhibitors that was observed in cells with high TIMP-1 levels. Pathway analysis showed an enrichment of proteins from functional categories such as apoptosis, cell cycle, DNA repair, transcription factors, drug targets and proteins associated......Tissue inhibitor of metalloproteinase 1 (TIMP-1) is a protein with a potential biological role in drug resistance. To elucidate the unknown molecular mechanisms underlying the association between high TIMP-1 levels and increased chemotherapy resistance, we employed SILAC-based quantitative mass...... spectrometry to analyze global proteome and phosphoproteome differences of MCF-7 breast cancer cells expressing high or low levels of TIMP-1. In TIMP-1 high expressing cells, 312 proteins and 452 phosphorylation sites were up-regulated. Among these were the cancer drug targets topoisomerase 1, 2A and 2B, which...

  2. Increasing the protein content of meals and its effect on daily energy intake.

    Science.gov (United States)

    Blatt, Alexandria D; Roe, Liane S; Rolls, Barbara J

    2011-02-01

    High-protein preloads have been shown to enhance satiety, but little is known about the satiating effects of protein in more typical situations when meals are consumed ad libitum. To investigate the effects of protein in amounts commonly consumed over a day, a crossover study was conducted in 2008. In this experiment, 18 normal-weight women consumed ad libitum lunch and dinner entrées 1 day a week that were covertly varied in protein content (10%, 15%, 20%, 25%, or 30% energy). Entrées were manipulated by substituting animal protein for starchy ingredients and were matched for energy density, fat content, palatability, and appearance. Unmanipulated breakfasts and evening snacks were consumed ad libitum. Participants rated their hunger and fullness before and after meals as well as the taste and appearance of entrées. Data were analyzed using a mixed linear model. Results showed that mean 24-hour protein intake increased significantly across conditions, from 44±2 g/day in the 10% protein condition to 82±6 g/day in the 30% condition. Daily energy intake did not differ significantly across the 10% to 30% protein conditions (means 1,870±93, 1,887±93, 1,848±111, 1,876±100, and 1,807±98 kcal in the 10%, 15%, 20%, 25%, and 30% energy groups, respectively). There were no significant differences in hunger and fullness ratings across conditions or in taste and appearance ratings of the manipulated entrées. This study showed that varying the protein content of several entrées consumed ad libitum did not differentially influence daily energy intake or affect ratings of satiety. Copyright © 2011 American Dietetic Association. Published by Elsevier Inc. All rights reserved.

  3. Increased expression of pyruvate carboxylase and biotin protein ligase increases lysine production in a biotin prototrophic Corynebacterium glutamicum strain

    DEFF Research Database (Denmark)

    Wang, Zhihao; Moslehi-Jenabian, Soloomeh; Solem, Christian

    2015-01-01

    , and achieved biotin prototrophy. We found that AHP-3, containing pBIO, was able to produce lysine in a medium lacking biotin and that the lysine yield on glucose was similar to what is obtained when using a medium containing biotin. However, there was a decrease in specific growth rate of 20% when the strain...... pimeloyl-Acyl Carrier Protein [ACP]) formation. Pyruvate carboxylase (pycA), a biotin-dependent enzyme needed for lysine biosynthesis and biotin ligase (birA), which is responsible for attaching biotin to pyruvate carboxylase, were overexpressed by replacing the native promoters with the strong superoxide...... dismutase (sod) promoter, to see whether growth could be restored. Neither pycA nor birA overexpression, whether alone or in combination, had an effect on specific growth rate, but they did have a positive effect on lysine yield, which increased by 55% in the strain overexpressing both enzymes....

  4. Increased accumulation of dendritic cells in celiac disease associates with increased expression of autophagy protein LC3

    Directory of Open Access Journals (Sweden)

    Paramaguru Rajaguru

    2013-01-01

    Full Text Available Background: Celiac disease (CD an immune-mediated disorder associates with accumulation of dendritic cell (DC in duodenal mucosa. Autophagy has recently been implicated in autoantigen formation. However, its role in CD is still unknown. Aim: To examine role of autophagic protein LC3 expressed by activated DC in CD. Materials and Methods : Thirty CD patients were analyzed at initial presentation and after 6 months of gluten-free diet (GFD. Duodenal biopsies were studied for histological changes and CD11c, CD86, and MAP1LC3A expressions by double immunohistochemistry (IHC. Masson′s trichrome (MT staining was used to assess basement membrane (BM thickness and Oil Red O (ORO staining for mucosal lipid deposit. Polymerase chain reaction (PCR was performed for HLA-DQ system. Statistical analysis was done using paired and unpaired t test, chi-square test, Fisher′s exact test, and McNemar-Bowker test. A P-value <0.05 was considered statistically significant. Results: HLA-DQ2 and HLA-DQ8 alleles were present in all studied patients. Increased BM thickness was observed in 63% and 73% had ORO-positive lipid in surface lining epithelium. Pre-treatment biopsies showed increased DCs expressing LC3, which were significantly less in follow-up biopsies. The follow-up biopsies had shown significant reduction in BM thickness and ORO. Conclusion : Histological improvement in duodenal biopsies was associated with reduction in activated DCs expressing autophagic protein, which probably play important role in pathogenesis of an autoimmune disorder like CD.

  5. Increasing the genetic variance of rice protein through mutation breeding techniques

    International Nuclear Information System (INIS)

    Ismachin, M.

    1975-01-01

    Recommended rice variety in Indonesia, Pelita I/1 was treated with gamma rays at the doses of 20 krad, 30 krad, and 40 krad. The seeds were also treated with EMS 1%. In M 2 generation, the protein content of seeds from the visible mutants and from the normal looking plants were analyzed by DBC method. No significant increase in the genetic variance was found on the samples treated with 20 krad gamma, and on the normal looking plants treated by EMS 1%. The mean value of the treated samples were mostly significant decrease compared with the mean value of the protein distribution in untreated samples (control). Since significant increase in genetic variance was also found in M 2 normal looking plants - treated with gamma at the doses of 30 krad and 40 krad -selection of protein among these materials could be more valuable. (author)

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

    DEFF Research Database (Denmark)

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

    1998-01-01

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

  7. Lower Growth Temperature Increases Alternative Pathway Capacity and Alternative Oxidase Protein in Tobacco 1

    Science.gov (United States)

    Vanlerberghe, Greg C.; McIntosh, Lee

    1992-01-01

    Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow) have been used to study the effect of growth temperature on the CN-resistant, salicylhydroxamic acid-sensitive alternative pathway of respiration. Mitochondria isolated from cells maintained at 30°C had a low capacity to oxidize succinate via the alternative pathway, whereas mitochondria isolated from cells 24 h after transfer to 18°C displayed, on average, a 5-fold increase in this capacity (from 7 to 32 nanoatoms oxygen per milligram protein per minute). This represented an increase in alternative pathway capacity from 18 to 45% of the total capacity of electron transport. This increased capacity was lost upon transfer of cells back to 30°C. A monoclonal antibody to the terminal oxidase of the alternative pathway (the alternative oxidase) from Sauromatum guttatum (T.E. Elthon, R.L. Nickels, L. McIntosh [1989] Plant Physiology 89: 1311-1317) recognized a 35-kilodalton mitochondrial protein in tobacco. There was an excellent correlation between the capacity of the alternative path in isolated tobacco mitochondria and the levels of this 35-kilodalton alternative oxidase protein. Cycloheximide could inhibit both the increased level of the 35-kilodalton alternative oxidase protein and the increased alternative pathway capacity normally seen upon transfer to 18°C. We conclude that transfer of tobacco cells to the lower temperature increases the capacity of the alternative pathway due, at least in part, to de novo synthesis of the 35-kilodalton alternative oxidase protein. Images Figure 3 Figure 4 PMID:16652932

  8. Highly increased detection of silver stained protein bands in polyacrylamide gels with thermo-optical methods

    Science.gov (United States)

    Mazza, Giulia; Posnicek, Thomas; Brandl, Martin

    2016-11-01

    Sodium dodecyl sulfate polyacrylamide gel electrophoresis is a well-known technique to separate proteins by their molecular weight. After electrophoresis, the gels are commonly stained for protein band analysis with silver stain; this allows the detection of protein loads to about 1 ng. To increase the detection sensitivity of the protein bands down in the subnanogram level, a sensor has been developed based on the thermal lens effect to scan and quantify protein loads which would remain undetected using the standard imaging systems. The thermal lens sensor is equipped with a 450 nm diode pump laser modulated at 1 Hz and a HeNe probe laser mounted in collinear geometry. The sensor could detect protein bands of 0.05 ng when the gel was soaked in methanol/water and 0.1 ng in water. The limit of detection ranged from 8 to 20 pg, depending on the soaking medium and the staining efficiency. Thus, the detection of silver stain by thermal lens effect results 10 to 20 times more sensitive than the standard colorimetric method.

  9. Increased adsorption of histidine-tagged proteins onto tissue culture polystyrene.

    Science.gov (United States)

    Holmberg, Maria; Hansen, Thomas Steen; Lind, Johan Ulrik; Hjortø, Gertrud Malene

    2012-04-01

    In this study we compare histidine-tagged and native proteins with regards to adsorption properties. We observe significantly increased adsorption of proteins with an incorporated polyhistidine amino acid motif (HIS-tag) onto tissue culture polystyrene (TCPS) compared to similar proteins without a HIS-tag. The effect is not observed on polystyrene (PS). Adsorption experiments have been performed at physiological pH (7.4) and the effect was only observed for the investigated proteins that have pI values below or around 7.4. Competitive adsorption experiments with imidazole and ethylenediaminetetraacetic acid (EDTA), as well as adsorption performed at different pH and ionic strength indicates that the high adsorption is caused by electrostatic interaction between negatively charged carboxylate groups on the TCPS surface and positively charged histidine residues in the proteins. Pre-adsorption of bovine serum albumin (BSA) does not decrease the adsorption of HIS-tagged proteins onto TCPS. Our findings identify a potential problem in using HIS-tagged signalling molecule in assays with cells cultured on TCPS, since the concentration of the molecule in solution might be affected and this could critically influence the assay outcome. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Protein energy malnutrition decreases immunity and increases susceptibility to influenza infection in mice.

    Science.gov (United States)

    Taylor, Andrew K; Cao, Weiping; Vora, Keyur P; De La Cruz, Juan; Shieh, Wun-Ju; Zaki, Sherif R; Katz, Jacqueline M; Sambhara, Suryaprakash; Gangappa, Shivaprakash

    2013-02-01

    Protein energy malnutrition (PEM), a common cause of secondary immune deficiency in children, is associated with an increased risk of infections. Very few studies have addressed the relevance of PEM as a risk factor for influenza. We investigated the influence of PEM on susceptibility to, and immune responses following, influenza virus infection using isocaloric diets providing either adequate protein (AP; 18%) or very low protein (VLP; 2%) in a mouse model. We found that mice maintained on the VLP diet, when compared to mice fed with the AP diet, exhibited more severe disease following influenza infection based on virus persistence, trafficking of inflammatory cell types to the lung tissue, and virus-induced mortality. Furthermore, groups of mice maintained on the VLP diet showed significantly lower virus-specific antibody response and a reduction in influenza nuclear protein-specific CD8(+) T cells compared with mice fed on the AP diet. Importantly, switching diets for the group maintained on the VLP diet to the AP diet improved virus clearance, as well as protective immunity to viral challenge. Our results highlight the impact of protein energy on immunity to influenza infection and suggest that balanced protein energy replenishment may be one strategy to boost immunity against influenza viral infections.

  11. Reduced Response to Activated Protein C Is Associated with Increased Risk for Cerebrovascular Disease

    NARCIS (Netherlands)

    Bom, J.G. van der; Bots, M.L.; Haverkate, F.; Slagboom, P.E.; Meijer, P.; Jong, P.T.V.M. de; Hofman, A.; Grobbee, D.E.; Kluft, C.

    1996-01-01

    Background: Resistance to activated protein C (APC), which results from various factors, including a mutation in the gene for coagulant factor V, has been associated with increased risk for venous thrombosis. However, its relation to arterial disease is still not well defined. Objective: To

  12. A Halotyrosine Antibody that Detects Increased Protein Modifications in Asthma Patients

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hongjun; Hallstrand, Teal S.; Daly, Don S.; Matzke, Melissa M.; Nair, Parameswaran; Bigelow, Diana J.; Pounds, Joel G.; Zangar, Richard C.

    2014-01-31

    Background-Airway inflammation plays an important pathophysiological role in asthma. Eosinophils produce hypobromite and bromotyrosine while neutrophils produce hypochlorite and chlorotyrosine. Objective-To evaluate halotyrosine modifications of individual airway proteins as a marker of inflammation in asthma using an antibody-based assay. Methods-We developed a novel monoclonal antibody (BTK-94C) that binds halogenated tyrosine residues, and used this antibody in a custom enzyme-linked immunosorbent assay (ELISA) microarray platform to examine halotyrosine levels in 23 proteins in three independent sets of sputum samples (52 samples total). Results-In 15 subjects with either no asthma, or with asthma characterized by high or low sputum eosinophil counts, we found associations between increased halotyrosine levels of at least three proteins and severity of airway hyperresponsiveness (AHR). Treatment with mepolizumab in 17 patients with sputum eosinophilia markedly reduced the sputum eosinophilia and significantly reduced halotyrosine levels in one sputum protein. Further analysis of 10 subjects with neutrophilic asthma and 10 health controls demonstrated a broad increase in halotyrosine in the patients with airway neutrophilia. Conclusions-Significantly higher levels of halotyrosine are associated with asthma in the asthma phenotypes we examined. The halotyrosine levels correlated with indirect AHR in the form of exercise-induced bronchoconstriction. Clinical Implication-An antibody-based assay for tyrosine halogenation in specific proteins may prove useful for assessing airway inflammation in asthma. Capsule Summary-An antibody to measure protein monobrominated tyrosine and other halotyrosine modifications was developed and used to evaluate halogenation in specific proteins in the airways for the first time. Associations were found between levels of halotyrosine and exercise-induced bronchoconstriction, and eosinophil and neutrophil inflammation in sputum from

  13. Enteral β-hydroxy-β-methylbutyrate supplementation increases protein synthesis in skeletal muscle of neonatal pigs

    Science.gov (United States)

    Kao, Michelle; Columbus, Daniel A.; Suryawan, Agus; Steinhoff-Wagner, Julia; Hernandez-Garcia, Adriana; Nguyen, Hanh V.; Fiorotto, Marta L.

    2016-01-01

    Many low-birth weight infants are at risk for poor growth due to an inability to achieve adequate protein intake. Administration of the amino acid leucine stimulates protein synthesis in skeletal muscle of neonates. To determine the effects of enteral supplementation of the leucine metabolite β-hydroxy-β-methylbutyrate (HMB) on protein synthesis and the regulation of translation initiation and degradation pathways, overnight-fasted neonatal pigs were studied immediately (F) or fed one of five diets for 24 h: low-protein (LP), high-protein (HP), or LP diet supplemented with 4 (HMB4), 40 (HMB40), or 80 (HMB80) μmol HMB·kg body wt−1·day−1. Cell replication was assessed from nuclear incorporation of BrdU in the longissimus dorsi (LD) muscle and jejunum crypt cells. Protein synthesis rates in LD, gastrocnemius, rhomboideus, and diaphragm muscles, lung, and brain were greater in HMB80 and HP and in brain were greater in HMB40 compared with LP and F groups. Formation of the eIF4E·eIF4G complex and S6K1 and 4E-BP1 phosphorylation in LD, gastrocnemius, and rhomboideus muscles were greater in HMB80 and HP than in LP and F groups. Phosphorylation of eIF2α and eEF2 and expression of SNAT2, LAT1, MuRF1, atrogin-1, and LC3-II were unchanged. Numbers of BrdU-positive myonuclei in the LD were greater in HMB80 and HP than in the LP and F groups; there were no differences in jejunum. The results suggest that enteral supplementation with HMB increases skeletal muscle protein anabolism in neonates by stimulation of protein synthesis and satellite cell proliferation. PMID:27143558

  14. Strong negative self regulation of Prokaryotic transcription factors increases the intrinsic noise of protein expression

    Directory of Open Access Journals (Sweden)

    Jenkins Dafyd J

    2008-01-01

    Full Text Available Abstract Background Many prokaryotic transcription factors repress their own transcription. It is often asserted that such regulation enables a cell to homeostatically maintain protein abundance. We explore the role of negative self regulation of transcription in regulating the variability of protein abundance using a variety of stochastic modeling techniques. Results We undertake a novel analysis of a classic model for negative self regulation. We demonstrate that, with standard approximations, protein variance relative to its mean should be independent of repressor strength in a physiological range. Consequently, in that range, the coefficient of variation would increase with repressor strength. However, stochastic computer simulations demonstrate that there is a greater increase in noise associated with strong repressors than predicted by theory. The discrepancies between the mathematical analysis and computer simulations arise because with strong repressors the approximation that leads to Michaelis-Menten-like hyperbolic repression terms ceases to be valid. Because we observe that strong negative feedback increases variability and so is unlikely to be a mechanism for noise control, we suggest instead that negative feedback is evolutionarily favoured because it allows the cell to minimize mRNA usage. To test this, we used in silico evolution to demonstrate that while negative feedback can achieve only a modest improvement in protein noise reduction compared with the unregulated system, it can achieve good improvement in protein response times and very substantial improvement in reducing mRNA levels. Conclusion Strong negative self regulation of transcription may not always be a mechanism for homeostatic control of protein abundance, but instead might be evolutionarily favoured as a mechanism to limit the use of mRNA. The use of hyperbolic terms derived from quasi-steady-state approximation should also be avoided in the analysis of stochastic

  15. Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis.

    Science.gov (United States)

    Gan, Qinglei; Fan, Chenguang

    2017-11-01

    Cell-free protein synthesis provides a robust platform for co-translational incorporation of noncanonical amino acid (ncAA) into proteins to facilitate biological studies and biotechnological applications. Recently, eliminating the activity of release factor 1 has been shown to increase ncAA incorporation in response to amber codons. However, this approach could promote mis-incorporation of canonical amino acids by near cognate suppression. We performed a facile protocol to remove near cognate tRNA isoacceptors of the amber codon from total tRNAs, and used the phosphoserine (Sep) incorporation system as validation. By manipulating codon usage of target genes and tRNA species introduced into the cell-free protein synthesis system, we increased the fidelity of Sep incorporation at a specific position. By removing three near cognate tRNA isoacceptors of the amber stop codon [tRNA Lys , tRNA Tyr , and tRNA Gln (CUG)] from the total tRNA, the near cognate suppression decreased by 5-fold without impairing normal protein synthesis in the cell-free protein synthesis system. Mass spectrometry analyses indicated that the fidelity of ncAA incorporation was improved. Removal of near cognate tRNA isoacceptors of the amber codon could increase ncAA incorporation fidelity towards the amber stop codon in release factor deficiency systems. We provide a general strategy to improve fidelity of ncAA incorporation towards stop, quadruplet and sense codons in cell-free protein synthesis systems. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Moderate expression of SEC16 increases protein secretion by Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Bao, Jichen; Huang, Mingtao; Petranovic, Dina

    2017-01-01

    in yeast, by moderately overexpressing SEC16, which is involved in protein translocation from the endoplasmic reticulum to the Golgi apparatus. The moderate overexpression of SEC16 increased α-amylase secretion by generating more endoplasmic reticulum exit sites. The production of reactive oxygen species...... were observed. Finally, the moderate overexpression of SEC16 was shown to improve the secretion of two other recombinant proteins, Trichoderma reesei endoglucanase I and Rhizopus oryzae glucan-1,4-α-glucosidase, indicating that this mechanism is of general relevance....

  17. Blood parameters in growing pigs fed increasing levels of bacterial protein meal

    DEFF Research Database (Denmark)

    Hellwing, Anne Louise Frydendahl; Tauson, Anne-Helene; Skrede, Anders

    2007-01-01

    The experiment investigated the effects of increasing dietary levels of bacterial protein meal (BPM) on various blood parameters reflecting protein and fat metabolism, liver function, and purine base metabolism in growing pigs. Sixteen barrows were allocated to four different experimental diets....... The control diet was based on soybean meal. In the other three diets soybean meal was replaced with increasing levels of BPM, approximately 17%, 35%, and 50% of the nitrogen being derived from BPM. Blood samples from the jugular vein were taken when the body weights of the pigs were approximately 10 kg, 21 kg......, 45 kg, and 77 kg. The blood parameters reflecting fat metabolism and liver funtion were not affected by diet. Both the plasma albumin and uric acid concentrations tended to decrease (P = 0.07 and 0.01, respectively) with increasing dietary BPM content, whereas the plasma glucose concentration tended...

  18. Methamphetamine increases Prion Protein and induces dopamine-dependent expression of protease resistant PrPsc.

    Science.gov (United States)

    Ferrucci, M; Ryskalin, L; Biagioni, F; Gambardella, S; Busceti, C L; Falleni, A; Lazzeri, G; Fornai, F

    2017-07-01

    The cellular prion protein (PrPc) is physiologically expressed within selective brain areas of mammals. Alterations in the secondary structure of this protein lead to scrapie-like prion protein (PrPsc), which precipitates in the cell. PrPsc has been detected in infectious, inherited or sporadic neurodegenerative disorders. Prion protein metabolism is dependent on autophagy and ubiquitin proteasome. Despite not being fully elucidated, the physiological role of prion protein relates to chaperones which rescue cells under stressful conditions.Methamphetamine (METH) is a widely abused drug which produces oxidative stress in various brain areas causing mitochondrial alterations and protein misfolding. These effects produce a compensatory increase of chaperones while clogging cell clearing pathways. In the present study, we explored whether METH administration modifies the amount of PrPc. Since high levels of PrPc when the clearing systems are clogged may lead to its misfolding into PrPsc, we further tested whether METH exposure triggers the appearance of PrPsc. We analysed the effects of METH and dopamine administration in PC12 and striatal cells by using SDS-PAGE Coomassie blue, immune- histochemistry and immune-gold electron microscopy. To analyze whether METH administration produces PrPsc aggregates we used antibodies directed against PrP following exposure to proteinase K or sarkosyl which digest folded PrPc but misfolded PrPsc. We fond that METH triggers PrPsc aggregates in DA-containing cells while METH is not effective in primary striatal neurons which do not produce DA. In the latter cells exogenous DA is needed to trigger PrPsc accumulation similarly to what happens in DA containing cells under the effects of METH. The present findings, while fostering novel molecular mechanisms involving prion proteins, indicate that, cell pathology similar to prion disorders can be mimicked via a DA-dependent mechanism by a drug of abuse.

  19. Increased helix and protein stability through the introduction of a new tertiary hydrogen bond.

    Science.gov (United States)

    Peterson, R W; Nicholson, E M; Thapar, R; Klevit, R E; Scholtz, J M

    1999-03-12

    In an effort to quantify the importance of hydrogen bonding and alpha-helix formation to protein stability, a capping box motif was introduced into the small phosphocarrier protein HPr. Previous studies had confirmed that Ser46, at the N-cap position of the short helix-B in HPr, serves as an N-cap in solution. Thus, only a single-site mutation was required to produce a canonical S-X-X-E capping box: Lys49 at the N3 position was substituted with a glutamic acid residue. Thermal and chemical denaturation studies on the resulting K49E HPr show that the designed variant is approximately 2 kcal mol-1 more stable than the wild-type protein. However, NMR studies indicate that the side-chain of Glu49 does not participate in the expected capping H-bond interaction, but instead forms a new tertiary H-bond that links helix-B to the four-stranded beta-sheet of HPr. Here, we demonstrate that a strategy in which new non-native H-bonds are introduced can generate proteins with increased stability. We discuss why the original capping box design failed, and compare the energetic consequences of the new tertiary side-chain to main-chain H-bond with a local (helix-capping) side-chain to main-chain H-bond on the protein's global stability. Copyright 1999 Academic Press.

  20. Expression of a truncated ATHB17 protein in maize increases ear weight at silking.

    Directory of Open Access Journals (Sweden)

    Elena A Rice

    Full Text Available ATHB17 (AT2G01430 is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize.

  1. Expression of a truncated ATHB17 protein in maize increases ear weight at silking.

    Science.gov (United States)

    Rice, Elena A; Khandelwal, Abha; Creelman, Robert A; Griffith, Cara; Ahrens, Jeffrey E; Taylor, J Philip; Murphy, Lesley R; Manjunath, Siva; Thompson, Rebecca L; Lingard, Matthew J; Back, Stephanie L; Larue, Huachun; Brayton, Bonnie R; Burek, Amanda J; Tiwari, Shiv; Adam, Luc; Morrell, James A; Caldo, Rico A; Huai, Qing; Kouadio, Jean-Louis K; Kuehn, Rosemarie; Sant, Anagha M; Wingbermuehle, William J; Sala, Rodrigo; Foster, Matt; Kinser, Josh D; Mohanty, Radha; Jiang, Dongming; Ziegler, Todd E; Huang, Mingya G; Kuriakose, Saritha V; Skottke, Kyle; Repetti, Peter P; Reuber, T Lynne; Ruff, Thomas G; Petracek, Marie E; Loida, Paul J

    2014-01-01

    ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize.

  2. Expression of a Truncated ATHB17 Protein in Maize Increases Ear Weight at Silking

    Science.gov (United States)

    Creelman, Robert A.; Griffith, Cara; Ahrens, Jeffrey E.; Taylor, J. Philip; Murphy, Lesley R.; Manjunath, Siva; Thompson, Rebecca L.; Lingard, Matthew J.; Back, Stephanie L.; Larue, Huachun; Brayton, Bonnie R.; Burek, Amanda J.; Tiwari, Shiv; Adam, Luc; Morrell, James A.; Caldo, Rico A.; Huai, Qing; Kouadio, Jean-Louis K.; Kuehn, Rosemarie; Sant, Anagha M.; Wingbermuehle, William J.; Sala, Rodrigo; Foster, Matt; Kinser, Josh D.; Mohanty, Radha; Jiang, Dongming; Ziegler, Todd E.; Huang, Mingya G.; Kuriakose, Saritha V.; Skottke, Kyle; Repetti, Peter P.; Reuber, T. Lynne; Ruff, Thomas G.; Petracek, Marie E.; Loida, Paul J.

    2014-01-01

    ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize. PMID:24736658

  3. Gestational Protein Restriction Increases Cardiac Connexin 43 mRNA levels in male adult rat offspring

    Science.gov (United States)

    Rossini, Kamila Fernanda; de Oliveira, Camila Andrea; Rebelato, Hércules Jonas; Esquisatto, Marcelo Augusto Marreto; Catisti, Rosana

    2017-01-01

    Background The dietary limitation during pregnancy influences the growth and development of the fetus and offspring and their health into adult life. The mechanisms underlying the adverse effects of gestational protein restriction (GPR) in the development of the offspring hearts are not well understood. Objectives The aim of this study was to evaluate the effects of GPR on cardiac structure in male rat offspring at day 60 after birth (d60). Methods Pregnant Wistar rats were fed a normal-protein (NP, 17% casein) or low-protein (LP, 6% casein) diet. Blood pressure (BP) values from 60-day-old male offspring were measured by an indirect tail-cuff method using an electro sphygmomanometer. Hearts (d60) were collected for assessment of connexin 43 (Cx43) mRNA expression and morphological and morphometric analysis. Results LP offspring showed no difference in body weight, although they were born lighter than NP offspring. BP levels were significantly higher in the LP group. We observed a significant increase in the area occupied by collagen fibers, a decrease in the number of cardiomyocytes by 104 µm2, and an increase in cardiomyocyte area associated with an increased Cx43 expression. Conclusion GPR changes myocardial levels of Cx43 mRNA in male young adult rats, suggesting that this mechanism aims to compensate the fibrotic process by the accumulation of collagen fibers in the heart interstitium. PMID:28678925

  4. Fermentation of rapeseed meal, sunflower meal and faba beans in combination with wheat bran increases solubility of protein and phosphorus

    DEFF Research Database (Denmark)

    Poulsen, Hanne Damgaard; Blaabjerg, Karoline

    2017-01-01

    BACKGROUND To increase self-supply of protein and phosphorus (P) in European pig and poultry diets and reduce nitrogen (N) and P excretion, attention is directed to approaches increasing protein and P digestibility of rapeseed, sunflower and faba beans. Wheat bran is rich in enzymes degrading...... and solubilizing protein and phytate. Herein, solubilization of protein, N and P was investigated when increasing ratios of wheat bran were fermented with rapeseed meal (RSM), sunflower meal (SFM), faba beans (FB) or a combination of these (RSM/SFM/FB). RESULTS Protein, N and P solubility was greater, for all...

  5. The Common Inhalational Anesthetic Sevoflurane Induces Apoptosis and Increases β-Amyloid Protein Levels

    Science.gov (United States)

    Dong, Yuanlin; Zhang, Guohua; Zhang, Bin; Moir, Robert D.; Xia, Weiming; Marcantonio, Edward R.; Culley, Deborah J.; Crosby, Gregory; Tanzi, Rudolph E.; Xie, Zhongcong

    2009-01-01

    Objective: To assess the effects of sevoflurane, the most commonly used inhalation anesthetic, on apoptosis and β-amyloid protein (Aβ) levels in vitro and in vivo. Subjects: Naive mice, H4 human neuroglioma cells, and H4 human neuroglioma cells stably transfected to express full-length amyloid precursor protein. Interventions: Human H4 neuroglioma cells stably transfected to express full-length amyloid precursor protein were exposed to 4.1% sevoflurane for 6 hours. Mice received 2.5% sevoflurane for 2 hours. Caspase-3 activation, apoptosis, and Aβ levels were assessed. Results: Sevoflurane induced apoptosis and elevated levels of β-site amyloid precursor protein-cleaving enzyme and Aβ in vitro and in vivo. The caspase inhibitor Z-VAD decreased the effects of sevoflurane on apoptosis and Aβ. Sevoflurane-induced caspase-3 activation was attenuated by the γ-secretase inhibitor L-685,458 and was potentiated by Aβ. These results suggest that sevoflurane induces caspase activation which, in turn, enhances β-site amyloid precursor protein–cleaving enzyme and Aβ levels. Increased Aβ levels then induce further rounds of apoptosis. Conclusions: These results suggest that inhalational anesthetic sevoflurane may promote Alzheimer disease neuropathogenesis. If confirmed in human subjects, it may be prudent to caution against the use of sevoflurane as an anesthetic, especially in those suspected of possessing excessive levels of cerebral Aβ. PMID:19433662

  6. Light-load resistance exercise increases muscle protein synthesis and hypertrophy signaling in elderly men

    DEFF Research Database (Denmark)

    Agergaard, Jakob; Bülow, Jacob; Jensen, Jacob K

    2017-01-01

    to 13 h of supine rest. After 2.5 h of rest, unilateral LL-RE, consisting of leg extensions (10 sets, 36 repetitions) at 16% of 1 repetition maximum (RM), was conducted. Subsequently, the subjects were randomized to oral intake of 4 g of whey protein per hour (PULSE, n = 10), 28 g of whey protein at 0 h...... and 12 g of whey protein at 7 h postexercise (BOLUS, n = 10), or 4 g of maltodextrin per hour (placebo, n = 10). Quadriceps muscle biopsies were taken at 0, 3, 7, and 10 h postexercise from the resting and the exercised leg of each subject. Myofibrillar FSR and activity of select targets from...... persisted in the placebo group only. Levels of phosphorylated (T37/46) eukaryotic translation initiation factor 4E-binding protein 1 increased throughout the postexercise period in the exercised leg in the placebo and BOLUS groups and peaked at 7 h. In all three groups, phosphorylated (T56) eukaryotic...

  7. Elevated maternal C-reactive protein and increased risk of schizophrenia in a national birth cohort.

    Science.gov (United States)

    Canetta, Sarah; Sourander, Andre; Surcel, Heljä-Marja; Hinkka-Yli-Salomäki, Susanna; Leiviskä, Jaana; Kellendonk, Christoph; McKeague, Ian W; Brown, Alan S

    2014-09-01

    The objective of the present study was to investigate an association between early gestational C-reactive protein, an established inflammatory biomarker, prospectively assayed in maternal sera, and schizophrenia in a large, national birth cohort with an extensive serum biobank. A nested case-control design from the Finnish Prenatal Study of Schizophrenia cohort was utilized. A total of 777 schizophrenia cases (schizophrenia, N=630; schizoaffective disorder, N=147) with maternal sera available for C-reactive protein testing were identified and matched to 777 control subjects in the analysis. Maternal C-reactive protein levels were assessed using a latex immunoassay from archived maternal serum specimens. Increasing maternal C-reactive protein levels, classified as a continuous variable, were significantly associated with schizophrenia in offspring (adjusted odds ratio=1.31, 95% confidence interval=1.10-1.56). This finding remained significant after adjusting for potential confounders, including maternal and parental history of psychiatric disorders, twin/singleton birth, urbanicity, province of birth, and maternal socioeconomic status. This finding provides the most robust evidence to date that maternal inflammation may play a significant role in schizophrenia, with possible implications for identifying preventive strategies and pathogenic mechanisms in schizophrenia and other neurodevelopmental disorders.

  8. Protein oxidative stress markers in peritoneal fluids of women with deep infiltrating endometriosis are increased.

    Science.gov (United States)

    Santulli, Pietro; Chouzenoux, Sandrine; Fiorese, Mauro; Marcellin, Louis; Lemarechal, Herve; Millischer, Anne-Elodie; Batteux, Frédéric; Borderie, Didier; Chapron, Charles

    2015-01-01

    Are protein oxidative stress markers [thiols, advanced oxidation protein products (AOPP), protein carbonyls and nitrates/nitrites] in perioperative peritoneal fluid higher in women with histologically proven endometriosis when compared with endometriosis-free controls? Protein oxidative stress markers are significantly increased in peritoneal fluids from women with deep infiltrating endometriosis with intestinal involvement when compared with endometriosis-free controls. Endometriosis is a common gynaecologic condition characterized by an important inflammatory process. Various source of evidence support the role of oxidative stress in the development of endometriosis. We conducted a prospective laboratory study in a tertiary-care university hospital between January 2011 and December 2012, and included 235 non-pregnant women, younger than 42 year old, undergoing surgery for a benign gynaecological condition. After complete surgical exploration of the abdomino-pelvic cavity, 150 women with histologically proven endometriosis and 85 endometriosis-free controls women were enrolled. Women with endometriosis were staged according to a surgical classification in three different phenotypes of endometriosis: superficial peritoneal endometriosis (SUP), ovarian endometrioma (OMA) and deeply infiltrating endometriosis (DIE). Perioperative peritoneal fluids samples were obtained from all study participants. Thiols, AOPP, protein carbonyls and nitrates/nitrites were assayed in all peritoneal samples. Concentrations of peritoneal AOPP were significantly higher in endometriosis patients than in the control group (median, 128.9 µmol/l; range, 0.3-1180.1 versus median, 77.8 µmol/l; range, 0.8-616.1; P peritoneal nitrates/nitrites were higher in endometriosis patients than in the control group (median, 24.8 µmol/l; range, 1.6-681.6 versus median, 18.5 µmol/l; range, 1.6-184.5; P peritoneal fluids protein AOPP and nitrates/nitrites were significantly increased only in DIE samples

  9. Phenotypic Screening Identifies Protein Synthesis Inhibitors as H-Ras-Nanocluster-Increasing Tumor Growth Inducers.

    Science.gov (United States)

    Najumudeen, Arafath K; Posada, Itziar M D; Lectez, Benoit; Zhou, Yong; Landor, Sebastian K-J; Fallarero, Adyary; Vuorela, Pia; Hancock, John; Abankwa, Daniel

    2015-12-15

    Ras isoforms H-, N-, and K-ras are each mutated in specific cancer types at varying frequencies and have different activities in cell fate control. On the plasma membrane, Ras proteins are laterally segregated into isoform-specific nanoscale signaling hubs, termed nanoclusters. As Ras nanoclusters are required for Ras signaling, chemical modulators of nanoclusters represent ideal candidates for the specific modulation of Ras activity in cancer drug development. We therefore conducted a chemical screen with commercial and in-house natural product libraries using a cell-based H-ras-nanoclustering FRET assay. Next to established Ras inhibitors, such as a statin and farnesyl-transferase inhibitor, we surprisingly identified five protein synthesis inhibitors as positive regulators. Using commonly employed cycloheximide as a representative compound, we show that protein synthesis inhibition increased nanoclustering and effector recruitment specifically of active H-ras but not of K-ras. Consistent with these data, cycloheximide treatment activated both Erk and Akt kinases and specifically promoted H-rasG12V-induced, but not K-rasG12V-induced, PC12 cell differentiation. Intriguingly, cycloheximide increased the number of mammospheres, which are enriched for cancer stem cells. Depletion of H-ras in combination with cycloheximide significantly reduced mammosphere formation, suggesting an exquisite synthetic lethality. The potential of cycloheximide to promote tumor cell growth was also reflected in its ability to increase breast cancer cell tumors grown in ovo. These results illustrate the possibility of identifying Ras-isoform-specific modulators using nanocluster-directed screening. They also suggest an unexpected feedback from protein synthesis inhibition to Ras signaling, which might present a vulnerability in certain tumor cell types.

  10. Increased signaling entropy in cancer requires the scale-free property of protein interaction networks

    Science.gov (United States)

    Teschendorff, Andrew E.; Banerji, Christopher R. S.; Severini, Simone; Kuehn, Reimer; Sollich, Peter

    2015-01-01

    One of the key characteristics of cancer cells is an increased phenotypic plasticity, driven by underlying genetic and epigenetic perturbations. However, at a systems-level it is unclear how these perturbations give rise to the observed increased plasticity. Elucidating such systems-level principles is key for an improved understanding of cancer. Recently, it has been shown that signaling entropy, an overall measure of signaling pathway promiscuity, and computable from integrating a sample's gene expression profile with a protein interaction network, correlates with phenotypic plasticity and is increased in cancer compared to normal tissue. Here we develop a computational framework for studying the effects of network perturbations on signaling entropy. We demonstrate that the increased signaling entropy of cancer is driven by two factors: (i) the scale-free (or near scale-free) topology of the interaction network, and (ii) a subtle positive correlation between differential gene expression and node connectivity. Indeed, we show that if protein interaction networks were random graphs, described by Poisson degree distributions, that cancer would generally not exhibit an increased signaling entropy. In summary, this work exposes a deep connection between cancer, signaling entropy and interaction network topology. PMID:25919796

  11. Blood parameters in growing pigs fed increasing levels of bacterial protein meal

    Directory of Open Access Journals (Sweden)

    Tauson Anne-Helene

    2007-11-01

    Full Text Available Abstract The experiment investigated the effects of increasing dietary levels of bacterial protein meal (BPM on various blood parameters reflecting protein and fat metabolism, liver function, and purine base metabolism in growing pigs. Sixteen barrows were allocated to four different experimental diets. The control diet was based on soybean meal. In the other three diets soybean meal was replaced with increasing levels of BPM, approximately 17%, 35%, and 50% of the nitrogen being derived from BPM. Blood samples from the jugular vein were taken when the body weights of the pigs were approximately 10 kg, 21 kg, 45 kg, and 77 kg. The blood parameters reflecting fat metabolism and liver function were not affected by diet. Both the plasma albumin and uric acid concentrations tended to decrease (P = 0.07 and 0.01, respectively with increasing dietary BPM content, whereas the plasma glucose concentration tended to increase (P = 0.07 with increasing dietary BPM content. It was concluded that up to 50% of the nitrogen could be derived from BPM without affecting metabolic function, as reflected in the measured blood parameters.

  12. Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems

    Science.gov (United States)

    Badenes, Sara M.; Fernandes, Tiago G.; Cordeiro, Cláudia S. M.; Boucher, Shayne; Kuninger, David; Vemuri, Mohan C.; Diogo, Maria Margarida; Cabral, Joaquim M. S.

    2016-01-01

    Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x106 cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells. PMID:26999816

  13. Vitronectin Binds to a Specific Stretch within the Head Region of Yersinia Adhesin A and Thereby Modulates Yersinia enterocolitica Host Interaction.

    Science.gov (United States)

    Mühlenkamp, Melanie C; Hallström, Teresia; Autenrieth, Ingo B; Bohn, Erwin; Linke, Dirk; Rinker, Janina; Riesbeck, Kristian; Singh, Birendra; Leo, Jack C; Hammerschmidt, Sven; Zipfel, Peter F; Schütz, Monika S

    2017-01-01

    Complement resistance is an important virulence trait of Yersinia enterocolitica (Ye). The predominant virulence factor expressed by Ye is Yersinia adhesin A (YadA), which enables bacterial attachment to host cells and extracellular matrix and additionally allows the acquisition of soluble serum factors. The serum glycoprotein vitronectin (Vn) acts as an inhibitory regulator of the terminal complement complex by inhibiting the lytic pore formation. Here, we show YadA-mediated direct interaction of Ye with Vn and investigated the role of this Vn binding during mouse infection in vivo. Using different Yersinia strains, we identified a short stretch in the YadA head domain of Ye O:9 E40, similar to the 'uptake region' of Y. pseudotuberculosis YPIII YadA, as crucial for efficient Vn binding. Using recombinant fragments of Vn, we found the C-terminal part of Vn, including heparin-binding domain 3, to be responsible for binding to YadA. Moreover, we found that Vn bound to the bacterial surface is still functionally active and thus inhibits C5b-9 formation. In a mouse infection model, we demonstrate that Vn reduces complement-mediated killing of Ye O:9 E40 and, thus, improved bacterial survival. Taken together, these findings show that YadA-mediated Vn binding influences Ye pathogenesis. © 2016 S. Karger AG, Basel.

  14. Engineering of an E. coli outer membrane protein FhuA with increased channel diameter

    Directory of Open Access Journals (Sweden)

    Dworeck Tamara

    2011-08-01

    Full Text Available Abstract Background Channel proteins like FhuA can be an alternative to artificial chemically synthesized nanopores. To reach such goals, channel proteins must be flexible enough to be modified in their geometry, i.e. length and diameter. As continuation of a previous study in which we addressed the lengthening of the channel, here we report the increasing of the channel diameter by genetic engineering. Results The FhuA Δ1-159 diameter increase has been obtained by doubling the amino acid sequence of the first two N-terminal β-strands, resulting in variant FhuA Δ1-159 Exp. The total number of β-strands increased from 22 to 24 and the channel surface area is expected to increase by ~16%. The secondary structure analysis by circular dichroism (CD spectroscopy shows a high β-sheet content, suggesting the correct folding of FhuA Δ1-159 Exp. To further prove the FhuA Δ1-159 Exp channel functionality, kinetic measurement using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine were conducted. The results indicated a 17% faster diffusion kinetic for FhuA Δ1-159 Exp as compared to FhuA Δ1-159, well correlated to the expected channel surface area increase of ~16%. Conclusion In this study using a simple "semi rational" approach the FhuA Δ1-159 diameter was enlarged. By combining the actual results with the previous ones on the FhuA Δ1-159 lengthening a new set of synthetic nanochannels with desired lengths and diameters can be produced, broadening the FhuA Δ1-159 applications. As large scale protein production is possible our approach can give a contribution to nanochannel industrial applications.

  15. Increasing the thermal stability of cellulase C using rules learned from thermophilic proteins: a pilot study.

    Science.gov (United States)

    Németh, Attila; Kamondi, Szilárd; Szilágyi, András; Magyar, Csaba; Kovári, Zoltán; Závodszky, Péter

    2002-05-02

    Some structural features underlying the increased thermostability of enzymes from thermophilic organisms relative to their homologues from mesophiles are known from earlier studies. We used cellulase C from Clostridium thermocellum to test whether thermostability can be increased by mutations designed using rules learned from thermophilic proteins. Cellulase C has a TIM barrel fold with an additional helical subdomain. We designed and produced a number of mutants with the aim to increase its thermostability. Five mutants were designed to create new electrostatic interactions. They all retained catalytic activity but exhibited decreased thermostability relative to the wild-type enzyme. Here, the stabilizing contributions are obviously smaller than the destabilization caused by the introduction of the new side chains. In another mutant, the small helical subdomain was deleted. This mutant lost activity but its melting point was only 3 degrees C lower than that of the wild-type enzyme, which suggests that the subdomain is an independent folding unit and is important for catalytic function. A double mutant was designed to introduce a new disulfide bridge into the enzyme. This mutant is active and has an increased stability (deltaT(m)=3 degrees C, delta(deltaG(u))=1.73 kcal/mol) relative to the wild-type enzyme. Reduction of the disulfide bridge results in destabilization and an altered thermal denaturation behavior. We conclude that rules learned from thermophilic proteins cannot be used in a straightforward way to increase the thermostability of a protein. Creating a crosslink such as a disulfide bond is a relatively sure-fire method but the stabilization may be smaller than calculated due to coupled destabilizing effects.

  16. Variable-Intensity Simulated Team-Sport Exercise Increases Daily Protein Requirements in Active Males.

    Science.gov (United States)

    Packer, Jeffrey E; Wooding, Denise J; Kato, Hiroyuki; Courtney-Martin, Glenda; Pencharz, Paul B; Moore, Daniel R

    2017-01-01

    Protein requirements are generally increased in strength and endurance trained athletes relative to their sedentary peers. However, less is known about the daily requirement for this important macronutrient in individuals performing variable intensity, stop-and-go type exercise that is typical for team sport athletes. The objective of the present study was to determine protein requirements in active, trained adult males performing a simulated soccer match using the minimally invasive indicator amino acid oxidation (IAAO) method. After 2 days of controlled diet (1.2 g⋅kg -1 ⋅day -1 protein), seven trained males (23 ± 1 years; 177.5 ± 6.7 cm; 82.3 ± 6.1 kg; 13.5% ± 4.7% body fat; 52.3 ± 5.9 ml O 2 ⋅kg -1 ⋅min -1 ; mean ± SD) performed an acute bout of variable intensity exercise in the form of a modified Loughborough Intermittent Shuttle Test (4 × 15 min of exercise over 75 min). Immediately after exercise, hourly meals were consumed providing a variable amount of protein (0.2-2.6 g⋅kg -1 ⋅day -1 ) and sufficient energy and carbohydrate (6 g⋅kg -1 ⋅day -1 ). Protein was provided as a crystalline amino acids modeled after egg protein with the exception of phenylalanine and tyrosine, which were provided in excess to ensure the metabolic partitioning of the indicator amino acid (i.e., [1- 13 C]phenylalanine included within the phenylalanine intake) was directed toward oxidation when protein intake was limiting. Whole body phenylalanine flux and 13 CO 2 excretion (F 13 CO 2 ) were determined at metabolic and isotopic steady state from urine and breath samples, respectively. Biphasic linear regression analysis was performed on F 13 CO 2 to determine the estimated average requirement (EAR) for protein with a safe intake defined as the upper 95% confidence interval. Phenylalanine flux was not impacted by protein intake ( P  = 0.45). Bi-phase linear regression ( R 2  = 0.64) of F 13 CO 2 resulted

  17. Variable-Intensity Simulated Team-Sport Exercise Increases Daily Protein Requirements in Active Males

    Directory of Open Access Journals (Sweden)

    Jeffrey E. Packer

    2017-12-01

    Full Text Available Protein requirements are generally increased in strength and endurance trained athletes relative to their sedentary peers. However, less is known about the daily requirement for this important macronutrient in individuals performing variable intensity, stop-and-go type exercise that is typical for team sport athletes. The objective of the present study was to determine protein requirements in active, trained adult males performing a simulated soccer match using the minimally invasive indicator amino acid oxidation (IAAO method. After 2 days of controlled diet (1.2 g⋅kg−1⋅day−1 protein, seven trained males (23 ± 1 years; 177.5 ± 6.7 cm; 82.3 ± 6.1 kg; 13.5% ± 4.7% body fat; 52.3 ± 5.9 ml O2⋅kg−1⋅min-1; mean ± SD performed an acute bout of variable intensity exercise in the form of a modified Loughborough Intermittent Shuttle Test (4 × 15 min of exercise over 75 min. Immediately after exercise, hourly meals were consumed providing a variable amount of protein (0.2–2.6 g⋅kg−1⋅day−1 and sufficient energy and carbohydrate (6 g⋅kg−1⋅day−1. Protein was provided as a crystalline amino acids modeled after egg protein with the exception of phenylalanine and tyrosine, which were provided in excess to ensure the metabolic partitioning of the indicator amino acid (i.e., [1-13C]phenylalanine included within the phenylalanine intake was directed toward oxidation when protein intake was limiting. Whole body phenylalanine flux and 13CO2 excretion (F13CO2 were determined at metabolic and isotopic steady state from urine and breath samples, respectively. Biphasic linear regression analysis was performed on F13CO2 to determine the estimated average requirement (EAR for protein with a safe intake defined as the upper 95% confidence interval. Phenylalanine flux was not impacted by protein intake (P = 0.45. Bi-phase linear regression (R2 = 0.64 of F13CO2 resulted in an EAR

  18. Insulin increases phosphorylation of mitochondrial proteins in human skeletal muscle in vivo

    DEFF Research Database (Denmark)

    Zhao, Xiaolu; Bak, Steffen; Pedersen, Andreas James Thestrup

    2014-01-01

    , we investigated the effect of insulin on the phosphorylation of mitochondrial proteins in human skeletal muscle in vivo. Using a combination of TiO2 phosphopeptide-enrichment, HILIC fractionation, and LC−MS/MS, we compared the phosphoproteomes of isolated mitochondria from skeletal muscle samples...... obtained from healthy individuals before and after 4 h of insulin infusion. In total, we identified 207 phosphorylation sites in 95 mitochondrial proteins. Of these phosphorylation sites, 45% were identified in both basal and insulin-stimulated samples. Insulin caused a 2-fold increase in the number...... of different mitochondrial phosphopeptides (87 ± 7 vs 40 ± 7, p = 0.015) and phosphoproteins (46 ± 2 vs 26 ± 3, p = 0.005) identified in each mitochondrial preparation. Almost half of the mitochondrial phosphorylation sites (n = 94) were exclusively identified in the insulin-stimulated state and included...

  19. Brillouin spectroscopy as a new method of screening for increased CSF total protein during bacterial meningitis.

    Science.gov (United States)

    Steelman, Zachary; Meng, Zhaokai; Traverso, Andrew J; Yakovlev, Vladislav V

    2015-05-01

    Bacterial meningitis is a disease of pronounced clinical significance, especially in the developing world. Immediate treatment with antibiotics is essential, and no single test can provide a conclusive diagnosis. It is well established that elevated total protein in cerebrospinal fluid (CSF) is associated with bacterial meningitis. Brillouin spectroscopy is a widely used optical technique for noninvasive determination of the elastic moduli of materials. We found that elevated protein levels in CSF alter the fluid elasticity sufficiently to be measurable by Brillouin spectroscopy, with model healthy and diseased fluids distinguishable to marked significance (P = 0.014), which increases with sample concentration by dialysis. Typical raw output of a 2-stage VIPA Brillouin spectrometer: inelastically scattered Brillouin peaks (arrows) and elastically scattered incident radiation (center cross). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Energy dense, protein restricted diet increases adiposity and perturbs metabolism in young, genetically lean pigs.

    Science.gov (United States)

    Fisher, Kimberly D; Scheffler, Tracy L; Kasten, Steven C; Reinholt, Brad M; van Eyk, Gregory R; Escobar, Jeffery; Scheffler, Jason M; Gerrard, David E

    2013-01-01

    Animal models of obesity and metabolic dysregulation during growth (or childhood) are lacking. Our objective was to increase adiposity and induce metabolic syndrome in young, genetically lean pigs. Pre-pubertal female pigs, age 35 d, were fed a high-energy diet (HED; n = 12), containing 15% tallow, 35% refined sugars and 9.1-12.9% crude protein, or a control corn-based diet (n = 11) with 12.2-19.2% crude protein for 16 wk. Initially, HED pigs self-regulated energy intake similar to controls, but by wk 5, consumed more (Pblood glucose increased (Pblood glucose did not return to baseline (P = 0.01), even 4 h post-challenge. During OGTT, glucose area under the curve (AUC) was higher and insulin AUC was lower in HED pigs compared to controls (P = 0.001). Chronic HED intake increased (PAUC and insulin AUC did not improve, supporting that dietary intervention was not sufficient to recover glucose tolerance or insulin production. These data suggest a HED may be used to increase adiposity and disrupt glucose homeostasis in young, growing pigs.

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

    Science.gov (United States)

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

    2014-06-01

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

  2. Iron Loading Selectively Increases Hippocampal Levels of Ubiquitinated Proteins and Impairs Hippocampus-Dependent Memory.

    Science.gov (United States)

    Figueiredo, Luciana Silva; de Freitas, Betânia Souza; Garcia, Vanessa Athaíde; Dargél, Vinícius Ayub; Köbe, Luiza Machado; Kist, Luiza Wilges; Bogo, Maurício Reis; Schröder, Nadja

    2016-11-01

    Alterations of brain iron levels have been observed in a number of neurodegenerative disorders. We have previously demonstrated that iron overload in the neonatal period results in severe and persistent memory deficits in the adulthood. Protein degradation mediated by the ubiquitin-proteasome system (UPS) plays a central regulatory role in several cellular processes. Impairment of the UPS has been implicated in the pathogenesis of neurodegenerative disorders. Here, we examined the effects of iron exposure in the neonatal period (12th-14th day of postnatal life) on the expression of proteasome β-1, β-2, and β-5 subunits, and ubiquitinated proteins in brains of 15-day-old rats, to evaluate the immediate effect of the treatment, and in adulthood to assess long-lasting effects. Two different memory types, emotionally motivated conditioning and object recognition were assessed in adult animals. We found that iron administered in the neonatal period impairs both emotionally motivated and recognition memory. Polyubiquitinated protein levels were increased in the hippocampus, but not in the cortex, of adult animals treated with iron. Gene expression of subunits β1 and β5 was affected by age, being higher in the early stages of development in the hippocampus, accompanied by an age-related increase in polyubiquitinated protein levels in adults. In the cortex, gene expression of the three proteasome subunits was significantly higher in adulthood than in the neonatal period. These findings suggest that expression of proteasome subunits and activity are age-dependently regulated. Iron exposure in the neonatal period produces long-lasting harmful effects on the UPS functioning, which may be related with iron-induced memory impairment.

  3. Semi-solid microbial fermentation of rice and wheat straw for protein enrichment and increased digestibility

    Energy Technology Data Exchange (ETDEWEB)

    Balasubramanya, R.H.; Bhatawdekar, S.P.

    1980-12-01

    Rice and wheat straws were hydrolyzed in various concentrations of sulfuric acid at different temperatures and different water: substrate ratios. The maximum amount of sugars of about 30-34% was released when heated at 121 degrees C with 0.5 N H2SO4 at a water: substrate ratio of 3:1. The pH of the hydrolyzed straws was raised to 5.0-5.5 with 5 N NH4OH. Such ammoniated straws were inoculated with the cultures of Penicillium funiculosum Thom. and Candida utilis (Henneb.) Lodder and Kreger-van Rij, and fermentation was carried out on semi-solid substrate for 5-7 days at room temperature. The fermentation resulted in 37-180% increase in crude protein, 23-100% increase in crude fat and 20-30% increase in the digestibility. (Refs. 29).

  4. The heat shock proteins at the increasing of the radioresistance of silkworm embryo Bombyx Moril

    International Nuclear Information System (INIS)

    Agaev, F.A.; Garibov, A.A.; Aliev, D.I.; Alieva, I.N.

    2002-01-01

    The aim of this study is revealing the role of Heat Shock Proteins (HSP) at the radio-modification effect of Heat Shock (HS) on the silkworm embryo. Our preliminary study was indicated that 3-daily silkworm embryo is more than 10-12 times sensitive to gamma-radiation in comparative to 7-daily embryo. Investigation of the HS effect on the radiosensitivity of the embryo indicates that thermal treatment (40 deg. C) of 3 daily embryo during 60 min before irradiation leads the increasing of its radioresistance. At the same time, the HS treatment immediately after irradiation capable increases strike affect of irradiation. LD 50 for heat treated embryo before and after irradiation consists of 46.7 and 19.0 Gy, correspondingly. For embryo irradiated without heat treatment LD 50 is 29.5 Gy. Identical effects are observed for 7-daily embryo. Increasing of the radioresistance by HS before irradiation, obviously, may be explained by the embryo cell reply to the stress factor and capable initiated by different biochemical shifts, for example, by induction of HPS synthesis. According to the results of carried out experiments HS treatment leads to the induction or increasing of the HSP synthesis into this embryo. Protein with molecular mass 70 kDa (HSP-70 kDa) has been synthesised de novo. The synthesis of the other two proteins (HSP-83 kDa and HSP-68 kDa) significantly increases at the high temperature. It is noted, that HSP-70 kDa consists of 55-60 % of whole included radioactive mark. Identical induction was observed in the experiments at the combined effect both HS and gamma-radiation on the embryo. At the post-radiation heat treatment the induction of HSP synthesis is observed, too. It was concluded that damages induced by irradiation can not prevent HSP induction into embryo. The result of comparative analysis was shown that in 3-daily embryo in spite of 7-daily embryo the synthesis of HSP is more intensive and correlated with the radio- modification effect of HS. The

  5. Investigation of New Morpholino Oligomers to Increase Survival Motor Neuron Protein Levels in Spinal Muscular Atrophy.

    Science.gov (United States)

    Ramirez, Agnese; Crisafulli, Sebastiano G; Rizzuti, Mafalda; Bresolin, Nereo; Comi, Giacomo P; Corti, Stefania; Nizzardo, Monica

    2018-01-06

    Spinal muscular atrophy (SMA) is an autosomal-recessive childhood motor neuron disease and the main genetic cause of infant mortality. SMA is caused by deletions or mutations in the survival motor neuron 1 ( SMN1 ) gene, which results in SMN protein deficiency. Only one approved drug has recently become available and allows for the correction of aberrant splicing of the paralogous SMN2 gene by antisense oligonucleotides (ASOs), leading to production of full-length SMN protein. We have already demonstrated that a sequence of an ASO variant, Morpholino (MO), is particularly suitable because of its safety and efficacy profile and is both able to increase SMN levels and rescue the murine SMA phenotype. Here, we optimized this strategy by testing the efficacy of four new MO sequences targeting SMN2 . Two out of the four new MO sequences showed better efficacy in terms of SMN protein production both in SMA induced pluripotent stem cells (iPSCs) and SMAΔ7 mice. Further, the effect was enhanced when different MO sequences were administered in combination. Our data provide an important insight for MO-based treatment for SMA. Optimization of the target sequence and validation of a treatment based on a combination of different MO sequences could support further pre-clinical studies and the progression toward future clinical trials.

  6. Single amino acid substitutions on the needle tip protein IpaD increased Shigella virulence.

    Science.gov (United States)

    Meghraoui, Alaeddine; Schiavolin, Lionel; Allaoui, Abdelmounaaïm

    2014-07-01

    Infection of colonic epithelial cells by Shigella is associated with the type III secretion system, which serves as a molecular syringe to inject effectors into host cells. This system includes an extracellular needle used as a conduit for secreted proteins. Two of these proteins, IpaB and IpaD, dock at the needle tip to control secretion and are also involved in the insertion of a translocation pore into host cell membrane allowing effector delivery. To better understand the function of IpaD, we substituted thirteen residues conserved among homologous proteins in other bacterial species. Generated variants were tested for their ability to surface expose IpaB and IpaD, to control secretion, to insert the translocation pore, and to invade host cells. In addition to a first group of seven ipaD variants that behaved similarly to the wild-type strain, we identified a second group with mutations V314D and I319D that deregulated secretion of all effectors, but remained fully invasive. Moreover, we identified a third group with mutations Y153A, T161D, Q165L and Y276A, that exhibited increased levels of translocators secretion, pore formation, and cell entry. Altogether, our results offer a better understanding of the role of IpaD in the control of Shigella virulence. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  7. Selection for a Zinc-Finger Protein Contributes to Seed Oil Increase during Soybean Domestication.

    Science.gov (United States)

    Li, Qing-Tian; Lu, Xiang; Song, Qing-Xin; Chen, Hao-Wei; Wei, Wei; Tao, Jian-Jun; Bian, Xiao-Hua; Shen, Ming; Ma, Biao; Zhang, Wan-Ke; Bi, Ying-Dong; Li, Wei; Lai, Yong-Cai; Lam, Sin-Man; Shui, Guang-Hou; Chen, Shou-Yi; Zhang, Jin-Song

    2017-04-01

    Seed oil is a momentous agronomical trait of soybean ( Glycine max ) targeted by domestication in breeding. Although multiple oil-related genes have been uncovered, knowledge of the regulatory mechanism of seed oil biosynthesis is currently limited. We demonstrate that the seed-preferred gene GmZF351 , encoding a tandem CCCH zinc finger protein, is selected during domestication. Further analysis shows that GmZF351 facilitates oil accumulation by directly activating WRINKLED1 , BIOTIN CARBOXYL CARRIER PROTEIN2 , 3-KETOACYL-ACYL CARRIER PROTEIN SYNTHASE III , DIACYLGLYCEROL O-ACYLTRANSFERASE1 , and OLEOSIN2 in transgenic Arabidopsis ( Arabidopsis thaliana ) seeds. Overexpression of GmZF351 in transgenic soybean also activates lipid biosynthesis genes, thereby accelerating seed oil accumulation. The ZF351 haplotype from the cultivated soybean group and the wild soybean ( Glycine soja ) subgroup III correlates well with high gene expression level, seed oil contents and promoter activity, suggesting that selection of GmZF351 expression leads to increased seed oil content in cultivated soybean. Our study provides novel insights into the regulatory mechanism for seed oil accumulation, and the manipulation of GmZF351 may have great potential in the improvement of oil production in soybean and other related crops. © 2017 American Society of Plant Biologists. All Rights Reserved.

  8. Taste sensitivity for monosodium glutamate and an increased liking of dietary protein.

    Science.gov (United States)

    Luscombe-Marsh, Natalie D; Smeets, Astrid J P G; Westerterp-Plantenga, Margriet S

    2008-04-01

    The aim of the present study was to determine individuals' taste threshold for monosodium glutamate (MSG) alone and in combination with inosine 5'-monophosphate (IMP-5) and to examine if this threshold was related to an increase in sensory properties (including pleasantness of taste) and/or to one's preference for dietary protein over carbohydrate and fat. Using the triangle tasting method, the taste threshold was determined for thirty-six women and twenty-four men. Thresholds varied from zero to infinite as determined using a clear soup with added MSG in the concentration range of 0.1 to 0.8 % (w/w) MSG. Subjects rated fourteen sensory properties of the soup and also their 'liking', 'eating frequency' and 'preference' of twenty-two common high-protein, high-carbohydrate and high-fat food items. The taste threshold (and therefore sensitivity) of MSG was lowered from 0.33 (sem 0.24) to 0.26 (sem 0.22) % MSG when 0.25 % (w/w) IMP-5 was added. None of the sensory properties assessed was associated with the taste threshold of MSG +/- 0.25 % IMP-5 in the overall study population. However, the taste descriptor 'meatiness' was associated with the threshold data for individuals who could taste concentrations of protein were found to be related to the threshold of MSG +/- 0.25 % IMP-5. From this study population we conclude that the taste threshold of MSG in combination with IMP-5 does appear to predict one's 'liking' of as well as 'preference' for high-protein foods.

  9. Carotenoid crystal formation in Arabidopsis and carrot roots caused by increased phytoene synthase protein levels.

    Directory of Open Access Journals (Sweden)

    Dirk Maass

    Full Text Available BACKGROUND: As the first pathway-specific enzyme in carotenoid biosynthesis, phytoene synthase (PSY is a prime regulatory target. This includes a number of biotechnological approaches that have successfully increased the carotenoid content in agronomically relevant non-green plant tissues through tissue-specific PSY overexpression. We investigated the differential effects of constitutive AtPSY overexpression in green and non-green cells of transgenic Arabidopsis lines. This revealed striking similarities to the situation found in orange carrot roots with respect to carotenoid amounts and sequestration mechanism. METHODOLOGY/PRINCIPAL FINDINGS: In Arabidopsis seedlings, carotenoid content remained unaffected by increased AtPSY levels although the protein was almost quantitatively imported into plastids, as shown by western blot analyses. In contrast, non-photosynthetic calli and roots overexpressing AtPSY accumulated carotenoids 10 and 100-fold above the corresponding wild-type tissues and contained 1800 and 500 microg carotenoids per g dry weight, respectively. This increase coincided with a change of the pattern of accumulated carotenoids, as xanthophylls decreased relative to beta-carotene and carotene intermediates accumulated. As shown by polarization microscopy, carotenoids were found deposited in crystals, similar to crystalline-type chromoplasts of non-green tissues present in several other taxa. In fact, orange-colored carrots showed a similar situation with increased PSY protein as well as carotenoid levels and accumulation patterns whereas wild white-rooted carrots were similar to Arabidopsis wild type roots in this respect. Initiation of carotenoid crystal formation by increased PSY protein amounts was further confirmed by overexpressing crtB, a bacterial PSY gene, in white carrots, resulting in increased carotenoid amounts deposited in crystals. CONCLUSIONS: The sequestration of carotenoids into crystals can be driven by the

  10. Age-associated increase of the active zone protein Bruchpilot within the honeybee mushroom body.

    Directory of Open Access Journals (Sweden)

    Katrin B Gehring

    Full Text Available In honeybees, age-associated structural modifications can be observed in the mushroom bodies. Prominent examples are the synaptic complexes (microglomeruli, MG in the mushroom body calyces, which were shown to alter their size and density with age. It is not known whether the amount of intracellular synaptic proteins in the MG is altered as well. The presynaptic protein Bruchpilot (BRP is localized at active zones and is involved in regulating the probability of neurotransmitter release in the fruit fly, Drosophila melanogaster. Here, we explored the localization of the honeybee BRP (Apis mellifera BRP, AmBRP in the bee brain and examined age-related changes in the AmBRP abundance in the central bee brain and in microglomeruli of the mushroom body calyces. We report predominant AmBRP localization near the membrane of presynaptic boutons within the mushroom body MG. The relative amount of AmBRP was increased in the central brain of two-week old bees whereas the amount of Synapsin, another presynaptic protein involved in the regulation of neurotransmitter release, shows an increase during the first two weeks followed by a decrease. In addition, we demonstrate an age-associated modulation of AmBRP located near the membrane of presynaptic boutons within MG located in mushroom body calyces where sensory input is conveyed to mushroom body intrinsic neurons. We discuss that the observed age-associated AmBRP modulation might be related to maturation processes or to homeostatic mechanisms that might help to maintain synaptic functionality in old animals.

  11. Epithelial Cell Damage Activates Bactericidal/Permeability Increasing-Protein (BPI Expression in Intestinal Epithelium

    Directory of Open Access Journals (Sweden)

    Arjun Balakrishnan

    2017-08-01

    Full Text Available As the first line of defense against invading pathogen, intestinal epithelium produces various antimicrobial proteins (AMP that help in clearance of pathogen. Bactericidal/permeability-increasing protein (BPI is a 55 kDa AMP that is expressed in intestinal epithelium. Dysregulation of BPI in intestinal epithelium is associated with various inflammatory diseases like Crohn’s Disease, Ulcerative colitis, and Infectious enteritis’s. In this paper, we report a direct correlation between intestinal damage and BPI expression. In Caco-2 cells, we see a significant increase in BPI levels upon membrane damage mediated by S. aureus infection and pore-forming toxins (Streptolysin and Listeriolysin. Cells detect changes in potassium level as a Danger-associated molecular pattern associated with cell damage and induce BPI expression in a p38 dependent manner. These results are further supported by in vivo findings that the BPI expression in murine intestinal epithelium is induced upon infection with bacteria which cause intestinal damage (Salmonella Typhimurium and Shigella flexneri whereas mutants that do not cause intestinal damage (STM ΔfliC and STM ΔinvC did not induce BPI expression. Our results suggest that epithelial damage associated with infection act as a signal to induce BPI expression.

  12. Oral nutritional supplementation increases caloric and protein intake in peritoneal dialysis patients.

    Science.gov (United States)

    Boudville, Neil; Rangan, Anna; Moody, Harry

    2003-03-01

    Malnutrition is highly prevalent in peritoneal dialysis (PD) patients and is associated with a poor prognosis. Attempts to improve nutritional status with enteral supplements have yielded poor results. We performed a crossover-design trial on 13 PD patients to investigate whether these patients reduce their food intake after drinking oral nutritional supplements. Patients attended three visits in which they were administered a standard oral nutritional supplement either 2 hours or 30 minutes before lunch or a placebo drink 30 minutes before lunch. Lunch was provided as a self-select buffet-style meal, and food intake was measured. Total intake was calculated by adding the nutritional content of the oral supplement. Patients showed poor food intake, with mean values equaling only 18% of the recommended daily intake for calories and 34% for protein. Drinking the supplement 2 hours before lunch resulted in a significant increase compared with the placebo visit in total caloric (430 to 843 kcal; P lunch. These results indicate that oral nutritional supplements administered before a meal may significantly increase caloric and protein intakes of PD patients. Copyright 2003 by the National Kidney Foundation, Inc.

  13. Increasing levels of crude protein in multiple supplements for grazing beef heifers in rainy season

    Directory of Open Access Journals (Sweden)

    Lívia Vieira de Barros

    2015-06-01

    Full Text Available The objective was to evaluate the effect of multiple supplements with differents levels of crude protein (CP or mineral supplements on the nutritional parameters and performance of beef heifers grazing Uruchloa decumbens in the rainy season. A complete random design was employed. The treatments were made up of increasing levels of CP in the multiple supplements and a control treatment (MM in which animals were offered only mineral mixture. Multiple supplements contained 17; 30; 43 and 56% of CP, for treatments CP17; CP30; CP43 and CP56, respectively. Average daily gain (ADG (g was 447.7; 554.6; 638.4; 587.9; 590.4, for treatments MM, CP17; CP30; CP43 and CP56, respectively. A quadratic effect of the levels of crude protein was found (p< 0.10 on ADG. A greater intake of dry matter (DM, organic matter (OM, CP, ether extract (EE, non-fibrous carbohydrates (NFC, total digestible nutrients (TDN, and digested dry matter (p< 0.10 was found in animals supplemented with multiple supplements. Multiple supplements increased the apparent digestibility coefficient of DM, CP, EE and NFC. Supply of multiple multiple supplements for heifers grazing in medium to high quality pastures in the rainy season improves the performance of the animals.

  14. Universal method for protein bioconjugation with nanocellulose scaffolds for increased cell adhesion.

    Science.gov (United States)

    Kuzmenko, Volodymyr; Sämfors, Sanna; Hägg, Daniel; Gatenholm, Paul

    2013-12-01

    Bacterial nanocellulose (BNC) is an emerging biomaterial since it is biocompatible, integrates well with host tissue and can be biosynthesized in desired architecture. However, being a hydrogel, it exhibits low affinity for cell attachment, which is crucial for the cellular fate process. To increase cell attachment, the surface of BNC scaffolds was modified with two proteins, fibronectin and collagen type I, using an effective bioconjugation method applying 1-cyano-4-dimethylaminopyridinium (CDAP) tetrafluoroborate as the intermediate catalytic agent. The effect of CDAP treatment on cell adhesion to the BNC surface is shown for human umbilical vein endothelial cells and the mouse mesenchymal stem cell line C3H10T1/2. In both cases, the surface modification increased the number of cells attached to the surfaces. In addition, the morphology of the cells indicated more healthy and viable cells. CDAP activation of bacterial nanocellulose is shown to be a convenient method to conjugate extracellular proteins to the scaffold surfaces. CDAP treatment can be performed in a short period of time in an aqueous environment under heterogeneous and mild conditions preserving the nanofibrillar network of cellulose. © 2013.

  15. Expression of HIV gp120 protein increases sensitivity to the rewarding properties of methamphetamine in mice

    Science.gov (United States)

    Kesby, James P.; Hubbard, David T.; Markou, Athina; Semenova, Svetlana

    2012-01-01

    Methamphetamine abuse and human immunodeficiency virus (HIV) infection induce neuropathological changes in corticolimbic brain areas involved in reward and cognitive function. Little is known about the combined effects of methamphetamine and HIV infection on cognitive and reward processes. The HIV/gp120 protein induces neurodegeneration in mice, similar to HIV-induced pathology in humans. We investigated the effects of gp120 expression on associative learning, preference for methamphetamine and non-drug reinforcers, and sensitivity to the conditioned rewarding properties of methamphetamine in transgenic (tg) mice expressing HIV/gp120 protein (gp120-tg). gp120-tg mice learned the operant response for food at the same rate as non-tg mice. In the two-bottle choice procedure with restricted access to drugs, gp120-tg mice exhibited greater preference for methamphetamine and saccharin than non-tg mice, whereas preference for quinine was similar between genotypes. Under conditions of unrestricted access to methamphetamine, the mice exhibited a decreased preference for increasing methamphetamine concentrations. However, male gp120-tg mice showed a decreased preference for methamphetamine at lower concentrations than non-tg male mice. gp120-tg mice developed methamphetamine-induced conditioned place preference at lower methamphetamine doses compared with non-tg mice. No differences in methamphetamine pharmacokinetics were found between genotypes. These results indicate that gp120-tg mice exhibit no deficits in associative learning or reward/motivational function for a natural reinforcer. Interestingly, gp120 expression resulted in increased preference for methamphetamine and a highly palatable non-drug reinforcer (saccharin) and increased sensitivity to methamphetamine-induced conditioned reward. These data suggest that HIV-positive individuals may have increased sensitivity to methamphetamine, leading to high methamphetamine abuse potential in this population. PMID

  16. Energy dense, protein restricted diet increases adiposity and perturbs metabolism in young, genetically lean pigs.

    Directory of Open Access Journals (Sweden)

    Kimberly D Fisher

    Full Text Available Animal models of obesity and metabolic dysregulation during growth (or childhood are lacking. Our objective was to increase adiposity and induce metabolic syndrome in young, genetically lean pigs. Pre-pubertal female pigs, age 35 d, were fed a high-energy diet (HED; n = 12, containing 15% tallow, 35% refined sugars and 9.1-12.9% crude protein, or a control corn-based diet (n = 11 with 12.2-19.2% crude protein for 16 wk. Initially, HED pigs self-regulated energy intake similar to controls, but by wk 5, consumed more (P<0.001 energy per kg body weight. At wk 15, pigs were subjected to an oral glucose tolerance test (OGTT; blood glucose increased (P<0.05 in control pigs and returned to baseline levels within 60 min. HED pigs were hyperglycemic at time 0, and blood glucose did not return to baseline (P = 0.01, even 4 h post-challenge. During OGTT, glucose area under the curve (AUC was higher and insulin AUC was lower in HED pigs compared to controls (P = 0.001. Chronic HED intake increased (P<0.05 subcutaneous, intramuscular, and perirenal fat deposition, and induced hyperglycemia, hypoinsulinemia, and low-density lipoprotein hypercholesterolemia. A subset of HED pigs (n = 7 was transitioned back to a control diet for an additional six weeks. These pigs were subjected to an additional OGTT at 22 wk. Glucose AUC and insulin AUC did not improve, supporting that dietary intervention was not sufficient to recover glucose tolerance or insulin production. These data suggest a HED may be used to increase adiposity and disrupt glucose homeostasis in young, growing pigs.

  17. Increased bone morphogenetic protein signaling contributes to age-related declines in neurogenesis and cognition.

    Science.gov (United States)

    Meyers, Emily A; Gobeske, Kevin T; Bond, Allison M; Jarrett, Jennifer C; Peng, Chian-Yu; Kessler, John A

    2016-02-01

    Aging is associated with decreased neurogenesis in the hippocampus and diminished hippocampus-dependent cognitive functions. Expression of bone morphogenetic protein 4 (BMP4) increases with age by more than 10-fold in the mouse dentate gyrus while levels of the BMP inhibitor, noggin, decrease. This results in a profound 30-fold increase in phosphorylated-SMAD1/5/8, the effector of canonical BMP signaling. Just as observed in mice, a profound increase in expression of BMP4 is observed in the dentate gyrus of humans with no known cognitive abnormalities. Inhibition of BMP signaling either by overexpression of noggin or transgenic manipulation not only increases neurogenesis in aging mice, but remarkably, is associated with a rescue of cognitive deficits to levels comparable to young mice. Additive benefits are observed when combining inhibition of BMP signaling and environmental enrichment. These findings indicate that increased BMP signaling contributes significantly to impairments in neurogenesis and to cognitive decline associated with aging, and identify this pathway as a potential druggable target for reversing age-related changes in cognition. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. The molecular mechanism of mediation of adsorbed serum proteins to endothelial cells adhesion and growth on biomaterials.

    Science.gov (United States)

    Yang, Dayun; Lü, Xiaoying; Hong, Ying; Xi, Tingfei; Zhang, Deyuan

    2013-07-01

    To explore molecular mechanism of mediation of adsorbed proteins to cell adhesion and growth on biomaterials, this study examined endothelial cell adhesion, morphology and viability on bare and titanium nitride (TiN) coated nickel titanium (NiTi) alloys and chitosan film firstly, and then identified the type and amount of serum proteins adsorbed on the three surfaces by proteomic technology. Subsequently, the mediation role of the identified proteins to cell adhesion and growth was investigated with bioinformatics analyses, and further confirmed by a series of cellular and molecular biological experiments. Results showed that the type and amount of adsorbed serum proteins associated with cell adhesion and growth was obviously higher on the alloys than on the chitosan film, and these proteins mediated endothelial cell adhesion and growth on the alloys via four ways. First, proteins such as adiponectin in the adsorbed protein layer bound with cell surface receptors to generate signal transduction, which activated cell surface integrins through increasing intracellular calcium level. Another way, thrombospondin 1 in the adsorbed protein layer promoted TGF-β signaling pathway activation and enhanced integrins expression. The third, RGD sequence containing proteins such as fibronectin 1, vitronectin and thrombospondin 1 in the adsorbed protein layer bound with activated integrins to activate focal adhesion pathway, increased focal adhesion formation and actin cytoskeleton organization and mediated cell adhesion and spreading. In addition, the activated focal adhesion pathway promoted the expression of cell growth related genes and resulted in cell proliferation. The fourth route, coagulation factor II (F2) and fibronectin 1 in the adsorbed protein layer bound with cell surface F2 receptor and integrin, activated regulation of actin cytoskeleton pathway and regulated actin cytoskeleton organization. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. MECHANISMS IN ENDOCRINOLOGY: Exogenous insulin does not increase muscle protein synthesis rate when administered systemically: a systematic review.

    Science.gov (United States)

    Trommelen, Jorn; Groen, Bart B L; Hamer, Henrike M; de Groot, Lisette C P G M; van Loon, Luc J C

    2015-07-01

    Though it is well appreciated that insulin plays an important role in the regulation of muscle protein metabolism, there is much discrepancy in the literature on the capacity of exogenous insulin administration to increase muscle protein synthesis rates in vivo in humans. To assess whether exogenous insulin administration increases muscle protein synthesis rates in young and older adults. A systematic review of clinical trials was performed and the presence or absence of an increase in muscle protein synthesis rate was reported for each individual study arm. In a stepwise manner, multiple models were constructed that excluded study arms based on the following conditions: model 1, concurrent hyperaminoacidemia; model 2, insulin-induced hypoaminoacidemia; model 3, supraphysiological insulin concentrations; and model 4, older, more insulin resistant, subjects. From the presented data in the current systematic review, we conclude that: i) exogenous insulin and amino acid administration effectively increase muscle protein synthesis, but this effect is attributed to the hyperaminoacidemia; ii) exogenous insulin administered systemically induces hypoaminoacidemia which obviates any insulin-stimulatory effect on muscle protein synthesis; iii) exogenous insulin resulting in supraphysiological insulin levels exceeding 50, 000  pmol/l may effectively augment muscle protein synthesis; iv) exogenous insulin may have a diminished effect on muscle protein synthesis in older adults due to age-related anabolic resistance; and v) exogenous insulin administered systemically does not increase muscle protein synthesis in healthy, young adults. © 2015 European Society of Endocrinology.

  20. Dynamic Contact Angle Analysis of Protein Adsorption on Polysaccharide Multilayer’s Films for Biomaterial Reendothelialization

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    Safiya Benni

    2014-01-01

    Full Text Available Atherosclerosis is a major cardiovascular disease. One of the side effects is restenosis. The aim of this work was to study the coating of stents by dextran derivates based polyelectrolyte’s multilayer (PEM films in order to increase endothelialization of injured arterial wall after stent implantation. Films were composed with diethylaminoethyl dextran (DEAE as polycation and dextran sulphate (DS as polyanion. One film was composed with 4 bilayers of (DEAE-DS4 and was labeled D−. The other film was the same as D− but with an added terminal layer of DEAE polycation: (DEAE-DS4-DEAE (labeled D+. The dynamic adsorption/desorption of proteins on the films were characterized by dynamic contact angle (DCA and atomic force microscopy (AFM. Human endothelial cell (HUVEC adhesion and proliferation were quantified and correlated to protein adsorption analyzed by DCA for fibronectin, vitronectin, and bovine serum albumin (BSA. Our results showed that the endothelial cell response was optimal for films composed of DS as external layer. Fibronectin was found to be the only protein to exhibit a reversible change in conformation after desorption test. This behavior was only observed for (DEAE-DS4 films. (DEAE-DS4 films could enhance HUVEC proliferation in agreement with fibronectin ability to easily change from conformation.

  1. Soy-dairy protein blend and whey protein ingestion after resistance exercise increases amino acid transport and transporter expression in human skeletal muscle

    Science.gov (United States)

    Reidy, P. T.; Walker, D. K.; Dickinson, J. M.; Gundermann, D. M.; Drummond, M. J.; Timmerman, K. L.; Cope, M. B.; Mukherjea, R.; Jennings, K.; Volpi, E.

    2014-01-01

    Increasing amino acid availability (via infusion or ingestion) at rest or postexercise enhances amino acid transport into human skeletal muscle. It is unknown whether alterations in amino acid availability, from ingesting different dietary proteins, can enhance amino acid transport rates and amino acid transporter (AAT) mRNA expression. We hypothesized that the prolonged hyperaminoacidemia from ingesting a blend of proteins with different digestion rates postexercise would enhance amino acid transport into muscle and AAT expression compared with the ingestion of a rapidly digested protein. In a double-blind, randomized clinical trial, we studied 16 young adults at rest and after acute resistance exercise coupled with postexercise (1 h) ingestion of either a (soy-dairy) protein blend or whey protein. Phenylalanine net balance and transport rate into skeletal muscle were measured using stable isotopic methods in combination with femoral arteriovenous blood sampling and muscle biopsies obtained at rest and 3 and 5 h postexercise. Phenylalanine transport into muscle and mRNA expression of select AATs [system L amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, system A amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, cationic amino acid transporter 1/SLC7A1] increased to a similar extent in both groups (P protein blend resulted in a prolonged and positive net phenylalanine balance during postexercise recovery compared with whey protein (P protein synthesis increased similarly between groups. We conclude that, while both protein sources enhanced postexercise AAT expression, transport into muscle, and myofibrillar protein synthesis, postexercise ingestion of a protein blend results in a slightly prolonged net amino acid balance across the leg compared with whey protein. PMID:24699854

  2. Low carbohydrate, high fat diet increases C-reactive protein during weight loss.

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    Rankin, Janet W; Turpyn, Abigail D

    2007-04-01

    Chronic inflammation is associated with elevated risk of heart disease and may be linked to oxidative stress in obesity. Our objective was to evaluate the effect of weight loss diet composition (low carbohydrate, high fat, LC or high carbohydrate, low fat, HC) on inflammation and to determine whether this was related to oxidative stress. Twenty nine overweight women, BMI 32.1 +/- 5.4 kg/m(2), were randomly assigned to a self-selected LC or HC diet for 4 wks. Weekly group sessions and diet record collections helped enhance compliance. Body weight, markers of inflammation (serum interleukin-6, IL-6; C-reactive protein, CRP) oxidative stress (urinary 8-epi-prostaglandin F2alpha, 8-epi) and fasting blood glucose and free fatty acids were measured weekly. The diets were similar in caloric intake (1357 kcal/d LC vs. 1361 HC, p=0.94), but differed in macronutrients (58, 12, 30 and 24, 59, 18 for percent of energy as fat, carbohydrate, and protein for LC and HC, respectively). Although LC lost more weight (3.8 +/- 1.2 kg LC vs. 2.6 +/- 1.7 HC, p=0.04), CRP increased 25%; this factor was reduced 43% in HC (p=0.02). For both groups, glucose decreased with weight loss (85.4 vs. 82.1 mg/dl for baseline and wk 4, p<0.01), while IL-6 increased (1.39 to 1.62 pg/mL, p=0.04). Urinary 8-epi varied differently over time between groups (p<0.05) with no consistent pattern. Diet composition of the weight loss diet influenced a key marker of inflammation in that LC increased while HC reduced serum CRP but evidence did not support that this was related to oxidative stress.

  3. Lack of Plasma Protein Hemopexin Results in Increased Duodenal Iron Uptake.

    Science.gov (United States)

    Fiorito, Veronica; Geninatti Crich, Simonetta; Silengo, Lorenzo; Aime, Silvio; Altruda, Fiorella; Tolosano, Emanuela

    2013-01-01

    The body concentration of iron is regulated by a fine equilibrium between absorption and losses of iron. Iron can be absorbed from diet as inorganic iron or as heme. Hemopexin is an acute phase protein that limits iron access to microorganisms. Moreover, it is the plasma protein with the highest binding affinity for heme and thus it mediates heme-iron recycling. Considering its involvement in iron homeostasis, it was postulated that hemopexin may play a role in the physiological absorption of inorganic iron. Hemopexin-null mice showed elevated iron deposits in enterocytes, associated with higher duodenal H-Ferritin levels and a significant increase in duodenal expression and activity of heme oxygenase. The expression of heme-iron and inorganic iron transporters was normal. The rate of iron absorption was assessed by measuring the amount of (57)Fe retained in tissues from hemopexin-null and wild-type animals after administration of an oral dose of (57)FeSO4 or of (57)Fe-labelled heme. Higher iron retention in the duodenum of hemopexin-null mice was observed as compared with normal mice. Conversely, iron transfer from enterocytes to liver and bone marrow was unaffected in hemopexin-null mice. The increased iron level in hemopexin-null duodenum can be accounted for by an increased iron uptake by enterocytes and storage in ferritins. These data indicate that the lack of hemopexin under physiological conditions leads to an enhanced duodenal iron uptake thus providing new insights to our understanding of body iron homeostasis.

  4. Zinc finger protein ZBTB20 expression is increased in hepatocellular carcinoma and associated with poor prognosis

    International Nuclear Information System (INIS)

    Wang, Qing; Wang, Hong-yang; Tan, Ye-xiong; Ren, Yi-bin; Dong, Li-wei; Xie, Zhi-fang; Tang, Liang; Cao, Dan; Zhang, Wei-ping; Hu, He-ping

    2011-01-01

    Our previous studies showed that ZBTB20, a new BTB/POZ-domain gene, could negatively regulate α feto-protein and other liver-specific genes, concerning such as bio-transformation, glucose metabolism and the regulation of the somatotropic hormonal axis. The aim of this study is to determine the potential clinical implications of ZBTB20 in hepatocellular carcinoma (HCC). Quantitative real-time RT-PCR and Western blot analyses were used to detect expression levels of ZBTB20 in 50 paired HCC tumorous and nontumorous tissues and in 20 normal liver tissues. Moreover, expression of ZBTB20 was assessed by immunohistochemistry of paired tumor and peritumoral liver tissue from 102 patients who had undergone hepatectomy for histologically proven HCC. And its relationship with clinicopathological parameters and prognosis was investigated. Both messenger RNA and protein expression levels of ZBTB20 were elevated significantly in HCC tissues compared with the paired non-tumor tissues and normal liver tissues. Overexpressed ZBTB20 protein in HCC was significantly associated with vein invasion (P = 0.016). Importantly, the recurrence or metastasis rates of HCCs with higher ZBTB20 expression were markedly greater than those of HCCs with lower expression (P = 0.003, P = 0.00015, respectively). Univariate and multivariate analyses revealed that ZBTB20 overexpression was an independent prognostic factor for HCC. The disease-free survival period and over-all survival period in patients with overexpressed ZBTB20 in HCC was significantly reduced. The expression of ZBTB20 is increased in HCC and associated with poor prognosis in patients with HCC, implicating ZBTB20 as a candidate prognostic marker in HCC

  5. Intrauterine Growth Restriction Increases TNFα and Activates the Unfolded Protein Response in Male Rat Pups

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    Emily S. Riddle

    2014-01-01

    Full Text Available Intrauterine growth restriction (IUGR programs adult disease, including obesity and insulin resistance. Our group previously demonstrated that IUGR dysregulates adipose deposition in male, but not female, weanling rats. Dysregulated adipose deposition is often accompanied by the release of proinflammatory signaling molecules, such as tumor necrosis factor alpha (TNFα. TNFα contributes to adipocyte inflammation and impaired insulin signaling. TNFα has also been implicated in the activation of the unfolded protein response (UPR, which impairs insulin signaling. We hypothesized that, in male rat pups, IUGR would increase TNFα, TNFR1, and components of the UPR (Hspa5, ATF6, p-eIF2α, and Ddit3 prior to the onset of obesity. We further hypothesized that impaired glucose tolerance would occur after the onset of adipose dysfunction in male IUGR rats. To test this hypothesis, we used a well-characterized rat model of uteroplacental insufficiency-induced IUGR. Our primary findings are that, in male rats, IUGR (1 increased circulating and adipose TNFα, (2 increased mRNA levels of UPR components as well as p-eIF2a, and (3 impaired glucose tolerance after observed TNFα increased and after UPR activation. We speculate that programmed dysregulation of TNFα and UPR contributed to the development of glucose intolerance in male IUGR rats.

  6. Potential of beta-adrenergic agonists for increasing protein deposition in ruminants in developing countries

    International Nuclear Information System (INIS)

    Berschauer, F.

    1989-01-01

    Various substituted phenylethanolamines, acting on the sympathetic nervous system, have been shown to increase protein retention (via decreased proteolysis) and reduce fat deposition (via increased lipolysis and reduced lipogenesis) in ruminants and monogastrics. Research with finishing lambs in developed countries show various beta-adrenergic agonists to improve growth rate (by 18%), feed conversion (by 12%) and carcass quality (28% increase in area of longissimus dorsi and 33% reduction in subcutaneous fat). Similar effects of beta-agonists on carcass composition of well fed cattle have been reported. The effects of beta-agonists on livestock in developing countries of the tropics have not yet been investigated, but their effects in increasing metabolic rate suggest that treated ruminants would be more vulnerable to hot environments. Beta-agonists appear to improve nitrogen retention to a greater extent in breeds with a lower potential for muscle growth. In view of this, they might be particularly effective in improving nitrogen retention in tropical breeds which have a low growth potential. This aspect, together with the response of undernourished animals in the developing countries, needs investigation. Beta-adrenergic agonists are not yet registered for use in animal production, but product licenses for some of them are expected to be granted soon. (author). 31 refs, 1 fig., 12 tabs

  7. Increased CSF levels of phosphorylated neurofilament heavy protein following bout in amateur boxers.

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    Sanna Neselius

    Full Text Available INTRODUCTION: Diagnosis of mild TBI is hampered by the lack of imaging or biochemical measurements for identifying or quantifying mild TBI in a clinical setting. We have previously shown increased biomarker levels of protein reflecting axonal (neurofilament light protein and tau and glial (GFAP and S-100B damage in cerebrospinal fluid (CSF after a boxing bout. The aims of this study were to find other biomarkers of mild TBI, which may help clinicians diagnose and monitor mild TBI, and to calculate the role of APOE ε4 allele genotype which has been associated with poor outcome after TBI. MATERIALS AND METHODS: Thirty amateur boxers with a minimum of 45 bouts and 25 non-boxing matched controls were included in a prospective cohort study. CSF and blood were collected at one occasion between 1 and 6 days after a bout, and after a rest period for at least 14 days (follow up. The controls were tested once. CSF levels of neurofilament heavy (pNFH, amyloid precursor proteins (sAPPα and sAPPβ, ApoE and ApoA1 were analyzed. In blood, plasma levels of Aβ42 and ApoE genotype were analyzed. RESULTS: CSF levels of pNFH were significantly increased between 1 and 6 days after boxing as compared with controls (p<0.001. The concentrations decreased at follow up but were still significantly increased compared to controls (p = 0.018. CSF pNFH concentrations correlated with NFL (r =  0.57 after bout and 0.64 at follow up, p<0.001. No significant change was found in the other biomarkers, as compared to controls. Boxers carrying the APOE ε4 allele had similar biomarker concentrations as non-carriers. CONCLUSIONS: Subconcussive repetitive trauma in amateur boxing causes a mild TBI that may be diagnosed by CSF analysis of pNFH, even without unconsciousness or concussion symptoms. Possession of the APOE ε4 allele was not found to influence biomarker levels after acute TBI.

  8. Protein-enhanced soups: a consumer-accepted food for increasing dietary protein provision among older adults.

    Science.gov (United States)

    Donahue, Elizabeth; Crowe, Kristi Michele; Lawrence, Jeannine

    2015-02-01

    Protein-enhanced soups (PES) may improve protein intake among older adults. This study examined sensory attributes (aroma, texture, taste, and overall acceptability) and preferences of PES (chicken noodle and cheddar broccoli) compared with flavor-matched control soups (FCS) among older adults (≥65 years) and evaluated dietary profile changes of a standard menu based on the substitution of one PES serving/d for a standard soup. Modified paired preference tests and 5-point facial hedonic scales were administered to participants (n = 44). No significant differences in sensory attributes between either PES compared with FCS were identified, but significant gender- and age-related differences (p preferred protein-enhanced chicken noodle soup while only 38% preferred protein-enhanced cheddar broccoli soup to their respective FCS. Substituting one PES serving for one non-fortified soup serving per day resulted in significantly higher (p < 0.001) protein profile. Results suggest that all attributes of PES were consistent with sensory expectations and PES substitution could improve protein provision.

  9. Nicotinic Acid Increases Adiponectin Secretion from Differentiated Bovine Preadipocytes through G-Protein Coupled Receptor Signaling

    Directory of Open Access Journals (Sweden)

    Christina Kopp

    2014-11-01

    Full Text Available The transition period in dairy cows (3 weeks prepartum until 3 weeks postpartum is associated with substantial mobilization of energy stores, which is often associated with metabolic diseases. Nicotinic acid (NA is an antilipolytic and lipid-lowering compound used to treat dyslipidaemia in humans, and it also reduces non-esterified fatty acids in cattle. In mice the G-protein coupled receptor 109A (GPR109A ligand NA positively affects the secretion of adiponectin, an important modulator of glucose and fat metabolism. In cattle, the corresponding data linking NA to adiponectin are missing. Our objective was to examine the effects of NA on adiponectin and AMPK protein abundance and the expression of mRNAs of related genes such as chemerin, an adipokine that enhances adiponectin secretion in vitro. Differentiated bovine adipocytes were incubated with pertussis toxin (PTX to verify the involvement of GPR signaling, and treated with 10 or 15 µM NA for 12 or 24 h. NA increased adiponectin concentrations (p ≤ 0.001 and the mRNA abundances of GPR109A (p ≤ 0.05 and chemerin (p ≤ 0.01. Pre-incubation with PTX reduced the adiponectin response to NA (p ≤ 0.001. The NA-stimulated secretion of adiponectin and the mRNA expression of chemerin in the bovine adipocytes were suggestive of GPR signaling-dependent improved insulin sensitivity and/or adipocyte metabolism in dairy cows.

  10. Nonuniform Internal Structure of Fibrin Fibers: Protein Density and Bond Density Strongly Decrease with Increasing Diameter

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    Wei Li

    2017-01-01

    Full Text Available The major structural component of a blood clot is a meshwork of fibrin fibers. It has long been thought that the internal structure of fibrin fibers is homogeneous; that is, the protein density and the bond density between protofibrils are uniform and do not depend on fiber diameter. We performed experiments to investigate the internal structure of fibrin fibers. We formed fibrin fibers with fluorescently labeled fibrinogen and determined the light intensity of a fiber, I, as a function of fiber diameter, D. The intensity and, thus, the total number of fibrin molecules in a cross-section scaled as D1.4. This means that the protein density (fibrin per cross-sectional area, ρp, is not homogeneous but instead strongly decreases with fiber diameter as D-0.6. Thinner fibers are denser than thicker fibers. We also determined Young’s modulus, Y, as a function of fiber diameter. Y decreased strongly with increasing D; Y scaled as D-1.5. This implies that the bond density, ρb, also scales as D-1.5. Thinner fibers are stiffer than thicker fibers. Our data suggest that fibrin fibers have a dense, well-connected core and a sparse, loosely connected periphery. In contrast, electrospun fibrinogen fibers, used as a control, have a homogeneous cross-section.

  11. Role of Hypothalamic Melanocortin System in Adaptation of Food Intake to Food Protein Increase in Mice

    Science.gov (United States)

    Pillot, Bruno; Duraffourd, Céline; Bégeot, Martine; Joly, Aurélie; Luquet, Serge; Houberdon, Isabelle; Naville, Danielle; Vigier, Michèle; Gautier-Stein, Amandine; Magnan, Christophe; Mithieux, Gilles

    2011-01-01

    The hypothalamic melanocortin system—the melanocortin receptor of type 4 (MC4R) and its ligands: α-melanin-stimulating hormone (α-MSH, agonist, inducing hypophagia), and agouti-related protein (AgRP, antagonist, inducing hyperphagia)—is considered to play a central role in the control of food intake. We tested its implication in the mediation of the hunger-curbing effects of protein-enriched diets (PED) in mice. Whereas there was a 20% decrease in food intake in mice fed on the PED, compared to mice fed on an isocaloric starch-enriched diet, there was a paradoxical decrease in expression of the hypothalamic proopiomelanocortin gene, precursor of α-MSH, and increase in expression of the gene encoding AgRP. The hypophagia effect of PED took place in mice with invalidation of either MC4R or POMC, and was even strengthened in mice with ablation of the AgRP-expressing neurons. These data strongly suggest that the hypothalamic melanocortin system does not mediate the hunger-curbing effects induced by changes in the macronutrient composition of food. Rather, the role of this system might be to defend the body against the variations in food intake generated by the nutritional environment. PMID:21544212

  12. Role of hypothalamic melanocortin system in adaptation of food intake to food protein increase in mice.

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    Bruno Pillot

    Full Text Available The hypothalamic melanocortin system--the melanocortin receptor of type 4 (MC4R and its ligands: α-melanin-stimulating hormone (α-MSH, agonist, inducing hypophagia, and agouti-related protein (AgRP, antagonist, inducing hyperphagia--is considered to play a central role in the control of food intake. We tested its implication in the mediation of the hunger-curbing effects of protein-enriched diets (PED in mice. Whereas there was a 20% decrease in food intake in mice fed on the PED, compared to mice fed on an isocaloric starch-enriched diet, there was a paradoxical decrease in expression of the hypothalamic proopiomelanocortin gene, precursor of α-MSH, and increase in expression of the gene encoding AgRP. The hypophagia effect of PED took place in mice with invalidation of either MC4R or POMC, and was even strengthened in mice with ablation of the AgRP-expressing neurons. These data strongly suggest that the hypothalamic melanocortin system does not mediate the hunger-curbing effects induced by changes in the macronutrient composition of food. Rather, the role of this system might be to defend the body against the variations in food intake generated by the nutritional environment.

  13. Hunting Increases Phosphorylation of Calcium/Calmodulin-Dependent Protein Kinase Type II in Adult Barn Owls

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    Grant S. Nichols

    2015-01-01

    Full Text Available Juvenile barn owls readily adapt to prismatic spectacles, whereas adult owls living under standard aviary conditions do not. We previously demonstrated that phosphorylation of the cyclic-AMP response element-binding protein (CREB provides a readout of the instructive signals that guide plasticity in juveniles. Here we investigated phosphorylation of calcium/calmodulin-dependent protein kinase II (pCaMKII in both juveniles and adults. In contrast to CREB, we found no differences in pCaMKII expression between prism-wearing and control juveniles within the external nucleus of the inferior colliculus (ICX, the major site of plasticity. For prism-wearing adults that hunted live mice and are capable of adaptation, expression of pCaMKII was increased relative to prism-wearing adults that fed passively on dead mice and are not capable of adaptation. This effect did not bear the hallmarks of instructive information: it was not localized to rostral ICX and did not exhibit a patchy distribution reflecting discrete bimodal stimuli. These data are consistent with a role for CaMKII as a permissive rather than an instructive factor. In addition, the paucity of pCaMKII expression in passively fed adults suggests that the permissive default setting is “off” in adults.

  14. Aerobic Exercise Training Adaptations Are Increased by Postexercise Carbohydrate-Protein Supplementation

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    Lisa Ferguson-Stegall

    2011-01-01

    Full Text Available Carbohydrate-protein supplementation has been found to increase the rate of training adaptation when provided postresistance exercise. The present study compared the effects of a carbohydrate and protein supplement in the form of chocolate milk (CM, isocaloric carbohydrate (CHO, and placebo on training adaptations occurring over 4.5 weeks of aerobic exercise training. Thirty-two untrained subjects cycled 60 min/d, 5 d/wk for 4.5 wks at 75–80% of maximal oxygen consumption (VO2 max. Supplements were ingested immediately and 1 h after each exercise session. VO2 max and body composition were assessed before the start and end of training. VO2 max improvements were significantly greater in CM than CHO and placebo. Greater improvements in body composition, represented by a calculated lean and fat mass differential for whole body and trunk, were found in the CM group compared to CHO. We conclude supplementing with CM postexercise improves aerobic power and body composition more effectively than CHO alone.

  15. Membrane proteins involved in transport, vesicle traffic and Ca(2+) signaling increase in beetroots grown in saline soils.

    Science.gov (United States)

    Lino, Bárbara; Chagolla, Alicia; E González de la Vara, Luis

    2016-07-01

    By separating plasma membrane proteins according to their hydropathy from beetroots grown in saline soils, several proteins probably involved in salt tolerance were identified by mass spectrometry. Beetroots, as a salt-tolerant crop, have developed mechanisms to cope with stresses associated with saline soils. To observe which plasma membrane (PM) proteins were more abundant in beet roots grown in saline soils, beet root plants were irrigated with water or 0.2 M NaCl. PM-enriched membrane preparations were obtained from these plants, and their proteins were separated according to their hydropathy by serial phase partitioning with Triton X-114. Some proteins whose abundance increased visibly in membranes from salt-grown beetroots were identified by mass spectrometry. Among them, there was a V-type H(+)-ATPase (probably from contaminating vacuolar membranes), which increased with salt at all stages of beetroots' development. Proteins involved in solute transport (an H(+)-transporting PPase and annexins), vesicle traffic (clathrin and synaptotagmins), signal perception and transduction (protein kinases and phospholipases, mostly involved in calcium signaling) and metabolism, appeared to increase in salt-grown beetroot PM-enriched membranes. These results suggest that PM and vacuolar proteins involved in transport, metabolism and signal transduction increase in beet roots adapted to saline soils. In addition, these results show that serial phase partitioning with Triton X-114 is a useful method to separate membrane proteins for their identification by mass spectrometry.

  16. Intestine-specific deletion of microsomal triglyceride transfer protein increases mortality in aged mice.

    Science.gov (United States)

    Liang, Zhe; Xie, Yan; Dominguez, Jessica A; Breed, Elise R; Yoseph, Benyam P; Burd, Eileen M; Farris, Alton B; Davidson, Nicholas O; Coopersmith, Craig M

    2014-01-01

    Mice with conditional, intestine-specific deletion of microsomal triglyceride transfer protein (Mttp-IKO) exhibit a complete block in chylomicron assembly together with lipid malabsorption. Young (8-10 week) Mttp-IKO mice have improved survival when subjected to a murine model of Pseudomonas aeruginosa-induced sepsis. However, 80% of deaths in sepsis occur in patients over age 65. The purpose of this study was to determine whether age impacts outcome in Mttp-IKO mice subjected to sepsis. Aged (20-24 months) Mttp-IKO mice and WT mice underwent intratracheal injection with P. aeruginosa. Mice were either sacrificed 24 hours post-operatively for mechanistic studies or followed seven days for survival. In contrast to young septic Mttp-IKO mice, aged septic Mttp-IKO mice had a significantly higher mortality than aged septic WT mice (80% vs. 39%, p = 0.005). Aged septic Mttp-IKO mice exhibited increased gut epithelial apoptosis, increased jejunal Bax/Bcl-2 and Bax/Bcl-XL ratios yet simultaneously demonstrated increased crypt proliferation and villus length. Aged septic Mttp-IKO mice also manifested increased pulmonary myeloperoxidase levels, suggesting increased neutrophil infiltration, as well as decreased systemic TNFα compared to aged septic WT mice. Blocking intestinal chylomicron secretion alters mortality following sepsis in an age-dependent manner. Increases in gut apoptosis and pulmonary neutrophil infiltration, and decreased systemic TNFα represent potential mechanisms for why intestine-specific Mttp deletion is beneficial in young septic mice but harmful in aged mice as each of these parameters are altered differently in young and aged septic WT and Mttp-IKO mice.

  17. Intestine-specific deletion of microsomal triglyceride transfer protein increases mortality in aged mice.

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    Zhe Liang

    Full Text Available Mice with conditional, intestine-specific deletion of microsomal triglyceride transfer protein (Mttp-IKO exhibit a complete block in chylomicron assembly together with lipid malabsorption. Young (8-10 week Mttp-IKO mice have improved survival when subjected to a murine model of Pseudomonas aeruginosa-induced sepsis. However, 80% of deaths in sepsis occur in patients over age 65. The purpose of this study was to determine whether age impacts outcome in Mttp-IKO mice subjected to sepsis.Aged (20-24 months Mttp-IKO mice and WT mice underwent intratracheal injection with P. aeruginosa. Mice were either sacrificed 24 hours post-operatively for mechanistic studies or followed seven days for survival.In contrast to young septic Mttp-IKO mice, aged septic Mttp-IKO mice had a significantly higher mortality than aged septic WT mice (80% vs. 39%, p = 0.005. Aged septic Mttp-IKO mice exhibited increased gut epithelial apoptosis, increased jejunal Bax/Bcl-2 and Bax/Bcl-XL ratios yet simultaneously demonstrated increased crypt proliferation and villus length. Aged septic Mttp-IKO mice also manifested increased pulmonary myeloperoxidase levels, suggesting increased neutrophil infiltration, as well as decreased systemic TNFα compared to aged septic WT mice.Blocking intestinal chylomicron secretion alters mortality following sepsis in an age-dependent manner. Increases in gut apoptosis and pulmonary neutrophil infiltration, and decreased systemic TNFα represent potential mechanisms for why intestine-specific Mttp deletion is beneficial in young septic mice but harmful in aged mice as each of these parameters are altered differently in young and aged septic WT and Mttp-IKO mice.

  18. Health effects of an increased protein intake on kidney function and colorectal cancer risk factors, including the role of animal and plant protein sources – the PREVIEW project

    DEFF Research Database (Denmark)

    Møller, Grith

    intake, including the role of animal and plant protein in pre-diabetic, overweight or obese individuals on health outcomes: markers of kidney function and putative risk factors for colorectal cancer as well as insulin sensitivity and kidney function in healthy individuals. The thesis is based on PREVIEW......, especially plant protein, on insulin sensitivity and kidney function. In paper II, the aim of the study was to assess the effect after one year of a higher protein intake on kidney function, measured by in creatinine clearance. This was investigated in pre-diabetic older adults based on a sub-group of 310...... pre-diabetic individuals included in the PREVIEW RCT. We found that a higher protein intake was associated with a significant increase in urea to creatinine ratio and serum urea after one year. There were no associations between increased protein intake and creatinine clearance, estimated glomerular...

  19. Increased expression of argininosuccinate synthetase protein predicts poor prognosis in human gastric cancer

    Science.gov (United States)

    SHAN, YAN-SHEN; HSU, HUI-PING; LAI, MING-DERG; YEN, MENG-CHI; LUO, YI-PEY; CHEN, YI-LING

    2015-01-01

    Aberrant expression of argininosuccinate synthetase (ASS1, also known as ASS) has been found in cancer cells and is involved in the carcinogenesis of gastric cancer. The aim of the present study was to investigate the level of ASS expression in human gastric cancer and to determine the possible correlations between ASS expression and clinicopathological findings. Immunohistochemistry was performed on paraffin-embedded tissues to determine whether ASS was expressed in 11 of 11 specimens from patients with gastric cancer. The protein was localized primarily to the cytoplasm of cancer cells and normal epithelium. In the Oncomine cancer microarray database, expression of the ASS gene was significantly increased in gastric cancer tissues. To investigate the clinicopathological and prognostic roles of ASS expression, we performed western blot analysis of 35 matched specimens of gastric adenocarcinomas and normal tissue obtained from patients treated at the National Cheng Kung University Hospital. The ratio of relative ASS expression (expressed as the ASS/β-actin ratio) in tumor tissues to that in normal tissues was correlated with large tumor size (P=0.007) and with the tumor, node, metastasis (TNM) stage of the American Joint Committee on Cancer staging system (P=0.031). Patients whose cancer had increased the relative expression of ASS were positive for perineural invasion and had poor recurrence-free survival. In summary, ASS expression in gastric cancer was associated with a poor prognosis. Further study of mechanisms to silence the ASS gene or decrease the enzymatic activity of ASS protein has the potential to provide new treatments for patients with gastric cancer. PMID:25333458

  20. Gamete therapeutics: recombinant protein adsorption by sperm for increasing fertility via artificial insemination.

    Science.gov (United States)

    Alvarez-Gallardo, Horacio; Kjelland, Michael E; Moreno, Juan F; Welsh, Thomas H; Randel, Ronald D; Lammoglia, Miguel A; Pérez-Martínez, Mario; Lara-Sagahón, Alma V; Esperón-Sumano, A Enrique; Romo, Salvador

    2013-01-01

    A decrease in fertility can have a negative economic impact, both locally and over a broader geographical scope, and this is especially the case with regard to the cattle industry. Therefore, much interest exists in evaluating proteins that might be able to increase the fertility of sperm. Heparin binding proteins (HBPs), specifically the fertility associated antigen (FAA) and the Type-2 tissue inhibitor of metalloproteinase (TIMP-2), act to favor the capacitation and acrosome reaction and perhaps even modulate the immune system's response toward the sperm. The objective of this research was to determine the effect on fertility of adding recombinant FAA (rFAA) and recombinant TIMP-2 (rTIMP-2) to bovine semen before cryopreservation for use in an artificial insemination (AI) program in a tropical environment. For this experiment, 100 crossbred (Bos taurus x Bos indicus) heifers were selected based on their estrus cycle, body condition score (BCS), of 4 to 6 on a scale of 1 to 9, and adequate anatomical conformation evaluated by pelvic and genital (normal) measurements. Heifers were synchronized using estradiol benzoate (EB), Celosil® (PGF2α) (Shering-Plough) and a controlled internal drug release (CIDR) device was inserted that contained progesterone. Inseminations were performed in two groups at random, 50 animals per group. The control group was inseminated with conventional semen. The treatment group was inseminated with semen containing rFAA (25 µg/mL) and rTIMP-2 (25 µg/mL). In the control group a 16% pregnancy rate was obtained versus a 40% pregnancy rate for the HBP treatment group, resulting in a significant difference (P = 0.0037). Given the results herein, one may conclude that the HBPs can increase fertility and could be an option for cattle in tropical conditions; however, one needs to consider the environment, nutrition, and the genetic interaction affecting the final result in whatever reproductive program that is implemented.

  1. Gamete therapeutics: recombinant protein adsorption by sperm for increasing fertility via artificial insemination.

    Directory of Open Access Journals (Sweden)

    Horacio Alvarez-Gallardo

    Full Text Available A decrease in fertility can have a negative economic impact, both locally and over a broader geographical scope, and this is especially the case with regard to the cattle industry. Therefore, much interest exists in evaluating proteins that might be able to increase the fertility of sperm. Heparin binding proteins (HBPs, specifically the fertility associated antigen (FAA and the Type-2 tissue inhibitor of metalloproteinase (TIMP-2, act to favor the capacitation and acrosome reaction and perhaps even modulate the immune system's response toward the sperm. The objective of this research was to determine the effect on fertility of adding recombinant FAA (rFAA and recombinant TIMP-2 (rTIMP-2 to bovine semen before cryopreservation for use in an artificial insemination (AI program in a tropical environment. For this experiment, 100 crossbred (Bos taurus x Bos indicus heifers were selected based on their estrus cycle, body condition score (BCS, of 4 to 6 on a scale of 1 to 9, and adequate anatomical conformation evaluated by pelvic and genital (normal measurements. Heifers were synchronized using estradiol benzoate (EB, Celosil® (PGF2α (Shering-Plough and a controlled internal drug release (CIDR device was inserted that contained progesterone. Inseminations were performed in two groups at random, 50 animals per group. The control group was inseminated with conventional semen. The treatment group was inseminated with semen containing rFAA (25 µg/mL and rTIMP-2 (25 µg/mL. In the control group a 16% pregnancy rate was obtained versus a 40% pregnancy rate for the HBP treatment group, resulting in a significant difference (P = 0.0037. Given the results herein, one may conclude that the HBPs can increase fertility and could be an option for cattle in tropical conditions; however, one needs to consider the environment, nutrition, and the genetic interaction affecting the final result in whatever reproductive program that is implemented.

  2. Increased expression of G-protein-coupled receptor kinases 3 and 4 in hyperfunctioning thyroid nodules.

    Science.gov (United States)

    Voigt, Carsten; Holzapfel, Hans-Peter; Meyer, Silke; Paschke, Ralf

    2004-07-01

    G-protein-coupled receptor kinases (GRKs) are implicated in the pathophysiology of human diseases such as arterial hypertension, heart failure and rheumatoid arthritis. While G-protein-coupled receptor kinases 2 and 5 have been shown to be involved in the desensitization of the rat thyrotropin receptor (TSHR), their role in the pathophysiology of hyperfunctioning thyroid nodules (HTNs) is unknown. Therefore, we analyzed the expression pattern of the known GRKs in human thyroid tissue and investigated their function in the pathology of HTNs. The expression of different GRKs in human thyroid and HTNs was measured by Western blotting. The influence of GRK expression on TSHR function was analyzed by coexpression experiments in HEK 293 cells. We demonstrate that in addition to GRKs 2, 5 and 6, GRKs 3 and 4 are also expressed in the human thyroid. GRKs 2, 3, 5 and 6 are able to desensitize the TSHR in vitro. This GRK-induced desensitization is amplified by the additional over-expression of beta-arrestin 1 or 2. We did not find any mutations in the GRKs 2, 3 and 5 from 14 HTNs without TSHR mutations and Gsalpha mutations. The expression of GRKs 3 and 4 was increased in HTNs independently from the existence of TSHR mutations or Gsalpha mutations. In conclusion, the increased expression of GRK 3 in HTNs and the ability of GRK 3 to desensitize the TSHR in vitro, suggest a potential role for GRK 3 as a negative feedback regulator for the constitutively activated cAMP pathway in HTNs.

  3. Efficient protein-repelling thin films regulated by chain mobility of low-Tg polymers with increased stability via crosslinking

    Science.gov (United States)

    Zhang, Jinghui; Huang, Zhiwei; Liu, Dan

    2017-12-01

    Polymer thin films are generally employed as coatings on implants to prevent protein adsorption. Polymer chain mobility and surface softness have been found to contribute to the protein resistance, but also bring film instability in a liquid protein medium. We investigated the protein resistance ability of three low-Tg polymers, including hydrophobic polymers polyisoprene (PI), poly(n-butyl methacrylate) (PnBMA) and hydrophilic polyethylene oxide (PEO), by overcoming the instability issue with crosslinking. We found that the Tgs of PI and PEO can be increased to around 0 °C after crosslinking. The remained strong chain mobility of both films can still resist protein adsorption regardless the hydrophobicity, yet greatly increases the film stability under an aqueous circumstance. The PnBMA film increased its Tg to around room temperature after crosslinking, which deteriorated the protein-resistance ability having the surface covered by BSA molecules. Our results support that the chain mobility of a polymer film plays an important role in resisting protein adsorption due to the increased entropy associated with more mobile polymer chains. By tune the degree of crosslinking, the stability of polymer in aqueous environment can be increased while the protein resistant ability can be remained. Our results provide a new strategy to design polymer materials for effective antifouling.

  4. Overexpression of amyloid precursor protein increases copper content in HEK293 cells

    International Nuclear Information System (INIS)

    Suazo, Miriam; Hodar, Christian; Morgan, Carlos; Cerpa, Waldo; Cambiazo, Veronica; Inestrosa, Nibaldo C.; Gonzalez, Mauricio

    2009-01-01

    Amyloid precursor protein (APP) is a transmembrane glycoprotein widely expressed in mammalian tissues and plays a central role in Alzheimer's disease. However, its physiological function remains elusive. Cu 2+ binding and reduction activities have been described in the extracellular APP135-156 region, which might be relevant for cellular copper uptake and homeostasis. Here, we assessed Cu 2+ reduction and 64 Cu uptake in two human HEK293 cell lines overexpressing APP. Our results indicate that Cu 2+ reduction increased and cells accumulated larger levels of copper, maintaining cell viability at supra-physiological levels of Cu 2+ ions. Moreover, wild-type cells exposed to both Cu 2+ ions and APP135-155 synthetic peptides increased copper reduction and uptake. Complementation of function studies in human APP751 transformed Fre1 defective Saccharomyces cerevisiae cells rescued low Cu 2+ reductase activity and increased 64 Cu uptake. We conclude that Cu 2+ reduction activity of APP facilitates copper uptake and may represent an early step in cellular copper homeostasis.

  5. Increased CD69 Expression on Peripheral Eosinophils from Patients with Food Protein-Induced Enterocolitis Syndrome.

    Science.gov (United States)

    Wada, Taizo; Matsuda, Yusuke; Toma, Tomoko; Koizumi, Eiko; Okamoto, Hiroyuki; Yachie, Akihiro

    2016-01-01

    Food protein-induced enterocolitis syndrome (FPIES) is an uncommon, non-IgE-mediated food allergy. We recently described a significant increase in fecal eosinophil-derived neurotoxin (EDN) after ingestion of the causative food. However, little is known about the activation status of circulating eosinophils in patients with an acute FPIES reaction. Surface CD69 expression was assessed by flow cytometry on peripheral eosinophils from 5 patients with FPIES before and after ingestion of the causative food. Fecal EDN was measured by enzyme-linked immunosorbent assay. No eosinophil activation was observed before ingestion; however, a significant increase in CD69 expression on eosinophils after an acute FIPES reaction was demonstrated in all of the patients. There was no significant change in absolute eosinophil counts in the peripheral blood. The levels of fecal EDN increased on the day after ingestion of the causative food in all patients. These results suggest that circulating eosinophils as well as eosinophils in the intestinal mucosal tissue are activated in acute FPIES reactions and might be associated with systemic immune events in FPIES. © 2016 S. Karger AG, Basel.

  6. Circadian Misalignment Increases C-Reactive Protein and Blood Pressure in Chronic Shift Workers.

    Science.gov (United States)

    Morris, Christopher J; Purvis, Taylor E; Mistretta, Joseph; Hu, Kun; Scheer, Frank A J L

    2017-04-01

    Shift work is a risk factor for inflammation, hypertension, and cardiovascular disease. This increased risk cannot be fully explained by classical risk factors. Shift workers' behavioral and environmental cycles are typically misaligned relative to their endogenous circadian system. However, there is little information on the impact of acute circadian misalignment on cardiovascular disease risk in shift workers, independent of differences in work stress, food quality, and other factors that are likely to differ between night and day shifts. Thus, our objectives were to determine the independent effect of circadian misalignment on 24-h high-sensitivity C-reactive protein (hs-CRP; a marker of systemic inflammation) and blood pressure levels-cardiovascular disease risk factors-in chronic shift workers. Chronic shift workers undertook two 3-day laboratory protocols that simulated night work, comprising 12-hour inverted behavioral and environmental cycles (circadian misalignment) or simulated day work (circadian alignment), using a randomized, crossover design. Circadian misalignment increased 24-h hs-CRP by 11% ( p shift workers. This may help explain the increased inflammation, hypertension, and cardiovascular disease risk in shift workers.

  7. Using a verbal prompt to increase protein consumption in a hospital setting: a field study

    NARCIS (Netherlands)

    Zanden, van der L.D.T.; Essen, van H.; Kleef, van E.; Wijk, de R.A.; Trijp, van J.C.M.

    2015-01-01

    Background: Sufficient protein intake among hospitalized patients may contribute to faster recovery and a decrease in healthcare costs. Nevertheless, hospitalized patients are often found to consume too little protein. This field study explored the success of a small, inexpensive intervention

  8. The effects of increased dietary protein yogurt snack in the afternoon on appetite control and eating initiation in healthy women

    OpenAIRE

    Ortinau, Laura C; Culp, Julie M; Hoertel, Heather A; Douglas, Steve M; Leidy, Heather J

    2013-01-01

    Background A large portion of daily intake comes from snacking. One of the increasingly common, healthier snacks includes Greek-style yogurt, which is typically higher in protein than regular yogurt. This study evaluated whether a 160?kcal higher-protein (HP) Greek-style yogurt snack improves appetite control, satiety, and delays subsequent eating compared to an isocaloric normal protein (NP) regular yogurt in healthy women. This study also identified the factors that predict the onset of eat...

  9. MEMBRANE TECHNOLOGIES — AN INNOVATIVE METHOD OF PROTEIN BIOLOGICAL VALUE INCREASING IN YOUNG CHILDREN FEEDING

    Directory of Open Access Journals (Sweden)

    I. V. Gmoshinskii

    2012-01-01

    Full Text Available A qualitatively new approach to protein production for milk formulas for infants is discussed in this article. The advantage of membrane technologies usage is that they allow preserving protein biological value and make it possible to control the levels of amino-acids in protein by optimizing their proportion and quantity.

  10. Using affinity capillary electrophoresis and computational models for binding studies of heparinoids with p-selectin and other proteins.

    Science.gov (United States)

    Mozafari, Mona; Balasupramaniam, Shantheya; Preu, Lutz; El Deeb, Sami; Reiter, Christian G; Wätzig, Hermann

    2017-06-01

    A fast and precise affinity capillary electrophoresis (ACE) method has been developed and applied for the investigation of the binding interactions between P-selectin and heparinoids as potential P-selectin inhibitors in the presence and absence of calcium ions. Furthermore, model proteins and vitronectin were used to appraise the binding behavior of P-selectin. The normalized mobility ratios (∆R/R f ), which provided information about the binding strength and the overall charge of the protein-ligand complex, were used to evaluate the binding affinities. It was found that P-selectin interacts more strongly with heparinoids in the presence of calcium ions. P-selectin was affected by heparinoids at the concentration of 3 mg/L. In addition, the results of the ACE experiments showed that among other investigated proteins, albumins and vitronectin exhibited strong interactions with heparinoids. Especially with P-selectin and vitronectin, the interaction may additionally induce conformational changes. Subsequently, computational models were applied to interpret the ACE experiments. Docking experiments explained that the binding of heparinoids on P-selectin is promoted by calcium ions. These docking models proved to be particularly well suited to investigate the interaction of charged compounds, and are therefore complementary to ACE experiments. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. PROTEOSE INTOXICATIONS AND INJURY OF BODY PROTEIN : V. THE INCREASE IN NON-PROTEIN NITROGEN OF THE BLOOD IN ACUTE INFLAMMATORY PROCESSES AND ACUTE INTOXICATIONS.

    Science.gov (United States)

    Cooke, J V; Whipple, G H

    1918-08-01

    Sterile abscess formation in the dog is accompanied by a large increase in output of urinary nitrogen and also by a small but definite increase in the blood non-protein nitrogen. All this nitrogenous material of course is derived from body protein injury and autolysis. Septic inflammation in the dog (pleurisy, pneumonia, peritonitis, etc.) likewise shows a distinct rise in the blood non-protein nitrogen. This rise is not often so great as that frequently observed in the intoxication of intestinal obstruction. Many acute infections in man (septicemia, peritonitis, pneumonia, etc.) show a definite rise in the non-protein nitrogen and urea nitrogen of the blood; some cases show a very great rise above normal (over 100 mg. of non-protein nitrogen per 100 cc. of blood). There may be no anatomical change in the kidney beyond the familiar picture of cloudy swelling. This does not exclude the possibility of some transient functional derangement of the kidney epithelium. Certain obscure intoxications in man may show a considerable rise in the non-protein nitrogen of the blood, indicating a large amount of protein disintegration. These findings must be taken into account in any clinical analysis and interpretation of high non-protein nitrogen of the blood in pathological conditions.

  12. Increased protein expression of LHCG receptor and 17a-hydroxylase/17,20-lyase in human polycystic ovaries

    NARCIS (Netherlands)

    Comim, F.V.; Teerds, K.J.; Hardy, K.; Franks, S.

    2013-01-01

    STUDY QUESTION Does the expression of LHCG receptor (LHCGR) protein and key enzymes in the androgen biosynthetic pathway differ in normal human versus polycystic ovarian tissue? SUMMARY ANSWER LHCGR and 17a-hydroxylase/17-20-lyase (CYP17A1) protein levels are increased in polycystic ovaries (PCOs).

  13. Increasing Sucrose Uptake Capacity of Wheat Grains Stimulates Storage Protein Synthesis1[W

    Science.gov (United States)

    Weichert, Nicola; Saalbach, Isolde; Weichert, Heiko; Kohl, Stefan; Erban, Alexander; Kopka, Joachim; Hause, Bettina; Varshney, Alok; Sreenivasulu, Nese; Strickert, Marc; Kumlehn, Jochen; Weschke, Winfriede; Weber, Hans

    2010-01-01

    Increasing grain sink strength by improving assimilate uptake capacity could be a promising approach toward getting higher yield. The barley (Hordeum vulgare) sucrose transporter HvSUT1 (SUT) was expressed under control of the endosperm-specific Hordein B1 promoter (HO). Compared with the wild type, transgenic HOSUT grains take up more sucrose (Suc) in vitro, showing that the transgene is functional. Grain Suc levels are not altered, indicating that Suc fluxes are influenced rather than steady-state levels. HOSUT grains have increased percentages of total nitrogen and prolamins, which is reflected in increased levels of phenylalanine, tyrosine, tryptophan, isoleucine, and leucine at late grain development. Transcript profiling indicates specific stimulation of prolamin gene expression at the onset of storage phase. Changes in gene expression and metabolite levels related to carbon metabolism and amino acid biosynthesis suggest deregulated carbon-nitrogen balance, which together indicate carbon sufficiency and relative depletion of nitrogen. Genes, deregulated together with prolamin genes, might represent candidates, which respond positively to assimilate supply and are related to sugar-starch metabolism, cytokinin and brassinosteroid functions, cell proliferation, and sugar/abscisic acid signaling. Genes showing inverse expression patterns represent potential negative regulators. It is concluded that HvSUT1 overexpression increases grain protein content but also deregulates the metabolic status of wheat (Triticum aestivum) grains, accompanied by up-regulated gene expression of positive and negative regulators related to sugar signaling and assimilate supply. In HOSUT grains, alternating stimulation of positive and negative regulators causes oscillatory patterns of gene expression and highlights the capacity and great flexibility to adjust wheat grain storage metabolism in response to metabolic alterations. PMID:20018590

  14. Increased plasma ghrelin suppresses insulin release in wethers fed with a high-protein diet.

    Science.gov (United States)

    Takahashi, T; Sato, K; Kato, S; Yonezawa, T; Kobayashi, Y; Ohtani, Y; Ohwada, S; Aso, H; Yamaguchi, T; Roh, S G; Katoh, K

    2014-06-01

    Ghrelin is a multifunctional peptide that promotes an increase of food intake and stimulates GH secretion. Ghrelin secretion is regulated by nutritional status and nutrients. Although a high-protein (HP) diet increases plasma ghrelin secretion in mammals, the mechanisms and the roles of the elevated ghrelin concentrations due to a HP diet have not been fully established. To clarify the roles of elevated acylated ghrelin upon intake of a HP diet, we investigated the regulation of ghrelin concentrations in plasma and tissues in wethers fed with either the HP diet or the control (CNT) diet for 14 days, and examined the action of the elevated plasma ghrelin by using a ghrelin-receptor antagonist. The HP diet gradually increased the plasma acylated-ghrelin concentrations, but the CNT diet did not. Although the GH concentrations did not vary significantly across the groups, an injection of ghrelin-receptor antagonist enhanced insulin levels in circulation in the HP diet group. In the fundus region of the stomach, the ghrelin levels did not differ between the HP and CNT diet groups, whereas ghrelin O-acyltransferase mRNA levels were higher in the group fed with HP diet than those of the CNT diet group were. These results indicate that the HP diet elevated the plasma ghrelin levels by increasing its synthesis; this elevation strongly suppresses the appearance of insulin in the circulation of wethers, but it is not involved in GH secretion. Overall, our findings indicate a role of endogenous ghrelin action in secretion of insulin, which acts as a regulator after the consumption of a HP diet. © 2014 Society for Endocrinology.

  15. Increased infectivity of anchorless mouse scrapie prions in transgenic mice overexpressing human prion protein.

    Science.gov (United States)

    Race, Brent; Phillips, Katie; Meade-White, Kimberly; Striebel, James; Chesebro, Bruce

    2015-06-01

    Prion protein (PrP) is found in all mammals, mostly as a glycoprotein anchored to the plasma membrane by a C-terminal glycosylphosphatidylinositol (GPI) linkage. Following prion infection, host protease-sensitive prion protein (PrPsen or PrPC) is converted into an abnormal, disease-associated, protease-resistant form (PrPres). Biochemical characteristics, such as the PrP amino acid sequence, and posttranslational modifications, such as glycosylation and GPI anchoring, can affect the transmissibility of prions as well as the biochemical properties of the PrPres generated. Previous in vivo studies on the effects of GPI anchoring on prion infectivity have not examined cross-species transmission. In this study, we tested the effect of lack of GPI anchoring on a species barrier model using mice expressing human PrP. In this model, anchorless 22L prions derived from tg44 mice were more infectious than 22L prions derived from C57BL/10 mice when tested in tg66 transgenic mice, which expressed wild-type anchored human PrP at 8- to 16-fold above normal. Thus, the lack of the GPI anchor on the PrPres from tg44 mice appeared to reduce the effect of the mouse-human PrP species barrier. In contrast, neither source of prions induced disease in tgRM transgenic mice, which expressed human PrP at 2- to 4-fold above normal. Prion protein (PrP) is found in all mammals, usually attached to cells by an anchor molecule called GPI. Following prion infection, PrP is converted into a disease-associated form (PrPres). While most prion diseases are species specific, this finding is not consistent, and species barriers differ in strength. The amino acid sequence of PrP varies among species, and this variability affects prion species barriers. However, other PrP modifications, including glycosylation and GPI anchoring, may also influence cross-species infectivity. We studied the effect of PrP GPI anchoring using a mouse-to-human species barrier model. Experiments showed that prions produced by

  16. Increased liver pathology in hepatitis C virus transgenic mice expressing the hepatitis B virus X protein

    International Nuclear Information System (INIS)

    Keasler, Victor V.; Lerat, Herve; Madden, Charles R.; Finegold, Milton J.; McGarvey, Michael J.; Mohammed, Essam M.A.; Forbes, Stuart J.; Lemon, Stanley M.; Hadsell, Darryl L.; Grona, Shala J.; Hollinger, F. Blaine; Slagle, Betty L.

    2006-01-01

    Transgenic mice expressing the full-length HCV coding sequence were crossed with mice that express the HBV X gene-encoded regulatory protein HBx (ATX mice) to test the hypothesis that HBx expression accelerates HCV-induced liver pathogenesis. At 16 months (mo) of age, hepatocellular carcinoma was identified in 21% of HCV/ATX mice, but in none of the single transgenic animals. Analysis of 8-mo animals revealed that, relative to HCV/WT mice, HCV/ATX mice had more severe steatosis, greater liver-to-body weight ratios, and a significant increase in the percentage of hepatocytes staining for proliferating cell nuclear antigen. Furthermore, primary hepatocytes from HCV, ATX, and HCV/ATX transgenic mice were more resistant to fas-mediated apoptosis than hepatocytes from nontransgenic littermates. These results indicate that HBx expression contributes to increased liver pathogenesis in HCV transgenic mice by a mechanism that involves an imbalance in hepatocyte death and regeneration within the context of severe steatosis

  17. ALDH2(E487K) mutation increases protein turnover and promotes murine hepatocarcinogenesis.

    Science.gov (United States)

    Jin, Shengfang; Chen, Jiang; Chen, Lizao; Histen, Gavin; Lin, Zhizhong; Gross, Stefan; Hixon, Jeffrey; Chen, Yue; Kung, Charles; Chen, Yiwei; Fu, Yufei; Lu, Yuxuan; Lin, Hui; Cai, Xiujun; Yang, Hua; Cairns, Rob A; Dorsch, Marion; Su, Shinsan M; Biller, Scott; Mak, Tak W; Cang, Yong

    2015-07-21

    Mitochondrial aldehyde dehydrogenase 2 (ALDH2) in the liver removes toxic aldehydes including acetaldehyde, an intermediate of ethanol metabolism. Nearly 40% of East Asians inherit an inactive ALDH2*2 variant, which has a lysine-for-glutamate substitution at position 487 (E487K), and show a characteristic alcohol flush reaction after drinking and a higher risk for gastrointestinal cancers. Here we report the characterization of knockin mice in which the ALDH2(E487K) mutation is inserted into the endogenous murine Aldh2 locus. These mutants recapitulate essentially all human phenotypes including impaired clearance of acetaldehyde, increased sensitivity to acute or chronic alcohol-induced toxicity, and reduced ALDH2 expression due to a dominant-negative effect of the mutation. When treated with a chemical carcinogen, these mutants exhibit increased DNA damage response in hepatocytes, pronounced liver injury, and accelerated development of hepatocellular carcinoma (HCC). Importantly, ALDH2 protein levels are also significantly lower in patient HCC than in peritumor or normal liver tissues. Our results reveal that ALDH2 functions as a tumor suppressor by maintaining genomic stability in the liver, and the common human ALDH2 variant would present a significant risk factor for hepatocarcinogenesis. Our study suggests that the ALDH2*2 allele-alcohol interaction may be an even greater human public health hazard than previously appreciated.

  18. Interleukin-6 markedly decreases skeletal muscle protein turnover and increases nonmuscle amino acid utilization in healthy individuals

    DEFF Research Database (Denmark)

    van Hall, Gerrit; Steensberg, Adam; Fischer, Christian

    2008-01-01

    CONTEXT: IL-6 is a key modulator of immune function and suggested to be involved in skeletal muscle wasting as seen in sepsis. OBJECTIVE: Our objective was to determine the role of IL-6 in human in vivo systemic and skeletal muscle amino acid metabolism and protein turnover. SUBJECTS AND METHODS...... synthesis was more suppressed than breakdown, causing a small increase in net muscle protein breakdown. Furthermore, rhIL-6 decreased arterial amino acid concentration with 20-40%, despite the increase net release from muscle. CONCLUSIONS: We demonstrated that IL-6 profoundly alters amino acid turnover....... A substantial decrease in plasma amino acids was observed with a concomitant 50% decrease in muscle protein turnover, however, modest increase in net muscle degradation. We hypothesize that the profound reduction in muscle protein turnover and modest increase in net degradation are primarily caused...

  19. Gestational Protein Restriction Increases Cardiac Connexin 43 mRNA levels in male adult rat offspring.

    Science.gov (United States)

    Rossini, Kamila Fernanda; Oliveira, Camila Andrea de; Rebelato, Hércules Jonas; Esquisatto, Marcelo Augusto Marreto; Catisti, Rosana

    2017-07-01

    The dietary limitation during pregnancy influences the growth and development of the fetus and offspring and their health into adult life. The mechanisms underlying the adverse effects of gestational protein restriction (GPR) in the development of the offspring hearts are not well understood. The aim of this study was to evaluate the effects of GPR on cardiac structure in male rat offspring at day 60 after birth (d60). Pregnant Wistar rats were fed a normal-protein (NP, 17% casein) or low-protein (LP, 6% casein) diet. Blood pressure (BP) values from 60-day-old male offspring were measured by an indirect tail-cuff method using an electro sphygmomanometer. Hearts (d60) were collected for assessment of connexin 43 (Cx43) mRNA expression and morphological and morphometric analysis. LP offspring showed no difference in body weight, although they were born lighter than NP offspring. BP levels were significantly higher in the LP group. We observed a significant increase in the area occupied by collagen fibers, a decrease in the number of cardiomyocytes by 104 µm2, and an increase in cardiomyocyte area associated with an increased Cx43 expression. GPR changes myocardial levels of Cx43 mRNA in male young adult rats, suggesting that this mechanism aims to compensate the fibrotic process by the accumulation of collagen fibers in the heart interstitium. A limitação dietética durante a gravidez influencia o crescimento e desenvolvimento do feto e da prole e sua saúde na vida adulta. Os mecanismos subjacentes dos efeitos adversos da restrição proteica gestacional (RPG) no desenvolvimento dos corações da prole não são bem compreendidos. Avaliar os efeitos da RPG sobre a estrutura cardíaca em filhotes machos de ratas aos 60 dias após o nascimento (d60). Ratos fêmeas Wistar grávidas foram alimentadas com uma dieta de proteína normal (PN, 17% caseína) ou de baixa proteína (BP, caseína 6%). Os valores de pressão arterial (PA) de descendentes do sexo masculino de

  20. A Lipid Transfer Protein Increases the Glutathione Content and Enhances Arabidopsis Resistance to a Trichothecene Mycotoxin.

    Directory of Open Access Journals (Sweden)

    John E McLaughlin

    Full Text Available Fusarium head blight (FHB or scab is one of the most important plant diseases worldwide, affecting wheat, barley and other small grains. Trichothecene mycotoxins such as deoxynivalenol (DON accumulate in the grain, presenting a food safety risk and health hazard to humans and animals. Despite considerable breeding efforts, highly resistant wheat or barley cultivars are not available. We screened an activation tagged Arabidopsis thaliana population for resistance to trichothecin (Tcin, a type B trichothecene in the same class as DON. Here we show that one of the resistant lines identified, trichothecene resistant 1 (trr1 contains a T-DNA insertion upstream of two nonspecific lipid transfer protein (nsLTP genes, AtLTP4.4 and AtLTP4.5. Expression of both nsLTP genes was induced in trr1 over 10-fold relative to wild type. Overexpression of AtLTP4.4 provided greater resistance to Tcin than AtLTP4.5 in Arabidopsis thaliana and in Saccharomyces cerevisiae relative to wild type or vector transformed lines, suggesting a conserved protection mechanism. Tcin treatment increased reactive oxygen species (ROS production in Arabidopsis and ROS stain was associated with the chloroplast, the cell wall and the apoplast. ROS levels were attenuated in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls. Exogenous addition of glutathione and other antioxidants enhanced resistance of Arabidopsis to Tcin while the addition of buthionine sulfoximine, an inhibitor of glutathione synthesis, increased sensitivity, suggesting that resistance was mediated by glutathione. Total glutathione content was significantly higher in Arabidopsis and in yeast overexpressing AtLTP4.4 relative to the controls, highlighting the importance of AtLTP4.4 in maintaining the redox state. These results demonstrate that trichothecenes cause ROS accumulation and overexpression of AtLTP4.4 protects against trichothecene-induced oxidative stress by increasing the glutathione

  1. The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1alpha protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis.

    Science.gov (United States)

    Welsh, Sarah J; Bellamy, William T; Briehl, Margaret M; Powis, Garth

    2002-09-01

    Hypoxia-inducible factor 1 (HIF-1), a heterodimer of HIF-1alpha and HIF-1beta subunits, is a transcriptional activator central to the cellular response to low oxygen that includes metabolic adaptation, angiogenesis, metastasis, and inhibited apoptosis. Thioredoxin-1 (Trx-1) is a small redox protein overexpressed in a number of human primary tumors. We have examined the effects of Trx-1 on HIF activity and the activation of downstream genes. Stable transfection of human breast carcinoma MCF-7 cells with human Trx-1 caused a significant increase in HIF-1alpha protein levels under both normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. Trx-1 increased hypoxia-induced HIF-1 transactivation activity measured using a luciferase reporter under the control of the hypoxia response element. Changes in HIF-1alpha mRNA levels did not account for the changes observed at the protein level, and HIF-1beta protein levels did not change. Trx-1 transfection also caused a significant increase in the protein products of hypoxia-responsive genes, including vascular endothelial growth factor (VEGF) and nitric oxide synthase 2 in a number of different cell lines (MCF-7 human breast and HT29 human colon carcinomas and WEHI7.2 mouse lymphoma cells) under both normoxic and hypoxic conditions. The pattern of expression of the different isoforms of VEGF was not changed by Trx-1. Transfection of a redox-inactive Trx-1 (C32S/C35S) markedly decreased levels of HIF-1alpha protein, HIF-1 transactivating activity, and VEGF protein in MCF-7 cells compared with empty vector controls. In vivo studies using WEHI7.2 cells transfected with Trx-1 showed significantly increased tumor VEGF and angiogenesis. The results suggest that Trx-1 increases HIF-1alpha protein levels in cancer cells and increases VEGF production and tumor angiogenesis.

  2. The transient nature of bunyamwera orthobunyavirus NSs protein expression : effects of increased stability of NSs protein on virus replication

    OpenAIRE

    van Knippenberg, Ingeborg; Fragkoudis, Rennos; Elliott, Richard M.

    2013-01-01

    The NSs proteins of bunyaviruses are the viral interferon antagonists, counteracting the host's antiviral response to infection. During high-multiplicity infection of cultured mammalian cells with Bunyamwera orthobunyavirus (BUNV), NSs is rapidly degraded after reaching peak levels of expression at 12hpi. Through the use of inhibitors this was shown to be the result of proteasomal degradation. A recombinant virus (rBUN4KR), in which all four lysine residues in NSs were replaced by arginine re...

  3. Acute high-caffeine exposure increases autophagic flux and reduces protein synthesis in C2C12 skeletal myotubes.

    Science.gov (United States)

    Hughes, M A; Downs, R M; Webb, G W; Crocker, C L; Kinsey, S T; Baumgarner, Bradley L

    2017-04-01

    Caffeine is a highly catabolic dietary stimulant. High caffeine concentrations (1-10 mM) have previously been shown to inhibit protein synthesis and increase protein degradation in various mammalian cell lines. The purpose of this study was to examine the effect of short-term caffeine exposure on cell signaling pathways that regulate protein metabolism in mammalian skeletal muscle cells. Fully differentiated C2C12 skeletal myotubes either received vehicle (DMSO) or 5 mM caffeine for 6 h. Our analysis revealed that caffeine promoted a 40% increase in autolysosome formation and a 25% increase in autophagic flux. In contrast, caffeine treatment did not significantly increase the expression of the skeletal muscle specific ubiquitin ligases MAFbx and MuRF1 or 20S proteasome activity. Caffeine treatment significantly reduced mTORC1 signaling, total protein synthesis and myotube diameter in a CaMKKβ/AMPK-dependent manner. Further, caffeine promoted a CaMKII-dependent increase in myostatin mRNA expression that did not significantly contribute to the caffeine-dependent reduction in protein synthesis. Our results indicate that short-term caffeine exposure significantly reduced skeletal myotube diameter by increasing autophagic flux and promoting a CaMKKβ/AMPK-dependent reduction in protein synthesis.

  4. Concentrations of lipopolysaccharide-binding protein, bactericidal/permeability-increasing protein, soluble CD14 and plasma lipids in relation to endotoxaemia in patients with alcoholic liver disease

    DEFF Research Database (Denmark)

    Schäfer, C.; Parlesak, Alexandr; Schütt, C.

    2002-01-01

    of endotoxin on its target cells (LPS-binding protein and sCD14) were increased. Endotoxin antagonists, such as bactericidal/permeability-increasing protein and high-density lipoprotein, were increased in the pre-cirrhotic stages, whereas a significant reduction of the latter was observed in cirrhosis. Low......-density lipoprotein remained unchanged. The elevation of binding factors in the pre-cirrhotic stages of alcoholic liver disease might attenuate the effects of endotoxaemia, whereas in cirrhosis the reduction of high density lipoprotein, to which large quantities of endotoxin bind, may contribute to its pro...

  5. Fed levels of amino acids are required for the somatotropin-induced increase in muscle protein synthesis.

    Science.gov (United States)

    Wilson, Fiona A; Suryawan, Agus; Orellana, Renán A; Nguyen, Hanh V; Jeyapalan, Asumthia S; Gazzaneo, Maria C; Davis, Teresa A

    2008-10-01

    Chronic somatotropin (pST) treatment in pigs increases muscle protein synthesis and circulating insulin, a known promoter of protein synthesis. Previously, we showed that the pST-mediated rise in insulin could not account for the pST-induced increase in muscle protein synthesis when amino acids were maintained at fasting levels. This study aimed to determine whether the pST-induced increase in insulin promotes skeletal muscle protein synthesis when amino acids are provided at fed levels and whether the response is associated with enhanced translation initiation factor activation. Growing pigs were treated with pST (0 or 180 microg x kg(-1) x day(-1)) for 7 days, and then pancreatic-glucose-amino acid clamps were performed. Amino acids were raised to fed levels in the presence of either fasted or fed insulin concentrations; glucose was maintained at fasting throughout. Muscle protein synthesis was increased by pST treatment and by amino acids (with or without insulin) (P<0.001). In pST-treated pigs, fed, but not fasting, amino acid concentrations further increased muscle protein synthesis rates irrespective of insulin level (P<0.02). Fed amino acids, with or without raised insulin concentrations, increased the phosphorylation of S6 kinase (S6K1) and eukaryotic initiation factor (eIF) 4E-binding protein 1 (4EBP1), decreased inactive 4EBP1.eIF4E complex association, and increased active eIF4E.eIF4G complex formation (P<0.02). pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of muscle protein synthesis requires fed amino acid levels, but not fed insulin levels. However, under the current conditions, the response to amino acids is not mediated by the activation of translation initiation factors that regulate mRNA binding to the ribosomal complex.

  6. Increased serum levels of high mobility group box 1 protein in patients with autistic disorder.

    Science.gov (United States)

    Emanuele, Enzo; Boso, Marianna; Brondino, Natascia; Pietra, Stefania; Barale, Francesco; Ucelli di Nemi, Stefania; Politi, Pierluigi

    2010-05-30

    High mobility group box 1 (HMGB1) is a highly conserved, ubiquitous protein that functions as an activator for inducing the immune response and can be released from neurons after glutamate excitotoxicity. The objective of the present study was to measure serum levels of HMGB1 in patients with autistic disorder and to study their relationship with clinical characteristics. We enrolled 22 adult patients with autistic disorder (mean age: 28.1+/-7.7 years) and 28 age- and gender-matched healthy controls (mean age: 28.7+/-8.1 years). Serum levels of HMGB1 were measured by enzyme-linked immunosorbent assay (ELISA). Compared with healthy subjects, serum levels of HMGB1 were significantly higher in patients with autistic disorder (10.8+/-2.6 ng/mL versus 5.6+/-2.5 ng/mL, respectively, Pautistic disorder. Increased HMGB1 may be a biological correlate of the impaired reciprocal social interactions in this neurodevelopmental disorder. Copyright 2010 Elsevier Inc. All rights reserved.

  7. Xanthene derivatives increase glucose utilization through activation of LKB1-dependent AMP-activated protein kinase.

    Directory of Open Access Journals (Sweden)

    Yonghoon Kwon

    Full Text Available 5' AMP-activated protein kinase (AMPK is a highly conserved serine-threonine kinase that regulates energy expenditure by activating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. Therefore AMPK activators are considered to be drug targets for treatment of metabolic diseases such as diabetes mellitus. To identify novel AMPK activators, we screened xanthene derivatives. We determined that the AMPK activators 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-nitro-phenyl-thioureido]-ethyl}-amide (Xn and 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-cyano-phenyl-thioureido]-ethyl}-amide (Xc elevated glucose uptake in L6 myotubes by stimulating translocation of glucose transporter type 4 (GLUT4. Treatment with the chemical AMPK inhibitor compound C and infection with dominant-negative AMPKa2-virus inhibited AMPK phosphorylation and glucose uptake in myotubes induced by either Xn or Xc. Of the two major upstream kinases of AMPK, we found that Xn and Xc showed LKB1 dependency by knockdown of STK11, an ortholog of human LKB1. Single intravenous administration of Xn and Xc to high-fat diet-induced diabetic mice stimulated AMPK phosphorylation of skeletal muscle and improved glucose tolerance. Taken together, these results suggest that Xn and Xc regulate glucose homeostasis through LKB1-dependent AMPK activation and that the compounds are potential candidate drugs for the treatment of type 2 diabetes mellitus.

  8. Protein energy malnutrition associates with different types of hearing impairments in toddlers: Anemia increases cochlear dysfunction.

    Science.gov (United States)

    Kamel, Terez Boshra; Deraz, Tharwat Ezzat; Elkabarity, Rasha H; Ahmed, Rasha K

    2016-06-01

    This work aimed to highlight a challenging asymptomatic problem which is early detection of hearing impairment in toddlers with protein energy malnutrition (PEM) as a neuro-cognitive effect of PEM on developing brain in relation to hemoglobin level. 100 toddlers, aged 6-24 months, fifty with moderate/severe PEM and fifty healthy children, were included in study. Both TEOAEs and ABR testing were used to assess auditory function. Study reported an association between malnutrition and hearing impairment, 26% of cases had conductive deafness secondary to otitis media with effusion using tympanometry; 84.6% showed type B and 15.4% type C which may suggest developing or resolving otitis media. Their ABR showed 46% mild and 53% moderate impairment. 32% of PEM cases had sensory neural hearing loss and with type (A) tympanometry. Those were assessed using ABR; 58% had mild, 34% moderate and 8% profound impairment. 10% of PEM cases had mixed hearing loss with 50% type B and 50% type C tympanometry and their ABR showed moderate to profound impairment. TEOAEs latencies at different frequencies correlate negatively with hemoglobin level. Toddlers with moderate/severe PEM had hearing impairments of different types and degrees. Neuro-physiological methods could be early and safe detectors of auditory disorders especially in high-risk toddlers. Anemia increases risk for auditory dysfunction. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  9. Heterogeneity of elderly depression: increased risk of Alzheimer's disease and Aβ protein metabolism.

    Science.gov (United States)

    Namekawa, Yuki; Baba, Hajime; Maeshima, Hitoshi; Nakano, Yoshiyuki; Satomura, Emi; Takebayashi, Naoko; Nomoto, Hiroshi; Suzuki, Toshihito; Arai, Heii

    2013-06-03

    Epidemiological studies have proposed that depression may increase the risk for Alzheimer's disease (AD), even in patients with early-onset depression. Although metabolism of amyloid β protein (Aβ) in elderly depression received attention in terms of their correlation, there is a serious heterogeneity in elderly depression in terms of age at onset of depression. Moreover, it is unknown whether early-onset major depressive disorder (MDD) has a long-term effect on the involvement of Aβ metabolism and later development of AD. Thus, we evaluated serum Aβ40 and Aβ42 levels, the Aβ40/Aβ42 ratio in 89 elderly (≥60 years of age) inpatients with MDD and 81 age-matched healthy controls, and compared them among patients with early-onset (great interest that the serum Aβ40/Aβ42 ratio was negatively correlated with the age at MDD onset (R=-0.201, p=0.032). These results suggest that an earlier onset of MDD may have a more serious abnormality in Aβ metabolism, possibly explaining a biological mechanism underlying the link between depression and AD. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Adaptive aneuploidy protects against thiol peroxidase deficiency by increasing respiration via key mitochondrial proteins.

    Science.gov (United States)

    Kaya, Alaattin; Gerashchenko, Maxim V; Seim, Inge; Labarre, Jean; Toledano, Michel B; Gladyshev, Vadim N

    2015-08-25

    Aerobic respiration is a fundamental energy-generating process; however, there is cost associated with living in an oxygen-rich environment, because partially reduced oxygen species can damage cellular components. Organisms evolved enzymes that alleviate this damage and protect the intracellular milieu, most notably thiol peroxidases, which are abundant and conserved enzymes that mediate hydrogen peroxide signaling and act as the first line of defense against oxidants in nearly all living organisms. Deletion of all eight thiol peroxidase genes in yeast (∆8 strain) is not lethal, but results in slow growth and a high mutation rate. Here we characterized mechanisms that allow yeast cells to survive under conditions of thiol peroxidase deficiency. Two independent ∆8 strains increased mitochondrial content, altered mitochondrial distribution, and became dependent on respiration for growth but they were not hypersensitive to H2O2. In addition, both strains independently acquired a second copy of chromosome XI and increased expression of genes encoded by it. Survival of ∆8 cells was dependent on mitochondrial cytochrome-c peroxidase (CCP1) and UTH1, present on chromosome XI. Coexpression of these genes in ∆8 cells led to the elimination of the extra copy of chromosome XI and improved cell growth, whereas deletion of either gene was lethal. Thus, thiol peroxidase deficiency requires dosage compensation of CCP1 and UTH1 via chromosome XI aneuploidy, wherein these proteins support hydroperoxide removal with the reducing equivalents generated by the electron transport chain. To our knowledge, this is the first evidence of adaptive aneuploidy counteracting oxidative stress.

  11. AMP-activated protein kinase controls exercise training- and AICAR-induced increases in SIRT3 and MnSOD

    DEFF Research Database (Denmark)

    Brandauer, Josef; Andersen, Marianne A; Kellezi, Holti

    2015-01-01

    , the acetylation status of SIRT3 target lysine residues on MnSOD (K122) or oligomycin-sensitivity conferring protein (OSCP; K139) was not altered in either mouse or human skeletal muscle in response to acute exercise. We propose an important role for AMPK in regulating mitochondrial function and ROS handling......The mitochondrial protein deacetylase sirtuin (SIRT) 3 may mediate exercise training-induced increases in mitochondrial biogenesis and improvements in reactive oxygen species (ROS) handling. We determined the requirement of AMP-activated protein kinase (AMPK) for exercise training-induced increases...... in skeletal muscle abundance of SIRT3 and other mitochondrial proteins. Exercise training for 6.5 weeks increased SIRT3 (p

  12. Maternal low protein diet and postnatal high fat diet increases adipose imprinted gene expression

    Science.gov (United States)

    Maternal and postnatal diet can alter Igf2 gene expression and DNA methylation. To test whether maternal low protein and postnatal high fat (HF) diet result in alteration in Igf2 expression and obesity, we fed obese-prone Sprague-Dawley rats 8% (LP) or 20% (NP) protein for 3 wk prior to breeding and...

  13. Enteral B-hydroxy-B-methylbutyrate supplementation increases protein synthesis in skeletal muscle of neonatal pigs

    Science.gov (United States)

    Many low-birth weight infants are at risk for poor growth due to an inability to achieve adequate protein intake. Administration of the amino acid leucine stimulates protein synthesis in skeletal muscle of neonates. To determine the effects of enteral supplementation of the leucine metabolite B-hydr...

  14. Intramolecular three-colour single pair FRET of intrinsically disordered proteins with increased dynamic range.

    Science.gov (United States)

    Milles, Sigrid; Koehler, Christine; Gambin, Yann; Deniz, Ashok A; Lemke, Edward A

    2012-10-01

    Single molecule observation of fluorescence resonance energy transfer can be used to provide insight into the structure and dynamics of proteins. Using a straightforward triple-colour labelling strategy, we present a measurement and analysis scheme that can simultaneously study multiple regions within single intrinsically disordered proteins.

  15. Plasma intestinal fatty acid binding protein (I-FABP) concentrations increase following intestinal ischemia in pigs

    NARCIS (Netherlands)

    Niewold, T.A.; Meinen, M.; Meulen, van der J.

    2004-01-01

    Intestinal fatty acid binding protein (I-FABP) is an intracellular epithelial protein in the intestinal mucosa of many animals. IFABP appears in the circulation following epithelial damage, and in humans, is proven to be a parameter for damage to the mucosa. In this paper, an ELISA test designed for

  16. Increased Body Mass Index, Elevated C-reactive Protein, and Short Telomere Length

    DEFF Research Database (Denmark)

    Rode, Line; Nordestgaard, Børge G; Weischer, Maren

    2014-01-01

    -reactive protein. SETTING AND DESIGN: We studied 45,069 individuals from the Copenhagen General Population Study with measurements of leukocyte telomere length, BMI, and C-reactive protein in a Mendelian randomization study. Using the three obesity-associated polymorphisms FTO rs9939609, MC4R rs17782313, and TMEM...

  17. Increased Levels of Antinutritional and/or Defense Proteins Reduced the Protein Quality of a Disease-Resistant Soybean Cultivar.

    Science.gov (United States)

    Sousa, Daniele O B; Carvalho, Ana F U; Oliveira, José Tadeu A; Farias, Davi F; Castelar, Ivan; Oliveira, Henrique P; Vasconcelos, Ilka M

    2015-07-22

    The biochemical and nutritional attributes of two soybean (Glycine max (L.) Merr.) cultivars, one susceptible (Seridó) and the other resistant (Seridó-RCH) to stem canker, were examined to assess whether the resistance to pathogens was related to levels of antinutritional and/or defense proteins in the plant and subsequently affected the nutritional quality. Lectin, urease, trypsin inhibitor, peroxidase and chitinase activities were higher in the resistant cultivar. Growing rats were fed with isocaloric and isoproteic diets prepared with defatted raw soybean meals. Those on the Seridó-RCH diet showed the worst performance in terms of protein quality indicators. Based on regression analysis, lectin, trypsin inhibitor, peroxidase and chitinase appear to be involved in the resistance trait but also in the poorer nutritional quality of Seridó-RCH. Thus, the development of cultivars for disease resistance may lead to higher concentrations of antinutritional compounds, affecting the quality of soybean seeds. Further research that includes the assessment of more cultivars/genotypes is needed.

  18. Increased tolerance to two oomycete pathogens in transgenic tobacco expressing pathogenesis-related protein 1a.

    OpenAIRE

    Alexander, D; Goodman, R M; Gut-Rella, M; Glascock, C; Weymann, K; Friedrich, L; Maddox, D; Ahl-Goy, P; Luntz, T; Ward, E

    1993-01-01

    Expression of pathogenesis-related protein 1a (PR-1a), a protein of unknown biochemical function, is induced to high levels in tobacco in response to pathogen infection. The induction of PR-1a expression is tightly correlated with the onset of systemic acquired resistance (SAR), a defense response effective against a variety of fungal, viral, and bacterial pathogens. While PR-1a has been postulated to be involved in SAR, and is the most highly expressed of the PR proteins, evidence for its ro...

  19. Reduced hemoglobin and increased C-reactive protein are associated with upper gastrointestinal bleeding.

    Science.gov (United States)

    Tomizawa, Minoru; Shinozaki, Fuminobu; Hasegawa, Rumiko; Togawa, Akira; Shirai, Yoshinori; Ichiki, Noboru; Motoyoshi, Yasufumi; Sugiyama, Takao; Yamamoto, Shigenori; Sueishi, Makoto

    2014-02-07

    To investigate the early upper gastrointestinal endoscopy (endoscopy) significantly reduces mortality resulting from upper gastrointestinal (GI) bleeding. Upper GI bleeding was defined as 1a, 1b, 2a, and 2b according to the Forrest classification. The hemoglobin (Hb), and C-reactive protein (CRP) were examined at around the day of endoscopy and 3 mo prior to endoscopy. The rate of change was calculated as follows: (the result of blood examination on the day of endoscopy - the results of blood examination 3 mo prior to endoscopy)/(results of blood examination 3 mo prior to endoscopy). Receiver operating characteristic curves were created to determine threshold values. Seventy-nine men and 77 women were enrolled. There were 17 patients with upper GI bleeding: 12 with a gastric ulcer, 3 with a duodenal ulcer, 1 with an acute gastric mucosal lesion, and 1 with gastric cancer. The area under the curve (AUC), threshold, sensitivity, and specificity of Hb around the day of endoscopy were 0.902, 11.7 g/dL, 94.1%, and 77.1%, respectively, while those of CRP were 0.722, 0.5 mg/dL, 70.5%, and 73%, respectively. The AUC, threshold, sensitivity, and specificity of the rate of change of Hb were 0.851, -21.3%, 76.4%, and 82.6%, respectively, while those of CRP were 0.901, 100%, 100%, and 82.5%, respectively. Predictors for upper GI bleeding were Hb 21.3% and an increase in the CRP > 100%, 3 mo before endoscopy.

  20. Mycobacterium tuberculosis PPE18 Protein Reduces Inflammation and Increases Survival in Animal Model of Sepsis.

    Science.gov (United States)

    Ahmed, Asma; Dolasia, Komal; Mukhopadhyay, Sangita

    2018-04-18

    Mycobacterium tuberculosis PPE18 is a member of the PPE family. Previous studies have shown that recombinant PPE18 (rPPE18) protein binds to TLR2 and triggers a signaling cascade which reduces levels of TNF-α and IL-12, and increases IL-10 in macrophages. Because TNF-α is a major mediator of the pathophysiology of sepsis and blocking inflammation is a possible line of therapy in such circumstances, we tested the efficacy of rPPE18 in reducing symptoms of sepsis in a mouse model of Escherichia coli- induced septic peritonitis. rPPE18 significantly decreased levels of serum TNF-α, IL-1β, IL-6, and IL-12 and reduced organ damage in mice injected i.p. with high doses of E. coli Peritoneal cells isolated from rPPE18-treated mice had characteristics of M2 macrophages which are protective in excessive inflammation. Additionally, rPPE18 inhibited disseminated intravascular coagulation, which can cause organ damage resulting in death. rPPE18 was able to reduce sepsis-induced mortality when given prophylactically or therapeutically. Additionally, in a mouse model of cecal ligation and puncture-induced sepsis, rPPE18 reduced TNF-α, alanine transaminase, and creatinine, attenuated organ damage, prevented depletion of monocytes and lymphocytes, and improved survival. Our studies show that rPPE18 has potent anti-inflammatory properties and can serve as a novel therapeutic to control sepsis. Copyright © 2018 by The American Association of Immunologists, Inc.

  1. Testosterone increases urinary calcium excretion and inhibits expression of renal calcium transport proteins.

    NARCIS (Netherlands)

    Hsu, Y.J.; Dimke, H.; Schoeber, J.P.H.; Hsu, S.C.; Lin, S.H.; Chu, P.; Hoenderop, J.G.J.; Bindels, R.J.M.

    2010-01-01

    Although gender differences in the renal handling of calcium have been reported, the overall contribution of androgens to these differences remains uncertain. We determined here whether testosterone affects active renal calcium reabsorption by regulating calcium transport proteins. Male mice had

  2. Human plasma phospholipid transfer protein increases the antiatherogenic potential of high density lipoproteins in transgenic mice

    NARCIS (Netherlands)

    M.J. van Haperen (Rien); A. van Tol (Arie); P. Vermeulen; M. Jauhiainen; T. van Gent (Teus); P.M. van den Berg (Paul); S. Ehnholm (Sonja); A.W.M. van der Kamp (Arthur); M.P.G. de Crom (Rini); F.G. Grosveld (Frank)

    2000-01-01

    textabstractPlasma phospholipid transfer protein (PLTP) transfers phospholipids between lipoprotein particles and alters high density lipoprotein (HDL) subfraction patterns in vitro, but its physiological function is poorly understood. Transgenic mice that overexpress

  3. Increased KPI containing amyloid precursor protein in experimental autoimmune encephalomyelitis brains.

    Science.gov (United States)

    Beilin, Orit; Karussis, Dimitrios M; Korczyn, Amos D; Gurwitz, David; Aronovich, Ramona; Mizrachi-Kol, Rachel; Chapman, Joab

    2007-04-16

    Amyloid precursor protein can be translated from three alternatively spliced mRNAs. We measured levels of amyloid precursor protein isoforms containing the Kunitz protease inhibitor domain (KPIAPP), and amyloid precursor protein without the Kunitz protease inhibitor domain (KPIAPP) in brain homogenates of acute experimental autoimmune encephalomyelitis mice. At the preclinical phase of the disease, both KPIAPP and KPIAPP levels were significantly higher in homogenates from brains of autoimmune encephalomyelitis mice, whereas at the acute phase of the disease only KPIAPP remained significantly elevated compared with controls. At the recovery phase, no differences were observed between the groups. The early and isoform-specific elevation of KPIAPP in autoimmune encephalomyelitis mice suggests a possible role for amyloid precursor protein in the immune response mediating the disease.

  4. Potassium Bicarbonate Attenuates the Urinary Nitrogen Excretion That Accompanies an Increase in Dietary Protein and May Promote Calcium Absorption

    Science.gov (United States)

    Ceglia, Lisa; Harris, Susan S.; Abrams, Steven A.; Rasmussen, Helen M.; Dallal, Gerard E.; Dawson-Hughes, Bess

    2009-01-01

    Context: Protein is an essential component of muscle and bone. However, the acidic byproducts of protein metabolism may have a negative impact on the musculoskeletal system, particularly in older individuals with declining renal function. Objective: We sought to determine whether adding an alkaline salt, potassium bicarbonate (KHCO3), allows protein to have a more favorable net impact on intermediary indices of muscle and bone conservation than it does in the usual acidic environment. Design: We conducted a 41-d randomized, placebo-controlled, double-blind study of KHCO3 or placebo with a 16-d phase-in and two successive 10-d metabolic diets containing low (0.5 g/kg) or high (1.5 g/kg) protein in random order with a 5-d washout between diets. Setting: The study was conducted in a metabolic research unit. Participants: Nineteen healthy subjects ages 54–82 yr participated. Intervention: KHCO3 (up to 90 mmol/d) or placebo was administered for 41 d. Main Outcome Measures: We measured 24-h urinary nitrogen excretion, IGF-I, 24-h urinary calcium excretion, and fractional calcium absorption. Results: KHCO3 reduced the rise in urinary nitrogen excretion that accompanied an increase in protein intake (P = 0.015) and was associated with higher IGF-I levels on the low-protein diet (P = 0.027) with a similar trend on the high-protein diet (P = 0.050). KHCO3 was also associated with higher fractional calcium absorption on the low-protein diet (P = 0.041) with a similar trend on the high-protein diet (P = 0.064). Conclusions: In older adults, KHCO3 attenuates the protein-induced rise in urinary nitrogen excretion, and this may be mediated by IGF-I. KHCO3 may also promote calcium absorption independent of the dietary protein content. PMID:19050051

  5. Modulating secretory pathway pH by proton channel co-expression can increase recombinant protein stability in plants.

    Science.gov (United States)

    Jutras, Philippe V; D'Aoust, Marc-André; Couture, Manon M-J; Vézina, Louis-Philippe; Goulet, Marie-Claire; Michaud, Dominique; Sainsbury, Frank

    2015-09-01

    Eukaryotic expression systems are used for the production of complex secreted proteins. However, recombinant proteins face considerable biochemical challenges along the secretory pathway, including proteolysis and pH variation between organelles. As the use of synthetic biology matures into solutions for protein production, various host-cell engineering approaches are being developed to ameliorate host-cell factors that can limit recombinant protein quality and yield. We report the potential of the influenza M2 ion channel as a novel tool to neutralize the pH in acidic subcellular compartments. Using transient expression in the plant host, Nicotiana benthamiana, we show that ion channel expression can significantly raise pH in the Golgi apparatus and that this can have a strong stabilizing effect on a fusion protein separated by an acid-susceptible linker peptide. We exemplify the utility of this effect in recombinant protein production using influenza hemagglutinin subtypes differentially stable at low pH; the expression of hemagglutinins prone to conformational change in mildly acidic conditions is considerably enhanced by M2 co-expression. The co-expression of a heterologous ion channel to stabilize acid-labile proteins and peptides represents a novel approach to increasing the yield and quality of secreted recombinant proteins in plants and, possibly, in other eukaryotic expression hosts. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Aspirin-mediated acetylation of haemoglobin increases in presence of high glucose concentration and decreases protein glycation

    Directory of Open Access Journals (Sweden)

    Francesco Finamore

    2015-09-01

    Full Text Available Glycation represents the first stage in the development of diabetic complications. Aspirin was shown to prevent sugars reacting with proteins, but the exact mechanism of this interaction was not well defined. We performed a quantitative analysis to calculate the levels of acetylation and glycation of haemoglobin, among others red blood cell (RBC proteins, using a label free approach. After glucose incubation, increases in the acetylation levels were seen for several haemoglobin subunits, while a parallel decrease of their glycation levels was observed after aspirin incubation. These results suggest that, a mutual influence between these two modifications, occur at protein level.

  7. Stimulation of muscle protein synthesis by somatotropin in pigs is independent of the somatotropin-induced increase in circulating insulin.

    Science.gov (United States)

    Wilson, Fiona A; Orellana, Renán A; Suryawan, Agus; Nguyen, Hanh V; Jeyapalan, Asumthia S; Frank, Jason; Davis, Teresa A

    2008-07-01

    Chronic treatment of growing pigs with porcine somatotropin (pST) promotes protein synthesis and doubles postprandial levels of insulin, a hormone that stimulates translation initiation. This study aimed to determine whether the pST-induced increase in skeletal muscle protein synthesis was mediated through an insulin-induced stimulation of translation initiation. After 7-10 days of pST (150 microg x kg(-1) x day(-1)) or control saline treatment, pancreatic glucose-amino acid clamps were performed in overnight-fasted pigs to reproduce 1) fasted (5 microU/ml), 2) fed control (25 microU/ml), and 3) fed pST-treated (50 microU/ml) insulin levels while glucose and amino acids were maintained at baseline fasting levels. Fractional protein synthesis rates and indexes of translation initiation were examined in skeletal muscle. Effectiveness of pST treatment was confirmed by reduced urea nitrogen and elevated insulin-like growth factor I levels in plasma. Skeletal muscle protein synthesis was independently increased by both insulin and pST. Insulin increased the phosphorylation of protein kinase B and the downstream effectors of the mammalian target of rapamycin, ribosomal protein S6 kinase, and eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1). Furthermore, insulin reduced inactive 4E-BP1.eIF4E complex association and increased active eIF4E.eIF4G complex formation, indicating enhanced eIF4F complex assembly. However, pST treatment did not alter translation initiation factor activation. We conclude that the pST-induced stimulation of skeletal muscle protein synthesis in growing pigs is independent of the insulin-associated activation of translation initiation.

  8. Can fortified foods and snacks increase the energy and protein intake of hospitalised older patients? A systematic review.

    Science.gov (United States)

    Mills, S R; Wilcox, C R; Ibrahim, K; Roberts, H C

    2018-01-10

    Undernutrition affects over 44% of hospitalised older people, who often dislike oral nutritional supplements (ONS). This review summarises the evidence for an alternative strategy, using energy and protein dense meals (via fortification) or snacks (supplementation) to increase the dietary energy and protein intake of older inpatients. A search was conducted through PubMed, EMBASE, CINAHL and the Cochrane database of systematic reviews (May 1996 to May 2016) that used fortification or supplementation to increase the energy or protein intake of patients (mean age ≥60 years) in hospitals or rehabilitation centres. Ten articles (546 patients, mean age 60-83 years) were identified. Compared with usual nutritional care, six studies using either energy or protein based fortification and supplementation significantly increased intake of energy (250-450 kcal day -1 ) or protein (12-16 g day -1 ). Two studies enriched menus with both energy and protein, and significantly increased both energy (698 kcal day -1 and 21 kJ kg -1 ) and protein (16 g and 0.2 g kg -1 ) intake compared to usual care. ONS was similar to supplementation in one study but superior to fortification in another. Four studies reported good acceptability of enriched products and two studies that found they were cost-effective. Compared with usual nutritional care, energy- and protein-based fortification and supplementation could be employed as an effective, well-tolerated and cost-effective intervention to improve dietary intake amongst older inpatients. This strategy may be particularly useful for patients with cognitive impairment who struggle with ONS, and clinical trials are required to compare these approaches and establish their impact on functional outcomes. © 2018 The British Dietetic Association Ltd.

  9. Betulinic acid selectively increases protein degradation and enhances prostate cancer-specific apoptosis: possible role for inhibition of deubiquitinase activity.

    Directory of Open Access Journals (Sweden)

    Teresita Reiner

    Full Text Available Inhibition of the ubiquitin-proteasome system (UPS of protein degradation is a valid anti-cancer strategy and has led to the approval of bortezomib for the treatment of multiple myeloma. However, the alternative approach of enhancing the degradation of oncoproteins that are frequently overexpressed in cancers is less developed. Betulinic acid (BA is a plant-derived small molecule that can increase apoptosis specifically in cancer but not in normal cells, making it an attractive anti-cancer agent. Our results in prostate cancer suggested that BA inhibited multiple deubiquitinases (DUBs, which resulted in the accumulation of poly-ubiquitinated proteins, decreased levels of oncoproteins, and increased apoptotic cell death. In normal fibroblasts, however, BA did not inhibit DUB activity nor increased total poly-ubiquitinated proteins, which was associated with a lack of effect on cell death. In the TRAMP transgenic mouse model of prostate cancer, treatment with BA (10 mg/kg inhibited primary tumors, increased apoptosis, decreased angiogenesis and proliferation, and lowered androgen receptor and cyclin D1 protein. BA treatment also inhibited DUB activity and increased ubiquitinated proteins in TRAMP prostate cancer but had no effect on apoptosis or ubiquitination in normal mouse tissues. Overall, our data suggests that BA-mediated inhibition of DUBs and induction of apoptotic cell death specifically in prostate cancer but not in normal cells and tissues may provide an effective non-toxic and clinically selective agent for chemotherapy.

  10. Ablation of the mitochondrial complex IV assembly protein Surf1 leads to increased expression of the UPRMT and increased resistance to oxidative stress in primary cultures of fibroblasts

    Directory of Open Access Journals (Sweden)

    Gavin Pharaoh

    2016-08-01

    Full Text Available Mice deficient in the electron transport chain (ETC complex IV assembly protein SURF1 have reduced assembly and activity of cytochrome c oxidase that is associated with an upregulation of components of the mitochondrial unfolded protein response (UPRMT and increased mitochondrial number. We hypothesized that the upregulation of proteins associated with the UPRMT in response to reduced cytochrome c oxidase activity in Surf1−/− mice might contribute to increased stress resistance. To test this hypothesis we asked whether primary cultures of fibroblasts from Surf1−/− mice exhibit enhanced resistance to stressors compared to wild-type fibroblasts. Here we show that primary dermal fibroblasts isolated from Surf1−/− mice have increased expression of UPRMT components ClpP and Hsp60, and increased expression of Lon protease. Fibroblasts from Surf1−/− mice are significantly more resistant to cell death caused by oxidative stress induced by paraquat or tert-Butyl hydroperoxide compared to cells from wild-type mice. In contrast, Surf1−/− fibroblasts show no difference in sensitivity to hydrogen peroxide stress. The enhanced cell survival in response to paraquat or tert-Butyl hydroperoxide in Surf1−/− fibroblasts compared to wild-type fibroblasts is associated with induced expression of Lon, ClpP, and Hsp60, increased maximal respiration, and increased reserve capacity as measured using the Seahorse Extracellular Flux Analyzer. Overall these data support a protective role for the activation of the UPRMT in cell survival.

  11. Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy

    International Nuclear Information System (INIS)

    Zanzoni, Serena; D’Onofrio, Mariapina; Molinari, Henriette; Assfalg, Michael

    2012-01-01

    Highlights: ► Bile acid binding proteins from different constructs retain structural integrity. ► NMR 15 N-T 1 relaxation data of BABPs show differences if LVPR extension is present. ► Deviations from a 15 N-T 1 /molecular-weight calibration curve indicate aggregation. -- Abstract: The use of a recombinant protein to investigate the function of the native molecule requires that the former be obtained with the same amino acid sequence as the template. However, in many cases few additional residues are artificially introduced for cloning or purification purposes, possibly resulting in altered physico-chemical properties that may escape routine characterization. For example, increased aggregation propensity without visible protein precipitation is hardly detected by most analytical techniques but its investigation may be of great importance for optimizing the yield of recombinant protein production in biotechnological and structural biology applications. In this work we show that bile acid binding proteins incorporating the common C-terminal LeuValProArg extension display different hydrodynamic properties from those of the corresponding molecules without such additional amino acids. The proteins were produced enriched in nitrogen-15 for analysis via heteronuclear NMR spectroscopy. Residue-specific spin relaxation rates were measured and related to rotational tumbling time and molecular size. While the native-like recombinant proteins show spin-relaxation rates in agreement with those expected for monomeric globular proteins of their mass, our data indicate the presence of larger adducts for samples of proteins with very short amino acid extensions. The used approach is proposed as a further screening method for the quality assessment of biotechnological protein products.

  12. Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Zanzoni, Serena; D' Onofrio, Mariapina; Molinari, Henriette [Department of Biotechnology, University of Verona, 37134 Verona (Italy); Assfalg, Michael, E-mail: michael.assfalg@univr.it [Department of Biotechnology, University of Verona, 37134 Verona (Italy)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer Bile acid binding proteins from different constructs retain structural integrity. Black-Right-Pointing-Pointer NMR {sup 15}N-T{sub 1} relaxation data of BABPs show differences if LVPR extension is present. Black-Right-Pointing-Pointer Deviations from a {sup 15}N-T{sub 1}/molecular-weight calibration curve indicate aggregation. -- Abstract: The use of a recombinant protein to investigate the function of the native molecule requires that the former be obtained with the same amino acid sequence as the template. However, in many cases few additional residues are artificially introduced for cloning or purification purposes, possibly resulting in altered physico-chemical properties that may escape routine characterization. For example, increased aggregation propensity without visible protein precipitation is hardly detected by most analytical techniques but its investigation may be of great importance for optimizing the yield of recombinant protein production in biotechnological and structural biology applications. In this work we show that bile acid binding proteins incorporating the common C-terminal LeuValProArg extension display different hydrodynamic properties from those of the corresponding molecules without such additional amino acids. The proteins were produced enriched in nitrogen-15 for analysis via heteronuclear NMR spectroscopy. Residue-specific spin relaxation rates were measured and related to rotational tumbling time and molecular size. While the native-like recombinant proteins show spin-relaxation rates in agreement with those expected for monomeric globular proteins of their mass, our data indicate the presence of larger adducts for samples of proteins with very short amino acid extensions. The used approach is proposed as a further screening method for the quality assessment of biotechnological protein products.

  13. A low-protein, high-carbohydrate diet increases browning in perirenal adipose tissue but not in inguinal adipose tissue.

    Science.gov (United States)

    Pereira, Mayara P; Ferreira, Laís A A; da Silva, Flávia H S; Christoffolete, Marcelo A; Metsios, George S; Chaves, Valéria E; de França, Suélem A; Damazo, Amílcar S; Flouris, Andreas D; Kawashita, Nair H

    2017-10-01

    The aim of this study was to evaluate the browning and origin of fatty acids (FAs) in the maintenance of triacylglycerol (TG) storage and/or as fuel for thermogenesis in perirenal adipose tissue (periWAT) and inguinal adipose tissue (ingWAT) of rats fed a low-protein, high-carbohydrate (LPHC) diet. LPHC (6% protein, 74% carbohydrate) or control (C; 17% protein, 63% carbohydrate) diets were administered to rats for 15 d. The tissues were stained with hematoxylin and eosin for histologic analysis. The content of uncoupling protein 1 (UCP1) was determined by immunofluorescence. Levels of T-box transcription factor (TBX1), PR domain containing 16 (PRDM16), adipose triacylglycerol lipase (ATGL), hormone-sensitive lipase, lipoprotein lipase (LPL), glycerokinase, phosphoenolpyruvate carboxykinase (PEPCK), glucose transporter 4, β 3 -adrenergic receptor (AR), β 1 -AR, protein kinase A (PKA), adenosine-monophosphate-activated protein kinase (AMPK), and phospho-AMPK were determined by immunoblotting. Serum fibroblast growth factor 21 (FGF21) was measured using a commercial kit (Student's t tests, P diet increased FGF21 levels by 150-fold. The presence of multilocular adipocytes, combined with the increased contents of UCP1, TBX1, and PRDM16 in periWAT of LPHC-fed rats, suggested the occurrence of browning. The contents of β 1 -AR and LPL were increased in the periWAT. The ingWAT showed higher ATGL and PEPCK levels, phospho-AMPK/AMPK ratio, and reduced β 3 -AR and PKA levels. These findings suggest that browning occurred only in the periWAT and that higher utilization of FAs from blood lipoproteins acted as fuel for thermogenesis. Increased glycerol 3-phosphate generation by glyceroneogenesis increased FAs reesterification from lipolysis, explaining the increased TG storage in the ingWAT. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Growth performance of sea bass fed increasing levels of pea-wheat protein in diets varying in fish meal quality

    Directory of Open Access Journals (Sweden)

    E. Tibaldi

    2010-04-01

    Full Text Available A 11-week trial was carried out to compare the growth performance of sea bass (D. labrax fed six isonitrogenous isocaloric diets where protein from two fish meals of different nutritive value was replaced with graded levels (0, 50 or 75% of a mixture made up by a pea protein concentrate and wheat gluten. Fish meal quality did not affect (P>0.05 weight gain or feed efficiency in fish fed graded levels of plant protein in the diet. Feed intake decreased (P<0.05 as the level of plant protein was increased in the diet but this did not led to impaired growth or feed conversion rate. Protein efficiency and retention were equally improved (P<0.05 only with diets where a poor quality fish meal was substituted by protein rich-plant ingredients. Calculations based on the mass balance of nutrients of sea bass proven the inclusion of a mixture of highly purified plant-protein derivatives in complete diets for the sea bass, to be beneficial in reducing pollution load.

  15. Increased Milk Protein Concentration in a Rehydration Drink Enhances Fluid Retention Caused by Water Reabsorption in Rats.

    Science.gov (United States)

    Ito, Kentaro; Saito, Yuri; Ashida, Kinya; Yamaji, Taketo; Itoh, Hiroyuki; Oda, Munehiro

    2015-01-01

    A fluid-retention effect is required for beverages that are designed to prevent dehydration. That is, fluid absorbed from the intestines should not be excreted quickly; long-term retention is desirable. Here, we focused on the effect of milk protein on fluid retention, and propose a new effective oral rehydration method that can be used daily for preventing dehydration. We first evaluated the effects of different concentrations of milk protein on fluid retention by measuring the urinary volumes of rats fed fluid containing milk protein at concentrations of 1, 5, and 10%. We next compared the fluid-retention effect of milk protein-enriched drink (MPD) with those of distilled water (DW) and a sports drink (SD) by the same method. Third, to investigate the mechanism of fluid retention, we measured plasma insulin changes in rats after ingesting these three drinks. We found that the addition of milk protein at 5 or 10% reduced urinary volume in a dose-dependent manner. Ingestion of the MPD containing 4.6% milk protein resulted in lower urinary volumes than DW and SD. MPD also showed a higher water reabsorption rate in the kidneys and higher concentrations of plasma insulin than DW and SD. These results suggest that increasing milk protein concentration in a beverage enhances fluid retention, which may allow the possibility to develop rehydration beverages that are more effective than SDs. In addition, insulin-modifying renal water reabsorption may contribute to the fluid-retention effect of MPD.

  16. Missense mutation Lys18Asn in dystrophin that triggers X-linked dilated cardiomyopathy decreases protein stability, increases protein unfolding, and perturbs protein structure, but does not affect protein function.

    Directory of Open Access Journals (Sweden)

    Surinder M Singh

    Full Text Available Genetic mutations in a vital muscle protein dystrophin trigger X-linked dilated cardiomyopathy (XLDCM. However, disease mechanisms at the fundamental protein level are not understood. Such molecular knowledge is essential for developing therapies for XLDCM. Our main objective is to understand the effect of disease-causing mutations on the structure and function of dystrophin. This study is on a missense mutation K18N. The K18N mutation occurs in the N-terminal actin binding domain (N-ABD. We created and expressed the wild-type (WT N-ABD and its K18N mutant, and purified to homogeneity. Reversible folding experiments demonstrated that both mutant and WT did not aggregate upon refolding. Mutation did not affect the protein's overall secondary structure, as indicated by no changes in circular dichroism of the protein. However, the mutant is thermodynamically less stable than the WT (denaturant melts, and unfolds faster than the WT (stopped-flow kinetics. Despite having global secondary structure similar to that of the WT, mutant showed significant local structural changes at many amino acids when compared with the WT (heteronuclear NMR experiments. These structural changes indicate that the effect of mutation is propagated over long distances in the protein structure. Contrary to these structural and stability changes, the mutant had no significant effect on the actin-binding function as evident from co-sedimentation and depolymerization assays. These results summarize that the K18N mutation decreases thermodynamic stability, accelerates unfolding, perturbs protein structure, but does not affect the function. Therefore, K18N is a stability defect rather than a functional defect. Decrease in stability and increase in unfolding decrease the net population of dystrophin molecules available for function, which might trigger XLDCM. Consistently, XLDCM patients have decreased levels of dystrophin in cardiac muscle.

  17. Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway.

    Science.gov (United States)

    Kosalková, Katarina; García-Estrada, Carlos; Barreiro, Carlos; Flórez, Martha G; Jami, Mohammad S; Paniagua, Miguel A; Martín, Juan F

    2012-01-10

    The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold) in cells grown with casein or casein phosphopeptides (CPPs). CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs), whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase), which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.

  18. Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes

    International Nuclear Information System (INIS)

    Csermely, P.; Szamel, M.; Resch, K.; Somogyi, J.

    1988-01-01

    In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested. In the present report, the authors demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes,and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn 2+ , while Fe 2+ and Mn 2+ are only partially counteractive. The results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca 2+ , phorbol ester, or antigen

  19. Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Csermely, P.; Szamel, M.; Resch, K.; Somogyi, J.

    1988-05-15

    In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested. In the present report, the authors demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes,and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn/sup 2 +/, while Fe/sup 2 +/ and Mn/sup 2 +/ are only partially counteractive. The results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca/sup 2 +/, phorbol ester, or antigen.

  20. Genetic control of soybean seed oil: II. QTL and genes that increase oil concentration without decreasing protein or with increased seed yield.

    Science.gov (United States)

    Eskandari, Mehrzad; Cober, Elroy R; Rajcan, Istvan

    2013-06-01

    Soybean [Glycine max (L.) Merrill] seed oil is the primary global source of edible oil and a major renewable and sustainable feedstock for biodiesel production. Therefore, increasing the relative oil concentration in soybean is desirable; however, that goal is complex due to the quantitative nature of the oil concentration trait and possible effects on major agronomic traits such as seed yield or protein concentration. The objectives of the present study were to study the relationship between seed oil concentration and important agronomic and seed quality traits, including seed yield, 100-seed weight, protein concentration, plant height, and days to maturity, and to identify oil quantitative trait loci (QTL) that are co-localized with the traits evaluated. A population of 203 F4:6 recombinant inbred lines, derived from a cross between moderately high oil soybean genotypes OAC Wallace and OAC Glencoe, was developed and grown across multiple environments in Ontario, Canada, in 2009 and 2010. Among the 11 QTL associated with seed oil concentration in the population, which were detected using either single-factor ANOVA or multiple QTL mapping methods, the number of QTL that were co-localized with other important traits QTL were six for protein concentration, four for seed yield, two for 100-seed weight, one for days to maturity, and one for plant height. The oil-beneficial allele of the QTL tagged by marker Sat_020 was positively associated with seed protein concentration. The oil favorable alleles of markers Satt001 and GmDGAT2B were positively correlated with seed yield. In addition, significant two-way epistatic interactions, where one of the interacting markers was solely associated with seed oil concentration, were identified for the selected traits in this study. The number of significant epistatic interactions was seven for yield, four for days to maturity, two for 100-seed weight, one for protein concentration, and one for plant height. The identified molecular

  1. Low cut-off values increase diagnostic performance of protein S assays

    NARCIS (Netherlands)

    Mulder, Rene; ten Kate, Min Ki; Kluin-Nelemans, Hanneke C.; Mulder, Andre B.

    Conflicting data have been reported on the accuracy of protein S (PS) assays for detection of hereditary PS deficiency. In this study we assessed the diagnostic performance of two total PS antigen assays, four free PS assays and three PS activity assays in a group of 28 heterozygous carriers of

  2. Allergen-induced increase of eosinophil cationic protein in nasal lavage fluid

    DEFF Research Database (Denmark)

    Bisgaard, H; Grønborg, H; Mygind, N

    1990-01-01

    It was our aim to study the effect of nasal allergen provocation on the concentration of eosinophil cationic protein (ECP) in nasal lavage fluid, with and without glucocorticoid pretreatment. Twenty grass-pollen sensitive volunteers were provoked outside the pollen season on 2 consecutive days...

  3. Increasing intake of soybean protein or casein, but not cod meal, reduces nephrocalcinosis in female rats.

    NARCIS (Netherlands)

    Zhang, X.; Beynen, A.C.

    1992-01-01

    Female weanling rats were fed diets with soybean protein, casein or cod meal at 171, 342 or 513 mmol nitrogen/100 g for 3 wk. The diets were isonitrogenous and balanced for fat, cholesterol, calcium, magnesium and phosphorus. Cod meal feeding at 171 and 342 mmol nitrogen/100 g diet produced lower

  4. Dystropathology increases energy expenditure and protein turnover in the Mdx mouse model of Duchenne muscular dystrophy

    Science.gov (United States)

    The skeletal muscles in Duchenne muscular dystrophy and the mdx mouse model lack functional dystrophin and undergo repeated bouts of necrosis, regeneration, and growth. These processes have a high metabolic cost. However, the consequences for whole body energy and protein metabolism, and on the diet...

  5. Hemin-Graphene Derivatives with Increased Peroxidase Activities Restrain Protein Tyrosine Nitration.

    Science.gov (United States)

    Xu, Huan; Yang, Zhen; Li, Hailing; Gao, Zhonghong

    2017-12-14

    Protein tyrosine nitration is implicated in the occurrence and progression of pathological conditions involving free radical reactions. It is well recognized that hemin can catalyze protein tyrosine nitration in the presence of nitrite and hydrogen peroxide. Generally, the catalytic efficiency is positively correlated to its peroxidase activity. In this study, however, it is found that the efficiency of hemin in catalyzing protein tyrosine nitration is largely suppressed after functionalization with graphene derivatives, even though its peroxidase-like activity is more than quadrupled. Further studies show that the oxidation of tyrosine is still observed for these composites; dityrosine formation, however, is greatly inhibited. Furthermore, these composites also exhibit strong effects on the oxidation of nitrite into nitrate. Therefore, we propose a mechanism in which hemin-graphene derivatives facilitate the oxidation of tyrosine and nitrite to produce tyrosyl radicals and nitrogen dioxide radicals in the presence of hydrogen peroxide, but graphene interlayers serve as barriers that hinder radical-radical coupling reactions; consequently, protein tyrosine nitration is restrained. This property of hemin-graphene derivatives, by which they catalyze substrate oxidation but suppress radical-radical coupling reactions, shows their great potential in selective oxidation procedures for byproduct removal. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Method Development to Increase Protein Enrichment During Dry Fractionation of Starch-Rich Legumes

    NARCIS (Netherlands)

    Pelgrom, P.J.M.; Boom, R.M.; Schutyser, M.A.I.

    2015-01-01

    A facile method was developed to establish milling settings that optimally separate starch granules from protein bodies and cell wall fibres for starch-rich legumes. Optimal separation was obtained for pea, bean, lentil and chickpea when the particle size distribution curve of flour and isolated

  7. Estrogens increase expression of bone morphogenetic protein 8b in brown adipose tissue of mice

    NARCIS (Netherlands)

    A. Grefhorst (Aldo); J.C. van den Beukel (Anneke); A.F. van Houten (A.); J. Steenbergen (Jacobie); J.A. Visser (Jenny); A.P.N. Themmen (Axel)

    2015-01-01

    textabstractBackground: In mammals, white adipose tissue (WAT) stores fat and brown adipose tissue (BAT) dissipates fat to produce heat. Several studies showed that females have more active BAT. Members of the bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) families are expressed

  8. Mechanisms of edema formation in myxedema--increased protein extravasation and relatively slow lymphatic drainage

    DEFF Research Database (Denmark)

    Parving, H H; Hansen, J M; Nielsen, S L

    1979-01-01

    of generalized edema (P less than 0.05). All variables returned to normal during l-thyroxine treatment. The extravascular accumulation of albumin, and presumably of all other plasma proteins, is important in the generalized edema typically found in myxedema. Inadequate lymphatic drainage may also explain...

  9. Deletion of PTEN Produces Deficits in Conditioned Fear and Increases Fragile X Mental Retardation Protein

    Science.gov (United States)

    Lugo, Joaquin N.; Smith, Gregory D.; Morrison, Jessica B.; White, Jessika

    2013-01-01

    The phosphatase and tensin homolog detected on chromosome 10 (PTEN) gene product modulates activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. The PI3K pathway has been found to be involved in the regulation of the fragile X mental retardation protein, which is important for long-term depression and in the formation of new…

  10. Increased Protein Requirements in Female Athletes after Variable-Intensity Exercise.

    Science.gov (United States)

    Wooding, Denise J; Packer, Jeff E; Kato, Hiroyuki; West, Daniel W D; Courtney-Martin, Glenda; Pencharz, Paul B; Moore, Daniel R

    2017-11-01

    Protein requirements are primarily studied in the context of resistance or endurance exercise with little research devoted to variable-intensity intermittent exercise characteristic of many team sports. Further, female populations are underrepresented in dietary sports science studies. We aimed to determine a dietary protein requirement in active females performing variable-intensity intermittent exercise using the indicator amino acid oxidation (IAAO) method. We hypothesized that these requirements would be greater than current IAAO-derived estimates in nonactive adult males. Six females (21.2 ± 0.8 yr, 68.8 ± 4.1 kg, 47.1 ± 1.2 mL O2·kg·min; mean ± SE) completed five to seven metabolic trials during the luteal phase of the menstrual cycle. Participants performed a modified Loughborough Intermittent Shuttle Test before consuming eight hourly mixed meals providing the test protein intake (0.2-2.66 g·kg·d), 6 g·kg·d CHO and sufficient energy for resting and exercise-induced energy expenditure. Protein was provided as crystalline amino acid modeling egg protein with [C]phenylalanine as the indicator amino acid. Phenylalanine turnover (Q) was determined from urinary [C]phenylalanine enrichment. Breath CO2 excretion (FCO2) was analyzed using mixed effects biphase linear regression with the breakpoint and upper 95% confidence interval approximating the estimated average requirement and recommended dietary allowance, respectively. Protein intake had no effect on Q (68.7 ± 7.3 μmol·kg·h; mean ± SE). FCO2 displayed a robust biphase response (R = 0.66) with an estimated average requirement of 1.41 g·kg·d and recommended dietary allowance of 1.71 g·kg·d. The protein requirement estimate of 1.41 and 1.71 g·kg·d for females performing variable-intensity intermittent exercise is greater than the IAAO-derived estimates of adult males (0.93 and 1.2 g·kg·d) and at the upper range of the American College of Sports Medicine athlete recommendations (1.2-2.0 g·kg·d).

  11. Increased protein intake reduces lean body mass loss during weight loss in athletes.

    Science.gov (United States)

    Mettler, Samuel; Mitchell, Nigel; Tipton, Kevin D

    2010-02-01

    To examine the influence of dietary protein on lean body mass loss and performance during short-term hypoenergetic weight loss in athletes. In a parallel design, 20 young healthy resistance-trained athletes were examined for energy expenditure for 1 wk and fed a mixed diet (15% protein, 100% energy) in the second week followed by a hypoenergetic diet (60% of the habitual energy intake), containing either 15% (approximately 1.0 g x kg(-1)) protein (control group, n = 10; CP) or 35% (approximately 2.3 g x kg(-1)) protein (high-protein group, n = 10; HP) for 2 wk. Subjects continued their habitual training throughout the study. Total, lean body, and fat mass, performance (squat jump, maximal isometric leg extension, one-repetition maximum (1RM) bench press, muscle endurance bench press, and 30-s Wingate test) and fasting blood samples (glucose, nonesterified fatty acids (NEFA), glycerol, urea, cortisol, free testosterone, free Insulin-like growth factor-1 (IGF-1), and growth hormone), and psychologic measures were examined at the end of each of the 4 wk. Total (-3.0 +/- 0.4 and -1.5 +/- 0.3 kg for the CP and HP, respectively, P = 0.036) and lean body mass loss (-1.6 +/- 0.3 and -0.3 +/- 0.3 kg, P = 0.006) were significantly larger in the CP compared with those in the HP. Fat loss, performance, and most blood parameters were not influenced by the diet. Urea was higher in HP, and NEFA and urea showed a group x time interaction. Fatigue ratings and "worse than normal" scores on the Daily Analysis of Life Demands for Athletes were higher in HP. These results indicate that approximately 2.3 g x kg(-1) or approximately 35% protein was significantly superior to approximately 1.0 g x kg(-1) or approximately 15% energy protein for maintenance of lean body mass in young healthy athletes during short-term hypoenergetic weight loss.

  12. A randomized trial of protein supplementation compared with extra fast food on the effects of resistance training to increase metabolism.

    Science.gov (United States)

    Hambre, David; Vergara, Marta; Lood, Yvonne; Bachrach-Lindström, Margareta; Lindström, Torbjörn; Nystrom, Fredrik H

    2012-10-01

    To prospectively evaluate the effects of resistance training combined with increased energy intake or protein-supplementation on lean body-mass, resting metabolic-rate (RMR) and cardiovascular risk factors. Twenty-four healthy males (aged 19-32 years) performed resistance exercise for 12 weeks aiming for at least 1 hour training-sessions 3 times a week. The participants were randomized to consume extra protein (33 g whey protein/day) or a meal of fast-food/day (1350 kcal, 41 g protein). Body-composition was measured with Dual-Energy X-ray Absorptiometry (DEXA) and RMR by indirect calorimetry. Fasting blood samples were drawn before and after the 3-month training period and after 12 months. The body weight increased from 75.1 ± 6.9 kg to 78.7 ± 7.2 kg (p < 0.0001), without differences between the groups. RMR increased from 1787 ± 143 kcal/24 h to 1954 ± 187 kcal/24 h (p < 0.0001, N = 24), which was more than expected from the increase in lean body-mass (increase from 59.7 ± 4.3 kg to 61.8 ± 4.1 kg p = 0.004). Fasting serum-insulin levels increased in the fast-food group compared with the extra-protein group (p = 0.03). ApoB increased from 0.691 ± 0.14 g/L to 0.768 ± 0.17 g/L, p = 0.004, in the fast-food group only. Long-term follow up after 12 months showed that RMR, body weight, total fat and lean body-masses did not differ from baseline (n = 19). Resistance training for 12 weeks increased RMR and lean body-mass similarly when based on either an increased energy-intake or protein supplement. However, the increase in RMR was higher than expected from the increase in lean body-mass. Thus resistance training could potentially decrease the risk of obesity by induction of increased RMR.

  13. Marked increase in rat red blood cell membrane protein glycosylation by one-month treatment with a cafeteria diet

    Directory of Open Access Journals (Sweden)

    Laia Oliva

    2015-07-01

    Full Text Available Background and Objectives. Glucose, an aldose, spontaneously reacts with protein amino acids yielding glycosylated proteins. The compounds may reorganize to produce advanced glycosylation products, which regulatory importance is increasingly being recognized. Protein glycosylation is produced without the direct intervention of enzymes and results in the loss of function. Glycosylated plasma albumin, and glycosylated haemoglobin are currently used as index of mean plasma glucose levels, since higher glucose availability results in higher glycosylation rates. In this study we intended to detect the early changes in blood protein glycosylation elicited by an obesogenic diet.Experimental Design. Since albumin is in constant direct contact with plasma glucose, as are the red blood cell (RBC membranes, we analyzed their degree or glycosylation in female and male rats, either fed a standard diet or subjected to a hyper-energetic self-selected cafeteria diet for 30 days. This model produces a small increase in basal glycaemia and a significant increase in body fat, leaving the animals in the initial stages of development of metabolic syndrome. We also measured the degree of glycosylation of hemoglobin, and the concentration of glucose in contact with this protein, that within the RBC. Glycosylation was measured by colorimetric estimation of the hydroxymethylfurfural liberated from glycosyl residues by incubation with oxalate.Results. Plasma glucose was higher in cafeteria diet and in male rats, both independent effects. However, there were no significant differences induced by sex or diet in either hemoglobin or plasma proteins. Purified RBC membranes showed a marked effect of diet: higher glycosylation in cafeteria rats, which was more marked in females (not in controls. In any case, the number of glycosyl residues per molecule were higher in hemoglobin than in plasma proteins (after correction for molecular weight. The detected levels of glucose in

  14. Instability of buried hydration sites increases protein subdomains fluctuations in the human prion protein by the pathogenic mutation T188R

    Science.gov (United States)

    Tomobe, Katsufumi; Yamamoto, Eiji; Akimoto, Takuma; Yasui, Masato; Yasuoka, Kenji

    2016-05-01

    The conformational change from the cellular prion protein (PrPc) to scrapie prion protein (PrPsc) is a key process in prion diseases. The prion protein has buried water molecules which significantly contribute to the stability of the protein; however, there has been no report investigating the influence on the buried hydration sites by a pathogenic mutation not adjacent to the buried hydration sites. Here, we perform molecular dynamics simulations of wild type (WT) PrPc and pathogenic point mutant T188R to investigate conformational changes and the buried hydration sites. In WT-PrPc, four buried hydration sites are identified by residence time and rotational relaxation analysis. However, there are no stable buried hydration sites in one of T188R simulations, which indicates that T188R sometimes makes the buried hydration sites fragile. We also find that fluctuations of subdomains S1-H1-S2 and H1-H2 increase in T188R when the buried hydration sites become unstable. Since the side chain of arginine which is replaced from threonine in T188R is larger than of threonine, the side chain cannot be embedded in the protein, which is one of the causes of the instability of subdomains. These results show correlations between the buried hydration sites and the mutation which is far from them, and provide a possible explanation for the instability by mutation.

  15. Instability of buried hydration sites increases protein subdomains fluctuations in the human prion protein by the pathogenic mutation T188R

    Directory of Open Access Journals (Sweden)

    Katsufumi Tomobe

    2016-05-01

    Full Text Available The conformational change from the cellular prion protein (PrPc to scrapie prion protein (PrPsc is a key process in prion diseases. The prion protein has buried water molecules which significantly contribute to the stability of the protein; however, there has been no report investigating the influence on the buried hydration sites by a pathogenic mutation not adjacent to the buried hydration sites. Here, we perform molecular dynamics simulations of wild type (WT PrPc and pathogenic point mutant T188R to investigate conformational changes and the buried hydration sites. In WT-PrPc, four buried hydration sites are identified by residence time and rotational relaxation analysis. However, there are no stable buried hydration sites in one of T188R simulations, which indicates that T188R sometimes makes the buried hydration sites fragile. We also find that fluctuations of subdomains S1-H1-S2 and H1-H2 increase in T188R when the buried hydration sites become unstable. Since the side chain of arginine which is replaced from threonine in T188R is larger than of threonine, the side chain cannot be embedded in the protein, which is one of the causes of the instability of subdomains. These results show correlations between the buried hydration sites and the mutation which is far from them, and provide a possible explanation for the instability by mutation.

  16. AMP-activated protein kinase controls exercise training- and AICAR-induced increases in SIRT3 and MnSOD

    Directory of Open Access Journals (Sweden)

    Josef eBrandauer

    2015-03-01

    Full Text Available The mitochondrial protein deacetylase sirtuin (SIRT 3 may mediate exercise training-induced increases in mitochondrial biogenesis and improvements in reactive oxygen species (ROS handling. We determined the requirement of AMP-activated protein kinase (AMPK for exercise training-induced increases in skeletal muscle abundance of SIRT3 and other mitochondrial proteins. Exercise training for 6.5 weeks increased SIRT3 (p<0.01 and superoxide dismutase 2 (MnSOD; p<0.05 protein abundance in quadriceps muscle of wild-type (WT; n=13-15, but not AMPK α2 kinase dead (KD; n=12-13 mice. We also observed a strong trend for increased MnSOD abundance in exercise-trained skeletal muscle of healthy humans (p=0.051; n=6. To further elucidate a role for AMPK in mediating these effects, we treated WT (n=7-8 and AMPK α2 KD (n=7-9 mice with 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR. Four weeks of daily AICAR injections (500 mg/kg resulted in AMPK-dependent increases in SIRT3 (p<0.05 and MnSOD (p<0.01 in WT, but not AMPK α2 KD mice. We also tested the effect of repeated AICAR treatment on mitochondrial protein levels in mice lacking the transcriptional coactivator peroxisome proliferator-activated receptor γ-coactivator 1α (PGC-1α KO; n=9-10. Skeletal muscle SIRT3 and MnSOD protein abundance was reduced in sedentary PGC-1α KO mice (p<0.01 and AICAR-induced increases in SIRT3 and MnSOD protein abundance was only observed in WT mice (p<0.05. Finally, the acetylation status of SIRT3 target lysine residues on MnSOD (K122 or oligomycin-sensitivity conferring protein (OSCP; K139 was not altered in either mouse or human skeletal muscle in response to acute exercise. We propose an important role for AMPK in regulating mitochondrial function and ROS handling in skeletal muscle in response to exercise training.

  17. Breakfasts Higher in Protein Increase Postprandial Energy Expenditure, Increase Fat Oxidation, and Reduce Hunger in Overweight Children from 8 to 12 Years of Age.

    Science.gov (United States)

    Baum, Jamie I; Gray, Michelle; Binns, Ashley

    2015-10-01

    Currently 1 in every 3 children aged 2-19 y is overweight or obese. Breakfast is a key component of a healthy diet and has the potential to affect children's health. The objective of this study was to determine whether consumption of a protein-based breakfast (PRO) increases postprandial energy metabolism and substrate oxidation, reduces hunger, and reduces food intake at lunch compared with a carbohydrate-based breakfast (CHO) in normal weight (NW) vs. overweight/obese (OW) children. A randomized, crossover-design study was conducted in NW (n = 16; 33 ± 1 kg) and OW (n = 13; 46 ± 2 kg) children (10 ± 1 y). Participants were served either a PRO [344 kcal, 21% protein (18 g), 52% carbohydrate, and 27% fat] or CHO [327 kcal, 4% protein (3 g), 67% carbohydrate, and 29% fat]. Energy expenditure (EE), substrate oxidation, appetite, and blood glucose were measured over a 4 h period. Four hour postprandial participants were provided with access to a lunch buffet and food intake was recorded. After breakfast, OW children in the PRO group had higher (P fat oxidation over the 4 h period than did the NW children in the CHO and PRO groups. There was no difference in postprandial EE or carbohydrate oxidation between the CHO and PRO groups over the 4 h period; however, fat oxidation was 16% higher (P children. A PRO increases postprandial EE and fat oxidation, reduces hunger, and increases satiety when compared with a carbohydrate-based breakfast. © 2015 American Society for Nutrition.

  18. Increase of bcl-2 Protein Expression in Aggressive Basal Cell Carcinoma of Head and Neck

    OpenAIRE

    Cláudia CAZAL; Mariana Roesch ELY; Ana Paula Veras SOBRAL; Wilton Wilney Nascimento PADILHA

    2006-01-01

    Objective: The aim of this study was to verify the bcl-2 protein expression in 22 cutaneous basal cell carcinomas (BCC) of the head and neck, and to compare it with its aggressive behavior. Method: Tumors were histologically classified in non-aggressive (BCC 1) and aggressive (BCC 2) and then submitted to the immunohistochemistry technique with the streptavidin-biotin peroxidase method using the anti-bcl-2 antibody. Results: After proceeding to morphological analysis, sixteen tumors (72.7%) w...

  19. Increased protein intake and meal frequency reduces abdominal fat during energy balance and energy deficit.

    Science.gov (United States)

    Arciero, Paul J; Ormsbee, Michael J; Gentile, Christopher L; Nindl, Bradley C; Brestoff, Jonathan R; Ruby, Maxwell

    2013-07-01

    Unrefined, complex carbohydrates and lean protein diets are used to combat obesity, although it's unknown whether more frequent meals may improve this response. The effects of consuming traditional (~15%) versus higher (~35%) protein intakes as three or six meals/day on abdominal fat, postprandial thermogenesis (TEM), and cardiometabolic biomarkers in overweight individuals during 28 days of energy balance (BAL) and deficit (NEG), respectively were compared. Overweight individuals (n = 30) were randomized into three groups: two high-protein groups (35% of energy) consumed as three (HP3) or six (HP6) meals/day and one group consumed three meals/day of a traditional intake (TD3). Following a 5-day baseline control (CON), subjects consumed their respective diets throughout a 56-day intervention consisting of two, 28 day phases: a BAL followed by a NEG phase (75% of energy needs). Total body fat (BF) and abdominal BF (ABF), body weight (BW), TEM, and fasting biomarkers were assessed at the end of CON, BAL, and NEG phases. BW remained stable throughout CON and BAL in all groups, whereas BF (P meals/day in overweight individuals during both BAL and NEG. Copyright © 2013 The Obesity Society.

  20. Human Protein C produces anticoagulation and increased fibrinolytic activity in the cat

    International Nuclear Information System (INIS)

    Burdick, M.D.; Schaub, R.G.

    1986-01-01

    The effect of activated human Protein C (PCa) infusion on the coagulation and fibrinolytic systems of the Nembutal anesthetized cat was assessed. Human Protein C was activated by incubation with thrombin or by passage over a column of thrombin immobilized on CNBr Sepharose 4B. Cats were given bolus i.v. injections of either vehicle or PCa in a dose range of 3-16 μg/mL of calculated whole body volume. Citrated blood samples (9:1) were taken from a femoral vein prior to and at 5, 10, 20, 30, 60, 120, and 180 min. after PCa. Activated partial thromboplastin time (APTT), thrombin time (TT) euglobulin clot lysis (ECLT) and I-125 fibrin release (FR) was measured. Vehicle treated cats had no change in any parameter. PCa produced a dose and time dependent prolongation of APTT while TT was unchanged. Anticoagulation was evident immediately after PCa infusion and began to normalize within 20 min. Fibrinolytic activity measured by ECLT and FR was also stimulated by PCa but was not evident until 40-60 minutes after PCa injection. The results show that human PCa induces anticoagulation effects in the cat similar to other species. However, stimulation of fibrinolysis requires a longer period of time before expression. This delay of fibrinolytic stimulation should be considered when assessing the effects of human Protein C in other species

  1. Increased signaling by the autism-related Engrailed-2 protein enhances dendritic branching and spine density, alters synaptic structural matching, and exaggerates protein synthesis.

    Science.gov (United States)

    Soltani, Asma; Lebrun, Solène; Carpentier, Gilles; Zunino, Giulia; Chantepie, Sandrine; Maïza, Auriane; Bozzi, Yuri; Desnos, Claire; Darchen, François; Stettler, Olivier

    2017-01-01

    Engrailed 1 (En1) and 2 (En2) code for closely related homeoproteins acting as transcription factors and as signaling molecules that contribute to midbrain and hindbrain patterning, to development and maintenance of monoaminergic pathways, and to retinotectal wiring. En2 has been suggested to be an autism susceptibility gene and individuals with autism display an overexpression of this homeogene but the mechanisms remain unclear. We addressed in the present study the effect of exogenously added En2 on the morphology of hippocampal cells that normally express only low levels of Engrailed proteins. By means of RT-qPCR, we confirmed that En1 and En2 were expressed at low levels in hippocampus and hippocampal neurons, and observed a pronounced decrease in En2 expression at birth and during the first postnatal week, a period characterized by intense synaptogenesis. To address a putative effect of Engrailed in dendritogenesis or synaptogenesis, we added recombinant En1 or En2 proteins to hippocampal cell cultures. Both En1 and En2 treatment increased the complexity of the dendritic tree of glutamatergic neurons, but only En2 increased that of GABAergic cells. En1 increased the density of dendritic spines both in vitro and in vivo. En2 had similar but less pronounced effect on spine density. The number of mature synapses remained unchanged upon En1 treatment but was reduced by En2 treatment, as well as the area of post-synaptic densities. Finally, both En1 and En2 elevated mTORC1 activity and protein synthesis in hippocampal cells, suggesting that some effects of Engrailed proteins may require mRNA translation. Our results indicate that Engrailed proteins can play, even at low concentrations, an active role in the morphogenesis of hippocampal cells. Further, they emphasize the over-regulation of GABA cell morphology and the vulnerability of excitatory synapses in a pathological context of En2 overexpression.

  2. Increased signaling by the autism-related Engrailed-2 protein enhances dendritic branching and spine density, alters synaptic structural matching, and exaggerates protein synthesis.

    Directory of Open Access Journals (Sweden)

    Asma Soltani

    Full Text Available Engrailed 1 (En1 and 2 (En2 code for closely related homeoproteins acting as transcription factors and as signaling molecules that contribute to midbrain and hindbrain patterning, to development and maintenance of monoaminergic pathways, and to retinotectal wiring. En2 has been suggested to be an autism susceptibility gene and individuals with autism display an overexpression of this homeogene but the mechanisms remain unclear. We addressed in the present study the effect of exogenously added En2 on the morphology of hippocampal cells that normally express only low levels of Engrailed proteins. By means of RT-qPCR, we confirmed that En1 and En2 were expressed at low levels in hippocampus and hippocampal neurons, and observed a pronounced decrease in En2 expression at birth and during the first postnatal week, a period characterized by intense synaptogenesis. To address a putative effect of Engrailed in dendritogenesis or synaptogenesis, we added recombinant En1 or En2 proteins to hippocampal cell cultures. Both En1 and En2 treatment increased the complexity of the dendritic tree of glutamatergic neurons, but only En2 increased that of GABAergic cells. En1 increased the density of dendritic spines both in vitro and in vivo. En2 had similar but less pronounced effect on spine density. The number of mature synapses remained unchanged upon En1 treatment but was reduced by En2 treatment, as well as the area of post-synaptic densities. Finally, both En1 and En2 elevated mTORC1 activity and protein synthesis in hippocampal cells, suggesting that some effects of Engrailed proteins may require mRNA translation. Our results indicate that Engrailed proteins can play, even at low concentrations, an active role in the morphogenesis of hippocampal cells. Further, they emphasize the over-regulation of GABA cell morphology and the vulnerability of excitatory synapses in a pathological context of En2 overexpression.

  3. [High-level expression of heterologous protein based on increased copy number in Saccharomyces cerevisiae].

    Science.gov (United States)

    Zhang, Xinjie; He, Peng; Tao, Yong; Yang, Yi

    2013-11-04

    High-level expression system of heterologous protein mediated by internal ribosome entry site (IRES) in Saccharomyces cerevisiae was constructed, which could be used for other applications of S. cerevisiae in metabolic engineering. We constructed co-expression cassette (promoter-mCherry-TIF4631 IRES-URA3) containing promoters Pilv5, Padh2 and Ptdh3 and recombined the co-expression cassette into the genome of W303-1B-A. The URA3+ transformants were selected. By comparing the difference in the mean florescence value of mCherry in transformants, the effect of three promoters was detected in the co-expression cassette. The copy numbers of the interested genes in the genome were determined by Real-Time PCR. We analyzed genetic stability by continuous subculturing transformants in the absence of selection pressure. To verify the application of co-expression cassette, the ORF of mCherry was replaced by beta-galactosidase (LACZ) and xylose reductase (XYL1). The enzyme activities and production of beta-galactosidase and xylose reductase were detected. mCherry has been expressed in the highest-level in transformants with co-expression cassette containing Pilv5 promoter. The highest copy number of DNA fragment integrating in the genome was 47 in transformants containing Pilv5. The engineering strains showed good genetic stability. Xylose reductase was successfully expressed in the co-expression cassette containing Pilv5 promoter and TIF4631 IRES. The highest enzyme activity was 0. 209 U/mg crude protein in the transformants WIX-10. Beta-galactosidase was also expressed successfully. The transformants that had the highest enzyme activity was WIL-1 and the enzyme activity was 12.58 U/mg crude protein. The system mediated by Pilv5 promoter and TIF4631 IRES could express heterologous protein efficiently in S. cerevisiae. This study offered a new strategy for expression of heterologous protein in S. cerevisiae and provided sufficient experimental evidence for metabolic engineering

  4. Macroautophagy-generated increase of lysosomal amyloid β-protein mediates oxidant-induced apoptosis of cultured neuroblastoma cells

    DEFF Research Database (Denmark)

    Zheng, Lin; Terman, Alexei; Hallbeck, Martin

    2011-01-01

    and accumulation of Aβ within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aβ monomers and oligomers, (2) increased reactive oxygen species production...... and resulting lysosomal Aβ accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide additional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration....

  5. Spontaneous nisin-resistant Listeria monocytogenes mutants with increased expression of a putative penicillin-binding protein and their sensitivity to various antibiotics

    DEFF Research Database (Denmark)

    Gravesen, Anne; Sorensen, K.; Aarestrup, Frank Møller

    2001-01-01

    -molecular-weight penicillin-binding proteins (PBPs), a histidine protein kinase, a protein of unknown function, and ClpB (putative functions from homology), The three former proteins had increased expression in a total of six out of 10 independent mutants originating from five different wildtype strains, indicating...

  6. Short term exercise induces PGC-1α, ameliorates inflammation and increases mitochondrial membrane proteins but fails to increase respiratory enzymes in aging diabetic hearts.

    Science.gov (United States)

    Botta, Amy; Laher, Ismail; Beam, Julianne; Decoffe, Daniella; Brown, Kirsty; Halder, Swagata; Devlin, Angela; Gibson, Deanna L; Ghosh, Sanjoy

    2013-01-01

    PGC-1α, a transcriptional coactivator, controls inflammation and mitochondrial gene expression in insulin-sensitive tissues following exercise intervention. However, attributing such effects to PGC-1α is counfounded by exercise-induced fluctuations in blood glucose, insulin or bodyweight in diabetic patients. The goal of this study was to investigate the role of PGC-1α on inflammation and mitochondrial protein expressions in aging db/db mice hearts, independent of changes in glycemic parameters. In 8-month-old db/db mice hearts with diabetes lasting over 22 weeks, short-term, moderate-intensity exercise upregulated PGC-1α without altering body weight or glycemic parameters. Nonetheless, such a regimen lowered both cardiac (macrophage infiltration, iNOS and TNFα) and systemic (circulating chemokines and cytokines) inflammation. Curiously, such an anti-inflammatory effect was also linked to attenuated expression of downstream transcription factors of PGC-1α such as NRF-1 and several respiratory genes. Such mismatch between PGC-1α and its downstream targets was associated with elevated mitochondrial membrane proteins like Tom70 but a concurrent reduction in oxidative phosphorylation protein expressions in exercised db/db hearts. As mitochondrial oxidative stress was predominant in these hearts, in support of our in vivo data, increasing concentrations of H2O2 dose-dependently increased PGC-1α expression while inhibiting expression of inflammatory genes and downstream transcription factors in H9c2 cardiomyocytes in vitro. We conclude that short-term exercise-induced oxidative stress may be key in attenuating cardiac inflammatory genes and impairing PGC-1α mediated gene transcription of downstream transcription factors in type 2 diabetic hearts at an advanced age.

  7. Increased expression of Myosin binding protein H in the skeletal muscle of amyotrophic lateral sclerosis patients

    KAUST Repository

    Conti, Antonio

    2014-01-01

    Amyotrophic lateral sclerosis (ALS) is a severe and fatal neurodegenerative disease of still unknown pathogenesis. Recent findings suggest that the skeletal muscle may play an active pathogenetic role. To investigate ALS\\'s pathogenesis and to seek diagnostic markers, we analyzed skeletal muscle biopsies with the differential expression proteomic approach. We studied skeletal muscle biopsies from healthy controls (CN), sporadic ALS (sALS), motor neuropathies (MN) and myopathies (M). Pre-eminently among several differentially expressed proteins, Myosin binding protein H (MyBP-H) expression in ALS samples was anomalously high. MyBP-H is a component of the thick filaments of the skeletal muscle and has strong affinity for myosin, but its function is still unclear. High MyBP-H expression level was associated with abnormal expression of Rho kinase 2 (ROCK2), LIM domain kinase 1 (LIMK1) and cofilin2, that might affect the actin-myosin interaction. We propose that MyBP-H expression level serves, as a putative biomarker in the skeletal muscle, to discriminate ALS from motor neuropathies, and that it signals the onset of dysregulation in actin-myosin interaction; this in turn might contribute to the pathogenesis of ALS. © 2013 Elsevier B.V.

  8. Increased expression of Myosin binding protein H in the skeletal muscle of amyotrophic lateral sclerosis patients.

    Science.gov (United States)

    Conti, Antonio; Riva, Nilo; Pesca, Mariasabina; Iannaccone, Sandro; Cannistraci, Carlo V; Corbo, Massimo; Previtali, Stefano C; Quattrini, Angelo; Alessio, Massimo

    2014-01-01

    Amyotrophic lateral sclerosis (ALS) is a severe and fatal neurodegenerative disease of still unknown pathogenesis. Recent findings suggest that the skeletal muscle may play an active pathogenetic role. To investigate ALS's pathogenesis and to seek diagnostic markers, we analyzed skeletal muscle biopsies with the differential expression proteomic approach. We studied skeletal muscle biopsies from healthy controls (CN), sporadic ALS (sALS), motor neuropathies (MN) and myopathies (M). Pre-eminently among several differentially expressed proteins, Myosin binding protein H (MyBP-H) expression in ALS samples was anomalously high. MyBP-H is a component of the thick filaments of the skeletal muscle and has strong affinity for myosin, but its function is still unclear. High MyBP-H expression level was associated with abnormal expression of Rho kinase 2 (ROCK2), LIM domain kinase 1 (LIMK1) and cofilin2, that might affect the actin-myosin interaction. We propose that MyBP-H expression level serves, as a putative biomarker in the skeletal muscle, to discriminate ALS from motor neuropathies, and that it signals the onset of dysregulation in actin-myosin interaction; this in turn might contribute to the pathogenesis of ALS. © 2013 Elsevier B.V. All rights reserved.

  9. Apoptosis and necrosis increase antigenicity of proteins recognized by antinuclear antibodies

    Directory of Open Access Journals (Sweden)

    J.J. Bollain-y-Goitia

    2011-09-01

    Full Text Available Obiettivo. Lo scopo di questo studio è quello di indagare se l’apoptosis e la necrosi aumentano l’antigenicità delle proteine riconosciute da anticorpi antinucleo. Materiale e metodi. Cellule HEp-2 sono state coltivate in condizioni standard; l’apoptosis è stata indotta con camptecina e la necrosi con cloruro di mercurio. L’antigenicità delle proteine estratte dalle cellule è stato testata su membrane di nitrocellulosa e sondata con sieri positivi o negativi per anticorpi antinucleo utilizzando un sistema ELISA a luminescenza (luminescent. Risultati. Le alterazioni apoptotiche nelle cellule HEp-2 sono apparse entro 24 ore dall’esposizione alla camptoicina, mentre i segni di necrosi si sono evidenziati più precocemente. La luminescenza si è dimostrata significativamente superiore nei sieri ANA positivi che nei controlli ANA negativi. Gli antcorpi antinucleari sieirici riconoscono meglio gli antigeni da cellule apoptotiche e necrotiche rispetto ai controlli che non hanno subito trattamenti chimici. Conclusioni. L’apoptosi e la necrosi incrementano la capacità legante degli ANA attraverso una migliore disponibilità di antigeni intracellulari o svelando epitopi criptici.

  10. Increased immunogenicity and protective efficacy of influenza M2e fused to a tetramerizing protein.

    Directory of Open Access Journals (Sweden)

    Anne-Marie Carola Andersson

    Full Text Available The ectodomain of the matrix 2 protein (M2e of influenza A virus represents an attractive target for developing a universal influenza A vaccine, with its sequence being highly conserved amongst human variants of this virus. With the aim of targeting conformational epitopes presumably shared by diverse influenza A viruses, a vaccine (M2e-NSP4 was constructed linking M2e (in its consensus sequence to the rotavirus fragment NSP4(98-135; due to its coiled-coil region this fragment is known to form tetramers in aqueous solution and in this manner we hoped to mimick the natural configuration of M2e as presented in membranes. M2e-NSP4 was then evaluated side-by-side with synthetic M2e peptide for its immunogenicity and protective efficacy in a murine influenza challenge model. Here we demonstrate that M2e fused to the tetramerizing protein induces an accelerated, augmented and more broadly reactive antibody response than does M2e peptide as measured in two different assays. Most importantly, vaccination with M2e-NSP4 caused a significant decrease in lung virus load early after challenge with influenza A virus and maintained its efficacy against a lethal challenge even at very low vaccine doses. Based on the results presented in this study M2e-NSP4 merits further investigation as a candidate for or as a component of a universal influenza A vaccine.

  11. A mutation in the centriole-associated protein centrin causes genomic instability via increased chromosome loss in Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Marshall Wallace F

    2005-05-01

    Full Text Available Abstract Background The role of centrioles in mitotic spindle function remains unclear. One approach to investigate mitotic centriole function is to ask whether mutation of centriole-associated proteins can cause genomic instability. Results We addressed the role of the centriole-associated EF-hand protein centrin in genomic stability using a Chlamydomonas reinhardtii centrin mutant that forms acentriolar bipolar spindles and lacks the centrin-based rhizoplast structures that join centrioles to the nucleus. Using a genetic assay for loss of heterozygosity, we found that this centrin mutant showed increased genomic instability compared to wild-type cells, and we determined that the increase in genomic instability was due to a 100-fold increase in chromosome loss rates compared to wild type. Live cell imaging reveals an increased rate in cell death during G1 in haploid cells that is consistent with an elevated rate of chromosome loss, and analysis of cell death versus centriole copy number argues against a role for multipolar spindles in this process. Conclusion The increased chromosome loss rates observed in a centrin mutant that forms acentriolar spindles suggests a role for centrin protein, and possibly centrioles, in mitotic fidelity.

  12. In utero exposure to benzene increases embryonic c-Myb and Pim-1 protein levels in CD-1 mice

    International Nuclear Information System (INIS)

    Wan, Joanne; Winn, Louise M.

    2008-01-01

    Benzene is a known human leukemogen, but its role as an in utero leukemogen remains controversial. Epidemiological studies have correlated parental exposure to benzene with an increased incidence of childhood leukemias. We hypothesize that in utero exposure to benzene may cause leukemogenesis by affecting the embryonic c-Myb/Pim-1 signaling pathway and that this is mediated by oxidative stress. To investigate this hypothesis, pregnant CD-1 mice were treated with either 800 mg/kg of benzene or corn oil (i.p.) on days 10 and 11 of gestation and in some cases pretreated with 25 kU/kg of PEG-catalase. Phosphorylated and total embryonic c-Myb and Pim-1 protein levels were assessed using Western blotting and maternal and embryonic oxidative stress were assessed by measuring reduced to oxidized glutathione ratios. Our results show increased oxidative stress at 4 and 24 h after exposure, increased phosphorylated Pim-1 protein levels 4 h after benzene exposure, and increased Pim-1 levels at 24 and 48 h after benzene exposure. Embryonic c-Myb levels were elevated at 24 h after exposure. PEG-catalase pretreatment prevented benzene-mediated increases in embryonic c-Myb and Pim-1 protein levels, and benzene-induced oxidative stress. These results support a role for ROS in c-Myb and Pim-1 alterations after in utero benzene exposure

  13. Increasing the sampling efficiency of protein conformational transition using velocity-scaling optimized hybrid explicit/implicit solvent REMD simulation

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Yuqi; Wang, Jinan; Shao, Qiang, E-mail: qshao@mail.shcnc.ac.cn, E-mail: Jiye.Shi@ucb.com, E-mail: wlzhu@mail.shcnc.ac.cn; Zhu, Weiliang, E-mail: qshao@mail.shcnc.ac.cn, E-mail: Jiye.Shi@ucb.com, E-mail: wlzhu@mail.shcnc.ac.cn [ACS Key Laboratory of Receptor Research, Drug Discovery and Design Center, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203 (China); Shi, Jiye, E-mail: qshao@mail.shcnc.ac.cn, E-mail: Jiye.Shi@ucb.com, E-mail: wlzhu@mail.shcnc.ac.cn [UCB Pharma, 216 Bath Road, Slough SL1 4EN (United Kingdom)

    2015-03-28

    The application of temperature replica exchange molecular dynamics (REMD) simulation on protein motion is limited by its huge requirement of computational resource, particularly when explicit solvent model is implemented. In the previous study, we developed a velocity-scaling optimized hybrid explicit/implicit solvent REMD method with the hope to reduce the temperature (replica) number on the premise of maintaining high sampling efficiency. In this study, we utilized this method to characterize and energetically identify the conformational transition pathway of a protein model, the N-terminal domain of calmodulin. In comparison to the standard explicit solvent REMD simulation, the hybrid REMD is much less computationally expensive but, meanwhile, gives accurate evaluation of the structural and thermodynamic properties of the conformational transition which are in well agreement with the standard REMD simulation. Therefore, the hybrid REMD could highly increase the computational efficiency and thus expand the application of REMD simulation to larger-size protein systems.

  14. Increase in skeletal muscle protein content by the ß-2 selective adrenergic agonist clenbuterol exacerbates hypoalbuminemia in rats fed a low-protein diet

    Directory of Open Access Journals (Sweden)

    A.L. Sawaya

    1998-06-01

    Full Text Available This investigation examined how the nutritional status of rats fed a low-protein diet was affected when the animals were treated with the ß-2 selective agonist clenbuterol (CL. Males (4 weeks old from an inbred, specific-pathogen-free strain of hooded rats maintained at the Dunn Nutritional Laboratory were used in the experiments (N = 6 rats per group. CL treatment (Ventipulmin, Boehringer-Ingelheim Ltd., 3.2 mg/kg diet for 2 weeks caused an exacerbation of the symptoms associated with protein deficiency in rats. Plasma albumin concentrations, already low in rats fed a low-protein diet (group A, were further reduced in CL rats (A = 25.05 ± 0.31 vs CL = 23.64 ± 0.30 g/l, P<0.05. Total liver protein decreased below the level seen in either pair-fed animals (group P or animals with free access to the low-protein diet (A = 736.56 ± 26 vs CL = 535.41 ± 54 mg, P<0.05, whereas gastrocnemius muscle protein was higher than the values normally described for control (C animals (C = 210.88 ± 3.2 vs CL = 227.14 ± 1.7 mg/g, P<0.05. Clenbuterol-treated rats also showed a reduction in growth when compared to P rats (P = 3.2 ± 1.1 vs CL = -10.2 ± 1.9 g, P<0.05. This was associated with a marked decrease in fat stores (P = 5.35 ± 0.81 vs CL = 2.02 ± 0.16 g, P<0.05. Brown adipose tissue (BAT cytochrome oxidase activity, although slightly lower than in P rats (P = 469.96 ± 16.20 vs CL = 414.48 ± 11.32 U/BAT x kg body weight, P<0.05, was still much higher than in control rats (C = 159.55 ± 11.54 vs CL = 414.48 ± 11.32 U/BAT x kg body weight, P<0.05. The present findings support the hypothesis that an increased muscle protein content due to clenbuterol stimulation worsened amino acid availability to the liver and further reduced albumin synthesis causing exacerbation of hypoalbuminemia in rats fed a low-protein diet.

  15. Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway

    Directory of Open Access Journals (Sweden)

    Kosalková Katarina

    2012-01-01

    Full Text Available Abstract Background The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Results Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold in cells grown with casein or casein phosphopeptides (CPPs. CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% using partially dephosphorylated casein and is not exerted by casamino acids. The phosphopeptides effect was not exerted at transcriptional level, but instead, it was clearly observed on the secretion of chymosin by immunodetection analysis. Proteomics studies revealed very interesting metabolic changes in response to phosphopeptides supplementation. The oxidative metabolism was reduced, since enzymes involved in fermentative processes were overrepresented. An oxygen-binding hemoglobin-like protein was overrepresented in the proteome following phosphopeptides addition. Most interestingly, the intracellular pre-protein enzymes, including pre-prochymosin, were depleted (most of them are underrepresented in the intracellular proteome after the addition of CPPs, whereas the extracellular mature form of several of these secretable proteins and cell-wall biosynthetic enzymes was greatly overrepresented in the secretome of phosphopeptides-supplemented cells. Another important 'moonlighting' protein (glyceraldehyde-3-phosphate dehydrogenase, which has been described to have vesicle fusogenic and cytoskeleton formation modulating activities, was clearly overrepresented in phosphopeptides-supplemented cells. Conclusions In summary, CPPs cause the reprogramming of cellular metabolism, which leads to massive secretion of extracellular proteins.

  16. Multiple reaction monitoring targeted LC-MS analysis of potential cell death marker proteins for increased bioprocess control.

    Science.gov (United States)

    Albrecht, Simone; Kaisermayer, Christian; Reinhart, David; Ambrose, Monica; Kunert, Renate; Lindeberg, Anna; Bones, Jonathan

    2018-05-01

    The monitoring of protein biomarkers for the early prediction of cell stress and death is a valuable tool for process characterization and efficient biomanufacturing control. A representative set of six proteins, namely GPDH, PRDX1, LGALS1, CFL1, TAGLN2 and MDH, which were identified in a previous CHO-K1 cell death model using discovery LC-MS E was translated into a targeted liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM-MS) platform and verified. The universality of the markers was confirmed in a cell growth model for which three Chinese hamster ovary host cell lines (CHO-K1, CHO-S, CHO-DG44) were grown in batch culture in two different types of basal media. LC-MRM-MS was also applied to spent media (n = 39) from four perfusion biomanufacturing series. Stable isotope-labelled peptide analogues and a stable isotope-labelled monoclonal antibody were used for improved protein quantitation and simultaneous monitoring of the workflow reproducibility. Significant increases in protein concentrations were observed for all viability marker proteins upon increased dead cell numbers and allowed for discrimination of spent media with dead cell densities below and above 1 × 10 6  dead cells/mL which highlights the potential of the selected viability marker proteins in bioprocess control. Graphical abstract Overview of the LC-MRM-MS workflow for the determination of proteomic markers in conditioned media from the bioreactor that correlate with CHO cell death.

  17. Increase in tendon protein synthesis in response to insulin-like growth factor-I is preserved in elderly men

    DEFF Research Database (Denmark)

    Nielsen, Rie Harboe; Holm, Lars; Malmgaard-Clausen, Nikolaj Mølkjær

    2014-01-01

    the in vivo IGF-I stimulation of tendon protein synthesis in elderly compared with young men. We injected IGF-I in the patellar tendons of young (n = 11, 20-30 yr of age) and old (n = 11, 66-75 yr of age) men, and the acute fractional synthesis rate (FSR) of tendon protein was measured with the stable isotope.......01). This increase in protein synthesis was seen in both young and old men, with no differences between age groups. The old group had markedly lower serum IGF-I levels compared with young (165 ± 17 vs. 281 ± 27 ng/ml, P protein synthesis in both young and old men...... technique and compared with the contralateral side (injected with saline as control). We found that tendons injected with IGF-I had significantly higher protein FSR compared with controls (old group: 0.018 ± 0.015 vs. 0.008 ± 0.008, young group: 0.016 ± 0.009 vs. 0.009 ± 0.006%/h, mean ± SE, P

  18. Anchoring tick salivary anti-complement proteins IRAC I and IRAC II to membrane increases their immunogenicity.

    Science.gov (United States)

    Gillet, Laurent; Schroeder, Hélène; Mast, Jan; Thirion, Muriel; Renauld, Jean-Christophe; Dewals, Benjamin; Vanderplasschen, Alain

    2009-01-01

    Tick salivary proteins are promising targets for the development of anti-tick vaccines. Recently, we described two paralogous anti-complement proteins, called Ixodes ricinus anti-complement (IRAC) proteins I and II, that are co-expressed in tick I. ricinus salivary glands. However, our previous attempts to immunize rabbits against IRAC via infection with recombinant Bovine herpesvirus 4 (BoHV-4) vectors invariably failed although both recombinants expressed high levels of functional IRAC proteins in vitro. As IRAC are soluble monovalent antigens, one of the possible explanations is that monovalent ligation of the B-cell receptor induces receptor activation but fails to promote antigen presentation, a phenomenon that is thought to induce a state of B-cell tolerance. In the present study, we tried to increase IRAC immunogenicity by expressing them as oligovalent antigens. To this end, IRAC were fused to membrane anchors and BoHV-4 vectors expressing these recombinant forms were produced. The immunization potentials of recombinant viruses expressing either secreted or transmembrane IRAC proteins were then compared. While the former did not induce a detectable immune response against IRAC, the latter led to high titres of anti-IRAC antibodies that only marginally affected tick blood feeding. All together, the data presented in this study demonstrate that the immunogenicity of a soluble antigen can be greatly improved by anchoring it in membrane.

  19. Perinatal nicotine treatment induces transient increases in NACHO protein levels in the rat frontal cortex

    DEFF Research Database (Denmark)

    Wichern, Franziska; Jensen, Majbrit M; Christensen, Ditte Z

    2017-01-01

    The nicotinic acetylcholine receptor (nAChR) regulator chaperone (NACHO) was recently identified as an important regulator of nAChR maturation and surface expression. Here we show that NACHO levels decrease during early postnatal development in rats. This decrease occurs earlier and to a greater...... degree in the frontal cortex (FC) compared with the hippocampus (HIP). We further show that rats exposed to nicotine during pre- and postnatal development exhibit significantly higher NACHO levels in the FC at postnatal day (PND) 21, but not at PND60. Repeated exposure to nicotine selectively during...... a single exposure to a combination of nicotine and the type II α7 nAChR positive allosteric modulator (PAM) PNU-120596, but not the type I PAM AVL-3288. These findings suggest that exposure to nAChR agonism affects NACHO protein levels, and that this effect is more pronounced during pre- or early postnatal...

  20. Effect of increased CRM₁₉₇ carrier protein dose on meningococcal C bactericidal antibody response.

    Science.gov (United States)

    Lee, Lucia H; Blake, Milan S

    2012-04-01

    New multivalent CRM(197)-based conjugate vaccines are available for childhood immunization. Clinical studies were reviewed to assess meningococcal group C (MenC) antibody responses following MenC-CRM(197) coadministration with CRM(197)-based pneumococcal or Haemophilus influenzae type b conjugate vaccines. Infants receiving a total CRM(197) carrier protein dose of ∼50 μg and concomitant diphtheria-tetanus-acellular pertussis (DTaP)-containing vaccine tended to have lower MenC geometric mean antibody titers and continued to have low titers after the toddler dose. Nevertheless, at least 95% of children in the reported studies achieved a MenC serum bactericidal antibody (SBA) titer of ≥ 1:8 after the last infant or toddler dose. SBA was measured using an assay with a baby rabbit or human complement source. Additional studies are needed to assess long-term antibody persistence and MenC CRM(197) conjugate vaccine immunogenicity using alternative dosing schedules.

  1. Ingestive behavior of grazing steers fed increasing levels of concentrate supplementation with different crude protein contents.

    Science.gov (United States)

    Mendes, Fabrício Bacelar Lima; Silva, Robério Rodrigues; de Carvalho, Gleidson Giordano Pinto; da Silva, Fabiano Ferreira; Lins, Túlio Otávio Jardim D Almeida; da Silva, Anderson Luiz Nascimento; Macedo, Venício; Abreu Filho, George; de Souza, Sinvaldo Oliveira; Guimarães, Joanderson Oliveira

    2015-02-01

    This study aimed to evaluate the ingestive behavior of steers on Brachiaria brizantha pasture fed diets with increasing levels of concentrate supplementation. Thirty-two crossbred steers in the finishing phase with average weight of 420 ± 8 kg were distributed in a completely randomized design with four treatments and eight replicates per treatment. Their behavior was assessed every 5 min for 24 h, in the middle of the experimental period. Variance and regression analyses at 0.05 % probability were adopted. The times spent grazing and ruminating reduced linearly (P <0.05), whereas the times spent at the trough (eating) and on other activities increased linearly (P <0.05) as the supplementation levels were elevated. The total feeding and chewing times decreased linearly (P <0.05) as the concentrate levels in the diet were elevated. By increasing the supplementation levels, the number of bites per day decreased linearly (P <0.05), and the feed efficiency of dry matter increased quadratically. Rumination efficiency of dry matter increased linearly (P <0.05) with increasing levels of concentrate supplementation. Grazing and rumination activities are reduced when the time devoted to other activities and at the trough are increased, as a result of the substitution effect.

  2. Deleting the 14-3-3 protein Bmh1 extends life span in Saccharomyces cerevisiae by increasing stress response.

    Science.gov (United States)

    Wang, Chen; Skinner, Craig; Easlon, Erin; Lin, Su-Ju

    2009-12-01

    Enhanced stress response has been suggested to promote longevity in many species. Calorie restriction (CR) and conserved nutrient-sensing target of rapamycin (TOR) and protein kinase A (PKA) pathways have also been suggested to extend life span by increasing stress response, which protects cells from age-dependent accumulation of oxidative damages. Here we show that deleting the yeast 14-3-3 protein, Bmh1, extends chronological life span (CLS) by activating the stress response. 14-3-3 proteins are highly conserved chaperone-like proteins that play important roles in many cellular processes. bmh1Delta-induced heat resistance and CLS extension require the general stress-response transcription factors Msn2, Msn4, and Rim15. The bmh1Delta mutant also displays a decreased reactive oxygen species level and increased heat-shock-element-driven transcription activity. We also show that BMH1 genetically interacts with CR and conserved nutrient-sensing TOR- and PKA-signaling pathways to regulate life span. Interestingly, the level of phosphorylated Ser238 on Bmh1 increases during chronological aging, which is delayed by CR or by reduced TOR activities. In addition, we demonstrate that PKA can directly phosphorylate Ser238 on Bmh1. The status of Bmh1 phosphorylation is therefore likely to play important roles in life-span regulation. Together, our studies suggest that phosphorylated Bmh1 may cause inhibitory effects on downstream longevity factors, including stress-response proteins. Deleting Bmh1 may eliminate the inhibitory effects of Bmh1 on these longevity factors and therefore extends life span.

  3. Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

    International Nuclear Information System (INIS)

    Chen, Yi; Pirisi, Lucia; Creek, Kim E.

    2013-01-01

    We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer. - Highlights: • Ski oncoprotein levels increase during progression of HPV16-transformed cells. • Ski and phospho-Ski protein levels are cell cycle dependent. • Ski knock-down in HPV16-transformed keratinocytes inhibited cell proliferation. • Cervical cancer samples overexpress Ski

  4. Effects of Increasing Prepartum Dietary Protein Level Using Poultry by-Product Meal on Productive Performance and Health of Multiparous

    Directory of Open Access Journals (Sweden)

    M Hossein Yazdi

    2011-12-01

    Full Text Available The aim of this study was to compare the effects of two levels of crude protein using poultry by-product meals (PBPM fed during late gestation on the performance, blood metabolites, and colostrum composition of Holstein dairy cows. Sixteen multiparous cows 26±6 d before expected calving were assigned randomly to two treatments containing 1 14% and 2 16% crude protein. The cow’s BCS was 3.56 ± 0.5 on average, at the beginning of the trial. Yields of milk, protein, lactose, fat, and SNF were not affected by prepartum dietary CP level. Colostrum composition (fat, CP and Total solids, blood metabolites (Ca, Glucose, Total protein, Albumin, Globulin and Urea N, and metabolic diseases incidence were not influenced by prepartum dietary CP level. There was no significant difference between treatments in body weight and BCS changes. As expected, blood urea N before calving was higher in the cows fed 16% CP diets. Serum cholesterol during prepartum and postpartum periods was significantly decreased as the CP increased in the diet. In general, although postpartum glucose level increased in cows which received 16% CP in the diet, it seems that no other obvious advantages over feeding the 14% CP diet are apparent. So feeding this last diet is recommended to close up cows.

  5. Chemokine (C-X-C) ligand 1 (CXCL1) protein expression is increased in aggressive bladder cancers

    International Nuclear Information System (INIS)

    Miyake, Makito; Lawton, Adrienne; Goodison, Steve; Urquidi, Virginia; Gomes-Giacoia, Evan; Zhang, Ge; Ross, Shanti; Kim, Jeongsoon; Rosser, Charles J

    2013-01-01

    Chemokines, including chemokine (C-X-C motif) ligand 1 (CXCL1), may regulate tumor epithelial-stromal interactions that facilitate tumor growth and invasion. Studies have linked CXCL1 expression to gastric, colon and skin cancers, but limited studies to date have described CXCL1 protein expression in human bladder cancer (BCa). CXCL1 protein expression was examined in 152 bladder tissue specimens (142 BCa) by immunohistochemical staining. The expression of CXCL1 was scored by assigning a combined score based on the proportion of cells staining and intensity of staining. CXCL1 expression patterns were correlated with clinicopathological features and follow-up data. CXCL1 protein expression was present in cancerous tissues, but was entirely absent in benign tissue. CXCL1 combined immunostaining score was significantly higher in high-grade tumors relative to low-grade tumors (p = 0.012). Similarly, CXCL1 combined immunostaining score was higher in high stage tumors (T2-T4) than in low stage tumors (Ta-T1) (p < 0.0001). An increase in the combined immunostaining score of CXCL1 was also associated with reduced disease-specific survival. To date, this is the largest study describing increased CXCL1 protein expression in more aggressive phenotypes in human BCa. Further studies are warranted to define the role CXCL1 plays in bladder carcinogenesis and progression

  6. Methods for increasing production of 3-methyl-2-butenol using fusion proteins

    Energy Technology Data Exchange (ETDEWEB)

    Chou, Howard; Keasling, Jay D.

    2017-04-25

    The present invention relates to methods and compositions for increasing production of 3-methyl-2-butenol in a de novo synthetic pathway in a genetically modified host cell using isopentenyl disphosphate (IPP) as a substrate.

  7. Gestational Age-Dependent Increase of Survival Motor Neuron Protein in Umbilical Cord-Derived Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Sota Iwatani

    2017-09-01

    Full Text Available BackgroundSpinal muscular atrophy (SMA is the most common genetic neurological disease leading to infant death. It is caused by loss of survival motor neuron (SMN 1 gene and subsequent reduction of SMN protein in motor neurons. Because SMN is ubiquitously expressed and functionally linked to general RNA metabolism pathway, fibroblasts (FBs are most widely used for the assessment of SMN expression in SMA patients but usually isolated from skin biopsy samples after the onset of overt symptoms. Although recent translational studies of SMN-targeted therapies have revealed the very limited time window for effective SMA therapies during perinatal period, the exact time point when SMN shortage became evident is unknown in human samples. In this study, we analyzed SMN mRNA and protein expression during perinatal period by using umbilical cord-derived mesenchymal stem cells (UC-MSCs obtained from preterm and term infants.MethodsUC-MSCs were isolated from 16 control infants delivered at 22–40 weeks of gestation and SMA fetus aborted at 19 weeks of gestation (UC-MSC-Control and UC-MSC-SMA. FBs were isolated from control volunteer and SMA patient (FB-Control and FB-SMA. SMN mRNA and protein expression in UC-MSCs and FBs was determined by RT-qPCR and Western blot.ResultsUC-MSC-Control and UC-MSC-SMA expressed the comparable level of MSC markers on their cell surface and were able to differentiate into adipocytes, osteocytes, and chondrocytes. At steady state, SMN mRNA and protein expression was decreased in UC-MSC-SMA compared to UC-MSC-Control, as observed in FB-SMA and FB-Control. In response to histone deacetylase inhibitor valproic acid, SMN mRNA and protein expression in UC-MSC-SMA and FB-SMA was increased. During perinatal development from 22 to 40 weeks of gestation, SMN mRNA and protein expression in UC-MSC-Control was positively correlated with gestational age.ConclusionUC-MSCs isolated from 17 fetus/infant of 19–40 weeks of gestation

  8. The effects of increased dietary protein yogurt snack in the afternoon on appetite control and eating initiation in healthy women.

    Science.gov (United States)

    Ortinau, Laura C; Culp, Julie M; Hoertel, Heather A; Douglas, Steve M; Leidy, Heather J

    2013-06-06

    A large portion of daily intake comes from snacking. One of the increasingly common, healthier snacks includes Greek-style yogurt, which is typically higher in protein than regular yogurt. This study evaluated whether a 160 kcal higher-protein (HP) Greek-style yogurt snack improves appetite control, satiety, and delays subsequent eating compared to an isocaloric normal protein (NP) regular yogurt in healthy women. This study also identified the factors that predict the onset of eating. Thirty-two healthy women (age: 27 ± 2y; BMI: 23.0 ± 0.4 kg/m2) completed the acute, randomized crossover-design study. On separate days, participants came to our facility to consume a standardized lunch followed by the consumption of the NP (5.0 g protein) or HP (14.0 g protein) yogurt at 3 h post-lunch. Perceived hunger and fullness were assessed throughout the afternoon until dinner was voluntarily requested; ad libitum dinner was then provided. Snacking led to reductions in hunger and increases in fullness. No differences in post-snack perceived hunger or fullness were observed between the NP and HP yogurt snacks. Dinner was voluntarily requested at approximately 2:40 ± 0:05 h post-snack with no differences between the HP vs. NP yogurts. Ad libitum dinner intake was not different between the snacks (NP: 686 ± 33 kcal vs. HP: 709 ± 34 kcal; p = 0.324). In identifying key factors that predict eating initiation, perceived hunger, fullness, and habitual dinner time accounted for 30% of the variability of time to dinner request (r = 0.55; p < 0.001). The additional 9 g of protein contained in the high protein Greek yogurt was insufficient to elicit protein-related improvements in markers of energy intake regulation.

  9. The effects of increased dietary protein yogurt snack in the afternoon on appetite control and eating initiation in healthy women

    Science.gov (United States)

    2013-01-01

    Background A large portion of daily intake comes from snacking. One of the increasingly common, healthier snacks includes Greek-style yogurt, which is typically higher in protein than regular yogurt. This study evaluated whether a 160 kcal higher-protein (HP) Greek-style yogurt snack improves appetite control, satiety, and delays subsequent eating compared to an isocaloric normal protein (NP) regular yogurt in healthy women. This study also identified the factors that predict the onset of eating. Findings Thirty-two healthy women (age: 27 ± 2y; BMI: 23.0 ± 0.4 kg/m2) completed the acute, randomized crossover-design study. On separate days, participants came to our facility to consume a standardized lunch followed by the consumption of the NP (5.0 g protein) or HP (14.0 g protein) yogurt at 3 h post-lunch. Perceived hunger and fullness were assessed throughout the afternoon until dinner was voluntarily requested; ad libitum dinner was then provided. Snacking led to reductions in hunger and increases in fullness. No differences in post-snack perceived hunger or fullness were observed between the NP and HP yogurt snacks. Dinner was voluntarily requested at approximately 2:40 ± 0:05 h post-snack with no differences between the HP vs. NP yogurts. Ad libitum dinner intake was not different between the snacks (NP: 686 ± 33 kcal vs. HP: 709 ± 34 kcal; p = 0.324). In identifying key factors that predict eating initiation, perceived hunger, fullness, and habitual dinner time accounted for 30% of the variability of time to dinner request (r = 0.55; p < 0.001). Conclusions The additional 9 g of protein contained in the high protein Greek yogurt was insufficient to elicit protein-related improvements in markers of energy intake regulation. PMID:23742659

  10. Protein and glucogenic precursor supplementation: a nutritional strategy to increase reproductive and economic output.

    Science.gov (United States)

    Mulliniks, J T; Cox, S H; Kemp, M E; Endecott, R L; Waterman, R C; Vanleeuwen, D M; Torell, L A; Petersen, M K

    2011-10-01

    Reproductive performance in young beef cows is often compromised due to a mismatch of physiological demands and suboptimal environmental conditions. Studies conducted at the Corona Range and Livestock Research Center from 2000 to 2007 evaluated 3 postpartum supplement strategies that varied in the amount of glucogenic potential (GP) supplied. Reproductive variables, milk production, and serum metabolites were used to assess supplement effectiveness and economics associated with 2- and 3-yr-old beef cows (n = 379) grazing native range. Supplements were individually fed twice/week at 1,135 g/d (2003 to 2004) or 908 g/d (all other years) and provided 1) 327 g of CP, 109 to 118 g of RUP (CON); 2) 327 to 341 g of CP, 142 to 157 g of RUP (RUP); or 3) 327 g of CP, 151 to 173 g of RUP + 40 to 100 g of propionate salt (PS; RUP+PS). Ultimately, total GP for CON, RUP, and RUP+PS was 44 to 47, 57 to 70, and 93 to 141 g, respectively. Blood samples were collected once/week (2000) or twice/week (2001 to 2007) for progesterone analysis to estimate days to resumption of estrus. Cows were exposed to bulls for 60 d or less, and pregnancy was confirmed by rectal palpation at weaning. Days to resumption of estrus after calving decreased linearly (P = 0.02), resulting in an increased pregnancy rate (P = 0.03) with increasing GP. Milk production exhibited a quadratic (P = 0.04) response to increasing GP, with cows fed RUP producing the most amount of milk. However, a linear decrease (P = 0.07) in days from BW nadir to estrus was found with increasing GP. Total kilograms of calf weaned per cow exposed for the supplemental year and subsequent year was increased linearly (P = 0.07) with increased GP. The improvement in pregnancy rate by supplementing RUP+PS resulted in an increase in total revenue of 18% compared with CON-fed cows and 9.5% compared with RUP-fed cows in the subsequent year after supplementation. These data suggest feeding young cows additional GP in the form of PS allows

  11. Increased friction coefficient and superficial zone protein expression in patients with advanced osteoarthritis.

    Science.gov (United States)

    Neu, C P; Reddi, A H; Komvopoulos, K; Schmid, T M; Di Cesare, P E

    2010-09-01

    To quantify the concentration of superficial zone protein (SZP) in the articular cartilage and synovial fluid of patients with advanced osteoarthritis (OA) and to further correlate the SZP content with the friction coefficient, OA severity, and levels of proinflammatory cytokines. Samples of articular cartilage and synovial fluid were obtained from patients undergoing elective total knee replacement surgery. Additional normal samples were obtained from donated body program and tissue bank sources. Regional SZP expression in cartilage obtained from the femoral condyles was quantified by enzyme-linked immunosorbent assay (ELISA) and visualized by immunohistochemistry. Friction coefficient measurements of cartilage plugs slid in the boundary lubrication system were obtained. OA severity was graded using histochemical analyses. The concentrations of SZP and proinflammatory cytokines in synovial fluid were determined by ELISA. A pattern of SZP localization in knee cartilage was identified, with load-bearing regions exhibiting high SZP expression. SZP expression patterns were correlated with friction coefficient and OA severity; however, SZP expression was observed in all samples at the articular surface, regardless of OA severity. SZP expression and aspirate volume of synovial fluid were higher in OA patients than in normal controls. Expression of cytokines was elevated in the synovial fluid of some patients. Our findings indicate a mechanochemical coupling in which physical forces regulate OA severity and joint lubrication. The findings of this study also suggest that SZP may be ineffective in reducing joint friction in the boundary lubrication mode at an advanced stage of OA, where other mechanisms may dominate the observed tribological behavior.

  12. Formoterol attenuates increased oxidative stress and myosin protein loss in respiratory and limb muscles of cancer cachectic rats

    Directory of Open Access Journals (Sweden)

    Anna Salazar-Degracia

    2017-12-01

    Full Text Available Muscle mass loss and wasting are characteristic features of patients with chronic conditions including cancer. Therapeutic options are still scarce. We hypothesized that cachexia-induced muscle oxidative stress may be attenuated in response to treatment with beta2-adrenoceptor-selective agonist formoterol in rats. In diaphragm and gastrocnemius of tumor-bearing rats (108 AH-130 Yoshida ascites hepatoma cells inoculated intraperitoneally with and without treatment with formoterol (0.3 mg/kg body weight/day for seven days, daily subcutaneous injection, redox balance (protein oxidation and nitration and antioxidants and muscle proteins (1-dimensional immunoblots, carbonylated proteins (2-dimensional immunoblots, inflammatory cells (immunohistochemistry, and mitochondrial respiratory chain (MRC complex activities were explored. In the gastrocnemius, but not the diaphragm, of cancer cachectic rats compared to the controls, protein oxidation and nitration levels were increased, several functional and structural proteins were carbonylated, and in both study muscles, myosin content was reduced, inflammatory cell counts were greater, while no significant differences were seen in MRC complex activities (I, II, and IV. Treatment of cachectic rats with formoterol attenuated all the events in both respiratory and limb muscles. In this in vivo model of cancer-cachectic rats, the diaphragm is more resistant to oxidative stress. Formoterol treatment attenuated the rise in oxidative stress in the limb muscles, inflammatory cell infiltration, and the loss of myosin content seen in both study muscles, whereas no effects were observed in the MRC complex activities. These findings have therapeutic implications as they demonstrate beneficial effects of the beta2 agonist through decreased protein oxidation and inflammation in cachectic muscles, especially the gastrocnemius.

  13. ROLE OF TYROSINE-SULFATED PROTEINS IN RETINAL STRUCTURE AND FUNCTION

    Science.gov (United States)

    Kanan, Y.; Al-Ubaidi, M.R.

    2014-01-01

    The extracellular matrix (ECM) plays a significant role in cellular and retinal health. The study of retinal tyrosine-sulfated proteins is an important first step toward understanding the role of ECM in retinal health and diseases. These secreted proteins are members of the retinal ECM. Tyrosine sulfation was shown to be necessary for the development of proper retinal structure and function. The importance of tyrosine sulfation is further demonstrated by the evolutionary presence of tyrosylprotein sulfotransferases, enzymes that catalyze proteins’ tyrosine sulfation, and the compensatory abilities of these enzymes. Research has identified four tyrosine-sulfated retinal proteins: fibulin 2, vitronectin, complement factor H (CFH), and opticin. Vitronectin and CFH regulate the activation of the complement system and are involved in the etiology of some cases of age-related macular degeneration. Analysis of the role of tyrosine sulfation in fibulin function showed that sulfation influences the protein's ability to regulate growth and migration. Although opticin was recently shown to exhibit anti-angiogenic properties, it is not yet determined what role sulfation plays in that function. Future studies focusing on identifying all of the tyrosine-sulfated retinal proteins would be instrumental in determining the impact of sulfation on retinal protein function in retinal homeostasis and diseases. PMID:25819460

  14. Micro-RNA 10a Is Increased in Feline T Regulatory Cells and Increases Foxp3 Protein Expression Following In Vitro Transfection

    Directory of Open Access Journals (Sweden)

    Yan Wang

    2017-02-01

    Full Text Available CD4+CD25+Foxp3+ T regulatory (Treg cells are activated during the course of lentiviral infection and exhibit heightened suppressor function when compared to Treg cells from uninfected controls. Foxp3 is essential to Treg cell function and multiple studies have documented that lentivirus-activated Treg cells exhibit heightened Foxp3 expression when compared to Treg cells from uninfected controls. Our hypothesis was that lentivirus-induced micro-RNAs (miRNAs contribute to heightened Treg cell suppressor function by stabilizing Foxp3 expression. We demonstrated that CD4+CD25+ T cells from both feline immunodeficiency virus infected (FIV+ cats and uninfected control cats exhibit increased miRNA 10a and 21 levels compared to autologous CD4+CD25− T cells but there was no difference in the levels of these miRNAs when Treg cells from FIV+ cats were compared to Treg cells from uninfected controls. Further, there was no increase in Foxp3 mRNA following transfection of miRNA 10a or 21 into a feline cell line. However, transfection with miRNA 10a resulted in increased Foxp3 protein expression.

  15. In anemia of multiple myeloma hepcidin is induced by increased bone-morphogenetic protein-2

    Science.gov (United States)

    Hepcidin is the principal iron-regulatory hormone and pathogenic factor in anemia of inflammation. Patients with multiple myeloma (MM) frequently present with anemia. We showed that MM patients had increased serum hepcidin, which inversely correlated with hemoglobin, suggesting that hepcidin contrib...

  16. Does high C-reactive protein concentration increase atherosclerosis? The Whitehall II Study.

    Directory of Open Access Journals (Sweden)

    Mika Kivimäki

    Full Text Available BACKGROUND: C-reactive protein (CRP, a marker of systemic inflammation, is associated with risk of coronary events and sub-clinical measures of atherosclerosis. Evidence in support of this link being causal would include an association robust to adjustments for confounders (multivariable standard regression analysis and the association of CRP gene polymorphisms with atherosclerosis (Mendelian randomization analysis. METHODOLOGY/PRINCIPAL FINDINGS: We genotyped 3 tag single nucleotide polymorphisms (SNPs [+1444T>C (rs1130864; +2303G>A (rs1205 and +4899T>G (rs 3093077] in the CRP gene and assessed CRP and carotid intima-media thickness (CIMT, a structural marker of atherosclerosis, in 4941 men and women aged 50-74 (mean 61 years (the Whitehall II Study. The 4 major haplotypes from the SNPs were consistently associated with CRP level, but not with other risk factors that might confound the association between CRP and CIMT. CRP, assessed both at mean age 49 and at mean age 61, was associated both with CIMT in age and sex adjusted standard regression analyses and with potential confounding factors. However, the association of CRP with CIMT attenuated to the null with adjustment for confounding factors in both prospective and cross-sectional analyses. When examined using genetic variants as the instrument for serum CRP, there was no inferred association between CRP and CIMT. CONCLUSIONS/SIGNIFICANCE: Both multivariable standard regression analysis and Mendelian randomization analysis suggest that the association of CRP with carotid atheroma indexed by CIMT may not be causal.

  17. Mutant HFE H63D Protein Is Associated with Prolonged Endoplasmic Reticulum Stress and Increased Neuronal Vulnerability*

    Science.gov (United States)

    Liu, Yiting; Lee, Sang Y.; Neely, Elizabeth; Nandar, Wint; Moyo, Mthabisi; Simmons, Zachary; Connor, James R.

    2011-01-01

    A specific polymorphism in the hemochromatosis (HFE) gene, H63D, is over-represented in neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer disease. Mutations of HFE are best known as being associated with cellular iron overload, but the mechanism by which HFE H63D might increase the risk of neuron degeneration is unclear. Here, using an inducible expression cell model developed from a human neuronal cell line SH-SY5Y, we reported that the presence of the HFE H63D protein activated the unfolded protein response (UPR). This response was followed by a persistent endoplasmic reticulum (ER) stress, as the signals of UPR sensors attenuated and followed by up-regulation of caspase-3 cleavage and activity. Our in vitro findings were recapitulated in a transgenic mouse model carrying Hfe H67D, the mouse equivalent of the human H63D mutation. In this model, UPR activation was detected in the lumbar spinal cord at 6 months then declined at 12 months in association with increased caspase-3 cleavage. Moreover, upon the prolonged ER stress, the number of cells expressing HFE H63D in early apoptosis was increased moderately. Cell proliferation was decreased without increased cell death. Additionally, despite increased iron level in cells carrying HFE H63D, it appeared that ER stress was not responsive to the change of cellular iron status. Overall, our studies indicate that the HFE H63D mutant protein is associated with prolonged ER stress and chronically increased neuronal vulnerability. PMID:21349849

  18. Sterol regulatory element binding protein-1 (SREBP1) gene expression is similarly increased in polycystic ovary syndrome and endometrial cancer.

    Science.gov (United States)

    Shafiee, Mohamad N; Mongan, Nigel; Seedhouse, Claire; Chapman, Caroline; Deen, Suha; Abu, Jafaru; Atiomo, William

    2017-05-01

    Women with polycystic ovary syndrome have a three-fold higher risk of endometrial cancer. Insulin resistance and hyperlipidemia may be pertinent factors in the pathogenesis of both conditions. The aim of this study was to investigate endometrial sterol regulatory element binding protein-1 gene expression in polycystic ovary syndrome and endometrial cancer endometrium, and to correlate endometrial sterol regulatory element binding protein-1 gene expression with serum lipid profiles. A cross-sectional study was performed at Nottingham University Hospital, UK. A total of 102 women (polycystic ovary syndrome, endometrial cancer and controls; 34 participants in each group) were recruited. Clinical and biochemical assessments were performed before endometrial biopsies were obtained from all participants. Taqman real-time polymerase chain reaction for endometrial sterol regulatory element binding protein-1 gene and its systemic protein expression were analyzed. The body mass indices of women with polycystic ovary syndrome (29.28 ± 2.91 kg/m 2 ) and controls (28.58 ± 2.62 kg/m 2 ) were not significantly different. Women with endometrial cancer had a higher mean body mass index (32.22 ± 5.70 kg/m 2 ). Sterol regulatory element binding protein-1 gene expression was significantly increased in polycystic ovary syndrome and endometrial cancer endometrium compared with controls (p ovary syndrome, but this was not statistically significant. Similarly, statistically insignificant positive correlations were found between endometrial sterol regulatory element binding protein-1 gene expression and body mass index in endometrial cancer (r = 0.643, p = 0.06) and waist-hip ratio (r = 0.096, p = 0.073). Sterol regulatory element binding protein-1 gene expression was significantly positively correlated with triglyceride in both polycystic ovary syndrome and endometrial cancer (p = 0.028 and p = 0.027, respectively). Quantitative serum sterol regulatory element

  19. Increased expression of p62/SQSTM1 in prion diseases and its association with pathogenic prion protein.

    Science.gov (United States)

    Homma, Takujiro; Ishibashi, Daisuke; Nakagaki, Takehiro; Satoh, Katsuya; Sano, Kazunori; Atarashi, Ryuichiro; Nishida, Noriyuki

    2014-03-28

    Prion diseases are neurodegenerative disorders characterized by the aggregation of abnormally folded prion protein (PrP(Sc)). In this study, we focused on the mechanism of clearance of PrP(Sc), which remains unclear. p62 is a cytosolic protein known to mediate both the formation and degradation of aggregates of abnormal proteins. The levels of p62 protein increased in prion-infected brains and persistently infected cell cultures. Upon proteasome inhibition, p62 co-localized with PrP(Sc), forming a large aggregate in the perinuclear region, hereafter referred to as PrP(Sc)-aggresome. These aggregates were surrounded with autophagosome marker LC3 and lysosomes in prion-infected cells. Moreover, transient expression of the phosphomimic form of p62, which has enhanced ubiquitin-binding activity, reduced the amount of PrP(Sc) in prion-infected cells, indicating that the activation of p62 could accelerate the clearance of PrP(Sc). Our findings would thus suggest that p62 could be a target for the therapeutic control of prion diseases.

  20. Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes.

    Science.gov (United States)

    Csermely, P; Szamel, M; Resch, K; Somogyi, J

    1988-05-15

    In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested (Parker, P.J., Coussens, L., Totty, N., Rhee, L., Young, S., Chen, E., Stabel, S., Waterfield, M.D., and Ullrich, A. (1986) Science 233, 853-859). In the present report, we demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes, and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn2+, while Fe2+ and Mn2+ are only partially counteractive. Our results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca2+, phorbol ester, or antigen.

  1. 11C-PBR28 binding to translocator protein increases with progression of Alzheimer’s disease

    OpenAIRE

    Kreisl, William C.; Lyoo, Chul Hyoung; Liow, Jeih-San; Wei, Monica; Snow, Joseph; Page, Emily; Jenko, Kimberly J.; Morse, Cheryl L.; Zoghbi, Sami S.; Pike, Victor W.; Turner, R. Scott; Innis, Robert B.

    2016-01-01

    This longitudinal study sought to determine whether the 18 kDa translocator protein (TSPO), a marker of neuroinflammation, increases over time in Alzheimer’s disease. Positron emission tomography (PET) imaging with the TSPO radioligand 11C-PBR28 imaging was performed at baseline and after a median follow-up of 2.7 years in 14 amyloid-positive patients and eight amyloid-negative controls. Patients had a greater increase in TSPO binding than controls in inferior parietal lobul...

  2. Increased protein expression of LHCG receptor and 17α-hydroxylase/17-20-lyase in human polycystic ovaries.

    Science.gov (United States)

    Comim, F V; Teerds, K; Hardy, K; Franks, S

    2013-11-01

    Does the expression of LHCG receptor (LHCGR) protein and key enzymes in the androgen biosynthetic pathway differ in normal human versus polycystic ovarian tissue? LHCGR and 17α-hydroxylase/17-20-lyase (CYP17A1) protein levels are increased in polycystic ovaries (PCOs). The predominant source of excess androgen secretion in women with polycystic ovary syndrome (PCOS) is ovarian theca cells but few studies have directly assessed the presence and abundance of protein for key molecules involved in androgen production by theca, including LHCGR and the rate-limiting enzyme in androgen production, CYP17A1. This is a laboratory-based, cross-sectional study comparing protein expression of key molecules in the androgen biosynthetic pathway in archived ovarian tissue from women with normal ovaries (n = 10) with those with PCOs (n = 16). A quantitative morphometric study was performed using sections of archived human ovaries (n = 26) previously characterized as normal or polycystic. The distribution and abundance of LHCGR, CYP17A1, 3β-hydroxysteroid dehydrogenase type 2 (3βHSDII) and 17β-hydroxysteroid dehydrogenase type 5 (17βHSD5) proteins were evaluated by immunohistochemistry and quantified. A higher proportion of theca cells from anovulatory PCO expressed LHCGR protein when compared with control ovaries (P = 0.01). A significant increase in the intensity of immunostaining for CYP17A1 was identified in antral follicles in sections of PCO compared with ovaries from normal women (P = 0.04). As the study used formalin-fixed ovarian tissue sections, it was not possible to carry out studies 'in vitro' using the same ovarian tissues in order to also demonstrate increased functional activity of LHCGR and CYP17A1. The data are in keeping with the results of previous studies in isolated theca cells and support the notion of an intrinsic abnormality of theca cell androgen production in women with PCOS. The research was supported by a Programme Grant, G0802782, from the Medical

  3. Childhood maltreatment is associated with increased body mass index and increased C-reactive protein levels in first-episode psychosis patients.

    Science.gov (United States)

    Hepgul, N; Pariante, C M; Dipasquale, S; DiForti, M; Taylor, H; Marques, T R; Morgan, C; Dazzan, P; Murray, R M; Mondelli, V

    2012-09-01

    The high incidence of the metabolic syndrome in patients with psychosis is mainly attributed to antipsychotic treatment. However, it is also possible that psychological stress plays a role, inducing a chronic inflammatory process that may predispose to the development of metabolic abnormalities. We investigated the association between childhood maltreatment and inflammatory and metabolic biomarkers in subjects with first-episode psychosis and healthy controls. Body mass index (BMI), weight and waist circumference were measured in 95 first-episode psychosis patients and 97 healthy controls. Inflammatory and metabolic markers were measured in a subsample of 28 patients and 45 controls. In all the subjects we collected information on childhood maltreatment and recent stressors. Patients with childhood maltreatment had higher BMI [25.0 (S.E.=0.6) kg/m2] and C-reactive protein (CRP) levels [1.1 (S.E.=0.6) mg/dl] when compared with healthy controls [23.4 (S.E.=0.4) kg/m2, p=0.030 and 0.2 (S.E.=0.1) mg/dl, p=0.009, respectively]. In contrast, patients without childhood maltreatment were not significantly different from healthy controls for either BMI [24.7 (S.E.=0.6) kg/m2, p=0.07] or CRP levels [0.5 (S.E.=0.2) mg/dl, p=0.25]. After controlling for the effect of BMI, the difference in CRP levels across the three groups remained significant (F 2,58=3.6, p=0.035), suggesting that the increase in inflammation was not driven by an increase in adipose tissue. Childhood maltreatment is associated with higher BMI, and increased CRP levels, in patients with a first-episode psychosis. Further studies need to confirm the mechanisms underlying the putative causal relationship between childhood maltreatment and higher BMI, and whether this is indeed mediated by increased inflammation.

  4. Kinetoplastid membrane protein-11 is present in promastigotes and amastigotes of Leishmania amazonensis and its surface expression increases during metacyclogenesis

    Directory of Open Access Journals (Sweden)

    Denise CS Matos

    2010-05-01

    Full Text Available Kinetoplastid membrane protein-11 (KMP-11, a protein present in all kinetoplastid protozoa, is considered a potential candidate for a leishmaniasis vaccine. A suitable leishmaniasis vaccine candidate molecule must be expressed in amastigotes, the infective stage for mammals. However, the expression of KMP-11 in Leishmania amastigotes has been a subject of controversy. We evaluated the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, of Leishmania amazonensis by immunoblotting, flow cytometry and immunocytochemistry, using a monoclonal antibody against KMP-11. We found that KMP-11 is present in promastigotes and amastigotes. In both stages, the protein was found in association with membrane structures (at the cell surface, flagellar pocket and intracellular vesicles. More importantly, its surface expression is higher in amastigotes than in promastigotes and increases during metacyclogenesis. The increased expression of KMP-11 in metacyclic promastigotes, and especially in amastigotes, indicates a role for this molecule in the parasite relationship with the mammalian host. The presence of this molecule in amastigotes is consistent with the previously demonstrated immunoprotective capacity of vaccine prototypes based on the KMP-11-coding gene and the presence of humoral and cellular immune responses to KMP-11 in Leishmania-infected humans and animals.

  5. Activated protein C (APC) can increase bone anabolism via a protease-activated receptor (PAR)1/2 dependent mechanism.

    Science.gov (United States)

    Shen, Kaitlin; Murphy, Ciara M; Chan, Ben; Kolind, Mille; Cheng, Tegan L; Mikulec, Kathy; Peacock, Lauren; Xue, Meilang; Park, Sang-Youel; Little, David G; Jackson, Chris J; Schindeler, Aaron

    2014-12-01

    Activated Protein C (APC) is an anticoagulant with strong cytoprotective properties that has been shown to promote wound healing. In this study APC was investigated for its potential orthopedic application using a Bone Morphogenetic Protein 2 (rhBMP-2) induced ectopic bone formation model. Local co-administration of 10 µg rhBMP-2 with 10 µg or 25 µg APC increased bone volume at 3 weeks by 32% (N.S.) and 74% (pAPC are largely mediated by its receptors endothelial protein C receptor (EPCR) and protease-activated receptors (PARs). Cultured pre-osteoblasts and bone nodule tissue sections were shown to express PAR1/2 and EPCR. When pre-osteoblasts were treated with APC, cell viability and phosphorylation of ERK1/2, Akt, and p38 were increased. Inhibition with PAR1 and sometimes PAR2 antagonists, but not with EPCR blocking antibodies, ameliorated the effects of APC on cell viability and kinase phosphorylation. These data indicate that APC can affect osteoblast viability and signaling, and may have in vivo applications with rhBMP-2 for bone repair. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  6. Simple clinical means of documenting increased pulmonary endothelial permeability to protein

    International Nuclear Information System (INIS)

    Mishkin, F.S.; Niden, A.; Kumar, A.; Thomas, A.; Reese, I.C.; Vasinrapee, P.

    1987-01-01

    The authors investigated a simple method that can be used at the bedside for documenting the net accumulation of albumin in the lung. The technique employs measurement with a computer-linked gamma camera of the activity ratio in an area of the right lung compared with the same-sized area in the heart at 20 minutes and three hours following intravenous injection of technetium Tc 99m albumin. They applied this measurement to three groups of patients: a control group and patients with roentgenographic evidence of edema classified according to clinically available criteria as either hydrostatic edema or permeability edema to see if they could document differences among these groups. In control patients this ratio did not increase by more than seven units between the 20-minute and three-hour measurements. Of 18 patients classified by other routine clinical means as having hydrostatic pulmonary edema, 89% showed no increase in lung albumin accumulation. In 29 patients with permeability edema associated with the so-called adult respiratory distress syndrome, 31% showed evidence of net pulmonary albumin accumulation. These findings suggest that some patients otherwise classified as having hydrostatic edema have concomitant permeability changes in the microvasculature and that permeability edema represents a spectrum of endothelial damage

  7. Simple clinical means of documenting increased pulmonary endothelial permeability to protein

    Energy Technology Data Exchange (ETDEWEB)

    Mishkin, F.S.; Niden, A.; Kumar, A.; Thomas, A.; Reese, I.C.; Vasinrapee, P.

    1987-02-20

    The authors investigated a simple method that can be used at the bedside for documenting the net accumulation of albumin in the lung. The technique employs measurement with a computer-linked gamma camera of the activity ratio in an area of the right lung compared with the same-sized area in the heart at 20 minutes and three hours following intravenous injection of technetium Tc 99m albumin. They applied this measurement to three groups of patients: a control group and patients with roentgenographic evidence of edema classified according to clinically available criteria as either hydrostatic edema or permeability edema to see if they could document differences among these groups. In control patients this ratio did not increase by more than seven units between the 20-minute and three-hour measurements. Of 18 patients classified by other routine clinical means as having hydrostatic pulmonary edema, 89% showed no increase in lung albumin accumulation. In 29 patients with permeability edema associated with the so-called adult respiratory distress syndrome, 31% showed evidence of net pulmonary albumin accumulation. These findings suggest that some patients otherwise classified as having hydrostatic edema have concomitant permeability changes in the microvasculature and that permeability edema represents a spectrum of endothelial damage.

  8. Filtration as the main mechanism of increased protein extravasation in liver cirrhosis

    DEFF Research Database (Denmark)

    Henriksen, J H; Parving, H H; Lassen, N A

    1980-01-01

    Transvascular escape rates of albumin and immunoglobulin-G, IgG (TERalb and TERIgG, i.e, the fractions of intravascular mass of albumin and IgG passing to the extravascular space per unit time) were determined simultaneously from the disappearance of intravenously injected 131I-labelled human serum...... albumin and 125I-labelled human IgG in eight patients with cirrhosis of the liver. The mean wedged hepatic venous pressure was 22 mmHg (range 13-34). TERalb and and TERIgG/TERalb ratio was on average 8.4 +/- 0.8%/h (SD), and 7.4 +/- 1.9%/h (SD), respectively and these values are significantly increased...

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

    Science.gov (United States)

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

    2014-01-01

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

  10. Increasing protein intake modulates lipid metabolism in healthy young men and women consuming a high-fat hypercaloric diet.

    Science.gov (United States)

    Rietman, Annemarie; Schwarz, Jessica; Blokker, Britt A; Siebelink, Els; Kok, Frans J; Afman, Lydia A; Tomé, Daniel; Mensink, Marco

    2014-08-01

    The objective of this study was to evaluate the effect of increasing protein intake, at the expense of carbohydrates, on intrahepatic lipids (IHLs), circulating triglycerides (TGs), and body composition in healthy humans consuming a high-fat, hypercaloric diet. A crossover randomized trial with a parallel control group was performed. After a 2-wk run-in period, participants were assigned to either the control diet [n = 10; 27.8 energy percent (en%) fat, 16.9 en% protein, 55.3 en% carbohydrates] for 4 wk or a high-fat, hypercaloric diet (n = 17; >2 MJ/d) crossover trial with 2 periods of 2 wk, with either high-protein (HP) (37.7 en% fat, 25.7 en% protein, 36.6 en% carbohydrates) or normal-protein (NP) (39.4 en% fat, 15.4 en% protein, 45.2 en% carbohydrates) content. Measurements were performed after 2 wk of run-in (baseline), 2 wk of intervention (period 1), and 4 wk of intervention (period 2). A trend toward lower IHL and plasma TG concentrations during the HP condition compared with the NP condition was observed (IHL: 0.35 ± 0.04% vs. 0.51 ± 0.08%, P = 0.08; TG: 0.65 ± 0.03 vs. 0.77 ± 0.05 mmol/L, P = 0.07, for HP and NP, respectively). Fat mass was significantly lower (10.6 ± 1.72 vs. 10.9 ± 1.73 kg; P = 0.02) with the HP diet than with the NP diet, whereas fat-free mass was higher (55.7 ± 2.79 vs. 55.2 ± 2.80 kg; P = 0.003). This study indicated that an HP, high-fat, hypercaloric diet affects lipid metabolism. It tends to lower the IHL and circulating TG concentrations and significantly lowers fat mass and increases fat-free mass compared with an NP, high-fat, hypercaloric diet. This trail was registered at www.clinicaltrials.gov as NCT01354626. © 2014 American Society for Nutrition.

  11. The common inhalation anesthetic isoflurane induces caspase activation and increases amyloid beta-protein level in vivo.

    Science.gov (United States)

    Xie, Zhongcong; Culley, Deborah J; Dong, Yuanlin; Zhang, Guohua; Zhang, Bin; Moir, Robert D; Frosch, Matthew P; Crosby, Gregory; Tanzi, Rudolph E

    2008-12-01

    An estimated 200 million patients worldwide have surgery each year. Anesthesia and surgery have been reported to facilitate emergence of Alzheimer's disease. The commonly used inhalation anesthetic isoflurane has previously been reported to induce apoptosis, and to increase levels and aggregation of Alzheimer's disease-associated amyloid beta-protein (Abeta) in cultured cells. However, the in vivo relevance has not been addressed. We therefore set out to determine effects of isoflurane on caspase activation and levels of beta-site amyloid precursor protein-cleaving enzyme (BACE) and Abeta in naive mice, using Western blot, immunohistochemistry, and reverse transcriptase polymerase chain reaction. Here we show for the first time that a clinically relevant isoflurane anesthesia (1.4% isoflurane for 2 hours) leads to caspase activation and modest increases in levels of BACE 6 hours after anesthesia in mouse brain. Isoflurane anesthesia induces caspase activation, and increases levels of BACE and Abeta up to 24 hours after anesthesia. Isoflurane may increase BACE levels by reducing BACE degradation. Moreover, the Abeta aggregation inhibitor, clioquinol, was able to attenuate isoflurane-induced caspase-3 activation in vivo. Given that transient insults to brain may lead to long-term brain damage, these findings suggest that isoflurane may promote Alzheimer's disease neuropathogenesis and, as such, have implications for use of isoflurane in humans, pending human study confirmation.

  12. A single amino acid substitution in the core protein of West Nile virus increases resistance to acidotropic compounds.

    Directory of Open Access Journals (Sweden)

    Miguel A Martín-Acebes

    Full Text Available West Nile virus (WNV is a worldwide distributed mosquito-borne flavivirus that naturally cycles between birds and mosquitoes, although it can infect multiple vertebrate hosts including horses and humans. This virus is responsible for recurrent epidemics of febrile illness and encephalitis, and has recently become a global concern. WNV requires to transit through intracellular acidic compartments at two different steps to complete its infectious cycle. These include fusion between the viral envelope and the membrane of endosomes during viral entry, and virus maturation in the trans-Golgi network. In this study, we followed a genetic approach to study the connections between viral components and acidic pH. A WNV mutant with increased resistance to the acidotropic compound NH4Cl, which blocks organelle acidification and inhibits WNV infection, was selected. Nucleotide sequencing revealed that this mutant displayed a single amino acid substitution (Lys 3 to Glu on the highly basic internal capsid or core (C protein. The functional role of this replacement was confirmed by its introduction into a WNV infectious clone. This single amino acid substitution also increased resistance to other acidification inhibitor (concanamycin A and induced a reduction of the neurovirulence in mice. Interestingly, a naturally occurring accompanying mutation found on prM protein abolished the resistant phenotype, supporting the idea of a genetic crosstalk between the internal C protein and the external glycoproteins of the virion. The findings here reported unveil a non-previously assessed connection between the C viral protein and the acidic pH necessary for entry and proper exit of flaviviruses.

  13. A single amino acid substitution in the core protein of West Nile virus increases resistance to acidotropic compounds.

    Science.gov (United States)

    Martín-Acebes, Miguel A; Blázquez, Ana-Belén; de Oya, Nereida Jiménez; Escribano-Romero, Estela; Shi, Pei-Yong; Saiz, Juan-Carlos

    2013-01-01

    West Nile virus (WNV) is a worldwide distributed mosquito-borne flavivirus that naturally cycles between birds and mosquitoes, although it can infect multiple vertebrate hosts including horses and humans. This virus is responsible for recurrent epidemics of febrile illness and encephalitis, and has recently become a global concern. WNV requires to transit through intracellular acidic compartments at two different steps to complete its infectious cycle. These include fusion between the viral envelope and the membrane of endosomes during viral entry, and virus maturation in the trans-Golgi network. In this study, we followed a genetic approach to study the connections between viral components and acidic pH. A WNV mutant with increased resistance to the acidotropic compound NH4Cl, which blocks organelle acidification and inhibits WNV infection, was selected. Nucleotide sequencing revealed that this mutant displayed a single amino acid substitution (Lys 3 to Glu) on the highly basic internal capsid or core (C) protein. The functional role of this replacement was confirmed by its introduction into a WNV infectious clone. This single amino acid substitution also increased resistance to other acidification inhibitor (concanamycin A) and induced a reduction of the neurovirulence in mice. Interestingly, a naturally occurring accompanying mutation found on prM protein abolished the resistant phenotype, supporting the idea of a genetic crosstalk between the internal C protein and the external glycoproteins of the virion. The findings here reported unveil a non-previously assessed connection between the C viral protein and the acidic pH necessary for entry and proper exit of flaviviruses.

  14. Glucose, fructose and sucrose increase the solubility of protein-tannin complexes and at high concentration, glucose and sucrose interfere with bisulphite bleaching of wine pigments.

    Science.gov (United States)

    Harbertson, James F; Yuan, Chunlong; Mireles, Maria S; Hanlin, Rachel L; Downey, Mark O

    2013-05-01

    Wines were modified with increasing sugar concentrations and decreasing tannin concentrations and analysed by a combination of protein precipitation and bisulphite bleaching. Increasing sugar concentration decreased the precipitation of tannin and protein-precipitable polymeric pigments (PPP). The use of a hydrogen bond disruptor (urea) to reduce protein-tannin and protein-pigment complex formation showed that the effect of sugar concentration occurred by increasing the solubility of the tannin-protein complex, not by interfering with protein-tannin complex formation. By increasing the solubility of pigment-protein complexes, non-protein-precipitable polymeric pigments (nPPP) appeared to increase. There was also an increase in total polymeric pigments at each tannin concentration with increasing glucose and sucrose concentration, indicating that sugar concentration might also affect bisulphite bleaching of wine pigments. While a significant effect of sugar concentration on tannin-protein complex solubility was observed, these effects were greatest at sugar concentrations far in excess of normal wine making conditions. Under normal wine making conditions, sugar concentration will have a negligible effect on protein-precipitable tannin, PPP and nPPP concentrations. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. Increase in serum pregnancy-associated plasma protein-A is correlated with increase in cardiovascular risk factors in adult patients with growth hormone deficiency.

    Science.gov (United States)

    Li, Linman; Ren, Wei; Li, Jinchao; Liu, Jingjing; Wang, Lingli; Zheng, Xiaoya; Liu, Dezhen; Li, Sufang; Souvenir, Rhonda; Tang, Jiping

    2012-10-01

    Adult Growth Hormone Deficiency (AGHD) is correlated to many adverse effects on metabolism and increased cardiovascular risk. Pregnancy-associated plasma protein-A (PAPP-A) is a protease that promotes IGF-I availability in vascular tissues in recent study, and PAPP-A levels have been proposed as an early predictor of cardiac events. The aim of our study was to compare PAPP-A levels in AGHD patients with that of healthy adult subjects to determine if there is a relationship between serum PAPP-A and glucose and lipid metabolism. Twenty AGHD patients and 20 healthy, age-matched and weight-matched persons were chosen for the study. Their weight, height, blood pressure, body mass index (BMI), body fat percentage, waist and hip circumference, and waist-hips ratio were assessed. An oral glucose tolerance test was performed and venous blood was collected from the each patient's cubital vein for biochemical analysis. Serum PAPP-A level in AGHD patients was significantly higher than that of the control group [(7.62 ± 1.62 vs. 6.54 ± 1.31) p < 0.05], and PAPP-A was positively correlated to age, BMI, waist circumference and so on. After adjusting for the waist circumference, waist-hip ratio, 2 h postprandial blood glucose, triglycerides, the serum PAPP-A in AGHD patients was positively correlated to the BMI (r = 0.728, p < 0.05) and fasting insulin (r = 0.433, p < 0.05). In a multiple step-wise regression analysis, BMI, 2 h postprandial glucose, fasting insulin, HOMA-IR were independently associated with serum PAPP-A in AGHD patients. The increase in serum PAPP-A levels is associated with abnormal glucose metabolism and increased risk of atherosclerosis in AGHD patients.

  16. The thermal structural transition of alpha-crystallin modulates subunit interactions and increases protein solubility.

    Directory of Open Access Journals (Sweden)

    Giuseppe Maulucci

    Full Text Available BACKGROUND: Alpha crystallin is an oligomer composed of two types of subunits, alpha-A and alpha-B crystallin, and is the major constituent of human lens. The temperature induced condensation of alpha-crystallin, the main cause for eye lens opacification (cataract, is a two step-process, a nucleation followed by an aggregation phase, and a protective effect towards the aggregation is exhibited over the alpha crystallin phase transition temperature (Tc = 318.16 K. METHODS/RESULTS: To investigate if a modulation of the subunit interactions over Tc could trigger the protective mechanism towards the aggregation, we followed, by using simultaneously static and dynamic light scattering, the temperature induced condensation of alpha-crystallin. By developing a mathematical model able to uncouple the nucleation and aggregation processes, we find a previously unobserved transition in the nucleation rate constant. Its temperature dependence allows to determine fundamental structural parameters, the chemical potential (Δμ and the interfacial tension (γ of the aggregating phase, that characterize subunit interactions. CONCLUSIONS/GENERAL SIGNIFICANCE: The decrease of both Δμ and γ at Tc, and a relative increase in solubility, reveal a significative decrease in the strenght of alpha-crystallin subunits interactions, which protects from supramolecolar condensation in hypertermic conditions. On the whole, we suggest a general approach able to understand the structural and kinetic mechanisms involved in aggregation-related diseases and in drugs development and testing.

  17. SINEUPs are modular antisense long-non coding RNAs that increase synthesis of target proteins in cells

    Directory of Open Access Journals (Sweden)

    Silvia eZucchelli

    2015-05-01

    Full Text Available Despite recent efforts in discovering novel long non-coding RNAs (lncRNAs and unveiling their functions in a wide range of biological processes their applications as biotechnological or therapeutic tools are still at their infancy. We have recently shown that AS Uchl1, a natural lncRNA antisense to the Parkinson’s disease-associated gene Ubiquitin carboxyl-terminal esterase L1 (Uchl1, is able to increase UchL1 protein synthesis at post-transcriptional level. Its activity requires two RNA elements: an embedded inverted SINEB2 sequence to increase translation and the overlapping region to target its sense mRNA. This functional organization is shared with several mouse lncRNAs antisense to protein coding genes. The potential use of AS Uchl1-derived lncRNAs as enhancers of target mRNA translation remains unexplored. Here we define AS Uchl1 as the representative member of a new functional class of natural and synthetic antisense lncRNAs that activate translation. We named this class of RNAs SINEUPs for their requirement of the inverted SINEB2 sequence to UP-regulate translation in a gene-specific manner. The overlapping region is indicated as the Binding Doman (BD while the embedded inverted SINEB2 element is the Effector Domain (ED. By swapping BD, synthetic SINEUPs are designed targeting mRNAs of interest. SINEUPs function in an array of cell lines and can be efficiently directed towards N-terminally tagged proteins. Their biological activity is retained in a miniaturized version within the range of small RNAs length. Its modular structure was exploited to successfully design synthetic SINEUPs targeting endogenous Parkinson’s disease-associated DJ-1 and proved to be active in different neuronal cell lines.In summary, SINEUPs represent the first scalable tool to increase synthesis of proteins of interest. We propose SINEUPs as reagents for molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies.

  18. Mechanical stimulation of C2C12 cells increases m-calpain expression, focal adhesion plaque protein degradation

    DEFF Research Database (Denmark)

    Grossi, Alberto; Karlsson, Anders H; Lawson, Moira Ann

    2008-01-01

    . Stimulation due to stretch- or load-induced signaling is now beginning to be understood as a factor which affects gene sequences, protein synthesis and an increase in Ca2+ influx in myocytes. Evidence of the involvement of Ca2+ -dependent activity in myoblast fusion, cell membrane and cytoskeleton component...... reorganization due to the activity of the ubiquitous proteolytic enzymes, calpains, has been reported. Whether there is a link between stretch- or load-induced signaling and calpain expression and activation is not known. Using a magnetic bead stimulation assay and C2C12 mouse myoblasts cell population, we have...... demonstrated that mechanical stimulation via laminin receptors leads to an increase in m-calpain expression, but no increase in the expression of other calpain isoforms. Our study revealed that after a short period of stimulation, m-calpain relocates into focal adhesion complexes and is followed by a breakdown...

  19. The chitinase-like protein YKL-40 increases mucin5AC production in human bronchial epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Chunyi; Li, Qi [Division of Respiratory Medicine, Second Affiliated Hospital, Chongqing Medical University, No. 74, Linjiang Road, Yuzhong District, Chongqing 400010 (China); Zhou, Xiangdong, E-mail: zxd999@263.net [Division of Respiratory Medicine, Second Affiliated Hospital, Chongqing Medical University, No. 74, Linjiang Road, Yuzhong District, Chongqing 400010 (China); Kolosov, Victor P.; Perelman, Juliy M. [Far Eastern Scientific Center of Physiology and Pathology of Respiration, Siberian Branch, Russian Academy of Medical Sciences, Blagoveshchensk (Russian Federation)

    2013-11-01

    Mucus overproduction is an important feature in patients with chronic inflammatory airway diseases. However, the regulatory mechanisms that mediate excessive mucin production remain elusive. Recently, the level of YKL-40, a chitinase-like protein, has been found to be significantly increased in chronic inflammatory airway diseases and has been shown to be associated with the severity of these diseases. In this study, we sought to explore the effect of YKL-40 on mucin5AC (MUC5AC) production in chronic inflammatory airway diseases and the potential signaling pathways involved in this process. We found that elevated YKL-40 levels increased the mRNA and protein expression of MUC5AC in a dose- and time-dependent manner, in association with the phosphorylation of extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB), reflecting their activation. These responses were significantly suppressed by the knockdown of protease-activating receptor 2 (PAR2) with specific small interfering RNA or the inhibitors of ERK and NF-κB. YKL-40-induced MUC5AC overproduction was also effectively attenuated by the inhibitor of focal adhesion kinase (FAK). Taken together, these results imply that YKL-40 can stimulate excessive MUC5AC production through PAR2- and FAK-mediated mechanisms. - Highlights: • MUC5AC is the major secreted mucin in chronic inflammatory airway diseases. • YKL-40 is a prototype of the chitinase-like protein in mammals. • YKL-40 is an active player in chronic inflammatory airway diseases. • YKL-40 can increase MUC5AC production via PAR2-mediated pathway. • FAK is another candidate to mediate YKL-40-induced MUC5AC overexpression.

  20. Low-protein, high-carbohydrate diet increases glucose uptake and fatty acid synthesis in brown adipose tissue of rats.

    Science.gov (United States)

    Aparecida de França, Suélem; Pavani Dos Santos, Maísa; Nunes Queiroz da Costa, Roger Vinícius; Froelich, Mendalli; Buzelle, Samyra Lopes; Chaves, Valéria Ernestânia; Giordani, Morenna Alana; Pereira, Mayara Peron; Colodel, Edson Moleta; Marlise Balbinotti Andrade, Cláudia; Kawashita, Nair Honda

    2014-04-01

    The aim of this study was to evaluate glucose uptake and the contribution of glucose to fatty acid (FA) synthesis and the glycerol-3-phosphate (G3P) of triacylglycerol synthesis by interscapular brown adipose tissue (IBAT) of low-protein, high-carbohydrate (LPHC) diet-fed rats. LPHC (6% protein; 74% carbohydrate) or control (17% protein; 63% carbohydrate) diets were administered to rats (∼ 100 g) for 15 d. Total FA and G3P synthesis and the synthesis of FA and G3P from glucose were evaluated in vivo by (3)H2O and (14)C-glucose. Sympathetic neural contribution for FA synthesis was evaluated by comparing the synthesis in denervated (7 d before) IBAT with that of the contralateral innervated side. The insulin signaling and β3 adrenergic receptor (β3-AR) contents, as well as others, were determined by Western blot (Student's t test or analysis of variance; P ≤ 0.05). Total FA synthesis in IBAT was 133% higher in the LPHC group and was reduced 85% and 70% by denervation for the LPHC and control groups, respectively. Glucose uptake was 3.5-fold higher in the IBAT of LPHC rats than in that of the control rats, and the contribution of glucose to the total FA synthesis increased by 12% in control rats compared with 18% in LPHC rats. The LPHC diet increased the G3P generation from glucose by 270% and the insulin receptor content and the p-AKT insulin stimulation in IBAT by 120% and reduced the β3-AR content by 50%. The LPHC diet stimulated glucose uptake, both the total rates and the rates derived from glucose-dependent FA and G3P synthesis, by increasing the insulin sensitivity and the sympathetic flux, despite a reduction in the β3-AR content. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. The chitinase-like protein YKL-40 increases mucin5AC production in human bronchial epithelial cells

    International Nuclear Information System (INIS)

    Liu, Chunyi; Li, Qi; Zhou, Xiangdong; Kolosov, Victor P.; Perelman, Juliy M.

    2013-01-01

    Mucus overproduction is an important feature in patients with chronic inflammatory airway diseases. However, the regulatory mechanisms that mediate excessive mucin production remain elusive. Recently, the level of YKL-40, a chitinase-like protein, has been found to be significantly increased in chronic inflammatory airway diseases and has been shown to be associated with the severity of these diseases. In this study, we sought to explore the effect of YKL-40 on mucin5AC (MUC5AC) production in chronic inflammatory airway diseases and the potential signaling pathways involved in this process. We found that elevated YKL-40 levels increased the mRNA and protein expression of MUC5AC in a dose- and time-dependent manner, in association with the phosphorylation of extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB), reflecting their activation. These responses were significantly suppressed by the knockdown of protease-activating receptor 2 (PAR2) with specific small interfering RNA or the inhibitors of ERK and NF-κB. YKL-40-induced MUC5AC overproduction was also effectively attenuated by the inhibitor of focal adhesion kinase (FAK). Taken together, these results imply that YKL-40 can stimulate excessive MUC5AC production through PAR2- and FAK-mediated mechanisms. - Highlights: • MUC5AC is the major secreted mucin in chronic inflammatory airway diseases. • YKL-40 is a prototype of the chitinase-like protein in mammals. • YKL-40 is an active player in chronic inflammatory airway diseases. • YKL-40 can increase MUC5AC production via PAR2-mediated pathway. • FAK is another candidate to mediate YKL-40-induced MUC5AC overexpression

  2. Suppression of lipin-1 expression increases monocyte chemoattractant protein-1 expression in 3T3-L1 adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Nobuhiko, E-mail: ntkhs@hoku-iryo-u.ac.jp [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1 Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan); Yoshizaki, Takayuki [Innovation Center, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065 (Japan); Hiranaka, Natsumi; Suzuki, Takeshi [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Yui, Tomoo; Akanuma, Masayasu; Oka, Kazuya [Department of Fixed Prosthodontics and Oral Implantology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Kanazawa, Kaoru [Department of Dental Anesthesiology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Yoshida, Mika; Naito, Sumiyoshi [Department of Clinical Laboratory, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Fujiya, Mikihiro; Kohgo, Yutaka [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1 Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan); Ieko, Masahiro [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer Lipin-1 affects lipid metabolism, adipocyte differentiation, and transcription. Black-Right-Pointing-Pointer Adipose lipin-1 expression is reduced in obesity. Black-Right-Pointing-Pointer Lipin-1 depletion using siRNA in 3T3-L1 adipocytes increased MCP-1 expression. Black-Right-Pointing-Pointer Lipin-1 is involved in adipose inflammation. -- Abstract: Lipin-1 plays a crucial role in the regulation of lipid metabolism and cell differentiation in adipocytes. Expression of adipose lipin-1 is reduced in obesity, and metabolic syndrome. However, the significance of this reduction remains unclear. This study investigated if and how reduced lipin-1 expression affected metabolism. We assessed mRNA expression levels of various genes related to adipocyte metabolism in lipin-1-depleted 3T3-L1 adipocytes by introducing its specific small interfering RNA. In lipin-1-depleted adipocytes, mRNA and protein expression levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased, although the other genes tested were not altered. The conditioned media from the cells promoted monocyte chemotaxis. The increase in MCP-1 expression was prevented by treatment with quinazoline or salicylate, inhibitors of nuclear factor-{kappa}B activation. Because MCP-1 is related to adipose inflammation and systemic insulin resistance, these results suggest that a reduction in adipose lipin-1 in obesity may exacerbate adipose inflammation and metabolism.

  3. Suppression of lipin-1 expression increases monocyte chemoattractant protein-1 expression in 3T3-L1 adipocytes

    International Nuclear Information System (INIS)

    Takahashi, Nobuhiko; Yoshizaki, Takayuki; Hiranaka, Natsumi; Suzuki, Takeshi; Yui, Tomoo; Akanuma, Masayasu; Oka, Kazuya; Kanazawa, Kaoru; Yoshida, Mika; Naito, Sumiyoshi; Fujiya, Mikihiro; Kohgo, Yutaka; Ieko, Masahiro

    2011-01-01

    Highlights: ► Lipin-1 affects lipid metabolism, adipocyte differentiation, and transcription. ► Adipose lipin-1 expression is reduced in obesity. ► Lipin-1 depletion using siRNA in 3T3-L1 adipocytes increased MCP-1 expression. ► Lipin-1 is involved in adipose inflammation. -- Abstract: Lipin-1 plays a crucial role in the regulation of lipid metabolism and cell differentiation in adipocytes. Expression of adipose lipin-1 is reduced in obesity, and metabolic syndrome. However, the significance of this reduction remains unclear. This study investigated if and how reduced lipin-1 expression affected metabolism. We assessed mRNA expression levels of various genes related to adipocyte metabolism in lipin-1-depleted 3T3-L1 adipocytes by introducing its specific small interfering RNA. In lipin-1-depleted adipocytes, mRNA and protein expression levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased, although the other genes tested were not altered. The conditioned media from the cells promoted monocyte chemotaxis. The increase in MCP-1 expression was prevented by treatment with quinazoline or salicylate, inhibitors of nuclear factor-κB activation. Because MCP-1 is related to adipose inflammation and systemic insulin resistance, these results suggest that a reduction in adipose lipin-1 in obesity may exacerbate adipose inflammation and metabolism.

  4. Human extrahepatic portal vein obstruction correlates with decreased factor VII and protein C transcription but increased hepatocyte proliferation.

    Science.gov (United States)

    Chiu, Bill; Melin-Aldana, Hector; Superina, Riccardo A

    2007-10-01

    A 3-year-old girl developed extrahepatic portal vein obstruction (EHPVO) after a liver transplant. She had sequelae of portal hypertension that required another transplantation. The circumstances allowed for comparison of liver-dependent coagulation factor production between the second donor liver and the explanted liver with EHPVO. Liver samples from the explanted first graft and the second transplant were obtained. Fresh tissue was used to perform reverse transcription-polymerase chain reaction with primers against factors V, VII, as well as VIII, protein C, and paraffin-embedded sections for hepatocyte proliferation using Ki-67 antibody as well as for apoptosis using TUNEL assay. The transcription of factor VII and that of protein C were decreased in the explant as compared with the newly transplanted liver (factor VII, 77% of the donor; protein C, 88% of the donor). The transcription of factor V and that of factor VIII were unchanged. The explant had a greater percentage of proliferating hepatocytes than the new organ (0.85% +/- 0.75% vs 0.11% +/- 0.21%). The percentage of apoptotic cells was similar between the 2 livers (0.09% +/- 0.13% vs 0.09% +/- 0.13%). Idiopathic EHPVO is associated with a reduction in liver-dependent coagulation factor transcription and an increase in hepatocyte proliferation. Portal blood flow deprivation alters hepatic homeostasis and initiates mechanisms that attempt to restore liver-dependent coagulation factors.

  5. Mild prenatal protein malnutrition increases alpha 2C-adrenoceptor expression in the rat cerebral cortex during postnatal life.

    Science.gov (United States)

    Sierralta, Walter; Hernández, Alejandro; Valladares, Luis; Pérez, Hernán; Mondaca, Mauricio; Soto-Moyano, Rubén

    2006-05-15

    Mild reduction in the protein content in the diet of pregnant rats from 25 to 8% casein, calorically compensated by carbohydrates, does not alter body and brain weights of rat pups at birth, but results in significant changes of the concentration and release of cortical noradrenaline during postnatal life, together with impaired long-term potentiation and memory formation. Since some central noradrenergic receptors are critically involved in neuroplasticity, the present study evaluated, by utilizing immunohistochemical methods, the effect of mild prenatal protein malnutrition on the alpha 2C-adrenoceptor expression in the frontal and occipital cortices of 8- and 60-day-old rats. At day 8 of postnatal age, prenatally malnourished rats exhibited a three-fold increase of alpha 2C-adrenoceptor expression in both the frontal and the occipital cortices, as compared to well-nourished controls. At 60 days of age, prenatally malnourished rats showed normal expression levels scores of alpha 2C-adrenoceptor in the neocortex. Results suggest that overexpression of neocortical alpha 2C-adrenoceptors during early postnatal life, subsequent to mild prenatal protein malnutrition, could in part be responsible for neural and behavioral disturbances showing prenatally malnourished animals during the postnatal life.

  6. Protein S-glutathionylation induced by hypoxia increases hypoxia-inducible factor-1α in human colon cancer cells.

    Science.gov (United States)

    Jeon, Daun; Park, Heon Joo; Kim, Hong Seok

    2018-01-01

    Hypoxia is a common characteristic of many types of solid tumors. Intratumoral hypoxia selects for tumor cells that survive in a low oxygen environment, undergo epithelial-mesenchymal transition, are more motile and invasive, and show gene expression changes driven by hypoxia-inducible factor-1α (HIF-1α) activation. Therefore, targeting HIF-1α is an attractive strategy for disrupting multiple pathways crucial for tumor growth. In the present study, we demonstrated that hypoxia increases the S-glutathionylation of HIF-1α and its protein levels in colon cancer cells. This effect is significantly prevented by decreasing oxidized glutathione as well as glutathione depletion, indicating that S-glutathionylation and the formation of protein-glutathione mixed disulfides is related to HIF-1α protein levels. Moreover, colon cancer cells expressing glutaredoxin 1 are resistant to inducing HIF-1α and expressing hypoxia-responsive genes under hypoxic conditions. Therefore, S-glutathionylation of HIF-1α induced by tumor hypoxia may be a novel therapeutic target for the development of new drugs. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Selection for a Zinc-Finger Protein Contributes to Seed Oil Increase during Soybean Domestication1[OPEN

    Science.gov (United States)

    Li, Qing-Tian; Lu, Xiang; Song, Qing-Xin; Chen, Hao-Wei; Wei, Wei; Tao, Jian-Jun; Ma, Biao; Bi, Ying-Dong; Li, Wei; Lai, Yong-Cai; Shui, Guang-Hou; Chen, Shou-Yi

    2017-01-01

    Seed oil is a momentous agronomical trait of soybean (Glycine max) targeted by domestication in breeding. Although multiple oil-related genes have been uncovered, knowledge of the regulatory mechanism of seed oil biosynthesis is currently limited. We demonstrate that the seed-preferred gene GmZF351, encoding a tandem CCCH zinc finger protein, is selected during domestication. Further analysis shows that GmZF351 facilitates oil accumulation by directly activating WRINKLED1, BIOTIN CARBOXYL CARRIER PROTEIN2, 3-KETOACYL-ACYL CARRIER PROTEIN SYNTHASE III, DIACYLGLYCEROL O-ACYLTRANSFERASE1, and OLEOSIN2 in transgenic Arabidopsis (Arabidopsis thaliana) seeds. Overexpression of GmZF351 in transgenic soybean also activates lipid biosynthesis genes, thereby accelerating seed oil accumulation. The ZF351 haplotype from the cultivated soybean group and the wild soybean (Glycine soja) subgroup III correlates well with high gene expression level, seed oil contents and promoter activity, suggesting that selection of GmZF351 expression leads to increased seed oil content in cultivated soybean. Our study provides novel insights into the regulatory mechanism for seed oil accumulation, and the manipulation of GmZF351 may have great potential in the improvement of oil production in soybean and other related crops. PMID:28184009

  8. Whey protein and essential amino acids promote the reduction of adipose tissue and increased muscle protein synthesis during caloric restriction-induced weight loss in elderly, obese individuals

    Directory of Open Access Journals (Sweden)

    Coker Robert H

    2012-12-01

    Full Text Available Abstract Background Excess adipose tissue and sarcopenia presents a multifaceted clinical challenge that promotes morbidity and mortality in the obese, elderly population. Unfortunately, the mortality risks of muscle loss may outweigh the potential benefits of weight loss in the elderly. We have previously demonstrated the effectiveness of whey protein and essential amino acids towards the preservation of lean tissue, even under the conditions of strict bedrest in the elderly. Methods In the context of caloric restriction-based weight loss, we hypothesized that a similar formulation given as a meal replacement (EAAMR would foster the retention of lean tissue through an increase in the skeletal muscle fractional synthesis rate (FSR. We also proposed that EAAMR would promote the preferential loss of adipose tissue through the increased energy cost of skeletal muscle FSR. We recruited and randomized 12 elderly individuals to an 8 week, caloric restriction diet utilizing equivalent caloric meal replacements (800 kcal/day: 1 EAAMR or a 2 competitive meal replacement (CMR in conjunction with 400 kcal of solid food that totaled 1200 kcal/day designed to induce 7% weight loss. Combined with weekly measurements of total body weight and body composition, we also measured the acute change in the skeletal muscle FSR to EAAMR and CMR. Results By design, both groups lost ~7% of total body weight. While EAAMR did not promote a significant preservation of lean tissue, the reduction in adipose tissue was greater in EAAMR compared to CMR. Interestingly, these results corresponded to an increase in the acute skeletal muscle protein FSR. Conclusion The provision of EAAMR during caloric restriction-induced weight loss promotes the preferential reduction of adipose tissue and the modest loss of lean tissue in the elderly population.

  9. Electronegative Low-Density Lipoprotein Increases C-Reactive Protein Expression in Vascular Endothelial Cells through the LOX-1 Receptor

    OpenAIRE

    Chu, Chih-Sheng; Wang, Yu-Chen; Lu, Long-Sheng; Walton, Brian; Yilmaz, H. Ramazan; Huang, Roger Y.; Sawamura, Tatsuya; Dixon, Richard A. F.; Lai, Wen-Ter; Chen, Chu-Huang; Lu, Jonathan

    2013-01-01

    Objectives Increased plasma C-reactive protein (CRP) levels are associated with the occurrence and severity of acute coronary syndrome. We investigated whether CRP can be generated in vascular endothelial cells (ECs) after exposure to the most electronegative subfraction of low-density lipoprotein (LDL), L5, which is atherogenic to ECs. Because L5 and CRP are both ligands for the lectin-like oxidized LDL receptor-1 (LOX-1), we also examined the role of LOX-1. Methods and Results Plasma LDL sa...

  10. Performance of repetitive tasks induces decreased grip strength and increased fibrogenic proteins in skeletal muscle: role of force and inflammation.

    Directory of Open Access Journals (Sweden)

    Samir M Abdelmagid

    Full Text Available This study elucidates exposure-response relationships between performance of repetitive tasks, grip strength declines, and fibrogenic-related protein changes in muscles, and their link to inflammation. Specifically, we examined forearm flexor digitorum muscles for changes in connective tissue growth factor (CTGF; a matrix protein associated with fibrosis, collagen type I (Col1; a matrix component, and transforming growth factor beta 1 (TGFB1; an upstream modulator of CTGF and collagen, in rats performing one of two repetitive tasks, with or without anti-inflammatory drugs.To examine the roles of force versus repetition, rats performed either a high repetition negligible force food retrieval task (HRNF, or a high repetition high force handle-pulling task (HRHF, for up to 9 weeks, with results compared to trained only (TR-NF or TR-HF and normal control rats. Grip strength declined with both tasks, with the greatest declines in 9-week HRHF rats. Quantitative PCR (qPCR analyses of HRNF muscles showed increased expression of Col1 in weeks 3-9, and CTGF in weeks 6 and 9. Immunohistochemistry confirmed PCR results, and also showed greater increases of CTGF and collagen matrix in 9-week HRHF rats than 9-week HRNF rats. ELISA, and immunohistochemistry revealed greater increases of TGFB1 in TR-HF and 6-week HRHF, compared to 6-week HRNF rats. To examine the role of inflammation, results from 6-week HRHF rats were compared to rats receiving ibuprofen or anti-TNF-α treatment in HRHF weeks 4-6. Both treatments attenuated HRHF-induced increases in CTGF and fibrosis by 6 weeks of task performance. Ibuprofen attenuated TGFB1 increases and grip strength declines, matching our prior results with anti-TNFα.Performance of highly repetitive tasks was associated with force-dependent declines in grip strength and increased fibrogenic-related proteins in flexor digitorum muscles. These changes were attenuated, at least short-term, by anti-inflammatory treatments.

  11. Rumen-protected lysine, methionine, and histidine increase milk protein yield in dairy cows fed a metabolizable protein-deficient diet.

    Science.gov (United States)

    Lee, C; Hristov, A N; Cassidy, T W; Heyler, K S; Lapierre, H; Varga, G A; de Veth, M J; Patton, R A; Parys, C

    2012-10-01

    The objective of this experiment was to evaluate the effect of supplementing a metabolizable protein (MP)-deficient diet with rumen-protected (RP) Lys, Met, and specifically His on dairy cow performance. The experiment was conducted for 12 wk with 48 Holstein cows. Following a 2-wk covariate period, cows were blocked by DIM and milk yield and randomly assigned to 1 of 4 diets, based on corn silage and alfalfa haylage: control, MP-adequate diet (ADMP; MP balance: +9 g/d); MP-deficient diet (DMP; MP balance: -317 g/d); DMP supplemented with RPLys (AminoShure-L, Balchem Corp., New Hampton, NY) and RPMet (Mepron; Evonik Industries AG, Hanau, Germany; DMPLM); and DMPLM supplemented with an experimental RPHis preparation (DMPLMH). The analyzed crude protein content of the ADMP and DMP diets was 15.7 and 13.5 to 13.6%, respectively. The apparent total-tract digestibility of all measured nutrients, plasma urea-N, and urinary N excretion were decreased by the DMP diets compared with ADMP. Milk N secretion as a proportion of N intake was greater for the DMP diets compared with ADMP. Compared with ADMP, dry matter intake (DMI) tended to be lower for DMP, but was similar for DMPLM and DMPLMH (24.5, 23.0, 23.7, and 24.3 kg/d, respectively). Milk yield was decreased by DMP (35.2 kg/d), but was similar to ADMP (38.8 kg/d) for DMPLM and DMPLMH (36.9 and 38.5kg/d, respectively), paralleling the trend in DMI. The National Research Council 2001model underpredicted milk yield of the DMP cows by an average (±SE) of 10.3 ± 0.75 kg/d. Milk fat and true protein content did not differ among treatments, but milk protein yield was increased by DMPLM and DMPLMH compared with DMP and was not different from ADMP. Plasma essential amino acids (AA), Lys, and His were lower for DMP compared with ADMP. Supplementation of the DMP diets with RP AA increased plasma Lys, Met, and His. In conclusion, MP deficiency, approximately 15% below the National Research Council requirements from 2001, decreased

  12. A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).

    Science.gov (United States)

    Ruth, P; Kamm, S; Nau, U; Pfeifer, A; Hofmann, F

    1996-01-01

    Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

  13. Increased protein kinase C gamma activity induces Purkinje cell pathology in a mouse model of spinocerebellar ataxia 14.

    Science.gov (United States)

    Ji, Jingmin; Hassler, Melanie L; Shimobayashi, Etsuko; Paka, Nagendher; Streit, Raphael; Kapfhammer, Josef P

    2014-10-01

    Spinocerebellar ataxias (SCAs) are hereditary diseases leading to Purkinje cell degeneration and cerebellar dysfunction. Most forms of SCA are caused by expansion of CAG repeats similar to other polyglutamine disorders such as Huntington's disease. In contrast, in the autosomal dominant SCA-14 the disease is caused by mutations in the protein kinase C gamma (PKCγ) gene which is a well characterized signaling molecule in cerebellar Purkinje cells. The study of SCA-14, therefore, offers the unique opportunity to reveal the molecular and pathological mechanism eventually leading to Purkinje cell dysfunction and degeneration. We have created a mouse model of SCA-14 in which PKCγ protein with a mutation found in SCA-14 is specifically expressed in cerebellar Purkinje cells. We find that in mice expressing the mutated PKCγ protein the morphology of Purkinje cells in cerebellar slice cultures is drastically altered and mimics closely the morphology seen after pharmacological PKC activation. Similar morphological abnormalities were seen in localized areas of the cerebellum of juvenile transgenic mice in vivo. In adult transgenic mice there is evidence for some localized loss of Purkinje cells but there is no overall cerebellar atrophy. Transgenic mice show a mild cerebellar ataxia revealed by testing on the rotarod and on the walking beam. Our findings provide evidence for both an increased PKCγ activity in Purkinje cells in vivo and for pathological changes typical for cerebellar disease thus linking the increased and dysregulated activity of PKCγ tightly to the development of cerebellar disease in SCA-14 and possibly also in other forms of SCA. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. 17-AAG improves cognitive process and increases heat shock protein response in a model lesion with Aβ25-35.

    Science.gov (United States)

    Ortega, Laura; Calvillo, Minerva; Luna, Félix; Pérez-Severiano, Francisca; Rubio-Osornio, Moisés; Guevara, Jorge; Limón, Ilhuicamina Daniel

    2014-08-01

    Molecular chaperones, or heat shock proteins (HSP), have been implicated in numerous neurodegenerative disorders characterized by the accumulation of protein aggregates, such as Alzheimer disease. The agglomeration of insoluble structures of Aβ is thought to be responsible for neuronal death, which in turn leads to the loss of cognitive functions. Recent findings have shown that the induction of HSP decreases the level of abnormal protein aggregates, as well as demonstrating that 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), an analogue of geldanamycin (GA), increases Aβ clearance through the induction of molecular chaperones in cell culture. In light of this discovery that HSP overexpression can be neuroprotective, the search for a way to pharmacologically induce the overexpression of HSP and other associated chaperones may lead to a promising approach for the treatment of neurodegenerative diseases. The aim of our study was to evaluate both the effect of 17-AAG on the cognitive process and the HSP response in rats injected with Aβ25-35 into the CA1 of the hippocampus. The results show that the injection of Aβ caused a significant increase in the expression of the HSP involved in the regulation of cellular proteostasis. While the HSP did not reverse excitotoxic damage, given that experimental subjects showed learning and memory deficits, the administration of 17-AAG prior to the injection of Aβ25-35 did show an improvement in the behavioral assessment that correlated with the upregulation of HSP70 in subjects injured with Aβ. Overall, our data shows that the pharmacological induction of HSP using 17-AAG may be an alternative treatment of neurodegenerative diseases. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Does elevated C-reactive protein increase atrial fibrillation risk? A Mendelian randomization of 47,000 individuals from the general population

    DEFF Research Database (Denmark)

    Marott, Sarah C W; Nordestgaard, Børge G; Zacho, Jeppe

    2010-01-01

    The purpose of this study was to test whether the association of C-reactive protein (CRP) with increased risk of atrial fibrillation is a robust and perhaps even causal association.......The purpose of this study was to test whether the association of C-reactive protein (CRP) with increased risk of atrial fibrillation is a robust and perhaps even causal association....

  16. Loss of Oca2 disrupts the unfolded protein response and increases resistance to endoplasmic reticulum stress in melanocytes.

    Science.gov (United States)

    Cheng, Tsing; Orlow, Seth J; Manga, Prashiela

    2013-11-01

    Accumulation of proteins in the endoplasmic reticulum (ER) typically induces stress and initiates the unfolded protein response (UPR) to facilitate recovery. If homeostasis is not restored, apoptosis is induced. However, adaptation to chronic UPR activation can increase resistance to subsequent acute ER stress. We therefore investigated adaptive mechanisms in Oculocutaneous albinism type 2 (Oca2)-null melanocytes where UPR signaling is arrested despite continued tyrosinase accumulation leading to resistance to the chemical ER stressor thapsigargin. Although thapsigargin triggers UPR activation, instead of Perk-mediated phosphorylation of eIF2α, in Oca2-null melanocytes, eIF2α was rapidly dephosphorylated upon treatment. Dephosphorylation was mediated by the Gadd34-PP1α phosphatase complex. Gadd34-complex inhibition blocked eIF2α dephosphorylation and significantly increased Oca2-null melanocyte sensitivity to thapsigargin. Thus, Oca2-null melanocytes adapt to acute ER stress by disruption of pro-apoptotic Perk signaling, which promotes cell survival. This is the first study to demonstrate rapid eIF2α dephosphorylation as an adaptive mechanism to ER stress. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Bactericidal/Permeability-increasing protein fold-containing family member A1 in airway host protection and respiratory disease.

    Science.gov (United States)

    Britto, Clemente J; Cohn, Lauren

    2015-05-01

    Bactericidal/permeability-increasing protein fold-containing family member A1 (BPIFA1), formerly known as SPLUNC1, is one of the most abundant proteins in respiratory secretions and has been identified with increasing frequency in studies of pulmonary disease. Its expression is largely restricted to the respiratory tract, being highly concentrated in the upper airways and proximal trachea. BPIFA1 is highly responsive to airborne pathogens, allergens, and irritants. BPIFA1 actively participates in host protection through antimicrobial, surfactant, airway surface liquid regulation, and immunomodulatory properties. Its expression is modulated in multiple lung diseases, including cystic fibrosis, chronic obstructive pulmonary disease, respiratory malignancies, and idiopathic pulmonary fibrosis. However, the role of BPIFA1 in pulmonary pathogenesis remains to be elucidated. This review highlights the versatile properties of BPIFA1 in antimicrobial protection and its roles as a sensor of environmental exposure and regulator of immune cell function. A greater understanding of the contribution of BPIFA1 to disease pathogenesis and activity may clarify if BPIFA1 is a biomarker and potential drug target in pulmonary disease.

  18. Phenobarbital increases monkey in vivo nicotine disposition and induces liver and brain CYP2B6 protein

    Science.gov (United States)

    Lee, Anna M; Miksys, Sharon; Tyndale, Rachel F

    2006-01-01

    CYP2B6 is a drug-metabolizing enzyme expressed in the liver and brain that can metabolize bupropion (Zyban®, a smoking cessation drug), activate tobacco-smoke nitrosamines, and inactivate nicotine. Hepatic CYP2B6 is induced by phenobarbital and induction may affect in vivo nicotine disposition, while brain CYP2B6 induction may affect local levels of centrally acting substrates. We investigated the effect of chronic phenobarbital treatment on induction of in vivo nicotine disposition and CYP2B6 expression in the liver and brain of African Green (Vervet) monkeys. Monkeys were split into two groups (n=6 each) and given oral saccharin daily for 22 days; one group was supplemented with 20 mg kg−1 phenobarbital. Monkeys were given a 0.1 mg kg−1 nicotine dose subcutaneously before and after treatment. Phenobarbital treatment resulted in a significant, 56%, decrease (P=0.04) in the maximum nicotine plasma concentration and a 46% decrease (P=0.003) in the area under the concentration–time curve. Phenobarbital also increased hepatic CYP2B6 protein expression. In monkey brain, significant induction (Pphenobarbital treatment in monkeys resulted in increased in vivo nicotine disposition, and induced hepatic and brain CYP2B6 protein levels and cellular expression. This induction may alter the metabolism of CYP2B6 substrates including peripherally acting drugs such as cyclophosphamide and centrally acting drugs such as bupropion, ecstasy and phencyclidine. PMID:16751792

  19. Exhaustive Training Increases Uncoupling Protein 2 Expression and Decreases Bcl-2/Bax Ratio in Rat Skeletal Muscle

    Directory of Open Access Journals (Sweden)

    W. Y. Liu

    2013-01-01

    Full Text Available This work investigates the effects of oxidative stress due to exhaustive training on uncoupling protein 2 (UCP2 and Bcl-2/Bax in rat skeletal muscles. A total of 18 Sprague-Dawley female rats were randomly divided into three groups: the control group (CON, the trained control group (TC, and the exhaustive trained group (ET. Malondialdehyde (MDA, superoxide dismutase (SOD, xanthine oxidase (XOD, ATPase, UCP2, and Bcl-2/Bax ratio in red gastrocnemius muscles were measured. Exhaustive training induced ROS increase in red gastrocnemius muscles, which led to a decrease in the cell antiapoptotic ability (Bcl-2/Bax ratio. An increase in UCP2 expression can reduce ROS production and affect mitochondrial energy production. Thus, oxidative stress plays a significant role in overtraining.

  20. A common haplotype in the G-protein-coupled receptor gene GPR74 is associated with leanness and increased lipolysis

    DEFF Research Database (Denmark)

    Dahlman, Ingrid; Dicker, Andrea; Jiao, Hong

    2007-01-01

    0.36; P=.036) among those selected for obese or lean phenotypes. The ATAG haplotype was associated with increased adipocyte lipid mobilization (lipolysis) in vivo and in vitro. In human fat cells, GPR74 receptor stimulation and inhibition caused a significant and marked decrease and increase......, respectively, of lipolysis, which could be linked to catecholamine stimulation of adipocytes through beta -adrenergic receptors. These findings suggest that a common haplotype in the GPR74 gene protects against obesity, which, at least in part, is caused by a relief of inhibition of lipid mobilization from......The G-protein-coupled receptor GPR74 is a novel candidate gene for body weight regulation. In humans, it is predominantly expressed in brain, heart, and adipose tissue. We report a haplotype in the GPR74 gene, ATAG, with allele frequency ~4% in Scandinavian cohorts, which was associated...

  1. Periodontitis increases rheumatic factor serum levels and citrullinated proteins in gingival tissues and alter cytokine balance in arthritic rats.

    Directory of Open Access Journals (Sweden)

    Mônica G Corrêa

    Full Text Available This study investigated some immunological features by experimental periodontitis (EP and rheumatoid arthritis (RA disease interact in destructive processes in arthritic rats. Rats were assigned to the following groups: EP +RA; RA; EP; and Negative Control. RA was induced by immunizations with type-II collagen and a local immunization with Complete Freund's adjuvant in the paw. Periodontitis was induced by ligating the right first molars. The serum level of rheumatoid factor (RF and anti-citrullinated protein antibody (ACCPA were measured before the induction of EP (T1 and at 28 days after (T2 by ELISA assay. ACCPA levels were also measured in the gingival tissue at T2. The specimens were processed for morphometric analysis of bone loss, and the gingival tissue surrounding the first molar was collected for the quantification of interleukin IL-1β, IL-4, IL-6, IL-17 and TNF-α using a Luminex/MAGpix assay. Paw edema was analyzed using a plethysmometer. Periodontitis increased the RF and ACCPA levels in the serum and in the gingival tissue, respectively. Besides, the level of paw swelling was increased by EP and remained in progress until the end of the experiment, when EP was associated with RA. Greater values of IL-17 were observed only when RA was present, in spite of PE. It can be concluded that periodontitis increases rheumatic factor serum levels and citrullinated proteins level in gingival tissues and alter cytokine balance in arthritic rats; at the same time, arthritis increases periodontal destruction, confirming the bidirectional interaction between diseases.

  2. Prolonged seizure activity leads to increased Protein Kinase A activation in the rat pilocarpine model of status epilepticus.

    Science.gov (United States)

    Bracey, James M; Kurz, Jonathan E; Low, Brian; Churn, Severn B

    2009-08-04

    Status epilepticus is a life-threatening form of seizure activity that represents a major medical emergency associated with significant morbidity and mortality. Protein Kinase A is an important regulator of synaptic strength that may play an important role in the development of status epilepticus-induced neuronal pathology. This study demonstrated an increase in PKA activity against exogenous and endogenous substrates during later stages of SE. As SE progressed, a significant increase in PKA-mediated phosphorylation of an exogenous peptide substrate was demonstrated in cortical structures. The increased activity was not due to altered expression of either regulatory or catalytic subunits of the enzyme. Through the use of phospho-specific antibodies, this study also investigated the effects of SE on the phosphorylation of the GluR1 subunit of the AMPA subtype of glutamate receptor. After the onset of continuous seizure activity, an increase in phosphorylation of the PKA site on the GluR1 subunit of the AMPA receptor was observed. These data suggest a potential mechanism by which SE may increase neuronal excitability in the cortex, potentially leading to maintenance of seizure activity or long-term neuronal pathology.

  3. Protein O-GlcNAc Modification Increases in White Blood Cells After a Single Bout of Physical Exercise.

    Science.gov (United States)

    Nagy, Tamás; Kátai, Emese; Fisi, Viktória; Takács, Tamás Tibor; Stréda, Antal; Wittmann, István; Miseta, Attila

    2018-01-01

    Protein O-linked N -acetylglucosamine (O-GlcNAc) is a dynamic posttranslational modification influencing the function of many intracellular proteins. Recently it was revealed that O-GlcNAc regulation is modified under various stress states, including ischemia and oxidative stress. Aside from a few contradictory studies based on animal models, the effect of exercise on O-GlcNAc is unexplored. To evaluate O-GlcNAc levels in white blood cells (WBC) of human volunteers following physical exercise. Young (age 30 ± 5.2), healthy male volunteers ( n  = 6) were enlisted for the study. Blood parameters including metabolites, ions, "necro"-enzymes, and cell counts were measured before and after a single bout of exercise (2-mile run). From WBC samples, we performed western blots to detect O-GlcNAc modified proteins. The distribution of O-GlcNAc in WBC subpopulations was assessed by flow cytometry. Elevation of serum lactic acid (increased from 1.3 ± 0.4 to 6.9 ± 1.7 mM), creatinine (from 77.5 ± 6.3 U/L to 102.2 ± 7.0 μM), and lactate dehydrogenase (from 318.5 ± 26.2 to 380.5 ± 33.2 U/L) confirmed the effect of exercise. WBC count also significantly increased (from 6.6 ± 1.0 to 8.4 ± 1.4 G/L). The level of O-GlcNAc modified proteins in WBCs showed significant elevation after exercise (85 ± 51%, p  O-GlcNAc status of WBCs. O-GlcNAc modification could be a natural process by which physical activity modulates the immune system. Further research could elucidate the role of O-GlcNAc during exercise and validate O-GlcNAc as a biomarker for fitness assessment.

  4. The human papillomavirus type 16 E6 oncoprotein activates mTORC1 signaling and increases protein synthesis.

    Science.gov (United States)

    Spangle, Jennifer M; Münger, Karl

    2010-09-01

    The mammalian target of rapamycin (mTOR) kinase acts as a cellular rheostat that integrates signals from a variety of cellular signal transduction pathways that sense growth factor and nutrient availability as well as intracellular energy status. It was previously reported that the human papillomavirus type 16 (HPV16) E6 oncoprotein may activate the S6 protein kinase (S6K) through binding and E6AP-mediated degradation of the mTOR inhibitor tuberous sclerosis complex 2 (TSC2) (Z. Lu, X. Hu, Y. Li, L. Zheng, Y. Zhou, H. Jiang, T. Ning, Z. Basang, C. Zhang, and Y. Ke, J. Biol. Chem. 279:35664-35670, 2004; L. Zheng, H. Ding, Z. Lu, Y. Li, Y. Pan, T. Ning, and Y. Ke, Genes Cells 13:285-294, 2008). Our results confirmed that HPV16 E6 expression causes an increase in mTORC1 activity through enhanced phosphorylation of mTOR and activation of downstream signaling pathways S6K and eukaryotic initiation factor binding protein 1 (4E-BP1). However, we did not detect a decrease in TSC2 levels in HPV16 E6-expressing cells. We discovered, however, that HPV16 E6 expression causes AKT activation through the upstream kinases PDK1 and mTORC2 under conditions of nutrient deprivation. We show that HPV16 E6 expression causes an increase in protein synthesis by enhancing translation initiation complex assembly at the 5' mRNA cap and an increase in cap-dependent translation. The increase in cap-dependent translation likely results from HPV16 E6-induced AKT/mTORC1 activation, as the assembly of the translation initiation complex and cap-dependent translation are rapamycin sensitive. Lastly, coexpression of the HPV16 E6 and E7 oncoproteins does not affect HPV16 E6-induced activation of mTORC1 and cap-dependent translation. HPV16 E6-mediated activation of mTORC1 signaling and cap-dependent translation may be a mechanism to promote viral replication under conditions of limited nutrient supply in differentiated, HPV oncoprotein-expressing proliferating cells.

  5. Increased binding of [3H] colchicine to visual cortex proteins of dark-reared rats on first exposure to light

    International Nuclear Information System (INIS)

    Stewart, M.G.; Rose, S.P.R.

    1978-01-01

    The binding of [ 3 H] colchicine (or a functionally similar metabolite) to acid-insoluble material in vivo was measured in the motor and visual cortices of littermate rats which were either dark-reared (D), exposed to light for 3 h or 24 h (L), or raised normally (N) in 12 h light/12 h dark animal house conditions. Significant differences were found in the binding in the motor cortex of the 3 h or 24 h L, D or N animals, but in the visual cortex after 3 h of light exposure a 23% elevation in binding was measured in L compared with D animals and a small though non-significant (10%) increase in binding was also observed in this region in L compared with N animals. After 24 h of light exposure, binding of the label in the L animals fell near to that of the N and D animals. The results of vinblastine precipitation experiments suggested that much of the radioactivity was bound to the protein tubulin, and this was confirmed when no increased binding of an analogue of colchicine, lumi-colchicine, was observed after 3 h of light exposure in L compared with D animals. It is suggested that these experiments show that colchicine can be used as a marker for changes in the tubulin population in light exposed animals, and demonstrate the transient nature of the increase in tubulin quantity, as opposed to a lasting effect on its synthesis. Further, they argue strongly in support of the idea that a component of protein flow from neuronal cell body to axons and dendrites in light exposed animals, is subject to environmental modification. (author)

  6. Production of Fibronectin Binding Protein A at the surface of Lactococcus lactis increases plasmid transfer in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Daniela Pontes

    Full Text Available Lactococci are noninvasive lactic acid bacteria frequently used as protein delivery vectors and, more recently, as DNA delivery vehicles. We previously showed that Lactococcus lactis (LL expressing the Fibronectin-Binding Protein A of Staphylococcus aureus (LL-FnBPA+ showed higher internalization rates in vitro in Caco-2 cells than the native (wt lactococci and were able to deliver a eukaryotic Green Fluorescent Protein (GFP expression plasmid in 1% of human Caco-2 cells. Here, using the bovine beta-lactoglobulin (BLG, one of the major cow's milk allergen, and GFP we characterized the potential of LL-FnBPA+ as an in vivo DNA vaccine delivery vehicle. We first showed that the invasive strain LL-FnBPA+ carrying the plasmid pValac:BLG (LL-FnBPA+ BLG was more invasive than LL-BLG and showed the same invasivity as LL-FnBPA+. Then we demonstrated that the Caco-2 cells, co-incubated with LL-FnBPA+ BLG produced up to 30 times more BLG than the Caco-2 cells co-incubated with the non invasive LL-BLG. Using two different gene reporters, BLG and GFP, and two different methods of detection, EIA and fluorescence microscopy, we showed in vivo that: i in order to be effective, LL-FnBPA+ required a pre-coating with Fetal Calf Serum before oral administration; ii plasmid transfer occurred in enterocytes without regard to the strains used (invasive or not; iii the use of LL-FnBPA+ increased the number of mice producing BLG, but not the level of BLG produced. We thus confirmed the good potential of invasive recombinant lactic acid bacteria as DNA delivery vector in vivo.

  7. Caspase activation increases beta-amyloid generation independently of caspase cleavage of the beta-amyloid precursor protein (APP).

    Science.gov (United States)

    Tesco, Giuseppina; Koh, Young Ho; Tanzi, Rudolph E

    2003-11-14

    The amyloid precursor protein (APP) undergoes "alternative" proteolysis mediated by caspases. Three major caspase recognition sites have been identified in the APP, i.e. one at the C terminus (Asp720) and two at the N terminus (Asp197 and Asp219). Caspase cleavage at Asp720 has been suggested as leading to increased production of Abeta. Thus, we set out to determine which putative caspase sites in APP, if any, are cleaved in Chinese hamster ovary cell lines concurrently with the increased Abeta production that occurs during apoptosis. We found that cleavage at Asp720 occurred concurrently with caspase 3 activation and the increased production of total secreted Abeta and Abeta1-42 in association with staurosporine- and etoposide-induced apoptosis. To investigate the contribution of caspase cleavage of APP to Abeta generation, we expressed an APP mutant truncated at Asp720 that mimics APP caspase cleavage at the C-terminal site. This did not increase Abeta generation but, in contrast, dramatically decreased Abeta production in Chinese hamster ovary cells. Furthermore, the ablation of caspase-dependent cleavage at Asp720, Asp197, and Asp219 (by site-directed mutagenesis) did not prevent enhanced Abeta production following etoposide-induced apoptosis. These findings indicate that the enhanced Abeta generation associated with apoptosis does not require cleavage of APP at its C-terminal (Asp720) and/or N-terminal caspase sites.

  8. Increased cerebrospinal fluid levels of cytokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1β (MIP-1β) in patients with amyotrophic lateral sclerosis.

    Science.gov (United States)

    Martínez, H R; Escamilla-Ocañas, C E; Camara-Lemarroy, C R; González-Garza, M T; Moreno-Cuevas, J; García Sarreón, M A

    2017-10-10

    Neuroinflammation has recently been described in amyotrophic lateral sclerosis (ALS). However, the precise role of such proinflammatory cytokines as monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1β (MIP-1β) in ALS has not yet been determined. In this study, we determined cerebrospinal fluid (CSF) MCP-1 and MIP-1β levels and assessed their association with the duration and severity of ALS. Concentrations of MCP-1 and MIP-1β were determined in the CSF of 77 patients diagnosed with ALS and 13 controls. Cytokine levels were analysed in relation to ALS duration (12months) and severity (30points on the ALS Functional Rating Scale administered at hospital admission). Higher CSF MIP-1β (10.68pg/mL vs. 4.69pg/mL, P<.0001) and MCP-1 (234.89pg/mL vs. 160.95pg/mL, P=.011) levels were found in the 77 patients with ALS compared to controls. There were no differences in levels of either cytokine in relation to disease duration or severity. However, we did observe a significant positive correlation between MIP-1β and MCP-1 in patients with ALS. The increase in MIP-1β and MCP-1 levels suggests that these cytokines may have a synergistic effect on ALS pathogenesis. However, in our cohort, no association was found with either the duration or the clinical severity of the disease. Copyright © 2017 Sociedad Española de Neurología. Publicado por Elsevier España, S.L.U. All rights reserved.

  9. Increased systemic elastase and C-reactive protein in aggressive periodontitis (CLOI-D-00160R2).

    Science.gov (United States)

    Wohlfeil, Martin; Scharf, Susanne; Siegelin, Yasemin; Schacher, Beate; Oremek, Gerhard M; Sauer-Eppel, Hildegund; Schubert, Ralf; Eickholz, Peter

    2012-08-01

    The inflammatory mediators, serum elastase and C-reactive protein (CRP), are associated with an increased risk for coronary heart disease. Thus, the aim of this study is to compare systemic inflammatory mediators in periodontally healthy controls (C), patients with untreated aggressive (AgP) and chronic (ChP) periodontitis. C [periodontal pocket probing depth (PPD)  30% of sites; age >35 years), and AgP (clinically healthy; PDD ≥ 3.6 mm at >30% of sites, bone loss ≥50% at ≥2 teeth; age ≤35 years) were examined clinically, and the body mass index was assessed. Blood was sampled for assessment of serum levels of elastase, CRP, lipopolysaccharide binding protein (LBP), interleukin (IL) 6, 8, and leukocyte counts. Thirty C, 31 ChP, and 29 AgP were analyzed. Elastase, CRP, LBP, and IL-6 levels were elevated in AgP compared to C (p C. AgP patients exhibit a stronger systemic inflammatory burden than C patients.

  10. Increased levels of proteins of the acute inflammatory phase in the peritoneal fluid of women with advanced stages of endometriosis.

    Science.gov (United States)

    Polak, Grzegorz; Barczyński, Bartłomiej; Bednarek, Wiesława; Kwaśniewski, Wojciech; Wertell, Iwona; Derewianka-Polak, Magdalena; Makara-Studzińska, Marta; Kotarski, Jan

    2015-06-01

    Most investigators agree that endometriosis is associated with a state of subclinical, non-infectious peritoneal inflammation. The objective of the study was to assess concentrations of two markers of the acute inflammatory phase proteins, haptoglobin and ceruloplasmin, in peritoneal fluid of endometriotic women. 229 women who underwent diagnostic or therapeutic laparoscopy were included in the study Minimal, mild, moderate and severe endometriosis according to ASRM was confirmed in 119 women (study groups), whereas 110 patients suffered from simple serous or dermoid ovarian cysts (reference groups). Haptoglobin and ceruloplasmin concentrations in the peritoneal fluid samples aspirated during laparoscopy were measured using commercially available radial immunodiffusion kits. The concentration of haptoglobin in the peritoneal fluid of women with endometriosis was significantly higher as compared to patients with serous and dermoid ovarian cysts. Significantly higher haptoglobin level was observed in patients with severe and moderate endometriosis as compared to women from both reference groups. No significant difference in the peritoneal fluid ceruloplasmin levels was found between patients with endometriosis and women from reference groups. However, it was noted that ceruloplasmin levels are higher in the subgroup of patients with severe endometriosis as compared to both reference groups and women with mild disease. Our results support the hypothesis that endometriosis is associated with subclinical inflammation within the peritoneal cavity It may be speculated that pro-inflammatory stimuli strong enough to cause an increase in acute inflammatory phase proteins peritoneal fluid concentrations are observed only in the advanced stages of the disease.

  11. Self-assembling protein nanoparticles with built-in flagellin domains increases protective efficacy of a Plasmodium falciparum based vaccine.

    Science.gov (United States)

    Kaba, Stephen A; Karch, Christopher P; Seth, Labdhi; Ferlez, Karen M B; Storme, Casey K; Pesavento, Danielle M; Laughlin, Paige Y; Bergmann-Leitner, Elke S; Burkhard, Peter; Lanar, David E

    2018-02-01

    To eliminate the problems associated with the use of extraneous adjuvants we have designed a Self-Assembling Protein Nanoparticle (SAPN) containing epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) (designated FMP014) and portions of the TLR5 agonist flagellin (designated FMP014 D0D1 ) as an intrinsic adjuvant. By combining different molar ratios of FMP014 to FMP014 D0D1 monomers before self-assembly, we generated multiple nanoparticles and investigated their biophysical characteristics, immunogenicity and protective efficacy. Immunization with the construct formulated with the ratio 58:2 of FMP014 to FMP014 D0D1 had the highest protective efficacy against a challenge with a transgenic P. berghei sporozoite expressing PfCSP. Increasing the proportion of flagellin per particle resulted in an inverse relationship with levels of both antibody titers and protection. The cytokine profiles of the various immunization groups were evaluated and quantitative amounts of the cytokines IL-2, IFN-γ, IL-12/p70 (Th1); IL4, IL5 (Th2); TNF-α, IL1β, IL-6, KC/GRO (pro-inflammatory), and IL-10 (immunomodulatory) were measured. The relationship of the cytokines to each other revealed a strong immunomodulatory effect depending on the proportion of flagellin in the construct. Our results demonstrate that SAPNs with flagellin may be a promising strategy for the development and delivery of a safe vaccine for infectious diseases. Published by Elsevier Ltd.

  12. CCAAT/enhancer binding protein β deletion increases mitochondrial function and protects mice from LXR-induced hepatic steatosis

    International Nuclear Information System (INIS)

    Rahman, Shaikh M.; Choudhury, Mahua; Janssen, Rachel C.; Baquero, Karalee C.; Miyazaki, Makoto; Friedman, Jacob E.

    2013-01-01

    Highlights: ► LXR agonist activation increases liver TG accumulation by increasing lipogenesis. ► C/EBPβ −/− mouse prevents LXR activation-mediated induction of hepatic lipogenesis. ► C/EBPβ deletion increases mitochondrial transport chain function. ► Beneficial effects of LXR activation on liver cholesterol metabolism did not change. ► C/EBPβ inhibition might have important therapeutic potential. -- Abstract: Drugs designed specifically to activate liver X receptors (LXRs) have beneficial effects on lowering cholesterol metabolism and inflammation but unfortunately lead to severe hepatic steatosis. The transcription factor CCAAT/enhancer binding protein beta (C/EBPβ) is an important regulator of liver gene expression but little is known about its involvement in LXR-based steatosis and cholesterol metabolism. The present study investigated the role of C/EBPβ expression in LXR agonist (T0901317)-mediated alteration of hepatic triglyceride (TG) and lipogenesis in mice. C/EBPβ deletion in mice prevented LXR agonist-mediated induction of lipogenic gene expression in liver in conjunction with significant reduction of liver TG accumulation. Surprisingly, C/EBPβ −/− mice showed a major increase in liver mitochondrial electron chain function compared to WT mice. Furthermore, LXR activation in C/EBPβ −/− mice increased the expression of liver ATP-binding cassette transporter ABCG1, a gene implicated in cholesterol efflux and reducing blood levels of total and LDL-cholesterol. Together, these findings establish a central role for C/EBPβ in the LXR-mediated steatosis and mitochondrial function, without impairing the influence of LXR activation on lowering LDL and increasing HDL-cholesterol. Inactivation of C/EBPβ might therefore be an important therapeutic strategy to prevent LXR activation-mediated adverse effects on liver TG metabolism without disrupting its beneficial effects on cholesterol metabolism.

  13. CCAAT/enhancer binding protein {beta} deletion increases mitochondrial function and protects mice from LXR-induced hepatic steatosis

    Energy Technology Data Exchange (ETDEWEB)

    Rahman, Shaikh M., E-mail: rmizanoor@hotmail.com [Department of Pediatrics, School of Medicine, University of Colorado Denver, Aurora, CO 80045 (United States); Choudhury, Mahua; Janssen, Rachel C.; Baquero, Karalee C. [Department of Pediatrics, School of Medicine, University of Colorado Denver, Aurora, CO 80045 (United States); Miyazaki, Makoto [Division of Renal Diseases and Hypertension, School of Medicine, University of Colorado Denver, Aurora, CO 80045 (United States); Friedman, Jacob E. [Department of Pediatrics, School of Medicine, University of Colorado Denver, Aurora, CO 80045 (United States); Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Denver, Aurora, CO 80045 (United States)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer LXR agonist activation increases liver TG accumulation by increasing lipogenesis. Black-Right-Pointing-Pointer C/EBP{beta}{sup -/-} mouse prevents LXR activation-mediated induction of hepatic lipogenesis. Black-Right-Pointing-Pointer C/EBP{beta} deletion increases mitochondrial transport chain function. Black-Right-Pointing-Pointer Beneficial effects of LXR activation on liver cholesterol metabolism did not change. Black-Right-Pointing-Pointer C/EBP{beta} inhibition might have important therapeutic potential. -- Abstract: Drugs designed specifically to activate liver X receptors (LXRs) have beneficial effects on lowering cholesterol metabolism and inflammation but unfortunately lead to severe hepatic steatosis. The transcription factor CCAAT/enhancer binding protein beta (C/EBP{beta}) is an important regulator of liver gene expression but little is known about its involvement in LXR-based steatosis and cholesterol metabolism. The present study investigated the role of C/EBP{beta} expression in LXR agonist (T0901317)-mediated alteration of hepatic triglyceride (TG) and lipogenesis in mice. C/EBP{beta} deletion in mice prevented LXR agonist-mediated induction of lipogenic gene expression in liver in conjunction with significant reduction of liver TG accumulation. Surprisingly, C/EBP{beta}{sup -/-} mice showed a major increase in liver mitochondrial electron chain function compared to WT mice. Furthermore, LXR activation in C/EBP{beta}{sup -/-} mice increased the expression of liver ATP-binding cassette transporter ABCG1, a gene implicated in cholesterol efflux and reducing blood levels of total and LDL-cholesterol. Together, these findings establish a central role for C/EBP{beta} in the LXR-mediated steatosis and mitochondrial function, without impairing the influence of LXR activation on lowering LDL and increasing HDL-cholesterol. Inactivation of C/EBP{beta} might therefore be an important therapeutic strategy to prevent LXR

  14. Opioid withdrawal increases transient receptor potential vanilloid 1 activity in a protein kinase A-dependent manner.

    Science.gov (United States)

    Spahn, Viola; Fischer, Oliver; Endres-Becker, Jeannette; Schäfer, Michael; Stein, Christoph; Zöllner, Christian

    2013-04-01

    Hyperalgesia is a cardinal symptom of opioid withdrawal. The transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated ion channel expressed on sensory neurons responding to noxious heat, protons, and chemical stimuli such as capsaicin. TRPV1 can be inhibited via μ-opioid receptor (MOR)-mediated reduced activity of adenylyl cyclases (ACs) and decreased cyclic adenosine monophosphate (cAMP) levels. In contrast, opioid withdrawal following chronic activation of MOR uncovers AC superactivation and subsequent increases in cAMP and protein kinase A (PKA) activity. Here we investigated (1) whether an increase in cAMP during opioid withdrawal increases the activity of TRPV1 and (2) how opioid withdrawal modulates capsaicin-induced nocifensive behavior in rats. We applied whole-cell patch clamp, microfluorimetry, cAMP assays, radioligand binding, site-directed mutagenesis, and behavioral experiments. Opioid withdrawal significantly increased cAMP levels and capsaicin-induced TRPV1 activity in both transfected human embryonic kidney 293 cells and dissociated dorsal root ganglion (DRG) neurons. Inhibition of AC and PKA, as well as mutations of the PKA phosphorylation sites threonine 144 and serine 774, prevented the enhanced TRPV1 activity. Finally, capsaicin-induced nocifensive behavior was increased during opioid withdrawal in vivo. In summary, our results demonstrate an increased activity of TRPV1 in DRG neurons as a new mechanism contributing to opioid withdrawal-induced hyperalgesia. Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2008-11-01

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

  16. Secretion of the endoplasmic reticulum stress protein, GRP78, into the BALF is increased in cigarette smokers.

    Science.gov (United States)

    Aksoy, Mark O; Kim, Victor; Cornwell, William D; Rogers, Thomas J; Kosmider, Beata; Bahmed, Karim; Barrero, Carlos; Merali, Salim; Shetty, Neena; Kelsen, Steven G

    2017-05-02

    Identification of biomarkers of cigarette smoke -induced lung damage and early COPD is an area of intense interest. Glucose regulated protein of 78 kD (i.e., GRP78), a multi-functional protein which mediates cell responses to oxidant stress, is increased in the lungs of cigarette smokers and in the serum of subjects with COPD. We have suggested that secretion of GRP78 by lung cells may explain the increase in serum GRP78 in COPD. To assess GRP78 secretion by the lung, we assayed GRP78 in bronchoalveolar lavage fluid (BALF) in chronic smokers and non-smokers. We also directly assessed the acute effect of cigarette smoke material on GRP78 secretion in isolated human airway epithelial cells (HAEC). GRP78 was measured in BALF of smokers (S; n = 13) and non-smokers (NS; n = 11) by Western blotting. GRP78 secretion by HAEC was assessed by comparing its concentration in cell culture medium and cell lysates. Cells were treated for 24 h with either the volatile phase of cigarette smoke (cigarette smoke extract (CSE) or the particulate phase (cigarette smoke condensate (CSC)). GRP78 was present in the BALF of both NS and S but levels were significantly greater in S (p = 0.04). GRP78 was secreted constitutively in HAEC. CSE 15% X 24 h increased GRP78 in cell-conditioned medium without affecting its intracellular concentration. In contrast, CSC X 24 h increased intracellular GRP78 expression but did not affect GRP78 secretion. Brefeldin A, an inhibitor of classical Golgi secretion pathways, did not inhibit GRP78 secretion indicating that non-classical pathways were involved. The present study indicates that GRP78 is increased in BALF in cigarette smokers; that HAEC secrete GRP78; and that GRP78 secretion by HAEC is augmented by cigarette smoke particulates. Enhanced secretion of GRP78 by lung cells makes it a potential biomarker of cigarette smoke-induced lung injury.

  17. Mutation of the key residue for extraribosomal function of ribosomal protein S19 cause increased grooming behaviors in mice.

    Science.gov (United States)

    Chen, Jun; Kaitsuka, Taku; Fujino, Rika; Araki, Kimi; Tomizawa, Kazuhito; Yamamoto, Tetsuro

    2016-08-26

    Ribosomal protein S19 (RP S19) possesses ribosomal function as RP S19 monomer and extraribosomal function as cross-linked RP S19 oligomers which function as a ligand of the complement 5a (C5a) receptor (CD88). We have generated a Gln137Glu-RP S19 knock-in (KI) mouse, which is shown to possess the weakened extraribosomal function of RP S19. Because whether the extraribosomal function of RP S19 has a role in brain function had been unclear, we performed behavioral analysis on these mice and demonstrated that KI mice displayed an increased grooming behavior during open-field test and elevated plus maze test and an enhanced freezing behavior in contextual fear conditioning test. These results suggest an involvement of RP S19 oligomers in some anxiety-like behavior, especially grooming behavior. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. Increased serum C-reactive protein concentrations in dogs with congestive heart failure due to myxomatous mitral valve disease

    DEFF Research Database (Denmark)

    Reimann, M. J.; Ljungvall, I.; Hillstrom, A.

    2016-01-01

    Cardiovascular disease in humans and dogs is associated with mildly increased circulating concentrations of C-reactive protein (CRP). Few studies have evaluated associations between circulating CRP and canine myxomatous mitral valve disease (MMVD) and the results reported have been divergent....... The aim of this study was to investigate whether serum concentrations of CRP, determined using a novel automated canine-specific high -sensitivity CRP assay (Gentian hsCRP), were associated with severity of MMVD and selected clinical variables in dogs. The study included 188 client-owned dogs...... with different severities of MMVD. Dogs were classified based on ACVIM consensus statement guidelines (group A, n = 58; group BI, n = 56; group B2, n = 38; group C, n = 36). Data were analysed using descriptive statistics and multiple regression analysis. Dogs with congestive heart failure (CHF; group C) had...

  19. Mutation of praR in Rhizobium leguminosarum enhances root biofilms, improving nodulation competitiveness by increased expression of attachment proteins.

    Science.gov (United States)

    Frederix, Marijke; Edwards, Anne; Swiderska, Anna; Stanger, Andrew; Karunakaran, Ramakrishnan; Williams, Alan; Abbruscato, Pamela; Sanchez-Contreras, Maria; Poole, Philip S; Downie, J Allan

    2014-08-01

    In Rhizobium leguminosarum bv. viciae, quorum-sensing is regulated by CinR, which induces the cinIS operon. CinI synthesizes an AHL, whereas CinS inactivates PraR, a repressor. Mutation of praR enhanced biofilms in vitro. We developed a light (lux)-dependent assay of rhizobial attachment to roots and demonstrated that mutation of praR increased biofilms on pea roots. The praR mutant out-competed wild-type for infection of pea nodules in mixed inoculations. Analysis of gene expression by microarrays and promoter fusions revealed that PraR represses its own transcription and mutation of praR increased expression of several genes including those encoding secreted proteins (the adhesins RapA2, RapB and RapC, two cadherins and the glycanase PlyB), the polysaccharide regulator RosR, and another protein similar to PraR. PraR bound to the promoters of several of these genes indicating direct repression. Mutations in rapA2, rapB, rapC, plyB, the cadherins or rosR did not affect the enhanced root attachment or nodule competitiveness of the praR mutant. However combinations of mutations in rapA, rapB and rapC abolished the enhanced attachment and nodule competitiveness. We conclude that relief of PraR-mediated repression determines a lifestyle switch allowing the expression of genes that are important for biofilm formation on roots and the subsequent initiation of infection of legume roots. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

  20. Activation of extrasynaptic, but not synaptic, NMDA receptors modifies amyloid precursor protein expression pattern and increases amyloid-ß production.

    Science.gov (United States)

    Bordji, Karim; Becerril-Ortega, Javier; Nicole, Olivier; Buisson, Alain

    2010-11-24

    Calcium is a key mediator controlling essential neuronal functions depending on electrical activity. Altered neuronal calcium homeostasis affects metabolism of amyloid precursor protein (APP), leading to increased production of β-amyloid (Aβ), and contributing to the initiation of Alzheimer's disease (AD). A linkage between excessive glutamate receptor activation and neuronal Aβ release was established, and recent reports suggest that synaptic and extrasynaptic NMDA receptor (NMDAR) activation may have distinct consequences in plasticity, gene regulation, and neuronal death. Here, we report for the first time that prolonged activation of extrasynaptic NMDAR, but not synaptic NMDAR, dramatically increased the neuronal production of Aβ. This effect was preceded by a shift from APP695 to Kunitz protease inhibitory domain (KPI) containing APPs (KPI-APPs), isoforms exhibiting an important amyloidogenic potential. Conversely, after synaptic NMDAR activation, we failed to detect any KPI-APP expression and neuronal Aβ production was not modified. Calcium imaging data showed that intracellular calcium concentration after extrasynaptic NMDAR stimulation was lower than after synaptic activation. This suggests distinct signaling pathways for each pool of receptors. We found that modification of neuronal APP expression pattern triggered by extrasynaptic NMDAR activation was regulated at an alternative splicing level involving calcium-/calmodulin-dependent protein kinase IV, but overall APP expression remained identical. Finally, memantine dose-dependently inhibited extrasynaptic NMDAR-induced KPI-APPs expression as well as neuronal Aβ release. Altogether, these data suggest that a chronic activation of extrasynaptic NMDAR promotes amyloidogenic KPI-APP expression leading to neuronal Aβ release, representing a causal risk factor for developing AD.

  1. Overexpression of Cholesteryl Ester Transfer Protein Increases Macrophage-Derived Foam Cell Accumulation in Atherosclerotic Lesions of Transgenic Rabbits

    Directory of Open Access Journals (Sweden)

    Shoucui Gao

    2017-01-01

    Full Text Available High levels of plasma high-density lipoprotein-cholesterol (HDL-C are inversely associated with the risk of atherosclerosis and other cardiovascular diseases; thus, pharmacological inhibition of cholesteryl ester transfer protein (CETP is considered to be a therapeutic method of raising HDL-C levels. However, many CETP inhibitors have failed to achieve a clinical benefit despite raising HDL-C. In the study, we generated transgenic (Tg rabbits that overexpressed the human CETP gene to examine the influence of CETP on the development of atherosclerosis. Both Tg rabbits and their non-Tg littermates were fed a high cholesterol diet for 16 weeks. Plasma lipids and body weight were measured every 4 weeks. Gross lesion areas of the aortic atherosclerosis along with lesional cellular components were quantitatively analyzed. Overexpression of human CETP did not significantly alter the gross atherosclerotic lesion area, but the number of macrophages in lesions was significantly increased. Overexpression of human CETP did not change the plasma levels of total cholesterol or low-density lipoprotein cholesterol but lowered plasma HDL-C and increased triglycerides. These data revealed that human CETP may play an important role in the development of atherosclerosis mainly by decreasing HDL-C levels and increasing the accumulation of macrophage-derived foam cells.

  2. Uncoupling protein and ATP/ADP carrier increase mitochondrial proton conductance after cold adaptation of king penguins.

    Science.gov (United States)

    Talbot, Darren A; Duchamp, Claude; Rey, Benjamin; Hanuise, Nicolas; Rouanet, Jean Louis; Sibille, Brigitte; Brand, Martin D

    2004-07-01

    Juvenile king penguins develop adaptive thermogenesis after repeated immersion in cold water. However, the mechanisms of such metabolic adaptation in birds are unknown, as they lack brown adipose tissue and uncoupling protein-1 (UCP1), which mediate adaptive non-shivering thermogenesis in mammals. We used three different groups of juvenile king penguins to investigate the mitochondrial basis of avian adaptive thermogenesis in vitro. Skeletal muscle mitochondria isolated from penguins that had never been immersed in cold water showed no superoxide-stimulated proton conductance, indicating no functional avian UCP. Skeletal muscle mitochondria from penguins that had been either experimentally immersed or naturally adapted to cold water did possess functional avian UCP, demonstrated by a superoxide-stimulated, GDP-inhibitable proton conductance across their inner membrane. This was associated with a markedly greater abundance of avian UCP mRNA. In the presence (but not the absence) of fatty acids, these mitochondria also showed a greater adenine nucleotide translocase-catalysed proton conductance than those from never-immersed penguins. This was due to an increase in the amount of adenine nucleotide translocase. Therefore, adaptive thermogenesis in juvenile king penguins is linked to two separate mechanisms of uncoupling of oxidative phosphorylation in skeletal muscle mitochondria: increased proton transport activity of avian UCP (dependent on superoxide and inhibited by GDP) and increased proton transport activity of the adenine nucleotide translocase (dependent on fatty acids and inhibited by carboxyatractylate).

  3. Protein kinase D is increased and activated in lung epithelial cells and macrophages in idiopathic pulmonary fibrosis.

    Science.gov (United States)

    Gan, Huachen; McKenzie, Raymond; Hao, Qin; Idell, Steven; Tang, Hua

    2014-01-01

    Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive and usually fatal lung disease of unknown etiology for which no effective treatments currently exist. Hence, there is a profound need for the identification of novel drugable targets to develop more specific and efficacious therapeutic intervention in IPF. In this study, we performed immunohistochemical analyses to assess the cell type-specific expression and activation of protein kinase D (PKD) family kinases in normal and IPF lung tissue sections. We also analyzed PKD activation and function in human lung epithelial cells. We found that PKD family kinases (PKD1, PKD2 and PKD3) were increased and activated in the hyperplastic and regenerative alveolar epithelial cells lining remodeled fibrotic alveolar septa and/or fibroblast foci in IPF lungs compared with normal controls. We also found that PKD family kinases were increased and activated in alveolar macrophages, bronchiolar epithelium, and honeycomb cysts in IPF lungs. Interestingly, PKD1 was highly expressed and activated in the cilia of IPF bronchiolar epithelial cells, while PKD2 and PKD3 were expressed in the cell cytoplasm and nuclei. In contrast, PKD family kinases were not apparently increased and activated in IPF fibroblasts or myofibroblasts. We lastly found that PKD was predominantly activated by poly-L-arginine, lysophosphatidic acid and thrombin in human lung epithelial cells and that PKD promoted epithelial barrier dysfunction. These findings suggest that PKD may participate in the pathogenesis of IPF and may be a novel target for therapeutic intervention in this disease.

  4. Dietary carbohydrate deprivation increases 24-hour nitrogen excretion without affecting postabsorptive hepatic or whole body protein metabolism in healthy men

    NARCIS (Netherlands)

    Bisschop, PH; de Sain-van der Velden, MGM; Stellaard, F; Kuipers, F; Meijer, AJ; Sauerwein, HP; Romijn, JA

    Because insulin is an important regulator of protein metabolism, we hypothesized that physiological modulation of insulin secretion, by means of extreme variations in dietary carbohydrate content, affects postabsorptive protein metabolism. Therefore, we studied the effects of three isocaloric diets

  5. Dietary carbohydrate deprivation increases 24-hour nitrogen excretion without affecting postabsorptive hepatic or whole body protein metabolism in healthy men

    NARCIS (Netherlands)

    Bisschop, P. H.; de Sain-van der Velden, M. G. M.; Stellaard, F.; Kuipers, F.; Meijer, A. J.; Sauerwein, H. P.; Romijn, J. A.

    2003-01-01

    Because insulin is an important regulator of protein metabolism, we hypothesized that physiological modulation of insulin secretion, by means of extreme variations in dietary carbohydrate content, affects postabsorptive protein metabolism. Therefore, we studied the effects of three isocaloric diets

  6. Phosphodiesterase inhibitor KMUP-3 displays cardioprotection via protein kinase G and increases cardiac output via G-protein-coupled receptor agonist activity and Ca2+ sensitization

    Directory of Open Access Journals (Sweden)

    Chung-Pin Liu

    2016-02-01

    Full Text Available KMUP-3 (7-{2-[4-(4-nitrobenzene piperazinyl]ethyl}-1, 3-dimethylxanthine displays cardioprotection and increases cardiac output, and is suggested to increase cardiac performance and improve myocardial infarction. To determine whether KMUP-3 improves outcomes in hypoperfused myocardium by inducing Ca2+ sensitization to oppose protein kinase (PKG-mediated Ca2+ blockade, we measured left ventricular systolic blood pressure, maximal rates of pressure development, mean arterial pressure and heart rate in rats, and measured contractility and expression of PKs/RhoA/Rho kinase (ROCKII in beating guinea pig left atria. Hemodynamic changes induced by KMUP-3 (0.5–3.0 mg/kg, intravenously were inhibited by Y27632 [(R-(+-trans-4-1-aminoethyl-N-(4-Pyridyl cyclohexane carboxamide] and ketanserin (1 mg/kg, intravenously. In electrically stimulated left guinea pig atria, positive inotropy induced by KMUP-3 (0.1–100μM was inhibited by the endothelial NO synthase (eNOS inhibitors N-nitro-l-arginine methyl ester (L-NAME and 7-nitroindazole, cyclic AMP antagonist SQ22536 [9-(terahydro-2-furanyl-9H-purin-6-amine], soluble guanylyl cyclase (sGC antagonist ODQ (1H-[1,2,4] oxadiazolo[4,3-a] quinoxalin-1-one, RhoA inhibitor C3 exoenzyme, β-blocker propranolol, 5-hydroxytryptamine 2A antagonist ketanserin, ROCK inhibitor Y27632 and KMUP-1 (7-{2-[4-(2-chlorobenzene piperazinyl]ethyl}-1, 3-dimethylxanthine at 10μM. Western blotting assays indicated that KMUP-3 (0.1–10μM increased PKA, RhoA/ROCKII, and PKC translocation and CIP-17 (an endogenous 17-kDa inhibitory protein activation. In spontaneous right atria, KMUP-3 induced negative chronotropy that was blunted by 7-nitroindazole and atropine. In neonatal myocytes, L-NAME inhibited KMUP-3-induced eNOS phosphorylation and RhoA/ROCK activation. In H9c2 cells, Y-27632 (50μM and PKG antagonist KT5823 [2,3,9,10,11,12-hexahydro-10R- methoxy-2,9-dimethyl-1-oxo-9S,12R-epoxy-1H-diindolo(1,2,3-fg:3′,2′,1

  7. Loss of function of ATXN1 increases amyloid beta-protein levels by potentiating beta-secretase processing of beta-amyloid precursor protein.

    Science.gov (United States)

    Zhang, Can; Browne, Andrew; Child, Daniel; Divito, Jason R; Stevenson, Jesse A; Tanzi, Rudolph E

    2010-03-19

    Alzheimer disease (AD) is a devastating neurodegenerative disease with complex and strong genetic inheritance. Four genes have been established to either cause familial early onset AD (APP, PSEN1, and PSEN2) or to increase susceptibility for late onset AD (APOE). To date approximately 80% of the late onset AD genetic variance remains elusive. Recently our genome-wide association screen identified four novel late onset AD candidate genes. Ataxin 1 (ATXN1) is one of these four AD candidate genes and has been indicated to be the disease gene for spinocerebellar ataxia type 1, which is also a neurodegenerative disease. Mounting evidence suggests that the excessive accumulation of Abeta, the proteolytic product of beta-amyloid precursor protein (APP), is the primary AD pathological event. In this study, we ask whether ATXN1 may lead to AD pathogenesis by affecting Abeta and APP processing utilizing RNA interference in a human neuronal cell model and mouse primary cortical neurons. We show that knock-down of ATXN1 significantly increases the levels of both Abeta40 and Abeta42. This effect could be rescued with concurrent overexpression of ATXN1. Moreover, overexpression of ATXN1 decreased Abeta levels. Regarding the underlying molecular mechanism, we show that the effect of ATXN1 expression on Abeta levels is modulated via beta-secretase cleavage of APP. Taken together, ATXN1 functions as a genetic risk modifier that contributes to AD pathogenesis through a loss-of-function mechanism by regulating beta-secretase cleavage of APP and Abeta levels.

  8. Loss of Function of ATXN1 Increases Amyloid β-Protein Levels by Potentiating β-Secretase Processing of β-Amyloid Precursor Protein*

    Science.gov (United States)

    Zhang, Can; Browne, Andrew; Child, Daniel; DiVito, Jason R.; Stevenson, Jesse A.; Tanzi, Rudolph E.

    2010-01-01

    Alzheimer disease (AD) is a devastating neurodegenerative disease with complex and strong genetic inheritance. Four genes have been established to either cause familial early onset AD (APP, PSEN1, and PSEN2) or to increase susceptibility for late onset AD (APOE). To date ∼80% of the late onset AD genetic variance remains elusive. Recently our genome-wide association screen identified four novel late onset AD candidate genes. Ataxin 1 (ATXN1) is one of these four AD candidate genes and has been indicated to be the disease gene for spinocerebellar ataxia type 1, which is also a neurodegenerative disease. Mounting evidence suggests that the excessive accumulation of Aβ, the proteolytic product of β-amyloid precursor protein (APP), is the primary AD pathological event. In this study, we ask whether ATXN1 may lead to AD pathogenesis by affecting Aβ and APP processing utilizing RNA interference in a human neuronal cell model and mouse primary cortical neurons. We show that knock-down of ATXN1 significantly increases the levels of both Aβ40 and Aβ42. This effect could be rescued with concurrent overexpression of ATXN1. Moreover, overexpression of ATXN1 decreased Aβ levels. Regarding the underlying molecular mechanism, we show that the effect of ATXN1 expression on Aβ levels is modulated via β-secretase cleavage of APP. Taken together, ATXN1 functions as a genetic risk modifier that contributes to AD pathogenesis through a loss-of-function mechanism by regulating β-secretase cleavage of APP and Aβ levels. PMID:20097758

  9. Increased protein-energy intake promotes anabolism in critically ill infants with viral bronchiolitis: a double-blind randomised controlled trial

    NARCIS (Netherlands)

    de Betue, C.T.; van Waardenburg, D.A.; Deutz, N.E.; van Eijk, H.M.; van Goudoever, J.B.; Luiking, Y.C.; Zimmermann, L.J.; Joosten, K.F.

    2011-01-01

    The preservation of nutritional status and growth is an important aim in critically ill infants, but difficult to achieve due to the metabolic stress response and inadequate nutritional intake, leading to negative protein balance. This study investigated whether increasing protein and energy intakes

  10. Association of increased radiocurability of murine carcinomas with low constitutive expression of p21{sup WAF1/CIP1} protein

    Energy Technology Data Exchange (ETDEWEB)

    Akimoto, Tetsuo; Seong, Jinsil; Hunter, Nancy R; Buchmiller, Lara; Mason, Kathy; Milas, Luka

    1999-05-01

    Purpose: The study investigated whether basal, constitutive levels of p21{sup WAF1/CIP1} protein in murine carcinomas are related to in vivo tumor radioresponse. The study is based on recent observations demonstrating that in vitro cancer cell lines are resistant to cytotoxic drugs when they express high basal levels of p21{sup WAF1/CIP1} protein, and that the loss of the p21 gene in the HCT116 human colorectal cancer cell line results in increased radioresponse of xenografts derived from that cell line. Methods and Materials: Protein levels of p21{sup WAF1/CIP1}, p53, bax, and bcl-2 were determined in 8 carcinomas (3 mammary carcinomas designated MCa-4, MCa-29, and MCa-35, 2 squamous cell carcinomas designated SCC-IV and SCC-VII, ovarian adenocarcinoma OCa-I, hepatocarcinoma HCa-I, and adenosquamous carcinoma ACa-SG) syngeneic to C3Hf/Kam mice using Western blot analysis. The tumors, growing in the right hind legs of mice, were 8 mm in diameter at the time of analysis. These tumors greatly differ in their radioresponse, assessed by TCD50 assay, and in their susceptibility to radiation-induced apoptosis. Results: Protein levels of these oncogenes varied among tumors, with p21{sup WAF1/CIP1} showing the greatest variation: its mean densitometric value ranged from 1 to 19. Bcl-2 levels also showed broad variation in densitometric values, from 1 to 10. In comparison, bax and p53 (7 of 8 tumors contained wild-type p53) varied much less among different tumor types; their variation was within a 5-fold range, and the level of p53 was similar in 6 of 8 tumors. Tumor radioresponse correlated significantly (R = 0.77, p = 0.02) only with the magnitude of p21{sup WAF1/CIP1}expression: tumors with high levels of p21{sup WAF1/CIP1}were less radiocurable than those with lower levels. Tumor radiocurability showed a significant positive correlation (p = 0.02) with the extent of radiation-induced apoptosis, indicating that tumors that responded to radiation with higher percentages

  11. Cardiac expression of microsomal triglyceride transfer protein is increased in obesity and serves to attenuate cardiac triglyceride accumulation.

    Directory of Open Access Journals (Sweden)

    Emil D Bartels

    Full Text Available Obesity causes lipid accumulation in the heart and may lead to lipotoxic heart disease. Traditionally, the size of the cardiac triglyceride pool is thought to reflect the balance between uptake and beta-oxidation of fatty acids. However, triglycerides can also be exported from cardiomyocytes via secretion of apolipoproteinB-containing (apoB lipoproteins. Lipoprotein formation depends on expression of microsomal triglyceride transfer protein (MTP; the mouse expresses two isoforms of MTP, A and B. Since many aspects of the link between obesity-induced cardiac disease and cardiac lipid metabolism remain unknown, we investigated how cardiac lipoprotein synthesis affects cardiac expression of triglyceride metabolism-controlling genes, insulin sensitivity, and function in obese mice. Heart-specific ablation of MTP-A in mice using Cre-loxP technology impaired upregulation of MTP expression in response to increased fatty acid availability during fasting and fat feeding. This resulted in cardiac triglyceride accumulation but unaffected cardiac insulin-stimulated glucose uptake. Long-term fat-feeding of male C57Bl/6 mice increased cardiac triglycerides, induced cardiac expression of triglyceride metabolism-controlling genes and attenuated heart function. Abolishing cardiac triglyceride accumulation in fat-fed mice by overexpression of an apoB transgene in the heart prevented the induction of triglyceride metabolism-controlling genes and improved heart function. The results suggest that in obesity, the physiological increase of cardiac MTP expression serves to attenuate cardiac triglyceride accumulation albeit without major effects on cardiac insulin sensitivity. Nevertheless, the data suggest that genetically increased lipoprotein secretion prevents development of obesity-induced lipotoxic heart disease.

  12. Molecular Stress-inducing Compounds Increase Osteoclast Formation in a Heat Shock Factor 1 Protein-dependent Manner*

    Science.gov (United States)

    Chai, Ryan C.; Kouspou, Michelle M.; Lang, Benjamin J.; Nguyen, Chau H.; van der Kraan, A. Gabrielle J.; Vieusseux, Jessica L.; Lim, Reece C.; Gillespie, Matthew T.; Benjamin, Ivor J.; Quinn, Julian M. W.; Price, John T.

    2014-01-01

    Many anticancer therapeutic agents cause bone loss, which increases the risk of fractures that severely reduce quality of life. Thus, in drug development, it is critical to identify and understand such effects. Anticancer therapeutic and HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) causes bone loss by increasing osteoclast formation, but the mechanism underlying this is not understood. 17-AAG activates heat shock factor 1 (Hsf1), the master transcriptional regulator of heat shock/cell stress responses, which may be involved in this negative action of 17-AAG upon bone. Using mouse bone marrow and RAW264.7 osteoclast differentiation models we found that HSP90 inhibitors that induced a heat shock response also enhanced osteoclast formation, whereas HSP90 inhibitors that did not (including coumermycin A1 and novobiocin) did not affect osteoclast formation. Pharmacological inhibition or shRNAmir knockdown of Hsf1 in RAW264.7 cells as well as the use of Hsf1 null mouse bone marrow cells demonstrated that 17-AAG-enhanced osteoclast formation was Hsf1-dependent. Moreover, ectopic overexpression of Hsf1 enhanced 17-AAG effects upon osteoclast formation. Consistent with these findings, protein levels of the essential osteoclast transcription factor microphthalmia-associated transcription factor were increased by 17-AAG in an Hsf1-dependent manner. In addition to HSP90 inhibitors, we also identified that other agents that induced cellular stress, such as ethanol, doxorubicin, and methotrexate, also directly increased osteoclast formation, potentially in an Hsf1-dependent manner. These results, therefore, indicate that cellular stress can enhance osteoclast differentiation via Hsf1-dependent mechanisms and may significantly contribute to pathological and therapeutic related bone loss. PMID:24692538

  13. Oral contraceptives increase insulin-like growth factor binding protein-1 concentration in women with polycystic ovarian disease.

    Science.gov (United States)

    Suikkari, A M; Tiitinen, A; Stenman, U H; Seppälä, M; Laatikainen, T

    1991-05-01

    Insulin-like growth factor-I (IGF-I) stimulates ovarian androgen production. Insulin-like growth factor binding protein-1 (IGFBP-1) inhibits IGF actions in vitro. To investigate the effect of oral contraceptive (OC) pills, given for 3 months, on serum gonadotropin, androgen, IGF-I, and IGFBP-1 concentrations, and glucose tolerance in seven women with polycystic ovarian disease (PCOD) and in five healthy control subjects. Seven women with PCOD and five healthy control subjects. An oral glucose tolerance test (OGTT) was performed before and after treatment with OC. After treatment with OC, serum luteinizing hormone, androstenedione, and free testosterone levels decreased, and sex hormone-binding globulin concentration increased in the women with PCOD as well as in the control subjects. The cumulative response of serum insulin to OGTT was larger in the women with PCOD than in the control subjects both before and after treatment. Serum IGF-I concentration, which was unchanged during OGTT, decreased from basal level of 326 +/- 70 micrograms/L to 199 +/- 28 micrograms/L after treatment with OC in the women with PCOD, whereas no change was found in the control subjects (from 235 +/- 11 micrograms/L to 226 +/- 11 micrograms/L). Treatment with OC caused an increase of the mean basal IGFBP-1 concentration from 24 +/- 7 micrograms/L to 73 +/- 14 micrograms/L in the women with PCOD. This increase was constant during the OGTT. In the control subjects, treatment with OC did not result in any significant change in IGFBP-1 concentrations (from 44 +/- 11 micrograms/L to 61 +/- 9 micrograms/L). The combination of decreased total IGF-I concentration and increased IGFBP-1 concentration induced by OC may decrease ovarian androgen production in PCOD.

  14. Molecular stress-inducing compounds increase osteoclast formation in a heat shock factor 1 protein-dependent manner.

    Science.gov (United States)

    Chai, Ryan C; Kouspou, Michelle M; Lang, Benjamin J; Nguyen, Chau H; van der Kraan, A Gabrielle J; Vieusseux, Jessica L; Lim, Reece C; Gillespie, Matthew T; Benjamin, Ivor J; Quinn, Julian M W; Price, John T

    2014-05-09

    Many anticancer therapeutic agents cause bone loss, which increases the risk of fractures that severely reduce quality of life. Thus, in drug development, it is critical to identify and understand such effects. Anticancer therapeutic and HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) causes bone loss by increasing osteoclast formation, but the mechanism underlying this is not understood. 17-AAG activates heat shock factor 1 (Hsf1), the master transcriptional regulator of heat shock/cell stress responses, which may be involved in this negative action of 17-AAG upon bone. Using mouse bone marrow and RAW264.7 osteoclast differentiation models we found that HSP90 inhibitors that induced a heat shock response also enhanced osteoclast formation, whereas HSP90 inhibitors that did not (including coumermycin A1 and novobiocin) did not affect osteoclast formation. Pharmacological inhibition or shRNAmir knockdown of Hsf1 in RAW264.7 cells as well as the use of Hsf1 null mouse bone marrow cells demonstrated that 17-AAG-enhanced osteoclast formation was Hsf1-dependent. Moreover, ectopic overexpression of Hsf1 enhanced 17-AAG effects upon osteoclast formation. Consistent with these findings, protein levels of the essential osteoclast transcription factor microphthalmia-associated transcription factor were increased by 17-AAG in an Hsf1-dependent manner. In addition to HSP90 inhibitors, we also identified that other agents that induced cellular stress, such as ethanol, doxorubicin, and methotrexate, also directly increased osteoclast formation, potentially in an Hsf1-dependent manner. These results, therefore, indicate that cellular stress can enhance osteoclast differentiation via Hsf1-dependent mechanisms and may significantly contribute to pathological and therapeutic related bone loss.

  15. Activated protein C plays no major roles in the inhibition of coagulation or increased fibrinolysis in acute coagulopathy of trauma-shock: a systematic review.

    Science.gov (United States)

    Gando, Satoshi; Mayumi, Toshihiko; Ukai, Tomohiko

    2018-01-01

    The pathophysiological mechanisms of acute coagulopathy of trauma-shock (ACOTS) are reported to include activated protein C-mediated suppression of thrombin generation via the proteolytic inactivation of activated Factor V (FVa) and FVIIIa; an increased fibrinolysis via neutralization of plasminogen activator inhibitor-1 (PAI-1) by activated protein C. The aims of this study are to review the evidences for the role of activated protein C in thrombin generation and fibrinolysis and to validate the diagnosis of ACOTS based on the activated protein C dynamics. We conducted systematic literature search (2007-2017) using PubMed, the Cochrane Database of Systematic Reviews (CDSR), and the Cochrane Central Register of Controlled Trials (CENTRAL). Clinical studies on trauma that measured activated protein C or the circulating levels of activated protein C-related coagulation and fibrinolysis markers were included in our study. Out of 7613 studies, 17 clinical studies met the inclusion criteria. The levels of activated protein C in ACOTS were inconsistently decreased, showed no change, or were increased in comparison to the control groups. Irrespective of the activated protein C levels, thrombin generation was always preserved or highly elevated. There was no report on the activated protein C-mediated neutralization of PAI-1 with increased fibrinolysis. No included studies used unified diagnostic criteria to diagnose ACOTS and those studies also used different terms to refer to the condition known as ACOTS. None of the studies showed direct cause and effect relationships between activated protein C and the suppression of coagulation and increased fibrinolysis. No definitive diagnostic criteria or unified terminology have been established for ACOTS based on the activated protein C dynamics.

  16. Angiopoietin-like protein 2 increases renal fibrosis by accelerating transforming growth factor-β signaling in chronic kidney disease.

    Science.gov (United States)

    Morinaga, Jun; Kadomatsu, Tsuyoshi; Miyata, Keishi; Endo, Motoyoshi; Terada, Kazutoyo; Tian, Zhe; Sugizaki, Taichi; Tanigawa, Hiroki; Zhao, Jiabin; Zhu, Shunshun; Sato, Michio; Araki, Kimi; Iyama, Ken-ichi; Tomita, Kengo; Mukoyama, Masashi; Tomita, Kimio; Kitamura, Kenichiro; Oike, Yuichi

    2016-02-01

    Renal fibrosis is a common pathological consequence of chronic kidney disease (CKD) with tissue fibrosis closely associated with chronic inflammation in numerous pathologies. However, molecular mechanisms underlying that association, particularly in the kidney, remain unclear. Here, we determine whether there is a molecular link between chronic inflammation and tissue fibrosis in CKD progression. Histological analysis of human kidneys indicated abundant expression of angiopoietin-like protein 2 (ANGPTL2) in renal tubule epithelial cells during progression of renal fibrosis. Numerous ANGPTL2-positive renal tubule epithelial cells colocalized with cells positive for transforming growth factor (TGF)-β1, a critical mediator of tissue fibrosis. Analysis of M1 collecting duct cells in culture showed that TGF-β1 increases ANGPTL2 expression by attenuating its repression through microRNA-221. Conversely, ANGPTL2 increased TGF-β1 expression through α5β1 integrin-mediated activation of extracellular signal-regulated kinase. Furthermore, ANGPTL2 deficiency in a mouse unilateral ureteral obstruction model significantly reduced renal fibrosis by decreasing TGF-β1 signal amplification in kidney. Thus, ANGPTL2 and TGF-β1 positively regulate each other as renal fibrosis progresses. Our study provides insight into molecular mechanisms underlying chronic inflammation and tissue fibrosis and identifies potential therapeutic targets for CKD treatment. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  17. Replacing dietary glucose with fructose increases ChREBP activity and SREBP-1 protein in rat liver nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Koo, Hyun-Young [Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, 905 S. Goodwin Avenue, Urbana, IL 61801 (United States); Miyashita, Michio [Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, 905 S. Goodwin Avenue, Urbana, IL 61801 (United States); Department of Pediatrics, Nihon University School of Medicine, Itabashi, Tokyo (Japan); Simon Cho, B.H. [Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, 905 S. Goodwin Avenue, Urbana, IL 61801 (United States); Harlan E. Moore Heart Research Foundation, 503 South Sixth Street, Champaign, IL 61820 (United States); Nakamura, Manabu T., E-mail: mtnakamu@illinois.edu [Division of Nutritional Sciences, University of Illinois at Urbana-Champaign, 905 S. Goodwin Avenue, Urbana, IL 61801 (United States)

    2009-12-11

    Diets high in fructose cause hypertriglyceridemia and insulin resistance in part due to simultaneous induction of gluconeogenic and lipogenic genes in liver. We investigated the mechanism underlying the unique pattern of gene induction by dietary fructose. Male Sprague-Dawley rats (n = 6 per group) were meal-fed (4 h/d) either 63% (w/w) glucose or 63% fructose diet. After two weeks, animals were killed at the end of the last meal. Nuclear SREBP-1 was 2.2 times higher in fructose-fed rats than glucose-fed rats. Nuclear FoxO1 was elevated 1.7 times in fructose group, but did not reach significance (P = 0.08). Unexpectedly, no difference was observed in nuclear ChREBP between two groups. However, ChREBP DNA binding was 3.9x higher in fructose-fed animals without an increase in xylulose-5-phospate, a proposed ChREBP activator. In conclusion, the gene induction by dietary fructose is likely to be mediated in part by simultaneously increased ChREBP activity, SREBP-1 and possibly FoxO1 protein in nucleus.

  18. Tocotrienols and Whey Protein Isolates Substantially Increase Exercise Endurance Capacity in Diet -Induced Obese Male Sprague-Dawley Rats

    Science.gov (United States)

    Aguila, Jay; McConell, Glenn K.; McAinch, Andrew J.; Mathai, Michael L.

    2016-01-01

    Background and Aims Obesity and impairments in metabolic health are associated with reductions in exercise capacity. Both whey protein isolates (WPIs) and vitamin E tocotrienols (TCTs) exert favorable effects on obesity-related metabolic parameters. This research sought to determine whether these supplements improved exercise capacity and increased glucose tolerance in diet-induced obese rats. Methods Six week old male rats (n = 35) weighing 187 ± 32g were allocated to either: Control (n = 9), TCT (n = 9), WPI (n = 8) or TCT + WPI (n = 9) and placed on a high-fat diet (40% of energy from fat) for 10 weeks. Animals received 50mg/kg body weight and 8% of total energy intake per day of TCTs and/or WPIs respectively. Food intake, body composition, glucose tolerance, insulin sensitivity, exercise capacity, skeletal muscle glycogen content and oxidative enzyme activity were determined. Results Both TCT and WPI groups ran >50% longer (2271 ± 185m and 2195 ± 265m respectively) than the Control group (1428 ± 139m) during the run to exhaustion test (Pexercise endurance (2068 ± 104m). WPIs increased the maximum in vitro activity of beta-hydroxyacyl-CoA in the soleus muscle (Pexercise endurance by 50% in sedentary, diet-induced obese rats. These positive effects of TCTs and WPIs were independent of body weight, adiposity or glucose tolerance. PMID:27058737

  19. Replacing dietary glucose with fructose increases ChREBP activity and SREBP-1 protein in rat liver nucleus

    International Nuclear Information System (INIS)

    Koo, Hyun-Young; Miyashita, Michio; Simon Cho, B.H.; Nakamura, Manabu T.

    2009-01-01

    Diets high in fructose cause hypertriglyceridemia and insulin resistance in part due to simultaneous induction of gluconeogenic and lipogenic genes in liver. We investigated the mechanism underlying the unique pattern of gene induction by dietary fructose. Male Sprague-Dawley rats (n = 6 per group) were meal-fed (4 h/d) either 63% (w/w) glucose or 63% fructose diet. After two weeks, animals were killed at the end of the last meal. Nuclear SREBP-1 was 2.2 times higher in fructose-fed rats than glucose-fed rats. Nuclear FoxO1 was elevated 1.7 times in fructose group, but did not reach significance (P = 0.08). Unexpectedly, no difference was observed in nuclear ChREBP between two groups. However, ChREBP DNA binding was 3.9x higher in fructose-fed animals without an increase in xylulose-5-phospate, a proposed ChREBP activator. In conclusion, the gene induction by dietary fructose is likely to be mediated in part by simultaneously increased ChREBP activity, SREBP-1 and possibly FoxO1 protein in nucleus.

  20. Hormone replacement therapy increases levels of antibodies against heat shock protein 65 and certain species of oxidized low density lipoprotein

    Directory of Open Access Journals (Sweden)

    Uint L.

    2003-01-01

    Full Text Available Hormone replacement therapy (HRT reduces cardiovascular risks, although the initiation of therapy may be associated with transient adverse ischemic and thrombotic events. Antibodies against heat shock protein (Hsp and oxidized low density lipoprotein (LDL have been found in atherosclerotic lesions and plasma of patients with coronary artery disease and may play an important role in the pathogenesis of atherosclerosis. The aim of the present study was to assess the effects of HRT on the immune response by measuring plasma levels of antibodies against Hsp 65 and LDL with a low and high degree of copper-mediated oxidative modification of 20 postmenopausal women before and 90 days after receiving orally 0.625 mg equine conjugate estrogen plus 2.5 mg medroxyprogesterone acetate per day. HRT significantly increased antibodies against Hsp 65 (0.316 ± 0.03 vs 0.558 ± 0.11 and against LDL with a low degree of oxidative modification (0.100 ± 0.01 vs 0.217 ± 0.02 (P<0.05 and P<0.001, respectively, ANOVA. The hormone-mediated immune response may trigger an inflammatory response within the vessel wall and potentially increase plaque burden. Whether or not this immune response is temporary or sustained and deleterious requires further investigation.

  1. Paroxetine Is a Direct Inhibitor of G Protein-Coupled Receptor Kinase 2 and Increases Myocardial Contractility

    Energy Technology Data Exchange (ETDEWEB)

    Thal, David M. [Univ. of Michigan, Ann Arbor, MI (United States); Homan, Kristoff T. [Univ. of Michigan, Ann Arbor, MI (United States); Chen, Jun [Univ. of New Mexico Health Sciences Center, Albuquerque, NM (United States); Wu, Emily K. [Univ. of Michigan, Ann Arbor, MI (United States); Hinkle, Patricia M. [Univ. of Rochester Medical Center, Rochester, NY (United States); Huang, Z. Maggie [Temple Univ. School of Medicine, Philadelphia, Pennsylvania (United States); Chuprun, J. Kurt [Temple Univ. School of Medicine, Philadelphia, Pennsylvania (United States); Song, Jianliang [Temple Univ. School of Medicine, Philadelphia, Pennsylvania (United States); Gao, Erhe [Temple Univ. School of Medicine, Philadelphia, Pennsylvania (United States); Cheung, Joseph Y. [Temple Univ. School of Medicine, Philadelphia, Pennsylvania (United States); Sklar, Larry A. [Univ. of New Mexico Health Sciences Center, Albuquerque, NM (United States); Koch, Walter J. [Temple Univ. School of Medicine, Philadelphia, Pennsylvania (United States); Tesmer, John J.G. [Univ. of Michigan, Ann Arbor, MI (United States)

    2012-08-10

    G protein-coupled receptor kinase 2 (GRK2) is a well-established therapeutic target for the treatment of heart failure. In this paper we identify the selective serotonin reuptake inhibitor (SSRI) paroxetine as a selective inhibitor of GRK2 activity both in vitro and in living cells. In the crystal structure of the GRK2·paroxetine–Gβγ complex, paroxetine binds in the active site of GRK2 and stabilizes the kinase domain in a novel conformation in which a unique regulatory loop forms part of the ligand binding site. Isolated cardiomyocytes show increased isoproterenol-induced shortening and contraction amplitude in the presence of paroxetine, and pretreatment of mice with paroxetine before isoproterenol significantly increases left ventricular inotropic reserve in vivo with no significant effect on heart rate. Neither is observed in the presence of the SSRI fluoxetine. Our structural and functional results validate a widely available drug as a selective chemical probe for GRK2 and represent a starting point for the rational design of more potent and specific GRK2 inhibitors.

  2. Bone morphogenetic protein 4 induces differentiation of colorectal cancer stem cells and increases their response to chemotherapy in mice.

    Science.gov (United States)

    Lombardo, Ylenia; Scopelliti, Alessandro; Cammareri, Patrizia; Todaro, Matilde; Iovino, Flora; Ricci-Vitiani, Lucia; Gulotta, Gaspare; Dieli, Francesco; de Maria, Ruggero; Stassi, Giorgio

    2011-01-01

    The limited clinical response observed in many patients with colorectal cancer may be related to the presence of chemoresistant colorectal cancer stem cells (CRC-SCs). Bone morphogenetic protein 4 (BMP4) promotes the differentiation of normal colonic stem cells. We investigated whether BMP4 might be used to induce differentiation of CRC-SCs and for therapeutic purposes. CRC-SCs were isolated from 25 tumor samples based on expression of CD133 or using a selection culture medium. BMP4 expression and activity on CRC-SCs were evaluated in vitro; progeny of the stem cells were evaluated by immunofluorescence, immunoblot, and flow cytometry analyses. The potential therapeutic effect of BMP4 was assessed in immunocompromised mice after injection of CRC-SCs that responded to chemotherapy (n = 4) or that did not (n = 2). CRC-SCs did not express BMP4 whereas differentiated cells did. Recombinant BMP4 promoted differentiation and apoptosis of CRC-SCs in 12 of 15 independent experiments; this effect did not depend on Small Mothers against decapentaplegic (Smad)4 expression level or microsatellite stability. BMP4 activated the canonical and noncanonical BMP signaling pathways, including phosphoInositide 3-kinase (PI3K) and PKB (protein kinase B)/AKT. Mutations in PI3K or loss of Phosphatase and Tensin homolog (PTEN) in Smad4-defective tumors made CRC-SCs unresponsive to BMP4. Administration of BMP4 to immunocompromised mice with tumors that arose from CRC-SCs increased the antitumor effects of 5-fluorouracil and oxaliplatin. BMP4 promotes terminal differentiation, apoptosis, and chemosensitization of CRC-SCs in tumors that do not have simultaneous mutations in Smad4 and constitutive activation of PI3K. BMP4 might be developed as a therapeutic agent against cancer stem cells in advanced colorectal tumors. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

  3. Aggregation of polyQ proteins is increased upon yeast aging and affected by Sir2 and Hsf1: novel quantitative biochemical and microscopic assays.

    Directory of Open Access Journals (Sweden)

    Aviv Cohen

    Full Text Available Aging-related neurodegenerative disorders, such as Parkinson's, Alzheimer's and Huntington's diseases, are characterized by accumulation of protein aggregates in distinct neuronal cells that eventually die. In Huntington's disease, the protein huntingtin forms aggregates, and the age of disease onset is inversely correlated to the length of the protein's poly-glutamine tract. Using quantitative assays to estimate microscopically and capture biochemically protein aggregates, here we study in Saccharomyces cerevisiae aging-related aggregation of GFP-tagged, huntingtin-derived proteins with different polyQ lengths. We find that the short 25Q protein never aggregates whereas the long 103Q version always aggregates. However, the mid-size 47Q protein is soluble in young logarithmically growing yeast but aggregates as the yeast cells enter the stationary phase and age, allowing us to plot an "aggregation timeline". This aging-dependent aggregation was associated with increased cytotoxicity. We also show that two aging-related genes, SIR2 and HSF1, affect aggregation of the polyQ proteins. In Δsir2 strain the aging-dependent aggregation of the 47Q protein is aggravated, while overexpression of the transcription factor Hsf1 attenuates aggregation. Thus, the mid-size 47Q protein and our quantitative aggregation assays provide valuable tools to unravel the roles of genes and environmental conditions that affect aging-related aggregation.

  4. Caffeine Increases Apolipoprotein A-1 and Paraoxonase-1 but not Paraoxonase-3 Protein Levels in Human-Derived Liver (HepG2) Cells.

    Science.gov (United States)

    Sayılan Özgün, Gülben; Özgün, Eray; Tabakçıoğlu, Kıymet; Süer Gökmen, Selma; Eskiocak, Sevgi; Çakır, Erol

    2017-12-01

    Apolipoprotein A-1, paraoxonase-1 and paraoxonase-3 are antioxidant and anti-atherosclerotic structural high-density lipoprotein proteins that are mainly synthesized by the liver. No study has ever been performed to specifically examine the effects of caffeine on paraoxonase enzymes and on liver apolipoprotein A-1 protein levels. To investigate the dose-dependent effects of caffeine on liver apolipoprotein A-1, paraoxonase-1 and paraoxonase-3 protein levels. In vitro experimental study. HepG2 cells were incubated with 0 (control), 10, 50 and 200 μM of caffeine for 24 hours. Cell viability was evaluated by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apolipoprotein A-1, paraoxonase-1 and paraoxonase-3 protein levels were measured by western blotting. We observed a significant increase on apolipoprotein A-1 and paraoxonase-1 protein levels in the cells incubated with 50 µM of caffeine and a significant increase on paraoxonase-1 protein level in the cells incubated with 200 µM of caffeine. Our study showed that caffeine does not change paraoxonase-3 protein level, but the higher doses used in our study do cause an increase in both apolipoprotein A-1 and paraoxonase-1 protein levels in liver cells.

  5. Body fat and insulin resistance independently predict increased serum C-reactive protein in hyperandrogenic women with polycystic ovary syndrome.

    Science.gov (United States)

    Tosi, Flavia; Dorizzi, Romolo; Castello, Roberto; Maffeis, Claudio; Spiazzi, Giovanna; Zoppini, Giacomo; Muggeo, Michele; Moghetti, Paolo

    2009-11-01

    Increased serum C-reactive protein (CRP), an independent predictor of coronary heart disease, was reported in women with polycystic ovary syndrome (PCOS). It remains unclear whether this finding is due to the association between PCOS and either insulin resistance, obesity, or androgen excess, which are all common features of this condition. The aims of this study were to assess whether increased serum CRP is a specific feature of PCOS and to investigate the mechanisms underlying this association. Serum high-sensitivity CRP (hs-CRP) was measured in 86 hyperandrogenic women (age 21.6+/-4.2 years, body mass index (BMI) 23.6+/-3.5 kg/m2), 50 with PCOS and 36 with idiopathic hyperandrogenism (HA). Thirty-five BMI-matched healthy women were also studied as controls. In these subjects, endocrine and metabolic profiles were assessed. In all hyperandrogenic subjects and 14 controls, insulin sensitivity was measured by the glucose clamp technique. Body fat was measured by bioelectrical impedance. Hs-CRP concentrations were higher in PCOS women (3.43+/-2.01 mg/l) than in HA subjects and healthy women (2.43+/-1.04, PPCOS). In multiple regression analyses, increased serum hs-CRP was independently predicted by higher body fat and lower insulin sensitivity. However, in lean women, serum-free testosterone was an additional, negative, predictive variable. PCOS is accompanied by a low-grade chronic inflammation. Body fat appears the main determining factor of this finding, which is only partly explained by insulin resistance. At least in lean women, androgen excess per se seems to play an additional, possibly protective, role in this association.

  6. Increasing brain serotonin corrects CO2 chemosensitivity in methyl-CpG-binding protein 2 (Mecp2)-deficient mice

    Science.gov (United States)

    Toward, Marie A.; Abdala, Ana P.; Knopp, Sharon J.; Paton, Julian F. R.; Bissonnette, John M.

    2013-01-01

    Mice deficient in the transcription factor methyl-CpG-binding protein 2 (Mecp2), a mouse model of Rett syndrome, display reduced CO2 chemosensitivity, which may contribute to their breathing abnormalities. In addition, patients with Rett syndrome and male mice that are null for Mecp2 show reduced levels of brain serotonin (5-HT). Serotonin is known to play a role in central chemosensitivity, and we hypothesized that increasing the availability of 5-HT in this mouse model would improve their respiratory response to CO2. Here we determined the apnoeic threshold in heterozygous Mecp2-deficient female mice and examined the effects of blocking 5-HT reuptake on the CO2 response in Mecp2-null male mice. Studies were performed in B6.129P2(C)-Mecp2τm1.1Bird null males and heterozygous females. In an in situ preparation, seven of eight Mecp2-deficient heterozygous females showed arrest of phrenic nerve activity when arterial CO2 was lowered to 3%, whereas the wild-types maintained phrenic nerve amplitude at 53 ± 3% of maximal. In vivo plethysmography studies were used to determine CO2 chemosensitivity in null males. These mice were exposed sequentially to 1, 3 and 5% CO2. The percentage increase in minute ventilation in response to increased inspired CO2 was less in Mecp2−/y than in Mecp2+/y mice. Pretreatment with citalopram, a selective 5-HT reuptake inhibitor (2.5 mg kg−1 I.P.), 40 min prior to CO2 exposure, in Mecp2−/y mice resulted in an improvement in CO2 chemosensitivity to wild-type levels. These results suggest that decreased 5-HT in Mecp2-deficient mice reduces CO2 chemosensitivity, and restoring 5-HT levels can reverse this effect. PMID:23180809

  7. Adenosine A1 receptor activation increases myocardial protein S-nitrosothiols and elicits protection from ischemia-reperfusion injury in male and female hearts.

    Directory of Open Access Journals (Sweden)

    Qin Shao

    Full Text Available Nitric oxide (NO plays an important role in cardioprotection, and recent work from our group and others has implicated protein S-nitrosylation (SNO as a critical component of NO-mediated protection in different models, including ischemic pre- and post-conditioning and sex-dependent cardioprotection. However, studies have yet to examine whether protein SNO levels are similarly increased with pharmacologic preconditioning in male and female hearts, and whether an increase in protein SNO levels, which is protective in male hearts, is sufficient to increase baseline protection in female hearts. Therefore, we pharmacologically preconditioned male and female hearts with the adenosine A1 receptor agonist N6-cyclohexyl adenosine (CHA. CHA administration prior to ischemia significantly improved functional recovery in both male and female hearts compared to baseline in a Langendorff-perfused heart model of ischemia-reperfusion injury (% of preischemic function ± SE: male baseline: 37.5±3.4% vs. male CHA: 55.3±3.2%; female baseline: 61.4±5.7% vs. female CHA: 76.0±6.2%. In a separate set of hearts, we found that CHA increased p-Akt and p-eNOS levels. We also used SNO-resin-assisted capture with LC-MS/MS to identify SNO proteins in male and female hearts, and determined that CHA perfusion induced a modest increase in protein SNO levels in both male (11.4% and female (12.3% hearts compared to baseline. These findings support a potential role for protein SNO in a model of pharmacologic preconditioning, and provide evidence to suggest that a modest increase in protein SNO levels is sufficient to protect both male and female hearts from ischemic injury. In addition, a number of the SNO proteins identified with CHA treatment were also observed with other forms of cardioprotective stimuli in prior studies, further supporting a role for protein SNO in cardioprotection.

  8. A lower isoelectric point increases signal sequence-mediated secretion of recombinant proteins through a bacterial ABC transporter.

    Science.gov (United States)

    Byun, Hyunjong; Park, Jiyeon; Kim, Sun Chang; Ahn, Jung Hoon

    2017-12-01

    Efficient protein production for industrial and academic purposes often involves engineering microorganisms to produce and secrete target proteins into the culture. Pseudomonas fluorescens has a TliDEF ATP-binding cassette transporter, a type I secretion system, which recognizes C-terminal LARD3 signal sequence of thermostable lipase TliA. Many proteins are secreted by TliDEF in vivo when recombined with LARD3, but there are still others that cannot be secreted by TliDEF even when LARD3 is attached. However, the factors that determine whether or not a recombinant protein can be secreted through TliDEF are still unknown. Here, we recombined LARD3 with several proteins and examined their secretion through TliDEF. We found that the proteins secreted via LARD3 are highly negatively charged with highly-acidic isoelectric points (pI) lower than 5.5. Attaching oligo-aspartate to lower the pI of negatively-charged recombinant proteins improved their secretion, and attaching oligo-arginine to negatively-charged proteins blocked their secretion by LARD3. In addition, negatively supercharged green fluorescent protein (GFP) showed improved secretion, whereas positively supercharged GFP did not secrete. These results disclosed that proteins' acidic pI and net negative charge are major factors that determine their secretion through TliDEF. Homology modeling for TliDEF revealed that TliD dimer forms evolutionarily-conserved positively-charged clusters in its pore and substrate entrance site, which also partially explains the pI dependence of the TliDEF-dependent secretions. In conclusion, lowering the isoelectric point improved LARD3-mediated protein secretion, both widening the range of protein targets for efficient production via secretion and signifying an important aspect of ABC transporter-mediated secretions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. N-epsilon-(carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins.

    OpenAIRE

    Ahmed, M U; Brinkmann Frye, E; Degenhardt, T P; Thorpe, S R; Baynes, J W

    1997-01-01

    Advanced glycation end-products and glycoxidation products, such as Nepsilon-(carboxymethyl)lysine (CML) and pentosidine, accumulate in long-lived tissue proteins with age and are implicated in the aging of tissue proteins and in the development of pathology in diabetes, atherosclerosis and other diseases. In this paper we describe a new advanced glycation end-product, Nepsilon-(carboxyethyl)lysine (CEL), which is formed during the reaction of methylglyoxal with lysine residues in model compo...

  10. Low dietary protein is associated with an increase in food intake and a decrease in the in vitro release of radiolabeled glutamate and GABA from the lateral hypothalamus.

    Science.gov (United States)

    White, B D; Du, F; Higginbotham, D A

    2003-12-01

    Moderately low-protein diets lead to a rapid increase in food intake and body fat. The increase in feeding is associated with a decrease in the concentration of serum urea nitrogen, suggesting that the low-protein-induced increase in food intake may be related to the decreased metabolism of nitrogen from amino acids. We hypothesized that low dietary protein would be associated with a decrease in the synaptic release of two nitrogen-containing neurotransmitters, GABA and glutamate, whose nitrogen can be derived from amino acids. In this study, we examined the effects of a low-protein diet (10% casein) in Sprague-Dawley rats on the in vitro release of 3H-GABA and 14C-glutamate from the lateral and medial hypothalamus. The low-protein diet increased food intake by about 25% after one day. After four days, the in vitro release of radiolabeled GABA and glutamate was assessed. The calcium-dependent, potassium-stimulated release of radiolabeled GABA and glutamate from the lateral hypothalamus was decreased in rats fed the low-protein diet. The magnitude of neurotransmitter release from the lateral hypothalamus inversely correlated with food intake. No dietary differences in the release of neurotransmitters from the medial hypothalamus were observed. These results support the contention that alterations in nitrogen metabolism are associated with low-protein-induced feeding.

  11. An amino acid substitution in the human intestinal fatty acid binding protein is associated with increased fatty acid binding, increased fat oxidation, and insulin resistance.

    OpenAIRE

    Baier, L J; Sacchettini, J C; Knowler, W C; Eads, J; Paolisso, G; Tataranni, P A; Mochizuki, H; Bennett, P H; Bogardus, C; Prochazka, M

    1995-01-01

    The intestinal fatty acid binding protein locus (FABP2) was investigated as a possible genetic factor in determining insulin action in the Pima Indian population. A polymorphism at codon 54 of FABP2 was identified that results in an alanine-encoding allele (frequency 0.71) and a threonine-encoding allele (frequency 0.29). Pimas who were homozygous or heterozygous for the threonine-encoding allele were found to have a higher mean fasting plasma insulin concentration, a lower mean insulin-stimu...

  12. Increased serum cartilage oligomeric matrix protein levels and decreased patellar bone mineral density in patients with chondromalacia patellae.

    Science.gov (United States)

    Murphy, E; FitzGerald, O; Saxne, T; Bresnihan, B

    2002-11-01

    Chondromalacia patellae is a potentially disabling disorder characterised by features of patellar cartilage degradation. To evaluate markers of cartilage and bone turnover in patients with chondromalacia patellae. 18 patients with chondromalacia patellae were studied. Serum cartilage oligomeric matrix protein (s-COMP) and bone sialoprotein (s-BSP) levels were measured by enzyme linked immunosorbent assay (ELISA) and compared with those of age and sex matched healthy control subjects. Periarticular bone mineral density (BMD) of both knee joints was assessed by dual energy x ray absorptiometry (DXA). s-COMP levels were significantly raised in all patients with chondromalacia patellae compared with healthy control subjects (p=0.0001). s-BSP levels did not differ significantly between the groups (p=0.41). BMD of the patella was significantly reduced in patients with chondromalacia patellae compared with the control subjects (p=0.016). In patients with bilateral chondromalacia patellae, BMD of the patella was lower in the more symptomatic knee joint (p=0.005). Changes in periarticular BMD were localised to the patella and were not present in femoral regions. Neither s-COMP (p=0.18) nor s-BSP (p=0.40) levels correlated with patellar BMD. Increased s-COMP levels, reflecting cartilage degradation, and reduced BMD localised to the patella may represent clinically useful markers in the diagnosis and monitoring of patients with chondromalacia patellae. Measures of cartilage degradation did not correlate with loss of patellar bone density, suggesting dissociated pathophysiological mechanisms.

  13. Phosphorylation of hormone-sensitive lipase by protein kinase A in vitro promotes an increase in its hydrophobic surface area

    DEFF Research Database (Denmark)

    Krintel, Christian; Mörgelin, Matthias; Logan, Derek T

    2009-01-01

    Hormone-sensitive lipase (EC 3.1.1.79; HSL) is a key enzyme in the mobilization of fatty acids from stored triacylglycerols. HSL activity is controlled by phosphorylation of at least four serines. In rat HSL, Ser563, Ser659 and Ser660 are phosphorylated by protein kinase A (PKA) in vitro as well......, the hydrophobic fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) was found to inhibit the hydrolysis of triolein by purified recombinant rat adipocyte HSL, with a decrease in the effect of bis-ANS upon PKA phosphorylation of HSL. The interaction of HSL with bis-ANS was found to have...... a Kd of 1 microM in binding assays. Upon PKA phosphorylation, the interactions of HSL with both bis-ANS and the alternative probe SYPRO Orange were increased. By negative stain transmission electron microscopy, phosphorylated HSL was found to have a closer interaction with phospholipid vesicles than...

  14. Overexpression of human sperm protein 17 increases migration and decreases the chemosensitivity of human epithelial ovarian cancer cells

    International Nuclear Information System (INIS)

    Li, Fang-qiu; Han, Yan-ling; Liu, Qun; Wu, Bo; Huang, Wen-bin; Zeng, Su-yun

    2009-01-01

    Most deaths from ovarian cancer are due to metastases that are resistant to conventional therapies. But the factors that regulate the metastatic process and chemoresistance of ovarian cancer are poorly understood. In the current study, we investigated the aberrant expression of human sperm protein 17 (HSp17) in human epithelial ovarian cancer cells and tried to analyze its influences on the cell behaviors like migration and chemoresistance. Immunohistochemistry and immunocytochemistry were used to identify HSp17 in paraffin embedded ovarian malignant tumor specimens and peritoneal metastatic malignant cells. Then we examined the effect of HSp17 overexpression on the proliferation, migration, and chemoresistance of ovarian cancer cells to carboplatin and cisplatin in a human ovarian carcinoma cell line, HO8910. We found that HSp17 was aberrantly expressed in 43% (30/70) of the patients with primary epithelial ovarian carcinomas, and in all of the metastatic cancer cells of ascites from 8 patients. The Sp17 expression was also detected in the metastatic lesions the same as in ovarian lesions. None of the 7 non-epithelial tumors primarily developed in the ovaries was immunopositive for HSp17. Overexpression of HSp17 increased the migration but decreased the chemosensitivity of ovarian carcinoma cells to carboplatin and cisplatin. HSp17 is aberrantly expressed in a significant proportion of epithelial ovarian carcinomas. Our results strongly suggest that HSp17 plays a role in metastatic disease and resistance of epithelial ovarian carcinoma to chemotherapy

  15. Expression of protein engineered NADP{sup +}-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Matsushika, Akinori; Inoue, Hiroyuki; Murakami, Katsuji; Takimura, Osamu; Sawayama, Shigeki [National Institute of Advanced Industrial Science and Technology, Hiroshima (Japan). Biomass Technology Research Center; Watanabe, Seiya; Kodaki, Tsutomu; Makino, Keisuke [Kyoto Univ. (Japan). Inst. of Advanced Energy

    2008-11-15

    A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD{sup +}-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP{sup +}. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP{sup +}-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP{sup +}-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain. (orig.)

  16. Elevated Maternal C-Reactive Protein is Associated with Increased Risk of Schizophrenia in a National Birth Cohort

    Science.gov (United States)

    Canetta, Sarah; Sourander, Andre; Surcel, Helja-Marja; Hinkka-Yli-Salomäki, Susanna; Leiviskä, Jaana; Kellendonk, Christoph; McKeague, Ian W.; Brown, Alan S.

    2014-01-01

    Objective The goal of the present study was to investigate an association between early gestational C-reactive protein (CRP), an established inflammatory biomarker, prospectively assayed in maternal sera, and schizophrenia in a large national birth cohort with an extensive serum biobank. Methods This study utilized a nested case-control design from the Finnish Prenatal Study of Schizophrenia cohort. 777 schizophrenia cases (630 with schizophrenia, 147 with schizoaffective disorder) that had maternal sera available for CRP testing were identified and matched to 777 controls in the analysis. Maternal CRP levels were assessed using a latex immunoassay from archived maternal serum specimens. Results Increasing maternal CRP levels, classified as a continuous variable, were significantly associated with schizophrenia in offspring (adjusted odds ratio (OR)=1.31, 95% confidence interval (CI)=1.10-1.56, p=0.003). This finding remained significant after adjusting for potential confounders including maternal and parental history of psychiatric disorders, twin/singleton birth, urbanicity, province of birth, and maternal socioeconomic status. Conclusion This finding provides the most robust evidence to date that maternal inflammation may play a significant role in schizophrenia, with possible implications for identifying preventive strategies and pathogenic mechanisms in schizophrenia and other neurodevelopmental disorders. PMID:24969261

  17. High C reactive protein associated with increased pulse wave velocity among urban men with metabolic syndrome in Malaysia.

    Science.gov (United States)

    Aminuddin, Amilia; Zakaria, Zaiton; Fuad, Ahmad F; Kamsiah, Jaarin; Othman, Faizah; Das, Srijit; Kamisah, Yusof; Qodriyah, Haji S; Jubri, Zakiah; Nordin, Nor Anita M; Ngah, Wan Z

    2013-03-01

    To determine the association between carotid femoral pulse wave velocity (PWVCF); augmentation index (AI); and high-sensitivity C reactive protein (hs-CRP) with metabolic syndrome (MetS), and to determine the influence of ethnicity on PWVCF and AI, and the association between high hs-CRP and increased PWV, and AI in MetS. A cross-sectional study was conducted at Universiti Kebangsaan Malaysia Medical Center, Kuala Lumpur, Malaysia from September 2009 to September 2011. Three hundred and eighty men (Chinese and Malays) were recruited from the study. The PWVCF and AI were measured by Vicorder (SMT Medical, Wuerzburg, Germany). The hs-CRP level was also determined. We defined MetS using the International Diabetes Federation (IDF) and harmonized criteria. Malays had higher AI compared to the Malaysian Chinese. Patients with MetS had higher PWVCF (IDF criteria: 8.5 [8.3-8.7] versus 8.2 [8.0-8.4] m/s, p=0.03; harmonized criteria: 8.5 [8.4-8.7] versus 8.2 [8.0-8.4] m/s, p=0.007) and hs-CRP (IDF criteria: 0.9+/-2.0 versus 0.4+/-1.1 mg/L, p=0.0007; harmonized criteria: 0.8+/-1.9 versus 0.4+/-1.1 mg/L, p=0.002) compared to non-MetS. In subjects with MetS, those with high hs-CRP (>3 mg/L) had higher PWVCF. Augmentation index values were significantly higher in Malays compared with Malaysian Chinese. Metabolic syndrome was associated with increased PWVCF and hs-CRP. Patients with MetS and high hs-CRP were associated with higher PWVCF. The measurement of hs-CRP reflects the degree of subclinical vascular damage in MetS.

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

    Science.gov (United States)

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

    2001-07-01

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

  19. Modulation of Thiol-Disulfide Oxidoreductases for Increased Production of Disulfide-Bond-Containing Proteins in Bacillus subtilis

    NARCIS (Netherlands)

    Kouwen, Thijs R. H. M.; Dubois, Jean-Yves F.; Freudl, Roland; Quax, Wim J.; van Dijl, Jan Maarten

    2008-01-01

    Disulfide bonds are important for the correct folding, structural integrity, and activity of many biotechnologically relevant proteins. For synthesis and subsequent secretion of these proteins in bacteria, such as the well-known "cell factory" Bacillus subtilis, it is often the correct formation of

  20. Polymorphisms in the prion protein gene and in the doppel gene increase susceptibility for Creutzfeldt-Jakob disease

    NARCIS (Netherlands)

    E.A. Croes (Esther); B.Z. Alizadeh (Behrooz); A.M. Bertoli Avella (Aida); T.A.M. Rademaker (Tessa); J. Vergeer-Drop (Jeannette); B. Dermaut (Bart); J.J. Houwing-Duistermaat (Jeanine); D.P.W.M. Wientjens (Dorothee); A. Hofman (Albert); C. van Broeckhoven (Christine); C.M. van Duijn (Cornelia)

    2004-01-01

    textabstractThe prion protein gene (PRNP) plays a central role in the origin of Creutzfeldt-Jakob disease (CJD), but there is growing interest in other polymorphisms that may be involved in CJD. Polymorphisms upstream of PRNP that may modulate the prion protein production as well as polymorphisms in

  1. Increased protein-energy intake promotes anabolism in critically ill infants with viral bronchiolitis: a double‑blind randomised controlled trial

    Science.gov (United States)

    de Betue, Carlijn T; van Waardenburg, Dick A; Deutz, Nicolaas E; van Eijk, Hans M; van Goudoever, Johannes B; Luiking, Yvette C; Zimmermann, Luc J; Joosten, Koen F

    2011-01-01

    Objective The preservation of nutritional status and growth is an important aim in critically ill infants, but difficult to achieve due to the metabolic stress response and inadequate nutritional intake, leading to negative protein balance. This study investigated whether increasing protein and energy intakes can promote anabolism. The primary outcome was whole body protein balance, and the secondary outcome was first pass splanchnic phenylalanine extraction (SPEPhe). Design This was a double-blind randomised controlled trial. Infants (n=18) admitted to the paediatric intensive care unit with respiratory failure due to viral bronchiolitis were randomised to continuous enteral feeding with protein and energy enriched formula (PE-formula) (n=8; 3.1±0.3 g protein/kg/24 h, 119±25 kcal/kg/24 h) or standard formula (S-formula) (n=10; 1.7±0.2 g protein/kg/24 h, 84±15 kcal/kg/24 h; equivalent to recommended intakes for healthy infants <6 months). A combined intravenous-enteral phenylalanine stable isotope protocol was used on day 5 after admission to determine whole body protein metabolism and SPEPhe. Results Protein balance was significantly higher with PE-formula than with S-formula (PE-formula: 0.73±0.5 vs S-formula: 0.02±0.6 g/kg/24 h) resulting from significantly increased protein synthesis (PE-formula: 9.6±4.4, S-formula: 5.2±2.3 g/kg/24 h), despite significantly increased protein breakdown (PE-formula: 8.9±4.3, S-formula: 5.2±2.6 g/kg/24 h). SPEPhe was not statistically different between the two groups (PE-formula: 39.8±18.3%, S-formula: 52.4±13.6%). Conclusions Increasing protein and energy intakes promotes protein anabolism in critically ill infants in the first days after admission. Since this is an important target of nutritional support, increased protein and energy intakes should be preferred above standard intakes in these infants. Dutch Trial Register number: NTR 515. PMID:21673183

  2. Increased protein damage in renal glomeruli, retina, nerve, plasma and urine and its prevention by thiamine and benfotiamine therapy in a rat model of diabetes.

    Science.gov (United States)

    Karachalias, N; Babaei-Jadidi, R; Rabbani, N; Thornalley, P J

    2010-07-01

    The aim of this study was to quantify protein damage by glycation, oxidation and nitration in a rat model of diabetes at the sites of development of microvascular complications, including the effects of thiamine and benfotiamine therapy. Diabetes was induced in male Sprague-Dawley rats by 55 mg/kg streptozotocin and moderated by insulin (2 U twice daily). Diabetic and control rats were given thiamine or benfotiamine (7 or 70 mg kg(-1) day(-1)) over 24 weeks. Plasma, urine and tissues were collected and analysed for protein damage by stable isotopic dilution analysis MS. There were two- to fourfold increases in fructosyl-lysine and AGE content of glomerular, retinal, sciatic nerve and plasma protein in diabetes. Increases in AGEs were reversed by thiamine and benfotiamine therapy but increases in fructosyl-lysine were not. Methionine sulfoxide content of plasma protein and 3-nitrotyrosine content of sciatic nerve protein were increased in diabetes. Plasma glycation free adducts were increased up to twofold in diabetes; the increases were reversed by thiamine. Urinary excretion of glycation, oxidation and nitration free adducts was increased by seven- to 27-fold in diabetes. These increases were reversed by thiamine and benfotiamine therapy. AGEs, particularly arginine-derived hydroimidazolones, accumulate at sites of microvascular complication development and have markedly increased urinary excretion rates in experimental diabetes. Thiamine and benfotiamine supplementation prevented tissue accumulation and increased urinary excretion of protein glycation, oxidation and nitration adducts. Similar effects may contribute to the reversal of early-stage clinical diabetic nephropathy by thiamine.

  3. Insulin-like growth factor binding protein-1 levels are increased in patients with IgA nephropathy

    International Nuclear Information System (INIS)

    Tokunaga, Koki; Uto, Hirofumi; Takami, Yoichiro; Mera, Kumiko; Nishida, Chika; Yoshimine, Yozo; Fukumoto, Mayumi; Oku, Manei; Sogabe, Atsushi; Nosaki, Tsuyoshi; Moriuchi, Akihiro; Oketani, Makoto; Ido, Akio; Tsubouchi, Hirohito

    2010-01-01

    Research highlights: → IGFBP-1 mRNA over express in kidneys obtained from mice model of IgA nephropathy. → Serum IGFBP-1 levels are high in patients with IgA nephropathy. → Serum IGFBP-1 levels correlate with renal function and the severity of renal injury. -- Abstract: The mechanisms underlying the pathogenesis of immunoglobulin A (IgA) nephropathy (IgAN) are not well understood. In this study, we examined gene expression profiles in kidneys obtained from mice with high serum IgA levels (HIGA mice), which exhibit features of human IgAN. Female inbred HIGA, established from the ddY line, were used in these experiments. Serum IgA levels, renal IgA deposition, mesangial proliferation, and glomerulosclerosis were increased in 32-week-old HIGA mice in comparison to ddY animals. By microarray analysis, five genes were observed to be increased by more than 2.5-fold in 32-week-old HIGA in comparison to 16-week-old HIGA; these same five genes were decreased more than 2.5-fold in 32-week-old ddY in comparison to 16-week-old ddY mice. Of these five genes, insulin-like growth factor (IGF) binding protein (IGFBP)-1 exhibited differential expression between these mouse lines, as confirmed by quantitative RT-PCR. In addition, serum IGFBP-1 levels were significantly higher in patients with IgAN than in healthy controls. In patients with IgAN, these levels correlated with measures of renal function, such as estimated glomerular filtration rate (eGFR), but not with sex, age, serum IgA, C3 levels, or IGF-1 levels. Pathologically, serum IGFBP-1 levels were significantly associated with the severity of renal injury, as assessed by mesangial cell proliferation and interstitial fibrosis. These results suggest that increased IGFBP-1 levels are associated with the severity of renal pathology in patients with IgAN.

  4. Insulin-like growth factor binding protein-1 levels are increased in patients with IgA nephropathy

    Energy Technology Data Exchange (ETDEWEB)

    Tokunaga, Koki [Department of Digestive and Life-Style Related Disease, Health Research Course, Human and Environmental Sciences, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8520 (Japan); Uto, Hirofumi, E-mail: hirouto@m2.kufm.kagoshima-u.ac.jp [Department of Digestive and Life-Style Related Disease, Health Research Course, Human and Environmental Sciences, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8520 (Japan); Takami, Yoichiro; Mera, Kumiko; Nishida, Chika; Yoshimine, Yozo; Fukumoto, Mayumi; Oku, Manei; Sogabe, Atsushi; Nosaki, Tsuyoshi; Moriuchi, Akihiro; Oketani, Makoto; Ido, Akio; Tsubouchi, Hirohito [Department of Digestive and Life-Style Related Disease, Health Research Course, Human and Environmental Sciences, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8520 (Japan)

    2010-08-20

    Research highlights: {yields} IGFBP-1 mRNA over express in kidneys obtained from mice model of IgA nephropathy. {yields} Serum IGFBP-1 levels are high in patients with IgA nephropathy. {yields} Serum IGFBP-1 levels correlate with renal function and the severity of renal injury. -- Abstract: The mechanisms underlying the pathogenesis of immunoglobulin A (IgA) nephropathy (IgAN) are not well understood. In this study, we examined gene expression profiles in kidneys obtained from mice with high serum IgA levels (HIGA mice), which exhibit features of human IgAN. Female inbred HIGA, established from the ddY line, were used in these experiments. Serum IgA levels, renal IgA deposition, mesangial proliferation, and glomerulosclerosis were increased in 32-week-old HIGA mice in comparison to ddY animals. By microarray analysis, five genes were observed to be increased by more than 2.5-fold in 32-week-old HIGA in comparison to 16-week-old HIGA; these same five genes were decreased more than 2.5-fold in 32-week-old ddY in comparison to 16-week-old ddY mice. Of these five genes, insulin-like growth factor (IGF) binding protein (IGFBP)-1 exhibited differential expression between these mouse lines, as confirmed by quantitative RT-PCR. In addition, serum IGFBP-1 levels were significantly higher in patients with IgAN than in healthy controls. In patients with IgAN, these levels correlated with measures of renal function, such as estimated glomerular filtration rate (eGFR), but not with sex, age, serum IgA, C3 levels, or IGF-1 levels. Pathologically, serum IGFBP-1 levels were significantly associated with the severity of renal injury, as assessed by mesangial cell proliferation and interstitial fibrosis. These results suggest that increased IGFBP-1 levels are associated with the severity of renal pathology in patients with IgAN.

  5. Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

    DEFF Research Database (Denmark)

    Beuchert Kallehauge, Thomas; Li, Shangzhong; Pedersen, Lasse Ebdrup

    2017-01-01

    Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as effici......Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated...... as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated...... as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we...

  6. Bacillus coagulans GBI-30, 6086 increases plant protein digestion in a dynamic, computer-controlled in vitro model of the small intestine (TIM-1).

    Science.gov (United States)

    Keller, D; Van Dinter, R; Cash, H; Farmer, S; Venema, K

    2017-05-30

    The aim of this study was to assess the potential of the probiotic Bacillus coagulans GBI-30, 6086 [GanedenBC 30 ] (BC30) to aid in protein digestion of alimentary plant proteins. To test this, three plant proteins, from pea, soy and rice, were digested in a validated in vitro model of the stomach and small intestine (TIM-1) in the absence and in the presence of BC30. Samples were taken from the TIM-1 fractions that mimic uptake of amino acids by the host and analysed for α-amino nitrogen (AAN) and total nitrogen (TN). Both were increased by BC30 for all three plant proteins sources. The ratio of TN/AAN indicated that for pea protein digestion was increased by BC30, but the degree of polymerisation of the liberated small peptides and free amino acids was not changed. For soy and rice, however, BC30 showed a 2-fold reduction in the TN/AAN ratio, indicating that the liberated digestion products formed during digestion in the presence of BC30 were shorter peptides and more free amino acids, than those liberated in the absence of BC30. As BC30 increased protein digestion and uptake in the upper gastrointestinal (GI) tract, it consequently also reduced the amount of protein that would be delivered to the colon, which could there be fermented into toxic metabolites by the gut microbiota. Thus, the enhanced protein digestion by BC30 showed a dual benefit: enhanced amino acid bioavailability from plant proteins in the upper GI tract, and a healthier environment in the colon.

  7. High-protein diet modifies colonic microbiota and luminal environment but not colonocyte metabolism in the rat model: the increased luminal bulk connection

    OpenAIRE

    LIU, Xinxin; BLOUIN, Jean-Marc; Santacruz, Arlette; Lan, Annaig; Andriamihaja, Mireille; Wilkanowicz, Sabina; Benetti, Pierre-Henri; Tomé, Daniel; Sanz, Yolanda; Blachier, Francois; Davila-Gay, Anne-Marie

    2014-01-01

    High-protein diets are used for body weight reduction, but consequences on the large intestine ecosystem are poorly known. Here, rats were fed for 15 days with either a normoproteic diet (NP, 14% protein) or a hyperproteic-hypoglucidic isocaloric diet (HP, 53% protein). Cecum and colon were recovered for analysis. Short- and branched-chain fatty acids, as well as lactate, succinate, formate, and ethanol contents, were markedly increased in the colonic luminal contents of HP rats (P < 0.05 or ...

  8. Phenylbutyrate improves nitrogen disposal via alternative pathway without eliciting an increase in protein breakdown and catabolism in control and ornithine transcarbamylace-deficient patients

    Science.gov (United States)

    Phenylbutyrate (PB) is a drug used in urea cycle disorder patients to elicit alternative pathways for nitrogen disposal. However, PB decreases plasma branched chain amino acid (BCAA) concentrations and prior research suggests that PB may increase leucine oxidation, indicating increased protein degra...

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

    Science.gov (United States)

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

    2017-06-16

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

  10. Increase and improvement of protein content of cereals and legumes. Part of a coordinated programme on the use of nuclear techniques for seed protein improvement

    International Nuclear Information System (INIS)

    Hadjichristodoulou, A.

    1979-11-01

    Mutation breeding was studied on barley (variety Athenais) and Italian drurum wheat (variety Capeiti). Gamma radiation and fast neutrons were used. The intention is to use high protein lines of barley and wheat in crosses with the high-yielding varieties grown in Cyprus, in order to develop new varieties with high seed protein, without reducing grain yield. The work on forage legumes was limited to the screening of varieties of forage vetches and medics, as part of the programme on improving dryland forage crops. Tables indicate the relative performance of M 6 -lines of Attiki barley (1977-79) and of M 4 Athenais barley (1978-79). A list of 8 publications by the author, some of them jointly with A. della, is given, relating to the project

  11. Protein A chromatography increases monoclonal antibody aggregation rate during subsequent low pH virus inactivation hold

    Science.gov (United States)

    Mazzer, Alice R.; Perraud, Xavier; Halley, Jennifer; O’Hara, John; Bracewell, Daniel G.

    2015-01-01

    Protein A chromatography is a near-ubiquitous method of mAb capture in bioprocesses. The use of low pH buffer for elution from protein A is known to contribute to product aggregation. Yet, a more limited set of evidence suggests that low pH may not be the sole cause of aggregation in protein A chromatography, rather, other facets of the process may contribute significantly. This paper presents a well-defined method for investigating this problem. An IgG4 was incubated in elution buffer after protein A chromatography (typical of the viral inactivation hold) and the quantity of monomer in neutralised samples was determined by size exclusion chromatography; elution buffers of different pH values predetermined to induce aggregation of the IgG4 were used. Rate constants for monomer decay over time were determined by fitting exponential decay functions to the data. Similar experiments were implemented in the absence of a chromatography step, i.e. IgG4 aggregation at low pH. Rate constants for aggregation after protein A chromatography were considerably higher than those from low pH exposure alone; a distinct shift in aggregation rates was apparent across the pH range tested. PMID:26346187

  12. Fish oil supplementation suppresses resistance exercise and feeding-induced increases in anabolic signaling without affecting myofibrillar protein synthesis in young men.

    Science.gov (United States)

    McGlory, Chris; Wardle, Sophie L; Macnaughton, Lindsay S; Witard, Oliver C; Scott, Fraser; Dick, James; Bell, J Gordon; Phillips, Stuart M; Galloway, Stuart D R; Hamilton, D Lee; Tipton, Kevin D

    2016-03-01

    Fish oil (FO) supplementation potentiates muscle protein synthesis (MPS) in response to a hyperaminoacidemic-hyperinsulinemic infusion. Whether FO supplementation potentiates MPS in response to protein ingestion or when protein ingestion is combined with resistance exercise (RE) remains unknown. In a randomized, parallel group design, 20 healthy males were randomized to receive 5 g/day of either FO or coconut oil control (CO) for 8 weeks. After supplementation, participants performed a bout of unilateral RE followed by ingestion of 30 g of whey protein. Skeletal muscle biopsies were obtained before and after supplementation for assessment of muscle lipid composition and relevant protein kinase activities. Infusion of L-[ring-(13)C6] phenylalanine was used to measure basal myofibrillar MP Sat rest (REST), in a nonexercised leg following protein ingestion (FED) and following RE and protein ingestion (FEDEX).MPS was significantly elevated above REST during FEDEX in both the FO and CO groups, but there was no effect of supplementation. There was a significant increase in MPS in both groups above REST during FED but no effect of supplementation. Supplementation significantly decreased pan PKB activity at RESTin the FO group but not the CO group. There was a significant increase from REST at post-RE for PKB and AMPKα2 activity in the CO group but not in the FO group. In FEDEX, there was a significant increase in p70S6K1 activity from REST at 3 h in the CO group only. These data highlight that 8 weeks of FO supplementation alters kinase signaling activity in response to RE plus protein ingestion without influencing MPS. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  13. Increased serum C-reactive protein level is associated with increased storage lower urinary tract symptoms in men with benign prostatic hyperplasia.

    Directory of Open Access Journals (Sweden)

    Shun-Fa Hung

    Full Text Available OBJECTIVE: Chronic inflammation is considered as one of the contributing mechanisms of lower urinary tract symptoms (LUTS. Serum C-reactive protein (CRP level is the widely used biomarker of inflammatory status. This study investigated the association between serum CRP level in men with benign prostatic hyperplasia (BPH and lower urinary tract symptoms (LUTS before and after medical treatment. METHODS: A total of 853 men with BPH and LUTS were enrolled. All patients completed the International Prostate Symptoms Score (IPSS questionnaire and urological examinations. The parameters of uroflowmetry (maximum flow rate, Qmax; voided volume, VV, post-void residual (PVR, total prostate volume (TPV and transition zone index (TZI, serum prostate specific antigen (PSA, and serum CRP levels were obtained. All patients were treated with alpha-blocker or antimuscarinic agent based on the IPSS voiding to storage subscore ratio (IPSS-V/S. Correlation analyses were performed between serum CRP levels with age, IPSS, TPV, TZI, Qmax, PVR, VV, PSA and between baseline and post treatment. RESULTS: The mean age was 66.9 ± 11.6 years old and the mean serum CRP levels were 0.31 ± 0.43 mg/dL. Univariate analyses revealed serum CRP levels were significantly associated with age (p<0.001, PSA levels (p = 0.005 and VV (p = 0.017, but not significantly associated with TPV (p = 0.854 or PVR (p = 0.068. CRP levels were positively associated with urgency (p<0.001 and nocturia (p<0.001 subscore of IPSS, total IPSS (p = 0.008 and storage IPSS (p<0.001 and negatively associated with IPSS- V/S ratio (p = 0.014. Multivariate analyses revealed that serum CRP levels were significantly associated with age (p = 0.004 and storage IPSS subscore p<0.001. Patients with IPSS-V/S<1 and treated with tolterodine for 3 months had significant decrease of CRP levels after treatment. CONCLUSION: Serum CRP levels are associated with storage LUTS and sensory bladder disorders, suggesting chronic

  14. High-protein diet modifies colonic microbiota and luminal environment but not colonocyte metabolism in the rat model: the increased luminal bulk connection.

    Science.gov (United States)

    Liu, Xinxin; Blouin, Jean-Marc; Santacruz, Arlette; Lan, Annaïg; Andriamihaja, Mireille; Wilkanowicz, Sabina; Benetti, Pierre-Henri; Tomé, Daniel; Sanz, Yolanda; Blachier, François; Davila, Anne-Marie

    2014-08-15

    High-protein diets are used for body weight reduction, but consequences on the large intestine ecosystem are poorly known. Here, rats were fed for 15 days with either a normoproteic diet (NP, 14% protein) or a hyperproteic-hypoglucidic isocaloric diet (HP, 53% protein). Cecum and colon were recovered for analysis. Short- and branched-chain fatty acids, as well as lactate, succinate, formate, and ethanol contents, were markedly increased in the colonic luminal contents of HP rats (P diet, whereas the amount of butyrate in feces was increased (P diet consumption allows maintenance in the luminal butyrate concentration and thus its metabolism in colonocytes despite modified microbiota composition and increased substrate availability. Copyright © 2014 the American Physiological Society.

  15. Long-term rates of mitochondrial protein synthesis are increased in mouse skeletal muscle with high-fat feeding regardless of insulin-sensitizing treatment.

    Science.gov (United States)

    Newsom, Sean A; Miller, Benjamin F; Hamilton, Karyn L; Ehrlicher, Sarah E; Stierwalt, Harrison D; Robinson, Matthew M

    2017-11-01

    Skeletal muscle mitochondrial protein synthesis is regulated in part by insulin. The development of insulin resistance with diet-induced obesity may therefore contribute to impairments to protein synthesis and decreased mitochondrial respiration. Yet the impact of diet-induced obesity and insulin resistance on mitochondrial energetics is controversial, with reports varying from decreases to increases in mitochondrial respiration. We investigated the impact of changes in insulin sensitivity on long-term rates of mitochondrial protein synthesis as a mechanism for changes to mitochondrial respiration in skeletal muscle. Insulin resistance was induced in C57BL/6J mice using 4 wk of a high-fat compared with a low-fat diet. For 8 additional weeks, diets were enriched with pioglitazone to restore insulin sensitivity compared with nonenriched control low-fat or high-fat diets. Skeletal muscle mitochondrial protein synthesis was measured using deuterium oxide labeling during weeks 10-12 High-resolution respirometry was performed using palmitoyl-l-carnitine, glutamate+malate, and glutamate+malate+succinate as substrates for mitochondria isolated from quadriceps. Mitochondrial protein synthesis and palmitoyl- l-carnitine oxidation were increased in mice consuming a high-fat diet, regardless of differences in insulin sensitivity with pioglitazone treatment. There was no effect of diet or pioglitazone treatment on ADP-stimulated respiration or H 2 O 2 emission using glutamate+malate or glutamate+malate+succinate. The results demonstrate no impairments to mitochondrial protein synthesis or respiration following induction of insulin resistance. Instead, mitochondrial protein synthesis was increased with a high-fat diet and may contribute to remodeling of the mitochondria to increase lipid oxidation capacity. Mitochondrial adaptations with a high-fat diet appear driven by nutrient availability, not intrinsic defects that contribute to insulin resistance. Copyright © 2017 the

  16. Actinidin from kiwifruit (Actinidia deliciosa cv. Hayward) increases the digestion and rate of gastric emptying of meat proteins in the growing pig.

    Science.gov (United States)

    Montoya, Carlos A; Rutherfurd, Shane M; Olson, Trent D; Purba, Ajitpal S; Drummond, Lynley N; Boland, Mike J; Moughan, Paul J

    2014-03-28

    The present study aimed to investigate the effect of dietary actinidin on the kinetics of gastric digestion of beef muscle proteins and on the rate of stomach emptying in growing pigs. For this purpose, 120 pigs (mean body weight 28 (sd 2·9) kg) were fed beef muscle protein-based diets containing either actinidin (fresh green kiwifruit pulp or gold kiwifruit pulp supplemented with purified actinidin) or no actinidin (fresh gold kiwifruit pulp or green kiwifruit pulp with inactivated actinidin). Additionally, fifteen pigs were fed with a protein-free diet to determine the endogenous protein flow. Pigs were euthanised at exactly 0·5, 1, 3, 5 and 7 h postprandially (n 6 per time point for each kiwifruit diet and n 3 for protein-free diet). Stomach chyme was collected for measuring gastric retention, actinidin activity, individual beef muscle protein digestion based on SDS-PAGE and the degree of hydrolysis based on the appearance of free amino groups. The stomach emptying of DM and N was faster when actinidin was present in the diet (P34 kDa; P< 0·05). In conclusion, dietary actinidin fed in the form of fresh green kiwifruit increased the rate of gastric emptying and the digestion of several beef muscle proteins.

  17. Insulin does not stimulate muscle protein synthesis during increased plasma branched-chain amino acids alone but still decreases whole body proteolysis in humans.

    Science.gov (United States)

    Everman, Sarah; Meyer, Christian; Tran, Lee; Hoffman, Nyssa; Carroll, Chad C; Dedmon, William L; Katsanos, Christos S

    2016-10-01

    Insulin stimulates muscle protein synthesis when the levels of total amino acids, or at least the essential amino acids, are at or above their postabsorptive concentrations. Among the essential amino acids, branched-chain amino acids (BCAA) have the primary role in stimulating muscle protein synthesis and are commonly sought alone to stimulate muscle protein synthesis in humans. Fourteen healthy young subjects were studied before and after insulin infusion to examine whether insulin stimulates muscle protein synthesis in relation to the availability of BCAA alone. One half of the subjects were studied in the presence of postabsorptive BCAA concentrations (control) and the other half in the presence of increased plasma BCAA (BCAA). Compared with that prior to the initiation of the insulin infusion, fractional synthesis rate of muscle protein (%/h) did not change (P > 0.05) during insulin in either the control (0.04 ± 0.01 vs 0.05 ± 0.01) or the BCAA (0.05 ± 0.02 vs. 0.05 ± 0.01) experiments. Insulin decreased (P BCAA (0.89 ± 0.07 vs 0.61 ± 0.03) experiments, but the change was not different between the two experiments (P > 0.05). In conclusion, insulin does not stimulate muscle protein synthesis in the presence of increased circulating levels of plasma BCAA alone. Insulin's suppressive effect on proteolysis is observed independently of the levels of circulating plasma BCAA. Copyright © 2016 the American Physiological Society.

  18. Serotype 3 pneumococci sequester platelet-derived human thrombospondin-1 via the adhesin and immune evasion protein Hic.

    Science.gov (United States)

    Binsker, Ulrike; Kohler, Thomas P; Krauel, Krystin; Kohler, Sylvia; Habermeyer, Johanna; Schwertz, Hansjörg; Hammerschmidt, Sven

    2017-04-07

    Streptococcus pneumoniae serotype 3 strains emerge frequently within clinical isolates of invasive diseases. Bacterial invasion into deeper tissues is associated with colonization and immune evasion mechanisms. Thus, pneumococci express a versatile repertoire of surface proteins sequestering and interacting specifically with components of the human extracellular matrix and serum. Hic, a PspC-like pneumococcal surface protein, possesses vitronectin and factor H binding activity. Here, we show that heterologously expressed Hic domains interact, similar to the classical PspC molecule, with human matricellular thrombospondin-1 (hTSP-1). Binding studies with isolated human thrombospondin-1 and various Hic domains suggest that the interaction between hTSP-1 and Hic differs from binding to vitronectin and factor H. Binding of Hic to hTSP-1 is inhibited by heparin and chondroitin sulfate A, indicating binding to the N-terminal globular domain or type I repeats of hTSP-1. Competitive inhibition experiments with other pneumococcal hTSP-1 adhesins demonstrated that PspC and PspC-like Hic recognize similar domains, whereas PavB and Hic can bind simultaneously to hTSP-1. In conclusion, Hic binds specifically hTSP-1; however, truncation in the N-terminal part of Hic decreases the binding activity, suggesting that the full length of the α-helical regions of Hic is required for an optimal interaction. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. NOS3 is involved in the increased protein and arginine metabolic response in muscle during early endotoxemia in mice

    NARCIS (Netherlands)

    Luiking, Yvette C.; Hallemeesch, Marcella M.; Lamers, Wouter H.; Deutz, Nicolaas E. P.

    2005-01-01

    Sepsis is a severe catabolic condition. The loss of skeletal muscle protein mass is characterized by enhanced release of the amino acids glutamine and arginine, which (in)directly affects interorgan arginine and the related nitric oxide (NO) synthesis. To establish whether changes in muscle amino

  20. Dietary supplementation with aromatic amino acids increases protein synthesis in children wHh severe acute malnutrition

    Science.gov (United States)

    Although 2 earlier studies reported that aromatic amino acid (AAA) supplementation of children with severe acute malnutrition (SAM) improved whole-body protein anabolism during the early postadmission (maintenance) phase of rehabilitation, it is not known whether this positive effect was maintained ...

  1. HER-2 protein concentrations in breast cancer cells increase before immunohistochemical and fluorescence in situ hybridization analysis turn positive

    DEFF Research Database (Denmark)

    Olsen, Dorte A; Østergaard, Birthe; Bokmand, Susanne

    2007-01-01

    BACKGROUND: The level of HER-2/neu in breast cancer cells is normally measured by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH). It determines whether patients should be treated with trastuzumab (Herceptin). In this study, HER-2 protein in breast cancer tissue...

  2. Increased levels of C-reactive protein and leukocyte count are poor predictors of anastomotic leakage following laparoscopic colorectal resection

    DEFF Research Database (Denmark)

    Pedersen, Torben; Roikjær, Ole; Jess, Per

    2012-01-01

    Laparoscopic procedure and fast-track regimen with short post-operative hospital stay are gaining ground in colorectal surgery. The aim of the present study was to determine whether the levels of C-reactive protein (CRP) and white blood cell counts (WBC) have a role as early predictors of post...

  3. Memory-enhancing intra-basolateral amygdala infusions of clenbuterol increase Arc and CaMKII-alpha protein expression in the rostral anterior cingulate cortex

    Directory of Open Access Journals (Sweden)

    Crystal M Holloway-Erickson

    2012-04-01

    Full Text Available Activation of β-adrenoceptors in the basolateral complex of the amygdala (BLA modulates memory through interactions with multiple memory systems. The cellular mechanisms for this interaction remain unresolved. Memory-modulating BLA manipulations influence expression of the protein product of the immediate early gene activity-regulated cytoskeletal-associated protein (Arc in the dorsal hippocampus, and hippocampal expression of Arc protein is critically involved in memory consolidation and long-term potentiation. The present studies examined whether this influence of the BLA is specific to the hippocampus and to Arc protein. Like the hippocampus, the rostral portion of the anterior cingulate cortex (rACC is involved in the consolidation of inhibitory avoidance (IA memory, and IA training increases Arc protein in the rACC. Because the BLA interacts with the rACC in the consolidation of IA memory, the rACC is a potential candidate for further studies of BLA modulation of synaptic plasticity. The alpha isoform of the Calcium/Calmodulin-dependent protein kinase II (CaMKIIα and the immediate early gene c-Fos are involved in long-term potentiation and memory. Both Arc and CaMKIIα proteins can be translated in isolated synapses, where the mRNA is localized, but c-Fos protein remains in the soma. To examine the influence of memory-modulating manipulations of the BLA on expression of these memory and plasticity-associated proteins in the rACC, male Sprague-Dawley rats were trained on an IA task and given intra-BLA infusions of either clenbuterol or lidocaine immediately after training. Findings suggest that noradrenergic stimulation of the BLA may modulate memory consolidation through effects on both synaptic proteins Arc and CaMKIIα, but not the somatic protein c-Fos. Furthermore, protein changes observed in the rACC following BLA manipulations suggest that the influence of the BLA on synaptic proteins is not limited to those in the dorsal

  4. Ultrafiltration of skimmed goat milk increases its nutritional value by concentrating nonfat solids such as proteins, Ca, P, Mg, and Zn.

    Science.gov (United States)

    Moreno-Montoro, Miriam; Olalla, Manuel; Giménez-Martínez, Rafael; Bergillos-Meca, Triana; Ruiz-López, María Dolores; Cabrera-Vique, Carmen; Artacho, Reyes; Navarro-Alarcón, Miguel

    2015-11-01

    Goat milk has been reported to possess good nutritional and health-promoting properties. Usually, it must be concentrated before fermented products can be obtained. The aim of this study was to compare physicochemical and nutritional variables among raw (RM), skimmed (SM), and ultrafiltration-concentrated skimmed (UFM) goat milk. The density, acidity, ash, protein, casein, whey protein, Ca, P, Mg, and Zn values were significantly higher in UFM than in RM or SM. Dry extract and fat levels were significantly higher in UFM than in SM, and Mg content was significantly higher in UFM than in RM. Ultrafiltration also increased the solubility of Ca and Mg, changing their distribution in the milk. The higher concentrations of minerals and proteins, especially caseins, increase the nutritional value of UFM, which may therefore be more appropriate for goat milk yogurt manufacturing in comparison to RM or SM. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  5. A quantitative analysis of 2-D gels identifies proteins in which labeling is increased following long-term sensitization in Aplysia

    International Nuclear Information System (INIS)

    Castellucci, V.F.; Kennedy, T.E.; Kandel, E.R.; Goelet, P.

    1988-01-01

    Long-term memory for sensitization of the gill- and siphon-withdrawal reflex in Aplysia, produced by 4 days of training, is associated with increased synaptic efficacy of the connection between the sensory and motor neurons. This training is also accompanied by neuronal growth; there is an increase in the number of synaptic varicosities per sensory neuron and in the number of active zones. Such structural changes may be due to changes in the rates of synthesis of certain proteins. We have searched for proteins in which the rates of [ 35 S]methionine labeling are altered during the maintenance phase of long-term memory for sensitization by using computer-assisted quantitative 2-D gel analysis. This method has allowed us to detect 4 proteins in which labeling is altered after 4 days of sensitization training

  6. Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position.

    Science.gov (United States)

    Honda, Yuki; Zang, Qian; Shimizu, Yasuhiro; Dadashipour, Mohammad; Zhang, Zilian; Kawarabayasi, Yutaka

    2017-02-01

    The ST0452 protein is a bifunctional protein exhibiting sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) and amino-sugar-1-phosphate acetyltransferase activities and was isolated from the thermophilic archaeon Sulfolobus tokodaii Based on the previous observation that five single mutations increased ST0452 sugar-1-P NTase activity, nine double-mutant ST0452 proteins were generated with the intent of obtaining enzymes exhibiting a further increase in catalysis, but all showed less than 15% of the wild-type N-acetyl-d-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase) activity. The Y97A mutant exhibited the highest activity of the single-mutant proteins, and thus site saturation mutagenesis of the 97th position (Tyr) was conducted. Six mutants showed both increased GlcNAc-1-P UTase and glucose-1-phosphate uridyltransferase activities, eight mutants showed only enhanced GlcNAc-1-P UTase activity, and six exhibited higher GlcNAc-1-P UTase activity than that of the Y97A mutant. Kinetic analyses of three typical mutants indicated that the increase in sugar-1-P NTase activity was mainly due to an increase in the apparent k cat value. We hypothesized that changing the 97th position (Tyr) to a smaller amino acid with similar electronic properties would increase activity, and thus the Tyr at the corresponding 103rd position of the Escherichia coli GlmU (EcGlmU) enzyme was replaced with the same residues. The Y103N mutant EcGlmU showed increased GlcNAc-1-P UTase activity, revealing that the Tyr at the 97th position of the ST0452 protein (103rd position in EcGlmU) plays an important role in catalysis. The present results provide useful information regarding how to improve the activity of natural enzymes and how to generate powerful enzymes for the industrial production of sugar nucleotides. It is typically difficult to increase enzymatic activity by introducing substitutions into a natural enzyme. However, it was previously found that the ST0452 protein

  7. Mutations of C19orf12, coding for a transmembrane glycine zipper containing mitochondrial protein, cause mis-localization of the protein, inability to respond to oxidative stress and increased mitochondrial Ca2+

    DEFF Research Database (Denmark)

    Venco, Paola; Bonora, Massimo; Giorgi, Carlotta

    2015-01-01

    19orf12 protein was not exclusively present in mitochondria, but also in the Endoplasmic Reticulum (ER) and MAM (Mitochondria Associated Membrane), while mutant C19orf12 variants presented a different localization. Moreover, after induction of oxidative stress, a GFP-tagged C19orf12 wild-type protein...... was able to relocate to the cytosol. On the contrary, mutant isoforms were not able to respond to oxidative stress. High mitochondrial calcium concentration and increased H2O2 induced apoptosis were found in fibroblasts derived from one patient as compared to controls. C19orf12 protein is a 17 k...... to rearrange in a structural domain, which is homologs to the N-terminal regulatory domain of the magnesium transporter MgtE, suggesting that C19orf12 may act as a regulatory protein for human MgtE transporters. The mutations here described affect respectively one glycine residue of the glycine zipper motifs...

  8. Engineered protein degradation of farnesyl pyrophosphate synthase is an effective regulatory mechanism to increase monoterpene production in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Peng, Bingyin; Nielsen, Lars K.; Kampranis, Sotirios C

    2018-01-01

    Monoterpene production in Saccharomyces cerevisae requires the introduction of heterologous monoterpene synthases (MTSs). The endogenous farnesyl pyrosphosphate synthase (FPPS; Erg20p) competes with MTSs for the precursor geranyl pyrophosphate (GPP), which limits the production of monoterpenes. ERG......20 is an essential gene that cannot be deleted and transcriptional down-regulation of ERG20 has failed to improve monoterpene production. Here, we investigated an N-degron-dependent protein degradation strategy to down-regulate Erg20p activity. Degron tagging decreased GFP protein half......-life drastically to 1 h (degron K3K15) or 15 min (degrons KN113 and KN119). Degron tagging of ERG20 was therefore paired with a sterol responsive promoter to ensure sufficient metabolic flux to essential downstream sterols despite the severe destabilisation effect of degron tagging. A dual monoterpene...

  9. Gestational Age-Dependent Increase of Survival Motor Neuron Protein in Umbilical Cord-Derived Mesenchymal Stem Cells

    OpenAIRE

    Iwatani, Sota; Harahap, Nur Imma Fatimah; Nurputra, Dian Kesumapramudya; Tairaku, Shinya; Shono, Akemi; Kurokawa, Daisuke; Yamana, Keiji; Thwin, Khin Kyae Mon; Yoshida, Makiko; Mizobuchi, Masami; Koda, Tsubasa; Fujioka, Kazumichi; Taniguchi-Ikeda, Mariko; Yamada, Hideto; Morioka, Ichiro

    2017-01-01

    Background: Spinal muscular atrophy (SMA) is the most common genetic neurological disease leading to infant death. It is caused by loss of survival motor neuron (SMN) 1 gene and subsequent reduction of SMN protein in motor neurons. Because SMN is ubiquitously expressed and functionally linked to general RNA metabolism pathway, fibroblasts (FBs) are most widely used for the assessment of SMN expression in SMA patients but usually isolated from skin biopsy samples after the onset of overt sympt...

  10. Senescence marker protein-30/superoxide dismutase 1 double knockout mice exhibit increased oxidative stress and hepatic steatosis

    Directory of Open Access Journals (Sweden)

    Yoshitaka Kondo

    2014-01-01

    Full Text Available Superoxide dismutase 1 (SOD1 is an antioxidant enzyme that converts superoxide anion radicals into hydrogen peroxide and molecular oxygen. The senescence marker protein-30 (SMP30 is a gluconolactonase that functions as an antioxidant protein in mammals due to its involvement in ascorbic acid (AA biosynthesis. SMP30 also participates in Ca2+ efflux by activating the calmodulin-dependent Ca2+-pump. To reveal the role of oxidative stress in lipid metabolism defects occurring in non-alcoholic fatty liver disease pathogenesis, we generated SMP30/SOD1-double knockout (SMP30/SOD1-DKO mice and investigated their survival curves, plasma and hepatic lipid profiles, amounts of hepatic oxidative stress, and hepatic protein levels expressed by genes related to lipid metabolism. While SMP30/SOD1-DKO pups had no growth retardation by 14 days of age, they did have low plasma and hepatic AA levels. Thereafter, 39% and 53% of male and female pups died by 15–24 and 89 days of age, respectively. Compared to wild type, SMP30-KO and SOD1-KO mice, by 14 days SMP30/SOD1-DKO mice exhibited: (1 higher plasma levels of triglyceride and aspartate aminotransferase; (2 severe accumulation of hepatic triglyceride and total cholesterol; (3 higher levels of superoxide anion radicals and thiobarbituric acid reactive substances in livers; and (4 decreased mRNA and protein levels of Apolipoprotein B (ApoB in livers – ApoB is an essential component of VLDL secretion. These results suggest that high levels of oxidative stress due to concomitant deficiency of SMP30 and/or AA, and SOD1 cause abnormal plasma lipid metabolism, hepatic lipid accumulation and premature death resulting from impaired VLDL secretion.

  11. Increased expression of heat shock protein 105 in rat uterus of early pregnancy and its significance in embryo implantation

    Directory of Open Access Journals (Sweden)

    Hu Zhao-Yuan

    2009-03-01

    Full Text Available Abstract Background Heat shock proteins (Hsps are a set of highly conserved proteins, Hsp105, has been suggested to play a role in reproduction. Methods Spatio-temporal expression of Hsp105 in rat uterus during peri-implantation period was examined by immunohistochemistry and Western blot, pseudopregnant uterus was used as control. Injection of antisense oligodeoxynucleotides to Hsp105 into pregnant rat uteri was carried out to look at effect of Hsp105 on embryo implantation. Results Expression of Hsp105 was mainly in the luminal epithelium on day 1 of pregnancy, and reached a peak level on day 5, whereas in stroma cells, adjacent to the implanting embryo, the strongest expression of Hsp105 was observed on day 6. The immunostaining profile in the uterus was consistent with that obtained by Western blot in the early pregnancy. In contrast, no obvious peak level of Hsp105 was observed in the uterus of pseudopregnant rat on day 5 or day 6. Furthermore, injection of antisense oligodeoxynucleotides to Hsp105 into the rat uterine horn on day 3 of pregnancy obviously suppressed the protein expression as expected and reduced number of the implanted embryos as compared with the control. Conclusion Temporal and spatial changes in Hsp105 expression in pregnant rat uterus may play a physiological role in regulating embryo implantation.

  12. Milk Fat Globule Membrane Attenuates High-Fat Diet-Induced Obesity by Inhibiting Adipogenesis and Increasing Uncoupling Protein 1 Expression in White Adipose Tissue of Mice

    Directory of Open Access Journals (Sweden)

    Tiange Li

    2018-03-01

    Full Text Available Milk fat globule membrane (MFGM, a protein-lipid complex surrounding the fat globules in milk, has many health benefits. The aim of the current study was to investigate whether MFGM could prevent obesity through inhibiting adipogenesis and promoting brown remodeling of white adipose tissue (WAT in mice fed with high-fat diet. C57BL/6 mice were fed a normal diet (ND, high-fat diet (HFD, HFD plus MFGM at 100 mg/kg BW, 200 mg/kg BW or 400 mg/kg BW for 8 weeks. Results showed that MFGM suppressed body weight gain induced by HFD, reduced white adipose tissue (WAT mass accompanied with the decrease in adipocyte sizes. MFGM was found to have partially improved serum lipid profiles, as well as to have suppressed HFD-induced adipogenesis as shown by reduced expression of peroxisome proliferators-activator receptor-γ (PPARγ, CCAAT/enhancer-binding protein-α (C/EBPα and sterol regulatory element-binding protein-1c (SREBP-1c. MFGM also markedly increased the phosphorylation of AMP-activated protein kinase (AMPK and acetyl-CoA carboxylase (ACC, showing activation of AMPK pathway. Moreover, MFGM promoted browning of inguinal WAT by upregulation the protein expression of uncoupling protein 1 (UCP1 in HFD mice. Taken together, these findings provide evidence that MFGM may protect against diet-induced adiposity by suppressing adipogenesis and promoting brown-like transformation in WAT.

  13. Inhibition of protein kinase CbetaII increases glucose uptake in 3T3-L1 adipocytes through elevated expression of glucose transporter 1 at the plasma membrane

    NARCIS (Netherlands)

    Bosch, Remko R.; Bazuine, Merlijn; Wake, Michelle M.; Span, Paul N.; Olthaar, André J.; Schürmann, Annette; Maassen, J. Antonie; Hermus, Ad R. M. M.; Willems, Peter H. G. M.; Sweep, C. G. J.

    2003-01-01

    The mechanism via which diacylglycerol-sensitive protein kinase Cs (PKCs) stimulate glucose transport in insulin-sensitive tissues is poorly defined. Phorbol esters, such as phorbol-12-myristate-13-acetate (PMA), are potent activators of conventional and novel PKCs. Addition of PMA increases the

  14. A high-protein diet increases postprandial but not fasting plasma total homocysteine concentrations: a dietary controlled, crossover trial in healthy volunteers

    NARCIS (Netherlands)

    Verhoef, P.; Vliet, van T.; Olthof, M.R.; Katan, M.B.

    2005-01-01

    Background: A high plasma concentration of total homocysteine (tHcy) is associated with increased risk of cardiovascular disease. A high protein intake and hence a high intake of methionine¿the sole dietary precursor of homocysteine¿may raise plasma tHcy concentrations. Objectives: We studied

  15. Effect of prescribing a high protein diet and increasing the dose of dialysis on nutrition in stable chronic haemodialysis patients : a randomized, controlled trial

    NARCIS (Netherlands)

    Kloppenburg, Wybe; Stegeman, CA; Kremer Hovinga, T; Vastenburg, G; Vos, P; de Jong, PE; Huisman, RM

    Background. Protein requirements in stable, adequately dialysed haemodialysis patients are not known and recommendations vary. It is not known whether increasing the dialysis dose above the accepted adequate level has a favourable effect on nutrition. The aim of this study was to determine whether

  16. A high-protein diet increases postprandial but not fasting plasma total homocysteine concentrations: A dietary controlled, crossover trial in healthy volunteers

    NARCIS (Netherlands)

    Verhoef, P.; Vliet, T. van; Olthof, M.R.; Katan, M.B.

    2005-01-01

    Background: A high plasma concentration of total homocysteine (tHcy) is associated with increased risk of cardiovascular disease. A high protein intake and hence a high intake of methionine-the sole dietary precursor of homocysteine-may raise plasma tHcy concentrations. Objectives: We studied

  17. A high-protein diet increases postprandial but not fasting plasma total homocysteine concentrations : A dietary controlled, crossover trial in healthy volunteers

    NARCIS (Netherlands)

    Verhoef, Petra; Van Vliet, Trinette; Olthof, Margreet R.; Katan, Martijn B.

    2005-01-01

    Background: A high plasma concentration of total homocysteine (tHcy) is associated with increased risk of cardiovascular disease. A high protein intake and hence a high intake of methionine-the sole dietary precursor of homocysteine-may raise plasma tHcy concentrations. Objectives: We studied

  18. Long-term intake of a high-protein diet increases liver triacylglycerol deposition pathways and hepatic signs of injury in rats

    NARCIS (Netherlands)

    Díaz-Rúa, Rubén; Keijer, Jaap; Palou, Andreu; Schothorst, van Evert M.; Oliver, Paula

    2017-01-01

    Intake of high-protein (HP) diets has increased over the last years, mainly due to their popularity for body weight control. Liver is the main organ handling ingested macronutrients and it is associated with the beginning of different pathologies. We aimed to deepen our knowledge on molecular

  19. Tetanic stimulation leads to increased accumulation of Ca2+ calmodulin-dependent protein kinase II via dendritic protein synthesis in hippocampal neurons

    OpenAIRE

    Rosenstein, Alan; Kreiman, Gabriel; Schuman, Erin M; Kennedy, Mary

    1999-01-01

    mRNA for the alpha-subunit of CaMKII is abundant in dendrites of neurons in the forebrain (Steward, 1997). Here we show that tetanic stimulation of the Schaffer collateral pathway causes an increase in the concentration of alpha-CaMKII in the dendrites of postsynaptic neurons. The increase is blocked by anisomycin and is detected by both quantitative immunoblot and semiquantitative immunocytochemistry. The increase in dendritic alpha-CaMKII can be measured 100-200 micrometer away from the neu...

  20. Long-term intake of a high protein diet increases liver triacylglycerol deposition pathways and hepatic signs of injury in rats

    KAUST Repository

    Diaz Rua, Ruben

    2017-04-19

    Intake of high-protein (HP) diets has increased over the last years, mainly due to their popularity for body weight control. Liver is the main organ handling ingested macronutrients and it is associated with the beginning of different pathologies. We aimed to deepen our knowledge on molecular pathways affected by long-term intake of an HP diet. We performed a transcriptome analysis on liver of rats chronically fed with a casein-rich HP diet and analyzed molecular parameters related to liver injury. Chronic increase in the dietary protein/carbohydrate ratio up-regulated processes related with amino acid uptake/metabolism and lipid synthesis, promoting a molecular environment indicative of hepatic triacylglycerol (TG) deposition. Moreover, changes in expression of genes involved in acid–base maintenance and oxidative stress indicate alterations in the pH balance due to the high acid load of the diet, which has been linked to liver/health damage. Up-regulation of immune-related genes was also observed. In concordance with changes at gene expression level, we observed increased liver TG content and increased serum markers of hepatic injury/inflammation (aspartate transaminase, C-reactive protein and TNF-alpha). Moreover, the HP diet strongly increased hepatic mRNA and protein levels of HSP90, a marker of liver injury. Thus, we show for the first time that long-term consumption of an HP diet, resulting in a high acid load, results in a hepatic transcriptome signature reflecting increased TG deposition and increased signs of health risk (increased inflammation, alterations in the acid–base equilibrium and oxidative stress). Persistence of this altered metabolic status could have unhealthy consequences.

  1. Long-term intake of a high protein diet increases liver triacylglycerol deposition pathways and hepatic signs of injury in rats

    KAUST Repository

    Diaz Rua, Ruben; Keijer, Jaap; Palou, Andreu; van Schothorst, Evert M.; Oliver, Paula

    2017-01-01

    Intake of high-protein (HP) diets has increased over the last years, mainly due to their popularity for body weight control. Liver is the main organ handling ingested macronutrients and it is associated with the beginning of different pathologies. We aimed to deepen our knowledge on molecular pathways affected by long-term intake of an HP diet. We performed a transcriptome analysis on liver of rats chronically fed with a casein-rich HP diet and analyzed molecular parameters related to liver injury. Chronic increase in the dietary protein/carbohydrate ratio up-regulated processes related with amino acid uptake/metabolism and lipid synthesis, promoting a molecular environment indicative of hepatic triacylglycerol (TG) deposition. Moreover, changes in expression of genes involved in acid–base maintenance and oxidative stress indicate alterations in the pH balance due to the high acid load of the diet, which has been linked to liver/health damage. Up-regulation of immune-related genes was also observed. In concordance with changes at gene expression level, we observed increased liver TG content and increased serum markers of hepatic injury/inflammation (aspartate transaminase, C-reactive protein and TNF-alpha). Moreover, the HP diet strongly increased hepatic mRNA and protein levels of HSP90, a marker of liver injury. Thus, we show for the first time that long-term consumption of an HP diet, resulting in a high acid load, results in a hepatic transcriptome signature reflecting increased TG deposition and increased signs of health risk (increased inflammation, alterations in the acid–base equilibrium and oxidative stress). Persistence of this altered metabolic status could have unhealthy consequences.

  2. Casein phosphopeptides drastically increase the secretion of extracellular proteins in Aspergillus awamori. Proteomics studies reveal changes in the secretory pathway

    OpenAIRE

    Kosalková Katarina; García-Estrada Carlos; Barreiro Carlos; Flórez Martha G; Jami Mohammad S; Paniagua Miguel A; Martín Juan F

    2012-01-01

    Abstract Background The secretion of heterologous animal proteins in filamentous fungi is usually limited by bottlenecks in the vesicle-mediated secretory pathway. Results Using the secretion of bovine chymosin in Aspergillus awamori as a model, we found a drastic increase (40 to 80-fold) in cells grown with casein or casein phosphopeptides (CPPs). CPPs are rich in phosphoserine, but phosphoserine itself did not increase the secretion of chymosin. The stimulatory effect is reduced about 50% u...

  3. Long-term intake of a high-protein diet increases liver triacylglycerol deposition pathways and hepatic signs of injury in rats.

    Science.gov (United States)

    Díaz-Rúa, Rubén; Keijer, Jaap; Palou, Andreu; van Schothorst, Evert M; Oliver, Paula

    2017-08-01

    Intake of high-protein (HP) diets has increased over the last years, mainly due to their popularity for body weight control. Liver is the main organ handling ingested macronutrients and it is associated with the beginning of different pathologies. We aimed to deepen our knowledge on molecular pathways affected by long-term intake of an HP diet. We performed a transcriptome analysis on liver of rats chronically fed with a casein-rich HP diet and analyzed molecular parameters related to liver injury. Chronic increase in the dietary protein/carbohydrate ratio up-regulated processes related with amino acid uptake/metabolism and lipid synthesis, promoting a molecular environment indicative of hepatic triacylglycerol (TG) deposition. Moreover, changes in expression of genes involved in acid-base maintenance and oxidative stress indicate alterations in the pH balance due to the high acid load of the diet, which has been linked to liver/health damage. Up-regulation of immune-related genes was also observed. In concordance with changes at gene expression level, we observed increased liver TG content and increased serum markers of hepatic injury/inflammation (aspartate transaminase, C-reactive protein and TNF-alpha). Moreover, the HP diet strongly increased hepatic mRNA and protein levels of HSP90, a marker of liver injury. Thus, we show for the first time that long-term consumption of an HP diet, resulting in a high acid load, results in a hepatic transcriptome signature reflecting increased TG deposition and increased signs of health risk (increased inflammation, alterations in the acid-base equilibrium and oxidative stress). Persistence of this altered metabolic status could have unhealthy consequences. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Alteration in cardiac uncoupling proteins and eNOS gene expression following high-intensity interval training in favor of increasing mechanical efficiency

    OpenAIRE

    Fallahi, Ali Asghar; Shekarfroush, Shahnaz; Rahimi, Mostafa; Jalali, Amirhossain; Khoshbaten, Ali

    2016-01-01

    Objective(s): High-intensity interval training (HIIT) increases energy expenditure and mechanical energy efficiency. Although both uncoupling proteins (UCPs) and endothelial nitric oxide synthase (eNOS) affect the mechanical efficiency and antioxidant capacity, their effects are inverse. The aim of this study was to determine whether the alterations of cardiac UCP2, UCP3, and eNOS mRNA expression following HIIT are in favor of increased mechanical efficiency or decreased oxidative stress. Mat...

  5. Krill protein hydrolysate reduces plasma triacylglycerol level with concurrent increase in plasma bile acid level and hepatic fatty acid catabolism in high-fat fed mice

    Directory of Open Access Journals (Sweden)

    Marie S. Ramsvik

    2013-11-01

    Full Text Available Background: Krill powder, consisting of both lipids and proteins, has been reported to modulate hepatic lipid catabolism in animals. Fish protein hydrolysate diets have also been reported to affect lipid metabolism and to elevate bile acid (BA level in plasma. BA interacts with a number of nuclear receptors and thus affects a variety of signaling pathways, including very low density lipoprotein (VLDL secretion. The aim of the present study was to investigate whether a krill protein hydrolysate (KPH could affect lipid and BA metabolism in mice. Method: C57BL/6 mice were fed a high-fat (21%, w/w diet containing 20% crude protein (w/w as casein (control group or KPH for 6 weeks. Lipids and fatty acid composition were measured from plasma, enzyme activity and gene expression were analyzed from liver samples, and BA was measured from plasma. Results: The effect of dietary treatment with KPH resulted in reduced levels of plasma triacylglycerols (TAG and non-esterified fatty acids (NEFAs. The KPH treated mice had also a marked increased plasma BA concentration. The increased plasma BA level was associated with induction of genes related to membrane canalicular exporter proteins (Abcc2, Abcb4 and to BA exporters to blood (Abcc3 and Abcc4. Of note, we observed a 2-fold increased nuclear farnesoid X receptor (Fxr mRNA levels in the liver of mice fed KPH. We also observed increased activity of the nuclear peroxiosme proliferator-activated receptor alpha (PPARα target gene carnitine plamitoyltransferase 2 (CPT-2. Conclusion: The KPH diet showed to influence lipid and BA metabolism in high-fat fed mice. Moreover, increased mitochondrial fatty acid oxidation and elevation of BA concentration may regulate the plasma level of TAGs and NEFAs.

  6. Increased mRNA expression of a laminin-binding protein in human colon carcinoma: Complete sequence of a full-length cDNA encoding the protein

    International Nuclear Information System (INIS)

    Yow, Hsiukang; Wong, Jau Min; Chen, Hai Shiene; Lee, C.; Steele, G.D. Jr.; Chen, Lanbo

    1988-01-01

    Reliable markers to distinguish human colon carcinoma from normal colonic epithelium are needed particularly for poorly differentiated tumors where no useful marker is currently available. To search for markers the authors constructed cDNA libraries from human colon carcinoma cell lines and screened for clones that hybridize to a greater degree with mRNAs of colon carcinomas than with their normal counterparts. Here they report one such cDNA clone that hybridizes with a 1.2-kilobase (kb) mRNA, the level of which is ∼9-fold greater in colon carcinoma than in adjacent normal colonic epithelium. Blot hybridization of total RNA from a variety of human colon carcinoma cell lines shows that the level of this 1.2-kb mRNA in poorly differentiated colon carcinomas is as high as or higher than that in well-differentiated carcinomas. Molecular cloning and complete sequencing of cDNA corresponding to the full-length open reading frame of this 1.2-kb mRNA unexpectedly show it to contain all the partial cDNA sequence encoding 135 amino acid residues previously reported for a human laminin receptor. The deduced amino acid sequence suggests that this putative laminin-binding protein from human colon carcinomas consists of 295 amino acid residues with interesting features. There is an unusual C-terminal 70-amino acid segment, which is trypsin-resistant and highly negatively charged

  7. The Human Papillomavirus Type 16 E6 Oncoprotein Activates mTORC1 Signaling and Increases Protein Synthesis ▿ †

    OpenAIRE

    Spangle, Jennifer M.; Münger, Karl

    2010-01-01

    The mammalian target of rapamycin (mTOR) kinase acts as a cellular rheostat that integrates signals from a variety of cellular signal transduction pathways that sense growth factor and nutrient availability as well as intracellular energy status. It was previously reported that the human papillomavirus type 16 (HPV16) E6 oncoprotein may activate the S6 protein kinase (S6K) through binding and E6AP-mediated degradation of the mTOR inhibitor tuberous sclerosis complex 2 (TSC2) (Z. Lu, X. Hu, Y....

  8. Hepatic nuclear sterol regulatory binding element protein 2 abundance is decreased and that of ABCG5 increased in male hamsters fed plant sterols.

    Science.gov (United States)

    Harding, Scott V; Rideout, Todd C; Jones, Peter J H

    2010-07-01

    The effect of dietary plant sterols on cholesterol homeostasis has been well characterized in the intestine, but how plant sterols affect lipid metabolism in other lipid-rich tissues is not known. Changes in hepatic cholesterol homeostasis in response to high dietary intakes of plant sterols were determined in male golden Syrian hamsters fed hypercholesterolemia-inducing diets with and without 2% plant sterols (wt:wt; Reducol, Forbes Meditech) for 28 d. Plasma and hepatic cholesterol concentrations, cholesterol biosynthesis and absorption, and changes in the expression of sterol response element binding protein 2 (SREBP2) and liver X receptor-beta (LXRbeta) and their target genes were measured. Plant sterol feeding reduced plasma total cholesterol, non-HDL cholesterol, and HDL cholesterol concentrations 43% (P 6-fold (P = 0.029) and >2-fold (P sterol-fed hamsters compared with controls. Plant sterol feeding also increased fractional cholesterol synthesis >2-fold (P sterol feeding increased hepatic protein expression of cytosolic (inactive) SREBP2, decreased nuclear (active) SREBP2, and tended to increase LXRbeta (P = 0.06) and ATP binding cassette transporter G5, indicating a differential modulation of the expression of proteins central to cholesterol metabolism. In conclusion, high-dose plant sterol feeding of hamsters changes hepatic protein abundance in favor of cholesterol excretion despite lower hepatic cholesterol concentrations and higher cholesterol fractional synthesis.

  9. The protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation and increases sarcoplasmic/endoplasmic reticulum calcium ATPase 2 levels

    International Nuclear Information System (INIS)

    King, Taj D.; Gandy, Johanna C.; Bijur, Gautam N.

    2006-01-01

    The ubiquitously expressed protein glycogen synthase kinase-3 (GSK3) is constitutively active, however its activity is markedly diminished following phosphorylation of Ser21 of GSK3α and Ser9 of GSK3β. Although several kinases are known to phosphorylate Ser21/9 of GSK3, for example Akt, relatively much less is known about the mechanisms that cause the dephosphorylation of GSK3 at Ser21/9. In the present study KCl-induced plasma membrane depolarization of SH-SY5Y cells, which increases intracellular calcium concentrations caused a transient decrease in the phosphorylation of Akt at Thr308 and Ser473, and GSK3 at Ser21/9. Overexpression of the selective protein phosphatase-1 inhibitor protein, inhibitor-2, increased basal GSK3 phosphorylation at Ser21/9 and significantly blocked the KCl-induced dephosphorylation of GSK3β, but not GSK3α. The phosphorylation of Akt was not affected by the overexpression of inhibitor-2. GSK3 activity is known to affect sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) levels. Overexpression of inhibitor-2 or treatment of cells with the GSK3 inhibitors lithium and SB216763 increased the levels of SERCA2. These results indicate that the protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation induced by KCl and that GSK3 activity regulates SERCA2 levels

  10. Increases of heat shock proteins and their mRNAs at high hydrostatic pressure in a deep-sea piezophilic bacterium, Shewanella violacea.

    Science.gov (United States)

    Sato, Hiroshi; Nakasone, Kaoru; Yoshida, Takao; Kato, Chiaki; Maruyama, Tadashi

    2015-07-01

    When non-extremophiles encounter extreme