Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

PubMed Central

van den Bremer, Ewald TJ; Beurskens, Frank J; Voorhorst, Marleen; Engelberts, Patrick J; de Jong, Rob N; van der Boom, Burt G; Cook, Erika M; Lindorfer, Margaret A; Taylor, Ronald P; van Berkel, Patrick HC; Parren, Paul WHI

2015-01-01

Human IgG is produced with C-terminal lysines that are cleaved off in circulation. The function of this modification was unknown and generally thought not to affect antibody function. We recently reported that efficient C1q binding and complement-dependent cytotoxicity (CDC) requires IgG hexamerization at the cell surface. Here we demonstrate that C-terminal lysines may interfere with this process, leading to suboptimal C1q binding and CDC of cells opsonized with C-terminal lysine-containing IgG. After we removed these lysines with a carboxypeptidase, maximal complement activation was observed. Interestingly, IgG1 mutants containing either a negative C-terminal charge or multiple positive charges lost CDC almost completely; however, CDC was fully restored by mixing C-terminal mutants of opposite charge. Our data indicate a novel post-translational control mechanism of human IgG: human IgG molecules are produced in a pro-form in which charged C-termini interfere with IgG hexamer formation, C1q binding and CDC. To allow maximal complement activation, C-terminal lysine processing is required to release the antibody's full cytotoxic potential. PMID:26037225

Multimarker Strategy for Heart Failure Prognostication. Value of Neurohormonal Biomarkers: Neprilysin vs NT-proBNP.

PubMed

Bayes-Genis, Antoni; Barallat, Jaume; Galán, Amparo; de Antonio, Marta; Domingo, Mar; Zamora, Elisabet; Gastelurrutia, Paloma; Vila, Joan; Peñafiel, Judith; Gálvez-Montón, Carolina; Lupón, Josep

2015-12-01

Neprilysin breaks down numerous vasoactive peptides. The soluble form of neprilysin, which was recently identified in heart failure, is associated with cardiovascular outcomes. Within a multibiomarker strategy, we directly compared soluble neprilysin and N-terminal pro-B-type natriuretic peptide as risk stratifiers in a real-life cohort of heart failure patients. Soluble neprilysin, N-terminal pro-B-type natriuretic peptide, ST2, and high-sensitivity troponin T levels were measured in 797 consecutive ambulatory heart failure patients followed up for 4.7 years. Comprehensive multivariable analyses and soluble neprilysin vs N-terminal pro-B-type natriuretic peptide head-to-head assessments of performance were performed. A primary composite endpoint included cardiovascular death or heart failure hospitalization. A secondary endpoint explored cardiovascular death alone. Median soluble neprilysin and N-terminal pro-B-type natriuretic peptide concentrations were 0.64ng/mL and 1187 ng/L, respectively. Both biomarkers significantly correlated with age (P<.001) and ST2 (P<.001), but only N-terminal pro-B-type natriuretic peptide significantly correlated with estimated glomerular filtration rate (P<.001), body mass index (P<.001), left ventricular ejection fraction (P=.02) and high-sensitivity troponin T (P<.001). In multivariable Cox regression analyses, soluble neprilysin remained independently associated with the composite endpoint (hazard ratio=1.14; 95% confidence interval, 1.02-1.27; P=.03) and cardiovascular death (hazard ratio=1.15; 95% confidence interval, 1.01-1.31; P=.04), but N-terminal pro-B-type natriuretic peptide did not. The head-to-head soluble neprilysin vs N-terminal pro-B-type natriuretic peptide comparison showed good calibration and similar discrimination and reclassification for both neurohormonal biomarkers, but only soluble neprilysin improved overall goodness-of-fit. When added to a multimarker strategy, soluble neprilysin remained an independent prognosticator, while N-terminal pro-B-type natriuretic peptide lost significance as a risk stratifier in ambulatory patients with heart failure. Both biomarkers performed similarly in head-to-head analyses. Copyright © 2015 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.

Bioinformatic mapping and production of recombinant N-terminal domains of human cardiac ryanodine receptor 2

PubMed Central

Bauerová-Hlinková, Vladena; Hostinová, Eva; Gašperík, Juraj; Beck, Konrad; Borko, Ľubomír; Lai, F. Anthony; Zahradníková, Alexandra; Ševčík, Jozef

2010-01-01

We report the domain analysis of the N-terminal region (residues 1–759) of the human cardiac ryanodine receptor (RyR2) that encompasses one of the discrete RyR2 mutation clusters associated with catecholaminergic polymorphic ventricular tachycardia (CPVT1) and arrhythmogenic right ventricular dysplasia (ARVD2). Our strategy utilizes a bioinformatics approach complemented by protein expression, solubility analysis and limited proteolytic digestion. Based on the bioinformatics analysis, we designed a series of specific RyR2 N-terminal fragments for cloning and overexpression in Escherichia coli. High yields of soluble proteins were achieved for fragments RyR21–606·His6, RyR2391–606·His6, RyR2409–606·His6, Trx·RyR2384–606·His6, Trx·RyR2391-606·His6 and Trx·RyR2409–606·His6. The folding of RyR21–606·His6 was analyzed by circular dichroism spectroscopy resulting in α-helix and β-sheet content of ∼23% and ∼29%, respectively, at temperatures up to 35 °C, which is in agreement with sequence based secondary structure predictions. Tryptic digestion of the largest recombinant protein, RyR21–606·His6, resulted in the appearance of two specific subfragments of ∼40 and 25 kDa. The 25 kDa fragment exhibited greater stability. Hybridization with anti-His6·Tag antibody indicated that RyR21–606·His6 is cleaved from the N-terminus and amino acid sequencing of the proteolytic fragments revealed that digestion occurred after residues 259 and 384, respectively. PMID:20045464

Classical Complement Pathway Activation in the Kidneys of Women With Preeclampsia.

PubMed

Penning, Marlies; Chua, Jamie S; van Kooten, Cees; Zandbergen, Malu; Buurma, Aletta; Schutte, Joke; Bruijn, Jan Anthonie; Khankin, Eliyahu V; Bloemenkamp, Kitty; Karumanchi, S Ananth; Baelde, Hans

2015-07-01

A growing body of evidence suggests that complement dysregulation plays a role in the pathogenesis of preeclampsia. The kidney is one of the major organs affected in preeclampsia. Because the kidney is highly susceptible to complement activation, we hypothesized that preeclampsia is associated with renal complement activation. We performed a nationwide search for renal autopsy material in the Netherlands using a computerized database (PALGA). Renal tissue was obtained from 11 women with preeclampsia, 25 pregnant controls, and 14 nonpregnant controls with hypertension. The samples were immunostained for C4d, C1q, mannose-binding lectin, properdin, C3d, C5b-9, IgA, IgG, and IgM. Preeclampsia was significantly associated with renal C4d-a stable marker of complement activation-and the classical pathway marker C1q. In addition, the prevalence of IgM was significantly higher in the kidneys of the preeclamptic women. No other complement markers studied differed between the groups. Our findings in human samples were validated using a soluble fms-like tyrosine kinase 1 mouse model of preeclampsia. The kidneys in the soluble fms-like tyrosine kinase 1-injected mice had significantly more C4 deposits than the control mice. The association between preeclampsia and renal C4d, C1q, and IgM levels suggests that the classical complement pathway is involved in the renal injury in preeclampsia. Moreover, our finding that soluble fms-like tyrosine kinase 1-injected mice develop excess C4 deposits indicates that angiogenic dysregulation may play a role in complement activation within the kidney. We suggest that inhibiting complement activation may be beneficial for preventing the renal manifestations of preeclampsia. © 2015 American Heart Association, Inc.

Coupling complement regulators to immunoglobulin domains generates effective anti-complement reagents with extended half-life in vivo

PubMed Central

HARRIS, C L; WILLIAMS, A S; LINTON, S M; MORGAN, B P

2002-01-01

Complement activation and subsequent generation of inflammatory molecules and membrane attack complex contributes to the pathology of a number of inflammatory and degenerative diseases, including arthritis, glomerulonephritis and demyelination. Agents that specifically inhibit complement activation might prove beneficial in the treatment of these diseases. Soluble recombinant forms of the naturally occurring membrane complement regulatory proteins (CRP) have been exploited for this purpose. We have undertaken to design better therapeutics based on CRP. Here we describe the generation of soluble, recombinant CRP comprising rat decay accelerating factor (DAF) or rat CD59 expressed as Fc fusion proteins, antibody-like molecules comprising two CRP moieties in place of the antibody Fab arms (CRP-Ig). Reagents bearing DAF on each arm (DAF-Ig), CD59 on each arm (CD59-Ig) and a hybrid reagent containing both DAF and CD59 were generated. All three reagents inhibited C activation in vitro. Compared with soluble CRP lacking Fc domains, activity was reduced, but was fully restored by enzymatic release of the regulator from the Ig moiety, implicating steric constraints in reducing functional activity. In vivo studies showed that DAF-Ig, when compared to soluble DAF, had a much extended half-life in the circulation in rats and concomitantly caused a sustained reduction in plasma complement activity. When given intra-articularly to rats in a model of arthritis, DAF-Ig significantly reduced severity of disease. The data demonstrate the potential of CRP-Ig as reagents for sustained therapy of inflammatory disorders, including arthritis, but emphasize the need for careful design of fusion proteins to retain function. PMID:12165074

Sex matters: Systemic complement activity of female C57BL/6J and BALB/cJ mice is limited by serum terminal pathway components.

PubMed

Kotimaa, Juha; Klar-Mohammad, Ngaisah; Gueler, Faikah; Schilders, Geurt; Jansen, Aswin; Rutjes, Helma; Daha, Mohamed R; van Kooten, Cees

2016-08-01

Experimental mouse models have been extensively used to elucidate the role of the complement system in different diseases and injuries. Contribution of gender has revealed an intriguing gender specific difference; female mice often show protection against most complement driven injuries such as ischemia/reperfusion injury, graft rejection and sepsis. Interestingly, early studies to the mouse complement system revealed that female mice have very low total complement activity (CH50), which is related to androgen regulation of hepatic complement synthesis. Here, our aim was to understand at which level the female specific differences in mouse complement resides. We have used recently developed complement assays to study the functional activities of female and male mice at the level of C3 and C9 activation, and furthermore assayed key complement factor levels in serum of age-matched female and male C57BL/6 mice. Our results show that the female mice have normal complement cascade functionality at the level of C3 activation, which was supported by determinations of early complement factors. However, all pathways are strongly reduced at the level of C9 activation, suggesting a terminal pathway specific difference. This was in line with C6 and C9 measurements, showing strongly decreased levels in females. Furthermore, similar gender differences were also found in BALB/cJ mice, but not in CD-1 mice. Our results clearly demonstrate that the complement system in females of frequently used mouse strains is restricted by the terminal pathway components and that the perceived female specific protection against experimental disease and injury might be in part explained by the inability promote inflammation through C5b-9. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Production of a highly active, soluble form of the cytochrome P450 reductase (CPR A) from Candida tropicalis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Donnelly, Mark

2006-08-01

The present invention provides soluble cytochrome p450 reductase (CPR) proteins from Candida sp. having an altered N-terminal region which results in reduced hydrophobicity of the N-terminal region. Also provided are host cells comprising the subject soluble CPR proteins. In addition, the present invention provides nucleotide and corresponding amino acid sequences for soluble CPR proteins and vectors comprising the nucleotide sequences. Methods for producing a soluble CPR, for increasing production of a dicarboxylic acid, and for detecting a cytochrome P450 are also provided.

STUDIES ON SCRUB TYPHUS

PubMed Central

Smadel, Joseph E.; Rights, Fred L.; Jackson, Elizabeth B.

1946-01-01

A complement-fixing antigen specific for scrub typhus occurs in the body fluids and tissues of infected mice, white rats, and cotton rats. The specific serological substance is demonstrable only in those animals which develop a rapidly fatal disease after an incubation period of a few days. Such an experimental infection is induced in mice and rats by the intravenous injection of suspensions of yolk sac rich in R. orientalis. Ether extraction is an important step in the preparation of a complement-fixing antigen from tissues of mice dying with scrub typhus. The Imphal No. 8 and Calcutta strains of R. orientalis are indistinguishable on the basis of complement fixation and cross-immunity tests. The complement-fixing antigen in body fluids of infected mice and rats and in our preparations of tissues from such animals occurs as a soluble antigen. Under the proper conditions the soluble antigen can be stored or dehydrated without loss of serological activity. PMID:19871518

The membrane attack complex of complement contributes to plasmin-induced synthesis of platelet-activating factor by endothelial cells and neutrophils

PubMed Central

Lupia, Enrico; Del Sorbo, Lorenzo; Bergerone, Serena; Emanuelli, Giorgio; Camussi, Giovanni; Montrucchio, Giuseppe

2003-01-01

Thrombolytic agents, used to restore blood flow to ischaemic tissues, activate several enzymatic systems with pro-inflammatory effects, thus potentially contributing to the pathogenesis of ischaemia–reperfusion injury. Platelet-activating factor (PAF), a phospholipid mediator of inflammation, has been implicated in the pathogenesis of this process. We previously showed that the infusion of streptokinase (SK) induces the intravascular release of PAF in patients with acute myocardial infarction (AMI), and that cultured human endothelial cells (EC) synthesized PAF in response to SK and plasmin (PLN). In the present study, we investigated the role of the membrane attack complex (MAC) of complement in the PLN-induced synthesis of PAF. In vivo, we showed a correlation between the levels of soluble terminal complement components (sC5b-9) and the concentrations of PAF detected in blood of patients with AMI infused with SK. In vitro both EC and polymorphonuclear neutrophils (PMN), incubated in the presence of PLN and normal human serum, showed an intense staining for the MAC neoepitope, while no staining was detected when they were incubated with PLN in the presence of heat-inactivated normal human serum. Moreover, the insertion of MAC on EC and PMN plasmamembrane elicited the synthesis of PAF. In conclusion, our results elucidate the mechanisms involved in PAF production during the activation of the fibrinolytic system, showing a role for complement products in this setting. The release of PAF may increase the inflammatory response, thus limiting the beneficial effects of thrombolytic therapy. Moreover, it may have a pathogenic role in other pathological conditions, such as transplant rejection, tumoral angiogenesis, and septic shock, where fibrinolysis is activated. PMID:12871223

The membrane attack complex of complement contributes to plasmin-induced synthesis of platelet-activating factor by endothelial cells and neutrophils.

PubMed

Lupia, Enrico; Del Sorbo, Lorenzo; Bergerone, Serena; Emanuelli, Giorgio; Camussi, Giovanni; Montrucchio, Giuseppe

2003-08-01

Thrombolytic agents, used to restore blood flow to ischaemic tissues, activate several enzymatic systems with pro-inflammatory effects, thus potentially contributing to the pathogenesis of ischaemia-reperfusion injury. Platelet-activating factor (PAF), a phospholipid mediator of inflammation, has been implicated in the pathogenesis of this process. We previously showed that the infusion of streptokinase (SK) induces the intravascular release of PAF in patients with acute myocardial infarction (AMI), and that cultured human endothelial cells (EC) synthesized PAF in response to SK and plasmin (PLN). In the present study, we investigated the role of the membrane attack complex (MAC) of complement in the PLN-induced synthesis of PAF. In vivo, we showed a correlation between the levels of soluble terminal complement components (sC5b-9) and the concentrations of PAF detected in blood of patients with AMI infused with SK. In vitro both EC and polymorphonuclear neutrophils (PMN), incubated in the presence of PLN and normal human serum, showed an intense staining for the MAC neoepitope, while no staining was detected when they were incubated with PLN in the presence of heat-inactivated normal human serum. Moreover, the insertion of MAC on EC and PMN plasmamembrane elicited the synthesis of PAF. In conclusion, our results elucidate the mechanisms involved in PAF production during the activation of the fibrinolytic system, showing a role for complement products in this setting. The release of PAF may increase the inflammatory response, thus limiting the beneficial effects of thrombolytic therapy. Moreover, it may have a pathogenic role in other pathological conditions, such as transplant rejection, tumoral angiogenesis, and septic shock, where fibrinolysis is activated.

Cloning, over-expression and purification of Pseudomonas aeruginosa murC encoding uridine diphosphate N-acetylmuramate: L-alanine ligase.

PubMed

El Zoeiby, A; Sanschagrin, F; Lamoureux, J; Darveau, A; Levesque, R C

2000-02-15

We cloned and sequenced the murC gene from Pseudomonas aeruginosa encoding a protein of 53 kDa. Multiple alignments with 20 MurC peptide sequences from different bacteria confirmed the presence of highly conserved regions having sequence identities ranging from 22-97% including conserved motifs for ATP-binding and the active site of the enzyme. Genetic complementation was done in Escherichia coli (murCts) suppressing the lethal phenotype. The murC gene was subcloned into the expression vector pET30a and overexpressed in E. coli BL21(lambdaDE3). Three PCR cloning strategies were used to obtain the three recombinant plasmids for expression of the native MurC, MurC His-tagged at N-terminal and at C-terminal, respectively. MurC His-tagged at C-terminal was chosen for large scale production and protein purification in the soluble form. The purification was done in a single chromatographic step on an affinity nickel column and obtained in mg quantities at 95% homogeneity. MurC protein was used to produce monoclonal antibodies for epitope mapping and for assay development in high throughput screenings. Detailed studies of MurC and other genes of the bacterial cell cycle will provide the reagents and strain constructs for high throughput screening and for design of novel antibacterials.

Functional analyses of rare genetic variants in complement component C9 identified in patients with age-related macular degeneration.

PubMed

Kremlitzka, Mariann; Geerlings, Maartje J; de Jong, Sarah; Bakker, Bjorn; Nilsson, Sara C; Fauser, Sascha; Hoyng, Carel B; de Jong, Eiko K; den Hollander, Anneke I; Blom, Anna M

2018-05-14

Age-related macular degeneration (AMD) is a progressive disease of the central retina and the leading cause of irreversible vision loss in the western world. The involvement of abnormal complement activation in AMD has been suggested by association of variants in genes encoding complement proteins with disease development. A low-frequency variant (p.P167S) in the complement component C9 (C9) gene was recently shown to be highly associated with AMD, however its functional outcome remains largely unexplored. In this study, we reveal five novel rare genetic variants (p.M45L, p.F62S, p.G126R, p.T170I and p.A529T) in C9 in AMD patients, and evaluate their functional effects in vitro together with the previously identified (p.R118W and p.P167S) C9 variants.Our results demonstrate that the concentration of C9 is significantly elevated in patients' sera carrying the p.M45L, p.F62S, p.P167S and p.A529T variants compared to non-carrier controls. However, no difference can be observed in soluble terminal complement complex levels between the carrier and non-carrier groups. Comparing the polymerization of the C9 variants we reveal that the p.P167S mutant spontaneously aggregates, while the other mutant proteins (except for C9 p.A529T) fail to polymerize in the presence of zinc. Altered polymerization of the p.F62S and p.P167S proteins associated with decreased lysis of sheep erythrocytes and ARPE-19 cells by carriers' sera. Our data suggest that the analysed C9 variants affect only the secretion and polymerization of C9, without influencing its classical lytic activity. Future studies need to be performed to understand the implications of the altered polymerization of C9 in AMD pathology.

Homologous species restriction of the complement-mediated killing of nucleated cells.

PubMed Central

Yamamoto, H; Blaas, P; Nicholson-Weller, A; Hänsch, G M

1990-01-01

The homologous restriction of complement (C) lysis is attributed to membrane proteins: decay-accelerating factor (DAF), C8 binding protein (C8bp) and P18/CD59. Since these proteins are also expressed on peripheral blood cells, species restriction was tested for in the complement-mediated killing of antibody-coated human leucocytes by human or rabbit complement. Killing was more efficient when rabbit complement was used. Preincubation of cells with an antibody to DAF abolished the difference. When C1-7 sites were first attached to the cells and either rabbit or human C8, C9 were added, the killing of monocytes and lymphocytes was equally efficient; only in polymorphonuclear neutrophils was a higher efficiency of rabbit C8, C9 seen. Thus, in contrast to haemolysis, restriction occurred predominantly at the C3 level and the action of the terminal complement components was not inhibited. Since C8bp isolated from peripheral blood cells showed essentially similar characteristics as the erythrocyte-derived C8bp, the failure of C8bp to inhibit the action of the terminal components on nucleated cells might reflect differences of the complement membrane interactions between erythrocytes or nucleated cells, respectively. Images Figure 5 PMID:1697561

Microfibrillar associated protein 4 mfap4 genes in catfish play a novel role in innate immune responses

USDA-ARS?s Scientific Manuscript database

The lectin pathway of the complement system is characterized by two groups of soluble pattern recognition molecules, mannose-binding lectins (MBLs) and ficolins. These molecules recognize and bind carbohydrates in pathogens and activate complement leading to opsonization, leukocyte activation, and d...

Positional effects of fusion partners on the yield and solubility of MBP fusion proteins.

PubMed

Raran-Kurussi, Sreejith; Keefe, Karina; Waugh, David S

2015-06-01

Escherichia coli maltose-binding protein (MBP) is exceptionally effective at promoting the solubility of its fusion partners. However, there are conflicting reports in the literature claiming that (1) MBP is an effective solubility enhancer only when it is joined to the N-terminus of an aggregation-prone passenger protein, and (2) MBP is equally effective when fused to either end of the passenger. Here, we endeavor to resolve this controversy by comparing the solubility of a diverse set of MBP fusion proteins that, unlike those analyzed in previous studies, are identical in every way except for the order of the two domains. The results indicate that fusion proteins with an N-terminal MBP provide an excellent solubility advantage along with more robust expression when compared to analogous fusions in which MBP is the C-terminal fusion partner. We find that only intrinsically soluble passenger proteins (i.e., those not requiring a solubility enhancer) are produced as soluble fusions when they precede MBP. We also report that even subtle differences in inter-domain linker sequences can influence the solubility of fusion proteins. Published by Elsevier Inc.

Positional effects of fusion partners on the yield and solubility of MBP fusion proteins

PubMed Central

Raran-Kurussi, Sreejith; Keefe, Karina; Waugh, David S.

2015-01-01

Escherichia coli maltose-binding protein (MBP) is exceptionally effective at promoting the solubility of its fusion partners. However, there are conflicting reports in the literature claiming that 1) MBP is an effective solubility enhancer only when it is joined to the N-terminus of an aggregation-prone passenger protein, and 2) MBP is equally effective when fused to either end of the passenger. Here, we endeavor to resolve this controversy by comparing the solubility of a diverse set of MBP fusion proteins that, unlike those analyzed in previous studies, are identical in every way except for the order of the two domains. The results indicate that fusion proteins with an N-terminal MBP provide an excellent solubility advantage along with more robust expression when compared to analogous fusions in which MBP is the C-terminal fusion partner. We find that only intrinsically soluble passenger proteins (i.e., those not requiring a solubility enhancer) are produced as soluble fusions when they precede MBP. We also report that even subtle differences in inter-domain linker sequences can influence the solubility of fusion proteins. PMID:25782741

Thermodynamically controlled preservation of organic carbon in floodplains

DOE PAGES

Boye, Kristin; Noel, Vincent; Tfaily, Malak M.; ...

2017-05-01

Organic matter decomposition in soils and terrestrial sediments has a prominent role in the global carbon cycle. Carbon stocks in anoxic environments, such as wetlands and the subsurface of floodplains, are large and presumed to decompose slowly. The degree of microbial respiration in anoxic environments is typically thought to depend on the energetics of available terminal electron acceptors such as nitrate or sulfate; microbes couple the reduction of these compounds to the oxidation of organic carbon. But, it is also possible that the energetics of the organic carbon itself can determine whether it is decomposed. We examined water-soluble organic carbonmore » by Fourier-transform ion-cyclotron-resonance mass spectrometry to compare the chemical composition and average nominal oxidation state of carbon—a metric reflecting whether microbial oxidation of organic matter is thermodynamically favourable—in anoxic (sulfidic) and oxic (non-sulfidic) floodplain sediments. We also observed distinct minima in the average nominal oxidation state of water-soluble carbon in sediments exhibiting anoxic, sulfate-reducing conditions, suggesting preservation of carbon compounds with nominal oxidation states below the threshold that makes microbial sulfate reduction thermodynamically favourable. Finally, we show that thermodynamic limitations constitute an important complement to other mechanisms of carbon preservation, such as enzymatic restrictions and mineral association, within anaerobic environments.« less

Thermodynamically controlled preservation of organic carbon in floodplains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boye, Kristin; Noel, Vincent; Tfaily, Malak M.

Organic matter decomposition in soils and terrestrial sediments has a prominent role in the global carbon cycle. Carbon stocks in anoxic environments, such as wetlands and the subsurface of floodplains, are large and presumed to decompose slowly. The degree of microbial respiration in anoxic environments is typically thought to depend on the energetics of available terminal electron acceptors such as nitrate or sulfate; microbes couple the reduction of these compounds to the oxidation of organic carbon. But, it is also possible that the energetics of the organic carbon itself can determine whether it is decomposed. We examined water-soluble organic carbonmore » by Fourier-transform ion-cyclotron-resonance mass spectrometry to compare the chemical composition and average nominal oxidation state of carbon—a metric reflecting whether microbial oxidation of organic matter is thermodynamically favourable—in anoxic (sulfidic) and oxic (non-sulfidic) floodplain sediments. We also observed distinct minima in the average nominal oxidation state of water-soluble carbon in sediments exhibiting anoxic, sulfate-reducing conditions, suggesting preservation of carbon compounds with nominal oxidation states below the threshold that makes microbial sulfate reduction thermodynamically favourable. Finally, we show that thermodynamic limitations constitute an important complement to other mechanisms of carbon preservation, such as enzymatic restrictions and mineral association, within anaerobic environments.« less

Thermodynamically controlled preservation of organic carbon in floodplains

NASA Astrophysics Data System (ADS)

Boye, Kristin; Noël, Vincent; Tfaily, Malak M.; Bone, Sharon E.; Williams, Kenneth H.; Bargar, John R.; Fendorf, Scott

2017-06-01

Organic matter decomposition in soils and terrestrial sediments has a prominent role in the global carbon cycle. Carbon stocks in anoxic environments, such as wetlands and the subsurface of floodplains, are large and presumed to decompose slowly. The degree of microbial respiration in anoxic environments is typically thought to depend on the energetics of available terminal electron acceptors such as nitrate or sulfate; microbes couple the reduction of these compounds to the oxidation of organic carbon. However, it is also possible that the energetics of the organic carbon itself can determine whether it is decomposed. Here we examined water-soluble organic carbon by Fourier-transform ion-cyclotron-resonance mass spectrometry to compare the chemical composition and average nominal oxidation state of carbon--a metric reflecting whether microbial oxidation of organic matter is thermodynamically favourable--in anoxic (sulfidic) and oxic (non-sulfidic) floodplain sediments. We observed distinct minima in the average nominal oxidation state of water-soluble carbon in sediments exhibiting anoxic, sulfate-reducing conditions, suggesting preservation of carbon compounds with nominal oxidation states below the threshold that makes microbial sulfate reduction thermodynamically favourable. We conclude that thermodynamic limitations constitute an important complement to other mechanisms of carbon preservation, such as enzymatic restrictions and mineral association, within anaerobic environments.

Factor H: A Complement Regulator in Health and Disease, and a Mediator of Cellular Interactions

PubMed Central

Kopp, Anne; Hebecker, Mario; Svobodová, Eliška; Józsi, Mihály

2012-01-01

Complement is an essential part of innate immunity as it participates in host defense against infections, disposal of cellular debris and apoptotic cells, inflammatory processes and modulation of adaptive immune responses. Several soluble and membrane-bound regulators protect the host from the potentially deleterious effects of uncontrolled and misdirected complement activation. Factor H is a major soluble regulator of the alternative complement pathway, but it can also bind to host cells and tissues, protecting them from complement attack. Interactions of factor H with various endogenous ligands, such as pentraxins, extracellular matrix proteins and DNA are important in limiting local complement-mediated inflammation. Impaired regulatory as well as ligand and cell recognition functions of factor H, caused by mutations or autoantibodies, are associated with the kidney diseases: atypical hemolytic uremic syndrome and dense deposit disease and the eye disorder: age-related macular degeneration. In addition, factor H binds to receptors on host cells and is involved in adhesion, phagocytosis and modulation of cell activation. In this review we discuss current concepts on the physiological and pathophysiological roles of factor H in light of new data and recent developments in our understanding of the versatile roles of factor H as an inhibitor of complement activation and inflammation, as well as a mediator of cellular interactions. A detailed knowledge of the functions of factor H in health and disease is expected to unravel novel therapeutic intervention possibilities and to facilitate the development or improvement of therapies. PMID:24970127

Combined Measurement of Soluble ST2 and Amino-Terminal Pro-B-Type Natriuretic Peptide Provides Early Assessment of Severity in Cardiogenic Shock Complicating Acute Coronary Syndrome.

PubMed

Tolppanen, Heli; Rivas-Lasarte, Mercedes; Lassus, Johan; Sadoune, Malha; Gayat, Etienne; Pulkki, Kari; Arrigo, Mattia; Krastinova, Evguenia; Sionis, Alessandro; Parissis, John; Spinar, Jindrich; Januzzi, James; Harjola, Veli-Pekka; Mebazaa, Alexandre

2017-07-01

Mortality in cardiogenic shock complicating acute coronary syndrome is high, and objective risk stratification is needed for rational use of advanced therapies such as mechanical circulatory support. Traditionally, clinical variables have been used to judge risk in cardiogenic shock. The aim of this study was to assess the added value of serial measurement of soluble ST2 and amino-terminal pro-B-type natriuretic peptide to clinical parameters for risk stratification in cardiogenic shock. CardShock (www.clinicaltrials.gov NCT01374867) is a prospective European multinational study of cardiogenic shock. The main study introduced CardShock risk score, which is calculated from seven clinical variables at baseline, and was associated with short-term mortality. Nine tertiary care university hospitals. Patients with cardiogenic shock caused by acute coronary syndrome (n=145). In this substudy, plasma samples from the study patients were analyzed at eight time points during the ICU or cardiac care unit stay. Additional prognostic value of the biomarkers was assessed with incremental discrimination improvement. The combination of soluble ST2 and amino-terminal pro-B-type natriuretic peptide showed excellent discrimination for 30-day mortality (area under the curve, 0.77 at 12 hr up to 0.93 at 5-10 d after cardiogenic shock onset). At 12 hours, patients with both biomarkers elevated (soluble ST2, ≥ 500 ng/mL and amino-terminal pro-B-type natriuretic peptide, ≥ 4,500 ng/L) had higher 30-day mortality (79%) compared to those with one or neither biomarkers elevated (31% or 10%, respectively; p < 0.001). Combined measurement of soluble ST2 and amino-terminal pro-B-type natriuretic peptide at 12 hours added value to CardShock risk score, correctly reclassifying 11% of patients. The combination of results for soluble ST2 and amino-terminal pro-B-type natriuretic peptide provides early risk assessment beyond clinical variables in patients with acute coronary syndrome-related cardiogenic shock and may help therapeutic decision making in these patients.

Protein- protein interaction detection system using fluorescent protein microdomains

DOEpatents

Waldo, Geoffrey S.; Cabantous, Stephanie

2010-02-23

The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

In vitro and in vivo mapping of the Prunus necrotic ringspot virus coat protein C-terminal dimerization domain by bimolecular fluorescence complementation.

PubMed

Aparicio, Frederic; Sánchez-Navarro, Jesús A; Pallás, Vicente

2006-06-01

Interactions between viral proteins are critical for virus viability. Bimolecular fluorescent complementation (BiFC) technique determines protein interactions in real-time under almost normal physiological conditions. The coat protein (CP) of Prunus necrotic ringspot virus is required for multiple functions in its replication cycle. In this study, the region involved in CP dimerization has been mapped by BiFC in both bacteria and plant tissue. Full-length and C-terminal deleted forms of the CP gene were fused in-frame to the N- and C-terminal fragments of the yellow fluorescent protein. The BiFC analysis showed that a domain located between residues 9 and 27 from the C-end plays a critical role in dimerization. The importance of this C-terminal region in dimer formation and the applicability of the BiFC technique to analyse viral protein interactions are discussed.

Nucleic acid encoding a self-assembling split-fluorescent protein system

DOEpatents

Waldo, Geoffrey S.; Cabantous, Stephanie

2014-04-01

The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

Nucleic acid encoding a self-assembling split-fluorescent protein system

DOEpatents

Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

2011-06-07

The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

Nucleic acid encoding a self-assembling split-fluorescent protein system

DOEpatents

Waldo, Geoffrey S.; Cabantous, Stephanie

2015-07-14

The invention provides a protein labeling and detection system based on self-complementing fragments of fluorescent and chromophoric proteins. The system of the invention is exemplified with various combinations of self-complementing fragments derived from Aequorea victoria Green Fluorescent Protein (GFP), which are used to detect and quantify protein solubility in multiple assay formats, both in vitro and in vivo.

Terminal Complement Blockade after Hematopoietic Stem Cell Transplantation Is Safe without Meningococcal Vaccination.

PubMed

Jodele, Sonata; Dandoy, Christopher E; Danziger-Isakov, Lara; Myers, Kasiani C; El-Bietar, Javier; Nelson, Adam; Wallace, Gregory; Teusink-Cross, Ashley; Davies, Stella M

2016-07-01

Eculizumab inhibits terminal complement-mediated intravascular hemolysis in patients with paroxysmal nocturnal hemoglobinuria and complement-mediated thrombotic microangiopathy (TMA) in patients with atypical hemolytic uremic syndrome and is now used as a first-line therapy in these diseases. Eculizumab is available only through a restricted program under a Risk Evaluation and Mitigation Strategy (REMS) because of an increased risk of meningococcal infections in persons without adequate functional complement. Administration of meningococcal vaccine is required at least 2 weeks before administering the first dose of eculizumab, and this advice is included in the product label. Eculizumab use for treatment of TMA in hematopoietic stem cell transplantation (HSCT) recipients brings a significant dilemma regarding REMS required meningococcal vaccination. TMA after HSCT usually occurs within the first 100 days after transplantation when patients are severely immunocompromised and are not able to mount a response to vaccines. We evaluated 30 HSCT recipients treated with eculizumab for high-risk TMA without meningococcal vaccine. All patients received antimicrobial prophylaxis adequate for Neisseria meningitides during eculizumab therapy and for 8 weeks after discontinuation of the drug. Median time to TMA diagnosis was 28 days after transplant (range, 13.8 to 48.5). Study subjects received a median of 14 eculizumab doses (range, 2 to 38 doses) for HSCT-associated TMA therapy. There were no incidences of meningococcal infections. The incidences of bacterial and fungal bloodstream infections were similar in patients treated with eculizumab (n = 30) as compared with those with HSCT-associated TMA who did not receive any complement blocking therapy (n = 39). Our data indicate that terminal complement blockade in the early post-transplant period can be performed without meningococcal vaccination while using appropriate antimicrobial prophylaxis until complement function is restored after therapy completion. Copyright © 2016 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Complement Evasion Strategies of Viruses: An Overview

PubMed Central

Agrawal, Palak; Nawadkar, Renuka; Ojha, Hina; Kumar, Jitendra; Sahu, Arvind

2017-01-01

Being a major first line of immune defense, the complement system keeps a constant vigil against viruses. Its ability to recognize large panoply of viruses and virus-infected cells, and trigger the effector pathways, results in neutralization of viruses and killing of the infected cells. This selection pressure exerted by complement on viruses has made them evolve a multitude of countermeasures. These include targeting the recognition molecules for the avoidance of detection, targeting key enzymes and complexes of the complement pathways like C3 convertases and C5b-9 formation – either by encoding complement regulators or by recruiting membrane-bound and soluble host complement regulators, cleaving complement proteins by encoding protease, and inhibiting the synthesis of complement proteins. Additionally, viruses also exploit the complement system for their own benefit. For example, they use complement receptors as well as membrane regulators for cellular entry as well as their spread. Here, we provide an overview on the complement subversion mechanisms adopted by the members of various viral families including Poxviridae, Herpesviridae, Adenoviridae, Flaviviridae, Retroviridae, Picornaviridae, Astroviridae, Togaviridae, Orthomyxoviridae and Paramyxoviridae. PMID:28670306

Mutants in three novel complementation groups inhibit membrane protein insertion into and soluble protein translocation across the endoplasmic reticulum membrane of Saccharomyces cerevisiae

PubMed Central

1992-01-01

We have isolated mutants that inhibit membrane protein insertion into the ER membrane of Saccharomyces cerevisiae. The mutants were contained in three complementation groups, which we have named SEC70, SEC71, and SEC72. The mutants also inhibited the translocation of soluble proteins into the lumen of the ER, indicating that they pleiotropically affect protein transport across and insertion into the ER membrane. Surprisingly, the mutants inhibited the translocation and insertion of different proteins to drastically different degrees. We have also shown that mutations in SEC61 and SEC63, which were previously isolated as mutants inhibiting the translocation of soluble proteins, also affect the insertion of membrane proteins into the ER. Taken together our data indicate that the process of protein translocation across the ER membrane involves a much larger number of gene products than previously appreciated. Moreover, different translocation substrates appear to have different requirements for components of the cellular targeting and translocation apparatus. PMID:1730771

A Parametric Rosetta Energy Function Analysis with LK Peptides on SAM Surfaces.

PubMed

Lubin, Joseph H; Pacella, Michael S; Gray, Jeffrey J

2018-05-08

Although structures have been determined for many soluble proteins and an increasing number of membrane proteins, experimental structure determination methods are limited for complexes of proteins and solid surfaces. An economical alternative or complement to experimental structure determination is molecular simulation. Rosetta is one software suite that models protein-surface interactions, but Rosetta is normally benchmarked on soluble proteins. For surface interactions, the validity of the energy function is uncertain because it is a combination of independent parameters from energy functions developed separately for solution proteins and mineral surfaces. Here, we assess the performance of the RosettaSurface algorithm and test the accuracy of its energy function by modeling the adsorption of leucine/lysine (LK)-repeat peptides on methyl- and carboxy-terminated self-assembled monolayers (SAMs). We investigated how RosettaSurface predictions for this system compare with the experimental results, which showed that on both surfaces, LK-α peptides folded into helices and LK-β peptides held extended structures. Utilizing this model system, we performed a parametric analysis of Rosetta's Talaris energy function and determined that adjusting solvation parameters offered improved predictive accuracy. Simultaneously increasing lysine carbon hydrophilicity and the hydrophobicity of the surface methyl head groups yielded computational predictions most closely matching the experimental results. De novo models still should be interpreted skeptically unless bolstered in an integrative approach with experimental data.

Interallelic complementation of mutations in propionic acidemia by microinjection of mutant cDNAs into fibroblasts of affected patients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Loyer, M.; Leclerc, D.; Gravel, R.A.

1994-09-01

Propionic acidemia is a rare autosomal recessive disorder resulting from defects of the {alpha} or {beta} subunit of biotin-dependent propionyl-CoA carboxylase (PCC). Mutations are assigned to defects of the PCCA ({alpha} subunit) or PCCB ({beta} subunit) gene through complementation studies after somatic fusion of patient cell lines. About two-thirds of patients with {beta} subunit defects (complementation group pccBC) show interallelic complementation in cell fusion experiments (subgroups pccB and pccC), monitored by the PCC-dependent metabolisms of {sup 14}C-propionate. Most patient cell lines are heteroallelic for two different mutations, leaving ambiguous the identity of the mutation participating in interallelic complementation. To identifymore » the complementing mutations, we have expressed {beta}-subunit cDNAs containing individual mutations by microinjection of the cDNAs in recipient cells from patients with {beta} subunit defects. Correction of the PCC defect was monitored by autoradiography of {sup 14}C-propionate incorporation. In some experiments, cDNAs were co-injected with a plasmid expressing the E. coli lacZ gene as a positive control for successful injection. Two mutations from the pccB subgroup showed complementation when injected into pccC cells; dupKICK140-143 and Pro228Leu. Similarly, two mutations from the pccC subgroup complemented after injection into pccB cells; {Delta}Ile408 and Arg410Trp. No mutation complemented with mutation of the pccBC group which are classified as non-complementing in cell fusion experiments. The results show that the complementing pccB mutations are found in the N-terminal half of the {beta} subunit, while the complementing pccC mutations cluxter at a site in the C-terminal half. The latter site is a candidate for the propionyl-CoA binding site based on sequence identity with a region of transcarboxylase from Propionibacterium shermanii.« less

Calcineurin inhibitor-induced complement system activation via ERK1/2 signalling is inhibited by SOCS-3 in human renal tubule cells.

PubMed

Loeschenberger, Beatrix; Niess, Lea; Würzner, Reinhard; Schwelberger, Hubert; Eder, Iris E; Puhr, Martin; Guenther, Julia; Troppmair, Jakob; Rudnicki, Michael; Neuwirt, Hannes

2018-02-01

One factor that significantly contributes to renal allograft loss is chronic calcineurin inhibitor (CNI) nephrotoxicity (CIN). Among other factors, the complement (C-) system has been proposed to be involved CIN development. Hence, we investigated the impact of CNIs on intracellular signalling and the effects on the C-system in human renal tubule cells. In a qPCR array, CNI treatment upregulated C-factors and downregulated SOCS-3 and the complement inhibitors CD46 and CD55. Additionally, ERK1/-2 was required for these regulations. Following knock-down and overexpression of SOCS-3, we found that SOCS-3 inhibits ERK1/-2 signalling. Finally, we assessed terminal complement complex formation, cell viability and apoptosis. Terminal complement complex formation was induced by CNIs. Cell viability was significantly decreased, whereas apoptosis was increased. Both effects were reversed under complement component-depleted conditions. In vivo, increased ERK1/-2 phosphorylation and SOCS-3 downregulation were observed at the time of transplantation in renal allograft patients who developed a progressive decline of renal function in the follow-up compared to stable patients. The progressive cohort also had lower total C3 levels, suggesting higher complement activity at baseline. In conclusion, our data suggest that SOCS-3 inhibits CNI-induced ERK1/-2 signalling, thereby blunting the negative control of C-system activation. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Novel Evasion Mechanisms of the Classical Complement Pathway

PubMed Central

Garcia, Brandon L.; Zwarthoff, Seline A.; Rooijakkers, Suzan H. M.; Geisbrecht, Brian V.

2016-01-01

Complement is a network of soluble and cell surface-associated proteins which gives rise to a self-amplifying, yet tightly regulated system with fundamental roles in immune surveillance and clearance. Complement becomes activated on the surface of ‘non-self’ cells by one of three initiating mechanisms known as the classical, lectin, or alternative pathways. Evasion of complement function is a hallmark of invasive pathogens and hematophagous organisms. While many complement inhibition strategies hinge on hijacking activities of endogenous complement regulatory proteins, an increasing number of uniquely evolved evasion molecules have been discovered over the past decade. In this review we focus on several recent investigations which have revealed mechanistically distinct inhibitors of the classical pathway. Because the classical pathway is an important and specific mediator of various autoimmune and inflammatory disorders, in-depth knowledge of novel evasion mechanisms could direct future development of therapeutic anti-inflammatory molecules. PMID:27591336

Novel Evasion Mechanisms of the Classical Complement Pathway.

PubMed

Garcia, Brandon L; Zwarthoff, Seline A; Rooijakkers, Suzan H M; Geisbrecht, Brian V

2016-09-15

Complement is a network of soluble and cell surface-associated proteins that gives rise to a self-amplifying, yet tightly regulated system with fundamental roles in immune surveillance and clearance. Complement becomes activated on the surface of nonself cells by one of three initiating mechanisms known as the classical, lectin, and alternative pathways. Evasion of complement function is a hallmark of invasive pathogens and hematophagous organisms. Although many complement-inhibition strategies hinge on hijacking activities of endogenous complement regulatory proteins, an increasing number of uniquely evolved evasion molecules have been discovered over the past decade. In this review, we focus on several recent investigations that revealed mechanistically distinct inhibitors of the classical pathway. Because the classical pathway is an important and specific mediator of various autoimmune and inflammatory disorders, in-depth knowledge of novel evasion mechanisms could direct future development of therapeutic anti-inflammatory molecules. Copyright © 2016 by The American Association of Immunologists, Inc.

Phenotypic characterization of ten methanol oxidation (Mox) mutant classes in methylobacterium AM1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nunn, D.N.; Lidstrom, M.E.

Twenty-five methanol oxidation mutants of the facultative methylotroph Methylobacterium strain AM1 have been characterized by complementation analysis and assigned to ten complementation groups, Mox A1,A2,A3 and B-H. We have characterized each of the mutants belonging to the ten Mox complementation groups by PMS-DCPIP dye linked methanol dehydrogenase activity, by methanol-dependent whole cell oxygen consumption, by the presence or absence of methanol dehydrogenase protein by SDS-polyacrylamide gels and Western blotting, by the absorption spectra of purified mutant methanol dehydrogenase proteins and by the presence or absence of the soluble cytochrome c proteins of Methylobacterium AM1. We propose functions for each ofmore » the genes deficient in the mutants of the ten Mox complementation groups. These functions include two linked genes that encode the methanol dehydrogenase structural protein and the soluble cytochrome c/sub L/, a gene encoding a secretion function essential for the synthesis and export of methanol dehydrogenase and cytochrome c/sub L/, three gene functions responsible for the proper association of the PQQ prosthetic group with the methanol dehydrogenase apoprotein and four positive regulatory gene functions controlling the expression of the ability to oxidize methanol. 24 refs., 5 figs., 2 tabs.« less

Secreted Amyloid β-Proteins in a Cell Culture Model Include N-Terminally Extended Peptides That Impair Synaptic Plasticity

PubMed Central

2014-01-01

Evidence for a central role of amyloid β-protein (Aβ) in the genesis of Alzheimer’s disease (AD) has led to advanced human trials of Aβ-lowering agents. The “amyloid hypothesis” of AD postulates deleterious effects of small, soluble forms of Aβ on synaptic form and function. Because selectively targeting synaptotoxic forms of soluble Aβ could be therapeutically advantageous, it is important to understand the full range of soluble Aβ derivatives. We previously described a Chinese hamster ovary (CHO) cell line (7PA2 cells) that stably expresses mutant human amyloid precursor protein (APP). Here, we extend this work by purifying an sodium dodecyl sulfate (SDS)-stable, ∼8 kDa Aβ species from the 7PA2 medium. Mass spectrometry confirmed its identity as a noncovalently bonded Aβ40 homodimer that impaired hippocampal long-term potentiation (LTP) in vivo. We further report the detection of Aβ-containing fragments of APP in the 7PA2 medium that extend N-terminal from Asp1 of Aβ. These N-terminally extended Aβ-containing monomeric fragments are distinct from soluble Aβ oligomers formed from Aβ1-40/42 monomers and are bioactive synaptotoxins secreted by 7PA2 cells. Importantly, decreasing β-secretase processing of APP elevated these alternative synaptotoxic APP fragments. We conclude that certain synaptotoxic Aβ-containing species can arise from APP processing events N-terminal to the classical β-secretase cleavage site. PMID:24840308

[Complement deficiencies and meningococcal disease in The Netherlands].

PubMed

Swart, A G; Fijen, C A; te Bulte, M T; Daha, M R; Dankert, J; Kuijper, E J

1993-06-05

To determine the prevalence of complement system deficiencies in patients who have survived a Neisseria meningitidis infection. Retrospective. Reference laboratory for bacterial meningitis of the University of Amsterdam and the National Institute of Public Health and Environmental Protection. Out of the files of the laboratory 187 patients who had experienced a meningococcal infection in the Netherlands between 1959-1990 were selected in two groups according to the infecting bacterial strain: 97 patients with a serogroup X, Y, Z, W135, 29E, or non-groupable strains and 90 patients with an infection due to serogroup A or C. The patients were asked for their cooperation by their family doctor and one of us visited the patients at home to take blood samples. The complement activity was studied with a haemolysis in gel test and with an assay of haemolytic activity in free solution. Complement deficiency was present in 18% of the 187 patients who had experienced a meningococcal infection. The highest prevalence was found in patients older than 10 years who had developed infections due to serogroups X, Y, W135, or non-groupable strains (45%). Of the patients with a serogroup A or C infection, 3% had an complement deficiency. Of the complement deficiencies, 42% concerned a component of the alternative pathway, 12% a deficiency of C3, and 46% a component of the terminal route. The most commonly found deficiencies were properdin deficiency (39%) and C8 deficiency (18%). 30% of the complement deficient patients reported other family members having experienced meningitis. Recurrent meningitis was only observed in patients with terminal route deficiencies. We recommend that patients with a meningococcal infection due to serogroups X, Y, W135 or non-groupable strains should be screened for complement deficiency.

Mapping the Complement Factor H-Related Protein 1 (CFHR1):C3b/C3d Interactions

PubMed Central

Laskowski, Jennifer; Thurman, Joshua M.; Hageman, Gregory S.; Holers, V. Michael

2016-01-01

Complement factor H-related protein 1 (CFHR1) is a complement regulator which has been reported to regulate complement by blocking C5 convertase activity and interfering with C5b surface association. CFHR1 also competes with complement factor H (CFH) for binding to C3b, and may act as an antagonist of CFH-directed regulation on cell surfaces. We have employed site-directed mutagenesis in conjunction with ELISA-based and functional assays to isolate the binding interaction that CFHR1 undertakes with complement components C3b and C3d to a single shared interface. The C3b/C3d:CFHR1 interface is identical to that which occurs between the two C-terminal domains (SCR19-20) of CFH and C3b. Moreover, we have been able to corroborate that dimerization of CFHR1 is necessary for this molecule to bind effectively to C3b and C3d, or compete with CFH. Finally, we have established that CFHR1 competes with complement factor H-like protein 1 (CFHL-1) for binding to C3b. CFHL-1 is a CFH gene splice variant, which is almost identical to the N-terminal 7 domains of CFH (SCR1-7). CFHR1, therefore, not only competes with the C-terminus of CFH for binding to C3b, but also sterically blocks the interaction that the N-terminus of CFH undertakes with C3b, and which is required for CFH-regulation. PMID:27814381

Control of total GFP expression by alterations to the 3′ region nucleotide sequence

PubMed Central

2013-01-01

Background Previously, we distinguished the Escherichia coli type II cytoplasmic membrane translocation pathways of Tat, Yid, and Sec for unfolded and folded soluble target proteins. The translocation of folded protein to the periplasm for soluble expression via the Tat pathway was controlled by an N-terminal hydrophilic leader sequence. In this study, we investigated the effect of the hydrophilic C-terminal end and its nucleotide sequence on total and soluble protein expression. Results The native hydrophilic C-terminal end of GFP was obtained by deleting the C-terminal peptide LeuGlu-6×His, derived from pET22b(+). The corresponding clones induced total and soluble GFP expression that was either slightly increased or dramatically reduced, apparently through reconstruction of the nucleotide sequence around the stop codon in the 3′ region. In the expression-induced clones, the hydrophilic C-terminus showed increased Tat pathway specificity for soluble expression. However, in the expression-reduced clone, after analyzing the role of the 5′ poly(A) coding sequence with a substituted synonymous codon, we proved that the longer 5′ poly(A) coding sequence interacted with the reconstructed 3′ region nucleotide sequence to create a new mRNA tertiary structure between the 5′ and 3′ regions, which resulted in reduced total GFP expression. Further, to recover the reduced expression by changing the 3′ nucleotide sequence, after replacing selected C-terminal 5′ codons and the stop codon in the ORF with synonymous codons, total GFP expression in most of the clones was recovered to the undeleted control level. The insertion of trinucleotides after the stop codon in the 3′-UTR recovered or reduced total GFP expression. RT-PCR revealed that the level of total protein expression was controlled by changes in translational or transcriptional regulation, which were induced or reduced by the substitution or insertion of 3′ region nucleotides. Conclusions We found that the hydrophilic C-terminal end of GFP increased Tat pathway specificity and that the 3′ nucleotide sequence played an important role in total protein expression through translational and transcriptional regulation. These findings may be useful for efficiently producing recombinant proteins as well as for potentially controlling the expression level of specific genes in the body for therapeutic purposes. PMID:23834827

Neutralization of soluble, synaptotoxic amyloid β species by antibodies is epitope specific.

PubMed

Zago, Wagner; Buttini, Manuel; Comery, Thomas A; Nishioka, Christopher; Gardai, Shyra J; Seubert, Peter; Games, Dora; Bard, Frédérique; Schenk, Dale; Kinney, Gene G

2012-02-22

Several anti-amyloid β (Aβ) antibodies are under evaluation for the treatment of Alzheimer's disease (AD). Clinical studies using the N-terminal-directed anti-Aβ antibody bapineuzumab have demonstrated reduced brain PET-Pittsburg-B signals, suggesting the reduction of Aβ plaques, and reduced levels of total and phosphorylated tau protein in the CSF of treated AD patients. Preclinical studies using 3D6 (the murine form of bapineuzumab) have demonstrated resolution of Aβ plaque and vascular burdens, neuritic dystrophy, and preservation of synaptic density in the transgenic APP mouse models. In contrast, few studies have evaluated the direct interaction of this antibody with synaptotoxic soluble Aβ species. In the current report, we demonstrated that 3D6 binds to soluble, synaptotoxic assemblies of Aβ(1-42) and prevents multiple downstream functional consequences in rat hippocampal neurons including changes in glutamate AMPA receptor trafficking, AD-type tau phosphorylation, and loss of dendritic spines. In vivo, we further demonstrated that 3D6 prevents synaptic loss and acutely reverses the behavioral deficit in the contextual fear conditioning task in transgenic mouse models of AD, two endpoints thought to be linked to synaptotoxic soluble Aβ moieties. Importantly C-terminal anti-Aβ antibodies were ineffective on these endpoints. These results, taken with prior studies, suggest that N-terminal anti-Aβ antibodies effectively interact with both soluble and insoluble forms of Aβ and therefore appear particularly well suited for testing the Aβ hypothesis of AD.

BINDING OF SOLUBLE IMMUNE COMPLEXES TO HUMAN LYMPHOBLASTOID CELLS

PubMed Central

Theofilopoulos, Argyrios N.; Dixon, Frank J.; Bokisch, Viktor A.

1974-01-01

In the present work we studied the expression of membrane-bound Ig (MBIg) as well as receptors for IgG Fc and complement on nine human lymphoblastoid cell lines. When MBIg and receptors for IgG Fc were compared, four categories of cell lines could be distinguished: (a) cell lines having both MBIg and receptors for IgG Fc, (b) cell lines having MBIg but lacking receptors for IgG Fc, (c) cell lines lacking MBIg but having receptors for IgG Fc, and (d) cell lines lacking both MBIg and receptors for IgG Fc. Two types of receptors for complement could be detected on the cell lines studied, one for C3-C3b and one for C3d. When sensitized red cells carrying C3b or C3d were used for rosette tests, three categories of cell lines could be distinguished: (a) cell lines having receptors for C3b and C3d, (b) cell lines having receptors only for C3d and (c) cell lines lacking both receptors. However, when a more sensitive immunofluorescent method was used instead of the rosette technique, it was found that cell lines unable to form rosettes with EAC1423bhu were able to bind soluble C3 or C3b which indicated the presence of these receptors on the cell surface. Inhibition experiments showed that receptors for C3-C3b and receptors for C3d are distinct and that receptors for C3-C3b and C3d are different from receptors for IgG Fc. A cell line (Raji) without MBIg but with receptors for IgG Fc, C3-C3b, and C3d was selected for use in studying the binding mechanism of soluble immune complexes to cell surface membrane. Aggregated human gamma globulin was used in place of immune complexes. Immune complexes containing complement bind to Raji cells only via receptors for complement, namely receptors for C3-C3b and C3d. Binding of immune complexes containing complement to cells is much greater than that of complexes without complement. Immune complexes bound to cells via receptors for complement can be partially released from the cell surface by addition of normal human serum as well as isolated human C3 or C3b. We postulate that such release is due to competition of immune complex bound C3b and free C3 or C3b for the receptors on Raji cells. PMID:4139225

The 26S Proteasome Degrades the Soluble but Not the Fibrillar Form of the Yeast Prion Ure2p In Vitro

PubMed Central

Wang, Kai; Redeker, Virginie; Madiona, Karine; Melki, Ronald; Kabani, Mehdi

2015-01-01

Yeast prions are self-perpetuating protein aggregates that cause heritable and transmissible phenotypic traits. Among these, [PSI +] and [URE3] stand out as the most studied yeast prions, and result from the self-assembly of the translation terminator Sup35p and the nitrogen catabolism regulator Ure2p, respectively, into insoluble fibrillar aggregates. Protein quality control systems are well known to govern the formation, propagation and transmission of these prions. However, little is known about the implication of the cellular proteolytic machineries in their turnover. We previously showed that the 26S proteasome degrades both the soluble and fibrillar forms of Sup35p and affects [PSI +] propagation. Here, we show that soluble native Ure2p is degraded by the proteasome in an ubiquitin-independent manner. Proteasomal degradation of Ure2p yields amyloidogenic N-terminal peptides and a C-terminal resistant fragment. In contrast to Sup35p, fibrillar Ure2p resists proteasomal degradation. Thus, structural variability within prions may dictate their ability to be degraded by the cellular proteolytic systems. PMID:26115123

Expression of recombinant CD59 with an N-terminal peptide epitope facilitates analysis of residues contributing to its complement-inhibitory function.

PubMed

Zhou, Q; Zhao, J; Hüsler, T; Sims, P J

1996-10-01

CD59 is a plasma membrane-anchored glycoprotein that serves to protect human cells from lysis by the C5b-9 complex of complement. The immunodominant epitopes of CD59 are known to be sensitive to disruption of native tertiary structure, complicating immunological measurement of expressed mutant constructs for structure function analysis. In order to quantify cell-surface expression of wild-type and mutant forms of this complement inhibitor, independent of CD59 antigen, an 11-residue peptide (TAG) recognized by monoclonal antibody (mAb) 9E10 was inserted before the N-terminal codon (L1) of mature CD59, in a pcDNA3 expression plasmid. SV-T2 cells were transfected with this plasmid, yielding cell lines expressing 0 to > 10(5) CD59/cell. The TAG-CD59 fusion protein was confirmed to be GPI-anchored, N-glycosylated and showed identical complement-inhibitory function to wild-type CD59, lacking the TAG peptide sequence. Using this construct, the contribution of each of four surface-localized aromatic residues (4Y, 47F, 61Y, and 62Y) to CD59's complement-inhibitory function was examined. These assays revealed normal surface expression with complete loss of complement-inhibitory function in the 4Y --> S, 47F --> G and 61Y --> S mutants. By contrast, 62Y --> S mutants retained approximately 40% of function of wild-type CD59. These studies confirmed the utility of the TAG-CD59 construct for quantifying CD59 surface expression and activity, and implicate surface aromatic residues 4Y, 47F, 61Y and 62Y as essential to maintenance of CD59's normal complement-regulatory function.

Kinetics of the isothermal decomposition of zirconium hydride: terminal solid solubility for precipitation and dissolution

NASA Astrophysics Data System (ADS)

Denisov, E. A.; Kompaniets, T. N.; Voyt, A. P.

2018-05-01

The hydrogen permeation technique in the surface-limited regime (SLR) was first used to study the isothermal decomposition of zirconium hydride. It is shown that under isothermal conditions, the hydrogen terminal solid solubility in the α-phase for hydride precipitation (TSSp) and dissolution (TSSd) differ only by 6%, in contrast to the 20-30% indicated in the available literature. It is demonstrated that even the minimum heating/cooling rate (1 C/min) used in the traditional methods of studying TSSp and TSSd is too high to exclude the effect of kinetics on the results obtained.

Systemic reduction of soluble complement receptor II/CD21 during pregnancy to levels reminiscent of autoimmune disease.

PubMed

Masilamani, Madhan; Rajasekaran, Narendiran; Singh, Anjana; Low, Hui-Zhi; Albus, Kerstin; Anders, Swantje; Behne, Frank; Eiermann, Peter; König, Katharina; Mindnich, Clarissa; Ribarska, Teodora; Illges, Harald

2008-09-01

Complement receptor type II/CD21 is the functional receptor for complement fragments such as C3d, iC3b and the Epstein Barr Virus. A soluble form of CD21 (sCD21) is shed from lymphocytes surface and is able to bind to its ligands found in the plasma. The amount of sCD21 in serum may modulate immunity as the plasma levels are correlated with autoimmune conditions, such as Systemic Lupus Erythematosus, Rheumatoid Arthritis and Sjoegren's Syndrome. Because of the fact that pregnancy may lead to remission of autoimmune diseases we determined the serum levels of sCD21 during pregnancy and postpartum. The serum sCD21 levels during pregnancy are significantly lower as compared to that of the healthy controls. There were no significant differences in sCD21 levels between the mother and the cord blood also immediately after parturition. Restoration of sCD21 levels to normal values takes between 6 weeks and 1 year after childbirth. Our study indicates that CD21-shedding is affected during pregnancy comparable to that of autoimmunity.

Solubility and Diffusivity: Important Metrics in the Search for the Root Cause of Light- and Elevated Temperature-Induced Degradation

DOE PAGES

Jensen, Mallory A.; Morishige, Ashley E.; Chakraborty, Sagnik; ...

2018-02-02

Light- and elevated temperature-induced degradation (LeTID) is a detrimental effect observed under operating conditions in p-type multicrystalline silicon (mc-Si) solar cells. In this paper, we employ synchrotron-based techniques to study the dissolution of precipitates due to different firing processes at grain boundaries in LeTID-affected mc-Si. The synchrotron measurements show clear dissolution of collocated metal precipitates during firing. We compare our observations with degradation behavior in the same wafers. The experimental results are complemented with process simulations to provide insight into the change in bulk point defect concentration due to firing. Several studies have proposed that LeTID is caused by metal-richmore » precipitate dissolution during contact firing, and we find that the solubility and diffusivity are promising screening metrics to identify metals that are compatible with this hypothesis. While slower and less soluble elements (e.g., Fe and Cr) are not compatible according to our simulations, the point defect concentrations of faster and more soluble elements (e.g., Cu and Ni) increase after a high-temperature firing process, primarily due to emitter segregation rather than precipitate dissolution. Finally, these results are a useful complement to lifetime spectroscopy techniques, and can be used to evaluate additional candidates in the search for the root cause of LeTID.« less

Solubility and Diffusivity: Important Metrics in the Search for the Root Cause of Light- and Elevated Temperature-Induced Degradation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jensen, Mallory A.; Morishige, Ashley E.; Chakraborty, Sagnik

Light- and elevated temperature-induced degradation (LeTID) is a detrimental effect observed under operating conditions in p-type multicrystalline silicon (mc-Si) solar cells. In this paper, we employ synchrotron-based techniques to study the dissolution of precipitates due to different firing processes at grain boundaries in LeTID-affected mc-Si. The synchrotron measurements show clear dissolution of collocated metal precipitates during firing. We compare our observations with degradation behavior in the same wafers. The experimental results are complemented with process simulations to provide insight into the change in bulk point defect concentration due to firing. Several studies have proposed that LeTID is caused by metal-richmore » precipitate dissolution during contact firing, and we find that the solubility and diffusivity are promising screening metrics to identify metals that are compatible with this hypothesis. While slower and less soluble elements (e.g., Fe and Cr) are not compatible according to our simulations, the point defect concentrations of faster and more soluble elements (e.g., Cu and Ni) increase after a high-temperature firing process, primarily due to emitter segregation rather than precipitate dissolution. Finally, these results are a useful complement to lifetime spectroscopy techniques, and can be used to evaluate additional candidates in the search for the root cause of LeTID.« less

Bioluminescent indicators for Ca2+ based on split Renilla luciferase complementation in living cells.

PubMed

Kaihara, Asami; Umezawa, Yoshio; Furukawa, Tetsushi

2008-01-01

Genetically encoded bioluminescent indicators for intracellular Ca2+ are described here with CaM-M13 interaction-induced complementation of split Renilla luciferase. The Ca2+-induced interaction between CaM and M13 leads to complementation of the N- and C-terminal halves of split Renilla luciferase in living cells. This intramolecular interaction results in the spontaneous and simultaneous emission of bioluminescence split Renilla luciferase. This is how intracellular Ca2+ is illuminated with the intramolecular complementation of split Renilla luciferase. The Ca2+-dependent spontaneous and simultaneous emission of bioluminescence promises to reveal Ca2+ dynamics in living cells, and also in vivo using the present indicators.

Complementation Studies of Bacteriophage λ O Amber Mutants by Allelic Forms of O Expressed from Plasmid, and O-P Interaction Phenotypes.

PubMed

Hayes, Sidney; Rajamanickam, Karthic; Hayes, Connie

2018-04-05

λ genes O and P are required for replication initiation from the bacteriophage λ origin site, ori λ, located within gene O . Questions have persisted for years about whether O-defects can indeed be complemented in trans . We show the effect of original null mutations in O and the influence of four origin mutations (three are in-frame deletions and one is a point mutation) on complementation. This is the first demonstration that O proteins with internal deletions can complement for O activity, and that expression of the N-terminal portion of gene P can completely prevent O complementation. We show that O-P co-expression can limit the lethal effect of P on cell growth. We explore the influence of the contiguous small RNA OOP on O complementation and P-lethality.

Expression, purification, crystallization and structure determination of the N terminal domain of Fhb, a factor H binding protein from Streptococcus suis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Chunmao; Yu, You; Yang, Maojun, E-mail: maojunyang@tsinghua.edu.cn

2015-10-23

Fhb is a surface virulence protein from Streptococcus suis, which could aid bacterial evasion of host innate immune defense by recruiting complement regulator factor H to inactivate C3b deposited on bacterial surface in blood. Here we successfully expressed and purified the N terminal domain of Fhb (N-Fhb) and obtained crystals of the N-Fhb by sitting-drop vapor diffusion method with a resolution of 1.50 Å. The crystals belong to space group C2 with unit cell parameters a = 127.1 Å, b = 77.3 Å, c = 131.6 Å, α = 90°, β = 115.9°, γ = 90°. The structure of N-Fhb was determined by SAD method and the core structure of N-Fhb is a β sandwich. Wemore » speculated that binding of Fhb to human factor H may be mainly mediated by surface amino acids with negative charges. - Highlights: • We expressed N-Fhb as the soluble protein in Escherichia coli. • Crystals of N-Fhb were grown by sitting drop vapor diffusion method. • Crystals of N-Fhb could diffracted to 1.5 Å. • The core structure of N-Fhb was a β sandwich. • A part of the surface of N-Fhb was rich with negative charges.« less

Functionality and Usability Assessment of the Complement Generation Tool (CGT): A Preliminary Review (Evaluation de la Fonctionnalite et de la Convivialite de Generation de Complements (CGT) - Examen Preliminaire)

DTIC Science & Technology

2011-04-01

Her Majesty the Queen in Right of Canada, as represented by the Minister of National Defence, 2011 © Sa Majesté la Reine (en droit du Canada), telle...l’utilité et la convivialité du CGT en ce qui concerne la détermination des besoins de main- d’œuvre au cours de la phase initiale de la conception d’une...lesquelles il a été conçu. L’évaluation a confirmé l’utilité du CGT en ce qui concerne la détermination des besoins de main-d’œuvre au cours de la phase

Subcutaneous injection of water-soluble multi-walled carbon nanotubes in tumor-bearing mice boosts the host immune activity

NASA Astrophysics Data System (ADS)

Meng, Jie; Yang, Man; Jia, Fumin; Kong, Hua; Zhang, Weiqi; Wang, Chaoying; Xing, Jianmin; Xie, Sishen; Xu, Haiyan

2010-04-01

The immunological responses induced by oxidized water-soluble multi-walled carbon nanotubes on a hepatocarcinoma tumor-bearing mice model via a local administration of subcutaneous injection were investigated. Experimental results show that the subcutaneously injected carbon nanotubes induced significant activation of the complement system, promoted inflammatory cytokines' production and stimulated macrophages' phagocytosis and activation. All of these responses increased the general activity of the host immune system and inhibited the progression of tumor growth.

Phenotypic characterization of 10 methanol oxidation mutant classes in Methylobacterium sp. strain AM1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nunn, D.N.; Lidstrom, M.E.

Twenty-five methanol oxidation mutants of the facultative methylotroph Methylobacterium sp. strain AM1 have been characterized by complementation analysis and assigned to 10 complementation groups, Mox A1, A2, A3, and B through H. In this study we have characterized each of the mutants belonging to the 10 Mox complementation groups for the following criteria: (i) phenazine methosulfate-dichlorophenolindophenol dye-linked methanol dehydrogenase activity; (ii) methanol-dependent whole-cell oxygen consumption; (iii) the presence or absence of methanol dehydrogenase protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting; (iv) the absorption spectra of purified mutant methanol dehydrogenase proteins; and (v) the presence or absence ofmore » the soluble cytochrome c proteins of Methylobacterium sp. strain AM1, as determined by reduced-oxidized difference spectra and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this information, we have proposed functions for each of the genes deficient in the mutants of the 10 Mox complementation groups. These proposed gene functions include two linked genes that encode the methanol dehydrogenase structural protein and the soluble cytochrome c/sub L/, a gene encoding a secretion function essential for the synthesis and export of methanol dehydrogenase and cytochrome c/sub L/, three gene functions responsible for the proper association of the pyrrolo-quinoline quinone prosthetic group with the methanol dehydrogenase apoprotein, and four positive regulatory gene functions controlling the expression of the ability to oxidize methanol.« less

The function of the yeast molecular chaperone Sse1 is mechanistically distinct from the closely related hsp70 family.

PubMed

Shaner, Lance; Trott, Amy; Goeckeler, Jennifer L; Brodsky, Jeffrey L; Morano, Kevin A

2004-05-21

The Sse1/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a C-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an N-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Delta yeast. Surprisingly, all mutants predicted to abolish ATP hydrolysis (D8N, K69Q, D174N, D203N) complemented the temperature sensitivity of sse1Delta and lethality of sse1Deltasse2Delta cells, whereas mutations in predicted ATP binding residues (G205D, G233D) were non-functional. Complementation ability correlated well with ATP binding assessed in vitro. The extreme C terminus of the Hsp70 family is required for substrate targeting and heterocomplex formation with other chaperones, but mutant Sse1 proteins with a truncation of up to 44 C-terminal residues that were not included in the PBD were active. Remarkably, the two domains of Sse1, when expressed in trans, functionally complement the sse1Delta growth phenotype and interact by coimmunoprecipitation analysis. In addition, a functional PBD was required to stabilize the Sse1 ATPase domain, and stabilization also occurred in trans. These data represent the first structure-function analysis of this abundant but ill defined chaperone, and establish several novel aspects of Sse1/Hsp110 function relative to Hsp70.

Complement factor H in host defense and immune evasion.

PubMed

Parente, Raffaella; Clark, Simon J; Inforzato, Antonio; Day, Anthony J

2017-05-01

Complement is the major humoral component of the innate immune system. It recognizes pathogen- and damage-associated molecular patterns, and initiates the immune response in coordination with innate and adaptive immunity. When activated, the complement system unleashes powerful cytotoxic and inflammatory mechanisms, and thus its tight control is crucial to prevent damage to host tissues and allow restoration of immune homeostasis. Factor H is the major soluble inhibitor of complement, where its binding to self markers (i.e., particular glycan structures) prevents complement activation and amplification on host surfaces. Not surprisingly, mutations and polymorphisms that affect recognition of self by factor H are associated with diseases of complement dysregulation, such as age-related macular degeneration and atypical haemolytic uremic syndrome. In addition, pathogens (i.e., non-self) and cancer cells (i.e., altered-self) can hijack factor H to evade the immune response. Here we review recent (and not so recent) literature on the structure and function of factor H, including the emerging roles of this protein in the pathophysiology of infectious diseases and cancer.

Comparative Characterization of Complete and Truncated Forms of Lactobacillus amylovorus α-Amylase and Role of the C-Terminal Direct Repeats in Raw-Starch Binding

PubMed Central

Rodriguez Sanoja, R.; Morlon-Guyot, J.; Jore, J.; Pintado, J.; Juge, N.; Guyot, J. P.

2000-01-01

Two constructs derived from the α-amylase gene (amyA) of Lactobacillus amylovorus were expressed in Lactobacillus plantarum, and their expression products were purified, characterized, and compared. These products correspond to the complete (AmyA) and truncated (AmyAΔ) forms of α-amylase; AmyAΔ lacks the 66-kDa carboxyl-terminal direct-repeating-unit region. AmyA and AmyAΔ exhibit similar amylase activities towards a range of soluble substrates (amylose, amylopectin and α-cyclodextrin, and soluble starch). The specific activities of the enzymes towards soluble starch are similar, but the KM and Vmax values of AmyAΔ were slightly higher than those of AmyA, whereas the thermal stability of AmyAΔ was lower than that of AmyA. In contrast to AmyA, AmyAΔ is unable to bind to β-cyclodextrin and is only weakly active towards glycogen. More striking is the fact that AmyAΔ cannot bind or hydrolyze raw starch, demonstrating that the carboxyl-terminal repeating-unit domain of AmyA is required for raw-starch binding activity. PMID:10919790

A Dual Protease Approach for Expression and Affinity Purification of Recombinant Proteins

PubMed Central

Raran-Kurussi, Sreejith; Waugh, David S.

2016-01-01

We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to “stick” to its fusion partners during affinity purification. PMID:27105777

A dual protease approach for expression and affinity purification of recombinant proteins.

PubMed

Raran-Kurussi, Sreejith; Waugh, David S

2016-07-01

We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to "stick" to its fusion partners during affinity purification. Published by Elsevier Inc.

Complement system biomarkers in epilepsy.

PubMed

Kopczynska, Maja; Zelek, Wioleta M; Vespa, Simone; Touchard, Samuel; Wardle, Mark; Loveless, Samantha; Thomas, Rhys H; Hamandi, Khalid; Morgan, B Paul

2018-05-24

To explore whether complement dysregulation occurs in a routinely recruited clinical cohort of epilepsy patients, and whether complement biomarkers have potential to be used as markers of disease severity and seizure control. Plasma samples from 157 epilepsy cases (106 with focal seizures, 46 generalised seizures, 5 unclassified) and 54 controls were analysed. Concentrations of 10 complement analytes (C1q, C3, C4, factor B [FB], terminal complement complex [TCC], iC3b, factor H [FH], Clusterin [Clu], Properdin, C1 Inhibitor [C1Inh] plus C-reactive protein [CRP]) were measured using enzyme linked immunosorbent assay (ELISA). Univariate and multivariate statistical analysis were used to test whether combinations of complement analytes were predictive of epilepsy diagnoses and seizure occurrence. Correlation between number and type of anti-epileptic drugs (AED) and complement analytes was also performed. We found: CONCLUSION: This study adds to evidence implicating complement in pathogenesis of epilepsy and may allow the development of better therapeutics and prognostic markers in the future. Replication in a larger sample set is needed to validate the findings of the study. Copyright © 2018. Published by Elsevier Ltd.

Selection for water-soluble carbohydrate accumulation and investigation of genetic × environment interactions in an elite wheat breeding population

USDA-ARS?s Scientific Manuscript database

The potential to increase the genetic capacity for water-soluble carbohydrate (WSC) accumulation is an opportunity to improve the drought tolerance capability of rainfed wheat varieties, particularly in Australia where terminal drought is a significant constraint to wheat production. A population of...

More than just immune evasion: Hijacking complement by Plasmodium falciparum.

PubMed

Schmidt, Christoph Q; Kennedy, Alexander T; Tham, Wai-Hong

2015-09-01

Malaria remains one of the world's deadliest diseases. Plasmodium falciparum is responsible for the most severe and lethal form of human malaria. P. falciparum's life cycle involves two obligate hosts: human and mosquito. From initial entry into these hosts, malaria parasites face the onslaught of the first line of host defence, the complement system. In this review, we discuss the complex interaction between complement and malaria infection in terms of hosts immune responses, parasite survival and pathogenesis of severe forms of malaria. We will focus on the role of complement receptor 1 and its associated polymorphisms in malaria immune complex clearance, as a mediator of parasite rosetting and as an entry receptor for P. falciparum invasion. Complement evasion strategies of P. falciparum parasites will also be highlighted. The sexual forms of the malaria parasites recruit the soluble human complement regulator Factor H to evade complement-mediated killing within the mosquito host. A novel evasion strategy is the deployment of parasite organelles to divert complement attack from infective blood stage parasites. Finally we outline the future challenge to understand the implications of these exploitation mechanisms in the interplay between successful infection of the host and pathogenesis observed in severe malaria. Copyright © 2015 Elsevier Ltd. All rights reserved.

NH2-Terminal Residues of Neurospora crassa Proteins

PubMed Central

Rho, Hyune Mo; DeBusk, A. Gib

1971-01-01

The NH2-terminal amino acid composition of the soluble and ribosomal proteins from Neurospora crassa mycelia and conidia was determined by the dinitrophenyl method. A nonrandom distribution of NH2-terminal amino acids was observed in the complex protein mixtures. Glycine, alanine, and serine accounted for 75% of the NH2-terminal amino acids, and glycine appeared most frequently in mature proteins of mycelia. The appearance of phenylalanine as one of the major NH2-termini in crude conidial fraction suggests that the composition of proteins may vary in different developmental stages. PMID:5095291

Plasma Biomarkers Reflecting Profibrotic Processes in Heart Failure With a Preserved Ejection Fraction

PubMed Central

Zile, Michael R.; Jhund, Pardeep S.; Baicu, Catalin F.; Claggett, Brian L.; Pieske, Burkert; Voors, Adriaan A.; Prescott, Margaret F.; Shi, Victor; Lefkowitz, Martin; McMurray, John J.V.; Solomon, Scott D.

2017-01-01

Background Heart failure with preserved ejection fraction is a clinical syndrome that has been associated with changes in the extracellular matrix. The purpose of this study was to determine whether profibrotic biomarkers accurately reflect the presence and severity of disease and underlying pathophysiology and modify response to therapy in patients with heart failure with preserved ejection fraction. Methods and Results Four biomarkers, soluble form of ST2 (an interleukin-1 receptor family member), galectin-3, matrix metalloproteinase-2, and collagen III N-terminal propeptide were measured in the Prospective Comparison of ARNI With ARB on Management of Heart Failure With Preserved Ejection Fraction (PARAMOUNT) trial at baseline, 12 and 36 weeks after randomization to valsartan or LCZ696. We examined the relationship between baseline biomarkers, demographic and echocardiographic characteristics, change in primary (change in N-terminal pro B-type natriuretic peptide) and secondary (change in left atrial volume) end points. The median (interquartile range) value for soluble form of ST2 (33 [24.6–48.1] ng/mL) and galectin 3 (17.8 [14.1–22.8] ng/mL) were higher, and for matrix metalloproteinase-2 (188 [155.5–230.6] ng/mL) lower, than in previously published referent controls; collagen III N-terminal propeptide (5.6 [4.3–6.9] ng/mL) was similar to referent control values. All 4 biomarkers correlated with severity of disease as indicated by N-terminal pro B-type natriuretic peptide, E/E′, and left atrial volume. Baseline biomarkers did not modify the response to LCZ696 for lowering N-terminal pro B-type natriuretic peptide; however, left atrial volume reduction varied by baseline level of soluble form of ST2 and galectin 3; patients with values less than the observed median (<33 ng/mL soluble form of ST2 and <17.8 ng/mL galectin 3) had reduction in left atrial volume, those above median did not. Although LCZ696 reduced N-terminal pro B-type natriuretic peptide, levels of the other 4 biomarkers were not affected over time. Conclusions In patients with heart failure with preserved ejection fraction, biomarkers that reflect collagen homeostasis correlated with the presence and severity of disease and underlying pathophysiology, and may modify the structural response to treatment. PMID:26754625

Complement-Mediated Enhancement of Monocyte Adhesion to Endothelial Cells by HLA Antibodies, and Blockade by a Specific Inhibitor of the Classical Complement Cascade, TNT003

PubMed Central

Valenzuela, Nicole M.; Thomas, Kimberly A.; Mulder, Arend; Parry, Graham C.; Panicker, Sandip; Reed, Elaine F.

2017-01-01

Background Antibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling. Methods Primary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003). Results Treatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement. Conclusions Despite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR. PMID:28640789

Mutations in the conserved carboxy-terminal hydrophobic region of glycoprotein gB affect infectivity of herpes simplex virus.

PubMed

Wanas, E; Efler, S; Ghosh, K; Ghosh, H P

1999-12-01

Glycoprotein gB is the most highly conserved glycoprotein in the herpesvirus family and plays a critical role in virus entry and fusion. Glycoprotein gB of herpes simplex virus type 1 contains a hydrophobic stretch of 69 aa near the carboxy terminus that is essential for its biological activity. To determine the role(s) of specific amino acids in the carboxy-terminal hydrophobic region, a number of amino acids were mutagenized that are highly conserved in this region within the gB homologues of the family HERPESVIRIDAE: Three conserved residues in the membrane anchor domain, namely A786, A790 and A791, as well as amino acids G743, G746, G766, G770 and P774, that are non-variant in Herpesviridae, were mutagenized. The ability of the mutant proteins to rescue the infectivity of the gB-null virus, K082, in trans was measured by a complementation assay. All of the mutant proteins formed dimers and were incorporated in virion particles produced in the complementation assay. Mutants G746N, G766N, F770S and P774L showed negligible complementation of K082, whereas mutant G743R showed a reduced activity. Virion particles containing these four mutant glycoproteins also showed a markedly reduced rate of entry compared to the wild-type. The results suggest that non-variant residues in the carboxy-terminal hydrophobic region of the gB protein may be important in virus infectivity.

Eculizumab in paroxysmal nocturnal hemoglobinuria with Budd-Chiari syndrome progressing despite anticoagulation

PubMed Central

2012-01-01

Paroxysmal nocturnal hemoglobinuria (PNH) is a progressive, life-threatening disorder characterized by chronic intravascular hemolysis caused by uncontrolled complement activation. Hepatic vein thrombosis (Budd-Chiari syndrome) is common in PNH patients. This case report describes the response to eculizumab (a humanized monoclonal antibody that inhibits terminal complement activation) in a 25-year-old male with progressive liver function deterioration despite standard anticoagulation therapy and transjugular intrahepatic porto-systemic shunt. The patient presented with anemia, severe thrombocytopenia, headache, abdominal pain, and distention. He was diagnosed with PNH, cerebral vein thrombosis, and Budd-Chiari syndrome. Despite adequate anticoagulation, diuretic administration, and placement of a transjugular shunt, additional thrombotic events and progressive liver damage were observed. Eculizumab therapy was initiated, resulting in rapid blockade of intravascular hemolysis, increased platelet counts, ascites resolution, and liver function recovery, all of which are presently sustained. Since starting eculizumab the patient has had no further thrombotic events and his quality of life has dramatically improved. This is the first report to confirm the role of complement-mediated injury in the progression of Budd-Chiari syndrome in a patient with PNH. This case shows that terminal complement blockade with eculizumab can reverse progressive thromboses and hepatic failure that is unresponsive to anticoagulation therapy and suggests that early initiation of eculizumab should be included in the therapeutic regimen of patients with PNH-related Budd-Chiari syndrome. PMID:23210433

Roles of N-glycans in the polymerization-dependent aggregation of mutant Ig-μ chains in the early secretory pathway.

PubMed

Giannone, Chiara; Fagioli, Claudio; Valetti, Caterina; Sitia, Roberto; Anelli, Tiziana

2017-02-03

The polymeric structure of secretory IgM allows efficient antigen binding and complement fixation. The available structural models place the N-glycans bound to asparagines 402 and 563 of Ig-μ chains within a densely packed core of native IgM. These glycans are found in the high mannose state also in secreted IgM, suggesting that polymerization hinders them to Golgi processing enzymes. Their absence alters polymerization. Here we investigate their role following the fate of aggregation-prone mutant μ chains lacking the Cμ1 domain (μ∆). Our data reveal that μ∆ lacking 563 glycans (μ∆5) form larger intracellular aggregates than μ∆ and are not secreted. Like μ∆, they sequester ERGIC-53, a lectin previously shown to promote polymerization. In contrast, μ∆ lacking 402 glycans (μ∆4) remain detergent soluble and accumulate in the ER, as does a double mutant devoid of both (μ∆4-5). These results suggest that the two C-terminal Ig-μ glycans shape the polymerization-dependent aggregation by engaging lectins and acting as spacers in the alignment of individual IgM subunits in native polymers.

Roles of N-glycans in the polymerization-dependent aggregation of mutant Ig-μ chains in the early secretory pathway

PubMed Central

Giannone, Chiara; Fagioli, Claudio; Valetti, Caterina; Sitia, Roberto; Anelli, Tiziana

2017-01-01

The polymeric structure of secretory IgM allows efficient antigen binding and complement fixation. The available structural models place the N-glycans bound to asparagines 402 and 563 of Ig-μ chains within a densely packed core of native IgM. These glycans are found in the high mannose state also in secreted IgM, suggesting that polymerization hinders them to Golgi processing enzymes. Their absence alters polymerization. Here we investigate their role following the fate of aggregation-prone mutant μ chains lacking the Cμ1 domain (μ∆). Our data reveal that μ∆ lacking 563 glycans (μ∆5) form larger intracellular aggregates than μ∆ and are not secreted. Like μ∆, they sequester ERGIC-53, a lectin previously shown to promote polymerization. In contrast, μ∆ lacking 402 glycans (μ∆4) remain detergent soluble and accumulate in the ER, as does a double mutant devoid of both (μ∆4–5). These results suggest that the two C-terminal Ig-μ glycans shape the polymerization-dependent aggregation by engaging lectins and acting as spacers in the alignment of individual IgM subunits in native polymers. PMID:28157181

The N-Terminal Residues 43 to 60 Form the Interface for Dopamine Mediated α-Synuclein Dimerisation

PubMed Central

Leong, Su Ling; Hinds, Mark G.; Connor, Andrea R.; Smith, David P.; Illes-Toth, Eva; Pham, Chi L. L.; Barnham, Kevin J.; Cappai, Roberto

2015-01-01

α-synuclein (α-syn) is a major component of the intracellular inclusions called Lewy bodies, which are a key pathological feature in the brains of Parkinson’s disease patients. The neurotransmitter dopamine (DA) inhibits the fibrillisation of α-syn into amyloid, and promotes α-syn aggregation into SDS-stable soluble oligomers. While this inhibition of amyloid formation requires the oxidation of both DA and the methionines in α-syn, the molecular basis for these processes is still unclear. This study sought to define the protein sequences required for the generation of oligomers. We tested N- (α-syn residues 43–140) and C-terminally (1–95) truncated α-syn, and found that similar to full-length protein both truncated species formed soluble DA:α-syn oligomers, albeit 1–95 had a different profile. Using nuclear magnetic resonance (NMR), and the N-terminally truncated α-syn 43–140 protein, we analysed the structural characteristics of the DA:α-syn 43–140 dimer and α-syn 43–140 monomer and found the dimerisation interface encompassed residues 43 to 60. Narrowing the interface to this small region will help define the mechanism by which DA mediates the formation of SDS-stable soluble DA:α-syn oligomers. PMID:25679387

EFFECT OF PROFLAVINE ON THE SYNTHESIS OF ADENOVIRUS, TYPE 5, AND ASSOCIATED SOLUBLE ANTIGENS

PubMed Central

Wilcox, Wesley C.; Ginsberg, Harold S.

1962-01-01

Wilcox, Wesley C. (University of Pennsylvania, Philadelphia) and Harold S. Ginsberg. Effect of proflavine on the synthesis of adenovirus, type 5, and associated soluble antigens. J. Bacteriol. 84:526–533. 1962.—The synthesis of type 5 adenovirus in HeLa cells was suppressed to a considerable extent by low concentrations of proflavine, an acridine dye. In comparison, the processes leading to the production of soluble complement-fixing antigens and toxin were less sensitive to the action of this chemical. Addition of proflavine to infected cells at different times during the virus growth cycle revealed that the processes leading to the synthesis of soluble antigens began prior to the first appearance of newly synthesized virus. This observation is compatible with the hypothesis that the soluble antigens may represent virus subunits or precursor materials. In addition, these data indicate that it is possible to interrupt the latter stages of the virus synthetic process by addition of proflavine late in the eclipse period. PMID:14000661

Host Soluble Mediators: Defying the Immunological Inertness of Aspergillus fumigatus Conidia.

PubMed

Wong, Sarah Sze Wah; Aimanianda, Vishukumar

2017-12-24

Aspergillus fumigatus produce airborne spores (conidia), which are inhaled in abundant quantity. In an immunocompromised population, the host immune system fails to clear the inhaled conidia, which then germinate and invade, leading to pulmonary aspergillosis. In an immunocompetent population, the inhaled conidia are efficiently cleared by the host immune system. Soluble mediators of the innate immunity, that involve the complement system, acute-phase proteins, antimicrobial peptides and cytokines, are often considered to play a complementary role in the defense of the fungal pathogen. In fact, the soluble mediators are essential in achieving an efficient clearance of the dormant conidia, which is the morphotype of the fungus upon inhalation by the host. Importantly, harnessing the host soluble mediators challenges the immunological inertness of the dormant conidia due to the presence of the rodlet and melanin layers. In the review, we summarized the major soluble mediators in the lung that are involved in the recognition of the dormant conidia. This knowledge is essential in the complete understanding of the immune defense against A. fumigatus .

Enhanced CDC of B cell chronic lymphocytic leukemia cells mediated by rituximab combined with a novel anti-complement factor H antibody.

PubMed

Winkler, Mark T; Bushey, Ryan T; Gottlin, Elizabeth B; Campa, Michael J; Guadalupe, Eross S; Volkheimer, Alicia D; Weinberg, J Brice; Patz, Edward F

2017-01-01

Rituximab therapy for B cell chronic lymphocytic leukemia (B-CLL) has met with mixed success. Among several factors to which resistance can be attributed is failure to activate complement dependent cytotoxicity (CDC) due to protective complement regulatory proteins, including the soluble regulator complement factor H (CFH). We hypothesized that rituximab killing of non-responsive B-CLL cells could be augmented by a novel human monoclonal antibody against CFH. The B cells from 11 patients with B-CLL were tested ex vivo in CDC assays with combinations of CFH monoclonal antibody, rituximab, and a negative control antibody. CDC of rituximab non-responsive malignant B cells from CLL patients could in some cases be augmented by the CFH monoclonal antibody. Antibody-mediated cytotoxicity of cells was dependent upon functional complement. In one case where B-CLL cells were refractory to CDC by the combination of rituximab plus CFH monoclonal antibody, additionally neutralizing the membrane complement regulatory protein CD59 allowed CDC to occur. Inhibiting CDC regulatory proteins such as CFH holds promise for overcoming resistance to rituximab therapy in B-CLL.

Systemic Lupus Erythematosus and Deficiencies of Early Components of the Complement Classical Pathway

PubMed Central

Macedo, Ana Catarina Lunz; Isaac, Lourdes

2016-01-01

The complement system plays an important role in the innate and acquired immune response against pathogens. It consists of more than 30 proteins found in soluble form or attached to cell membranes. Most complement proteins circulate in inactive forms and can be sequentially activated by the classical, alternative, or lectin pathways. Biological functions, such as opsonization, removal of apoptotic cells, adjuvant function, activation of B lymphocytes, degranulation of mast cells and basophils, and solubilization and clearance of immune complex and cell lysis, are dependent on complement activation. Although the activation of the complement system is important to avoid infections, it also can contribute to the inflammatory response triggered by immune complex deposition in tissues in autoimmune diseases. Paradoxically, the deficiency of early complement proteins from the classical pathway (CP) is strongly associated with development of systemic lupus erythematous (SLE) – mainly C1q deficiency (93%) and C4 deficiency (75%). The aim of this review is to focus on the deficiencies of early components of the CP (C1q, C1r, C1s, C4, and C2) proteins in SLE patients. PMID:26941740

Airway exchange of highly soluble gases.

PubMed

Hlastala, Michael P; Powell, Frank L; Anderson, Joseph C

2013-03-01

Highly blood soluble gases exchange with the bronchial circulation in the airways. On inhalation, air absorbs highly soluble gases from the airway mucosa and equilibrates with the blood before reaching the alveoli. Highly soluble gas partial pressure is identical throughout all alveoli. At the end of exhalation the partial pressure of a highly soluble gas decreases from the alveolar level in the terminal bronchioles to the end-exhaled partial pressure at the mouth. A mathematical model simulated the airway exchange of four gases (methyl isobutyl ketone, acetone, ethanol, and propylene glycol monomethyl ether) that have high water and blood solubility. The impact of solubility on the relative distribution of airway exchange was studied. We conclude that an increase in water solubility shifts the distribution of gas exchange toward the mouth. Of the four gases studied, ethanol had the greatest decrease in partial pressure from the alveolus to the mouth at end exhalation. Single exhalation breath tests are inappropriate for estimating alveolar levels of highly soluble gases, particularly for ethanol.

Airway exchange of highly soluble gases

PubMed Central

Powell, Frank L.; Anderson, Joseph C.

2013-01-01

Highly blood soluble gases exchange with the bronchial circulation in the airways. On inhalation, air absorbs highly soluble gases from the airway mucosa and equilibrates with the blood before reaching the alveoli. Highly soluble gas partial pressure is identical throughout all alveoli. At the end of exhalation the partial pressure of a highly soluble gas decreases from the alveolar level in the terminal bronchioles to the end-exhaled partial pressure at the mouth. A mathematical model simulated the airway exchange of four gases (methyl isobutyl ketone, acetone, ethanol, and propylene glycol monomethyl ether) that have high water and blood solubility. The impact of solubility on the relative distribution of airway exchange was studied. We conclude that an increase in water solubility shifts the distribution of gas exchange toward the mouth. Of the four gases studied, ethanol had the greatest decrease in partial pressure from the alveolus to the mouth at end exhalation. Single exhalation breath tests are inappropriate for estimating alveolar levels of highly soluble gases, particularly for ethanol. PMID:23305981

Synergistic Interactions between Chemokine Receptor Elements in Recognition of Interleukin-8 by Soluble Receptor Mimics†

PubMed Central

Barter, Emily F.; Stone, Martin J.

2012-01-01

Interleukin-8 (IL-8 or CXCL8), the archetypal member of the CXC chemokine subfamily, stimulates neutrophil chemotaxis by activation of the receptors CXCR1/IL8RA and CXCR2/IL8RB. Previous mutational studies have implicated both the N-terminal and third extracellular loop (E3) regions of these receptors in binding to IL-8. To investigate the interactions of these receptor elements with IL-8, we have constructed soluble proteins in which the N-terminal and E3 elements of either CXCR1 or CXCR2 are juxtaposed on a soluble scaffold protein; these are referred to as CROSS-NX1E3X1 and CROSS-NX2E3X2, respectively. Isothermal titration calorimetry (ITC) and NMR spectroscopy were used to compare the IL-8 binding properties of the receptor mimics to those of control proteins containing only the N-terminal or the E3 receptor element. CROSS-NX2E3X2 bound to monomeric IL-8 with the same affinity and induced the same chemical shift changes as the control protein containing only the N-terminal element of CXCR2, indicating that the E3 element of CXCR2 did not contribute to IL-8 binding. In contrast, CROSS-NX1E3X1 bound to IL-8 with ~10-fold increased affinity and induced different chemical shift changes compared to the control protein containing only the N-terminal element of CXCR1, suggesting that the E3 region of CXCR1 was interacting with IL-8. However, a chimeric protein containing the N-terminal region of CXCR1 and the E3 region of CXCR2 (CROSS-NX1E3X2) bound to IL-8 with thermodynamic properties and induced chemical shift changes indistinguishable from those of CROSS-NX1E3X1 and substantially different from those of CROSS-NX2E3X2. These results indicate that the N-terminal and E3 regions of CXCR1 interact synergistically to achieve optimal binding interactions with IL-8. PMID:22242662

Complement activation promotes muscle inflammation during modified muscle use

NASA Technical Reports Server (NTRS)

Frenette, J.; Cai, B.; Tidball, J. G.

2000-01-01

Modified muscle use can result in muscle inflammation that is triggered by unidentified events. In the present investigation, we tested whether the activation of the complement system is a component of muscle inflammation that results from changes in muscle loading. Modified rat hindlimb muscle loading was achieved by removing weight-bearing from the hindlimbs for 10 days followed by reloading through normal ambulation. Experimental animals were injected with the recombinant, soluble complement receptor sCR1 to inhibit complement activation. Assays for complement C4 or factor B in sera showed that sCR1 produced large reductions in the capacity for activation of the complement system through both the classical and alternative pathways. Analysis of complement C4 concentration in serum in untreated animals showed that the classical pathway was activated during the first 2 hours of reloading. Analysis of factor B concentration in untreated animals showed activation of the alternative pathway at 6 hours of reloading. Administration of sCR1 significantly attenuated the invasion of neutrophils (-49%) and ED1(+) macrophages (-52%) that occurred in nontreated animals after 6 hours of reloading. The presence of sCR1 also reduced significantly the degree of edema by 22% as compared to untreated animals. Together, these data show that increased muscle loading activated the complement system which then briefly contributes to the early recruitment of inflammatory cells during modified muscle loading.

Steroids and triterpenes from the fruit bodies of Ganoderma lucidum and their anti-complement activity.

PubMed

Seo, Hyo Won; Hung, Tran Manh; Na, MinKyun; Jung, Hyun Ju; Kim, Jin Cheol; Choi, Jae Sue; Kim, Jung Hee; Lee, Hyeong-Kyu; Lee, IkSoo; Bae, KiHwan; Hattori, Masao; Min, Byung Sun

2009-11-01

To determine the anti-complement activity of natural triterpenes, chromatographic separation of the EtOAc-soluble fraction from the fruiting body of Ganoderma lucidum led to the isolation of three steroids and five triterpenoids. They were identified as ergosterol peroxide (1), ergosterol (2), genoderic acid Sz (3), stella sterol (4), ganoderic aic C1 (5), ganoderic acid A (6), methyl ganoderate A (7), and lucidenic acid A (8) based on spectroscopic evidence and physicochemical properties. These compounds were examined for their anti-complement activity against the classical pathway of the complement system. Compounds 2 and 3 showed potent anti-complement activity with IC50 values of 52.0 and 44.6 microM, respectively. Compound 1 exhibited significant inhibitory activity with an IC50 value of 126.8 microM, whereas compounds 4-8 were inactive. Our findings suggested that in addition to the ketone group at C-3, the delta7(8), delta9(11)-lanostadiene type triterpene also plays an important role in inhibiting the hemolytic activity of human serum against erythrocytes.

Complement Effectors of Inflammation in Cystic Fibrosis Lung Fluid Correlate with Clinical Measures of Disease.

PubMed

Sass, Laura A; Hair, Pamela S; Perkins, Amy M; Shah, Tushar A; Krishna, Neel K; Cunnion, Kenji M

2015-01-01

In cystic fibrosis (CF), lung damage is mediated by a cycle of obstruction, infection, and inflammation. Here we explored complement inflammatory effectors in CF lung fluid. In this study soluble fractions (sols) from sputum samples of 15 CF patients were assayed for complement effectors and analyzed with clinical measurements. The pro-inflammatory peptide C5a was increased 4.8-fold (P = 0.04) in CF sols compared with controls. Incubation of CF sols with P. aeruginosa or S. aureus increased C5a concentration 2.3-fold (P = 0.02). A peptide inhibitor of complement C1 (PIC1) completely blocked the increase in C5a concentration from P. aeruginosa in CF sol in vitro (P = 0.001). C5a concentration in CF sol correlated inversely with body mass index (BMI) percentile in children (r = -0.77, P = 0.04). C3a, which has anti-inflammatory effects, correlated positively with FEV1% predicted (rs = 0.63, P = 0.02). These results suggest that complement effectors may significantly impact inflammation in CF lung fluid.

Evaluation of physicochemical properties of root-end filling materials using conventional and Micro-CT tests.

PubMed

Torres, Fernanda Ferrari Esteves; Bosso-Martelo, Roberta; Espir, Camila Galletti; Cirelli, Joni Augusto; Guerreiro-Tanomaru, Juliane Maria; Tanomaru-Filho, Mario

2017-01-01

To evaluate solubility, dimensional stability, filling ability and volumetric change of root-end filling materials using conventional tests and new Micro-CT-based methods. 7. The results suggested correlated or complementary data between the proposed tests. At 7 days, BIO showed higher solubility and at 30 days, showed higher volumetric change in comparison with MTA (p<0.05). With regard to volumetric change, the tested materials were similar (p>0.05) at 7 days. At 30 days, they presented similar solubility. BIO and MTA showed higher dimensional stability than ZOE (p<0.05). ZOE and BIO showed higher filling ability (p<0.05). ZOE presented a higher dimensional change, and BIO had greater solubility after 7 days. BIO presented filling ability and dimensional stability, but greater volumetric change than MTA after 30 days. Micro-CT can provide important data on the physicochemical properties of materials complementing conventional tests.

Characterization of Proteus vulgaris K80 lipase immobilized on amine-terminated magnetic microparticles.

PubMed

Natalia, Agnes; Kristiani, Lidya; Kim, Hyung Kwoun

2014-10-01

Proteus vulgaris K80 lipase was expressed in Escherichia coli BL21 (DE3) cells and immobilized on amine-terminated magnetic microparticles (Mag-MPs). The immobilization yield and activity retention were 84.15% and 7.87%, respectively. A homology model of lipase K80 was constructed using P. mirabilis lipase as the template. Many lysine residues were located on the protein surface, remote from active sites. The biochemical characteristics of immobilized lipase K80 were compared with the soluble free form of lipase K80. The optimum temperature of K80-Mag-MPs was 60°C, which was 20°C higher than that of the soluble form. K80-Mag-MPs also tended to be more stable than the soluble form at elevated temperatures and a broad range of pH. K80-Mag-MP maintained its stable form at up to 40°C and in a pH range of 5.0- 10.0, whereas soluble K80 maintained its activity up to 35°C and pH 6.0-10.0. K80-Mag-MPs had broader substrate specificity compared with that of soluble K80. K80-Mag-MPs showed about 80% residual relative activity after five recovery trials. These results indicate the potential benefit of K80-Mag-MPs as a biocatalyst in various industries.

Complement in Lupus Nephritis: New Perspectives.

PubMed

Bao, Lihua; Cunningham, Patrick N; Quigg, Richard J

2015-09-01

Systemic lupus erythematosus (SLE) is an autoimmune disorder caused by loss of tolerance to self-antigens, the production of autoantibodies and deposition of complement-fixing immune complexes (ICs) in injured tissues. SLE is characterized by a wide range of clinical manifestations and targeted organs, with lupus nephritis being one of the most serious complications. The complement system consists of three pathways and is tightly controlled by a set of regulatory proteins to prevent injudicious complement activation on host tissue. The involvement of the complement system in the pathogenesis of SLE is well accepted; yet, its exact role is still not clear. Complement plays dual roles in the pathogenesis of SLE. On the one hand, the complement system appears to have protective features in that hereditary homozygous deficiencies of classical pathway components, such as C1q and C4, are associated with an increased risk for SLE. On the other hand, IC-mediated activation of complement in affected tissues is clearly evident in both experimental and human SLE along with pathological features that are logical consequences of complement activation. Studies in genetically altered mice have shown that lack of complement inhibitors, such as complement factor H (CFH) or decay-accelerating factor (DAF) accelerates the development of experimental lupus nephritis, while treatment with recombinant protein inhibitors, such as Crry-Ig, CR2-Crry, CR2-DAF and CR2-CFH, ameliorates the disease development. Complement-targeted drugs, including soluble complement receptor 1 (TP10), C1 esterase inhibitor and a monoclonal anti-C5 antibody (eculizumab), have been shown to inhibit complement safely, and are now being investigated in a variety of clinical conditions. SLE is an autoimmune disorder which targets multiple systems. Complement is centrally involved and plays dual roles in the pathogenesis of SLE. Studies from experimental lupus models and clinical trials support the use of complement-targeted therapy in the treatment of SLE.

Anti-GM2 gangliosides IgM paraprotein induces neuromuscular block without neuromuscular damage.

PubMed

Santafé, Manel M; Sabaté, M Mar; Garcia, Neus; Ortiz, Nico; Lanuza, M Angel; Tomàs, Josep

2008-11-15

We analyzed the effect on the mouse neuromuscular synapses of a human monoclonal IgM, which binds specifically to gangliosides with the common epitope [GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-]. We focused on the role of the complement. Evoked neurotransmission was partially blocked by IgM both acutely (1 h) and chronically (10 days). Transmission electron microscopy shows important nerve terminal growth and retraction remodelling though axonal injury can be ruled out. Synapses did not show mouse C5b-9 immunofluorescence and were only immunolabelled when human complement was added. Therefore, the IgM-induced synaptic changes occur without complement-mediated membrane attack.

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

PubMed Central

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. PMID:19531344

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

PubMed

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.

Guilty as charged: all available evidence implicates complement's role in fetal demise.

PubMed

Girardi, Guillermina

2008-03-01

Appropriate complement inhibition is an absolute requirement for normal pregancy. Uncontrolled complement activation in the maternal-fetal interface leads to fetal death. Here we show that complement activation is a crucial and early mediator of pregnancy loss in two different mouse models of pregnancy loss. Using a mouse model of fetal loss and growth restriction (IUGR) induced by antiphospholipid antibodies (aPL), we examined the role of complement activation in fetal loss and IUGR. We found that C5a-C5aR interaction and neutrophils are key mediators of fetal injury. Treatment with heparin, the standard therapy for pregnant patients with aPL, prevents complement activation and protects mice from pregnancy complications induced by aPL, and anticoagulants that do not inhibit complement do not protect pregnancies. In an antibody-independent mouse model of spontaneous miscarriage and IUGR (CBA/JxDBA/2) we also identified C5a as an essential mediator. Complement activation caused dysregulation of the angiogenic factors required for normal placental development. In CBA/JxDBA/2 mice, we observed inflammatory infiltrates in placentas, functional deficiency of free vascular endothelial growth factor (VEGF), elevated levels of soluble VEGF receptor-1 (sVEGFR-1, also known as sFlt-1; a potent anti-angiogenic molecule), and defective placental development. Inhibition of complement activation blocked the increase in sVEGFR-1 and rescued pregnancies. Our studies in antibody-dependent and antibody-independent models of pregnancy complications identified complement activation as the key mediator of damage and will allow development of new interventions to prevent pregnancy loss and IUGR.

Evasion Mechanisms Used by Pathogens to Escape the Lectin Complement Pathway.

PubMed

Rosbjerg, Anne; Genster, Ninette; Pilely, Katrine; Garred, Peter

2017-01-01

The complement system is a crucial defensive network that protects the host against invading pathogens. It is part of the innate immune system and can be initiated via three pathways: the lectin, classical and alternative activation pathway. Overall the network compiles a group of recognition molecules that bind specific patterns on microbial surfaces, a group of associated proteases that initiates the complement cascade, and a group of proteins that interact in proteolytic complexes or the terminal pore-forming complex. In addition, various regulatory proteins are important for controlling the level of activity. The result is a pro-inflammatory response meant to combat foreign microbes. Microbial elimination is, however, not a straight forward procedure; pathogens have adapted to their environment by evolving a collection of evasion mechanisms that circumvent the human complement system. Complement evasion strategies features different ways of exploiting human complement proteins and moreover features different pathogen-derived proteins that interfere with the normal processes. Accumulated, these mechanisms target all three complement activation pathways as well as the final common part of the cascade. This review will cover the currently known lectin pathway evasion mechanisms and give examples of pathogens that operate these to increase their chance of invasion, survival and dissemination.

THE BIOLOGICAL ACTIVITY OF SOLUBLE ANTIGEN-ANTIBODY COMPLEXES

PubMed Central

Ishizaka, Kimishige; Ishizaka, Teruko; Campbell, Dan H.

1959-01-01

Soluble BSA-anti-BSA complexes, formed in antigen excess, give immediate skin reactions in normal guinea pigs. The mechanism of the reaction is not that of passive or reversed passive anaphylaxis. The complex itself is toxic. Skin activity of the complex depends on its composition. It has become obvious that the complex composed of two antigen molecules and one antibody molecule, (Ag2Ab), does not have the activity, whereas, Ag3Ab2 and more complicated complexes do. The role of complement as well as speculation on the structural changes of antibody-antigen complexes is presented. PMID:13620844

Complement activation in leprosy: a retrospective study shows elevated circulating terminal complement complex in reactional leprosy.

PubMed

Bahia El Idrissi, N; Hakobyan, S; Ramaglia, V; Geluk, A; Morgan, B Paul; Das, P Kumar; Baas, F

2016-06-01

Mycobacterium leprae infection gives rise to the immunologically and histopathologically classified spectrum of leprosy. At present, several tools for the stratification of patients are based on acquired immunity markers. However, the role of innate immunity, particularly the complement system, is largely unexplored. The present retrospective study was undertaken to explore whether the systemic levels of complement activation components and regulators can stratify leprosy patients, particularly in reference to the reactional state of the disease. Serum samples from two cohorts were analysed. The cohort from Bangladesh included multi-bacillary (MB) patients with (n = 12) or without (n = 46) reaction (R) at intake and endemic controls (n = 20). The cohort from Ethiopia included pauci-bacillary (PB) (n = 7) and MB (n = 23) patients without reaction and MB (n = 15) patients with reaction. The results showed that the activation products terminal complement complex (TCC) (P ≤ 0·01), C4d (P ≤ 0·05) and iC3b (P ≤ 0·05) were specifically elevated in Bangladeshi patients with reaction at intake compared to endemic controls. In addition, levels of the regulator clusterin (P ≤ 0·001 without R; P < 0·05 with R) were also elevated in MB patients, irrespective of a reaction. Similar analysis of the Ethiopian cohort confirmed that, irrespective of a reaction, serum TCC levels were increased significantly in patients with reactions compared to patients without reactions (P ≤ 0·05). Our findings suggests that serum TCC levels may prove to be a valuable tool in diagnosing patients at risk of developing reactions. © 2016 British Society for Immunology.

Study of the plant COPII vesicle coat subunits by functional complementation of yeast Saccharomyces cerevisiae mutants.

PubMed

De Craene, Johan-Owen; Courte, Fanny; Rinaldi, Bruno; Fitterer, Chantal; Herranz, Mari Carmen; Schmitt-Keichinger, Corinne; Ritzenthaler, Christophe; Friant, Sylvie

2014-01-01

The formation and budding of endoplasmic reticulum ER-derived vesicles depends on the COPII coat protein complex that was first identified in yeast Saccharomyces cerevisiae. The ER-associated Sec12 and the Sar1 GTPase initiate the COPII coat formation by recruiting the Sec23-Sec24 heterodimer following the subsequent recruitment of the Sec13-Sec31 heterotetramer. In yeast, there is usually one gene encoding each COPII protein and these proteins are essential for yeast viability, whereas the plant genome encodes multiple isoforms of all COPII subunits. Here, we used a systematic yeast complementation assay to assess the functionality of Arabidopsis thaliana COPII proteins. In this study, the different plant COPII subunits were expressed in their corresponding temperature-sensitive yeast mutant strain to complement their thermosensitivity and secretion phenotypes. Secretion was assessed using two different yeast cargos: the soluble α-factor pheromone and the membranous v-SNARE (vesicle-soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor) Snc1 involved in the fusion of the secretory vesicles with the plasma membrane. This complementation study allowed the identification of functional A. thaliana COPII proteins for the Sec12, Sar1, Sec24 and Sec13 subunits that could represent an active COPII complex in plant cells. Moreover, we found that AtSec12 and AtSec23 were co-immunoprecipitated with AtSar1 in total cell extract of 15 day-old seedlings of A. thaliana. This demonstrates that AtSar1, AtSec12 and AtSec23 can form a protein complex that might represent an active COPII complex in plant cells.

Gel-based and gel-free proteomic analysis of Nicotiana tabacum trichomes identifies proteins involved in secondary metabolism and in the (a)biotic stress response.

PubMed

Van Cutsem, Emmanuel; Simonart, Géraldine; Degand, Hervé; Faber, Anne-Marie; Morsomme, Pierre; Boutry, Marc

2011-02-01

Nicotiana tabacum leaves are covered by trichomes involved in the secretion of large amounts of secondary metabolites, some of which play a major role in plant defense. However, little is known about the metabolic pathways that operate in these structures. We undertook a proteomic analysis of N. tabacum trichomes in order to identify their protein complement. Efficient trichome isolation was obtained by abrading frozen leaves. After homogenization, soluble proteins and a microsomal fraction were prepared by centrifugation. Gel-based and gel-free proteomic analyses were then performed. 2-DE analysis of soluble proteins led to the identification of 1373 protein spots, which were digested and analyzed by MS/MS, leading to 680 unique identifications. Both soluble proteins and microsomal fraction were analyzed by LC MALDI-MS/MS after trypsin digestion, leading to 858 identifications, many of which had not been identified after 2-DE, indicating that the two methods complement each other. Many enzymes putatively involved in secondary metabolism were identified, including enzymes involved in the synthesis of terpenoid precursors and in acyl sugar production. Several transporters were also identified, some of which might be involved in secondary metabolite transport. Various (a)biotic stress response proteins were also detected, supporting the role of trichomes in plant defense. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Plasma Biomarkers Reflecting Profibrotic Processes in Heart Failure With a Preserved Ejection Fraction: Data From the Prospective Comparison of ARNI With ARB on Management of Heart Failure With Preserved Ejection Fraction Study.

PubMed

Zile, Michael R; Jhund, Pardeep S; Baicu, Catalin F; Claggett, Brian L; Pieske, Burkert; Voors, Adriaan A; Prescott, Margaret F; Shi, Victor; Lefkowitz, Martin; McMurray, John J V; Solomon, Scott D

2016-01-01

Heart failure with preserved ejection fraction is a clinical syndrome that has been associated with changes in the extracellular matrix. The purpose of this study was to determine whether profibrotic biomarkers accurately reflect the presence and severity of disease and underlying pathophysiology and modify response to therapy in patients with heart failure with preserved ejection fraction. Four biomarkers, soluble form of ST2 (an interleukin-1 receptor family member), galectin-3, matrix metalloproteinase-2, and collagen III N-terminal propeptide were measured in the Prospective Comparison of ARNI With ARB on Management of Heart Failure With Preserved Ejection Fraction (PARAMOUNT) trial at baseline, 12 and 36 weeks after randomization to valsartan or LCZ696. We examined the relationship between baseline biomarkers, demographic and echocardiographic characteristics, change in primary (change in N-terminal pro B-type natriuretic peptide) and secondary (change in left atrial volume) end points. The median (interquartile range) value for soluble form of ST2 (33 [24.6-48.1] ng/mL) and galectin 3 (17.8 [14.1-22.8] ng/mL) were higher, and for matrix metalloproteinase-2 (188 [155.5-230.6] ng/mL) lower, than in previously published referent controls; collagen III N-terminal propeptide (5.6 [4.3-6.9] ng/mL) was similar to referent control values. All 4 biomarkers correlated with severity of disease as indicated by N-terminal pro B-type natriuretic peptide, E/E', and left atrial volume. Baseline biomarkers did not modify the response to LCZ696 for lowering N-terminal pro B-type natriuretic peptide; however, left atrial volume reduction varied by baseline level of soluble form of ST2 and galectin 3; patients with values less than the observed median (<33 ng/mL soluble form of ST2 and <17.8 ng/mL galectin 3) had reduction in left atrial volume, those above median did not. Although LCZ696 reduced N-terminal pro B-type natriuretic peptide, levels of the other 4 biomarkers were not affected over time. In patients with heart failure with preserved ejection fraction, biomarkers that reflect collagen homeostasis correlated with the presence and severity of disease and underlying pathophysiology, and may modify the structural response to treatment. URL: http://www.clinicaltrials.gov. Unique identifier: NCT00887588. © 2016 American Heart Association, Inc.

Spectrum and management of complement immunodeficiencies (excluding hereditary angioedema) across Europe.

PubMed

Turley, A J; Gathmann, B; Bangs, C; Bradbury, M; Seneviratne, S; Gonzalez-Granado, L I; Hackett, S; Kutukculer, N; Alachkar, H; Hambleton, S; Ritterbusch, H; Kralickova, P; Marodi, L; Seidel, M G; Dueckers, G; Roesler, J; Huissoon, A; Baxendale, H; Litzman, J; Arkwright, P D

2015-02-01

Complement immunodeficiencies (excluding hereditary angioedema and mannose binding lectin deficiency) are rare. Published literature consists largely of case reports and small series. We collated data from 18 cities across Europe to provide an overview of primarily homozygous, rather than partial genotypes and their impact and management. Patients were recruited through the ESID registry. Clinical and laboratory information was collected onto standardized forms and analyzed using SPSS software. Seventy-seven patients aged 1 to 68 years were identified. 44 % presented in their first decade of life. 29 % had C2 deficiency, defects in 11 other complement factors were found. 50 (65 %) had serious invasive infections. 61 % of Neisseria meningitidis infections occurred in patients with terminal pathway defects, while 74 % of Streptococcus pneumoniae infections occurred in patients with classical pathway defects (p < 0.001). Physicians in the UK were more likely to prescribe antibiotic prophylaxis than colleagues on the Continent for patients with classical pathway defects. After diagnosis, 16 % of patients suffered serious bacterial infections. Age of the patient and use of prophylactic antibiotics were not associated with subsequent infection risk. Inflammatory/autoimmune diseases were not seen in patients with terminal pathway, but in one third of patients classical and alternative pathway defects. The clinical phenotypes of specific complement immunodeficiencies vary considerably both in terms of the predominant bacterial pathogen, and the risk and type of auto-inflammatory disease. Appreciation of these phenotypic differences should help both immunologists and other specialists in their diagnosis and management of these rare and complex patients.

Variola virus immune evasion proteins.

PubMed

Dunlop, Lance R; Oehlberg, Katherine A; Reid, Jeremy J; Avci, Dilek; Rosengard, Ariella M

2003-09-01

Variola virus, the causative agent of smallpox, encodes approximately 200 proteins. Over 80 of these proteins are located in the terminal regions of the genome, where proteins associated with host immune evasion are encoded. To date, only two variola proteins have been characterized. Both are located in the terminal regions and demonstrate immunoregulatory functions. One protein, the smallpox inhibitor of complement enzymes (SPICE), is homologous to a vaccinia virus virulence factor, the vaccinia virus complement-control protein (VCP), which has been found experimentally to be expressed early in the course of vaccinia infection. Both SPICE and VCP are similar in structure and function to the family of mammalian complement regulatory proteins, which function to prevent inadvertent injury to adjacent cells and tissues during complement activation. The second variola protein is the variola virus high-affinity secreted chemokine-binding protein type II (CKBP-II, CBP-II, vCCI), which binds CC-chemokine receptors. The vaccinia homologue of CKBP-II is secreted both early and late in infection. CKBP-II proteins are highly conserved among orthopoxviruses, sharing approximately 85% homology, but are absent in eukaryotes. This characteristic sets it apart from other known virulence factors in orthopoxviruses, which share sequence homology with known mammalian immune regulatory gene products. Future studies of additional variola proteins may help illuminate factors associated with its virulence, pathogenesis and strict human tropism. In addition, these studies may also assist in the development of targeted therapies for the treatment of both smallpox and human immune-related diseases.

Protein domains connect cell cycle stimulation directly to initiation of DNA replication.

PubMed Central

Gjørup, O V; Rose, P E; Holman, P S; Bockus, B J; Schaffhausen, B S

1994-01-01

Polyoma large T antigen (LT) is the only viral gene product required for viral DNA replication. LT can be divided into two domains, one N-terminal (NT) spanning residues 1-260 and one C-terminal (CT) comprising approximately residues 264-785. NT is known to immortalize primary cells in a manner dependent on binding of pRB/p107. Here a CT construct comprising residues 264-785 was shown to have independent function in DNA replication. CT is entirely sufficient for driving viral DNA replication in vivo in growing mouse cells at a level approaching that of full-length LT. In contrast, CT is strikingly deficient for replication in serum-starved cells. However, this deficiency can be complemented by coexpression of NT. BrdUrd incorporation in transfected, starved cells showed that NT was sufficient for inducing S phase, suggesting a mechanism for complementation. By contrast, CT was unable to induce S phase when tested in the same assay. NT also promotes phosphorylation of sites in CT that are likely to be important for replication. Other DNA tumor virus gene products such as adenovirus E1A 12S and human papillomavirus 16 E7 could also complement CT for replication. Although NT, E1A 12S, and E7 all bind the retinoblastoma gene product (pRB) and p107, genetic analysis demonstrates an additional function, independent of that binding, is responsible for complementation. Images PMID:7991595

The interrelationship of complement-activation fragments and angiogenesis-related factors in early pregnancy and their association with pre-eclampsia

PubMed Central

Lynch, AM; Murphy, JR; Gibbs, RS; Levine, RJ; Giclas, PC; Salmon, JE; Holers, VM

2016-01-01

Objective To determine the interrelationships during early pregnancy of complement-activation fragments Bb, C3a and sC5b-9, and angiogenesis-related factors placental growth factor (PiGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), and their associations with pre-eclampsia. Design Prospective cohort study. Setting Denver complement study (June 2005–June 2008). Population A total of 668 pregnant women with singleton gestations, recruited between 10 and 15 weeks of gestation. Methods Using univariable and multivariable logistic regression analysis, concentrations of complement-activation fragments and angiogenesis-related factors were compared between 10 and 15 weeks of gestation in women who subsequently did or did not develop pre-eclampsia. Interrelationships between these variables were tested using the non-parametric Spearman rank correlation coefficient. Main outcome measure Pre-eclampsia. The association of complement-activation fragments and angiogenesis-related factors with obesity was also examined. Results The mean (±SD) levels of complement Bb in early pregnancy among women who did and did not develop pre-eclampsia were 0.84 (±0.26) µg/ml and 0.69 (±0.2) µg/ml, respectively (P = 0.001). Concentrations of PiGF were significantly (P = 0.01) lower (31 ± 12 pg/ml) in early pregnancy in the pre-eclamptic group of women, as compared with the normotensive group (39 ± 32 pg/ml). The adjusted odds ratio (AOR) of Bb and PiGF were 2.1 (CI = 1.4–3.1, P < 0.0003) and 0.2 (CI = 0.07–0.7, P = 0.01), respectively. There was no significant difference in the levels of C3a, sC5b-9, sFlt-1 and sEng in early pregnancy among women who developed pre-eclampsia, compared with women who remained normotensive during pregnancy. Higher levels of Bb (P = 0.0001) and C3a (P = 0.03), and lower levels of sFlt-1 (P = 0.0002) and sEng (P = 0.0001) were found among women with obesity, compared with non-obese controls. No meaningful relationships were found between the complement-activation fragments and the angiogenesis-related factors. Conclusions In this cohort during early pregnancy, increased concentrations of complement-activation factor Bb and lower concentrations of PiGF were associated with the development of pre-eclampsia later in pregnancy. PMID:20074261

Complement factor H protects mice from ischemic acute kidney injury but is not critical for controlling complement activation by glomerular IgM.

PubMed

Goetz, Lindsey; Laskowski, Jennifer; Renner, Brandon; Pickering, Matthew C; Kulik, Liudmila; Klawitter, Jelena; Stites, Erik; Christians, Uwe; van der Vlag, Johan; Ravichandran, Kameswaran; Holers, V Michael; Thurman, Joshua M

2018-05-01

Natural IgM binds to glomerular epitopes in several progressive kidney diseases. Previous work has shown that IgM also binds within the glomerulus after ischemia/reperfusion (I/R) but does not fully activate the complement system. Factor H is a circulating complement regulatory protein, and congenital or acquired deficiency of factor H is a strong risk factor for several types of kidney disease. We hypothesized that factor H controls complement activation by IgM in the kidney after I/R, and that heterozygous factor H deficiency would permit IgM-mediated complement activation and injury at this location. We found that mice with targeted heterozygous deletion of the gene for factor H developed more severe kidney injury after I/R than wild-type controls, as expected, but that complement activation within the glomeruli remained well controlled. Furthermore, mice that are unable to generate soluble IgM were not protected from renal I/R, even in the setting of heterozygous factor H deficiency. These results demonstrate that factor H is important for limiting injury in the kidney after I/R, but it is not critical for controlling complement activation by immunoglobulin within the glomerulus in this setting. IgM binds to glomerular epitopes after I/R, but it is not a significant source of injury. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Arc1p is required for cytoplasmic confinement of synthetases and tRNA.

PubMed

Golinelli-Cohen, Marie-Pierre; Mirande, Marc

2007-06-01

In yeast, Arc1p interacts with ScMetRS and ScGluRS and operates as a tRNA-Interacting Factor (tIF) in trans of these two synthetases. Its N-terminal domain (N-Arc1p) binds the two synthetases and its C-terminal domain is an EMAPII-like domain organized around an OB-fold-based tIF. ARC1 is not an essential gene but its deletion (arc1- cells) is accompanied by a growth retardation phenotype. Here, we show that expression of N-Arc1p or of C-Arc1p alone palliates the growth defect of arc1- cells, and that bacterial Trbp111 or human p43, two proteins containing EMAPII-like domains, also improve the growth of an arc1- strain. The synthetic lethality of an arc1-los1- strain can be complemented with either ARC1 or LOS1. Expression of N-Arc1p or C-Arc1p alone does not complement an arc1-los1- phenotype, but coexpression of the two domains does. Our data demonstrate that Trbp111 or p43 may replace C-Arc1p to complement an arc1-los1- strain. The two functional domains of Arc1p (N-Arc1p and C-Arc1p) are required to get rid of the synthetic lethal phenotype but do not need to be physically linked. To get some clues to the discrete functions of N-Arc1p and C-Arc1p, we targeted ScMetRS or tIF domains to the nuclear compartment and analyzed their cellular localization by using GFP fusions, and their ability to sustain growth. Our results are consistent with a model according to which Arc1p is a bifunctional protein involved in the subcellular localization of ScMetRS and ScGluRS via its N-terminal domain and of tRNA via its C-terminal domain.

Trypanosoma cruzi H+-ATPase 1 (TcHA1) and 2 (TcHA2) genes complement yeast mutants defective in H+ pumps and encode plasma membrane P-type H+-ATPases with different enzymatic properties.

PubMed

Luo, Shuhong; Scott, David A; Docampo, Roberto

2002-11-15

Previous studies in Trypanosoma cruzi have shown that intracellular pH homeostasis requires ATP and is affected by H(+)-ATPase inhibitors, indicating a major role for ATP-driven proton pumps in intracellular pH control. In the present study, we report the cloning and sequencing of a pair of genes linked in tandem (TcHA1 and TcHA2) in T. cruzi which encode proteins with homology to fungal and plant P-type proton-pumping ATPases. The genes are expressed at the mRNA level in different developmental stages of T. cruzi: TcHA1 is expressed maximally in epimastigotes, whereas TcHA2 is expressed predominantly in trypomastigotes. The proteins predicted from the nucleotide sequence of the genes have 875 and 917 amino acids and molecular masses of 96.3 and 101.2 kDa, respectively. Full-length TcHA1 and an N-terminal truncated version of TcHA2 complemented a Saccharomyces cerevisiae strain deficient in P-type H(+)-ATPase activity, the proteins localized to the yeast plasma membrane, and ATP-driven proton pumping could be detected in proteoliposomes reconstituted from plasma membrane purified from transfected yeast. The reconstituted proton transport activity was reduced by inhibitors of P-type H(+)-ATPases. C-terminal truncation did not affect complementation of mutant yeast, suggesting the lack of C-terminal autoinhibitory domains in these proteins. ATPase activity in plasma membrane from TcHA1- and (N-terminal truncated) TcHA2-transfected yeast was inhibited to different extents by vanadate, whereas the latter yeast strain was more resistant to extremes of pH, suggesting that the native proteins may serve different functions at different stages in the T. cruzi life cycle.

NETosing Neutrophils Activate Complement Both on Their Own NETs and Bacteria via Alternative and Non-alternative Pathways

PubMed Central

Yuen, Joshua; Pluthero, Fred G.; Douda, David N.; Riedl, Magdalena; Cherry, Ahmed; Ulanova, Marina; Kahr, Walter H. A.; Palaniyar, Nades; Licht, Christoph

2016-01-01

Neutrophils deposit antimicrobial proteins, such as myeloperoxidase and proteases on chromatin, which they release as neutrophil extracellular traps (NETs). Neutrophils also carry key components of the complement alternative pathway (AP) such as properdin or complement factor P (CFP), complement factor B (CFB), and C3. However, the contribution of these complement components and complement activation during NET formation in the presence and absence of bacteria is poorly understood. We studied complement activation on NETs and a Gram-negative opportunistic bacterial pathogen Pseudomonas aeruginosa (PA01, PAKwt, and PAKgfp). Here, we show that anaphylatoxin C5a, formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA), which activates NADPH oxidase, induce the release of CFP, CFB, and C3 from neutrophils. In response to PMA or P. aeruginosa, neutrophils secrete CFP, deposit it on NETs and bacteria, and induce the formation of terminal complement complexes (C5b–9). A blocking anti-CFP antibody inhibited AP-mediated but not non-AP-mediated complement activation on NETs and P. aeruginosa. Therefore, NET-mediated complement activation occurs via both AP- and non AP-based mechanisms, and AP-mediated complement activation during NETosis is dependent on CFP. These findings suggest that neutrophils could use their “AP tool kit” to readily activate complement on NETs and Gram-negative bacteria, such as P. aeruginosa, whereas additional components present in the serum help to fix non-AP-mediated complement both on NETs and bacteria. This unique mechanism may play important roles in host defense and help to explain specific roles of complement activation in NET-related diseases. PMID:27148258

Lipid Sulfates and Sulfonates Are Allosteric Competitive Inhibitors of the N-Terminal Phosphatase Activity of the Mammalian Soluble Epoxide Hydrolase†

PubMed Central

Tran, Katherine L.; Aronov, Pavel A.; Tanaka, Hiromasa; Newman, John W.; Hammock, Bruce D.; Morisseau, Christophe

2006-01-01

The EPXH2 gene encodes for the soluble epoxide hydrolase (sEH), a homodimeric enzyme with each monomer containing two domains with distinct activities. The C-terminal domain, containing the epoxide hydrolase activity (Cterm-EH), is involved in the metabolism of arachidonic acid epoxides, endogenous chemical mediators that play important roles in blood pressure regulation, cell growth, and inflammation. We recently demonstrated that the N-terminal domain contains a Mg2+-dependent lipid phosphate phosphatase activity (Nterm-phos). However, the biological role of this activity is unknown. The inability of known phosphatase inhibitors to inhibit the Nterm-phos constitutes a significant barrier to the elucidation of its function. We describe herein sulfate, sulfonate, and phosphonate lipids as novel potent inhibitors of Nterm-phos. These compounds are allosteric competitive inhibitors with KI in the hundred nanomolar range. These inhibitors may provide a valuable tool to investigate the biological role of the Nterm-phos. We found that polyisoprenyl phosphates are substrates of Nterm-phos, suggesting a possible role in sterol synthesis or inflammation. Furthermore, some of these compounds inhibit the C-terminal sEH activity through a noncompetitive inhibition mechanism involving a new binding site on the C-terminal domain. This novel site may play a role in the natural in vivo regulation of epoxide hydrolysis by sEH. PMID:16142916

Chaperone protein HYPK interacts with the first 17 amino acid region of Huntingtin and modulates mutant HTT-mediated aggregation and cytotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choudhury, Kamalika Roy; Centre for Neuroscience, Indian Institute of Science, Bangalore 560012; Bhattacharyya, Nitai P., E-mail: nitai_sinp@yahoo.com

2015-01-02

Highlights: • HYPK reduces mutant HTT-mediated aggregate formation and cytotoxicity. • Interaction of HYPK with HTT requires N-terminal 17 amino acid of HTT (HTT-N17). • Deletion of HTT-N17 leads to SDS-soluble, smaller, nuclear aggregates. • These smaller aggregates do not associate with HYPK and are more cytotoxic. • Maybe, interaction of HYPK with amphipathic HTT-N17 block HTT aggregate formation. - Abstract: Huntington’s disease is a polyglutamine expansion disorder, characterized by mutant HTT-mediated aggregate formation and cytotoxicity. Many reports suggests roles of N-terminal 17 amino acid domain of HTT (HTT-N17) towards subcellular localization, aggregate formation and subsequent pathogenicity induced by N-terminalmore » HTT harboring polyQ stretch in pathogenic range. HYPK is a HTT-interacting chaperone which can reduce N-terminal mutant HTT-mediated aggregate formation and cytotoxicity in neuronal cell lines. However, how HYPK interacts with N-terminal fragment of HTT remained unknown. Here we report that specific interaction of HYPK with HTT-N17 is crucial for the chaperone activity of HYPK. Deletion of HTT-N17 leads to formation of tinier, SDS-soluble nuclear aggregates formed by N-terminal mutant HTT. The increased cytotoxicity imparted by these tiny aggregates might be contributed due to loss of interaction with HYPK.« less

Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin.

PubMed Central

Sasaki, T; Fukai, N; Mann, K; Göhring, W; Olsen, B R; Timpl, R

1998-01-01

The C-terminal domain NC1 of mouse collagen XVIII (38 kDa) and the shorter mouse and human endostatins (22 kDa) were prepared in recombinant form from transfected mammalian cells. The NC1 domain aggregated non-covalently into a globular trimer which was partially cleaved by endogenous proteolysis into several monomers (25-32 kDa) related to endostatin. Endostatins were obtained in a highly soluble, monomeric form and showed a single N-terminal sequence which, together with other data, indicated a compact folding. Endostatins and NC1 showed a comparable binding activity for the microfibrillar fibulin-1 and fibulin-2, and for heparin. Domain NC1, however, was a distinctly stronger ligand than endostatin for sulfatides and the basement membrane proteins laminin-1 and perlecan. Immunological assays demonstrated endostatin epitopes on several tissue components (22-38 kDa) and in serum (120-300 ng/ml), the latter representing the smaller variants. The data indicated that the NC1 domain consists of an N-terminal association region (approximately 50 residues), a central protease-sensitive hinge region (approximately 70 residues) and a C-terminal stable endostatin domain (approximately 180 residues). They also demonstrated that proteolytic release of endostatin can occur through several pathways, which may lead to a switch from a matrix-associated to a more soluble endocrine form. PMID:9687493

Elimination of soluble sup 123 I-labeled aggregates of IgG in patients with systemic lupus erythematosus. Effect of serum IgG and numbers of erythrocyte complement receptor type 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Halma, C.; Breedveld, F.C.; Daha, M.R.

1991-04-01

Using soluble {sup 123}I-labeled aggregates of human IgG ({sup 123}I-AHIgG) as a probe, we examined the function of the mononuclear phagocyte system in 22 patients with systemic lupus erythematosus (SLE) and 12 healthy controls. In SLE patients, a decreased number of erythrocyte complement receptor type 1 was associated with less binding of {sup 123}I-AHIgG to erythrocytes and a faster initial rate of elimination of {sup 123}I-AHIgG (mean +/- SEM half-maximal clearance time 5.23 +/- 0.2 minutes, versus 6.58 +/- 0.2 minutes in the controls), with possible spillover of the material outside the mononuclear phagocyte system of the liver and spleen.more » However, multiple regression analysis showed that serum concentrations of IgG were the most important factor predicting the rate of {sup 123}I-AHIgG elimination. IgG concentration may thus reflect immune complex clearance, which in turn, would influence the inflammatory reaction, in SLE.« less

Anopheles Midgut Epithelium Evades Human Complement Activity by Capturing Factor H from the Blood Meal

PubMed Central

Khattab, Ayman; Barroso, Marta; Miettinen, Tiera; Meri, Seppo

2015-01-01

Hematophagous vectors strictly require ingesting blood from their hosts to complete their life cycles. Exposure of the alimentary canal of these vectors to the host immune effectors necessitates efficient counteractive measures by hematophagous vectors. The Anopheles mosquito transmitting the malaria parasite is an example of hematophagous vectors that within seconds can ingest human blood double its weight. The innate immune defense mechanisms, like the complement system, in the human blood should thereby immediately react against foreign cells in the mosquito midgut. A prerequisite for complement activation is that the target cells lack complement regulators on their surfaces. In this work, we analyzed whether human complement is active in the mosquito midgut, and how the mosquito midgut cells protect themselves against complement attack. We found that complement remained active for a considerable time and was able to kill microbes within the mosquito midgut. However, the Anopheles mosquito midgut cells were not injured. These cells were found to protect themselves by capturing factor H, the main soluble inhibitor of the alternative complement pathway. Factor H inhibited complement on the midgut cells by promoting inactivation of C3b to iC3b and preventing the activity of the alternative pathway amplification C3 convertase enzyme. An interference of the FH regulatory activity by monoclonal antibodies, carried to the midgut via blood, resulted in increased mosquito mortality and reduced fecundity. By using a ligand blotting assay, a putative mosquito midgut FH receptor could be detected. Thereby, we have identified a novel mechanism whereby mosquitoes can tolerate human blood. PMID:25679788

Soluble Amyloid-beta Aggregates from Human Alzheimer’s Disease Brains

PubMed Central

Esparza, Thomas J.; Wildburger, Norelle C.; Jiang, Hao; Gangolli, Mihika; Cairns, Nigel J.; Bateman, Randall J.; Brody, David L.

2016-01-01

Soluble amyloid-beta (Aβ) aggregates likely contribute substantially to the dementia that characterizes Alzheimer’s disease. However, despite intensive study of in vitro preparations and animal models, little is known about the characteristics of soluble Aβ aggregates in the human Alzheimer’s disease brain. Here we present a new method for extracting soluble Aβ aggregates from human brains, separating them from insoluble aggregates and Aβ monomers using differential ultracentrifugation, and purifying them >6000 fold by dual antibody immunoprecipitation. The method resulted in <40% loss of starting material, no detectible ex vivo aggregation of monomeric Aβ, and no apparent ex vivo alterations in soluble aggregate sizes. By immunoelectron microscopy, soluble Aβ aggregates typically appear as clusters of 10–20 nanometer diameter ovoid structures with 2-3 amino-terminal Aβ antibody binding sites, distinct from previously characterized structures. This approach may facilitate investigation into the characteristics of native soluble Aβ aggregates, and deepen our understanding of Alzheimer’s dementia. PMID:27917876

UDP-glucose:glycoprotein glucosyltransferase (UGGT1) promotes substrate solubility in the endoplasmic reticulum

PubMed Central

Ferris, Sean P.; Jaber, Nikita S.; Molinari, Maurizio; Arvan, Peter; Kaufman, Randal J.

2013-01-01

Protein folding in the endoplasmic reticulum (ER) is error prone, and ER quality control (ERQC) processes ensure that only correctly folded proteins are exported from the ER. Glycoproteins can be retained in the ER by ERQC, and this retention contributes to multiple human diseases, termed ER storage diseases. UDP-glucose:glycoprotein glucosyltransferase (UGGT1) acts as a central component of glycoprotein ERQC, monoglucosylating deglucosylated N-glycans of incompletely folded glycoproteins and promoting subsequent reassociation with the lectin-like chaperones calreticulin and calnexin. The extent to which UGGT1 influences glycoprotein folding, however, has only been investigated for a few selected substrates. Using mouse embryonic fibroblasts lacking UGGT1 or those with UGGT1 complementation, we investigated the effect of monoglucosylation on the soluble/insoluble distribution of two misfolded α1-antitrypsin (AAT) variants responsible for AAT deficiency disease: null Hong Kong (NHK) and Z allele. Whereas substrate solubility increases directly with the number of N-linked glycosylation sites, our results indicate that additional solubility is conferred by UGGT1 enzymatic activity. Monoglucosylation-dependent solubility decreases both BiP association with NHK and unfolded protein response activation, and the solubility increase is blocked in cells deficient for calreticulin. These results suggest that UGGT1-dependent monoglucosylation of N-linked glycoproteins promotes substrate solubility in the ER. PMID:23864712

Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases.

PubMed

Kauffman, Karlina J; Yu, Shenliang; Jin, Jiaxin; Mugo, Brian; Nguyen, Nathan; O'Brien, Aidan; Nag, Shanta; Lystad, Alf Håkon; Melia, Thomas J

2018-04-10

During macroautophagy/autophagy, mammalian Atg8-family proteins undergo 2 proteolytic processing events. The first exposes a COOH-terminal glycine used in the conjugation of these proteins to lipids on the phagophore, the precursor to the autophagosome, whereas the second releases the lipid. The ATG4 family of proteases drives both cleavages, but how ATG4 proteins distinguish between soluble and lipid-anchored Atg8 proteins is not well understood. In a fully reconstituted delipidation assay, we establish that the physical anchoring of mammalian Atg8-family proteins in the membrane dramatically shifts the way ATG4 proteases recognize these substrates. Thus, while ATG4B is orders of magnitude faster at processing a soluble unprimed protein, all 4 ATG4 proteases can be activated to similar enzymatic activities on lipid-attached substrates. The recognition of lipidated but not soluble substrates is sensitive to a COOH-terminal LIR motif both in vitro and in cells. We suggest a model whereby ATG4B drives very fast priming of mammalian Atg8 proteins, whereas delipidation is inherently slow and regulated by all ATG4 homologs.

Markers of inflammation, oxidative stress, and endothelial dysfunction and the 20-year cumulative incidence of early age-related macular degeneration: the Beaver Dam Eye Study.

PubMed

Klein, Ronald; Myers, Chelsea E; Cruickshanks, Karen J; Gangnon, Ronald E; Danforth, Lorraine G; Sivakumaran, Theru A; Iyengar, Sudha K; Tsai, Michael Y; Klein, Barbara E K

2014-04-01

IMPORTANCE Modifying levels of factors associated with age-related macular degeneration (AMD) may decrease the risk for visual impairment in older persons. OBJECTIVE To examine the relationships of markers of inflammation, oxidative stress, and endothelial dysfunction to the 20-year cumulative incidence of early AMD. DESIGN, SETTING, AND PARTICIPANTS This longitudinal population-based cohort study involved a random sample of 975 persons in the Beaver Dam Eye Study without signs of AMD who participated in the baseline examination in 1988-1990 and up to 4 follow-up examinations in 1993-1995, 1998-2000, 2003-2005, and 2008-2010. EXPOSURES Serum markers of inflammation (high-sensitivity C-reactive protein, tumor necrosis factor-α receptor 2, interleukin-6, and white blood cell count), oxidative stress (8-isoprostane and total carbonyl content), and endothelial dysfunction (soluble vascular cell adhesion molecule-1 and soluble intercellular adhesion molecule-1) were measured. Interactions with complement factor H (rs1061170), age-related maculopathy susceptibility 2 (rs10490924), complement component 3 (rs2230199), and complement component 2/complement factor B (rs4151667) were examined using multiplicative models. Age-related macular degeneration was assessed from fundus photographs. MAIN OUTCOMES AND MEASURES Early AMD defined by the presence of any size drusen and the presence of pigmentary abnormalities or by the presence of large-sized drusen (≥125-μm diameter) in the absence of late AMD. RESULTS The 20-year cumulative incidence of early AMD was 23.0%. Adjusting for age, sex, and other risk factors, high-sensitivity C-reactive protein (odds ratio comparing fourth with first quartile, 2.18; P = .005), tumor necrosis factor-α receptor 2 (odds ratio, 1.78; P = .04), and interleukin-6 (odds ratio, 1.78; P = .03) were associated with the incidence of early AMD. Increased incidence of early AMD was associated with soluble vascular cell adhesion molecule-1 (odds ratio per SD on the logarithmic scale, 1.21; P = .04). CONCLUSIONS AND RELEVANCE We found modest evidence of relationships of serum high-sensitivity C-reactive protein, tumor necrosis factor-α receptor 2, interleukin-6, and soluble vascular cell adhesion molecule-1 to the 20-year cumulative incidence of early AMD independent of age, smoking status, and other factors. It is not known whether these associations represent a cause and effect relationship or whether other unknown confounders accounted for the findings. Even if inflammatory processes are a cause of early AMD, it is not known whether interventions that reduce systemic inflammatory processes will reduce the incidence of early AMD.

Moraxella catarrhalis synthesizes an autotransporter that is an acid phosphatase.

PubMed

Hoopman, Todd C; Wang, Wei; Brautigam, Chad A; Sedillo, Jennifer L; Reilly, Thomas J; Hansen, Eric J

2008-02-01

Moraxella catarrhalis O35E was shown to synthesize a 105-kDa protein that has similarity to both acid phosphatases and autotransporters. The N-terminal portion of the M. catarrhalis acid phosphatase A (MapA) was most similar (the BLAST probability score was 10(-10)) to bacterial class A nonspecific acid phosphatases. The central region of the MapA protein had similarity to passenger domains of other autotransporter proteins, whereas the C-terminal portion of MapA resembled the translocation domain of conventional autotransporters. Cloning and expression of the M. catarrhalis mapA gene in Escherichia coli confirmed the presence of acid phosphatase activity in the MapA protein. The MapA protein was shown to be localized to the outer membrane of M. catarrhalis and was not detected either in the soluble cytoplasmic fraction from disrupted M. catarrhalis cells or in the spent culture supernatant fluid from M. catarrhalis. Use of the predicted MapA translocation domain in a fusion construct with the passenger domain from another predicted M. catarrhalis autotransporter confirmed the translocation ability of this MapA domain. Inactivation of the mapA gene in M. catarrhalis strain O35E reduced the acid phosphatase activity expressed by this organism, and this mutation could be complemented in trans with the wild-type mapA gene. Nucleotide sequence analysis of the mapA gene from six M. catarrhalis strains showed that this protein was highly conserved among strains of this pathogen. Site-directed mutagenesis of a critical histidine residue (H233A) in the predicted active site of the acid phosphatase domain in MapA eliminated acid phosphatase activity in the recombinant MapA protein. This is the first description of an autotransporter protein that expresses acid phosphatase activity.

Moraxella catarrhalis Synthesizes an Autotransporter That Is an Acid Phosphatase▿

PubMed Central

Hoopman, Todd C.; Wang, Wei; Brautigam, Chad A.; Sedillo, Jennifer L.; Reilly, Thomas J.; Hansen, Eric J.

2008-01-01

Moraxella catarrhalis O35E was shown to synthesize a 105-kDa protein that has similarity to both acid phosphatases and autotransporters. The N-terminal portion of the M. catarrhalis acid phosphatase A (MapA) was most similar (the BLAST probability score was 10−10) to bacterial class A nonspecific acid phosphatases. The central region of the MapA protein had similarity to passenger domains of other autotransporter proteins, whereas the C-terminal portion of MapA resembled the translocation domain of conventional autotransporters. Cloning and expression of the M. catarrhalis mapA gene in Escherichia coli confirmed the presence of acid phosphatase activity in the MapA protein. The MapA protein was shown to be localized to the outer membrane of M. catarrhalis and was not detected either in the soluble cytoplasmic fraction from disrupted M. catarrhalis cells or in the spent culture supernatant fluid from M. catarrhalis. Use of the predicted MapA translocation domain in a fusion construct with the passenger domain from another predicted M. catarrhalis autotransporter confirmed the translocation ability of this MapA domain. Inactivation of the mapA gene in M. catarrhalis strain O35E reduced the acid phosphatase activity expressed by this organism, and this mutation could be complemented in trans with the wild-type mapA gene. Nucleotide sequence analysis of the mapA gene from six M. catarrhalis strains showed that this protein was highly conserved among strains of this pathogen. Site-directed mutagenesis of a critical histidine residue (H233A) in the predicted active site of the acid phosphatase domain in MapA eliminated acid phosphatase activity in the recombinant MapA protein. This is the first description of an autotransporter protein that expresses acid phosphatase activity. PMID:18065547

Evaluation of physicochemical properties of root-end filling materials using conventional and Micro-CT tests

PubMed Central

TORRES, Fernanda Ferrari Esteves; BOSSO-MARTELO, Roberta; ESPIR, Camila Galletti; CIRELLI, Joni Augusto; GUERREIRO-TANOMARU, Juliane Maria; TANOMARU-FILHO, Mario

2017-01-01

Abstract Objective To evaluate solubility, dimensional stability, filling ability and volumetric change of root-end filling materials using conventional tests and new Micro-CT-based methods. Material and Methods 7 Results The results suggested correlated or complementary data between the proposed tests. At 7 days, BIO showed higher solubility and at 30 days, showed higher volumetric change in comparison with MTA (p<0.05). With regard to volumetric change, the tested materials were similar (p>0.05) at 7 days. At 30 days, they presented similar solubility. BIO and MTA showed higher dimensional stability than ZOE (p<0.05). ZOE and BIO showed higher filling ability (p<0.05). Conclusions ZOE presented a higher dimensional change, and BIO had greater solubility after 7 days. BIO presented filling ability and dimensional stability, but greater volumetric change than MTA after 30 days. Micro-CT can provide important data on the physicochemical properties of materials complementing conventional tests. PMID:28877275

Evasion Mechanisms Used by Pathogens to Escape the Lectin Complement Pathway

PubMed Central

Rosbjerg, Anne; Genster, Ninette; Pilely, Katrine; Garred, Peter

2017-01-01

The complement system is a crucial defensive network that protects the host against invading pathogens. It is part of the innate immune system and can be initiated via three pathways: the lectin, classical and alternative activation pathway. Overall the network compiles a group of recognition molecules that bind specific patterns on microbial surfaces, a group of associated proteases that initiates the complement cascade, and a group of proteins that interact in proteolytic complexes or the terminal pore-forming complex. In addition, various regulatory proteins are important for controlling the level of activity. The result is a pro-inflammatory response meant to combat foreign microbes. Microbial elimination is, however, not a straight forward procedure; pathogens have adapted to their environment by evolving a collection of evasion mechanisms that circumvent the human complement system. Complement evasion strategies features different ways of exploiting human complement proteins and moreover features different pathogen-derived proteins that interfere with the normal processes. Accumulated, these mechanisms target all three complement activation pathways as well as the final common part of the cascade. This review will cover the currently known lectin pathway evasion mechanisms and give examples of pathogens that operate these to increase their chance of invasion, survival and dissemination. PMID:28553281

An efficient microwave-assisted synthesis method for the production of water soluble amine-terminated Si nanoparticles.

PubMed

Atkins, Tonya M; Louie, Angelique Y; Kauzlarich, Susan M

2012-07-27

Silicon nanoparticles can be considered a green material, especially when prepared via a microwave-assisted method without the use of highly reactive reducing agents or hydrofluoric acid. A simple solution synthesis of hydrogen-terminated Si- and Mn-doped Si nanoparticles via microwave-assisted synthesis is demonstrated. The reaction of the Zintl salt, Na(4)Si(4), or Mn-doped Na(4)Si(4), Na(4)Si(4(Mn)), with ammonium bromide, NH(4)Br, produces small dispersible nanoparticles along with larger particles that precipitate. Allylamine and 1-amino-10-undecene were reacted with the hydrogen-terminated Si nanoparticles to provide water solubility and stability. A one-pot, single-reaction process and a one-pot, two-step reaction process were investigated. Details of the microwave-assisted process are provided, with the optimal synthesis being the one-pot, two-step reaction procedure and a total time of about 15 min. The nanoparticles were characterized by transmission electron microscopy (TEM), x-ray diffraction, and fluorescence spectroscopies. The microwave-assisted method reliably produces a narrow size distribution of Si nanoparticles in solution.

HETEROGENETIC ANTIBODIES IN ACUTE HEPATITIS

PubMed Central

Eaton, Monroe D.; Murphy, William D.; Hanford, V. Lee

1944-01-01

A heterogenetic antibody showing fixation of complement with human liver and agglutination of sheep erythrocytes was found in certain cases of acute infective hepatitis. The antigen concerned in these reactions was apparently heat stable and alcohol soluble. Differences from other heterogenetic antigen-antibody systems have been noted. The possible relation of the heterogenetic antibody to liver damage was considered. PMID:19871386

Influence of aggregate size on the binding and activation of the first component of human complement by soluble IgG aggregates.

PubMed Central

Doekes, G; Vanes, L A; Daha, M R

1982-01-01

The interaction between small aggregates of human IgG and the first component of human complement was studied. Stabilized soluble IgG aggregates of restricted size were prepared by heat aggregation of human IgG, followed by sucrose-density ultracentrifugation. Human C1 was isolated in its precursor form by euglobulin precipitation, followed by gel filtration and immunoadsorption. A C1 preparation was obtained of which more than 90% was still in its unactivated form. Soluble aggregates containing 20, 10 or 5 molecules IgG, and monomeric IgG were tested for their ability to bind and to activate C1. The binding of C1 was determined by C1 consumption, whereas the activation of C1 was measured as the increased ability of the C1 preparation to consume purified human C4 after the incubation with the aggregates. The three aggregates tested and monomeric IgG were all able to bind and to activate C1, but the efficiency of both processes markedly increased with increasing aggregate-size. Furthermore, it was found that all four preparations activated an appreciable amount of C1 at concentrations that did not result in any detectable C1 fixation. These results confirm earlier suggestion that C1 can be activated during a short, transient binding to small aggregates or immune complexes that have a low avidity for C1, after which the activated form, C1, is released into the medium. PMID:7068172

Measurement of soluble CD59 in CSF in demyelinating disease: Evidence for an intrathecal source of soluble CD59.

PubMed

Zelek, Wioleta M; Watkins, Lewis M; Howell, Owain W; Evans, Rhian; Loveless, Sam; Robertson, Neil P; Beenes, Marijke; Willems, Loek; Brandwijk, Ricardo; Morgan, B Paul

2018-02-01

CD59, a broadly expressed glycosylphosphatidylinositol-anchored protein, is the principal cell inhibitor of complement membrane attack on cells. In the demyelinating disorders, multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD), elevated complement protein levels, including soluble CD59 (sCD59), were reported in cerebrospinal fluid (CSF). We compared sCD59 levels in CSF and matched plasma in controls and patients with MS, NMOSD and clinically isolated syndrome (CIS) and investigated the source of CSF sCD59 and whether it was microparticle associated. sCD59 was quantified using enzyme-linked immunosorbent assay (ELISA; Hycult; HK374-02). Patient and control CSF was subjected to western blotting to characterise anti-CD59-reactive materials. CD59 was localised by immunostaining and in situ hybridisation. CSF sCD59 levels were double those in plasma (CSF, 30.2 ng/mL; plasma, 16.3 ng/mL). Plasma but not CSF sCD59 levels differentiated MS from NMOSD, MS from CIS and NMOSD/CIS from controls. Elimination of microparticles confirmed that CSF sCD59 was not membrane anchored. CSF levels of sCD59 are not a biomarker of demyelinating diseases. High levels of sCD59 in CSF relative to plasma suggest an intrathecal source; CD59 expression in brain parenchyma was low, but expression was strong on choroid plexus (CP) epithelium, immediately adjacent the CSF, suggesting that this is the likely source.

Molecular Genetic Analysis of an Endotoxin Nonresponder Mutant Cell Line

PubMed Central

Schromm, Andra B.; Lien, Egil; Henneke, Philipp; Chow, Jesse C.; Yoshimura, Atsutoshi; Heine, Holger; Latz, Eicke; Monks, Brian G.; Schwartz, David A.; Miyake, Kensuke; Golenbock, Douglas T.

2001-01-01

Somatic cell mutagenesis is a powerful tool for characterizing receptor systems. We reported previously two complementation groups of mutant cell lines derived from CD14-transfected Chinese hamster ovary–K1 fibroblasts defective in responses to bacterial endotoxin. Both classes of mutants expressed a normal gene product for Toll-like receptor (TLR)4, and fully responded to stimulation by tumor necrosis factor (TNF)-α or interleukin (IL)-1β. We identified the lesion in one of the complementation groups in the gene for MD-2, a putative TLR4 coreceptor. The nonresponder phenotype of this mutant was reversed by transfection with MD-2. Cloning of MD-2 from the nonresponder cell line revealed a point mutation in a highly conserved region resulting in a C95Y amino acid exchange. Both forms of MD-2 colocalized with TLR4 on the cell surface after transfection, but only the wild-type cDNA reverted the lipopolysaccharide (LPS) nonresponder phenotype. Furthermore, soluble MD-2, but not soluble MD-2C95Y, functioned to enable LPS responses in cells that expressed TLR4. Thus, MD-2 is a required component of the LPS signaling complex and can function as a soluble receptor for cells that do not otherwise express it. We hypothesize that MD-2 conformationally affects the extracellular domain of TLR4, perhaps resulting in a change in affinity for LPS or functioning as a portion of the true ligand for TLR4. PMID:11435474

Formulation and evaluation of biodegradable nanoparticles for the oral delivery of fenretinide.

PubMed

Graves, Richard A; Ledet, Grace A; Glotser, Elena Y; Mitchner, Demaurian M; Bostanian, Levon A; Mandal, Tarun K

2015-08-30

Fenretinide is an anticancer drug with low water solubility and poor bioavailability. The goal of this study was to develop biodegradable polymeric nanoparticles of fenretinide with the intent of increasing its apparent aqueous solubility and intestinal permeability. Three biodegradable polymers were investigated for this purpose: two different poly lactide-co-glycolide (PLGA) polymers, one acid terminated and one ester terminated, and one poly lactide-co-glycolide/polyethylene glycol (PLGA/PEG) diblock copolymer. Nanoparticles were obtained by using an emulsification solvent evaporation technique. The formulations were characterized by differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and particle size analysis. Dissolution studies and Caco-2 cell permeation studies were also carried out for all formulations. Ultra high performance liquid chromatography coupled with mass spectrometry (UPLC/MS) and ultraviolet detection was used for the quantitative determination of fenretinide. Drug loading and the type of polymer affected the nanoparticles' physical properties, drug release rate, and cell permeability. While the acid terminated PLGA nanoparticles performed the best in drug release, the ester terminated PLGA nanoparticles performed the best in the Caco-2 cell permeability assays. The PLGA/PEG copolymer nanoparticles performed better than the formulations with ester terminated PLGA in terms of drug release but had the poorest performance in terms of cell permeation. All three categories of formulations performed better than the drug alone in both drug release and cell permeation studies. Copyright © 2015 Elsevier B.V. All rights reserved.

Acquisition of negative complement regulators by the saprophyte Leptospira biflexa expressing LigA or LigB confers enhanced survival in human serum

PubMed Central

Castiblanco-Valencia, Mónica M.; Fraga, Tatiana R.; Breda, Leandro C.D.; Vasconcellos, Sílvio A.; Figueira, Cláudio P.; Picardeau, Mathieu; Wunder, Elsio; Ko, Albert I.; Barbosa, Angela S.; Isaac, Lourdes

2017-01-01

Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules present in pathogenic but not in saprophytic Leptospira species. We have previously shown that Lig proteins interact with the soluble complement regulators Factor H (FH), FH like-1 (FHL-1), FH related-1 (FHR-1) and C4b Binding Protein (C4BP). In this study, we used the saprophyte L. biflexa serovar Patoc as a surrogate host to address the specific role of LigA and LigB proteins in leptospiral complement evasion. L. biflexa expressing LigA or LigB was able to acquire FH and C4BP. Bound complement regulators retained their cofactor activities of FI in the proteolytic cleavage of C3b and C4b. Moreover, heterologous expression of ligA and ligB genes in the saprophyte L. biflexa enhanced bacterial survival in human serum. Complement deposition on lig-transformed L. biflexa was assessed by flow cytometry analysis. With regard to MAC deposition, L. biflexa expressing LigA or LigB presented an intermediate profile: MAC deposition levels were greater than those found in the pathogenic L. interrogans, but lower than those observed for L. biflexa wildtype. In conclusion, Lig proteins contribute to in vitro control of complement activation on the leptospiral surface, promoting an increased bacterial survival in human serum. PMID:26976804

The profile of adsorbed plasma and serum proteins on methacrylic acid copolymer beads: Effect on complement activation.

PubMed

Wells, Laura A; Guo, Hongbo; Emili, Andrew; Sefton, Michael V

2017-02-01

Polymer beads made of 45% methacrylic acid co methyl methacrylate (MAA beads) promote vascular regenerative responses in contrast to control materials without methacrylic acid (here polymethyl methacrylate beads, PMMA). In vitro and in vivo studies suggest that MAA copolymers induce differences in macrophage phenotype and polarization and inflammatory responses, presumably due to protein adsorption differences between the beads. To explore differences in protein adsorption in an unbiased manner, we used high resolution shotgun mass spectrometry to identify and compare proteins that adsorb from human plasma or serum onto MAA and PMMA beads. From plasma, MAA beads adsorbed many complement proteins, such as C1q, C4-related proteins and the complement inhibitor factor H, while PMMA adsorbed proteins, such as albumin, C3 and apolipoproteins. Because of the differences in complement protein adsorption, follow-up studies focused on using ELISA to assess complement activation. When incubated in serum, MAA beads generated significantly lower levels of soluble C5b9 and C3a/C3a desarg in comparison to PMMA beads, indicating a decrease in complement activation with MAA beads. The differences in adsorbed protein on the two materials likely alter subsequent cell-material interactions that ultimately result in different host responses and local vascularization. Copyright © 2016 Elsevier Ltd. All rights reserved.

The use of small-molecule structures to complement protein–ligand crystal structures in drug discovery

PubMed Central

Cole, Jason C.

2017-01-01

Many ligand-discovery stories tell of the use of structures of protein–ligand complexes, but the contribution of structural chemistry is such a core part of finding and improving ligands that it is often overlooked. More than 800 000 crystal structures are available to the community through the Cambridge Structural Database (CSD). Individually, these structures can be of tremendous value and the collection of crystal structures is even more helpful. This article provides examples of how small-molecule crystal structures have been used to complement those of protein–ligand complexes to address challenges ranging from affinity, selectivity and bioavailability though to solubility. PMID:28291759

Solubility and dissolution kinetics of GaN in supercritical ammonia in presence of ammonoacidic and ammonobasic mineralizers

NASA Astrophysics Data System (ADS)

Schimmel, Saskia; Koch, Martina; Macher, Philipp; Kimmel, Anna-Carina L.; Steigerwald, Thomas G.; Alt, Nicolas S. A.; Schlücker, Eberhard; Wellmann, Peter

2017-12-01

Solubility and dissolution kinetics of GaN are investigated, as they represent essential parameters for ammonothermal crystal growth of GaN. In situ X-ray imaging is applied to monitor the dissolving crystal. Both ammonoacidic and ammonobasic conditions are investigated. Compared to NH4F, the dissolution is generally much slower using NaN3 mineralizer, leading to a much longer time needed to establish a saturated solution. The solubility of GaN at 540 °C and 260 MPa in supercritical ammonia with a molar concentration of NaN3 of 0.72 mmol/ml is determined to be 0.15 ± 0.01 mol%. This suggest a severe refinement of raw gravimetric literature data also for alkali metal based mineralizers, as we reported previously for ammonium halide mineralizers. The order of magnitude is in good agreement with refined gravimetric solubility data (Griffiths et al., 2016). The apparent discrepancy between the literature and this work regarding the temperature range in which retrograde solubility occurs is discussed. A possible reason for the occurrence of retrograde solubility at high temperatures is described. The paper is complemented by a section pointing out and partially quantifying potential, reactor-material-dependent sources of errors.

Genetic Map of Bacteriophage α

PubMed Central

Kejzlarovà, J.; Donini, P.; Eremenko-Volpe, T.; Graziosi, F.

1970-01-01

Temperature-sensitive mutants of phage α were obtained by means of various mutagens and assigned to 25 complementation groups. Temperature-sensitive mutants belonging to 21 complementation groups and a mutant giving turbid plaques were used to perform two- and three-factor crosses. Seventeen of the cistrons and the turbid mutant were shown to belong to the same linear linkage group, which showed no signs of circularity. The remaining four unlinked cistrons showed peculiarities in their recombination properties. Genes which are known to be expressed earlier apear to be grouped together in a terminal segment of the linkage group. PMID:4990532

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saha, Pritam; Anderson, Alan J.; Lee, Thomas

The solubility of tugarinovite (MoO 2) in pure water was investigated at temperatures between 400 and 800°C and at pressures ranging between 95 and 480 MPa by using in situ synchrotron X-ray fluorescence (SXRF) to separately analyze high temperature aqueous solutions in a hydrothermal diamond anvil cell (HDAC). The concentration of molybdenum in the fluid at 400 and 500°C was below detection; however, at temperatures between 600 and 800°C, the solubility of tugarinovite increased with increasing temperature by two orders of magnitude. The molybdenum concentration at 600°C and 800°C is 44 ppm and 658 ppm, respectively. The results complement themore » data of Kudrin (1985) and provide the first measurement of MoO 2solubility at pressure and temperature conditions comparable to intrusion-related Mo deposit formation. The data are also relevant to the study of water chemistry and corrosion product transport in supercritical-water-cooled reactors, where Mo-bearing steel alloys interact with aqueous solutions at temperatures greater than 600°C. The application of in situ SXRF to solubility measurements of sparingly soluble minerals is recommended because it circumvents analytical uncertainties inherent in determinations obtained by quenching and weight loss measurements.« less

Analysis of correlated domain motions in IgG light chain reveals possible mechanisms of immunological signal transduction.

PubMed

Król, Marcin; Roterman, Irena; Piekarska, Barbara; Konieczny, Leszek; Rybarska, Janina; Stopa, Barbara; Spólnik, Paweł

2005-05-15

It was shown experimentally that binding of a micelle composed of Congo red molecules to immunological complexes leads to the enhanced stability of the latter, and simultaneously prevents binding of a complement molecule (C1q). The dye binds in a cavity created by the removal of N-terminal polypeptide chain, as observed experimentally in a model system-immunoglobulin G (IgG) light chain dimer. Molecular Dynamics (MD) simulations of three forms of IgG light chain dimer, with and without the dye, were performed to investigate the role of N-terminal fragment and self-assembled ligand in coupling between V and C domains. Root-mean-square distance (RMSD) time profiles show that removal of N-terminal fragment leads to destabilization of V domain. A micelle composed of four self-assembled dye molecules stabilizes and fixes the domain. Analysis of root-mean-square fluctuation (RMSF) values and dynamic cross-correlation matrices (DCCM) reveals that removal of N-terminal fragment results in complete decoupling between V and C domains. Binding of self-assembled Congo red molecules improves the coupling, albeit slightly. The disruption of a small beta-sheet composed of N- and C-terminal fragments of the domain (NC sheet) is the most likely reason for the decoupling. Self-assembled ligand, bound in the place originally occupied by N-terminal fragment, is not able to take over the function of the beta-sheet. Lack of correlation of motions between residues in V and C domains denotes that light chain-Congo red complexes have hampered ability to transmit conformational changes between domains. This is a likely explanation of the lack of complement binding by immunological complexes, which bind Congo red, and supports the idea that the NC sheet is the key structural fragment taking part in immunological signal transduction. Copyright 2005 Wiley-Liss, Inc.

Electroluminescent TCC, C3dg and fB/Bb epitope assays for profiling complement cascade activation in vitro using an activated complement serum calibration standard.

PubMed

van Vuuren, B Jansen; Bergseth, G; Mollnes, T E; Shaw, A M

2014-01-15

Electroluminescent assays for epitopes on the complement components C3dg, terminal complement complex (TCC) and factor B/Bb (fB/Bb) have been developed with capture and detection antibodies to produce detection limits C3dg=91±9ng/mL, TCC=3±0.1ng/mL and fB=55.7±0.1ng/mL. The assay performance was assessed against a series of zymosan and heat aggregated IgG (HAIgG) in vitro activations of complement using a calibrated activated complement serum (ACS) as calibration standard. The ACS standard was stable within 20% accuracy over a 6-month period with freeze-thaw cycles as required. Differential activation of the complement cascade was observed for TCC showing a pseudo-first order formation half-life of 3.5h after activation with zymosan. The C3dg activation fragment indicates a 10% total activation for both activation agents. The kinetic-epitope analysis for fB indicates that the capture epitope is on the fB/Bb protein fragment which can then become covered by the formation of C3bBb or C3bBbP complexes during the time course of the cascade. Copyright © 2013 Elsevier B.V. All rights reserved.

Susceptibility of pathogenic and nonpathogenic Naegleria ssp

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whiteman, L.Y.

1988-01-01

The susceptibility of four species of Naegleria amoebae to complement-mediated lysis was determined. The amoebicidal activity of normal human serum (NHS) and normal guinea pig serum (NGPS) for Naegleria amoebae was measured by an in vitro cytotoxicity assay. Release of radioactivity from amoebae labeled with {sup 3}H-uridine and visual observation with a compound microscope were used as indices of lysis. Susceptibility or resistance to complement-mediated lysis in vitro correlated with the in vivo pathogenic potential. Nonpathogenic Naegleria amoebae were lysed at a faster rate and at higher cell concentrations than were pathogenic amoebae. Electrophoretic analysis of NHS incubated with pathogenicmore » or nonpathogenic Naegleria spp. demonstrated that amoebae activate the complement cascade resulting in the production of C3 and C5 complement cleavage products. Treatment with papain or trypsin for 1 h, but not with sialidase, increase the susceptibility of highly pathogenic, mouse-passaged N. fowleri to lysis. Treatment with actinomycin D, cycloheximide or various protease inhibitors for 4 h did not increase susceptibility to lysis. Neither a repair process involving de novo protein synthesis nor a complement-inactivating protease appear to account for the increase resistance of N. fowleri amoebae to complement-mediated lysis. A binding study with {sup 125}I radiolabeled C9 indicated that the terminal complement component does not remain stably bound to the membrane of pathogenic amoebae.« less

Protective responses to sublytic complement in the retinal pigment epithelium

PubMed Central

Tan, Li Xuan; Toops, Kimberly A.; Lakkaraju, Aparna

2016-01-01

The retinal pigment epithelium (RPE) is a key site of injury in inherited and age-related macular degenerations. Abnormal activation of the complement system is a feature of these blinding diseases, yet how the RPE combats complement attack is poorly understood. The complement cascade terminates in the cell-surface assembly of membrane attack complexes (MACs), which promote inflammation by causing aberrant signal transduction. Here, we investigated mechanisms crucial for limiting MAC assembly and preserving cellular integrity in the RPE and asked how these are compromised in models of macular degeneration. Using polarized primary RPE and the pigmented Abca4−/− Stargardt disease mouse model, we provide evidence for two protective responses occurring within minutes of complement attack, which are essential for maintaining mitochondrial health in the RPE. First, accelerated recycling of the membrane-bound complement regulator CD59 to the RPE cell surface inhibits MAC formation. Second, fusion of lysosomes with the RPE plasma membrane immediately after complement attack limits sustained elevations in intracellular calcium and prevents mitochondrial injury. Cholesterol accumulation in the RPE, induced by vitamin A dimers or oxidized LDL, inhibits these defense mechanisms by activating acid sphingomyelinase (ASMase), which increases tubulin acetylation and derails organelle traffic. Defective CD59 recycling and lysosome exocytosis after complement attack lead to mitochondrial fragmentation and oxidative stress in the RPE. Drugs that stimulate cholesterol efflux or inhibit ASMase restore both these critical safeguards in the RPE and avert complement-induced mitochondrial injury in vitro and in Abca4−/− mice, indicating that they could be effective therapeutic approaches for macular degenerations. PMID:27432952

Structural basis for complement evasion by Lyme disease pathogen Borrelia burgdorferi.

PubMed

Bhattacharjee, Arnab; Oeemig, Jesper S; Kolodziejczyk, Robert; Meri, Taru; Kajander, Tommi; Lehtinen, Markus J; Iwaï, Hideo; Jokiranta, T Sakari; Goldman, Adrian

2013-06-28

Borrelia burgdorferi spirochetes that cause Lyme borreliosis survive for a long time in human serum because they successfully evade the complement system, an important arm of innate immunity. The outer surface protein E (OspE) of B. burgdorferi is needed for this because it recruits complement regulator factor H (FH) onto the bacterial surface to evade complement-mediated cell lysis. To understand this process at the molecular level, we used a structural approach. First, we solved the solution structure of OspE by NMR, revealing a fold that has not been seen before in proteins involved in complement regulation. Next, we solved the x-ray structure of the complex between OspE and the FH C-terminal domains 19 and 20 (FH19-20) at 2.83 Å resolution. The structure shows that OspE binds FH19-20 in a way similar to, but not identical with, that used by endothelial cells to bind FH via glycosaminoglycans. The observed interaction of OspE with FH19-20 allows the full function of FH in down-regulation of complement activation on the bacteria. This reveals the molecular basis for how B. burgdorferi evades innate immunity and suggests how OspE could be used as a potential vaccine antigen.

Innate immune humoral factors, C1q and factor H, with differential pattern recognition properties, alter macrophage response to carbon nanotubes.

PubMed

Pondman, Kirsten M; Pednekar, Lina; Paudyal, Basudev; Tsolaki, Anthony G; Kouser, Lubna; Khan, Haseeb A; Shamji, Mohamed H; Ten Haken, Bennie; Stenbeck, Gudrun; Sim, Robert B; Kishore, Uday

2015-11-01

Interaction between the complement system and carbon nanotubes (CNTs) can modify their intended biomedical applications. Pristine and derivatised CNTs can activate complement primarily via the classical pathway which enhances uptake of CNTs and suppresses pro-inflammatory response by immune cells. Here, we report that the interaction of C1q, the classical pathway recognition molecule, with CNTs involves charge pattern and classical pathway activation that is partly inhibited by factor H, a complement regulator. C1q and its globular modules, but not factor H, enhanced uptake of CNTs by macrophages and modulated the pro-inflammatory immune response. Thus, soluble complement factors can interact differentially with CNTs and alter the immune response even without complement activation. Coating CNTs with recombinant C1q globular heads offers a novel way of controlling classical pathway activation in nanotherapeutics. Surprisingly, the globular heads also enhance clearance by phagocytes and down-regulate inflammation, suggesting unexpected complexity in receptor interaction. Carbon nanotubes (CNTs) maybe useful in the clinical setting as targeting drug carriers. However, it is also well known that they can interact and activate the complement system, which may have a negative impact on the applicability of CNTs. In this study, the authors functionalized multi-walled CNT (MWNT), and investigated the interaction with the complement pathway. These studies are important so as to gain further understanding of the underlying mechanism in preparation for future use of CNTs in the clinical setting. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Structural studies and molecular dynamics simulations suggest a processive mechanism of exolytic lytic transglycosylase from Campylobacter jejuni.

PubMed

Vijayaraghavan, Jagamya; Kumar, Vijay; Krishnan, Nikhil P; Kaufhold, Ross T; Zeng, Ximin; Lin, Jun; van den Akker, Focco

2018-01-01

The bacterial soluble lytic transglycosylase (LT) breaks down the peptidoglycan (PG) layer during processes such as cell division. We present here crystal structures of the soluble LT Cj0843 from Campylobacter jejuni with and without bulgecin A inhibitor in the active site. Cj0843 has a doughnut shape similar but not identical to that of E. coli SLT70. The C-terminal catalytic domain is preceded by an L-domain, a large helical U-domain, a flexible linker, and a small N-terminal NU-domain. The flexible linker allows the NU-domain to reach over and complete the circular shape, using residues conserved in the Epsilonproteobacteria LT family. The inner surface of the Cj0843 doughnut is mostly positively charged including a pocket that has 8 Arg/Lys residues. Molecular dynamics simulations with PG strands revealed a potential functional role for this pocket in anchoring the negatively charged terminal tetrapeptide of the PG during several steps in the reaction including homing and aligning the PG strand for exolytic cleavage, and subsequent ratcheting of the PG strand to enhance processivity in degrading PG strands.

The intermembrane space domain of Tim23 is intrinsically disordered with a distinct binding region for presequences

PubMed Central

de la Cruz, Laura; Bajaj, Rakhi; Becker, Stefan; Zweckstetter, Markus

2010-01-01

Proteins targeted to the mitochondrial matrix are translocated through the outer and the inner mitochondrial membranes by two protein complexes, the translocase of the outer membrane (TOM) and one of the translocases of the inner membrane (TIM23). The protein Tim23, the core component of TIM23, consists of an N-terminal, soluble domain in the intermembrane space (IMS) and a C-terminal domain that forms the import pore across the inner membrane. Before translocation proceeds, precursor proteins are recognized by the N-terminal domain of Tim23, Tim23N (residues 1–96). By using NMR spectroscopy, we show that Tim23N is a monomeric protein belonging to the family of intrinsically disordered proteins. Titrations of Tim23N with two presequences revealed a distinct binding region of Tim23N formed by residues 71–84. In a charge-hydropathy plot containing all soluble domains of TOM and TIM23, Tim23N was found to be the only domain with more than 40 residues in the IMS that is predicted to be intrinsically disordered, suggesting that Tim23N might function as hub in the mitochondrial import machinery protein network. PMID:20718036

Single point mutations distributed in 10 soluble and membrane regions of the Nicotiana plumbaginifolia plasma membrane PMA2 H+-ATPase activate the enzyme and modify the structure of the C-terminal region.

PubMed

Morsomme, P; Dambly, S; Maudoux, O; Boutry, M

1998-12-25

The Nicotiana plumbaginifolia pma2 (plasma membrane H+-ATPase) gene is capable of functionally replacing the H+-ATPase genes of the yeast Saccharomyces cerevisiae, provided that the external pH is kept above 5.0. Single point mutations within the pma2 gene were previously identified that improved H+-ATPase activity and allowed yeast growth at pH 4.0. The aim of the present study was to identify most of the PMA2 positions, the mutation of which would lead to improved growth and to determine whether all these mutations result in similar enzymatic and structural modifications. We selected additional mutants in total 42 distinct point mutations localized in 30 codons. They were distributed in 10 soluble and membrane regions of the enzyme. Most mutant PMA2 H+-ATPases were characterized by a higher specific activity, lower inhibition by ADP, and lower stimulation by lysophosphatidylcholine than wild-type PMA2. The mutants thus seem to be constitutively activated. Partial tryptic digestion and immunodetection showed that the PMA2 mutants had a conformational change making the C-terminal region more accessible. These data therefore support the hypothesis that point mutations in various H+-ATPase parts displace the inhibitory C-terminal region, resulting in enzyme activation. The high density of mutations within the first half of the C-terminal region suggests that this part is involved in the interaction between the inhibitory C-terminal region and the rest of the enzyme.

Acquisition of negative complement regulators by the saprophyte Leptospira biflexa expressing LigA or LigB confers enhanced survival in human serum.

PubMed

Castiblanco-Valencia, Mónica M; Fraga, Tatiana R; Breda, Leandro C D; Vasconcellos, Sílvio A; Figueira, Cláudio P; Picardeau, Mathieu; Wunder, Elsio; Ko, Albert I; Barbosa, Angela S; Isaac, Lourdes

2016-05-01

Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules present in pathogenic but not in saprophytic Leptospira species. We have previously shown that Lig proteins interact with the soluble complement regulators Factor H (FH), FH like-1 (FHL-1), FH related-1 (FHR-1) and C4b Binding Protein (C4BP). In this study, we used the saprophyte L. biflexa serovar Patoc as a surrogate host to address the specific role of LigA and LigB proteins in leptospiral complement evasion. L. biflexa expressing LigA or LigB was able to acquire FH and C4BP. Bound complement regulators retained their cofactor activities of FI in the proteolytic cleavage of C3b and C4b. Moreover, heterologous expression of ligA and ligB genes in the saprophyte L. biflexa enhanced bacterial survival in human serum. Complement deposition on lig-transformed L. biflexa was assessed by flow cytometry analysis. With regard to MAC deposition, L. biflexa expressing LigA or LigB presented an intermediate profile: MAC deposition levels were greater than those found in the pathogenic L. interrogans, but lower than those observed for L. biflexa wildtype. In conclusion, Lig proteins contribute to in vitro control of complement activation on the leptospiral surface, promoting an increased bacterial survival in human serum. Copyright © 2016 European Federation of Immunological Societies. All rights reserved.

Site-directed mutagenesis of the chemokine receptor CXCR6 suggests a novel paradigm for interactions with the ligand CXCL16.

PubMed

Petit, Sarah J; Chayen, Naomi E; Pease, James E

2008-08-01

Chemokine receptor CXCR6 mediates the chemotaxis and adhesion of leukocytes to soluble and membrane-anchored forms of CXCL16, and is an HIV-1 co-receptor. Here, we describe the effects of mutation of acidic extracellular CXCR6 residues on receptor function. Although most CXCR6 mutants examined were expressed at levels similar to wild-type (WT) CXCR6, an N-terminal E3Q mutant was poorly expressed, which may explain previously reported protective effects of a similar single nucleotide polymorphism, with respect to late-stage HIV-1 infection. In contrast to several other chemokine receptors, mutation of the CXCR6 N terminus and inhibition of post-translational modifications of this region were without effect on receptor function. Likewise, N-terminal extension of CXCL16 resulted in a protein with decent potency and efficacy in chemotaxis and not, as anticipated, a CXCR6 antagonist. D176N and E274Q CXCR6 mutants were unable to interact with soluble CXCL16, suggesting a critical role for D176 and E274 in ligand binding. Intriguingly, although unable to interact with soluble CXCL16, the E274Q mutant could promote robust adhesion to membrane-anchored CXCL16, suggesting that soluble and membrane-bound forms of CXCL16 possess distinct conformations. Collectively, our data suggest a novel paradigm for the CXCR6:CXCL16 interaction, a finding which may impact the discovery of small-molecule antagonists of CXCR6.

Tandem SUMO fusion vectors for improving soluble protein expression and purification.

PubMed

Guerrero, Fernando; Ciragan, Annika; Iwaï, Hideo

2015-12-01

Availability of highly purified proteins in quantity is crucial for detailed biochemical and structural investigations. Fusion tags are versatile tools to facilitate efficient protein purification and to improve soluble overexpression of proteins. Various purification and fusion tags have been widely used for overexpression in Escherichia coli. However, these tags might interfere with biological functions and/or structural investigations of the protein of interest. Therefore, an additional purification step to remove fusion tags by proteolytic digestion might be required. Here, we describe a set of new vectors in which yeast SUMO (SMT3) was used as the highly specific recognition sequence of ubiquitin-like protease 1, together with other commonly used solubility enhancing proteins, such as glutathione S-transferase, maltose binding protein, thioredoxin and trigger factor for optimizing soluble expression of protein of interest. This tandem SUMO (T-SUMO) fusion system was tested for soluble expression of the C-terminal domain of TonB from different organisms and for the antiviral protein scytovirin. Copyright © 2015 Elsevier Inc. All rights reserved.

NMR assignments of the N-terminal domain of Nephila clavipes spidroin 1

PubMed Central

Parnham, Stuart; Gaines, William A.; Duggan, Brendan M.; Marcotte, William R.

2011-01-01

The building blocks of spider dragline silk are two fibrous proteins secreted from the major ampullate gland named spidroins 1 and 2 (MaSp1, MaSp2). These proteins consist of a large central domain composed of approximately 100 tandem copies of a 35–40 amino acid repeat sequence. Non-repetitive N and C-terminal domains, of which the C-terminal domain has been implicated to transition from soluble and insoluble states during spinning, flank the repetitive core. The N-terminal domain until recently has been largely unknown due to difficulties in cloning and expression. Here, we report nearly complete assignment for all 1H, 13C, and 15N resonances in the 14 kDa N-terminal domain of major ampullate spidroin 1 (MaSp1-N) of the golden orb-web spider Nephila clavipes. PMID:21152998

Complementation analysis of mutants of nitric oxide synthase reveals that the active site requires two hemes.

PubMed Central

Xie, Q W; Leung, M; Fuortes, M; Sassa, S; Nathan, C

1996-01-01

For catalytic activity, nitric oxide synthases (NOSs) must be dimeric. Previous work revealed that the requirements for stable dimerization included binding of tetrahydrobiopterin (BH4), arginine, and heme. Here we asked what function is served by dimerization. We assessed the ability of individually inactive mutants of mouse inducible NOS (iNOS; NOS2), each deficient in binding a particular cofactor or cosubstrate, to complement each other by generating NO upon cotransfection into human epithelial cells. The ability of the mutants to homodimerize was gauged by gel filtration and/or PAGE under partially denaturing conditions, both followed by immunoblot. Their ability to heterodimerize was assessed by coimmunoprecipitation. Heterodimers that contained only one COOH-terminal hemimer and only one BH4-binding site could both form and function, even though the NADPH-, FAD-, and FMN-binding domains (in the COOH-terminal hemimer) and the BH4-binding sites (in the NH2-terminal hemimer) were contributed by opposite chains. Heterodimers that contained only one heme-binding site (Cys-194) could also form, either in cis or in trans to the nucleotide-binding domains. However, for NO production, both chains had to bind heme. Thus, NO production by iNOS requires dimerization because the active site requires two hemes. Images Fig. 2 Fig. 3 Fig. 4 Fig. 7 PMID:8643499

Structure and Function of Plasmodium falciparum malate dehydrogenase: Role of Critical Amino Acids in C-substrate Binding Procket

USDA-ARS?s Scientific Manuscript database

Malaria parasite thrives on anaerobic fermentation of glucose for energy. Earlier studies from our lab have demonstrated that a cytosolic malate dehydrogenase (PfMDH) with striking similarity to lactate dehydrogenase (PfLDH) might complement PfLDH function in Plasmodium falciparum. The N-terminal g...

Using liquid and solid state NMR and photoluminescence to study the synthesis and solubility properties of amine capped silicon nanoparticles.

PubMed

Giuliani, J R; Harley, S J; Carter, R S; Power, P P; Augustine, M P

2007-08-01

Water soluble silicon nanoparticles were prepared by the reaction of bromine terminated silicon nanoparticles with 3-(dimethylamino)propyl lithium and characterized with liquid and solid state nuclear magnetic resonance (NMR) and photoluminescence (PL) spectroscopies. The surface site dependent 29Si chemical shifts and the nuclear spin relaxation rates from an assortment of 1H-29Si heteronuclear solid state NMR experiments for the amine coated reaction product are consistent with both the 1H and 13C liquid state NMR results and routine transmission electron microscopy, ultra-violet/visible, and Fourier transform infrared measurements. PL was used to demonstrate the pH dependent solubility properties of the amine passivated silicon nanoparticles.

Distinct contributions of complement factors to platelet activation and fibrin formation in venous thrombus development

PubMed Central

Jäckel, Sven; Saffarzadeh, Mona; Langer, Florian

2017-01-01

Expanding evidence indicates multiple interactions between the hemostatic system and innate immunity, and the coagulation and complement cascades. Here we show in a tissue factor (TF)–dependent model of flow restriction-induced venous thrombosis that complement factors make distinct contributions to platelet activation and fibrin deposition. Complement factor 3 (C3) deficiency causes prolonged bleeding, reduced thrombus incidence, thrombus size, fibrin and platelet deposition in the ligated inferior vena cava, and diminished platelet activation in vitro. Initial fibrin deposition at the vessel wall over 6 hours in this model was dependent on protein disulfide isomerase (PDI) and TF expression by myeloid cells, but did not require neutrophil extracellular trap formation involving peptidyl arginine deiminase 4. In contrast to C3−/− mice, C5-deficient mice had no apparent defect in platelet activation in vitro, and vessel wall platelet deposition and initial hemostasis in vivo. However, fibrin formation, the exposure of negatively charged phosphatidylserine (PS) on adherent leukocytes, and clot burden after 48 hours were significantly reduced in C5−/− mice compared with wild-type controls. These results delineate that C3 plays specific roles in platelet activation independent of formation of the terminal complement complex and provide in vivo evidence for contributions of complement-dependent membrane perturbations to prothrombotic TF activation on myeloid cells. PMID:28223279

Translational Mini-Review Series on Complement Factor H: Structural and functional correlations for factor H

PubMed Central

Schmidt, C Q; Herbert, A P; Hocking, H G; Uhrín, D; Barlow, P N

2008-01-01

The 155-kDa glycoprotein, complement factor H (CFH), is a regulator of complement activation that is abundant in human plasma. Three-dimensional structures of over half the 20 complement control protein (CCP) modules in CFH have been solved in the context of single-, double- and triple-module segments. Proven binding sites for C3b occupy the N and C termini of this elongated molecule and may be brought together by a bend in CFH mediated by its central CCP modules. The C-terminal CCP 20 is key to the ability of the molecule to adhere to polyanionic markers on self-surfaces where CFH acts to regulate amplification of the alternative pathway of complement. The surface patch on CCP 20 that binds to model glycosaminoglycans has been mapped using nuclear magnetic resonance (NMR), as has a second glycosaminoglycan-binding patch on CCP 7. These patches include many of the residue positions at which sequence variations have been linked to three complement-mediated disorders: dense deposit disease, age-related macular degeneration and atypical haemolytic uraemic syndrome. In one plausible model, CCP 20 anchors CFH to self-surfaces via a C3b/polyanion composite binding site, CCP 7 acts as a ‘proof-reader’ to help discriminate self- from non-self patterns of sulphation, and CCPs 1–4 disrupt C3/C5 convertase formation and stability. PMID:18081691

Droplet-based microreactor for synthesis of water-soluble Ag₂S quantum dots.

PubMed

Shu, Yun; Jiang, Peng; Pang, Dai-Wen; Zhang, Zhi-Ling

2015-07-10

A droplet-based microreactor was used for synthesis of water-soluble Ag2S quantum dots (QDs). Monodispersed Ag2S nanoparticles with a surface of carboxylic acid-terminated were synthesized in the droplet microreactor. The x-ray powder diffraction results indicated products were monoclinic Ag2S nanocrystals. Furthermore, different-sized Ag2S QDs that were near-infrared-emitting or visible-emitting were continuously stably synthesized in droplet microreactors at different temperatures. We believe we offer a new method for obtaining different-sized Ag2S nanoparticles.

Droplet-based microreactor for synthesis of water-soluble Ag2S quantum dots

NASA Astrophysics Data System (ADS)

Shu, Yun; Jiang, Peng; Pang, Dai-Wen; Zhang, Zhi-Ling

2015-07-01

A droplet-based microreactor was used for synthesis of water-soluble Ag2S quantum dots (QDs). Monodispersed Ag2S nanoparticles with a surface of carboxylic acid-terminated were synthesized in the droplet microreactor. The x-ray powder diffraction results indicated products were monoclinic Ag2S nanocrystals. Furthermore, different-sized Ag2S QDs that were near-infrared-emitting or visible-emitting were continuously stably synthesized in droplet microreactors at different temperatures. We believe we offer a new method for obtaining different-sized Ag2S nanoparticles.

Synthetic Method for Oligonucleotide Block by Using Alkyl-Chain-Soluble Support.

PubMed

Matsuno, Yuki; Shoji, Takao; Kim, Shokaku; Chiba, Kazuhiro

2016-02-19

A straightforward method for the synthesis of oligonucleotide blocks using a Cbz-type alkyl-chain-soluble support (Z-ACSS) attached to the 3'-OH group of 3'-terminal nucleosides was developed. The Z-ACSS allowed for the preparation of fully protected deoxyribo- and ribo-oligonucleotides without chromatographic purification and released dimer- to tetramer-size oligonucleotide blocks via hydrogenation using a Pd/C catalyst without significant loss or migration of protective groups such as 5'-end 4,4'-dimethoxtrityl, 2-cyanoethyl on internucleotide bonds, or 2'-TBS.

Defective Pollen Wall is Required for Anther and Microspore Development in Rice and Encodes a Fatty Acyl Carrier Protein Reductase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, J.; Shanklin, J.; Tan, H.

Aliphatic alcohols naturally exist in many organisms as important cellular components; however, their roles in extracellular polymer biosynthesis are poorly defined. We report here the isolation and characterization of a rice (Oryza sativa) male-sterile mutant, defective pollen wall (dpw), which displays defective anther development and degenerated pollen grains with an irregular exine. Chemical analysis revealed that dpw anthers had a dramatic reduction in cutin monomers and an altered composition of cuticular wax, as well as soluble fatty acids and alcohols. Using map-based cloning, we identified the DPW gene, which is expressed in both tapetal cells and microspores during anther development.more » Biochemical analysis of the recombinant DPW enzyme shows that it is a novel fatty acid reductase that produces 1-hexadecanol and exhibits >270-fold higher specificity for palmiltoyl-acyl carrier protein than for C16:0 CoA substrates. DPW was predominantly targeted to plastids mediated by its N-terminal transit peptide. Moreover, we demonstrate that the monocot DPW from rice complements the dicot Arabidopsis thaliana male sterile2 (ms2) mutant and is the probable ortholog of MS2. These data suggest that DPWs participate in a conserved step in primary fatty alcohol synthesis for anther cuticle and pollen sporopollenin biosynthesis in monocots and dicots.« less

Simultaneously achieve soluble expression and biomimetic immobilization of Candida antarctica lipase B by introducing polyamine tags.

PubMed

Zhou, Xiaoxue; Han, Yu; Lv, Zheng; Tian, Xuemei; Li, Han; Xie, Panpan; Zheng, Liangyu

2017-05-10

Polyamine tags fused in Candida antarctica lipase B (CalB) can help achieve high soluble expression of CalB in E. coli and can directly mediate silicification, which leads to rapid formation of a CalB-silica particle complex through a one-step approach. After optimization experiments, the fused lipase CalB tagged with 6-histidine at the N terminal and 10-lysine at the C terminal (6His-CalB-10Lys) is effectively expressed with high solubility (0.1mg/mL) and specific activity (10.1U/mg), and easily cross-linked in silica particles with a high immobilization efficiency of 96.8% and activity recovery of 81.5%. The immobilized lipase 6His-CalB-10Lys exhibits excellent performance at broad temperature ranges, high thermal and storage stabilities, and superior reusability. Michaelis-Menten kinetics indicates that the affinity and enantioselectivity of the free and immobilized 6His-CalB-10Lys toward the substrate are better than that of commercial Novozym 435 in enantioselective resolution of (S)-N-(2-ethyl-6-methylphenyl) alanine ((S)-NEMPA). The strategies described in this paper are useful for the facile expression and construction of diverse enzyme systems with high efficiency and excellent recyclability. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression and purification of recombinant proteins in Escherichia coli tagged with a small metal-binding protein from Nitrosomonas europaea.

PubMed

Vargas-Cortez, Teresa; Morones-Ramirez, Jose Ruben; Balderas-Renteria, Isaias; Zarate, Xristo

2016-02-01

Escherichia coli is still the preferred organism for large-scale production of recombinant proteins. The use of fusion proteins has helped considerably in enhancing the solubility of heterologous proteins and their purification with affinity chromatography. Here, the use of a small metal-binding protein (SmbP) from Nitrosomonas europaea is described as a new fusion protein for protein expression and purification in E. coli. Fluorescent proteins tagged at the N-terminal with SmbP showed high levels of solubility, compared with those of maltose-binding protein and glutathione S-transferase, and low formation of inclusion bodies. Using commercially available IMAC resins charged with Ni(II), highly pure recombinant proteins were obtained after just one chromatography step. Proteins may be purified from the periplasm of E. coli if SmbP contains the signal sequence at the N-terminal. After removal of the SmbP tag from the protein of interest, high-yields are obtained since SmbP is a protein of just 9.9 kDa. The results here obtained suggest that SmbP is a good alternative as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli. Copyright © 2015 Elsevier Inc. All rights reserved.

Heterologous Expression of Der Homologs in an Escherichia coli der Mutant and Their Functional Complementation

PubMed Central

Choi, Eunsil; Kang, Nalae; Jeon, Young; Pai, Hyun-Sook

2016-01-01

ABSTRACT The unique Escherichia coli GTPase Der (double Era-like GTPase), which contains tandemly repeated GTP-binding domains, has been shown to play an essential role in 50S ribosomal subunit biogenesis. The depletion of Der results in the accumulation of precursors of 50S ribosomal subunits that are structurally unstable at low Mg2+ concentrations. Der homologs are ubiquitously found in eubacteria. Conversely, very few are conserved in eukaryotes, and none is conserved in archaea. In the present study, to verify their conserved role in bacterial 50S ribosomal subunit biogenesis, we cloned Der homologs from two gammaproteobacteria, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium; two pathogenic bacteria, Staphylococcus aureus and Neisseria gonorrhoeae; and the extremophile Deinococcus radiodurans and then evaluated whether they could functionally complement the E. coli der-null phenotype. Only K. pneumoniae and S. Typhimurium Der proteins enabled the E. coli der-null strain to grow under nonpermissive conditions. Sucrose density gradient experiments revealed that the expression of K. pneumoniae and S. Typhimurium Der proteins rescued the structural instability of 50S ribosomal subunits, which was caused by E. coli Der depletion. To determine what allows their complementation, we constructed Der chimeras. We found that only Der chimeras harboring both the linker and long C-terminal regions could reverse the growth defects of the der-null strain. Our findings suggest that ubiquitously conserved essential GTPase Der is involved in 50S ribosomal subunit biosynthesis in various bacteria and that the linker and C-terminal regions may participate in species-specific recognition or interaction with the 50S ribosomal subunit. IMPORTANCE In Escherichia coli, Der (double Era-like GTPase) is an essential GTPase that is important for the production of mature 50S ribosomal subunits. However, to date, its precise role in ribosome biogenesis has not been clarified. In this study, we used five Der homologs from gammaproteobacteria, pathogenic bacteria, and an extremophile to elucidate their conserved function in 50S ribosomal subunit biogenesis. Among them, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium Der homologs implicated the participation of Der in ribosome assembly in E. coli. Our results show that the linker and C-terminal regions of Der homologs are correlated with its functional complementation in E. coli der mutants, suggesting that they are involved in species-specific recognition or interaction with 50S ribosomal subunits. PMID:27297882

Molecular and expression analysis of complement component C5 in the nurse shark (Ginglymostoma cirratum) and its predicted functional role.

PubMed

Graham, Matthew; Shin, Dong-Ho; Smith, Sylvia L

2009-07-01

We present the complete cDNA sequence of shark (Ginglymostoma cirratum) pro-C5 and its molecular characterization with a descriptive analysis of the structural elements necessary for its potential functional role as a potent mediator of inflammation (fragment C5a) and initiator molecule (fragment C5b) for the assembly of the membrane attack complex (MAC) upon activation by C5 convertase. In mammals the three complement activation cascades, the classical, alternative and lectin pathways, converge at the activation of C3, a pivotal complement protein. It is, however, the subsequent activation of the next complement component, C5, which is the focal point at which the initiation of the terminal lytic pathway takes place and involves the stepwise assembly of the MAC. The effector cytolytic function of complement occurs with the insertion of MAC into target membranes causing dough-nut like holes and cell leakage. The lytic activity of shark complement results in structurally similar holes in target membranes suggesting the assembly of a shark MAC that likely involves a functional analogue of C5. The composition of shark MAC remains unresolved and to date conclusive evidence has been lacking for shark C5. The gene has not been cloned nor has the serum protein been characterized for any elasmobranch species. This report is the first to confirm the presence of C5 homologue in the shark. GcC5 is remarkably similar to human C5 in overall structure and domain arrangement. The GcC5 cDNA measured 5160-bp with 5' and 3' UTRs of 35 bp and 79 bp, respectively. Structural analysis of the derived protein sequence predicts a molecule that is a two-chain structure which lacks a thiolester bond and contains a C5 convertase cleavage site indicating that activation will generate two peptides, akin to C5b and C5a. The putative GcC5 molecule also contains the C-terminal C345C/Netrin module that characterizes C3, C4 and C5. Multiple alignment of deduced amino acid sequences shows that GcC5 shares more amino acid identities/similarities with mammals than that with bony fish. We conclude that at the time of emergence of sharks the elaborate mosaic structure of C5 had already evolved.

Molecular and expression analysis of complement component C5 in the nurse shark (Ginglymostoma cirratum) and its predicted functional role

PubMed Central

Graham, Matthew; Shin, Dong-Ho; Smith, Sylvia L.

2009-01-01

We present the complete cDNA sequence of shark (Ginglymostoma cirratum) pro-C5 and its molecular characterization with a descriptive analysis of the structural elements necessary for its potential functional role as a potent mediator of inflammation (fragment C5a) and initiator molecule (fragment C5b) for the assembly of the membrane attack complex (MAC) upon activation by C5 convertase. In mammals the three complement activation cascades, the classical, alternative and lectin pathways, converge at the activation of C3, a pivotal complement protein. It is, however, the subsequent activation of the next complement component, C5, which is the focal point at which the initiation of the terminal lytic pathway takes place and involves the stepwise assembly of the MAC. The effector cytolytic function of complement occurs with the insertion of MAC into target membranes causing dough-nut like holes and cell leakage. The lytic activity of shark complement results in structurally similar holes in target membranes suggesting the assembly of a shark MAC that likely involves a functional analogue of C5. The composition of shark MAC remains unresolved and to date conclusive evidence has been lacking for shark C5. The gene has not been cloned nor has the serum protein been characterized for any elasmobranch species. This report is the first to confirm the presence of C5 homologue in the shark. GcC5 is remarkably similar to human C5 in overall structure and domain arrangement. The GcC5 cDNA measured 5160-bp with 5′ and 3′ UTRs of 35bp and 79bp, respectively. Structural analysis of the derived protein sequence predicts a molecule that is a two-chain structure which lacks a thiolester bond and contains a C5 convertase cleavage site indicating that activation will generate two peptides, akin to C5b and C5a. The putative GcC5 molecule also contains the C-terminal C345C/Netrin module that characterizes C3, C4 and C5. Multiple alignment of deduced amino acid sequences show that GcC5 shares more amino acid identities/similarities with mammals than that with bony fish. We conclude that at the time of emergence of sharks the elaborate mosaic structure of C5 had already evolved. PMID:19410004

Comparative analysis of the XopD T3S effector family in plant pathogenic bacteria

PubMed Central

Kim, Jung-Gun; Taylor, Kyle W.; Mudgett, Mary Beth

2011-01-01

SUMMARY XopD is a type III effector protein that is required for Xanthomonas campestris pathovar vesicatoria (Xcv) growth in tomato. It is a modular protein consisting of an N-terminal DNA-binding domain, two EAR transcriptional repressor motifs, and a C-terminal SUMO protease. In tomato, XopD functions as a transcriptional repressor, resulting in the suppression of defense responses at late stages of infection. A survey of available genome sequences for phytopathogenic bacteria revealed that XopD homologs are limited to species within three Genera of Proteobacteria – Xanthomonas, Acidovorax, and Pseudomonas. While the EAR motif(s) and SUMO protease domain are conserved in all the XopD-like proteins, variation exists in the length and sequence identity of the N-terminal domains. Comparative analysis of the DNA sequences surrounding xopD and xopD-like genes led to revised annotation of the xopD gene. Edman degradation sequence analysis and functional complementation studies confirmed that the xopD gene from Xcv encodes a 760 amino acid protein with a longer N-terminal domain than previously predicted. None of the XopD-like proteins studied complemented Xcv ΔxopD mutant phenotypes in tomato leaves suggesting that the N-terminus of XopD defines functional specificity. Xcv ΔxopD strains expressing chimeric fusion proteins containing the N-terminus of XopD fused to the EAR motif(s) and SUMO protease domain of the XopD-like protein from Xanthomonas campestris pathovar campestris strain B100 were fully virulent in tomato demonstrating that the N-terminus of XopD controls specificity in tomato. PMID:21726373

On the value of therapeutic interventions targeting the complement system in acute myocardial infarction.

PubMed

Emmens, Reindert W; Wouters, Diana; Zeerleder, Sacha; van Ham, S Marieke; Niessen, Hans W M; Krijnen, Paul A J

2017-04-01

The complement system plays an important role in the inflammatory response subsequent to acute myocardial infarction (AMI). The aim of this study is to create a systematic overview of studies that have investigated therapeutic administration of complement inhibitors in both AMI animal models and human clinical trials. To enable extrapolation of observations from included animal studies toward post-AMI clinical trials, ex vivo studies on isolated hearts and proof-of-principle studies on inhibitor administration before experimental AMI induction were excluded. Positive therapeutic effects in AMI animal models have been described for cobra venom factor, soluble complement receptor 1, C1-esterase inhibitor (C1-inh), FUT-175, C1s-inhibitor, anti-C5, ADC-1004, clusterin, and glycosaminoglycans. Two types of complement inhibitors have been tested in clinical trials, being C1-inh and anti-C5. Pexelizumab (anti-C5) did not result in reproducible beneficial effects for AMI patients. Beneficial effects were reported in AMI patients for C1-inhibitor, albeit in small patient groups. In general, despite the absence of consistent positive effects in clinical trials thus far, the complement system remains a potentially interesting target for therapy in AMI patients. Based on the study designs of previous animal studies and clinical trials, we discuss several issues which require attention in the design of future studies: adjustment of clinical trial design to precise mechanism of action of administered inhibitor, optimizing the duration of therapy, and optimization of time point(s) on which therapeutic effects will be evaluated. Copyright © 2016 Elsevier Inc. All rights reserved.

Activities of sucrose and sorbitol metabolizing enzymes in vegetative sinks of peach and correlation with sink growth rate

Treesearch

Riccardo Lo Bianco; Mark Rieger; Shi-Jean S. Sung

1999-01-01

Terminal portions of 'Flordaguard' peach roots (Prunus persica ((L.) Batsch) were divided into six segments and the activities of NAD+-dependent sorbitol dehydrogenase (SDH), sorbitol oxidase (SOX), sucrose synthase (SS), soluble acid invertase (AI),...

Synthesis of Novel Cellulose Carbamates Possessing Terminal Amino Groups and Their Bioactivity.

PubMed

Ganske, Kristin; Wiegand, Cornelia; Hipler, Uta-Christina; Heinze, Thomas

2016-03-01

Cellulose phenyl carbonates are an excellent platform to synthesize a broad variety of soluble and functional cellulose carbamates. In this study, the synthesis of cellulose carbamates with terminal amino groups, namely ω-aminoethylcellulose- and ω-aminoethyl-p-aminobenzyl-cellulose carbamate, is discussed. The products are well soluble and their structures can be clearly described by NMR spectroscopy. The cellulose carbamates exhibit a bactericide and fungicide activity in vitro. The ω-aminoethylcellulose carbamate possesses a strong activity against Candida albicans and Staphylococcus aureus (IC50 of 0.02 mg mL(-1) and 0.05 mg mL(-1)). The antimicrobial activity and cytotoxicity can be improved by p-amino-benzylamine (ABA) as an additional substituent. The mixed cellulose carbamate exhibits a high biocompatibility (LC50 of 3.18 mg mL(-1)) and forms films on cotton and PES, which exhibit a strong activity against S. aureus and Klebsiella pneumoniae. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chemical composition of South American Burseraceae non-volatile oleoresins and preliminary solubility assessment of their commercial blend.

PubMed

Siani, A C; Nakamura, M J; Tappin, M R R; Monteiro, S S; Guimarães, A C; Ramos, M F S

2012-01-01

Non-volatile oleoresins from neotropical Burseraceae are traditionally used for craft, technological and medicinal purposes. The crude resin is usually sold in popular markets of the forest communities. Adding value to this rainforest raw material requires establishing its composition. To analyse the resin composition from different Burseraceae species and establish a minimally reproducible profile by gas chromatography, in order to parameterise its quality control. Crude oleoresin samples of 10 Protium and Trattinnickia species and a commercial blend were subjected to hydrodistillation to remove volatile compounds. The chloroform-soluble residues were methylated, analysed by GC-FID (flame ionisation detection), and individual components were identified by analysing their mass fragmentation pattern in GC-MS and comparison with data from the literature. The blend solubility was assayed in 30 non-chlorinated solvents at three different proportions. The resins consisted exclusively of triterpenes, showing a common predominance of four major compounds in all the samples, corresponding to α-amyrin, β-amyrin, α-amyrenone and β-amyrenone. This profile was complemented with minor amounts of the tetracyclic β-elemolic and α-elemolic acids, maniladiol, brein and other oxidised trace compounds. The better solvents for the resin were those chemically bearing more than four carbon atoms, as n-butyl acetate, 2-ethoxyethanol and isopropanol. The crude resin blend sold contained around 10% of insoluble material that was constituted by up to 70% inorganic residues mixed with humic acid derivatives, as attested by ash analysis and IR spectroscopy, respectively. The experimental results, complemented by a general inspection of the literature, demonstrated a systematically reproducible triterpene profile in Protium and Trattinnickia species. Copyright © 2012 John Wiley & Sons, Ltd.

Can antibodies with specificity for soluble antigens mimic the therapeutic effects of intravenous IgG in the treatment of autoimmune disease?

PubMed Central

Siragam, Vinayakumar; Brinc, Davor; Crow, Andrew R.; Song, Seng; Freedman, John; Lazarus, Alan H.

2005-01-01

Intravenous Ig (IVIg) mediates protection from the effects of immune thrombocytopenic purpura (ITP) as well as numerous other autoimmune states; however, the active antibodies within IVIg are unknown. There is some evidence that antibodies specific for a cell-associated antigen on erythrocytes are responsible, at least in part, for the therapeutic effect of IVIg in ITP. Yet whether an IVIg directed to a soluble antigen can likewise be beneficial in ITP or other autoimmune diseases is also unknown. A murine model of ITP was used to determine the effectiveness of IgG specific to soluble antigens in treating immune thrombocytopenic purpura. Mice experimentally treated with soluble OVA + anti-OVA versus mice treated with OVA conjugated to rbcs (OVA-rbcs) + anti-OVA were compared. In both situations, mice were protected from ITP. Both these experimental therapeutic regimes acted in a complement-independent fashion and both also blocked reticuloendothelial function. In contrast to OVA-rbcs + anti-OVA, soluble OVA + anti-OVA (as well as IVIg) did not have any effect on thrombocytopenia in mice lacking the inhibitory receptor FcγRIIB (FcγRIIB–/– mice). Similarly, antibodies reactive with the endogenous soluble antigens albumin and transferrin also ameliorated ITP in an FcγRIIB-dependent manner. Finally, broadening the significance of these experiments was the finding that anti-albumin was protective in a K/BxN serum–induced arthritis model. We conclude that IgG antibodies directed to soluble antigens ameliorated 2 disparate IVIg-treatable autoimmune diseases. PMID:15630455

Poly (amidoamine) dendrimer-mediated hybrid formulation for combination therapy of ramipril and hydrochlorothiazide.

PubMed

Singh, Mayank Kumar; Pooja, Deep; Kulhari, Hitesh; Jain, Sanjay Kumar; Sistla, Ramakrishna; Chauhan, Abhay Singh

2017-01-01

We present a dendrimer-based hybrid formulation strategy to explore the potential of poly (amidoamine) PAMAM dendrimers to be used as drug carriers for combination therapy of an anti-hypertensive drug ramipril (RAPL) and a diuretic hydrochlorothiazide (HCTZ). The drug-dendrimer complexes were prepared by phase-equilibration method. The results showed that the solubility of RAPL and HCTZ was dependent on dendrimer concentration and pH of dendrimer solution. The solubility profile of both RAPL and HCTZ dendrimer complexes illustrated a non-linear relationship with dendrimer concentration. At 0.8% (w/v) dendrimer concentration, solubility of RAPL was increased 4.91 folds with amine-terminated while for HCTZ, solubility enhancement was highest (3.72 folds) with carboxy-terminated. The complexes were characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance analysis and high performance liquid chromatography. In-vitro drug dissolution performance of pure drugs, individual drug loaded dendrimer formulations and hybrid formulations was studied in USP dissolution medium (pH7.0) and in simulated gastric fluid (pH1.2). Dendrimer mediated formulations showed faster and complete dissolution compared to pure RAPL or HCTZ. Surprisingly, similar pattern of dissolution profile was established with hybrid formulations as compared to individual drug loaded dendrimers. The dendrimer-based hybrid formulations were found to be stable at dark and refrigerated conditions up to 5weeks. Conclusively, the proposed formulation strategy establishes a novel multitasking platform using dendrimer for simultaneous loading and delivery of multiple drugs for pharmaceutical applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Information Theoretic Secret Key Generation: Structured Codes and Tree Packing

ERIC Educational Resources Information Center

Nitinawarat, Sirin

2010-01-01

This dissertation deals with a multiterminal source model for secret key generation by multiple network terminals with prior and privileged access to a set of correlated signals complemented by public discussion among themselves. Emphasis is placed on a characterization of secret key capacity, i.e., the largest rate of an achievable secret key,…

Genotypic and phenotypic diversity of Lactobacillus rhamnosus clinical isolates, their comparison with strain GG and their recognition by complement system

PubMed Central

Douillard, François P.; Ritari, Jarmo; Paulin, Lars; Järvinen, Hanna M.; Rasinkangas, Pia; Haapasalo, Karita; Meri, Seppo; Jarva, Hanna; de Vos, Willem M.

2017-01-01

Lactobacillus rhamnosus strains are ubiquitous in fermented foods, and in the human body where they are commensals naturally present in the normal microbiota composition of gut, vagina and skin. However, in some cases, Lactobacillus spp. have been implicated in bacteremia. The aim of the study was to examine the genomic and immunological properties of 16 clinical blood isolates of L. rhamnosus and to compare them to the well-studied L. rhamnosus probiotic strain GG. Blood cultures from bacteremic patients were collected at the Helsinki University Hospital laboratory in 2005–2011 and L. rhamnosus strains were isolated and characterized by genomic sequencing. The capacity of the L. rhamnosus strains to activate serum complement was studied using immunological assays for complement factor C3a and the terminal pathway complement complex (TCC). Binding of complement regulators factor H and C4bp was also determined using radioligand assays. Furthermore, the isolated strains were evaluated for their ability to aggregate platelets and to form biofilms in vitro. Genomic comparison between the clinical L. rhamnosus strains showed them to be clearly different from L. rhamnosus GG and to cluster in two distinct lineages. All L. rhamnosus strains activated complement in serum and none of them bound complement regulators. Four out of 16 clinical blood isolates induced platelet aggregation and/or formed more biofilms than L. rhamnosus GG, which did not display platelet aggregation activity nor showed strong biofilm formation. These findings suggest that clinical L. rhamnosus isolates show considerable heterogeneity but are clearly different from L. rhamnosus GG at the genomic level. All L. rhamnosus strains are still normally recognized by the human complement system. PMID:28493885

Siloxane containing addition polyimides

NASA Technical Reports Server (NTRS)

Maudgal, S.; St. Clair, T. L.

1984-01-01

Addition polyimide oligomers have been synthesized from bis(gamma-aminopropyl) tetramethyldisiloxane and 3, 3', 4, 4'-benzophenonetetracarboxylic dianhydride using a variety of latent crosslinking groups as endcappers. The prepolymers were isolated and characterized for solubility (in amide, chlorinated and ether solvents), melt flow and cure properties. The most promising systems, maleimide and acetylene terminated prepolymers, were selected for detailed study. Graphite cloth reinforced composites were prepared and properties compared with those of graphite/Kerimid 601, a commercially available bismaleimide. Mixtures of the maleimide terminated system with Kerimid 601, in varying proportions, were also studied.

Resin-Bound Crypto-Thioester for Native Chemical Ligation.

PubMed

Naruse, Naoto; Ohkawachi, Kento; Inokuma, Tsubasa; Shigenaga, Akira; Otaka, Akira

2018-04-20

The resin-bound N-sulfanylethylanilide (SEAlide) peptide was found to function as a crypto-thioester peptide. Exposure of the peptide resin to an aqueous solution under neutral conditions in the presence of thiols affords thioesters without accompanying racemization of C-terminal amino acids. Furthermore, the resin-bound SEAlide peptides react with N-terminal cysteinyl peptides in the absence of phosphate salts to afford ligated products, whereas soluble SEAlide peptides do not. This unexpected difference in reactivity of the SEAlide peptides allows for a one-pot/three-fragment ligation using resin-bound and unbound peptides.

The C-terminal region of translesion synthesis DNA polymerase η is partially unstructured and has high conformational flexibility

PubMed Central

Powers, Kyle T; Washington, M Todd

2018-01-01

Abstract Eukaryotic DNA polymerase η catalyzes translesion synthesis of thymine dimers and 8-oxoguanines. It is comprised of a polymerase domain and a C-terminal region, both of which are required for its biological function. The C-terminal region mediates interactions with proliferating cell nuclear antigen (PCNA) and other translesion synthesis proteins such as Rev1. This region contains a ubiquitin-binding/zinc-binding (UBZ) motif and a PCNA-interacting protein (PIP) motif. Currently little structural information is available for this region of polymerase η. Using a combination of approaches—including genetic complementation assays, X-ray crystallography, Langevin dynamics simulations, and small-angle X-ray scattering—we show that the C-terminal region is partially unstructured and has high conformational flexibility. This implies that the C-terminal region acts as a flexible tether linking the polymerase domain to PCNA thereby increasing its local concentration. Such tethering would facilitate the sampling of translesion synthesis polymerases to ensure that the most appropriate one is selected to bypass the lesion. PMID:29385534

Bothrops asper snake venom and its metalloproteinase BaP-1 activate the complement system. Role in leucocyte recruitment.

PubMed Central

Farsky, S H; Gonçalves, L R; Gutiérrez, J M; Correa, A P; Rucavado, A; Gasque, P; Tambourgi, D V

2000-01-01

The venom of the snake Bothrops asper, the most important poisonous snake in Central America, evokes an inflammatory response, the mechanisms of which are not well characterized. The objectives of this study were to investigate whether B. asper venom and its purified toxins--phospholipases and metalloproteinase--activate the complement system and the contribution of the effect on leucocyte recruitment. In vitro chemotaxis assays were performed using Boyden's chamber model to investigate the ability of serum incubated with venom and its purified toxins to induce neutrophil migration. The complement consumption by the venom was evaluated using an in vitro haemolytic assay. The importance of complement activation by the venom on neutrophil migration was investigated in vivo by injecting the venom into the peritoneal cavity of C5-deficient mice. Data obtained demonstrated that serum incubated with crude venom and its purified metalloproteinase BaP-1 are able to induce rat neutrophil chemotaxis, probably mediated by agent(s) derived from the complement system. This hypothesis was corroborated by the capacity of the venom to activate this system in vitro. The involvement of C5a in neutrophil chemotaxis induced by venom-activated serum was demonstrated by abolishing migration when neutrophils were pre-incubated with antirat C5a receptor antibody. The relevance of the complement system in in vivo leucocyte mobilization was further demonstrated by the drastic decrease of this response in C5-deficient mice. Pre-incubation of serum with the soluble human recombinant complement receptor type 1 (sCR 1) did not prevent the response induced by the venom, but abolished the migration evoked by metalloproteinase-activated serum. These data show the role of the complement system in bothropic envenomation and the participation of metalloproteinase in the effect. Also, they suggest that the venom may contain other component(s) which can cause direct activation of C5a. PMID:11200361

Anti-inflammatory and anti-fibrinolytic effects of thrombomodulin alfa through carboxypeptidase B2 in the presence of thrombin.

PubMed

Tawara, Shunsuke; Sakai, Takumi; Matsuzaki, Osamu

2016-11-01

Thrombomodulin (TM) alfa, a recombinant human soluble TM, enhances activation of pro-carboxypeptidase B2 (pro-CPB2) by thrombin. Activated pro-CPB2 (CPB2) exerts anti-inflammatory and anti-fibrinolytic activities. Therefore, TM alfa may also have anti-inflammatory and anti-fibrinolytic effects through CPB2. However, these effects of TM alfa have not been elucidated. In the present study, we investigated the effects of TM alfa on inactivation of complement component C5a as an anti-inflammatory effect and prolongation of clot lysis time as an anti-fibrinolytic effect via CPB2 in vitro. CPB2 activity and tissue factor-induced thrombin generation was examined by a chromogenic assay. C5a inactivation was evaluated by C-terminal cleavage of C5a and inhibition of C5a-induced human neutrophil migration. Clot lysis time prolongation was examined by a tissue-type plasminogen activator-induced clot lysis assay. CPB2 activity in human plasma was increased by TM alfa and thrombin in a concentration-dependent manner. TM alfa inhibited tissue factor-induced thrombin generation and enhanced pro-CPB2 activation in human plasma simultaneously. The mass spectrum of C5a treated with TM alfa, thrombin, and pro-CPB2 was decreased at 156m/z, indicating that TM alfa enhanced the processing of C5a to C-terminal-cleaved C5a, an inactive form of C5a. C5a-induced human neutrophil migration was decreased after C5a treatment with TM alfa, thrombin, and pro-CPB2. TM alfa prolonged the clot lysis time in human plasma, and this effect was completely abolished by addition of a CPB2 inhibitor. TM alfa exerts anti-inflammatory and anti-fibrinolytic effects through CPB2 in the presence of thrombin in vitro. Copyright © 2016 Elsevier Ltd. All rights reserved.

Prolidase is a critical enzyme for complete gliadin digestion in Tenebrio molitor larvae

USDA-ARS?s Scientific Manuscript database

Prolidase is a proline specific metallopeptidase that cleaves imidodipeptides with C-terminal Pro residue. Prolidase was purified and characterized from the Tenebrio molitor larval midgut. The enzyme was localized in the soluble fraction of posterior midgut tissues, corresponding to a predicted cyto...

Crosslinked Aspartic Acids as Helix-Nucleating Templates.

PubMed

Zhao, Hui; Liu, Qi-Song; Geng, Hao; Tian, Yuan; Cheng, Min; Jiang, Yan-Hong; Xie, Ming-Sheng; Niu, Xiao-Gang; Jiang, Fan; Zhang, Ya-Ou; Lao, Yuan-Zhi; Wu, Yun-Dong; Xu, Nai-Han; Li, Zi-Gang

2016-09-19

Described is a facile helix-nucleating template based on a tethered aspartic acid at the N-terminus [terminal aspartic acid (TD)]. The nucleating effect of the template is subtly influenced by the substituent at the end of the side-chain-end tether as indicated by circular dichroism, nuclear magnetic resonance, and molecular dynamics simulations. Unlike most nucleating strategies, the N-terminal amine is preserved, thus enabling further modification. Peptidomimetic estrogen receptor modulators (PERMs) constructed using this strategy show improved therapeutic properties. The current strategy can be regarded as a good complement to existing helix-stabilizing methods. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reduction in erythrocyte-bound complement activation products and titres of anti-C1q antibodies associate with clinical improvement in systemic lupus erythematosus.

PubMed

Buyon, Jill; Furie, Richard; Putterman, Chaim; Ramsey-Goldman, Rosalind; Kalunian, Kenneth; Barken, Derren; Conklin, John; Dervieux, Thierry

2016-01-01

The relationship between cell-bound complement activation products (CB-CAPs: EC4d, EC3d), anti-C1q, soluble complement C3/C4 and disease activity in systemic lupus erythematosus (SLE) was evaluated. Per protocol, at baseline all SLE subjects enrolled in this longitudinal study presented with active disease and elevated CB-CAPs. At each monthly visit, the non-serological (ns) Safety of Estrogens in Lupus Erythematosus: National Assessment (SELENA-SLEDAI) and the British Isles Lupus Assessment Group (BILAG)-2004 index scores were determined as was a random urinary protein to creatinine ratio (uPCR). Short-form 36 (SF-36) questionnaires were also collected. All soluble markers were determined using immunoassays, while EC4d and EC3d were determined using flow cytometry. Statistical analysis consisted of linear mixed models with random intercept and fixed slopes. A total of 36 SLE subjects (mean age 34 years; 94% female) were enrolled and evaluated monthly for an average 11 visits per subject. Clinical improvements were observed during the study, with significant decreases in ns-SELENA-SLEDAI scores, BILAG-2004 index scores and uPCR, and increases in all domains of SF-36 (p<0.01). The longitudinal decrease in ns-SELENA-SLEDAI and BILAG-2004 index scores was significantly associated with reduced EC4d and EC3d levels, reduced anti-C1q titres and increased serum complement C3/C4 (p<0.05). The changes in uPCR significantly correlated with C3, C4, anti-C1q and EC4d, with EC4d outperforming C3/C4 by a multivariate analysis. The reduced EC4d or EC3d was associated with improvements in at least six out of the eight domains of SF-36 and outperformed C3/C4. Anti-dsDNA titres did not correlate with changes in disease activity. These data indicate that CB-CAPs and anti-C1q are helpful in monitoring patients with SLE.

Identification of cis-acting elements on positive-strand subgenomic mRNA required for the synthesis of negative-strand counterpart in bovine coronavirus.

PubMed

Yeh, Po-Yuan; Wu, Hung-Yi

2014-07-30

It has been demonstrated that, in addition to genomic RNA, sgmRNA is able to serve as a template for the synthesis of the negative-strand [(-)-strand] complement. However, the cis-acting elements on the positive-strand [(+)-strand] sgmRNA required for (-)-strand sgmRNA synthesis have not yet been systematically identified. In this study, we employed real-time quantitative reverse transcription polymerase chain reaction to analyze the cis-acting elements on bovine coronavirus (BCoV) sgmRNA 7 required for the synthesis of its (-)-strand counterpart by deletion mutagenesis. The major findings are as follows. (1) Deletion of the 5'-terminal leader sequence on sgmRNA 7 decreased the synthesis of the (-)-strand sgmRNA complement. (2) Deletions of the 3' untranslated region (UTR) bulged stem-loop showed no effect on (-)-strand sgmRNA synthesis; however, deletion of the 3' UTR pseudoknot decreased the yield of (-)-strand sgmRNA. (3) Nucleotides positioned from -15 to -34 of the sgmRNA 7 3'-terminal region are required for efficient (-)-strand sgmRNA synthesis. (4) Nucleotide species at the 3'-most position (-1) of sgmRNA 7 is correlated to the efficiency of (-)-strand sgmRNA synthesis. These results together suggest, in principle, that the 5'- and 3'-terminal sequences on sgmRNA 7 harbor cis-acting elements are critical for efficient (-)-strand sgmRNA synthesis in BCoV.

Implication of the C terminus of the Prunus necrotic ringspot virus movement protein in cell-to-cell transport and in its interaction with the coat protein.

PubMed

Aparicio, Frederic; Pallás, Vicente; Sánchez-Navarro, Jesús

2010-07-01

The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for viral transport. Previous analysis with MPs of other members of the family Bromoviridae has shown that the C-terminal part of these MPs plays a critical role in the interaction with the cognate coat protein (CP) and in cell-to-cell transport. Bimolecular fluorescence complementation and overlay analysis confirm an interaction between the C-terminal 38 aa of PNRSV MP and its cognate CP. Mutational analysis of the C-terminal region of the PNRSV MP revealed that its C-terminal 38 aa are dispensable for virus transport, however, the 4 aa preceding the dispensable C terminus are necessary to target the MP to the plasmodesmata and for the functionality of the protein. The capacity of the PNRSV MP to use either a CP-dependent or a CP-independent cell-to-cell transport is discussed.

Hydrophobic benzyl amines as supports for liquid-phase C-terminal amidated peptide synthesis: application to the preparation of ABT-510.

PubMed

Matsumoto, Emiko; Fujita, Yuko; Okada, Yohei; Kauppinen, Esko I; Kamiya, Hidehiro; Chiba, Kazuhiro

2015-09-01

C-terminal amidation is one of the most common modification of peptides and frequently found in bioactive peptides. However, the C-terminal modification must be creative, because current chemical synthetic techniques of peptides are dominated by the use of C-terminal protecting supports. Therefore, it must be carried out after the removal of such supports, complicating reaction work-up and product isolation. In this context, hydrophobic benzyl amines were successfully added to the growing toolbox of soluble tag-assisted liquid-phase peptide synthesis as supports, leading to the total synthesis of ABT-510 (2). Although an ethyl amide-forming type was used in the present work, different types of hydrophobic benzyl amines could also be simply designed and prepared through versatile reductive aminations in one step. The standard acidic treatment used in the final deprotection step for peptide synthesis gave the desired C-terminal secondary amidated peptide with no epimerization. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

Plasmin cleaves fibrinogen and the human complement proteins C3b and C5 in the presence of Leptospira interrogans proteins: A new role of LigA and LigB in invasion and complement immune evasion.

PubMed

Castiblanco-Valencia, Mónica Marcela; Fraga, Tatiana Rodrigues; Pagotto, Ana Helena; Serrano, Solange Maria de Toledo; Abreu, Patricia Antonia Estima; Barbosa, Angela Silva; Isaac, Lourdes

2016-05-01

Plasminogen is a single-chain glycoprotein found in human plasma as the inactive precursor of plasmin. When converted to proteolytically active plasmin, plasmin(ogen) regulates both complement and coagulation cascades, thus representing an important target for pathogenic microorganisms. Leptospira interrogans binds plasminogen, which is converted to active plasmin. Leptospiral immunoglobulin-like (Lig) proteins are surface exposed molecules that interact with extracellular matrix components and complement regulators, including proteins of the FH family and C4BP. In this work, we demonstrate that these multifunctional molecules also bind plasminogen through both N- and C-terminal domains. These interactions are dependent on lysine residues and are affected by ionic strength. Competition assays suggest that plasminogen does not share binding sites with C4BP or FH on Lig proteins at physiological molar ratios. Plasminogen bound to Lig proteins is converted to proteolytic active plasmin in the presence of urokinase-type plasminogen activator (uPA). Lig-bound plasmin is able to cleave the physiological substrates fibrinogen and the complement proteins C3b and C5. Taken together, our data point to a new role of LigA and LigB in leptospiral invasion and complement immune evasion. Plasmin(ogen) acquisition by these versatile proteins may contribute to Leptospira infection, favoring bacterial survival and dissemination inside the host. Copyright © 2016. Published by Elsevier GmbH.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baraibar, Martin A.; Muhoberac, Barry B.; Garringer, Holly J.

Mutations in the coding sequence of the ferritin light chain (FTL) gene cause a neurodegenerative disease known as neuroferritinopathy or hereditary ferritinopathy, which is characterized by the presence of intracellular inclusion bodies containing the mutant FTL polypeptide and by abnormal accumulation of iron in the brain. Here, we describe the x-ray crystallographic structure and report functional studies of ferritin homopolymers formed from the mutant FTL polypeptide p.Phe167SerfsX26, which has a C terminus that is altered in amino acid sequence and length. The structure was determined and refined to 2.85 {angstrom} resolution and was very similar to the wild type betweenmore » residues Ile-5 and Arg-154. However, instead of the E-helices normally present in wild type ferritin, the C-terminal sequences of all 24 mutant subunits showed substantial amounts of disorder, leading to multiple C-terminal polypeptide conformations and a large disruption of the normally tiny 4-fold axis pores. Functional studies underscored the importance of the mutant C-terminal sequence in iron-induced precipitation and revealed iron mishandling by soluble mutant FTL homopolymers in that only wild type incorporated iron when in direct competition in solution with mutant ferritin. Even without competition, the amount of iron incorporation over the first few minutes differed severalfold. Our data suggest that disruption at the 4-fold pores may lead to direct iron mishandling through attenuated iron incorporation by the soluble form of mutant ferritin and that the disordered C-terminal polypeptides may play a major role in iron-induced precipitation and formation of ferritin inclusion bodies in hereditary ferritinopathy.« less

A hybrid two-component system protein from Azospirillum brasilense Sp7 was involved in chemotaxis.

PubMed

Cui, Yanhua; Tu, Ran; Wu, Lixian; Hong, Yuanyuan; Chen, Sanfeng

2011-09-20

We here report the sequence and functional analysis of org35 of Azospirillum brasilense Sp7, which was originally identified to be able to interact with NifA in yeast-two-hybrid system. The org35 encodes a hybrid two-component system protein, including N-terminal PAS domains, a histidine kinase (HPK) domain and a response regulator (RR) domain in C-terminal. To determine the function of the Org35, a deletion-insertion mutant in PAS domain [named Sp7353] and a complemental strain Sp7353C were constructed. The mutant had reduced chemotaxis ability compared to that of wild-type, and the complemental strain was similar to the wild-type strain. These data suggested that the A. brasilense org35 played a key role in chemotaxis. Variants containing different domains of the org35 were expressed, and the functions of these domains were studied in vitro. Phosphorylation assays in vitro demonstrated that the HPK domain of Org35 possessed the autokinase activity and that the phosphorylated HPK was able to transfer phosphate groups to the RR domain. The result indicated Org35 was a phosphorylation-communicating protein. Copyright © 2010 Elsevier GmbH. All rights reserved.

Structural insights into the functions of the FANCM-FAAP24 complex in DNA repair

PubMed Central

Yang, Hui; Zhang, Tianlong; Tao, Ye; Wang, Fang; Tong, Liang; Ding, Jianping

2013-01-01

Fanconi anemia (FA) is a genetically heterogeneous disorder associated with deficiencies in the FA complementation group network. FA complementation group M (FANCM) and FA-associated protein 24 kDa (FAAP24) form a stable complex to anchor the FA core complex to chromatin in repairing DNA interstrand crosslinks. Here, we report the first crystal structure of the C-terminal segment of FANCM in complex with FAAP24. The C-terminal segment of FANCM and FAAP24 both consist of a nuclease domain at the N-terminus and a tandem helix-hairpin-helix (HhH)2 domain at the C-terminus. The FANCM-FAAP24 complex exhibits a similar architecture as that of ApXPF. However, the variations of several key residues and the electrostatic property at the active-site region render a catalytically inactive nuclease domain of FANCM, accounting for the lack of nuclease activity. We also show that the first HhH motif of FAAP24 is a potential binding site for DNA, which plays a critical role in targeting FANCM-FAAP24 to chromatin. These results reveal the mechanistic insights into the functions of FANCM-FAAP24 in DNA repair. PMID:24003026

MG53-IRS-1 (Mitsugumin 53-Insulin Receptor Substrate-1) Interaction Disruptor Sensitizes Insulin Signaling in Skeletal Muscle.

PubMed

Lee, Hyun; Park, Jung-Jin; Nguyen, Nga; Park, Jun Sub; Hong, Jin; Kim, Seung-Hyeob; Song, Woon Young; Kim, Hak Joong; Choi, Kwangman; Cho, Sungchan; Lee, Jae-Seon; Kim, Bong-Woo; Ko, Young-Gyu

2016-12-23

Mitsugumin 53 (MG53) is an E3 ligase that interacts with and ubiquitinates insulin receptor substrate-1 (IRS-1) in skeletal muscle; thus, an MG53-IRS-1 interaction disruptor (MID), which potentially sensitizes insulin signaling with an elevated level of IRS-1 in skeletal muscle, is an excellent candidate for treating insulin resistance. To screen for an MID, we developed a bimolecular luminescence complementation system using an N-terminal luciferase fragment fused with IRS-1 and a C-terminal luciferase fragment fused with an MG53 C14A mutant that binds to IRS-1 but does not have E3 ligase activity. An MID, which was discovered using the bimolecular luminescence complementation system, disrupted the molecular association of MG53 with IRS-1, thus abolishing MG53-mediated IRS-1 ubiquitination and degradation. Thus, the MID sensitized insulin signaling and increased insulin-elicited glucose uptake with an elevated level of IRS-1 in C2C12 myotubes. These data indicate that this MID holds promise as a drug candidate for treating insulin resistance. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics.

PubMed

Sun, Kaiwen; Zheng, Yuyu; Zhu, Ziqiang

2017-11-20

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Rkr1/Ltn1 Ubiquitin Ligase-mediated Degradation of Translationally Stalled Endoplasmic Reticulum Proteins*

PubMed Central

Crowder, Justin J.; Geigges, Marco; Gibson, Ryan T.; Fults, Eric S.; Buchanan, Bryce W.; Sachs, Nadine; Schink, Andrea; Kreft, Stefan G.; Rubenstein, Eric M.

2015-01-01

Aberrant nonstop proteins arise from translation of mRNA molecules beyond the coding sequence into the 3′-untranslated region. If a stop codon is not encountered, translation continues into the poly(A) tail, resulting in C-terminal appendage of a polylysine tract and a terminally stalled ribosome. In Saccharomyces cerevisiae, the ubiquitin ligase Rkr1/Ltn1 has been implicated in the proteasomal degradation of soluble cytosolic nonstop and translationally stalled proteins. Rkr1 is essential for cellular fitness under conditions associated with increased prevalence of nonstop proteins. Mutation of the mammalian homolog causes significant neurological pathology, suggesting broad physiological significance of ribosome-associated quality control. It is not known whether and how soluble or transmembrane nonstop and translationally stalled proteins targeted to the endoplasmic reticulum (ER) are detected and degraded. We generated and characterized model soluble and transmembrane ER-targeted nonstop and translationally stalled proteins. We found that these proteins are indeed subject to proteasomal degradation. We tested three candidate ubiquitin ligases (Rkr1 and ER-associated Doa10 and Hrd1) for roles in regulating abundance of these proteins. Our results indicate that Rkr1 plays the primary role in targeting the tested model ER-targeted nonstop and translationally stalled proteins for degradation. These data expand the catalog of Rkr1 substrates and highlight a previously unappreciated role for this ubiquitin ligase at the ER membrane. PMID:26055716

Identification of hot spots in the variola virus complement inhibitor (SPICE) for human complement regulation.

PubMed

Yadav, Viveka Nand; Pyaram, Kalyani; Mullick, Jayati; Sahu, Arvind

2008-04-01

Variola virus, the causative agent of smallpox, encodes a soluble complement regulator named SPICE. Previously, SPICE has been shown to be much more potent in inactivating human complement than the vaccinia virus complement control protein (VCP), although they differ only in 11 amino acid residues. In the present study, we have expressed SPICE, VCP, and mutants of VCP by substituting each or more of the 11 non-variant VCP residues with the corresponding residue of SPICE to identify hot spots that impart functional advantage to SPICE over VCP. Our data indicate that (i) SPICE is approximately 90-fold more potent than VCP in inactivating human C3b, and the residues Y98, Y103, K108 and K120 are predominantly responsible for its enhanced activity; (ii) SPICE is 5.4-fold more potent in inactivating human C4b, and residues Y98, Y103, K108, K120 and L193 mainly dictate this increase; (iii) the classical pathway decay-accelerating activity of activity is only twofold higher than that of VCP, and the 11 mutations in SPICE do not significantly affect this activity; (iv) SPICE possesses significantly greater binding ability to human C3b compared to VCP, although its binding to human C4b is lower than that of VCP; (v) residue N144 is largely responsible for the increased binding of SPICE to human C3b; and (vi) the human specificity of SPICE is dictated primarily by residues Y98, Y103, K108, and K120 since these are enough to formulate VCP as potent as SPICE. Together, these results suggest that principally 4 of the 11 residues that differ between SPICE and VCP partake in its enhanced function against human complement.

Identification of Hot Spots in the Variola Virus Complement Inhibitor (SPICE) for Human Complement Regulation▿

PubMed Central

Yadav, Viveka Nand; Pyaram, Kalyani; Mullick, Jayati; Sahu, Arvind

2008-01-01

Variola virus, the causative agent of smallpox, encodes a soluble complement regulator named SPICE. Previously, SPICE has been shown to be much more potent in inactivating human complement than the vaccinia virus complement control protein (VCP), although they differ only in 11 amino acid residues. In the present study, we have expressed SPICE, VCP, and mutants of VCP by substituting each or more of the 11 non-variant VCP residues with the corresponding residue of SPICE to identify hot spots that impart functional advantage to SPICE over VCP. Our data indicate that (i) SPICE is ∼90-fold more potent than VCP in inactivating human C3b, and the residues Y98, Y103, K108 and K120 are predominantly responsible for its enhanced activity; (ii) SPICE is 5.4-fold more potent in inactivating human C4b, and residues Y98, Y103, K108, K120 and L193 mainly dictate this increase; (iii) the classical pathway decay-accelerating activity of activity is only twofold higher than that of VCP, and the 11 mutations in SPICE do not significantly affect this activity; (iv) SPICE possesses significantly greater binding ability to human C3b compared to VCP, although its binding to human C4b is lower than that of VCP; (v) residue N144 is largely responsible for the increased binding of SPICE to human C3b; and (vi) the human specificity of SPICE is dictated primarily by residues Y98, Y103, K108, and K120 since these are enough to formulate VCP as potent as SPICE. Together, these results suggest that principally 4 of the 11 residues that differ between SPICE and VCP partake in its enhanced function against human complement. PMID:18216095

Structure of genes and an insertion element in the methane producing archaebacterium Methanobrevibacter smithii.

PubMed

Hamilton, P T; Reeve, J N

1985-01-01

DNA fragments cloned from the methanogenic archaebacterium Methanobrevibacter smithii which complement mutations in the purE and proC genes of E. coli have been sequenced. Sequence analyses, transposon mutagenesis and expression in E. coli minicells indicate that purE and proC complementations result from the synthesis of M. smithii polypeptides with molecular weights of 36,697 and 27,836 respectively. The encoding genes appear to be located in operons. The M. smithii genome contains 69% A/T basepairs (bp) which is reflected in unusual codon usages and intergenic regions containing approximately 85% A/T bp. An insertion element, designated ISM1, was found within the cloned M. smithii DNA located adjacent to the proC complementing region. ISM1 is 1381 bp in length, has 29 bp terminal inverted repeat sequences and contains one major ORF encoded in 87% of the ISM1 sequence. ISM1 is mobile, present in approximately 10 copies per genome and integration duplicates 8 bp at the site of insertion. The duplicated sequences show homology with sequences within the 29 bp terminal repeat sequence of ISM1. Comparison of our data with sequences from halophilic archaebacteria suggests that 5'GAANTTTCA and 5'TTTTAATATAAA may be consensus promoter sequences for archaebacteria. These sequences closely resemble the consensus sequences which precede Drosophila heat-shock genes (Pelham 1982; Davidson et al. 1983). Methanogens appear to employ the eubacterial system of mRNA: 16SrRNA hybridization to ensure initiation of translation; the consensus ribosome binding sequence is 5'AGGTGA.

Heterologous expression of the Phycomyces blakesleeanus phytoene dehydrogenase gene (carB) in Mucor circinelloides.

PubMed

Ruiz-Hidalgo, M J; Eslava, A P; Alvarez, M I; Benito, E P

1999-11-01

A phytoene dehydrogenase-deficient mutant of Mucor circinelloides accumulating only phytoene was transformed with the gene encoding the corresponding enzyme (carB gene) of Phycomyces blakesleeanus. Carotenoids derived from phytoene were detected in the transformants showing that the P. blakesleeanus carB gene complements the M. circinelloides carB mutation. These newly formed carotenoids accumulated in low quantities, indicating that functional complementation was poor. carB mRNA molecules correctly transcribed were detected in the transformants, but they represented a small proportion of the total population of carB-derived mRNAs, mostly constituted by truncated transcripts and by transcripts longer than the transcript that is functional in Phycomyces. These results showed that the P. blakesleeanus carB gene was expressed in M. circinelloides and suggested that the poor complementation observed was owing, at least in part, to the lack of specificity in the recognition of the transcription initiation and termination signals of the P. blakesleeanus carB gene by the M. circinelloides transcriptional machinery.

Antibacterial activities of peptides from the water-soluble extracts of Italian cheese varieties.

PubMed

Rizzello, C G; Losito, I; Gobbetti, M; Carbonara, T; De Bari, M D; Zambonin, P G

2005-07-01

Water-soluble extracts of 9 Italian cheese varieties that differed mainly for type of cheese milk, starter, technology, and time of ripening were fractionated by reversed-phase fast protein liquid chromatography, and the antimicrobial activity of each fraction was first assayed toward Lactobacillus sakei A15 by well-diffusion assay. Active fractions were further analyzed by HPLC coupled to electrospray ionization-ion trap mass spectrometry, and peptide sequences were identified by comparison with a proteomic database. Parmigiano Reggiano, Fossa, and Gorgonzola water-soluble extracts did not show antibacterial peptides. Fractions of Pecorino Romano, Canestrato Pugliese, Crescenza, and Caprino del Piemonte contained a mixture of peptides with a high degree of homology. Pasta filata cheeses (Caciocavallo and Mozzarella) also had antibacterial peptides. Peptides showed high levels of homology with N-terminal, C-terminal, or whole fragments of well known antimicrobial or multifunctional peptides reported in the literature: alphaS1-casokinin (e.g., sheep alphaS1-casein (CN) f22-30 of Pecorino Romano and cow alphaS1-CN f24-33 of Canestrato Pugliese); isracidin (e.g., sheep alphaS1-CN f10-21 of Pecorino Romano); kappacin and casoplatelin (e.g., cow kappa-CN f106-115 of Canestrato Pugliese and Crescenza); and beta-casomorphin-11 (e.g., goat beta-CN f60-68 of Caprino del Piemonte). As shown by the broth microdilution technique, most of the water-soluble fractions had a large spectrum of inhibition (minimal inhibitory concentration of 20 to 200 microg/mL) toward gram-positive and gram-negative bacterial species, including potentially pathogenic bacteria of clinical interest. Cheeses manufactured from different types of cheese milk (cow, sheep, and goat) have the potential to generate similar peptides with antimicrobial activity.

Construction and applications of yellow fever virus replicons.

PubMed

Jones, Christopher T; Patkar, Chinmay G; Kuhn, Richard J

2005-01-20

Subgenomic replicons of yellow fever virus (YFV) were constructed to allow expression of heterologous reporter genes in a replication-dependent manner. Expression of the antibiotic resistance gene neomycin phosphotransferase II (Neo) from one of these YFV replicons allowed selection of a stable population of cells (BHK-REP cells) in which the YFV replicon persistently replicated. BHK-REP cells were successfully used to trans-complement replication-defective YFV replicons harboring large internal deletions within either the NS1 or NS3 proteins. Although replicons with large deletions in either NS1 or NS3 were trans-complemented in BHK-REP, replicons that contained deletions of NS3 were trans-complemented at lower levels. In addition, replicons that retained the N-terminal protease domain of NS3 in cis were trans-complemented with higher efficiency than replicons in which both the protease and helicase domains of NS3 were deleted. To study packaging of YFV replicons, Sindbis replicons were constructed that expressed the YFV structural proteins in trans. Using these Sindbis replicons, both replication-competent and trans-complemented, replication-defective YFV replicons could be packaged into pseudo-infectious particles (PIPs). Although these results eliminate a potential role of either NS1 or full-length NS3 in cis for packaging and assembly of the flavivirus virion, they do not preclude the possibility that these proteins may act in trans during these processes.

Antibody mediated rejection associated with complement factor h-related protein 3/1 deficiency successfully treated with eculizumab.

PubMed

Noone, D; Al-Matrafi, J; Tinckam, K; Zipfel, P F; Herzenberg, A M; Thorner, P S; Pluthero, F G; Kahr, W H A; Filler, G; Hebert, D; Harvey, E; Licht, C

2012-09-01

Antibody mediated rejection (AMR) activates the classical complement pathway and can be detrimental to graft survival. AMR can be accompanied by thrombotic microangiopathy (TMA). Eculizumab, a monoclonal C5 antibody prevents induction of the terminal complement cascade (TCC) and has recently emerged as a therapeutic option for AMR. We present a highly sensitized 13-year-old female with end-stage kidney disease secondary to spina bifida-associated reflux nephropathy, who developed severe steroid-, ATG- and plasmapheresis-resistant AMR with TMA 1 week post second kidney transplant despite previous desensitization therapy with immunoglobulin infusions. Eculizumab rescue therapy resulted in a dramatic improvement in biochemical (C3; creatinine) and hematological (platelets) parameters within 6 days. The patient was proven to be deficient in complement Factor H-related protein 3/1 (CFHR3/1), a plasma protein that regulates the complement cascade at the level of C5 conversion and has been involved in the pathogenesis of atypical hemolytic uremic syndrome caused by CFH autoantibodies (DEAP-HUS). CFHR1 deficiency may have worsened the severe clinical progression of AMR and possibly contributed to the development of donor-specific antibodies. Thus, screening for CFHR3/1 deficiency should be considered in patients with severe AMR associated with TMA. © Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.

A soluble acid invertase is directed to the vacuole by a signal anchor mechanism.

PubMed

Rae, Anne L; Casu, Rosanne E; Perroux, Jai M; Jackson, Mark A; Grof, Christopher P L

2011-06-15

Enzyme activities in the vacuole have an important impact on the net concentration of sucrose. In sugarcane (Saccharum hybrid), immunolabelling demonstrated that a soluble acid invertase (β-fructofuranosidase; EC 3.2.1.26) is present in the vacuole of storage parenchyma cells during sucrose accumulation. Examination of sequences from sugarcane, barley and rice showed that the N-terminus of the invertase sequence contains a signal anchor and a tyrosine motif, characteristic of single-pass membrane proteins destined for lysosomal compartments. The N-terminal peptide from the barley invertase was shown to be capable of directing the green fluorescent protein to the vacuole in sugarcane cells. The results suggest that soluble acid invertase is sorted to the vacuole in a membrane-bound form. Copyright © 2010 Elsevier GmbH. All rights reserved.

Implication of a Central Cysteine Residue and the HHCC Domain of Moloney Murine Leukemia Virus Integrase Protein in Functional Multimerization

PubMed Central

Donzella, George A.; Leon, Oscar; Roth, Monica J.

1998-01-01

Moloney murine leukemia virus (M-MuLV) IN-IN protein interactions important for catalysis of strand transfer and unimolecular and bimolecular disintegration reactions were investigated by using a panel of chemically modified M-MuLV IN proteins. Functional complementation of an HHCC-deleted protein (NΔ105) by an independent HHCC domain (CΔ232) was severely compromised by NEM modification of either subunit. Productive NΔ105 IN-DNA interactions with a disintegration substrate lacking a long terminal repeat 5′-single-stranded tail also required complementation by a functional HHCC domain. Virus encoding the C209A M-MuLV IN mutation exhibited delayed virion production and replication kinetics. PMID:9445080

Systemic human CR2-targeted complement alternative pathway inhibitor ameliorates mouse laser-induced choroidal neovascularization.

PubMed

Rohrer, Bärbel; Coughlin, Beth; Bandyopadhyay, Mausumi; Holers, V Michael

2012-08-01

Genetic associations and the presence of complement components within pathological structures of age-related macular degeneration (AMD) have generated the hypothesis that AMD is caused by chronic local complement activation. Since the majority of activity in the common terminal pathway results from engagement of the amplification loop, the alternative pathway has been proposed as a logical therapeutic target. We recently generated a factor H (fH)-based complement inhibitor (CR2-fH) with the capacity to be "targeted" to sites of complement C3 activation. We asked whether the human therapeutic (TT30) is effective in a mouse model of AMD. Choroidal neovascularization (CNV) was induced by argon laser photocoagulation of Bruch's membrane. Every other day, mice received intravenous injections of TT30 or vehicles, and after 6 days, the presence or absence of CNV and CNV-related changes were evaluated. Area of CNV, photoreceptor cell function, gene expression for complement components and cytokines, vascular endothelial growth factor (VEGF) protein levels, and TT30 bioavailability were determined. CNV development, which has previously been shown to require local complement activation, could be reduced by intravenous TT30 delivery. Specific inhibition of the alternative pathway not only reduced angiogenesis in CNV, but also ameliorated changes in several associated disease-related biomarkers, including diminished retinal function and molecular events known to be involved in AMD such as VEGF production. After intravenous injection, TT30 localized to CNV lesion sites in the retinal pigmented epithelium-choroid. Systemic administration of TT30 was found to reduce CNV pathology. These data may open new avenues for novel systemic AMD treatment strategies.

Extracellular truncated tau causes early presynaptic dysfunction associated with Alzheimer’s disease and other tauopathies

PubMed Central

Florenzano, Fulvio; Veronica, Corsetti; Ciasca, Gabriele; Ciotti, Maria Teresa; Pittaluga, Anna; Olivero, Gunedalina; Feligioni, Marco; Iannuzzi, Filomena; Latina, Valentina; Maria Sciacca, Michele Francesco; Sinopoli, Alessandro; Milardi, Danilo; Pappalardo, Giuseppe; Marco, De Spirito; Papi, Massimiliano; Atlante, Anna; Bobba, Antonella; Borreca, Antonella; Calissano, Pietro; Amadoro, Giuseppina

2017-01-01

The largest part of tau secreted from AD nerve terminals and released in cerebral spinal fluid (CSF) is C-terminally truncated, soluble and unaggregated supporting potential extracellular role(s) of NH2 -derived fragments of protein on synaptic dysfunction underlying neurodegenerative tauopathies, including Alzheimer’s disease (AD). Here we show that sub-toxic doses of extracellular-applied human NH2 tau 26-44 (aka NH 2 htau) -which is the minimal active moiety of neurotoxic 20-22kDa peptide accumulating in vivo at AD synapses and secreted into parenchyma- acutely provokes presynaptic deficit in K+ -evoked glutamate release on hippocampal synaptosomes along with alteration in local Ca2+ dynamics. Neuritic dystrophy, microtubules breakdown, deregulation in presynaptic proteins and loss of mitochondria located at nerve endings are detected in hippocampal cultures only after prolonged exposure to NH 2 htau. The specificity of these biological effects is supported by the lack of any significant change, either on neuronal activity or on cellular integrity, shown by administration of its reverse sequence counterpart which behaves as an inactive control, likely due to a poor conformational flexibility which makes it unable to dynamically perturb biomembrane-like environments. Our results demonstrate that one of the AD-relevant, soluble and secreted N-terminally truncated tau forms can early contribute to pathology outside of neurons causing alterations in synaptic activity at presynaptic level, independently of overt neurodegeneration. PMID:29029390

Proton Shuttles and Phosphatase Activity in Soluble Epoxide Hydrolase

PubMed Central

De Vivo, Marco; Ensing, Bernd; Peraro, Matteo Dal; Gomez, German A.; Christianson, David W.; Klein, Michael L.

2008-01-01

Recently, a novel metal (Mg2+)-dependent phosphatase activity has been discovered in the N-terminal domain of the soluble epoxide hydrolase (sEH), opening a new branch of fatty acid metabolism and providing an additional site for drug targeting. Importantly, the sEH N-terminal fold belongs to the haloacid dehalogenase (HAD) superfamily, which comprises a vast majority of phosphotransferases. Herein we present the results of a computational study of the sEH phosphatase activity, which includes classical molecular dynamics (MD) simulations and mixed quantum mechanical/molecular mechanics (QM/MM) calculations. Based on experimental results, a two-step mechanism has been proposed and herein investigated: 1) phosphoenzyme intermediate formation; 2) phosphoenzyme intermediate hydrolysis. Building on our earlier work, we now provide a detailed description of the reaction mechanism for the whole catalytic cycle along with its free energy profile. The present computations suggest metaphosphate-like transition states for these phosphoryl transfers. They also reveal that the enzyme promotes water deprotonation and facilitates shuttling of protons via a metal-ligand connecting water-bridge (WB). These WB mediated proton shuttles are crucial for the activation of the solvent nucleophile and for the stabilization of the leaving-group. Moreover, due to the conservation of structural features in the N-terminal catalytic site of sEH and other members of the HAD superfamily, we suggest a generalization of our findings to these other metal-dependent phosphatases. PMID:17212419

Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor-Derived Peptides for Regulation of Mast Cell Degranulation.

PubMed

Yang, Yoosoo; Kong, Byoungjae; Jung, Younghoon; Park, Joon-Bum; Oh, Jung-Mi; Hwang, Jaesung; Cho, Jae Youl; Kweon, Dae-Hyuk

2018-01-01

Vesicle-associated V-soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and target membrane-associated T-SNAREs (syntaxin 4 and SNAP-23) assemble into a core trans -SNARE complex that mediates membrane fusion during mast cell degranulation. This complex plays pivotal roles at various stages of exocytosis from the initial priming step to fusion pore opening and expansion, finally resulting in the release of the vesicle contents. In this study, peptides with the sequences of various SNARE motifs were investigated for their potential inhibitory effects against SNARE complex formation and mast cell degranulation. The peptides with the sequences of the N-terminal regions of vesicle-associated membrane protein 2 (VAMP2) and VAMP8 were found to reduce mast cell degranulation by inhibiting SNARE complex formation. The fusion of protein transduction domains to the N-terminal of each peptide enabled the internalization of the fusion peptides into the cells equally as efficiently as cell permeabilization by streptolysin-O without any loss of their inhibitory activities. Distinct subsets of mast cell granules could be selectively regulated by the N-terminal-mimicking peptides derived from VAMP2 and VAMP8, and they effectively decreased the symptoms of atopic dermatitis in mouse models. These results suggest that the cell membrane fusion machinery may represent a therapeutic target for atopic dermatitis.

Scabies Mite Peritrophins Are Potential Targets of Human Host Innate Immunity

PubMed Central

Holt, Deborah C.; Kemp, Dave J.; Fischer, Katja

2011-01-01

Background Pruritic scabies lesions caused by Sarcoptes scabiei burrowing in the stratum corneum of human skin facilitate opportunistic bacterial infections. Emerging resistance to current therapeutics emphasizes the need to identify novel targets for protective intervention. We have characterized several protein families located in the mite gut as crucial factors for host-parasite interactions. Among these multiple proteins inhibit human complement, presumably to avoid complement-mediated damage of gut epithelial cells. Peritrophins are major components of the peritrophic matrix often found in the gut of arthropods. We hypothesized that a peritrophin, if abundant in the scabies mite gut, could be an activator of complement. Methodology/Principal Findings A novel full length scabies mite peritrophin (SsPTP1) was identified in a cDNA library from scabies mites. The amino acid sequence revealed four putative chitin binding domains (CBD). Recombinant expression of one CBD of the highly repetitive SsPTP1 sequence as TSP-hexaHis-fusion protein resulted in soluble protein, which demonstrated chitin binding activity in affinity chromatography assays. Antibodies against a recombinant SsPTP1 fragment were used to immunohistochemically localize native SsPTP1 in the mite gut and in fecal pellets within the upper epidermis, co-localizing with serum components such as host IgG and complement. Enzymatic deglycosylation confirmed strong N- and O-glycosylation of the native peritrophin. Serum incubation followed by immunoblotting with a monoclonal antibody against mannan binding lectin (MBL), the recognition molecule of the lectin pathway of human complement activation, indicated that MBL may specifically bind to glycosylated SsPTP1. Conclusions/Significance This study adds a new aspect to the accumulating evidence that complement plays a major role in scabies mite biology. It identifies a novel peritrophin localized in the mite gut as a potential target of the lectin pathway of the complement cascade. These initial findings indicate a novel role of scabies mite peritrophins in triggering a host innate immune response within the mite gut. PMID:21980545

Solubility enhancement of simvastatin by arginine: thermodynamics, solute-solvent interactions, and spectral analysis.

PubMed

Meor Mohd Affandi, M M R; Tripathy, Minaketan; Shah, Syed Adnan Ali; Majeed, A B A

2016-01-01

We examined the solubility of simvastatin in water in 0.01 mol·dm(-3), 0.02 mol·dm(-3), 0.04 mol·dm(-3), 0.09 mol·dm(-3), 0.18 mol·dm(-3), 0.36 mol·dm(-3), and 0.73 mol·dm(-3) arginine (ARG) solutions. The investigated drug is termed the solute, whereas ARG the cosolute. Phase solubility studies illustrated a higher extent of solubility enhancement for simvastatin. The aforementioned system was subjected to conductometric and volumetric measurements at temperatures (T) of 298.15 K, 303.15 K, 308.15 K, and 313.15 K to illustrate the thermodynamics involved and related solute-solvent interactions. The conductance values were used to evaluate the limiting molar conductance and association constants. Thermodynamic parameters (ΔG (0), ΔH (0), ΔS (0), and E s) for the association process of the solute in the aqueous solutions of ARG were calculated. Limiting partial molar volumes and expansibilities were evaluated from the density values. These values are discussed in terms of the solute-solvent and solute-cosolute interactions. Further, these systems were analyzed using ultraviolet-visible analysis, Fourier-transform infrared spectroscopy, and (13)C, (1)H, and two-dimensional nuclear overhauser effect spectroscopy nuclear magnetic resonance to complement thermophysical explanation.

Differential pathways regulating innate and adaptive antitumor immune responses by particulate and soluble yeast-derived β-glucans

PubMed Central

Qi, Chunjian; Cai, Yihua; Gunn, Lacey; Ding, Chuanlin; Li, Bing; Kloecker, Goetz; Qian, Keqing; Vasilakos, John; Saijo, Shinobu; Iwakura, Yoichiro; Yannelli, John R.

2011-01-01

β-glucans have been reported to function as a potent adjuvant to stimulate innate and adaptive immune responses. However, β-glucans from different sources are differential in their structure, conformation, and thus biologic activity. Different preparations of β-glucans, soluble versus particulate, further complicate their mechanism of action. Here we show that yeast-derived particulate β-glucan activated dendritic cells (DCs) and macrophages via a C-type lectin receptor dectin-1 pathway. Activated DCs by particulate β-glucan promoted Th1 and cytotoxic T-lymphocyte priming and differentiation in vitro. Treatment of orally administered yeast-derived particulate β-glucan elicited potent antitumor immune responses and drastically down-regulated immunosuppressive cells, leading to the delayed tumor progression. Deficiency of the dectin-1 receptor completely abrogated particulate β-glucan–mediated antitumor effects. In contrast, yeast-derived soluble β-glucan bound to DCs and macrophages independent of the dectin-1 receptor and did not activate DCs. Soluble β-glucan alone had no therapeutic effect but significantly augmented antitumor monoclonal antibody-mediated therapeutic efficacy via a complement activation pathway but independent of dectin-1 receptor. These findings reveal the importance of different preparations of β-glucans in the adjuvant therapy and allow for the rational design of immunotherapeutic protocols usable in clinical trials. PMID:21531981

Solubility enhancement of simvastatin by arginine: thermodynamics, solute–solvent interactions, and spectral analysis

PubMed Central

Meor Mohd Affandi, MMR; Tripathy, Minaketan; Shah, Syed Adnan Ali; Majeed, ABA

2016-01-01

We examined the solubility of simvastatin in water in 0.01 mol·dm−3, 0.02 mol·dm−3, 0.04 mol·dm−3, 0.09 mol·dm−3, 0.18 mol·dm−3, 0.36 mol·dm−3, and 0.73 mol·dm−3 arginine (ARG) solutions. The investigated drug is termed the solute, whereas ARG the cosolute. Phase solubility studies illustrated a higher extent of solubility enhancement for simvastatin. The aforementioned system was subjected to conductometric and volumetric measurements at temperatures (T) of 298.15 K, 303.15 K, 308.15 K, and 313.15 K to illustrate the thermodynamics involved and related solute–solvent interactions. The conductance values were used to evaluate the limiting molar conductance and association constants. Thermodynamic parameters (ΔG0, ΔH0, ΔS0, and Es) for the association process of the solute in the aqueous solutions of ARG were calculated. Limiting partial molar volumes and expansibilities were evaluated from the density values. These values are discussed in terms of the solute–solvent and solute–cosolute interactions. Further, these systems were analyzed using ultraviolet–visible analysis, Fourier-transform infrared spectroscopy, and 13C, 1H, and two-dimensional nuclear overhauser effect spectroscopy nuclear magnetic resonance to complement thermophysical explanation. PMID:27041998

Directing an appropriate immune response: the role of defense collagens and other soluble pattern recognition molecules.

PubMed

Fraser, D A; Tenner, A J

2008-02-01

Defense collagens and other soluble pattern recognition receptors contain the ability to recognize and bind molecular patterns associated with pathogens (PAMPs) or apoptotic cells (ACAMPs) and signal appropriate effector-function responses. PAMP recognition by defense collagens C1q, MBL and ficolins leads to rapid containment of infection via complement activation. However, in the absence of danger, such as during the clearance of apoptotic cells, defense collagens such as C1q, MBL, ficolins, SP-A, SP-D and even adiponectin have all been shown to facilitate enhanced phagocytosis and modulate induction of cytokines towards an anti-inflammatory profile. In this way, cellular debris can be removed without provoking an inflammatory immune response which may be important in the prevention of autoimmunity and/or resolving inflammation. Indeed, deficiencies and/or knock-out mouse studies have highlighted critical roles for soluble pattern recognition receptors in the clearance of apoptotic bodies and protection from autoimmune diseases along with mediating protection from specific infections. Understanding the mechanisms involved in defense collagen and other soluble pattern recognition receptor modulation of the immune response may provide important novel insights into therapeutic targets for infectious and/or autoimmune diseases and additionally may identify avenues for more effective vaccine design.

THE ANTIBODY-FORMATION BY POLYSACCHARIDS.

PubMed

Nishimura, S

1929-09-30

1. By complement fixation tests, it has been clearly demonstrated that the sera of rabbits immunized with inulin, soluble starch and dextrine contain specific antibodies. 2. All these immune sera gave a negative precipitation reaction. 3. The kind of dextrine which has a construction very near to starch has an antigenic property, but those in a state of further decomposition do not give rise to antibodies. 4. All the three kinds of polysaccharids have power to produce antibodies without any vehicle. Dextrine is the only one of the three that gives rise to immune bodies more readily when pig serum is added to it. 5. Regarded as antigens, inulin stood first and soluble starch and dextrine next in order. 6. All three kinds of polysaccharids that were employed gave a negative protein color reaction. All of them, however, contained nitrogen. It has been proved that the large portion of the nitrogen contained in the soluble starch is derived from its protein contents. 7. It is suggested that in the production of immune bodies by these three kinds of polysaccharids, proteins might play the part of the vehicle. This is, however, still to be determined.

Purification, crystallization and preliminary x-ray crystallographic studies of RAIDD Death-Domain (DD).

PubMed

Jang, Tae-ho; Park, Hyun Ho

2009-06-03

Caspase-2 activation by formation of PIDDosome is critical for genotoxic stress induced apoptosis. PIDDosome is composed of three proteins, RAIDD, PIDD, and Caspase-2. RAIDD is an adaptor protein containing an N-terminal Caspase-Recruiting-Domain (CARD) and a C-terminal Death-Domain (DD). Its interactions with Caspase-2 and PIDD through CARD and DD respectively and formation of PIDDosome are important for the activation of Caspase-2. RAIDD DD cloned into pET26b vector was expressed in E. coli cells and purified by nickel affinity chromatography and gel filtration. Although it has been known that the most DDs are not soluble in physiological condition, RAIDD DD was soluble and interacts tightly with PIDD DD in physiological condition. The purified RAIDD DD alone has been crystallized. Crystals are trigonal and belong to space group P3(1)21 (or its enantiomorph P3(2)21) with unit-cell parameters a = 56.3, b = 56.3, c = 64.9 A and gamma = 120 degrees . The crystals were obtained at room temperature and diffracted to 2.0 A resolution.

Tadpole-like Conformations of Huntingtin Exon 1 Are Characterized by Conformational Heterogeneity that Persists regardless of Polyglutamine Length.

PubMed

Newcombe, Estella A; Ruff, Kiersten M; Sethi, Ashish; Ormsby, Angelique R; Ramdzan, Yasmin M; Fox, Archa; Purcell, Anthony W; Gooley, Paul R; Pappu, Rohit V; Hatters, Danny M

2018-05-11

Soluble huntingtin exon 1 (Httex1) with expanded polyglutamine (polyQ) engenders neurotoxicity in Huntington's disease. To uncover the physical basis of this toxicity, we performed structural studies of soluble Httex1 for wild-type and mutant polyQ lengths. Nuclear magnetic resonance experiments show evidence for conformational rigidity across the polyQ region. In contrast, hydrogen-deuterium exchange shows absence of backbone amide protection, suggesting negligible persistence of hydrogen bonds. The seemingly conflicting results are explained by all-atom simulations, which show that Httex1 adopts tadpole-like structures with a globular head encompassing the N-terminal amphipathic and polyQ regions and the tail encompassing the C-terminal proline-rich region. The surface area of the globular domain increases monotonically with polyQ length. This stimulates sharp increases in gain-of-function interactions in cells for expanded polyQ, and one of these interactions is with the stress-granule protein Fus. Our results highlight plausible connections between Httex1 structure and routes to neurotoxicity. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Targeted Lymphoma Cell Death by Novel Signal Transduction Modifications

DTIC Science & Technology

2007-07-01

monoclonal antibodies (mAbs) that bind the two NH2-terminal immunoglobulin domains of CD22 and specifically block the interaction of CD22 with its...ligand blocking mAbs that effectively crosslink CD22 have distinct functional properties and facilitate assembly of an effector protein complex. These...immune mechanisms such as antibody and complement dependent cellular cytotoxicity. We hypothesize that enhancing the intrinsic pro-apoptotic

Isolation and purification of C3 from human plasma.

PubMed

O'Rear, L D; Ross, G D

2001-05-01

The alternative pathway of complement shares its terminal components (C3 and C5 through 9) with the classical pathway, but has several unique components, including factors D, B, and P (properdin). This unit presents methods for assaying total alternative pathway activity and the activity of factors B and D. Radial immunodiffusion (RID) can also be used to measure factor D, B, and P concentrations.

Synthesis of uniform cyclodextrin thioethers to transport hydrophobic drugs

PubMed Central

Becker, Lisa F; Schwarz, Dennis H

2014-01-01

Summary Methyl and ethyl thioether groups were introduced at all primary positions of α-, β-, and γ-cyclodextrin by nucleophilic displacement reactions starting from the corresponding per-(6-deoxy-6-bromo)cyclodextrins. Further modification of all 2-OH positions by etherification with iodo terminated triethylene glycol monomethyl ether (and tetraethylene glycol monomethyl ether, respectively) furnished water-soluble hosts. Especially the β-cyclodextrin derivatives exhibit very high binding potentials towards the anaesthetic drugs sevoflurane and halothane. Since the resulting inclusion compounds are highly soluble in water at temperatures ≤37 °C they are good candidates for new aqueous dosage forms which would avoid inhalation anaesthesia. PMID:25550759

The catalytic chain of human complement subcomponent C1r. Purification and N-terminal amino acid sequences of the major cyanogen bromide-cleavage fragments.

PubMed

Arlaud, G J; Gagnon, J; Porter, R R

1982-01-01

1. The a- and b-chains of reduced and alkylated human complement subcomponent C1r were separated by high-pressure gel-permeation chromatography and isolated in good yield and in pure form. 2. CNBr cleavage of C1r b-chain yielded eight major peptides, which were purified by gel filtration and high-pressure reversed-phase chromatography. As determined from the sum of their amino acid compositions, these peptides accounted for a minimum molecular weight of 28 000, close to the value 29 100 calculated from the whole b-chain. 3. N-Terminal sequence determinations of C1r b-chain and its CNBr-cleavage peptides allowed the identification of about two-thirds of the amino acids of C1r b-chain. From our results, and on the basis of homology with other serine proteinases, an alignment of the eight CNBr-cleavage peptides from C1r b-chain is proposed. 4. The residues forming the 'charge-relay' system of the active site of serine proteinases (His-57, Asp-102 and Ser-195 in the chymotrypsinogen numbering) are found in the corresponding regions of C1r b-chain, and the amino acid sequence around these residues has been determined. 5. The N-terminal sequence of C1r b-chain has been extended to residue 60 and reveals that C1r b-chain lacks the 'histidine loop', a disulphide bond that is present in all other known serine proteinases.

Determining antibody-binding site of streptococcal pyrogenic exotoxin B to protect mice from group a streptococcus infection.

PubMed

Tsao, Nina; Cheng, Miao-Hui; Yang, Hsiu-Chen; Wang, Yu-Chieh; Liu, Yi-Ling; Kuo, Chih-Feng

2013-01-01

Streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, is an important virulence factor in group A streptococcal (GAS) infection. SPE B binds and cleaves antibody isotypes and further impairs the immune system by inhibiting complement activation. In this study, we examined the antibody-binding site of SPE B and used it to block SPE B actions during GAS infection. We constructed different segments of the spe B gene and induced them to express different recombinant fragments of SPE B. Using an enzyme-linked immunosorbent assay (ELISA), we found that residues 345-398 of the C-terminal domain of SPE B (rSPE B(345-398)), but not the N-terminal domain, was the major binding site for antibody isotypes. Using a competitive ELISA, we also found that rSPE B(345-398) bound to the Fc portion of IgG. The in vitro functional assays indicate that rSPE B(345-398) not only interfered with cleavage of antibody isotypes but also interfered with SPE B-induced inhibition of complement activation. Immunization of BALB/c mice using rSPE B(345-398) was able to induce production of a high titer of anti-rSPE B(345-398) antibodies and efficiently protected mice from GAS-induced death. These findings suggest that SPE B uses its C-terminal domain to bind the Fc portion of IgG and that immunization of mice with this binding domain (rSPE B(345-398)) could protect mice from GAS infection.

Terminal hospitalizations of nursing home residents: does facility increasing the rate of do not resuscitate orders reduce them?

PubMed

Teno, Joan M; Gozalo, Pedro; Mitchell, Susan L; Bynum, Julie P W; Dosa, David; Mor, Vincent

2011-06-01

Terminal hospitalizations are costly and often avoidable with appropriate advance care planning. This study examined the association between advance care planning, as measured by facility rate of do not resuscitate (DNR) orders in U.S. nursing homes (NHs) and changes in terminal hospitalization rates. Retrospective cohort study of the changing prevalence of DNR orders in U.S. NHs. Using a fixed effect multivariate model, we examined whether increasing facility rate of DNR orders correlates with reductions in terminal hospitalizations in the last week of life, controlling for changes in facility characteristics (staffing, use of NP/PA, case mix of nursing residents, admission volume, racial composition, payer mix). The average facility rate of terminal hospitalizations was 15.5%, fluctuating between 1999 (15.0%) and 2007 (14.8%). NHs starting with low rates of DNR orders that increased their rates had fewer terminal hospital admissions in 2007 (11.2%) than facilities with continuously low DNR usage. Even after applying a multivariate fixed effect model, the effect of changes in facility DNR order rate on terminal hospitalization was -0.056 (95% confidence interval: -0.061, -0.050), indicating that for every 10% increase in DNR orders there was 0.56% decrease in terminal hospitalizations. This rate can be compared with the increase of 0.70% in the terminal hospitalization rate when an NH became disproportionately dependent on Medicaid funding or the 0.40% decrease in terminal hospitalization rate associated with adding a nurse practitioner to the clinical staff complement. NHs that changed their culture of decision making by increasing their facility rate of DNR orders decreased their rate of terminal hospitalizations. Copyright © 2011 U.S. Cancer Pain Relief Committee. Published by Elsevier Inc. All rights reserved.

Expression, purification, and DNA-binding activity of the solubilized NtrC protein of Herbaspirillum seropedicae.

PubMed

Twerdochlib, Adriana L; Chubatsu, Leda S; Souza, Emanuel M; Pedrosa, Fábio O; Steffens, M Berenice R; Yates, M Geoffrey; Rigo, Liu U

2003-07-01

NtrC is a bacterial enhancer-binding protein (EBP) that activates transcription by the sigma54 RNA polymerase holoenzyme. NtrC has a three domain structure typical of EBP family. In Herbaspirillum seropedicae, an endophytic diazotroph, NtrC regulates several operons involved in nitrogen assimilation, including glnAntrBC. In order to over-express and purify the NtrC protein, DNA fragments containing the complete structural gene for the whole protein, and for the N-terminal+Central and Central+C-terminal domains were cloned into expression vectors. The NtrC and NtrC(N-terminal+Central) proteins were over-expressed as His-tag fusion proteins upon IPTG addition, solubilized using N-lauryl-sarcosyl and purified by metal affinity chromatography. The over-expressed His-tag-NtrC(Central+C-terminal) fusion protein was partially soluble and was also purified by affinity chromatography. DNA band-shift assays showed that the NtrC protein and the Central+C-terminal domains bound specifically to the H. seropedicae glnA promoter region. The C-terminal domain is presumably necessary for DNA-protein interaction and DNA-binding does not require a phosphorylated protein.

Use of LysoTracker to detect programmed cell death in embryos and differentiating embryonic stem cells.

PubMed

Fogel, Jennifer L; Thein, Thu Zan Tun; Mariani, Francesca V

2012-10-11

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs(1,2,6,7). During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects(3-5). To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells. Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it(8-10). After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death. To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome(11-13). The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible(12,13). In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis(14), is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase. In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media(12,13). Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

N-terminal lipid modification is required for the stable accumulation of CyanoQ in Synechocystis sp. PCC 6803

DOE PAGES

Juneau, Andrea D.; Frankel, Laurie K.; Bricker, Terry M.; ...

2016-09-22

Here, the CyanoQ protein has been demonstrated to be a component of cyanobacterial Photosystem II (PS II), but there exist a number of outstanding questions concerning its physical association with the complex. CyanoQ is a lipoprotein; upon cleavage of its transit peptide by Signal Peptidase II, which targets delivery of the mature protein to the thylakoid lumenal space, the N-terminal cysteinyl residue is lipid-modified. This modification appears to tether this otherwise soluble component to the thylakoid membrane. To probe the functional significance of the lipid anchor, mutants of the CyanoQ protein have been generated in Synechocystis sp. PCC 6803 tomore » eliminate the N-terminal cysteinyl residue, preventing lipid modification. Substitution of the N-terminal cysteinyl residue with serine (Q-C22S) resulted in a decrease in the amount of detectable CyanoQ protein to 17% that of the wild-type protein. Moreover, the physical properties of the accumulated Q-C22S protein were consistent with altered processing of the CyanoQ precursor. The Q-C22S protein was shifted to a higher apparent molecular mass and partitioned in the hydrophobic phase in TX-114 phase-partitioning experiments. These results suggest that the hydrophobic N-terminal 22 amino acids were not properly cleaved by a signal peptidase. Substitution of the entire CyanoQ transit peptide with the transit peptide of the soluble lumenal protein PsbO yielded the Q-SS mutant and resulted in no detectable accumulation of the modified CyanoQ protein. Finally, the CyanoQ protein was present at normal amounts in the PS II mutant strains ΔpsbB and ΔpsbO, indicating that an association with PS II was not a prerequisite for stable CyanoQ accumulation. Together these results indicate that CyanoQ accumulation in Synechocystis sp. PCC 6803 depends on the presence of the N-terminal lipid anchor, but not on the association of CyanoQ with the PS II complex.« less

N-terminal lipid modification is required for the stable accumulation of CyanoQ in Synechocystis sp. PCC 6803

DOE Office of Scientific and Technical Information (OSTI.GOV)

Juneau, Andrea D.; Frankel, Laurie K.; Bricker, Terry M.

Here, the CyanoQ protein has been demonstrated to be a component of cyanobacterial Photosystem II (PS II), but there exist a number of outstanding questions concerning its physical association with the complex. CyanoQ is a lipoprotein; upon cleavage of its transit peptide by Signal Peptidase II, which targets delivery of the mature protein to the thylakoid lumenal space, the N-terminal cysteinyl residue is lipid-modified. This modification appears to tether this otherwise soluble component to the thylakoid membrane. To probe the functional significance of the lipid anchor, mutants of the CyanoQ protein have been generated in Synechocystis sp. PCC 6803 tomore » eliminate the N-terminal cysteinyl residue, preventing lipid modification. Substitution of the N-terminal cysteinyl residue with serine (Q-C22S) resulted in a decrease in the amount of detectable CyanoQ protein to 17% that of the wild-type protein. Moreover, the physical properties of the accumulated Q-C22S protein were consistent with altered processing of the CyanoQ precursor. The Q-C22S protein was shifted to a higher apparent molecular mass and partitioned in the hydrophobic phase in TX-114 phase-partitioning experiments. These results suggest that the hydrophobic N-terminal 22 amino acids were not properly cleaved by a signal peptidase. Substitution of the entire CyanoQ transit peptide with the transit peptide of the soluble lumenal protein PsbO yielded the Q-SS mutant and resulted in no detectable accumulation of the modified CyanoQ protein. Finally, the CyanoQ protein was present at normal amounts in the PS II mutant strains ΔpsbB and ΔpsbO, indicating that an association with PS II was not a prerequisite for stable CyanoQ accumulation. Together these results indicate that CyanoQ accumulation in Synechocystis sp. PCC 6803 depends on the presence of the N-terminal lipid anchor, but not on the association of CyanoQ with the PS II complex.« less

Linked Production of Pyroglutamate-Modified Proteins via Self-Cleavage of Fusion Tags with TEV Protease and Autonomous N-Terminal Cyclization with Glutaminyl Cyclase In Vivo

PubMed Central

Shih, Yan-Ping; Chou, Chi-Chi; Chen, Yi-Ling; Huang, Kai-Fa; Wang, Andrew H.- J.

2014-01-01

Overproduction of N-terminal pyroglutamate (pGlu)-modified proteins utilizing Escherichia coli or eukaryotic cells is a challenging work owing to the fact that the recombinant proteins need to be recovered by proteolytic removal of fusion tags to expose the N-terminal glutaminyl or glutamyl residue, which is then converted into pGlu catalyzed by the enzyme glutaminyl cyclase. Herein we describe a new method for production of N-terminal pGlu-containing proteins in vivo via intracellular self-cleavage of fusion tags by tobacco etch virus (TEV) protease and then immediate N-terminal cyclization of passenger target proteins by a bacterial glutaminyl cyclase. To combine with the sticky-end PCR cloning strategy, this design allows the gene of target proteins to be efficiently inserted into the expression vector using two unique cloning sites (i.e., SnaB I and Xho I), and the soluble and N-terminal pGlu-containing proteins are then produced in vivo. Our method has been successfully applied to the production of pGlu-modified enhanced green fluorescence protein and monocyte chemoattractant proteins. This design will facilitate the production of protein drugs and drug target proteins that possess an N-terminal pGlu residue required for their physiological activities. PMID:24733552

A novel strategy for hemolytic uremic syndrome: successful treatment with thrombomodulin α.

PubMed

Honda, Takashi; Ogata, Shohei; Mineo, Eri; Nagamori, Yukako; Nakamura, Shinya; Bando, Yuki; Ishii, Masahiro

2013-03-01

Hemolytic uremic syndrome (HUS) is a life-threatening infectious disease in childhood for which there is no confirmed therapeutic strategy. Endothelial inflammation leading to microthrombosis formation via complement activation is the main pathology of HUS. Thrombomodulin is an endothelial membrane protein that has anticoagulation and anti-inflammatory effects, including the suppression of complement activity. Recombinant human soluble thrombomodulin (rTM) is a novel therapeutic medicine for disseminated intravascular coagulation. We administered rTM to 3 patients with HUS for 7 days and investigated the outcomes in view of the patients' prognoses, changes in biochemical markers, complications, and adverse effects of rTM. Symptoms and laboratory data improved after initiation of rTM in all 3 patients. Abnormal activation of complements was also dramatically suppressed in 1 patient. The patients recovered without any complications or adverse effects of rTM. They were discharged having normal neurologic status and with no renal dysfunction. To our knowledge, this is the first report of rTM being used to treat HUS. These case reports show the positive effect of rTM in patients with HUS. Randomized controlled studies should be performed to assess the efficacy and safety of rTM for children with HUS.

Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease

PubMed Central

1992-01-01

Serum mannose-binding protein (MBP) is a C-type lectin that binds to terminal mannose and N-acetylglucosamine moieties present on surfaces of certain pathogens and activates the classical complement pathway. In the present study, we describe the mechanism underlying the activation triggered by MBP. The human serum MBP fraction was obtained by sequential affinity chromatography on mannan-Sepharose, anti-IgM- Sepharose and anti-MBP-Sepharose in the presence of calcium ions. This fraction contained a C1s-like serine protease as assessed by C4 consumption. The C1s-like serine protease, designated MBP-associated serine protease (MASP), was separated from MBP by rechromatography on anti-MBP-Sepharose in the presence of ethylenediaminetetraacetic acid. MASP exhibited both C4- and C2-consuming activities. The molecular mass of MASP was estimated to be 83 kD with two polypeptides of heavy (66 kD) and light (L) (31 kD) chains linked by disulfide bonds. The serine residue responsible for protease activity is located on the L chain. Reconstitution experiments using MASP and MBP revealed that combination of the two components restores C4- and C2-activating capacity on mannan. Based on analyses of molecular size, antigenicity, and 11 NH2- terminal amino acid sequences of the L chain, we conclude that MASP is a novel protein different from C1r or C1s. Our findings are not in accord with a proposed mechanism by which MBP utilizes the C1r2-C1s2 complex to initiate the classical complement pathway. PMID:1460414

An alanine residue in human parainfluenza virus type 3 phosphoprotein is critical for restricting excessive N0-P interaction and maintaining N solubility.

PubMed

Zhang, Shengwei; Cheng, Qi; Luo, Chenxi; Yin, Lei; Qin, Yali; Chen, Mingzhou

2018-05-01

The phosphoprotein (P) of human parainfluenza virus type 3 (HPIV3) plays a pivotal role in viral RNA synthesis, which interacts with the nucleoprotein (N) to form a soluble N 0 -P complex (N 0 , free of RNAs) to prevent the nonspecific RNA binding and illegitimate aggregation of N. Functional regions within P have been studied intensively. However, the precise site (s) within P directly involved in N 0 -P interaction still remains unclear. In this study, using a series of deleted and truncated mutants of P of HPIV3, we demonstrate that amino-terminal 40 amino acids (aa) of P restrict and regulate N 0 -P interaction. Furthermore, using in vivo HPIV3 minigenome replicon assay, we identify a critical P mutant (P A28P ) located in amino-terminal 40 aa, which fails to support RNA synthesis of HPIV3 minigenome replicon. Although P A28P maintains an enhanced N-P interaction, it is unable to form N 0 -P complex and keep N soluble, thus, resulting in aggregation and functional abolishment of N-P complex. Moreover, we found that recombinant HPIV3 with mutation of A28P in P failed to be rescued. Taken together, we identified a residue within the extreme amino-terminus of P, which plays a critical role in restricting the excessively N-P interaction and keeping a functional N 0 -P complex formation. Copyright © 2018 Elsevier Inc. All rights reserved.

New ligation independent cloning vectors for expression of recombinant proteins with a self-cleaving CPD/6xHis-tag.

PubMed

Biancucci, Marco; Dolores, Jazel S; Wong, Jennifer; Grimshaw, Sarah; Anderson, Wayne F; Satchell, Karla J F; Kwon, Keehwan

2017-01-05

Recombinant protein purification is a crucial step for biochemistry and structural biology fields. Rapid robust purification methods utilize various peptide or protein tags fused to the target protein for affinity purification using corresponding matrices and to enhance solubility. However, affinity/solubility-tags often need to be removed in order to conduct functional and structural studies, adding complexities to purification protocols. In this work, the Vibrio cholerae MARTX toxin Cysteine Protease Domain (CPD) was inserted in a ligation-independent cloning (LIC) vector to create a C-terminal 6xHis-tagged inducible autoprocessing enzyme tag, called "the CPD-tag". The pCPD and alternative pCPD/ccdB cloning vectors allow for easy insertion of DNA and expression of the target protein fused to the CPD-tag, which is removed at the end of the purification step by addition of the inexpensive small molecule inositol hexakisphosphate to induce CPD autoprocessing. This process is demonstrated using a small bacterial membrane localization domain and for high yield purification of the eukaryotic small GTPase KRas. Subsequently, pCPD was tested with 40 proteins or sub-domains selected from a high throughput crystallization pipeline. pCPD vectors are easily used LIC compatible vectors for expression of recombinant proteins with a C-terminal CPD/6xHis-tag. Although intended only as a strategy for rapid tag removal, this pilot study revealed the CPD-tag may also increase expression and solubility of some recombinant proteins.

Impact of dialyzer membrane on apoptosis and function of polymorphonuclear cells and cytokine synthesis by peripheral blood mononuclear cells in hemodialysis patients.

PubMed

Andreoli, Maria C C; Dalboni, Maria A; Watanabe, Renato; Manfredi, Silvia R; Canziani, Maria E F; Kallás, Esper G; Sesso, Ricardo C; Draibe, Sergio A; Balakrishnan, Vaidyanathapuram S; Jaber, Bertrand L; Liangos, Orfeas; Cendoroglo, Miguel

2007-12-01

In an in vivo crossover trial, we compared a cellulosic with a synthetic dialyzer with respect to polymorphonuclear cells (PMN) function and apoptosis, cytokine serum levels and synthesis by peripheral blood mononuclear cells (PBMC), and complement activation. Twenty hemodialysis (HD) patients were assigned in alternate order to HD with cellulose acetate (CA) or polysulfone (PS) dialyzer. After 2 weeks, patients were crossed over to the second dialyzer and treated for another 2 weeks. Apoptosis was assessed by flow cytometry in freshly isolated PMN. Phagocytosis and production of peroxide by PMN were studied by flow cytometry in whole blood. PBMC were isolated from blood samples and incubated for 24 h with or without lipopolysaccharide (LPS). There was no impact of dialyzer biocompatibility on PMN apoptosis and function, cytokine synthesis by PBMC or on their serum levels, serum levels of C3a, and terminal complement complex (TCC). Nevertheless, after HD, serum levels of complement correlated negatively with PMN phagocytosis and peroxide production, and positively with PMN apoptosis and cytokine production by PBMC. Although the results did not show a dialyzer advantage on the immunologic parameters, complement activation may have modulated cell function and apoptosis after HD.

The HSP terminator of Arabidopsis thaliana induces a high level of miraculin accumulation in transgenic tomatoes.

PubMed

Hirai, Tadayoshi; Kurokawa, Natsuko; Duhita, Narendra; Hiwasa-Tanase, Kyoko; Kato, Kazuhisa; Kato, Ko; Ezura, Hiroshi

2011-09-28

High-level accumulation of the target recombinant protein is a significant issue in heterologous protein expression using transgenic plants. Miraculin, a taste-modifying protein, was accumulated in transgenic tomatoes using an expression cassette in which the miraculin gene was expressed by the cauliflower mosaic virus (CaMV) 35S promoter and the heat shock protein (HSP) terminator (MIR-HSP). The HSP terminator was derived from heat shock protein 18.2 in Arabidopsis thaliana . Using this HSP-containing cassette, the miraculin concentration in T0 transgenic tomato lines was 1.4-13.9% of the total soluble protein (TSP), and that in the T1 transgenic tomato line homozygous for the miraculin gene reached 17.1% of the TSP. The accumulation level of the target protein was comparable to levels observed with chloroplast transformation. The high-level accumulation of miraculin in T0 transgenic tomato lines achieved by the HSP terminator was maintained in the successive T1 generation, demonstrating the genetic stability of this accumulation system.

THE SEROLOGICAL SPECIFICITY OF PARTICULATE COMPONENTS DERIVED FROM VARIOUS NORMAL MAMMALIAN ORGANS

PubMed Central

Henle, Werner; Chambers, Leslie A.; Groupé, Vincent

1941-01-01

1. Particles derived from filtrates of organ suspensions by high speed centrifugation were serologically active as shown by agglutination and complement fixation techniques. Particles from brain, liver, lung, kidney, heart muscle, spleen, testicle, and pancreas of various species have been studied. 2. All particles showed a certain degree of organ specificity with the exception of pancreas. Cross-reactions occurred between the particles from various organs from one species, which were more marked when complement fixation technique was employed than by the agglutination test. However, agglutination always appeared earlier and was stronger, and complement fixation was positive in higher dilutions of antigen in the presence of homologous antiserum than with heterologous antisera. 3. The cross-reactions did not depend on the occasional precipitins for serum and the agglutinins for the red cells of the species from which the particles were derived, nor did they bear a relation to Wassermann and Forssman antibodies present in some of the sera. 4. The organ specific differentiation of the particles from various organs could more clearly be demonstrated by two means: The antiserum could be diluted in such a way that only the homologous reaction still showed a positive result while the cross-reactions had become negative; or the cross-reacting antibodies could be absorbed by heterologous particles and the homologous reaction was still more or less intact. 5. In addition to the organ specific differentiation, most particles were found to exhibit species specificity. While the particles derived from kidney, lung, testicle, and heart muscle aggregated only in the presence of the antiserum against the corresponding organ particles from the homologous species, brain particles reacted with brain antisera against both homologous and heterologous species alike. Absorption of brain particle antisera with brain preparations from a heterologous species removed all antibodies. Liver particle preparations showed an intermediate position in that all liver preparations with the exception of rabbit liver particles were aggregated by any liver particle antiserum. However, absorption with liver particles from a heterologous species left a distinct species specific reaction in the serum. 6. The antigens involved are all destroyed by heating to 100° C. for a few minutes with the exception of brain particles, which after 20 minutes at 100° C. still gave complement fixation almost to the same strength as the untreated controls. 7. Alcoholic and ether extracts of brain reacted with the brain particle antisera only. All alcoholic or ether extracts of other organs gave no complement fixation. None of the various other organ particle antisera tested contained antibodies for these extracts. 8. The relationship between the heat-stable and the alcohol-soluble brain particle antigen studied by absorption technique revealed that there were two antigens present, both organ specific and independent of the species, the one alcohol- and ether-soluble, the other not soluble in these solvents but heat stable. Some of the sera showed besides a few species specific antibodies. 9. Preliminary evidence has been gathered to show that no iso-immunization could be obtained with any one of the organ particles. As far as cytotoxic activity of the sera is concerned only the kidney particle antisera have been studied for nephrotoxins; these failed to reveal any such activity in the mouse. PMID:19871150

Phase Equilibria of Sn-Co-Cu Ternary System

NASA Astrophysics Data System (ADS)

Chen, Yu-Kai; Hsu, Chia-Ming; Chen, Sinn-Wen; Chen, Chih-Ming; Huang, Yu-Chih

2012-10-01

Sn-Co-Cu ternary alloys are promising lead-free solders, and isothermal sections of Sn-Co-Cu phase equilibria are fundamentally important for the alloys' development and applications. Sn-Co-Cu ternary alloys were prepared and equilibrated at 523 K, 1073 K, and 1273 K (250 °C, 800 °C, and 1000 °C), and the equilibrium phases were experimentally determined. In addition to the terminal solid solutions and binary intermetallic compounds, a new ternary compound, Sn3Co2Cu8, was found. The solubilities of Cu in the α-CoSn3 and CoSn2 phases at 523 K (250 °C) are 4.2 and 1.6 at. pct, respectively, while the Cu solubility in the α-Co3Sn2 phase is as high as 20.0 at. pct. The Cu solubility increases with temperature and is around 30.0 at. pct in the β-Co3Sn2 at 1073 K (800 °C). The Co solubility in the η-Cu6Sn5 phase is also significant and is 15.5 at. pct at 523 K (250 °C).

Expression and Purification of Recombinant Proteins in Escherichia coli with a His6 or Dual His6-MBP Tag.

PubMed

Raran-Kurussi, Sreejith; Waugh, David S

2017-01-01

Rapid advances in bioengineering and biotechnology over the past three decades have greatly facilitated the production of recombinant proteins in Escherichia coli. Affinity-based methods that employ protein or peptide based tags for protein purification have been instrumental in this progress. Yet insolubility of recombinant proteins in E. coli remains a persistent problem. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely acknowledged as a highly effective solubilizing agent. In this chapter, we describe how to construct either a His 6 - or a dual His 6 -MBP tagged fusion protein by Gateway ® recombinational cloning and how to evaluate their yield and solubility. We also describe a simple and rapid procedure to test the solubility of proteins after removing their N-terminal fusion tags by tobacco etch virus (TEV) protease digestion. The choice of whether to use a His 6 tag or a His 6 -MBP tag can be made on the basis of this solubility test.

Characterization and expression analysis of a complement component gene in sea cucumber ( Apostichopus japonicus)

NASA Astrophysics Data System (ADS)

Chen, Zhong; Zhou, Zunchun; Yang, Aifu; Dong, Ying; Guan, Xiaoyan; Jiang, Bei; Wang, Bai

2015-12-01

The complement system plays a crucial role in the innate immune system of animals. It can be activated by distinct yet overlapping classical, alternative and lectin pathways. In the alternative pathway, complement factor B (Bf) serves as the catalytic subunit of complement component 3 (C3) convertase, which plays the central role among three activation pathways. In this study, the Bf gene in sea cucumber ( Apostichopus japonicus), termed AjBf, was obtained by rapid amplification of cDNA ends (RACE). The full-length cDNA of AjBf was 3231 bp in length barring the poly (A) tail. It contained an open reading frame (ORF) of 2742 bp encoding 913 amino acids, a 105 bp 5'-UTR (5'-terminal untranslated region) and a 384 bp 3'-UTR. AjBf was a mosaic protein with six CCP (complement control protein) domains, a VWA (von Willebrand factor A) domain, and a serine protease domain. The deduced molecular weight of AjBf protein was 101 kDa. Quantitative real time PCR (qRT-PCR) analysis indicated that the expression level of AjBf in A. japonicus was obviously higher at larval stage than that at embryonic stage. Expression detection in different tissues showed that AjBf expressed higher in coelomocytes than in other four tissues. In addation, AjBf expression in different tissues was induced significantly after LPS or PolyI:C challenge. These results indicated that AjBf plays an important role in immune responses to pathogen infection.

Split luciferase complementation assay to detect regulated protein-protein interactions in rice protoplasts in a large-scale format

PubMed Central

2014-01-01

Background The rice interactome, in which a network of protein-protein interactions has been elucidated in rice, is a useful resource to identify functional modules of rice signal transduction pathways. Protein-protein interactions occur in cells in two ways, constitutive and regulative. While a yeast-based high-throughput method has been widely used to identify the constitutive interactions, a method to detect the regulated interactions is rarely developed for a large-scale analysis. Results A split luciferase complementation assay was applied to detect the regulated interactions in rice. A transformation method of rice protoplasts in a 96-well plate was first established for a large-scale analysis. In addition, an antibody that specifically recognizes a carboxyl-terminal fragment of Renilla luciferase was newly developed. A pair of antibodies that recognize amino- and carboxyl- terminal fragments of Renilla luciferase, respectively, was then used to monitor quality and quantity of interacting recombinant-proteins accumulated in the cells. For a proof-of-concept, the method was applied to detect the gibberellin-dependent interaction between GIBBERELLIN INSENSITIVE DWARF1 and SLENDER RICE 1. Conclusions A method to detect regulated protein-protein interactions was developed towards establishment of the rice interactome. PMID:24987490

Phototropin Influence on Eyespot Development and Regulation of Phototactic Behavior in Chlamydomonas reinhardtii[W

PubMed Central

Trippens, Jessica; Greiner, Andre; Schellwat, Jana; Neukam, Martin; Rottmann, Theresa; Lu, Yinghong; Kateriya, Suneel; Hegemann, Peter; Kreimer, Georg

2012-01-01

The eyespot of Chlamydomonas reinhardtii is a light-sensitive organelle important for phototactic orientation of the alga. Here, we found that eyespot size is strain specific and downregulated in light. In a strain in which the blue light photoreceptor phototropin was deleted by homologous recombination, the light regulation of the eyespot size was affected. We restored this dysfunction in different phototropin complementation experiments. Complementation with the phototropin kinase fragment reduced the eyespot size, independent of light. Interestingly, overexpression of the N-terminal light, oxygen or voltage sensing domains (LOV1+LOV2) alone also affected eyespot size and phototaxis, suggesting that aside from activation of the kinase domain, they fulfill an independent signaling function in the cell. Moreover, phototropin is involved in adjusting the level of channelrhodopsin-1, the dominant primary receptor for phototaxis within the eyespot. Both the level of channelrhodopsin-1 at the onset of illumination and its steady state level during the light period are downregulated by phototropin, whereas the level of channelrhodopsin-2 is not significantly altered. Furthermore, a light intensity–dependent formation of a C-terminal truncated phototropin form was observed. We propose that phototropin is a light regulator of phototaxis that desensitizes the eyespot when blue light intensities increase. PMID:23204408

Characterization and N-terminal sequencing of a calcium binding protein from the calcareous concretion organic matrix of the terrestrial crustacean Orchestia cavimana.

PubMed

Luquet, G; Testenière, O; Graf, F

1996-04-16

We extracted proteins from the organic matrix of calcareous concretions, which represents the calcium storage form in a terrestrial crustacean. Electrophoretic analyses of water-soluble organic-matrix proteinaceous components revealed 11 polypeptides, 6 of which are probably glycosylated. Among the unglycosylated proteins, we characterized a 23 kDa polypeptide, with an isoelectric point of 5.5, which is able to bind calcium. Its N-terminal sequence is rich in acidic amino acids (essentially aspartic acid). All these characteristics suggest its involvement in the calcium precipitation process within the successive layers of the organic matrix.

Novel Scabies Mite Serpins Inhibit the Three Pathways of the Human Complement System

PubMed Central

Mika, Angela; Reynolds, Simone L.; Mohlin, Frida C.; Willis, Charlene; Swe, Pearl M.; Pickering, Darren A.; Halilovic, Vanja; Wijeyewickrema, Lakshmi C.; Pike, Robert N.; Blom, Anna M.; Kemp, David J.; Fischer, Katja

2012-01-01

Scabies is a parasitic infestation of the skin by the mite Sarcoptes scabiei that causes significant morbidity worldwide, in particular within socially disadvantaged populations. In order to identify mechanisms that enable the scabies mite to evade human immune defenses, we have studied molecules associated with proteolytic systems in the mite, including two novel scabies mite serine protease inhibitors (SMSs) of the serpin superfamily. Immunohistochemical studies revealed that within mite-infected human skin SMSB4 (54 kDa) and SMSB3 (47 kDa) were both localized in the mite gut and feces. Recombinant purified SMSB3 and SMSB4 did not inhibit mite serine and cysteine proteases, but did inhibit mammalian serine proteases, such as chymotrypsin, albeit inefficiently. Detailed functional analysis revealed that both serpins interfered with all three pathways of the human complement system at different stages of their activation. SMSB4 inhibited mostly the initial and progressing steps of the cascades, while SMSB3 showed the strongest effects at the C9 level in the terminal pathway. Additive effects of both serpins were shown at the C9 level in the lectin pathway. Both SMSs were able to interfere with complement factors without protease function. A range of binding assays showed direct binding between SMSB4 and seven complement proteins (C1, properdin, MBL, C4, C3, C6 and C8), while significant binding of SMSB3 occurred exclusively to complement factors without protease function (C4, C3, C8). Direct binding was observed between SMSB4 and the complement proteases C1s and C1r. However no complex formation was observed between either mite serpin and the complement serine proteases C1r, C1s, MASP-1, MASP-2 and MASP-3. No catalytic inhibition by either serpin was observed for any of these enzymes. In summary, the SMSs were acting at several levels mediating overall inhibition of the complement system and thus we propose that they may protect scabies mites from complement-mediated gut damage. PMID:22792350

Plasma complement and vascular complement deposition in patients with coronary artery disease with and without inflammatory rheumatic diseases

PubMed Central

2017-01-01

Purpose Inflammatory rheumatic diseases (IRD) are associated with accelerated coronary artery disease (CAD), which may result from both systemic and vascular wall inflammation. There are indications that complement may be involved in the pathogenesis of CAD in Systemic Lupus Erythematosus (SLE) and Rheumatoid Arthritis (RA). This study aimed to evaluate the associations between circulating complement and complement activation products with mononuclear cell infiltrates (MCI, surrogate marker of vascular inflammation) in the aortic media and adventitia in IRDCAD and non-IRDCAD patients undergoing coronary artery bypass grafting (CABG). Furthermore, we compared complement activation product deposition patterns in rare aorta adventitial and medial biopsies from SLE, RA and non-IRD patients. Methods We examined plasma C3 (p-C3) and terminal complement complexes (p-TCC) in 28 IRDCAD (SLE = 3; RA = 25), 52 non-IRDCAD patients, and 32 IRDNo CAD (RA = 32) from the Feiring Heart Biopsy Study. Aortic biopsies taken from the CAD only patients during CABG were previously evaluated for adventitial MCIs. The rare aortic biopsies from 3 SLE, 3 RA and 3 non-IRDCAD were assessed for the presence of C3 and C3d using immunohistochemistry. Results IRDCAD patients had higher p-TCC than non-IRDCAD or IRDNo CAD patients (p<0.0001), but a similar p-C3 level (p = 0.42). Circulating C3 was associated with IRD duration (ρ, p-value: 0.46, 0.03). In multiple logistic regression analysis, IRD remained significantly related to the presence and size of MCI (p<0.05). C3 was present in all tissue samples. C3d was detected in the media of all patients and only in the adventitia of IRD patients (diffuse in all SLE and focal in one RA). Conclusion The independent association of IRD status with MCI and the observed C3d deposition supports the unique relationship between rheumatic disease, and, in particular, SLE with the complement system. Exaggerated systemic and vascular complement activation may accelerate CVD, serve as a CVD biomarker, and represent a target for new therapies. PMID:28362874

Two mannose-binding lectin homologues and an MBL-associated serine protease are expressed in the gut epithelia of the urochordate species Ciona intestinalis.

PubMed

Skjoedt, Mikkel-Ole; Palarasah, Yaseelan; Rasmussen, Karina; Vitved, Lars; Salomonsen, Jan; Kliem, Anette; Hansen, Soren; Koch, Claus; Skjodt, Karsten

2010-01-01

The lectin complement pathway has important functions in vertebrate host defence and accumulating evidence of primordial complement components trace its emergence to invertebrate phyla. We introduce two putative mannose-binding lectin homologues (CioMBLs) from the urochordate species Ciona intestinalis. The CioMBLs display similarities with vertebrate MBLs and comprise a collagen-like region, alpha-helical coiled-coils and a carbohydrate recognition domain (CRD) with conserved residues involved in calcium and carbohydrate binding. Structural analysis revealed an oligomerization through interchain disulphide bridges between N-terminal cysteine residues and cysteines located between the neck region and the CRD. RT-PCR showed a tissue specific expression of CioMBL in the gut and by immunohistochemistry analysis we also demonstrated that CioMBL co-localize with an MBL-associated serine protease in the epithelia cells lining the stomach and intestine. In conclusion we present two urochordate MBLs and identify an associated serine protease, which support the concept of an evolutionary ancient origin of the lectin complement pathway.

Small-molecule factor D inhibitors selectively block the alternative pathway of complement in paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome.

PubMed

Yuan, Xuan; Gavriilaki, Eleni; Thanassi, Jane A; Yang, Guangwei; Baines, Andrea C; Podos, Steven D; Huang, Yongqing; Huang, Mingjun; Brodsky, Robert A

2017-03-01

Paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome are diseases of excess activation of the alternative pathway of complement that are treated with eculizumab, a humanized monoclonal antibody against the terminal complement component C5. Eculizumab must be administered intravenously, and moreover some patients with paroxysmal nocturnal hemoglobinuria on eculizumab have symptomatic extravascular hemolysis, indicating an unmet need for additional therapeutic approaches. We report the activity of two novel small-molecule inhibitors of the alternative pathway component Factor D using in vitro correlates of both paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Both compounds bind human Factor D with high affinity and effectively inhibit its proteolytic activity against purified Factor B in complex with C3b. When tested using the traditional Ham test with cells from paroxysmal nocturnal hemoglobinuria patients, the Factor D inhibitors significantly reduced complement-mediated hemolysis at concentrations as low as 0.01 μM. Additionally the compound ACH-4471 significantly decreased C3 fragment deposition on paroxysmal nocturnal hemoglobinuria erythrocytes, indicating a reduced potential relative to eculizumab for extravascular hemolysis. Using the recently described modified Ham test with serum from patients with atypical hemolytic uremic syndrome, the compounds reduced the alternative pathway-mediated killing of PIGA -null reagent cells, thus establishing their potential utility for this disease of alternative pathway of complement dysregulation and validating the modified Ham test as a system for pre-clinical drug development for atypical hemolytic uremic syndrome. Finally, ACH-4471 blocked alternative pathway activity when administered orally to cynomolgus monkeys. In conclusion, the small-molecule Factor D inhibitors show potential as oral therapeutics for human diseases driven by the alternative pathway of complement, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Copyright© Ferrata Storti Foundation.

Growth and stability of Langmuir-Blodgett films on OH-, H-, or Br-terminated Si(001)

NASA Astrophysics Data System (ADS)

Bal, J. K.; Kundu, S.; Hazra, S.

2010-01-01

Growth of Langmuir-Blodgett (LB) films of nickel arachidate (NiA) on differently terminated (OH-, H-, or Br-terminated) Si(001) substrates and their structural evolution with time have been investigated by x-ray reflectivity technique and complemented by atomic force microscopy. Stable and strongly attached asymmetric monolayer (AML) of NiA is found to grow on freshly prepared oxide-covered Si substrate while unstable and weakly attached symmetric monolayer (SML) of NiA grows on H-terminated Si substrate, corresponding to stable hydrophilic and unstable hydrophobic natures of the substrates, respectively. The structure of LB film on Br-terminated Si substrate, however, shows intermediate behavior, namely, both AML and SML are present on the substrate, indicative of coexisting (hydrophilic and hydrophobic) nature of this terminated surface. Such coexisting nature of the substrate shows unusual growth behavior of LB films: (i) hydrophilic and hydrophobic attachments of NiA molecules in single up stroke of deposition and (ii) growth of few ring-shaped large-heights islands in subsequent deposition. These probably occur due to the presence of substrate-induced perturbation in the Langmuir monolayer and release of initially accumulated strain in the film structures near hydrophilic/hydrophobic interface, respectively, and provide the possibility to grow desired structures (AML or SML) of LB films by passivation-selective surface engineering.

Biocompatible silicon quantum dots by ultrasound-induced solution route

NASA Astrophysics Data System (ADS)

Lee, Soojin; Cho, Woon-Jo

2004-10-01

The water-soluble silicon quantum dots (QDs) of average diameter ~3 nm were prepared in organic solvent by ultrasound-induced solution route. This speedy rout produces the silicon QDs in the size range from 2 nm to 4 nm at room temperature and ambient pressure. The product yield of QDs was estimated to be higher than 60 % based on the initial NaSi weight. The surfaces of QDs were terminated with organic molecules including biocompatible ending groups (hydroxyl, amine and carboxyl) during simple preparation. Covalent attached molecules were characterized by FT-IR spectroscopy. These water-soluble passivation of QDs has just a little effect on the optical properties of original QDs.

The effects of certain glycols, substituted glycols and related organic solvents on the thermal stability of soluble collagen

PubMed Central

Hart, G. J.; Russell, A. E.; Cooper, D. R.

1971-01-01

The effects of a number of related diols, substituted diols and glycerol on the thermal stability of acid-soluble calf skin collagen were investigated. Thermal transition temperatures were determined by optical rotation measurement. Short-chain diols with terminal hydroxyl groups, i.e. ethylene glycol and propane-1,3-diol, stabilized the protein at all accessible concentrations. Stabilization was also observed with glycerol and diethylene glycol. Higher homologues in the diol series produced various effects, as did hydroxyl-group positional isomerism. Monoalkyl substitution of diols progressively lowered the denaturation temperature of collagen. Results are discussed in relation to possible mechanisms of perturbant action. PMID:5169191

Enhancement of Ebola Virus Infection via Ficolin-1 Interaction with the Mucin Domain of GP Glycoprotein.

PubMed

Favier, Anne-Laure; Gout, Evelyne; Reynard, Olivier; Ferraris, Olivier; Kleman, Jean-Philippe; Volchkov, Viktor; Peyrefitte, Christophe; Thielens, Nicole M

2016-06-01

Ebola virus infection requires the surface viral glycoprotein to initiate entry into the target cells. The trimeric glycoprotein is a highly glycosylated viral protein which has been shown to interact with host C-type lectin receptors and the soluble complement recognition protein mannose-binding lectin, thereby enhancing viral infection. Similarly to mannose-binding lectin, ficolins are soluble effectors of the innate immune system that recognize particular glycans at the pathogen surface. In this study, we demonstrate that ficolin-1 interacts with the Zaire Ebola virus (EBOV) glycoprotein, and we characterized this interaction by surface plasmon resonance spectroscopy. Ficolin-1 was shown to bind to the viral glycoprotein with a high affinity. This interaction was mediated by the fibrinogen-like recognition domain of ficolin-1 and the mucin-like domain of the viral glycoprotein. Using a ficolin-1 control mutant devoid of sialic acid-binding capacity, we identified sialylated moieties of the mucin domain to be potential ligands on the glycoprotein. In cell culture, using both pseudotyped viruses and EBOV, ficolin-1 was shown to enhance EBOV infection independently of the serum complement. We also observed that ficolin-1 enhanced EBOV infection on human monocyte-derived macrophages, described to be major viral target cells,. Competition experiments suggested that although ficolin-1 and mannose-binding lectin recognized different carbohydrate moieties on the EBOV glycoprotein, the observed enhancement of the infection likely depended on a common cellular receptor/partner. In conclusion, ficolin-1 could provide an alternative receptor-mediated mechanism for enhancing EBOV infection, thereby contributing to viral subversion of the host innate immune system. A specific interaction involving ficolin-1 (M-ficolin), a soluble effector of the innate immune response, and the glycoprotein (GP) of EBOV was identified. Ficolin-1 enhanced virus infection instead of tipping the balance toward its elimination. An interaction between the fibrinogen-like recognition domain of ficolin-1 and the mucin-like domain of Ebola virus GP occurred. In this model, the enhancement of infection was shown to be independent of the serum complement. The facilitation of EBOV entry into target host cells by the interaction with ficolin-1 and other host lectins shunts virus elimination, which likely facilitates the survival of the virus in infected host cells and contributes to the virus strategy to subvert the innate immune response. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A C3(H20) recycling pathway is a component of the intracellular complement system

PubMed Central

Elvington, Michelle; Bertram, Paula; Atkinson, John P.

2017-01-01

An intracellular complement system (ICS) has recently been described in immune and nonimmune human cells. This system can be activated in a convertase-independent manner from intracellular stores of the complement component C3. The source of these stores has not been rigorously investigated. In the present study, Western blotting identified a band corresponding to C3 in freshly isolated human peripheral blood cells that was absent in corresponding cell lines. One difference between native cells and cell lines was the time absent from a fluid-phase complement source; therefore, we hypothesized that loading C3 from plasma was a route of establishing intracellular C3 stores. We found that many types of human cells specifically internalized C3(H2O), the hydrolytic product of C3, and not native C3, from the extracellular milieu. Uptake was rapid, saturable, and sensitive to competition with unlabeled C3(H2O), indicating a specific mechanism of loading. Under steady-state conditions, approximately 80% of incorporated C3(H2O) was returned to the extracellular space. These studies identify an ICS recycling pathway for C3(H2O). The loaded C3(H2O) represents a source of C3a, and its uptake altered the cytokine profile of activated CD4+ T cells. Importantly, these results indicate that the impact of soluble plasma factors should be considered when performing in vitro studies assessing cellular immune function. PMID:28192370

THE ANTIBODY-FORMATION BY POLYSACCHARIDS

PubMed Central

Nishimura, Shoji

1929-01-01

1. By complement fixation tests, it has been clearly demonstrated that the sera of rabbits immunized with inulin, soluble starch and dextrine contain specific antibodies. 2. All these immune sera gave a negative precipitation reaction. 3. The kind of dextrine which has a construction very near to starch has an antigenic property, but those in a state of further decomposition do not give rise to antibodies. 4. All the three kinds of polysaccharids have power to produce antibodies without any vehicle. Dextrine is the only one of the three that gives rise to immune bodies more readily when pig serum is added to it. 5. Regarded as antigens, inulin stood first and soluble starch and dextrine next in order. 6. All three kinds of polysaccharids that were employed gave a negative protein color reaction. All of them, however, contained nitrogen. It has been proved that the large portion of the nitrogen contained in the soluble starch is derived from its protein contents. 7. It is suggested that in the production of immune bodies by these three kinds of polysaccharids, proteins might play the part of the vehicle. This is, however, still to be determined. PMID:19869634

Functional analysis of the putative peroxidase domain of FANCA, the Fanconi anemia complementation group A protein.

PubMed

Ren, J; Youssoufian, H

2001-01-01

Fanconi anemia (FA) is an autosomal recessive disorder manifested by chromosomal breakage, birth defects, and susceptibility to bone marrow failure and cancer. At least seven complementation groups have been identified, and the genes defective in four groups have been cloned. The most common subtype is complementation group A. Although the normal functions of the gene products defective in FA cells are not completely understood, a clue to the function of the FA group A gene product (FANCA) was provided by the detection of limited homology in the amino terminal region to a class of heme peroxidases. We evaluated this hypothesis by mutagenesis and functional complementation studies. We substituted alanine residues for the most conserved FANCA residues in the putative peroxidase domain and tested their effects on known biochemical and cellular functions of FANCA. While the substitution mutants were comparable to wild-type FANCA with regard to their stability, subcellular localization, and interaction with FANCG, only the Trp(183)-to-Ala substitution (W183A) abolished the ability of FANCA to complement the sensitivity of FA group A cells to mitomycin C. By contrast, TUNEL assays for apoptosis after exposure to H2O2 showed no differences between parental FA group A cells, cells complemented with wild-type FANCA, and cells complemented with the W183A of FANCA. Moreover, semiquantitative RT-PCR analysis for the expression of the peroxide-sensitive heme oxygenase gene showed appropriate induction after H2O2 exposure. Thus, W183A appears to be essential for the in vivo activity of FANCA in a manner independent of its interaction with FANCG. Moreover, neither wild-type FANCA nor the W183A mutation appears to alter the peroxide-induced apoptosisor peroxide-sensing ability of FA group A cells. Copyright 2001 Academic Press.

δ-COP contains a helix C-terminal to its longin domain key to COPI dynamics and function

PubMed Central

Arakel, Eric C.; Richter, Kora P.; Clancy, Anne; Schwappach, Blanche

2016-01-01

Membrane recruitment of coatomer and formation of coat protein I (COPI)-coated vesicles is crucial to homeostasis in the early secretory pathway. The conformational dynamics of COPI during cargo capture and vesicle formation is incompletely understood. By scanning the length of δ-COP via functional complementation in yeast, we dissect the domains of the δ-COP subunit. We show that the μ-homology domain is dispensable for COPI function in the early secretory pathway, whereas the N-terminal longin domain is essential. We map a previously uncharacterized helix, C-terminal to the longin domain, that is specifically required for the retrieval of HDEL-bearing endoplasmic reticulum-luminal residents. It is positionally analogous to an unstructured linker that becomes helical and membrane-facing in the open form of the AP2 clathrin adaptor complex. Based on the amphipathic nature of the critical helix it may probe the membrane for lipid packing defects or mediate interaction with cargo and thus contribute to stabilizing membrane-associated coatomer. PMID:27298352

Major proteins of yam bean tubers.

PubMed

Gomes, A V; Sirju-Charran, G; Barnes, J A

1997-09-01

The tuberous roots of the Mexican yam bean, jicama, (Pachyrhizus erosus L. Urban) contained large quantities of two acidic glycoproteins which accounted for more than 70% of the total soluble proteins (about 3 g per 100 g of tuber on a dry weight basis). The two major proteins, tentatively named YBG1 and YBG2, had apparent M(r)s of 28,000 and 26,000, respectively, by SDS-PAGE. A third protein named YBP22 which accounted for 2-5% of the total soluble proteins had an M(r) of 22,000. YBG1 and YBG2 exhibited great similarity on the basis of their amino acid composition and had identical N-terminal amino acid sequences. The first 23 amino acids in the N-terminal region of YBG2 were DDLPDYVDWRDYGAVTRIKNQGQ which showed strong homology with the papain class of cysteine proteases. YBG1 and YBG2 were found to bind to a Concanavalin A-Sepharose column and were also stained positively by a sensitive glycoprotein stain. Both glycoproteins exhibited cysteine proteolytic activity. In contrast, YBP22 showed sequence homology with several known protease inhibitors, and a polyclonal antibody raised against this protein cross reacted with soybean trypsin inhibitor.

The Nup153-Nup50 Protein Interface and Its Role in Nuclear Import*

PubMed Central

Makise, Masaki; Mackay, Douglas R.; Elgort, Suzanne; Shankaran, Sunita S.; Adam, Stephen A.; Ullman, Katharine S.

2012-01-01

Interactions between Nup50 and soluble transport factors underlie the efficiency of certain nucleocytoplasmic transport pathways. The platform on which these interactions take place is important to building a complete understanding of nucleocytoplasmic trafficking. Nup153 is the nucleoporin that provides this scaffold for Nup50. Here, we have delineated requirements for the interaction between Nup153 and Nup50, revealing a dual interface. An interaction between Nup50 and a region in the unique N-terminal region of Nup153 is critical for the nuclear pore localization of Nup50. A second site of interaction is at the distal tail of Nup153 and is dependent on importin α. Both of these interactions involve the N-terminal domain of Nup50. The configuration of the Nup153-Nup50 partnership suggests that the Nup153 scaffold provides not just a means of pore targeting for Nup50 but also serves to provide a local environment that facilitates bringing Nup50 and importin α together, as well as other soluble factors involved in transport. Consistent with this, disruption of the Nup153-Nup50 interface decreases efficiency of nuclear import. PMID:23007389

Fas Apoptosis Inhibitory Molecule (FAIM) Contains a Novel Beta Sandwich in Contact with a Partially Ordered Domain

PubMed Central

Hemond, Michael; Rothstein, Thomas L.; Wagner, Gerhard

2009-01-01

Summary Fas apoptosis inhibitory molecule (FAIM) is a soluble cytosolic protein inhibitor of programmed cell death and is found in organisms throughout the animal kingdom. A short isoform (FAIM-S) is expressed in all tissue types, while an alternatively spliced long isoform (FAIM-L) is specifically expressed in the brain. Here FAIM-S is shown to consist of two independently folding domains in contact with one another. The NMR solution structure of the C-terminal domain of murine FAIM is solved in isolation and revealed to be a novel protein fold, a noninterleaved seven-stranded beta sandwich. The structure and sequence reveal several residues that are likely to be involved in functionally significant interactions with the N-terminal domain or other binding partners. Chemical shift perturbation is used to elucidate contacts made between the N- and C-terminal domains. PMID:19168072

Platinum-Catalyzed, Terminal-Selective C(sp(3))-H Oxidation of Aliphatic Amines.

PubMed

Lee, Melissa; Sanford, Melanie S

2015-10-14

This Communication describes the terminal-selective, Pt-catalyzed C(sp(3))-H oxidation of aliphatic amines without the requirement for directing groups. CuCl2 is employed as a stoichiometric oxidant, and the reactions proceed in high yield at Pt loadings as low as 1 mol%. These transformations are conducted in the presence of sulfuric acid, which reacts with the amine substrates in situ to form ammonium salts. We propose that protonation of the amine serves at least three important roles: (i) it renders the substrates soluble in the aqueous reaction medium; (ii) it limits binding of the amine nitrogen to Pt or Cu; and (iii) it electronically deactivates the C-H bonds proximal to the nitrogen center. We demonstrate that this strategy is effective for the terminal-selective C(sp(3))-H oxidation of a variety of primary, secondary, and tertiary amines.

Platinum-Catalyzed Terminal-Selective C(sp3)–H Oxidation of Aliphatic Amines

PubMed Central

Lee, Melissa; Sanford, Melanie S.

2016-01-01

This paper describes the terminal-selective Pt-catalyzed C(sp3)–H oxidation of aliphatic amines without the requirement for directing groups. CuCl2 is employed as a stoichiometric oxidant, and the reactions proceed in high yield at Pt loadings as low as 1 mol %. These transformations are conducted in the presence of sulfuric acid, which reacts with the amine substrates in situ to form ammonium salts. We propose that protonation of the amine serves at least three important roles: (i) it renders the substrates soluble in the aqueous reaction medium; (ii) it limits binding of the amine nitrogen to Pt or Cu; and (ii) it electronically deactivates the C–H bonds proximal to the nitrogen center. We demonstrate that this strategy is effective for the terminal-selective C(sp3)–H oxidation of a variety of primary, secondary and tertiary amines. PMID:26439251

Immunogenic Characterization of the Dengue Virus Specified Nonstructural Glycoprotein GP48 (NV3, Soluble Complement Fixing Antigen)

DTIC Science & Technology

1988-09-09

Biochemlcals,.Gsnrbridge, UK), we synthesized all 404 possible over- lapping hexapeptideS of 17D YF NSI as well as a 98 amino acid segment of DEN 2 NS1, shown... acids from the NS1 amino terminus. In contrast, rabbit serum that we prepared to authentic YF NS1 was cytolytic and competed with the protective lytic Mab...precipitated. The method was modified y exposing NS1-containing acrylamide gel slices to CnBr vapors (10) in an effort to minimize formic acid -induced

Specific detection of soluble EphA2 fragments in blood as a new biomarker for pancreatic cancer.

PubMed

Koshikawa, Naohiko; Minegishi, Tomoko; Kiyokawa, Hirofumi; Seiki, Motoharu

2017-10-26

Because membrane type 1-matrix metalloproteinase 1 (MT1-MMP) and erythropoietin-producing hepatocellular receptor 2 (EphA2) expression are upregulated by the Ras/mitogen-activated protein kinase pathway, they are frequently coexpressed in malignant tumors. MT1-MMP cleaves the N-terminal ligand-binding domain of EphA2 and inactivates its ligand-dependent tumor-suppressing activity. Therefore, specific detection of the cleaved N-terminal EphA2 fragment in blood might be an effective biomarker to diagnose malignant tumors. To evaluate this possibility, we developed three monoclonal antibodies against the soluble EphA2 fragment. One of them recognized this fragment specifically, with negligible cross-reactivity to the intact form. We used the cleaved form-specific antibody to develop a quantitative enzyme-linked immunosorbent assay and confirmed the linear reactivity to the recombinant fragment. We applied this assay on commercially available serum specimens obtained from patients with several types of cancer including gastric, pancreatic, esophageal, gastroesophageal, and head-and-neck cancers, and healthy donors. Soluble EphA2 fragment levels in cancer-patient sera were higher than those in healthy donors (n=50). In particular, levels of eight out of nine (89%) pancreatic cancer patients and ten out of seventeen (59%) gastric cancer patients significantly exceeded cutoff values obtained from the healthy donors, whereas those of esophageal and head-and-neck cancer-patient sera were low. The preliminary receiver operating characteristic curve analysis for pancreatic cancer demonstrated that the sensitivity and specificity were 89.0% and 90.0%, respectively, whereas those of the conventional digestive tumor marker CA19-9 were 88.9% and 72.0%, respectively. These results indicated that specific detection of soluble EphA2 fragment levels in serum could be potentially useful as a biomarker to diagnose pancreatic cancer.

[Effect of N-terminal truncation of Bacillus acidopullulyticus pullulanase on enzyme properties and functions].

PubMed

Chen, A'na; Liu, Xiuxia; Dai, Xiaofeng; Zhan, Jinling; Peng, Feng; Li, Lu; Wang, Fen; Li, Song; Yang, Yankun; Bai, Zhonghu

2016-03-01

We constructed different N-terminal truncated variants based on Bacillus acidopullulyticus pullulanase 3D structure (PDB code 2WAN), and studied the effects of truncated mutation on soluble expression, enzymatic properties, and application in saccharification. Upon expression, the variants of X45 domain deletion existed as inclusion bodies, whereas deletion of CBM41 domain had an effective effect on soluble expression level. The variants that lack of CBM41 (M1), lack of X25 (M3), and lack both of CBM41 and X25 (M5) had the same optimal pH (5.0) and optimal temperature (60 degrees C) with the wild-type pullulanase (WT). The K(m) of M1 and M5 were 1.42 mg/mL and 1.85 mg/mL, respectively, 2.4- and 3.1-fold higher than that of the WT. k(cat)/K(m) value of M5 was 40% lower than that of the WT. Substrate specificity results show that the enzymes exhibited greater activity with the low-molecular-weight dextrin than with high-molecular-weight soluble starch. When pullulanases were added to the saccharification reaction system, the dextrose equivalent of the WT, M1, M3, and M5 were 93.6%, 94.7%, 94.5%, and93.1%, respectively. These results indicate that the deletion of CBM41 domain and/or X25 domain did not affect the practical application in starch saccharification process. Furthermore, low-molecular-weight variants facilitate the heterologous expression. Truncated variants may be more suitable for industrial production than the WT.

Programmable Electrochemical Rectifier Based on a Thin-Layer Cell.

PubMed

Park, Seungjin; Park, Jun Hui; Hwang, Seongpil; Kwak, Juhyoun

2017-06-21

A programmable electrochemical rectifier based on thin-layer electrochemistry is described here. Both the rectification ratio and the response time of the device are programmable by controlling the gap distance of the thin-layer electrochemical cell, which is easily controlled using commercially available beads. One of the electrodes was modified using a ferrocene-terminated self-assembled monolayer to offer unidirectional charge transfers via soluble redox species. The thin-layer configuration provided enhanced mass transport, which was determined by the gap thickness. The device with the smallest gap thickness (∼4 μm) showed an unprecedented, high rectification ratio (up to 160) with a fast response time in a two-terminal configuration using conventional electronics.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Young, D.A.; Smith, C.H.

A carboxyl-terminated butadiene/acrylonitrile (CTBN)/epoxy resin adduct, used to encapsulate electronic devices, was studied to improve its quality and reliability. The average physical and mechanical properties of the amine-cured product were obtained by testing 16 batches of adduct prepared from 13 separate lots of CTBN. It was found that by using a CTBN with a higher acrylonitrile content (or one in which the chemical structure includes carboxyl groups in the chain backbone, in addition to end termination), a clear, soluble liquid adduct that does not separate in storage or transit could be prepared. These materials also produced clear epoxy castings andmore » filled potting compounds with improved impact, flexural, compressive, and tensile strengths.« less

Structural basis for the stabilization of the complement alternative pathway C3 convertase by properdin

PubMed Central

Alcorlo, Martín; Tortajada, Agustín; Rodríguez de Córdoba, Santiago; Llorca, Oscar

2013-01-01

Complement is an essential component of innate immunity. Its activation results in the assembly of unstable protease complexes, denominated C3/C5 convertases, leading to inflammation and lysis. Regulatory proteins inactivate C3/C5 convertases on host surfaces to avoid collateral tissue damage. On pathogen surfaces, properdin stabilizes C3/C5 convertases to efficiently fight infection. How properdin performs this function is, however, unclear. Using electron microscopy we show that the N- and C-terminal ends of adjacent monomers in properdin oligomers conform a curly vertex that holds together the AP convertase, interacting with both the C345C and vWA domains of C3b and Bb, respectively. Properdin also promotes a large displacement of the TED (thioester-containing domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domains of C3b, which likely impairs C3-convertase inactivation by regulatory proteins. The combined effect of molecular cross-linking and structural reorganization increases stability of the C3 convertase and facilitates recruitment of fluid-phase C3 convertase to the cell surfaces. Our model explains how properdin mediates the assembly of stabilized C3/C5-convertase clusters, which helps to localize complement amplification to pathogen surfaces. PMID:23901101

Epitope and isotype specificities of antibodies to β-amyloid peptide for protection against Alzheimer's disease-like neuropathology

PubMed Central

Bard, Frédérique; Barbour, Robin; Cannon, Catherine; Carretto, Robert; Fox, Michael; Games, Dora; Guido, Teresa; Hoenow, Kathleen; Hu, Kang; Johnson-Wood, Kelly; Khan, Karen; Kholodenko, Dora; Lee, Celeste; Lee, Mike; Motter, Ruth; Nguyen, Minh; Reed, Amanda; Schenk, Dale; Tang, Pearl; Vasquez, Nicki; Seubert, Peter; Yednock, Ted

2003-01-01

Transgenic PDAPP mice, which express a disease-linked isoform of the human amyloid precursor protein, exhibit CNS pathology that is similar to Alzheimer's disease. In an age-dependent fashion, the mice develop plaques containing β-amyloid peptide (Aβ) and exhibit neuronal dystrophy and synaptic loss. It has been shown in previous studies that pathology can be prevented and even reversed by immunization of the mice with the Aβ peptide. Similar protection could be achieved by passive administration of some but not all monoclonal antibodies against Aβ. In the current studies we sought to define the optimal antibody response for reducing neuropathology. Immune sera with reactivity against different Aβ epitopes and monoclonal antibodies with different isotypes were examined for efficacy both ex vivo and in vivo. The studies showed that: (i) of the purified or elicited antibodies tested, only antibodies against the N-terminal regions of Aβ were able to invoke plaque clearance; (ii) plaque binding correlated with a clearance response and neuronal protection, whereas the ability of antibodies to capture soluble Aβ was not necessarily correlated with efficacy; (iii) the isotype of the antibody dramatically influenced the degree of plaque clearance and neuronal protection; (iv) high affinity of the antibody for Fc receptors on microglial cells seemed more important than high affinity for Aβ itself; and (v) complement activation was not required for plaque clearance. These results indicate that antibody Fc-mediated plaque clearance is a highly efficient and effective process for protection against neuropathology in an animal model of Alzheimer's disease. PMID:12566568

L-arginine mediated renaturation enhances yield of human, α6 type IV collagen non-collagenous domain from bacterial inclusion bodies

PubMed Central

Gunda, Venugopal; Boosani, Chandra Shekhar; Verma, Raj Kumar; Guda, Chittibabu; Akul Sudhakar, Yakkanti

2012-01-01

The anti-angiogenic, carboxy terminal non-collagenous domain (NC1) derived from human Collagen type IV alpha 6 chain, [α6(IV)NC1] or hexastatin, was earlier obtained using different recombinant methods of expression in bacterial systems. However, the effect of L-arginine mediated renaturation in enhancing the relative yields of this protein from bacterial inclusion bodies has not been evaluated. In the present study, direct stirring and on-column renaturation methods using L-arginine and different size exclusion chromatography matrices were applied for enhancing the solubility in purifying the recombinant α6(IV)NC1 from bacterial inclusion bodies. This methodology enabled purification of higher quantities of soluble protein from inclusion bodies, which inhibited endothelial cell proliferation, migration and tube formation. Thus, the scope for L-arginine mediated renaturation in obtaining higher yields of soluble, biologically active NC1 domain from bacterial inclusion bodies was evaluated. PMID:22512648

L-arginine mediated renaturation enhances yield of human, α6 Type IV collagen non-collagenous domain from bacterial inclusion bodies.

PubMed

Gunda, Venugopal; Boosani, Chandra Shekhar; Verma, Raj Kumar; Guda, Chittibabu; Sudhakar, Yakkanti Akul

2012-10-01

The anti-angiogenic, carboxy terminal non-collagenous domain (NC1) derived from human Collagen type IV alpha 6 chain, [α6(IV)NC1] or hexastatin, was earlier obtained using different recombinant methods of expression in bacterial systems. However, the effect of L-arginine mediated renaturation in enhancing the relative yields of this protein from bacterial inclusion bodies has not been evaluated. In the present study, direct stirring and on-column renaturation methods using L-arginine and different size exclusion chromatography matrices were applied for enhancing the solubility in purifying the recombinant α6(IV)NC1 from bacterial inclusion bodies. This methodology enabled purification of higher quantities of soluble protein from inclusion bodies, which inhibited endothelial cell proliferation, migration and tube formation. Thus, the scope for L-arginine mediated renaturation in obtaining higher yields of soluble, biologically active NC1 domain from bacterial inclusion bodies was evaluated.

The α-Secretase-derived N-terminal Product of Cellular Prion, N1, Displays Neuroprotective Function in Vitro and in Vivo*

PubMed Central

Guillot-Sestier, Marie-Victoire; Sunyach, Claire; Druon, Charlotte; Scarzello, Sabine; Checler, Frédéric

2009-01-01

Cellular prion protein (PrPc) undergoes a disintegrin-mediated physiological cleavage, generating a soluble amino-terminal fragment (N1), the function of which remained unknown. Recombinant N1 inhibits staurosporine-induced caspase-3 activation by modulating p53 transcription and activity, whereas the PrPc-derived pathological fragment (N2) remains biologically inert. Furthermore, N1 protects retinal ganglion cells from hypoxia-induced apoptosis, reduces the number of terminal deoxynucleotidyltransferase-mediated biotinylated UTP nick end labeling-positive and p53-immunoreactive neurons in a pressure-induced ischemia model of the rat retina and triggers a partial recovery of b-waves but not a-waves of rat electroretinograms. Our work is the first demonstration that the α-secretase-derived PrPc fragment N1, but not N2, displays in vivo and in vitro neuroprotective function by modulating p53 pathway. It further demonstrates that distinct N-terminal cleavage products of PrPc harbor different biological activities underlying the various phenotypes linking PrPc to cell survival. PMID:19850936

A homogeneous, recyclable polymer support for Rh(I)-catalyzed C-C bond formation.

PubMed

Jana, Ranjan; Tunge, Jon A

2011-10-21

A robust and practical polymer-supported, homogeneous, recyclable biphephos rhodium(I) catalyst has been developed for C-C bond formation reactions. Control of polymer molecular weight allowed tuning of the polymer solubility such that the polymer-supported catalyst is soluble in nonpolar solvents and insoluble in polar solvents. Using the supported rhodium catalysts, addition of aryl and vinylboronic acids to the electrophiles such as enones, aldehydes, N-sulfonyl aldimines, and alkynes occurs smoothly to provide products in high yields. Additions of terminal alkynes to enones and industrially relevant hydroformylation reactions have also been successfully carried out. Studies show that the leaching of Rh from the polymer support is low and catalyst recycle can be achieved by simple precipitation and filtration.

A Homogeneous, Recyclable Polymer Support for Rh(I)-Catalyzed C-C Bond Formation

PubMed Central

Jana, Ranjan; Tunge, Jon A.

2011-01-01

A robust and practical polymer-supported, homogeneous, recyclable biphephos rhodium(I) catalyst has been developed for C-C bond formation reactions. Control of polymer molecular weight allowed tuning of the polymer solubility such that the polymer-supported catalyst is soluble in nonpolar solvents and insoluble in polar solvents. Using the supported rhodium catalysts, addition of aryl and vinylboronic acids to the electrophiles such as enones, aldehydes, N-sulfonyl aldimines, and alkynes occurs smoothly to provide products in high yields. Additions of terminal alkynes to enones and industrially relevant hydroformylation reactions have also been successfully carried out. Studies show that the leaching of Rh from the polymer support is low and catalyst recycle can be achieved by simple precipitation and filtration. PMID:21895010

Growth of Rhodococcus sp. strain BCP1 on gaseous n-alkanes: new metabolic insights and transcriptional analysis of two soluble di-iron monooxygenase genes

PubMed Central

Cappelletti, Martina; Presentato, Alessandro; Milazzo, Giorgio; Turner, Raymond J.; Fedi, Stefano; Frascari, Dario; Zannoni, Davide

2015-01-01

Rhodococcus sp. strain BCP1 was initially isolated for its ability to grow on gaseous n-alkanes, which act as inducers for the co-metabolic degradation of low-chlorinated compounds. Here, both molecular and metabolic features of BCP1 cells grown on gaseous and short-chain n-alkanes (up to n-heptane) were examined in detail. We show that propane metabolism generated terminal and sub-terminal oxidation products such as 1- and 2-propanol, whereas 1-butanol was the only terminal oxidation product detected from n-butane metabolism. Two gene clusters, prmABCD and smoABCD—coding for Soluble Di-Iron Monooxgenases (SDIMOs) involved in gaseous n-alkanes oxidation—were detected in the BCP1 genome. By means of Reverse Transcriptase-quantitative PCR (RT-qPCR) analysis, a set of substrates inducing the expression of the sdimo genes in BCP1 were assessed as well as their transcriptional repression in the presence of sugars, organic acids, or during the cell growth on rich medium (Luria–Bertani broth). The transcriptional start sites of both the sdimo gene clusters were identified by means of primer extension experiments. Finally, proteomic studies revealed changes in the protein pattern induced by growth on gaseous- (n-butane) and/or liquid (n-hexane) short-chain n-alkanes as compared to growth on succinate. Among the differently expressed protein spots, two chaperonins and an isocytrate lyase were identified along with oxidoreductases involved in oxidation reactions downstream of the initial monooxygenase reaction step. PMID:26029173

Analysis of the interaction between respiratory syncytial virus and lipid-rafts in Hep2 cells during infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, Gaie; Jeffree, Chris E.; McDonald, Terence

2004-10-01

The assembly of respiratory syncytial virus (RSV) in lipid-rafts was examined in Hep2 cells. Confocal and electron microscopy showed that during RSV assembly, the cellular distribution of the complement regulatory proteins, decay accelerating factor (CD55) and CD59, changes and high levels of these cellular proteins are incorporated into mature virus filaments. The detergent-solubility properties of CD55, CD59, and the RSV fusion (F) protein were found to be consistent with each protein being located predominantly within lipid-raft structures. The levels of these proteins in cell-released virus were examined by immunoelectronmicroscopy and found to account for between 5% and 15% of themore » virus attachment (G) glycoprotein levels. Collectively, our findings suggest that an intimate association exists between RSV and lipid-raft membranes and that significant levels of these host-derived raft proteins, such as those regulating complement activation, are subsequently incorporated into the envelope of mature virus particles.« less

Molecular Interactions of the Hantaan Virus Nucleocapsid Protein

DTIC Science & Technology

1991-09-12

stomatitis virus (VSV; a rhabdovirus ), where the phosphoprotein NS (analogous to Sendai P protein) contains a single carboxyl-terminal domain...structures reminiscent of rhabdovirus capsomeres were clearly visible in the electron microscope (Blumberg et ai-, 1983). This innate ability of viral...passaged virus (Weiss et. al.., 1989) . In addition, a soluble form of VSV ( rhabdovirus ) N protein was found, in the presence of various RNAs, to

Formation of active inclusion bodies induced by hydrophobic self-assembling peptide GFIL8.

PubMed

Wang, Xu; Zhou, Bihong; Hu, Weike; Zhao, Qing; Lin, Zhanglin

2015-06-16

In the last few decades, several groups have observed that proteins expressed as inclusion bodies (IBs) in bacteria could still be biologically active when terminally fused to an appropriate aggregation-prone partner such as pyruvate oxidase from Paenibacillus polymyxa (PoxB). More recently, we have demonstrated that three amphipathic self-assembling peptides, an alpha helical peptide 18A, a beta-strand peptide ELK16, and a surfactant-like peptide L6KD, have properties that induce target proteins into active IBs. We have developed an efficient protein expression and purification approach for these active IBs by introducing a self-cleavable intein molecule. In this study, the self-assembling peptide GFIL8 (GFILGFIL) with only hydrophobic residues was analyzed, and this peptide effectively induced the formation of cytoplasmic IBs in Escherichia coli when terminally attached to lipase A and amadoriase II. The protein aggregates in cells were confirmed by transmission electron microscopy analysis and retained ~50% of their specific activities relative to the native counterparts. We constructed an expression and separation coupled tag (ESCT) by incorporating an intein molecule, the Mxe GyrA intein. Soluble target proteins were successfully released from active IBs upon cleavage of the intein between the GFIL8 tag and the target protein, which was mediated by dithiothreitol. A variant of GFIL8, GFIL16 (GFILGFILGFILGFIL), improved the ESCT scheme by efficiently eliminating interference from the soluble intein-GFIL8 molecule. The yields of target proteins at the laboratory scale were 3.0-7.5 μg/mg wet cell pellet, which is comparable to the yields from similar ESCT constructs using 18A, ELK16, or the elastin-like peptide tag scheme. The all-hydrophobic self-assembling peptide GFIL8 induced the formation of active IBs in E. coli when terminally attached to target proteins. GFIL8 and its variant GFIL16 can act as a "pull-down" tag to produce purified soluble proteins with reasonable quantity and purity from active aggregates. Owing to the structural simplicity, strong hydrophobicity, and high aggregating efficiency, these peptides can be further explored for enzyme production and immobilization.

Bioenergetics and the role of soluble cytochromes C for alkaline adaptation in gram-negative alkaliphilic Pseudomonas.

PubMed

Matsuno, T; Yumoto, I

2015-01-01

Very few studies have been conducted on alkaline adaptation of Gram-negative alkaliphiles. The reversed difference of H(+) concentration across the membrane will make energy production considerably difficult for Gram-negative as well as Gram-positive bacteria. Cells of the alkaliphilic Gram-negative bacterium Pseudomonas alcaliphila AL15-21(T) grown at pH 10 under low-aeration intensity have a soluble cytochrome c content that is 3.6-fold higher than that of the cells grown at pH 7 under high-aeration intensity. Cytochrome c-552 content was higher (64% in all soluble cytochromes c) than those of cytochrome c-554 and cytochrome c-551. In the cytochrome c-552-dificient mutant grown at pH 10 under low-aeration intensity showed a marked decrease in μ max⁡ [h(-1)] (40%) and maximum cell turbidity (25%) relative to those of the wild type. Considering the high electron-retaining abilities of the three soluble cytochromes c, the deteriorations in the growth of the cytochrome c-552-deficient mutant could be caused by the soluble cytochromes c acting as electron storages in the periplasmic space of the bacterium. These electron-retaining cytochromes c may play a role as electron and H(+) condenser, which facilitate terminal oxidation at high pH under air-limited conditions, which is difficult to respire owing to less oxygen and less H(+).

Bioenergetics and the Role of Soluble Cytochromes c for Alkaline Adaptation in Gram-Negative Alkaliphilic Pseudomonas

PubMed Central

Matsuno, T.; Yumoto, I.

2015-01-01

Very few studies have been conducted on alkaline adaptation of Gram-negative alkaliphiles. The reversed difference of H+ concentration across the membrane will make energy production considerably difficult for Gram-negative as well as Gram-positive bacteria. Cells of the alkaliphilic Gram-negative bacterium Pseudomonas alcaliphila AL15-21T grown at pH 10 under low-aeration intensity have a soluble cytochrome c content that is 3.6-fold higher than that of the cells grown at pH 7 under high-aeration intensity. Cytochrome c-552 content was higher (64% in all soluble cytochromes c) than those of cytochrome c-554 and cytochrome c-551. In the cytochrome c-552-dificient mutant grown at pH 10 under low-aeration intensity showed a marked decrease in μ max⁡ [h−1] (40%) and maximum cell turbidity (25%) relative to those of the wild type. Considering the high electron-retaining abilities of the three soluble cytochromes c, the deteriorations in the growth of the cytochrome c-552-deficient mutant could be caused by the soluble cytochromes c acting as electron storages in the periplasmic space of the bacterium. These electron-retaining cytochromes c may play a role as electron and H+ condenser, which facilitate terminal oxidation at high pH under air-limited conditions, which is difficult to respire owing to less oxygen and less H+. PMID:25705691

Identity of a peptide domain of human C9 that is bound by the cell-surface complement inhibitor, CD59.

PubMed

Chang, C P; Hüsler, T; Zhao, J; Wiedmer, T; Sims, P J

1994-10-21

The CD59 antigen is a plasma membrane glycoprotein that serves as an inhibitor of the C5b-9 complex of complement. This inhibitory activity appears related to the capacity of CD59 to bind with high affinity to sites that are nascently exposed in the alpha-chain subunit of human C8, as well as within the C9b domain (amino acid residues 245-538) of human C9, during assembly of the C5b-9 complex on the target membrane (Ninomiya, H., and Sims, P. J. (1992) J. Biol. Chem. 267, 13675-13680). The CD59 binding site in C9 was first investigated by N-terminal sequencing of CD59-binding peptides generated by limited digest of the isolated C9b domain. These experiments revealed a 17-kDa fragment (starting at C9 residue Thr-320) that retained affinity for CD59, suggesting the possibility for localizing the CD59 binding site by mapping with small C9-derived peptides. Peptides spanning the entire C9b sequence were expressed in Escherichia coli and then probed with CD59. CD59 bound specifically to all peptides starting N-terminal to C9 residue 359 with C termini extending beyond residue 411. Little to no CD59 binding was observed for various C9-derived peptides that started C-terminal to residue 359 or that were truncated N-terminal to residue 411. Affinity-purified antibody against C9 residues 320-411 inhibited CD59 binding to C9 by > 50% and completely inhibited its binding to the isolated C9b domain. Little to no specific binding of CD59 was detected for peptides restricted to the putative hinge domain within C9b (residues 245-271). These results indicate that a CD59 binding site is located between residues 320 and 411 of the C9 polypeptide and suggest that the affinity of this site is principally determined by residues 359-411.

[Cloning of cDNA for RNA polymerase subunit from the fission yeast Schizosaccharomyces pombe by heterospecific complementation in Saccharomyces cerevisiae].

PubMed

Shpakovskiĭ, G V; Lebedenko, E N; Thuriaux, P

1997-02-01

The rpb10 cDNA of the fission yeast Schizosaccharomyces pombe, encoding one of the five small subunits common to all three nuclear DNA-dependent RNA polymerases, was isolated from an expression cDNA library by two independent approaches: PCR-based screening and direct suppression by means of heterospecific complementation of a temperature-sensitive mutant defective in the corresponding gene of Saccharomyces cerevisiae. The cloned Sz. pombe cDNA encodes a protein Rpb10 of 71 amino acids with an M of 8,275 Da, sharing 51 amino acids (71% identity) with the subunit ABC10 beta of RNA polymerases I-III from S. cerevisiae. All eukaryotic members of this protein family have the same general organization featuring two highly conserved motifs (RCFT/SCGK and RYCCRRM) around an atypical zinc finger and an additional invariant HVDLIEK motif toward the C-terminal end. The last motif is only characteristics for homologs from eukaryotes. In keeping with this remarkable structural conservation, the Sz. pombe cDNA also fully complemented a S. cerevisiae deletion mutant lacking subunit ABC10 beta (null allele rpb10-delta 1::HIS3).

Functional anatomy of complement factor H.

PubMed

Makou, Elisavet; Herbert, Andrew P; Barlow, Paul N

2013-06-11

Factor H (FH) is a soluble regulator of the proteolytic cascade at the core of the evolutionarily ancient vertebrate complement system. Although FH consists of a single chain of similar protein modules, it has a demanding job description. Its chief role is to prevent complement-mediated injury to healthy host cells and tissues. This entails recognition of molecular patterns on host surfaces combined with control of one of nature's most dangerous examples of a positive-feedback loop. In this way, FH modulates, where and when needed, an amplification process that otherwise exponentially escalates the production of the pro-inflammatory, pro-phagocytic, and pro-cytolytic cleavage products of complement proteins C3 and C5. Mutations and single-nucleotide polymorphisms in the FH gene and autoantibodies against FH predispose individuals to diseases, including age-related macular degeneration, dense-deposit disease, and atypical hemolytic uremic syndrome. Moreover, deletions or variations of genes for FH-related proteins also influence the risk of disease. Numerous pathogens hijack FH and use it for self-defense. As reviewed herein, a molecular understanding of FH function is emerging. While its functional oligomeric status remains uncertain, progress has been achieved in characterizing its three-dimensional architecture and, to a lesser extent, its intermodular flexibility. Models are proposed, based on the reconciliation of older data with a wealth of recent evidence, in which a latent circulating form of FH is activated by its principal target, C3b tethered to a self-surface. Such models suggest hypotheses linking sequence variations to pathophysiology, but improved, more quantitative, functional assays and rigorous data analysis are required to test these ideas.

Complement Component 3C3 and C3a Receptor Are Required in Chitin-Dependent Allergic Sensitization to Aspergillus fumigatus but Dispensable in Chitin-Induced Innate Allergic Inflammation

PubMed Central

Roy, René M.; Paes, Hugo C.; Nanjappa, Som G.; Sorkness, Ron; Gasper, David; Sterkel, Alana; Wüthrich, Marcel; Klein, Bruce S.

2013-01-01

ABSTRACT Levels of the anaphylatoxin C3a are increased in patients with asthma compared with those in nonasthmatics and increase further still during asthma exacerbations. However, the role of C3a during sensitization to allergen is poorly understood. Sensitization to fungal allergens, such as Aspergillus fumigatus, is a strong risk factor for the development of asthma. Exposure to chitin, a structural polysaccharide of the fungal cell wall, induces innate allergic inflammation and may promote sensitization to fungal allergens. Here, we found that coincubation of chitin with serum or intratracheal administration of chitin in mice resulted in the generation of C3a. We established a model of chitin-dependent sensitization to soluble Aspergillus antigens to test the contribution of complement to these events. C3−/− and C3aR−/− mice were protected from chitin-dependent sensitization to Aspergillus and had reduced lung eosinophilia and type 2 cytokines and serum IgE. In contrast, complement-deficient mice were not protected against chitin-induced innate allergic inflammation. In sensitized mice, plasmacytoid dendritic cells from complement-deficient animals acquired a tolerogenic profile associated with enhanced regulatory T cell responses and suppressed Th2 and Th17 responses specific for Aspergillus. Thus, chitin induces the generation of C3a in the lung, and chitin-dependent allergic sensitization to Aspergillus requires C3aR signaling, which suppresses regulatory dendritic cells and T cells and induces allergy-promoting T cells. PMID:23549917

Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system

PubMed Central

Costa, Sofia; Almeida, André; Castro, António; Domingues, Lucília

2014-01-01

Proteins are now widely produced in diverse microbial cell factories. The Escherichia coli is still the dominant host for recombinant protein production but, as a bacterial cell, it also has its issues: the aggregation of foreign proteins into insoluble inclusion bodies is perhaps the main limiting factor of the E. coli expression system. Conversely, E. coli benefits of cost, ease of use and scale make it essential to design new approaches directed for improved recombinant protein production in this host cell. With the aid of genetic and protein engineering novel tailored-made strategies can be designed to suit user or process requirements. Gene fusion technology has been widely used for the improvement of soluble protein production and/or purification in E. coli, and for increasing peptide’s immunogenicity as well. New fusion partners are constantly emerging and complementing the traditional solutions, as for instance, the Fh8 fusion tag that has been recently studied and ranked among the best solubility enhancer partners. In this review, we provide an overview of current strategies to improve recombinant protein production in E. coli, including the key factors for successful protein production, highlighting soluble protein production, and a comprehensive summary of the latest available and traditionally used gene fusion technologies. A special emphasis is given to the recently discovered Fh8 fusion system that can be used for soluble protein production, purification, and immunogenicity in E. coli. The number of existing fusion tags will probably increase in the next few years, and efforts should be taken to better understand how fusion tags act in E. coli. This knowledge will undoubtedly drive the development of new tailored-made tools for protein production in this bacterial system. PMID:24600443

Solubility prediction, solvate and cocrystal screening as tools for rational crystal engineering.

PubMed

Loschen, Christoph; Klamt, Andreas

2015-06-01

The fact that novel drug candidates are becoming increasingly insoluble is a major problem of current drug development. Computational tools may address this issue by screening for suitable solvents or by identifying potential novel cocrystal formers that increase bioavailability. In contrast to other more specialized methods, the fluid phase thermodynamics approach COSMO-RS (conductor-like screening model for real solvents) allows for a comprehensive treatment of drug solubility, solvate and cocrystal formation and many other thermodynamics properties in liquids. This article gives an overview of recent COSMO-RS developments that are of interest for drug development and contains several new application examples for solubility prediction and solvate/cocrystal screening. For all property predictions COSMO-RS has been used. The basic concept of COSMO-RS consists of using the screening charge density as computed from first principles calculations in combination with fast statistical thermodynamics to compute the chemical potential of a compound in solution. The fast and accurate assessment of drug solubility and the identification of suitable solvents, solvate or cocrystal formers is nowadays possible and may be used to complement modern drug development. Efficiency is increased by avoiding costly quantum-chemical computations using a database of previously computed molecular fragments. COSMO-RS theory can be applied to a range of physico-chemical properties, which are of interest in rational crystal engineering. Most notably, in combination with experimental reference data, accurate quantitative solubility predictions in any solvent or solvent mixture are possible. Additionally, COSMO-RS can be extended to the prediction of cocrystal formation, which results in considerable predictive accuracy concerning coformer screening. In a recent variant costly quantum chemical calculations are avoided resulting in a significant speed-up and ease-of-use. © 2015 Royal Pharmaceutical Society.

The IgV domain of human B7-2 (CD86) is sufficient to co-stimulate T lymphocytes and induce cytokine secretion.

PubMed

Rennert, P; Furlong, K; Jellis, C; Greenfield, E; Freeman, G J; Ueda, Y; Levine, B; June, C H; Gray, G S

1997-06-01

B7-1 (CD80) and B7-2 (CD86) are genetically and structurally related molecules expressed on antigen-presenting cells. Both bind CD28 to co-stimulate T lymphocytes, resulting in proliferation and cytokine production. The extracellular portions of B7-1 and B7-2 which bind to CD28 and CTLA-4 are related to Ig variable (V) and Ig constant (C) domain sequences. Recent reports have described splice variant forms of B7 proteins which occur in vivo and are of unknown function. Here we describe soluble recombinant forms of B7-1 and B7-2 containing either both of the Ig-like extracellular domains or the individual IgV or IgC domains coupled to an Ig Fc tail. Soluble B7-1 and B7-2 bind to CD28 and CTLA-4, and effectively co-stimulate T lymphocytes resulting in their proliferation and the secretion of cytokines. Furthermore, the IgV domain of B7-2 binds CD28 and CTLA-4, competes with B7-1 and B7-2 for binding to these receptors, and co-stimulates T lymphocytes. Cross-linked soluble B7-2v was the most potent co-stimulatory molecule tested and was active at a concentration approximately 100-fold lower than cross-linked soluble B7-1 or B7-2 proteins. When bound to tosyl-activated beads, B7-2v was capable of sustaining multiple rounds of T cell expansion. These data complement the description of naturally occurring variants to suggest that T cell co-stimulation in vivo may be regulated by soluble or truncated forms of B7 proteins.

Mycophenolate Mofetil in Combination with Steroids for Treatment of C3 Glomerulopathy: A Case Series.

PubMed

Avasare, Rupali S; Canetta, Pietro A; Bomback, Andrew S; Marasa, Maddalena; Caliskan, Yasar; Ozluk, Yasemin; Li, Yifu; Gharavi, Ali G; Appel, Gerald B

2018-03-07

C3 glomerulopathy is a form of complement-mediated GN. Immunosuppressive therapy may be beneficial in the treatment of C3 glomerulopathy. Mycophenolate mofetil is an attractive treatment option given its role in the treatment of other complement-mediated diseases and the results of the Spanish Group for the Study of Glomerular Diseases C3 Study. Here, we study the outcomes of patients with C3 glomerulopathy treated with steroids and mycophenolate mofetil. We conducted a retrospective chart review of patients in the C3 glomerulopathy registry at Columbia University and identified patients treated with mycophenolate mofetil for at least 3 months and follow-up for at least 1 year. We studied clinical, histologic, and genetic data for the whole group and compared data for those who achieved complete or partial remission (responders) with those who did not achieve remission (nonresponders). We compared remission with mycophenolate mofetil with remission with other immunosuppressive regimens. We identified 30 patients who met inclusion criteria. Median age was 25 years old (interquartile range, 18-36), median creatinine was 1.07 mg/dl (interquartile range, 0.79-1.69), and median proteinuria was 3200 mg/g creatinine (interquartile range, 1720-6759). The median follow-up time was 32 months (interquartile range, 21-68). Twenty (67%) patients were classified as responders. There were no significant differences in baseline characteristics between responders and nonresponders, although initial proteinuria was lower (median 2468 mg/g creatinine) in responders compared with nonresponders (median 5000 mg/g creatinine) and soluble membrane attack complex levels were higher in responders compared with nonresponders. For those tapered off mycophenolate mofetil, relapse rate was 50%. Genome-wide analysis on complement genes was done, and in 12 patients, we found 18 variants predicted to be damaging. None of these variants were previously reported to be pathogenic. Mycophenolate mofetil with steroids outperformed other immunosuppressive regimens. Among patients who tolerated mycophenolate mofetil, combination therapy with steroids induced remission in 67% of this cohort. Heavier proteinuria at the start of therapy and lower soluble membrane attack complex levels were associated with treatment resistance. Copyright © 2018 by the American Society of Nephrology.

Comprehensive Study on the Impact of the Cation Alkyl Side Chain Length on the Solubility of Water in Ionic Liquids.

PubMed

Kurnia, Kiki A; Neves, Catarina M S S; Freire, Mara G; Santos, Luís M N B F; Coutinho, João A P

2015-10-01

A comprehensive study on the phase behaviour of two sets of ionic liquids (ILs) and their interactions with water is here presented through combining experimental and theoretical approaches. The impact of the alkyl side chain length and the cation symmetry on the water solubility in the asymmetric [C N- 1 C 1 im][NTf 2 ] and symmetric [C N- 1 C N- 1 im][NTf 2 ] series of ILs ( N up to 22), from 288.15 K to 318.15 K and at atmospheric pressure, was studied. The experimental data reveal that the solubility of water in ILs with an asymmetric cation is higher than in those with the symmetric isomer. Several trend shifts on the water solubility as a function of the alkyl side chain length were identified, namely at [C 6 C 1 im][NTf 2 ] for asymmetric ILs and at [C 4 C 4 im][NTf 2 ] and [C 7 C 7 im][NTf 2 ] for the symmetric ILs. To complement the experimental data and to further investigate the molecular-level mechanisms behind the dissolution process, Density Functional Theory calculations, using the Conductor-like Screening Model for Real Solvents (COSMO-RS) and the Electrostatic potential-derived CHelpG, were performed. The COSMO-RS model is able to qualitatively predict water solubility as function of temperature and alkyl chain lengths of both symmetric and asymmetric cations. Furthermore, the model is also capable to predict the somewhat higher water solubility in the asymmetric cation, as well as the trend shift as function of alkyl chain lengths experimentally observed. Both COSMO-RS and the electrostatic potential-derived CHelpG show that the interactions of water and the IL cation take place on the IL polar region, namely on the aromatic head and adjacent methylene groups what explains the differences in water solubility observed for cations with different chain lengths. Furthermore, the CHelpG calculations for the isolated cations in the gas phase indicates that the trend shift of water solubility as function of alkyl chain lengths and the difference of water solubility in symmetric may also result from the partial positive charge distribution/contribution of the cation.

Regulation of presynaptic Ca2+, synaptic plasticity and contextual fear conditioning by a N-terminal β-amyloid fragment.

PubMed

Lawrence, James L M; Tong, Mei; Alfulaij, Naghum; Sherrin, Tessi; Contarino, Mark; White, Michael M; Bellinger, Frederick P; Todorovic, Cedomir; Nichols, Robert A

2014-10-22

Soluble β-amyloid has been shown to regulate presynaptic Ca(2+) and synaptic plasticity. In particular, picomolar β-amyloid was found to have an agonist-like action on presynaptic nicotinic receptors and to augment long-term potentiation (LTP) in a manner dependent upon nicotinic receptors. Here, we report that a functional N-terminal domain exists within β-amyloid for its agonist-like activity. This sequence corresponds to a N-terminal fragment generated by the combined action of α- and β-secretases, and resident carboxypeptidase. The N-terminal β-amyloid fragment is present in the brains and CSF of healthy adults as well as in Alzheimer's patients. Unlike full-length β-amyloid, the N-terminal β-amyloid fragment is monomeric and nontoxic. In Ca(2+) imaging studies using a model reconstituted rodent neuroblastoma cell line and isolated mouse nerve terminals, the N-terminal β-amyloid fragment proved to be highly potent and more effective than full-length β-amyloid in its agonist-like action on nicotinic receptors. In addition, the N-terminal β-amyloid fragment augmented theta burst-induced post-tetanic potentiation and LTP in mouse hippocampal slices. The N-terminal fragment also rescued LTP inhibited by elevated levels of full-length β-amyloid. Contextual fear conditioning was also strongly augmented following bilateral injection of N-terminal β-amyloid fragment into the dorsal hippocampi of intact mice. The fragment-induced augmentation of fear conditioning was attenuated by coadministration of nicotinic antagonist. The activity of the N-terminal β-amyloid fragment appears to reside largely in a sequence surrounding a putative metal binding site, YEVHHQ. These findings suggest that the N-terminal β-amyloid fragment may serve as a potent and effective endogenous neuromodulator. Copyright © 2014 the authors 0270-6474/14/3414210-09$15.00/0.

Species Specificity of Vaccinia Virus Complement Control Protein for the Bovine Classical Pathway Is Governed Primarily by Direct Interaction of Its Acidic Residues with Factor I

PubMed Central

Kumar, Jitendra; Yadav, Viveka Nand; Phulera, Swastik; Kamble, Ashish; Gautam, Avneesh Kumar; Panwar, Hemendra Singh

2017-01-01

ABSTRACT Poxviruses display species tropism—variola virus is a human-specific virus, while vaccinia virus causes repeated outbreaks in dairy cattle. Consistent with this, variola virus complement regulator SPICE (smallpox inhibitor of complement enzymes) exhibits selectivity in inhibiting the human alternative complement pathway and vaccinia virus complement regulator VCP (vaccinia virus complement control protein) displays selectivity in inhibiting the bovine alternative complement pathway. In the present study, we examined the species specificity of VCP and SPICE for the classical pathway (CP). We observed that VCP is ∼43-fold superior to SPICE in inhibiting bovine CP. Further, functional assays revealed that increased inhibitory activity of VCP for bovine CP is solely due to its enhanced cofactor activity, with no effect on decay of bovine CP C3-convertase. To probe the structural basis of this specificity, we utilized single- and multi-amino-acid substitution mutants wherein 1 or more of the 11 variant VCP residues were substituted in the SPICE template. Examination of these mutants for their ability to inhibit bovine CP revealed that E108, E120, and E144 are primarily responsible for imparting the specificity and contribute to the enhanced cofactor activity of VCP. Binding and functional assays suggested that these residues interact with bovine factor I but not with bovine C4(H2O) (a moiety conformationally similar to C4b). Mapping of these residues onto the modeled structure of bovine C4b-VCP-bovine factor I supported the mutagenesis data. Taken together, our data help explain why the vaccine strain of vaccinia virus was able to gain a foothold in domesticated animals. IMPORTANCE Vaccinia virus was used for smallpox vaccination. The vaccine-derived virus is now circulating and causing outbreaks in dairy cattle in India and Brazil. However, the reason for this tropism is unknown. It is well recognized that the virus is susceptible to neutralization by the complement classical pathway (CP). Because the virus encodes a soluble complement regulator, VCP, we examined whether this protein displays selectivity in targeting bovine CP. Our data show that it does exhibit selectivity in inhibiting the bovine CP and that this is primarily determined by its amino acids E108, E120, and E144, which interact with bovine serine protease factor I to inactivate bovine C4b—one of the two subunits of CP C3-convertase. Of note, the variola complement regulator SPICE contains positively charged residues at these positions. Thus, these variant residues in VCP help enhance its potency against the bovine CP and thereby the fitness of the virus in cattle. PMID:28724763

On the intracellular trafficking of mouse S5 ribosomal protein from cytoplasm to nucleoli.

PubMed

Matragkou, Ch; Papachristou, H; Karetsou, Z; Papadopoulos, G; Papamarcaki, T; Vizirianakis, I S; Tsiftsoglou, A S; Choli-Papadopoulou, T

2009-10-09

The non-ribosomal functions of mammalian ribosomal proteins have recently attracted worldwide attention. The mouse ribosomal protein S5 (rpS5) derived from ribosomal material is an assembled non-phosphorylated protein. The free form of rpS5 protein, however, undergoes phosphorylation. In this study, we have (a) investigated the potential role of phosphorylation in rpS5 protein transport into the nucleus and then into nucleoli and (b) determined which of the domains of rpS5 are involved in this intracellular trafficking. In vitro PCR mutagenesis of mouse rpS5 cDNA, complemented by subsequent cloning and expression of rpS5 truncated recombinant forms, produced in fusion with green fluorescent protein, permitted the investigation of rpS5 intracellular trafficking in HeLa cells using confocal microscopy complemented by Western blot analysis. Our results indicate the following: (a) rpS5 protein enters the nucleus via the region 38-50 aa that forms a random coil as revealed by molecular dynamic simulation. (b) Immunoprecipitation of rpS5 with casein kinase II and immobilized metal affinity chromatography analysis complemented by in vitro kinase assay revealed that phosphorylation of rpS5 seems to be indispensable for its transport from nucleus to nucleoli; upon entering the nucleus, Thr-133 phosphorylation triggers Ser-24 phosphorylation by casein kinase II, thus promoting entrance of rpS5 into the nucleoli. Another important role of rpS5 N-terminal region is proposed to be the regulation of protein's cellular level. The repetitively co-appearance of a satellite C-terminal band below the entire rpS5 at the late stationary phase, and not at the early logarithmic phase, of cell growth suggests a specific degradation balancing probably the unassembled ribosomal protein molecules with those that are efficiently assembled to ribosomal subunits. Overall, these data provide new insights on the structural and functional domains within the rpS5 molecule that contribute to its cellular functions.

Non-ionic iodinated contrast media related immediate reactions: A mechanism study of 27 patients.

PubMed

Zhai, Liqin; Guo, Xiangjie; Zhang, Haoyue; Jin, Qianqian; Zeng, Qiang; Tang, Xiaoxian; Gao, Cairong

2017-01-01

The underlying mechanism of non-ionic iodinated contrast media-related immediate reactions was evaluated in this study. Patients presenting at least grade II immediate reactions after non-ionic iodinated contrast media injection were enrolled. Basophil activation was evaluated by flow cytometry. The plasma concentration of human terminal complement complex SC5b-9, as well as concentrations of serum chymase, tryptase, human mast cell carboxypeptidase A3, human prostaglandin D2, and total IgE were measured by enzyme-linked immunosorbent assay. The basophil activation percentage was significantly higher in the study group than in the control group (17.94±21.06% vs 3.45±1.49%). The plasma concentration of human terminal complement complex SC5b-9 and concentrations of serum chymase, human mast cell carboxypeptidase A3, prostaglandin D2, tryptase, and total IgE were also significantly increased (236.99±318.21 vs 49.70±30.41ng/mL, 0.41±0.49 vs 0.09±0.06ng/mL, 1.17±0.67 vs 0.30±0.17ng/mL, 203.52±137.27 vs 102.28±48.72pg/mL, 3.81±0.22 vs 2.70±0.16ng/mL, 102.00±51.84 vs 19.97±2.75ng/mL, respectively). Both mast cells and basophils were activated in non-ionic iodinated contrast media to mediate immediate hypersensitivity, and mast cells may be involved. Different mechanisms, including IgE-dependent, complement-dependent, and direct membrane effects, contributed to mast cell and basophil activation. Individual patients may use a single or combined mechanism involving single or combined mast cells and basophils. Immediate reactions following non-ionic iodinated contrast media injection may be a mechanically heterogenous disease. Copyright © 2016. Published by Elsevier B.V.

Pull-down Assay to Characterize Ca2+/Calmodulin Binding to Plant Receptor Kinases.

PubMed

Kaufmann, Christine; Sauter, Margret

2017-01-01

Plant receptor-like kinases (RLKs) are regulated by posttranscriptional modification and by interaction with regulatory proteins. A common modification of RLKs is (auto)phosphorylation, and a common regulatory protein is the calcium sensor calmodulin (CaM). We have developed protocols to detect the interaction of an RLK with CaM. The interaction with CaM was shown by bimolecular fluorescence complementation (BiFC) (see Chapter 14) and pull-down assay (this chapter). Both methods offer unique advantages. BiFC is useful in showing interaction of soluble as well as of membrane-bound proteins in planta. Pull-down assays are restricted to soluble proteins and provide in vitro data. The pull-down assay provides the advantage that proteins can be modified prior to binding and that experimental conditions such as the concentration of Ca 2+ or other divalent cations can be controlled. This chapter provides a pull-down protocol to study RLK-CaM interaction with optional steps to investigate the impact of RLK phosphorylation or of Ca 2+ .

Cultural Resource Investigations in the L’Anguille River Basin, Lee, St. Francis, Cross and Poinsett Counties, Arkansas.

DTIC Science & Technology

1989-01-01

Paleovegetational Record, Hood Lake, Poinsett County, Arkansas Pollen Diagram: Trees, Shrubs , and Vines . 20 Figure 7. Terminal Pleistocene/Holocene Paleovegetational...herbs and shrubs , as well as low percentages of vines (lianas), ferns, and fern allies. Among the herbs, sedge pollen rose from 5% to a peak of 25%, and...Rhus copallinum). The nonwoody complement of plants contained moderate abundances of shrubs , vines , herbs, and ferns (Figure 7). The life-form group of

(1-Adamantyl)methyl glycidyl ether: a versatile building block for living polymerization.

PubMed

Moers, Christian; Wrazidlo, Robert; Natalello, Adrian; Netz, Isabelle; Mondeshki, Mihail; Frey, Holger

2014-06-01

(1-Adamantyl)methyl glycidyl ether (AdaGE) is introduced as a versatile monomer for oxyanionic polymerization, enabling controlled incorporation of adamantyl moieties in aliphatic polyethers. Via copolymerization with ethoxyethyl glycidyl ether (EEGE) and subsequent cleavage of the acetal protection groups of EEGE, hydrophilic linear polyglycerols with an adjustable amount of pendant adamantyl moieties are obtained. The adamantyl unit permits control over thermal properties and solubility profile of these polymers (LCST). Additionally, AdaGE is utilized as a termination agent in carbanionic polymerization, affording adamantyl-terminated polymers. Using these structures as macroinitiators for the polymerization of ethylene oxide affords amphiphilic, in-chain adamantyl-functionalized block copolymers. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Use of eculizumab and plasma exchange in successful combined liver-kidney transplantation in a case of atypical HUS associated with complement factor H mutation.

PubMed

Tran, Ha; Chaudhuri, Abanti; Concepcion, Waldo; Grimm, Paul C

2014-03-01

Atypical hemolytic uremic syndrome (aHUS) evolves into end-stage renal failure in nearly half of affected patients and is associated with defective regulation of the alternative complement pathway. Patients with a complement factor H (CFH) mutation have a 30-100% risk of graft loss due to aHUS recurrence or graft thrombosis. Since CFH is produced predominantly by the liver, combined liver-kidney transplant is a curative treatment option. One major unexpected risk includes liver failure secondary to uncontrolled complement activation. We report a successful combined liver-kidney transplantation with perioperative plasma exchange and use of the humanized anti-C5 monoclonal antibody eculizumab. An 11-month-old female presented with oliguric renal failure after 3 weeks of flu-like symptoms in the absence of diarrhea. Following the identification of Escherichia coli 0157:H7 in her stool, she was discharged home on peritoneal dialysis with a diagnosis of Shiga toxin-associated HUS. Three months later, she developed severe anemia, thrombocytopenia, and neurological involvement. aHUS was diagnosed and confirmed, and genetic testing revealed a mutation in CFH SCR20. Once donor organs became available, she received preoperative plasma exchange followed by eculizumab infusion with intra-operative fresh frozen plasma prior to combined liver-kidney transplant. At 19 months post-transplant, she continues to have excellent allograft and liver function without signs of disease recurrence. Perioperative use of eculizumab in conjunction with plasma exchange during simultaneous liver-kidney transplant can be used to inhibit terminal complement activity, thereby optimizing successful transplantation by reducing the risk of graft thrombosis.

Cysteine proteinase from Streptococcus pyogenes enables evasion of innate immunity via degradation of complement factors.

PubMed

Honda-Ogawa, Mariko; Ogawa, Taiji; Terao, Yutaka; Sumitomo, Tomoko; Nakata, Masanobu; Ikebe, Kazunori; Maeda, Yoshinobu; Kawabata, Shigetada

2013-05-31

Streptococcus pyogenes is an important human pathogen that causes invasive diseases such as necrotizing fasciitis, sepsis, and streptococcal toxic shock syndrome. We investigated the function of a major cysteine protease from S. pyogenes that affects the amount of C1-esterase inhibitor (C1-INH) and other complement factors and aimed to elucidate the mechanism involved in occurrence of streptococcal toxic shock syndrome from the aspect of the complement system. First, we revealed that culture supernatant of a given S. pyogenes strain and recombinant SpeB degraded the C1-INH. Then, we determined the N-terminal sequence of the C1-INH fragment degraded by recombinant SpeB. Interestingly, the region containing one of the identified cleavage sites is not present in patients with C1-INH deficiency. Scanning electron microscopy of the speB mutant incubated in human serum showed the abnormal superficial architecture and irregular oval structure. Furthermore, unlike the wild-type strain, that mutant strain showed lower survival capacity than normal as compared with heat-inactivated serum, whereas it had a significantly higher survival rate in serum without the C1-INH than in normal serum. Also, SpeB degraded multiple complement factors and the membrane attack complex. Flow cytometric analyses revealed deposition of C9, one of the components of membrane the attack complex, in greater amounts on the surface of the speB mutant, whereas lower amounts of C9 were bound to the wild-type strain surface. These results suggest that SpeB can interrupt the human complement system via degrading the C1-INH, thus enabling S. pyogenes to evade eradication in a hostile environment.

PASSIVE HEMOLYSIS BY SERUM AND COBRA VENOM FACTOR: A NEW MECHANISM INDUCING MEMBRANE DAMAGE BY COMPLEMENT*

PubMed Central

Pickering, R. J.; Wolfson, M. R.; Good, R. A.; Gewurz, H.

1969-01-01

The studies presented here indicate that activation of the complement (C′) system by a foreign protein will cause membrane injury and passive lysis of unsensitized erythrocytes present at the time of the reaction. These observations suggest that in addition to the classical antibody-C′-induced cytolysis, there are alternative pathways or mechanisms for activation and participation of the terminal C′ components in the production of cell membrane injury. We have shown that a substance derived from cobra venom and eluted from a single protein band on polyacrylamide can promote lysis of unsensitized autologous or heterologous erythrocytes in the presence of fresh guinea pig serum and that this lysis-inducing activity and C′-inhibiting activity appear to reside in the same fractions. The lytic activity is prevented by several agents known to impair classical C′3 activity, but is unaffected by certain procedures which interfere with the function of C′ components C′1 and C′2, a suggestion that this reaction involves chiefly C′3-C′9. Further, the cobra venom (CV) factor depletes C′ activity in cobra serum, and the CV factor (with its 5S serum cofactor) converts purified C′3 to its inactive form,1 indicating that the reaction of this complex with the complement system occurs without participation of antibody. Therefore, since the lysis-inducing and C′-inhibiting activity of the CV factor appear to result from similar interactions with the complement system, these observations suggest that cell membrane damage and cell lysis can be accomplished through activation of the complement system by a mechanism involving little or no participation of classical antibody or C′ components C′1, 4, or 2. Images PMID:4978744

Cluster Analysis Identifies Distinct Pathogenetic Patterns in C3 Glomerulopathies/Immune Complex-Mediated Membranoproliferative GN.

PubMed

Iatropoulos, Paraskevas; Daina, Erica; Curreri, Manuela; Piras, Rossella; Valoti, Elisabetta; Mele, Caterina; Bresin, Elena; Gamba, Sara; Alberti, Marta; Breno, Matteo; Perna, Annalisa; Bettoni, Serena; Sabadini, Ettore; Murer, Luisa; Vivarelli, Marina; Noris, Marina; Remuzzi, Giuseppe

2018-01-01

Membranoproliferative GN (MPGN) was recently reclassified as alternative pathway complement-mediated C3 glomerulopathy (C3G) and immune complex-mediated membranoproliferative GN (IC-MPGN). However, genetic and acquired alternative pathway abnormalities are also observed in IC-MPGN. Here, we explored the presence of distinct disease entities characterized by specific pathophysiologic mechanisms. We performed unsupervised hierarchical clustering, a data-driven statistical approach, on histologic, genetic, and clinical data and data regarding serum/plasma complement parameters from 173 patients with C3G/IC-MPGN. This approach divided patients into four clusters, indicating the existence of four different pathogenetic patterns. Specifically, this analysis separated patients with fluid-phase complement activation (clusters 1-3) who had low serum C3 levels and a high prevalence of genetic and acquired alternative pathway abnormalities from patients with solid-phase complement activation (cluster 4) who had normal or mildly altered serum C3, late disease onset, and poor renal survival. In patients with fluid-phase complement activation, those in clusters 1 and 2 had massive activation of the alternative pathway, including activation of the terminal pathway, and the highest prevalence of subendothelial deposits, but those in cluster 2 had additional activation of the classic pathway and the highest prevalence of nephrotic syndrome at disease onset. Patients in cluster 3 had prevalent activation of C3 convertase and highly electron-dense intramembranous deposits. In addition, we provide a simple algorithm to assign patients with C3G/IC-MPGN to specific clusters. These distinct clusters may facilitate clarification of disease etiology, improve risk assessment for ESRD, and pave the way for personalized treatment. Copyright © 2018 by the American Society of Nephrology.

Effects of the Soluble Fiber Complex PolyGlycopleX® on Glucose Homeostasis and Body Weight in Young Zucker Diabetic Rats

PubMed Central

Grover, Gary James; Koetzner, Lee; Wicks, Joan; Gahler, Roland J.; Lyon, Michael R.; Reimer, Raylene A.; Wood, Simon

2011-01-01

Dietary fiber can reduce insulin resistance, body weight, and hyperlipidemia depending on fiber type, water solubility, and viscosity. PolyGlycopleX® (PGX®) is a natural, novel water soluble, non-starch polysaccharide complex that with water forms a highly viscous gel compared to other naturally occurring dietary fiber. We determined the effect of dietary PGX® vs. cellulose and inulin on the early development of insulin resistance, body weight, hyperlipidemia, and glycemia-induced tissue damage in young Zucker diabetic rats (ZDFs) in fasted and non-fasted states. ZDFs (5 weeks old) were fed a diet containing 5% (wgt/wgt) cellulose, inulin, or PGX® for 8 weeks. Body weight, lipids, insulin, and glucose levels were determined throughout the study and homeostasis model assessment (HOMA) was used to measure insulin sensitivity throughout the study in fasted animals. At study termination, insulin sensitivity (oral glucose tolerance test, OGTT) and kidney, liver, and pancreatic histopathology were determined. Body weight and food intake were significantly reduced by PGX® vs. inulin and cellulose. Serum insulin in fasted and non-fasted states was significantly reduced by PGX® as was non-fasted blood glucose. Insulin resistance, measured as a HOMA score, was significantly reduced by PGX® in weeks 5 through 8 as well as terminal OGTT scores in fed and fasted states. Serum total cholesterol was also significantly reduced by PGX®. PGX® significantly reduced histological kidney and hepatic damage in addition to reduced hepatic steatosis and cholestasis. A greater mass of pancreatic β-cells was found in the PGX® group. PGX® therefore may be a useful dietary additive in the control of the development of the early development of the metabolic syndrome. PMID:21922008

Expression and characterization of a novel truncated rotavirus VP4 for the development of a recombinant rotavirus vaccine.

PubMed

Li, Yijian; Xue, Miaoge; Yu, Linqi; Luo, Guoxing; Yang, Han; Jia, Lianzhi; Zeng, Yuanjun; Li, Tingdong; Ge, Shengxiang; Xia, Ningshao

2018-04-12

The outer capsid protein VP4 is an important target for the development of a recombinant rotavirus vaccine because it mediates the attachment and penetration of rotavirus. Due to the poor solubility of full-length VP4, VP8 was explored as candidate rotavirus vaccines in the past years. In previous studies, it has been found that the N-terminal truncated VP8 protein, VP8-1 (aa26-231), could be expressed in soluble form with improved immunogenicity compared to the core of VP8 (aa65-223). However, this protein stimulated only a weak immune response when aluminum hydroxide was used as an adjuvant. In addition, it should be noted that the protective efficacy of VP4 was higher than that of VP8 and VP5. In this study, it was found that when the N-terminal 25 amino acids were deleted, the truncated VP4 ∗ (aa26-476) containing VP8 and the stalk domain of VP5 could be expressed in soluble form in E. coli and purified to homogeneous trimers. Furthermore, the truncated VP4 could induce high titers of neutralizing antibodies when aluminum adjuvant was used and conferred high protective efficacy in reducing the severity of diarrhea and rotavirus shedding in stools in animal models. The immunogenicity of the truncated VP4 was significantly higher than that of VP8 ∗ and VP5 ∗ alone. Taken together, the truncated VP4 ∗ (aa26-476), with enhanced immunogenicity and immunoprotectivity, could be considered as a viable candidate for further development and has the potential to become a parenterally administered rotavirus vaccine. Copyright © 2018 Elsevier Ltd. All rights reserved.

The soluble fiber complex PolyGlycopleX lowers serum triglycerides and reduces hepatic steatosis in high-sucrose-fed rats.

PubMed

Reimer, Raylene A; Grover, Gary J; Koetzner, Lee; Gahler, Roland J; Lyon, Michael R; Wood, Simon

2011-04-01

Viscous soluble fibers have been shown to reduce risk factors associated with type 2 diabetes and cardiovascular disease. The novel functional fiber, PolyGlycopleX (PGX) (InovoBiologic Inc, Calgary, Alberta, Canada) displays greater viscosity than other currently identified soluble fibers. The objective of this study was to determine if PGX lowers serum and hepatic triglycerides (TGs) in a high-sucrose-fed rat model. In this rodent model, feeding a high-sucrose diet consistently increases serum TGs. We hypothesized that consumption of PGX would attenuate hypertriglyceridemia and reduce hepatic steatosis compared with cellulose in rats fed a high-sucrose background diet. Male Sprague-Dawley rats were fed diets containing 65% sucrose and supplemented with either 5% cellulose (control) or 5% PGX (wt/wt) for 43 weeks. At study termination, serum insulin and TGs, hepatic steatosis, and hepatocellular injury were assessed. Body weight increased over time in both groups, but weight gain was attenuated in rats fed PGX vs cellulose in weeks 2 through 22 (P < .05). Serum TGs did not differ from baseline for the first half of the study but consistently increased in the cellulose group thereafter. PolyGlycopleX significantly reduced serum TG to near-baseline levels. At study termination, rats fed PGX had significantly lower hepatic steatosis scores (measured by Sudan black staining) compared with rats fed cellulose. Hepatocellular injury scores did not differ between the groups. In conclusion, PGX reduced serum TG and lipid accumulation in the liver of sucrose-fed rats. Further examination of its potential as a fiber supplement aimed at lessening the burden of hepatic steatosis is warranted. Copyright © 2011 Elsevier Inc. All rights reserved.

Effects of the Soluble Fiber Complex PolyGlycopleX on Glucose Homeostasis and Body Weight in Young Zucker Diabetic Rats.

PubMed

Grover, Gary James; Koetzner, Lee; Wicks, Joan; Gahler, Roland J; Lyon, Michael R; Reimer, Raylene A; Wood, Simon

2011-01-01

Dietary fiber can reduce insulin resistance, body weight, and hyperlipidemia depending on fiber type, water solubility, and viscosity. PolyGlycopleX(®) (PGX(®)) is a natural, novel water soluble, non-starch polysaccharide complex that with water forms a highly viscous gel compared to other naturally occurring dietary fiber. We determined the effect of dietary PGX(®) vs. cellulose and inulin on the early development of insulin resistance, body weight, hyperlipidemia, and glycemia-induced tissue damage in young Zucker diabetic rats (ZDFs) in fasted and non-fasted states. ZDFs (5 weeks old) were fed a diet containing 5% (wgt/wgt) cellulose, inulin, or PGX(®) for 8 weeks. Body weight, lipids, insulin, and glucose levels were determined throughout the study and homeostasis model assessment (HOMA) was used to measure insulin sensitivity throughout the study in fasted animals. At study termination, insulin sensitivity (oral glucose tolerance test, OGTT) and kidney, liver, and pancreatic histopathology were determined. Body weight and food intake were significantly reduced by PGX(®) vs. inulin and cellulose. Serum insulin in fasted and non-fasted states was significantly reduced by PGX(®) as was non-fasted blood glucose. Insulin resistance, measured as a HOMA score, was significantly reduced by PGX(®) in weeks 5 through 8 as well as terminal OGTT scores in fed and fasted states. Serum total cholesterol was also significantly reduced by PGX(®). PGX(®) significantly reduced histological kidney and hepatic damage in addition to reduced hepatic steatosis and cholestasis. A greater mass of pancreatic β-cells was found in the PGX(®) group. PGX(®) therefore may be a useful dietary additive in the control of the development of the early development of the metabolic syndrome.

Periscope: quantitative prediction of soluble protein expression in the periplasm of Escherichia coli

NASA Astrophysics Data System (ADS)

Chang, Catherine Ching Han; Li, Chen; Webb, Geoffrey I.; Tey, Bengti; Song, Jiangning; Ramanan, Ramakrishnan Nagasundara

2016-03-01

Periplasmic expression of soluble proteins in Escherichia coli not only offers a much-simplified downstream purification process, but also enhances the probability of obtaining correctly folded and biologically active proteins. Different combinations of signal peptides and target proteins lead to different soluble protein expression levels, ranging from negligible to several grams per litre. Accurate algorithms for rational selection of promising candidates can serve as a powerful tool to complement with current trial-and-error approaches. Accordingly, proteomics studies can be conducted with greater efficiency and cost-effectiveness. Here, we developed a predictor with a two-stage architecture, to predict the real-valued expression level of target protein in the periplasm. The output of the first-stage support vector machine (SVM) classifier determines which second-stage support vector regression (SVR) classifier to be used. When tested on an independent test dataset, the predictor achieved an overall prediction accuracy of 78% and a Pearson’s correlation coefficient (PCC) of 0.77. We further illustrate the relative importance of various features with respect to different models. The results indicate that the occurrence of dipeptide glutamine and aspartic acid is the most important feature for the classification model. Finally, we provide access to the implemented predictor through the Periscope webserver, freely accessible at http://lightning.med.monash.edu/periscope/.

The solubility of quartz in water in the temperature interval from 25° to 300° C

USGS Publications Warehouse

Morey, G.W.; Fournier, R.O.; Rowe, J.J.

1962-01-01

the very slow rate at which dissolved silica polymerizes to species appropriate to act as nuclei for quartz growth. At the termination of the runs rotated at 75 rev/min, spikelike projections were present on many of the quartz grains. These are interpreted as indicating that abrasion was not the dominant cause for the great supersaturations which were obtained.  

Structures of Trypanosome Vacuolar Soluble Pyrophosphatases: Anti-Parasitic Drug Targets

PubMed Central

Yang, Yunyun; Ko, Tzu-Ping; Chen, Chun-Chi; Huang, Guozhong; Zheng, Yingying; Liu, Weidong; Wang, Iren; Ho, Meng-Ru; Danny Hsu, Shang-Te; O’Dowd, Bing; Huff, Hannah C.; Huang, Chun-Hsiang; Docampo, Roberto; Oldfield, Eric; Guo, Rey-Ting

2016-01-01

Trypanosomatid parasites are the causative agents of many neglected tropical diseases including the leishmaniases, Chagas disease, and human African trypanosomiasis. They exploit unusual vacuolar soluble pyrophosphatases (VSPs), absent in humans, for cell growth and virulence and as such, are drug targets. Here, we report the crystal structures of VSP1s from Trypanosoma cruzi and T. brucei, together with that of the T. cruzi protein bound to a bisphosphonate inhibitor. Both VSP1s form a hybrid structure containing an (N-terminal) EF-hand domain fused to a (C-terminal) pyrophosphatase domain. The two domains are connected via an extended loop of about 17 residues. Crystallographic analysis and size exclusion chromatography indicate that the VSP1s form tetramers containing head-to-tail dimers. Phosphate and diphosphate ligands bind in the PPase substrate-binding pocket and interact with several conserved residues, and a bisphosphonate inhibitor (BPH-1260) binds to the same site. Based on Cytoscape and other bioinformatics analyses it is apparent that similar folds will be found in most if not all trypanosomatid VSP1s, including those found in insects (Angomonas deanei, Strigomonas culicis), plant pathogens (Phytomonas spp.) and Leishmania spp. Overall, the results are of general interest since they open the way to structure-based drug design for many of the neglected tropical diseases. PMID:26907161

Alternative complement pathway activation during invasive coronary procedures in acute myocardial infarction and stable angina pectoris.

PubMed

Horváth, Zsófia; Csuka, Dorottya; Vargova, Katarina; Kovács, Andrea; Leé, Sarolta; Varga, Lilian; Préda, István; Tóth Zsámboki, Emese; Prohászka, Zoltán; Kiss, Róbert Gábor

2016-12-01

The effect of invasive percutaneous coronary procedures on complement activation has not been elucidated. We enrolled stable angina patients with elective percutaneous coronary intervention (SA-PCI, n=24), diagnostic coronary angiography (CA, n=52) and 23 patients with ST segment elevation myocardial infarction and primary PCI (STEMI-PCI). Complement activation products (C1rC1sC1inh, C3bBbP and SC5b-9) were measured on admission, 6 and 24h after coronary procedures. The alternative pathway product, C3bBbP significantly and reversibly increased 6h after elective PCI (baseline: 7.81AU/ml, 6h: 16.09AU/ml, 24h: 4.27AU/ml, p<0.01, n=23) and diagnostic angiography (baseline: 6.13AU/ml, 6h: 12.08AU/ml, 24h: 5.4AU/ml, p<0.01, n=52). Six hour C3bBbP values correlated with post-procedural CK, creatinine level and the applied contrast material volume (r=0.41, r=0.4, r=0.3, p<0.05, respectively). In STEMI-PCI, baseline C3bBbP level was higher, compared to SA-PCI or CA patients (11.33AU/ml vs. 7.81AU/ml or 6.13AU/ml, p<0.001). Similarly, the terminal complex (SC5b-9) level was already elevated at baseline compared to SA-PCI group (3.49AU/ml vs. 1.87AU/ml, p=0.011). Complement pathway products did not increase further after primary PCI. Elective coronary procedures induced transient alternative complement pathway activation, influenced by the applied contrast volume. In STEMI, the alternative complement pathway is promptly activated during the atherothrombotic event and PCI itself had no further detectable effect. Copyright © 2016 Elsevier B.V. All rights reserved.

Cloning and characterization of the Bacillus subtilis birA gene encoding a repressor of the biotin operon.

PubMed

Bower, S; Perkins, J; Yocum, R R; Serror, P; Sorokin, A; Rahaim, P; Howitt, C L; Prasad, N; Ehrlich, S D; Pero, J

1995-05-01

The Bacillus subtilis birA gene, which regulates biotin biosynthesis, has been cloned and characterized. The birA gene maps at 202 degrees on the B. subtilis chromosome and encodes a 36,200-Da protein that is 27% identical to Escherichia coli BirA protein. Three independent mutations in birA that lead to deregulation of biotin synthesis alter single amino acids in the amino-terminal end of the protein. The amino-terminal region that is affected by these three birA mutations shows sequence similarity to the helix-turn-helix DNA binding motif previously identified in E. coli BirA protein. B. subtilis BirA protein also possesses biotin-protein ligase activity, as judged by its ability to complement a conditional lethal birA mutant of E. coli.

Cloning and characterization of the Bacillus subtilis birA gene encoding a repressor of the biotin operon.

PubMed Central

Bower, S; Perkins, J; Yocum, R R; Serror, P; Sorokin, A; Rahaim, P; Howitt, C L; Prasad, N; Ehrlich, S D; Pero, J

1995-01-01

The Bacillus subtilis birA gene, which regulates biotin biosynthesis, has been cloned and characterized. The birA gene maps at 202 degrees on the B. subtilis chromosome and encodes a 36,200-Da protein that is 27% identical to Escherichia coli BirA protein. Three independent mutations in birA that lead to deregulation of biotin synthesis alter single amino acids in the amino-terminal end of the protein. The amino-terminal region that is affected by these three birA mutations shows sequence similarity to the helix-turn-helix DNA binding motif previously identified in E. coli BirA protein. B. subtilis BirA protein also possesses biotin-protein ligase activity, as judged by its ability to complement a conditional lethal birA mutant of E. coli. PMID:7730294

Improving solubility of Shewanella oneidensis MR-1 and Clostridium thermocellum JW-20 proteins expressed into Esherichia coli.

PubMed

Kataeva, Irina; Chang, Jessie; Xu, Hao; Luan, Chi-Hao; Zhou, Jizhong; Uversky, Vladimir N; Lin, Dawei; Horanyi, Peter; Liu, Z J; Ljungdahl, Lars G; Rose, John; Luo, Ming; Wang, Bi-Cheng

2005-01-01

Low solubility of proteins overexpressed in E. coli is a frequent problem in high-throughput structural genomics. To improve solubility of proteins from mesophilic Shewanella oneidensis MR-1 and thermophilic Clostridium thermocellum JW20, an approach was attempted that included a fusion of the target protein to a maltose-binding protein (MBP) and a decrease of induction temperature. The MBP was selected as the most efficient solubilizing carrier when compared to a glutathione S-transferase and a Nus A protein. A tobacco etch virus (TEV) protease recognition site was introduced between fused proteins using a double polymerase-chain reaction and four primers. In this way, 79 S. oneidensis proteins have been expressed in one case with an N-terminal 30-residue tag and in another case as a fusion protein with MBP. A foreign tag might significantly affect the properties of the target polypeptide. At 37 degrees C and 18 degrees C induction temperatures, only 5 and 17 tagged proteins were soluble, respectively. In fusion with MBP 4, 34, and 38 proteins were soluble upon induction at 37 degrees, 28 degrees, and 18 degrees C, respectively. The MBP is assumed to increase stability and solubility of a target protein by changing both the mechanism and the cooperativity of folding/unfolding. The 66 C. thermocellum proteins were expressed as fusion proteins with MBP. Induction at 37 degrees, 28 degrees, and 18 degrees C produced 34, 57, and 60 soluble proteins, respectively. The higher solubility of C. thermocellum proteins in comparison with the S. oneidensis proteins under similar conditions of induction correlates with the thermophilicity of the host. The two-factor Wilkinson-Harrison statistical model was used to identify soluble and insoluble proteins. Theoretical and experimental data showed good agreement for S. oneidensis proteins; however, the model failed to identify soluble/insoluble Clostridium proteins. A suggestion has been made that the Wilkinson-Harrison model is not applicable to C. thermocellum proteins because it did not account for the peculiarities of protein sequences from thermophiles.

C1q-targeted inhibition of the classical complement pathway prevents injury in a novel mouse model of acute motor axonal neuropathy.

PubMed

McGonigal, Rhona; Cunningham, Madeleine E; Yao, Denggao; Barrie, Jennifer A; Sankaranarayanan, Sethu; Fewou, Simon N; Furukawa, Koichi; Yednock, Ted A; Willison, Hugh J

2016-03-02

Guillain-Barré syndrome (GBS) is an autoimmune disease that results in acute paralysis through inflammatory attack on peripheral nerves, and currently has limited, non-specific treatment options. The pathogenesis of the acute motor axonal neuropathy (AMAN) variant is mediated by complement-fixing anti-ganglioside antibodies that directly bind and injure the axon at sites of vulnerability such as nodes of Ranvier and nerve terminals. Consequently, the complement cascade is an attractive target to reduce disease severity. Recently, C5 complement component inhibitors that block the formation of the membrane attack complex and subsequent downstream injury have been shown to be efficacious in an in vivo anti-GQ1b antibody-mediated mouse model of the GBS variant Miller Fisher syndrome (MFS). However, since gangliosides are widely expressed in neurons and glial cells, injury in this model was not targeted exclusively to the axon and there are currently no pure mouse models for AMAN. Additionally, C5 inhibition does not prevent the production of early complement fragments such as C3a and C3b that can be deleterious via their known role in immune cell and macrophage recruitment to sites of neuronal damage. In this study, we first developed a new in vivo transgenic mouse model of AMAN using mice that express complex gangliosides exclusively in neurons, thereby enabling specific targeting of axons with anti-ganglioside antibodies. Secondly, we have evaluated the efficacy of a novel anti-C1q antibody (M1) that blocks initiation of the classical complement cascade, in both the newly developed anti-GM1 antibody-mediated AMAN model and our established MFS model in vivo. Anti-C1q monoclonal antibody treatment attenuated complement cascade activation and deposition, reduced immune cell recruitment and axonal injury, in both mouse models of GBS, along with improvement in respiratory function. These results demonstrate that neutralising C1q function attenuates injury with a consequent neuroprotective effect in acute GBS models and promises to be a useful new target for human therapy.

Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator.

PubMed

Lee, Sooncheol; Nguyen, Huong Minh; Kang, Changwon

2010-10-01

No biological function has been identified for tiny RNA transcripts that are abortively and repetitiously released from initiation complexes of RNA polymerase in vitro and in vivo to date. In this study, we show that abortive initiation affects termination in transcription of bacteriophage T7 gene 10. Specifically, abortive transcripts produced from promoter phi 10 exert trans-acting antitermination activity on terminator T phi both in vitro and in vivo. Following abortive initiation cycling of T7 RNA polymerase at phi 10, short G-rich and oligo(G) RNAs were produced and both specifically sequestered 5- and 6-nt C + U stretch sequences, consequently interfering with terminator hairpin formation. This antitermination activity depended on sequence-specific hybridization of abortive transcripts with the 5' but not 3' half of T phi RNA. Antitermination was abolished when T phi was mutated to lack a C + U stretch, but restored when abortive transcript sequence was additionally modified to complement the mutation in T phi, both in vitro and in vivo. Antitermination was enhanced in vivo when the abortive transcript concentration was increased via overproduction of RNA polymerase or ribonuclease deficiency. Accordingly, antitermination activity exerted on T phi by abortive transcripts should facilitate expression of T phi-downstream promoter-less genes 11 and 12 in T7 infection of Escherichia coli.

A functional analysis of TOEFAZ1 uncovers protein domains essential for cytokinesis in Trypanosoma brucei.

PubMed

Sinclair-Davis, Amy N; McAllaster, Michael R; de Graffenried, Christopher L

2017-11-15

The parasite Trypanosoma brucei is highly polarized, including a flagellum that is attached along the cell surface by the flagellum attachment zone (FAZ). During cell division, the new FAZ positions the cleavage furrow, which ingresses from the anterior tip of the cell towards the posterior. We recently identified TOEFAZ1 (for 'Tip of the Extending FAZ protein 1') as an essential protein in trypanosome cytokinesis. Here, we analyzed the localization and function of TOEFAZ1 domains by performing overexpression and RNAi complementation experiments. TOEFAZ1 comprises three domains with separable functions: an N-terminal α-helical domain that may be involved in FAZ recruitment, a central intrinsically disordered domain that keeps the morphogenic kinase TbPLK at the new FAZ tip, and a C-terminal zinc finger domain necessary for TOEFAZ1 oligomerization. Both the N-terminal and C-terminal domains are essential for TOEFAZ1 function, but TbPLK retention at the FAZ is not necessary for cytokinesis. The feasibility of alternative cytokinetic pathways that do not employ TOEFAZ1 are also assessed. Our results show that TOEFAZ1 is a multimeric scaffold for recruiting proteins that control the timing and location of cleavage furrow ingression. © 2017. Published by The Company of Biologists Ltd.

Munc18-1-regulated stage-wise SNARE assembly underlying synaptic exocytosis.

PubMed

Ma, Lu; Rebane, Aleksander A; Yang, Guangcan; Xi, Zhiqun; Kang, Yuhao; Gao, Ying; Zhang, Yongli

2015-12-23

Synaptic-soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins couple their stage-wise folding/assembly to rapid exocytosis of neurotransmitters in a Munc18-1-dependent manner. The functions of the different assembly stages in exocytosis and the role of Munc18-1 in SNARE assembly are not well understood. Using optical tweezers, we observed four distinct stages of assembly in SNARE N-terminal, middle, C-terminal, and linker domains (or NTD, MD, CTD, and LD, respectively). We found that SNARE layer mutations differentially affect SNARE assembly. Comparison of their effects on SNARE assembly and on exocytosis reveals that NTD and CTD are responsible for vesicle docking and fusion, respectively, whereas MD regulates SNARE assembly and fusion. Munc18-1 initiates SNARE assembly and structures t-SNARE C-terminus independent of syntaxin N-terminal regulatory domain (NRD) and stabilizes the half-zippered SNARE complex dependent upon the NRD. Our observations demonstrate distinct functions of SNARE domains whose assembly is intimately chaperoned by Munc18-1.

Development of Iron Doped Silicon Nanoparticles as Bimodal Imaging Agents

PubMed Central

Singh, Mani P.; Atkins, Tonya M.; Muthuswamy, Elayaraja; Kamali, Saeed; Tu, Chuqiao; Louie, Angelique Y.; Kauzlarich, Susan M.

2012-01-01

We demonstrate the synthesis of water-soluble allylamine terminated Fe doped Si (SixFe) nanoparticles as bimodal agents for optical and magnetic imaging. The preparation involves the synthesis of a single source iron containing precursor, Na4Si4 with x% Fe (x = 1, 5, 10), and its subsequent reaction with NH4Br to produce hydrogen terminated SixFe nanoparticles. The hydrogen-capped nanoparticles are further terminated with allylamine via thermal hydrosilylation. Transmission electron microscopy (TEM) indicates that the average particle diameter is ~3.0±1.0 nm. The Si5Fe nanoparticles show strong photoluminescence quantum yield in water (~ 10 %) with significant T2 contrast (r2/r1value of 4.31). Electron paramagnetic resonance (EPR) and Mössbauer spectroscopies indicate that iron in the nanoparticles is in the +3 oxidation state. Analysis of cytotoxicity using the resazurin assay on HepG2 liver cells indicates that the particles have minimal toxicity. PMID:22616623

Development of iron-doped silicon nanoparticles as bimodal imaging agents.

PubMed

Singh, Mani P; Atkins, Tonya M; Muthuswamy, Elayaraja; Kamali, Saeed; Tu, Chuqiao; Louie, Angelique Y; Kauzlarich, Susan M

2012-06-26

We demonstrate the synthesis of water-soluble allylamine-terminated Fe-doped Si (Si(xFe)) nanoparticles as bimodal agents for optical and magnetic imaging. The preparation involves the synthesis of a single-source iron-containing precursor, Na(4)Si(4) with x% Fe (x = 1, 5, 10), and its subsequent reaction with NH(4)Br to produce hydrogen-terminated Si(xFe) nanoparticles. The hydrogen-capped nanoparticles are further terminated with allylamine via thermal hydrosilylation. Transmission electron microscopy indicates that the average particle diameter is ∼3.0 ± 1.0 nm. The Si(5Fe) nanoparticles show strong photoluminescence quantum yield in water (∼10%) with significant T(2) contrast (r(2)/r(1) value of 4.31). Electron paramagnetic resonance and Mössbauer spectroscopies indicate that iron in the nanoparticles is in the +3 oxidation state. Analysis of cytotoxicity using the resazurin assay on HepG2 liver cells indicates that the particles have minimal toxicity.

Structural Basis for Substrate Fatty Acyl Chain Specificity

PubMed Central

McAndrew, Ryan P.; Wang, Yudong; Mohsen, Al-Walid; He, Miao; Vockley, Jerry; Kim, Jung-Ja P.

2008-01-01

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is a member of the family of acyl-CoA dehydrogenases (ACADs). Unlike the other ACADs, which are soluble homotetramers, VLCAD is a homodimer associated with the mitochondrial membrane. VLCAD also possesses an additional 180 residues in the C terminus that are not present in the other ACADs. We have determined the crystal structure of VLCAD complexed with myristoyl-CoA, obtained by co-crystallization, to 1.91-Å resolution. The overall fold of the N-terminal ∼400 residues of VLCAD is similar to that of the soluble ACADs including medium-chain acyl-CoA dehydrogenase (MCAD). The novel C-terminal domain forms an α-helical bundle that is positioned perpendicular to the two N-terminal helical domains. The fatty acyl moiety of the bound substrate/product is deeply imbedded inside the protein; however, the adenosine pyrophosphate portion of the C14-CoA ligand is disordered because of partial hydrolysis of the thioester bond and high mobility of the CoA moiety. The location of Glu-422 with respect to the C2-C3 of the bound ligand and FAD confirms Glu-422 to be the catalytic base. In MCAD, Gln-95 and Glu-99 form the base of the substrate binding cavity. In VLCAD, these residues are glycines (Gly-175 and Gly-178), allowing the binding channel to extend for an additional 12Å and permitting substrate acyl chain lengths as long as 24 carbons to bind. VLCAD deficiency is among the more common defects of mitochondrial β-oxidation and, if left undiagnosed, can be fatal. This structure allows us to gain insight into how a variant VLCAD genotype results in a clinical phenotype. PMID:18227065

Expression of proteins in Escherichia coli as fusions with maltose-binding protein to rescue non-expressed targets in a high-throughput protein-expression and purification pipeline

PubMed Central

Hewitt, Stephen N.; Choi, Ryan; Kelley, Angela; Crowther, Gregory J.; Napuli, Alberto J.; Van Voorhis, Wesley C.

2011-01-01

Despite recent advances, the expression of heterologous proteins in Escherichia coli for crystallization remains a nontrivial challenge. The present study investigates the efficacy of maltose-binding protein (MBP) fusion as a general strategy for rescuing the expression of target proteins. From a group of sequence-verified clones with undetectable levels of protein expression in an E. coli T7 expression system, 95 clones representing 16 phylogenetically diverse organisms were selected for recloning into a chimeric expression vector with an N-terminal histidine-tagged MBP. PCR-amplified inserts were annealed into an identical ligation-independent cloning region in an MBP-fusion vector and were analyzed for expression and solubility by high-throughput nickel-affinity binding. This approach yielded detectable expression of 72% of the clones; soluble expression was visible in 62%. However, the solubility of most proteins was marginal to poor upon cleavage of the MBP tag. This study offers large-scale evidence that MBP can improve the soluble expression of previously non-expressing proteins from a variety of eukaryotic and prokaryotic organisms. While the behavior of the cleaved proteins was disappointing, further refinements in MBP tagging may permit the more widespread use of MBP-fusion proteins in crystallographic studies. PMID:21904041

PEG-lipid micelles as drug carriers: physiochemical attributes, formulation principles and biological implication.

PubMed

Gill, Kanwaldeep K; Kaddoumi, Amal; Nazzal, Sami

2015-04-01

PEG-lipid micelles, primarily conjugates of polyethylene glycol (PEG) and distearyl phosphatidylethanolamine (DSPE) or PEG-DSPE, have emerged as promising drug-delivery carriers to address the shortcomings associated with new molecular entities with suboptimal biopharmaceutical attributes. The flexibility in PEG-DSPE design coupled with the simplicity of physical drug entrapment have distinguished PEG-lipid micelles as versatile and effective drug carriers for cancer therapy. They were shown to overcome several limitations of poorly soluble drugs such as non-specific biodistribution and targeting, lack of water solubility and poor oral bioavailability. Therefore, considerable efforts have been made to exploit the full potential of these delivery systems; to entrap poorly soluble drugs and target pathological sites both passively through the enhanced permeability and retention (EPR) effect and actively by linking the terminal PEG groups with targeting ligands, which were shown to increase delivery efficiency and tissue specificity. This article reviews the current state of PEG-lipid micelles as delivery carriers for poorly soluble drugs, their biological implications and recent developments in exploring their active targeting potential. In addition, this review sheds light on the physical properties of PEG-lipid micelles and their relevance to the inherent advantages and applications of PEG-lipid micelles for drug delivery.

Effects of insulin, triiodothyronine and fat soluble vitamins on adipocyte differentiation and LPL gene expression in the stromal-vascular cells of red sea bream, Pagrus major.

PubMed

Oku, Hiromi; Tokuda, Masaharu; Okumura, Takuji; Umino, Tetsuya

2006-07-01

Various kinds of hormones including insulin, triiodothyronine (T(3)) and fat-soluble vitamins have been proposed as mediators of adipocyte differentiation in mammals. To investigate the factors which are responsible for fish adipocyte differentiation, we developed a serum-free culture system of stromal-vascular cells of red sea bream adipose tissue and examined the effects of bovine insulin, T(3), and fat-soluble vitamins (all-trans retinoic acid, retinyl acetate and 1,25-dihydroxyvitamin D(3)) on the differentiation-linked expression of the lipoprotein lipase (LPL) gene. As assessed by the increase in LPL gene expression after 3 day cultivation, like in mammalian adipocytes, insulin enhanced the adipocyte differentiation in a concentration-dependent manner. During 2 week cultivation, bovine insulin promoted lipid accumulation in differentiating adipocytes concentration-dependently until the terminal differentiation. These results indicate that the differentiation of fish adipocytes is inducible by insulin alone. T(3) alone had no effect but enhanced the differentiation-linked LPL gene expression in the presence of insulin. Fat-soluble vitamins, unlike in mammalian adipocytes, did not show any significant effects. The method developed in this study should be of interest for the characterization of factors involved in fish adipocyte differentiation.

Design of Fusion Proteins for Efficient and Soluble Production of Immunogenic Ebola Virus Glycoprotein in Escherichia coli.

PubMed

Ji, Yang; Lu, Yuan; Yan, Yishu; Liu, Xinxin; Su, Nan; Zhang, Chong; Bi, Shengli; Xing, Xin-Hui

2018-03-03

The Ebola hemorrhagic fever caused by Ebola virus is an extremely dangerous disease, and effective therapeutic agents are still lacking. Platforms for the efficient production of vaccines are crucial to ensure quick response against an Ebola virus outbreak. Ebola virus glycoprotein (EbolaGP) on the virion surface is responsible for membrane binding and virus entry, thus becoming the key target for vaccine development. However, heterologous expression of this protein still faces engineering challenges such as low production levels and insoluble aggregation. Here, the authors design and compare various fusion strategies, attaching great importance to the solubility-enhancing effect, and tag removal process. It is found that a C-terminal intein-based tag greatly enhances the solubility of EbolaGP and allows one-step chromatographic purification of the untagged EbolaGP through thiol-catalyzed self-cleavage. The purified untagged EbolaGP alone or with Freund's adjuvant are highly immunogenic, as confirmed in a mouse model. Consequently, the present study puts forward a new strategy for the efficient and soluble expression of untagged immunogenic EbolaGP. The intein-based protein fusion approach may be of importance for the large-scale production of Ebola virus subunit vaccine. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

STUDIES ON THE ORIGIN OF RIBOSOMES IN AMOEBA PROTEUS

PubMed Central

Craig, Nessly; Goldstein, Lester

1969-01-01

The origin of cytoplasmic RNA and ribosomes was studied in Amoeba proteus by transplantation of a radioactive nucleus into an unlabeled cell followed by examination of the cytoplasm of the recipient for the presence of label. When a RNA-labeled nucleus was used, label appeared in the ribosomes, ribosomal RNA, and soluble RNA. Since the kinetics of appearance of labeled RNA indicates that the nucleus was not injured during the transfer, and since the transferred nuclear pool of labeled acid-soluble RNA precursors is inadequate to account for the amount of cytoplasmic RNA label, it is concluded that cytoplasmic ribosomal RNA is derived from acid-insoluble nuclear RNA and is probably transported as an intact molecule. Likewise, cytoplasmic soluble RNA probably originated in the nucleus, although labeling by terminal exchange in the cytoplasm is also possible. The results were completely different when a protein-labeled nucleus was grafted into an unlabeled host. In this case, label was found only in soluble proteins in the host cell cytoplasm, and there were no (or very few) radioactive ribosomes. This suggests that the nuclear pool of ribosomal protein and ribosomal protein precursors is relatively small and perhaps nonexistent (and, furthermore, shows that there was no cytoplasmic ribosomal contamination of the transferred nucleus). PMID:5765758

Production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli

PubMed Central

Handa, Sumit; Spradling, Tyler J.; Dempsey, Daniel R.; Merkler, David J.

2013-01-01

Most mammalian bioactive peptides possess a C-terminal amino acid amide moiety. The presence of the C-terminal amide is a significant impediment to the recombinant production of α-amidated peptides. α-Amidated peptides are produced in vivo by the enzymatic cleavage of a precursor with a C-terminal glycine residue. Peptidylglycine α-hydroxylating monooxygenase catalyzes the key step in the oxidation of the glycine-extended precursors to the α-amidated peptide. Herein, we detail the production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli possessing a N-terminal fusion to thioredoxin (Trx). Trx was fused to hPHMcc to enhance the yield of the resulting 52 kDa protein as a soluble and catalytically active enzyme. The Trx-hPHMcc-His6 fusion was purified to homogeneity and exhibited steady-state kinetic parameters that were similar to purified rat PHMcc. The bacterial production of recombinant hPHMcc will foster efforts to generate α-amidated peptides by the co-expression of hPHMcc and the α-amidated peptide precursors in E. coli or the in vitro amidation of recombinantly expressed α-amidated peptide precursors. PMID:22554821

Gene expression in scrapie. Cloning of a new scrapie-responsive gene and the identification of increased levels of seven other mRNA transcripts.

PubMed

Dandoy-Dron, F; Guillo, F; Benboudjema, L; Deslys, J P; Lasmézas, C; Dormont, D; Tovey, M G; Dron, M

1998-03-27

To define genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies, we analyzed gene expression in scrapie-infected mouse brain using "mRNA differential display." The RNA transcripts of eight genes were increased 3-8-fold in the brains of scrapie-infected animals. Five of these genes have not previously been reported to exhibit increased expression in this disease: cathepsin S, the C1q B-chain of complement, apolipoprotein D, and two previously unidentified genes denominated scrapie-responsive gene (ScRG)-1 and ScRG-2, which are preferentially expressed in brain tissue. Increased expression of the three remaining genes, beta2 microglobulin, F4/80, and metallothionein II, has previously been reported to occur in experimental scrapie. Kinetic analysis revealed a concomitant increase in the levels of ScRG-1, cathepsin S, the C1q B-chain of complement, and beta2 microglobulin mRNA as well as glial fibrillary acidic protein and F4/80 transcripts, markers of astrocytosis and microglial activation, respectively. In contrast, the level of ScRG-2, apolipoprotein D, and metallothionein II mRNA was only increased at the terminal stage of the disease. ScRG-1 mRNA was found to be preferentially expressed in glial cells and to code for a short protein of 47 amino acids with a strong hydrophobic N-terminal region.

The N-terminal domain of the mammalian nucleoporin p62 interacts with other nucleoporins of the FXFG family during interphase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stochaj, Ursula; Banski, Piotr; Kodiha, Mohamed

2006-08-01

Nuclear pore complexes (NPCs) provide the only sites for macromolecular transport between nucleus and cytoplasm. The nucleoporin p62, a component of higher eukaryotic NPCs, is located at the central gated channel and involved in nuclear trafficking of various cargos. p62 is organized into an N-terminal segment that contains FXFG repeats and binds the soluble transport factor NTF2, whereas the C-terminal portion associates with other nucleoporins and importin-{beta}1. We have now identified new components that interact specifically with the p62 N-terminal domain. Using the p62 N-terminal segment as bait, we affinity-purified nucleoporins Nup358, Nup214 and Nup153 from crude cell extracts. Inmore » ligand binding assays, the N-terminal p62 segment associated with Nup358 and p62, suggesting their direct binding to the p62 N-terminal portion. Furthermore, p62 was isolated in complex with Nup358, Nup214 and Nup153 from growing HeLa cells, indicating that the interactions Nup358/p62, Nup214/p62 and p62/Nup153 also occur in vivo. The formation of Nup358/p62 and p62/Nup153 complexes was restricted to interphase cells, whereas Nup214/p62 binding was detected in interphase as well as during mitosis. Our results support a model of complex interactions between FXFG containing nucleoporins, and we propose that some of these interactions may contribute to the movement of cargo across the NPC.« less

Folding and stability of the isolated Greek key domains of the long-lived human lens proteins γD-crystallin and γS-crystallin

PubMed Central

Mills, Ishara A.; Flaugh, Shannon L.; Kosinski-Collins, Melissa S.; King, Jonathan A.

2007-01-01

The transparency of the eye lens depends on the high solubility and stability of the lens crystallin proteins. The monomeric γ-crystallins and oligomeric β-crystallins have paired homologous double Greek key domains, presumably evolved through gene duplication and fusion. Prior investigation of the refolding of human γD-crystallin revealed that the C-terminal domain folds first and nucleates the folding of the N-terminal domain. This result suggested that the human N-terminal domain might not be able to fold on its own. We constructed and expressed polypeptide chains corresponding to the isolated N- and C-terminal domains of human γD-crystallin, as well as the isolated domains of human γS-crystallin. Both circular dichroism and fluorescence spectroscopy indicated that the isolated domains purified from Escherichia coli were folded into native-like monomers. After denaturation, the isolated domains refolded efficiently at pH 7 and 37°C into native-like structures. The in vitro refolding of all four domains revealed two kinetic phases, identifying partially folded intermediates for the Greek key motifs. When subjected to thermal denaturation, the isolated N-terminal domains were less stable than the full-length proteins and less stable than the C-terminal domains, and this was confirmed in equilibrium unfolding/refolding experiments. The decrease in stability of the N-terminal domain of human γD-crystallin with respect to the complete protein indicated that the interdomain interface contributes of 4.2 kcal/mol to the overall stability of this very long-lived protein. PMID:17905830

Total chemical synthesis of human matrix Gla protein

PubMed Central

Hackeng, Tilman M.; Rosing, Jan; Spronk, Henri M.H.; Vermeer, Cees

2001-01-01

Human matrix Gla protein (MGP) is a vitamin K–dependent extracellular matrix protein that binds Ca2+ ions and that is involved in the prevention of vascular calcification. MGP is a 10.6-kD protein (84 amino acids) containing five γ-carboxyglutamic acid (Gla) residues and one disulfide bond. Studies of the mechanism by which MGP prevents calcification of the arterial media are hampered by the low solubility of the protein (<10 μg/mL). Because of solubility problems, processing of a recombinantly expressed MGP-fusion protein chimera to obtain MGP was unsuccessful. Here we describe the total chemical synthesis of MGP by tBoc solid-phase peptide synthesis (SPPS) and native chemical ligation. Peptide Tyr1-Ala53 was synthesized on a derivatized resin yielding a C-terminal thioester group. Peptide Cys54-Lys84 was synthesized on Lys-PAM resin yielding a C-terminal carboxylic acid. Subsequent native chemical ligation of the two peptides resulted in the formation of a native peptide bond between Ala53 and Cys54. Folding of the 1–84-polypeptide chain in 3 M guanidine (pH 8) resulted in a decrease of molecular mass from 10,605 to 10,603 (ESI-MS), representing the loss of two protons because of the formation of the Cys54-Cys60 internal disulfide bond. Like native MGP, synthetic MGP had the same low solubility when brought into aqueous buffer solutions with physiological salt concentrations, confirming its native like structure. However, the solubility of MGP markedly increased in borate buffer at pH 7.4 in the absence of sodium chloride. Ca2+-binding to MGP was confirmed by analytical HPLC, on which the retention time of MGP was reduced in the presence of CaCl2. Circular dichroism studies revealed a sharp increase in α-helicity at 0.2 mM CaCl2 that may explain the Ca2+-dependent shift in high-pressure liquid chromatography (HPLC)-retention time of MGP. In conclusion, facile and efficient chemical synthesis in combination with native chemical ligation yielded MGP preparations that can aid in unraveling the mechanism by which MGP prevents vascular calcification. PMID:11274477

Post monitoring of a cyclodextrin remeditated chlorinated solvent contaminated aquifer

NASA Astrophysics Data System (ADS)

Blanford, W. J.

2006-12-01

Hydroxypropyl-â-cyclodextrin (HPâCD) has been tested successfully in the laboratory and in the field for enhanced flushing of low-polarity contaminants from aquifers. The cyclodextrin molecule forms a toroidal structure, which has a hydrophobic cavity. Within this cavity, organic compounds of appropriate shape and size can form inclusion complexes, which is the basis for the use of cyclodextrin in groundwater remediation. The hydrophilic exterior of the molecule makes cyclodextrin highly water-soluble. The solubility of cyclodextrins can be further enhanced by adding functional groups, such as hydroxypropyl groups, to the cyclodextrin core. The aqueous solubility of HPâCD exceeds 950 g/L. These high solubilities are advantageous for field applications because they permit relatively high concentrations of the flushing agent. In order for cyclodextrin to become a feasible remediative alternative, it must be demonstrate a short term resistance to biodegradation during field application, but ultimately biodegrade so as not to pose a long term presence in the aquifer. The potential for degradation of cyclodextrin as well as changes in the chlorinated solvents and groundwater geochemistry were examined during the post monitoring of a field demonstration in a shallow aquifer at Little Creek Naval Amphibious Base in Virginia. It was found that a portion of the cyclodextrin remaining in the aquifer after the cessation of field activities biodegraded during the 425 days of post monitoring. This degradation also led to the degradation of the chlorinated solvents trichloroethylene and 1,1-trichloroethane through both biological and chemical processes. The aquifer remained anaerobic with average dissolved oxygen levels below 0.5 mg/L. Dissolved nitrate and sulfate concentrations within the cyclodextrin plume decreased due their being used as terminal electron acceptors during the degradation of the cyclodextrin. The concentrations of total iron at the field site showed no change over time. It can be concluded from this research that cyclodextrin remaining in the subsurface after cessation of active remediation will degrade due to microbial processes. The chlorinated solvents will also degrade through both chemical and biological processes to their daughter products. The terminal electron acceptors present within the cyclodextrin plume will also be used for energy during the degradation processes.

Male Sterile2 Encodes a Plastid-Localized Fatty Acyl Carrier Protein Reductase Required for Pollen Exine Development in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, W.; Shanklin, J.; Yu, X.-H.

Male Sterile2 (MS2) is predicted to encode a fatty acid reductase required for pollen wall development in Arabidopsis (Arabidopsis thaliana). Transient expression of MS2 in tobacco (Nicotiana benthamiana) leaves resulted in the accumulation of significant levels of C16 and C18 fatty alcohols. Expression of MS2 fused with green fluorescent protein revealed that an amino-terminal transit peptide targets the MS2 to plastids. The plastidial localization of MS2 is biologically important because genetic complementation of MS2 in ms2 homozygous plants was dependent on the presence of its amino-terminal transit peptide or that of the Rubisco small subunit protein amino-terminal transit peptide. Inmore » addition, two domains, NAD(P)H-binding domain and sterile domain, conserved in MS2 and its homologs were also shown to be essential for MS2 function in pollen exine development by genetic complementation testing. Direct biochemical analysis revealed that purified recombinant MS2 enzyme is able to convert palmitoyl-Acyl Carrier Protein to the corresponding C16:0 alcohol with NAD(P)H as the preferred electron donor. Using optimized reaction conditions (i.e. at pH 6.0 and 30 C), MS2 exhibits a K{sub m} for 16:0-Acyl Carrier Protein of 23.3 {+-} 4.0 {mu}m, a V{sub max} of 38.3 {+-} 4.5 nmol mg{sup -1} min{sup -1}, and a catalytic efficiency/K{sub m} of 1,873 m{sup -1} s{sup -1}. Based on the high homology of MS2 to other characterized fatty acid reductases, it was surprising that MS2 showed no activity against palmitoyl- or other acyl-coenzyme A; however, this is consistent with its plastidial localization. In summary, genetic and biochemical evidence demonstrate an MS2-mediated conserved plastidial pathway for the production of fatty alcohols that are essential for pollen wall biosynthesis in Arabidopsis.« less

Functional analysis of a frame-shift mutant of the dihydropyridine receptor pore subunit (α1S) expressing two complementary protein fragments

PubMed Central

Ahern, Chris A; Vallejo, Paola; Mortenson, Lindsay; Coronado, Roberto

2001-01-01

Background The L-type Ca2+ channel formed by the dihydropyridine receptor (DHPR) of skeletal muscle senses the membrane voltage and opens the ryanodine receptor (RyR1). This channel-to-channel coupling is essential for Ca2+ signaling but poorly understood. We characterized a single-base frame-shift mutant of α1S, the pore subunit of the DHPR, that has the unusual ability to function voltage sensor for excitation-contraction (EC) coupling by virtue of expressing two complementary hemi-Ca2+ channel fragments. Results Functional analysis of cDNA transfected dysgenic myotubes lacking α1S were carried out using voltage-clamp, confocal Ca2+ indicator fluoresence, epitope immunofluorescence and immunoblots of expressed proteins. The frame-shift mutant (fs-α1S) expressed the N-terminal half of α1S (M1 to L670) and the C-terminal half starting at M701 separately. The C-terminal fragment was generated by an unexpected restart of translation of the fs-α1S message at M701 and was eliminated by a M701I mutation. Protein-protein complementation between the two fragments produced recovery of skeletal-type EC coupling but not L-type Ca2+ current. Discussion A premature stop codon in the II-III loop may not necessarily cause a loss of DHPR function due to a restart of translation within the II-III loop, presumably by a mechanism involving leaky ribosomal scanning. In these cases, function is recovered by expression of complementary protein fragments from the same cDNA. DHPR-RyR1 interactions can be achieved via protein-protein complementation between hemi-Ca2+ channel proteins, hence an intact II-III loop is not essential for coupling the DHPR voltage sensor to the opening of RyR1 channel. PMID:11806762

Probing Conformational Dynamics of Tau Protein by Hydrogen/Deuterium Exchange Mass Spectrometry

NASA Astrophysics Data System (ADS)

Huang, Richard Y.-C.; Iacob, Roxana E.; Sankaranarayanan, Sethu; Yang, Ling; Ahlijanian, Michael; Tao, Li; Tymiak, Adrienne A.; Chen, Guodong

2018-01-01

Fibrillization of the microtubule-associated protein tau has been recognized as one of the signature pathologies of the nervous system in Alzheimer's disease, progressive supranuclear palsy, and other tauopathies. The conformational transition of tau in the fibrillization process, tau monomer to soluble aggregates to fibrils in particular, remains unclear. Here we report on the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) in combination with other biochemical approaches, including Thioflavin S fluorescence measurements, enzyme-linked immunosorbent assay (ELISA), and Western blotting to understand the heparin-induced tau's fibrillization. HDX-MS studies including anti-tau antibody epitope mapping experiments provided molecular level details of the full-length tau's conformational dynamics and its regional solvent accessibility upon soluble aggregates formation. The results demonstrate that R3 region in the full-length tau's microtubule binding repeat region (MTBR) is stabilized in the aggregation process, leaving both N and C terminal regions to be solvent exposed in the soluble aggregates and fibrils. The findings also illustrate the practical utility of orthogonal analytical methodologies for the characterization of protein higher order structure. [Figure not available: see fulltext.

Nogo goes in the pure water: solution structure of Nogo-60 and design of the structured and buffer-soluble Nogo-54 for enhancing CNS regeneration.

PubMed

Li, Minfen; Liu, Jingxian; Song, Jianxing

2006-08-01

The inability to determine the structure of the buffer-insoluble Nogo extracellular domain retarded further design of Nogo receptor (NgR) antagonists to treat CNS axonal injuries. Very surprisingly, we recently discovered that Nogo-60 was soluble and structured in salt-free water, thus allowing the determination of the first Nogo structure by heteronuclear NMR spectroscopy. Nogo-60 adopts an unusual helical structure with the N- and C-terminal helices connected by a long middle helix. While the N-helix has no contact with the rest of the molecule, the C-helix flips back to pack against the 20-residue middle helix. This packing appears to trigger the formation of the stable Nogo-60 structure because Nogo-40 with the last helix truncated is unstructured. The Nogo-60 structure offered us rationales for further design of the structured and buffer-soluble Nogo-54, which may be used as a novel NgR antagonist. Furthermore, our discovery may imply a general solution to solubilizing a category of buffer-insoluble proteins for urgent structural investigations.

Rubber-Modified Epoxies: Transitions and Morphology.

DTIC Science & Technology

1980-09-01

Hill. New Jersey 07974 i .i -2- INTRODUCTION Low levels of carboxyl-terminated reactive liquid rubber copolymers of butadiene and acrylonitrile ( CTBN ...parts per hundred parts resin (phr) of CTBN , and 5 phr piperidine is homo- geneous at the start of cure if the cure temperature is above some critical...solubility temperature (which is designated Tso). In the presence of piperidine there is a rapid reaction of the carboxyl end groups of the CTBN with

Anti-GK1 antibodies damage Taenia crassiceps cysticerci through complement activation.

PubMed

Núñez, Guadalupe; Villalobos, Nelly; Herrera, Cinthia P; Navarrete-Perea, José; Méndez, Adriana; Martinez-Maya, José J; Bobes, Raúl J; Fragoso, Gladis; Sciutto, Edda; Aguilar, Laura; Del Arenal, Irene P

2018-06-06

Taeniasis-cysticercosis, a zoonosis caused by Taenia solium, is prevalent in underdeveloped countries, where marginalization promotes its continued transmission. Pig cysticercosis, an essential stage for transmission, is preventable by vaccination. An efficient multiepitope vaccine against pig cysticercosis, S3Pvac, was developed. Previous studies showed that antibodies against one of the S3Pvac components, GK-1, are capable of damaging T. solium cysticerci, inhibiting their ability to transform into the adult stage in golden hamster gut. This study is aimed to evaluate one of the mechanisms that could mediate anti-GK-1 antibody-dependent protection. To this end, pig anti-GK-1 antibodies were produced and purified by using protein A. Proteomic analysis showed that the induced antibodies recognized the respective native cysticercal protein KE7 (Bobes et al. Infect Immun 85:e00395-17, 2017) and two additional T. solium proteins (endophilin B1 and Gp50). A new procedure to evaluate cysticercus viability, based on quantifying the cytochrome c released after parasite damage, was developed. Taenia crassiceps cysticerci were cultured in the presence of differing amounts of anti-GK-1 antibody and complement in a saturating concentration, along with the respective controls. Cysticercus viability was assessed by recording parasite motility, trypan blue exclusion, and cytochrome c levels in cysticercal soluble extract. Anti-GK-1 antibody significantly increased cysticercus damage as measured by all three methods. Parasite evaluation by electron microscopy after treatment with anti-GK-1 antibody plus complement demonstrated cysticercus damage as shorter, capsule-severed microtrichia; a decrease in glycocalyx length with respect to untreated cysts; and disaggregated desmosomes. These results demonstrate that anti-GK-1 antibodies damage cysticerci through classic complement activation.

Two Disease-Causing SNAP-25B Mutations Selectively Impair SNARE C-terminal Assembly.

PubMed

Rebane, Aleksander A; Wang, Bigeng; Ma, Lu; Qu, Hong; Coleman, Jeff; Krishnakumar, Shyam; Rothman, James E; Zhang, Yongli

2018-02-16

Synaptic exocytosis relies on assembly of three soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins into a parallel four-helix bundle to drive membrane fusion. SNARE assembly occurs by stepwise zippering of the vesicle-associated SNARE (v-SNARE) onto a binary SNARE complex on the target plasma membrane (t-SNARE). Zippering begins with slow N-terminal association followed by rapid C-terminal zippering, which serves as a power stroke to drive membrane fusion. SNARE mutations have been associated with numerous diseases, especially neurological disorders. It remains unclear how these mutations affect SNARE zippering, partly due to difficulties to quantify the energetics and kinetics of SNARE assembly. Here, we used single-molecule optical tweezers to measure the assembly energy and kinetics of SNARE complexes containing single mutations I67T/N in neuronal SNARE synaptosomal-associated protein of 25kDa (SNAP-25B), which disrupt neurotransmitter release and have been implicated in neurological disorders. We found that both mutations significantly reduced the energy of C-terminal zippering by ~10 k B T, but did not affect N-terminal assembly. In addition, we observed that both mutations lead to unfolding of the C-terminal region in the t-SNARE complex. Our findings suggest that both SNAP-25B mutations impair synaptic exocytosis by destabilizing SNARE assembly, rather than stabilizing SNARE assembly as previously proposed. Therefore, our measurements provide insights into the molecular mechanism of the disease caused by SNARE mutations. Copyright © 2017 Elsevier Ltd. All rights reserved.

A New Solution Route to Hydrogen Terminated Silicon Nanoparticles: Synthesis, Functionalization, and Water Stability.

PubMed

Zhang, Xiaoming; Neiner, Doinita; Wang, Shizhong; Louie, Angelique Y; Kauzlarich, Susan M

2007-01-24

Hydrogen capped silicon nanoparticles with strong blue photoluminescence were synthesized by the metathesis reaction of sodium silicide, NaSi, with NH 4 Br. The hydrogen capped Si nanoparticles were further terminated with octyl groups and then coated with a polymer to render them water soluble. The nanoparticles were characterized by TEM, FT-IR, UV-VIS absorption, and photoluminescence. The Si nanoparticles were shown to have an average diameter of 3.9 ±1.3 nm and exhibited room-temperature photoluminescence with a peak maximum at 438 nm with a quantum efficiency of 32% in hexane and 18% in water; the emission was stable in ambient air for up to 2 months. These nanoparticles could hold great potential as a non-heavy element containing quantum dot for applications in biology.

A new solution route to hydrogen-terminated silicon nanoparticles: synthesis, functionalization and water stability

NASA Astrophysics Data System (ADS)

Zhang, Xiaoming; Neiner, Doinita; Wang, Shizhong; Louie, Angelique Y.; Kauzlarich, Susan M.

2007-03-01

Hydrogen-capped silicon nanoparticles with strong blue photoluminescence were synthesized by the metathesis reaction of sodium silicide, NaSi, with NH4Br. The hydrogen-capped Si nanoparticles were further terminated with octyl groups and then coated with a polymer to render them water-soluble. The nanoparticles were characterized by TEM, FT-IR, UV-vis absorption and photoluminescence. The Si nanoparticles were shown to have an average diameter of 3.9 ± 1.3 nm and exhibited room temperature photoluminescence with a peak maximum at 438 nm with a quantum efficiency of 32% in hexane and 18% in water; the emission was stable in ambient air for up to 2 months. These nanoparticles could hold great potential as a non-heavy-element-containing quantum dot for applications in biology.

Isolation and characterization of the CNBr peptides from the proteolytically derived N-terminal fragment of ovine opsin.

PubMed Central

Brett, M; Findlay, J B

1983-01-01

Ovine rhodopsin may be cleaved in situ by Staphylococcus aureus V8 proteinase into two membrane-bound fragments designated V8-L (27 000 mol.wt.) and V8-S (12 000 mol.wt.). After purification of the proteolysed complex by affinity chromatography in detergent using concanavalin A immobilized on Sepharose 4B, the two polypeptide fragments may be separated by gel-permeation chromatography on Sephadex LH-60. Digestion of the N-terminal-derived V8-L fragment with CNBr in 70% (v/v) trifluoroacetic acid resulted in a peptide mixture that could be fractionated by procedures involving gel-permeation chromatography in organic and aqueous solvents and the use of differential solubility. The complete or partial sequences of all ten peptides are reported. PMID:6224479

Parasitism and the retrotransposon life cycle in plants: a hitchhiker's guide to the genome.

PubMed

Sabot, F; Schulman, A H

2006-12-01

LTR (long terminal repeat) retrotransposons are the main components of higher plant genomic DNA. They have shaped their host genomes through insertional mutagenesis and by effects on genome size, gene expression and recombination. These Class I transposable elements are closely related to retroviruses such as the HIV by their structure and presumptive life cycle. However, the retrotransposon life cycle has been closely investigated in few systems. For retroviruses and retrotransposons, individual defective copies can parasitize the activity of functional ones. However, some LTR retrotransposon groups as a whole, such as large retrotransposon derivatives and terminal repeats in miniature, are non-autonomous even though their genomic insertion patterns remain polymorphic between organismal accessions. Here, we examine what is known of the retrotransposon life cycle in plants, and in that context discuss the role of parasitism and complementation between and within retrotransposon groups.

Molecular dynamics in drug design: new generations of compstatin analogs.

PubMed

Tamamis, Phanourios; López de Victoria, Aliana; Gorham, Ronald D; Bellows-Peterson, Meghan L; Pierou, Panayiota; Floudas, Christodoulos A; Morikis, Dimitrios; Archontis, Georgios

2012-05-01

We report the computational and rational design of new generations of potential peptide-based inhibitors of the complement protein C3 from the compstatin family. The binding efficacy of the peptides is tested by extensive molecular dynamics-based structural and physicochemical analysis, using 32 atomic detail trajectories in explicit water for 22 peptides bound to human, rat or mouse target protein C3, with a total of 257 ns. The criteria for the new design are: (i) optimization for C3 affinity and for the balance between hydrophobicity and polarity to improve solubility compared to known compstatin analogs; and (ii) development of dual specificity, human-rat/mouse C3 inhibitors, which could be used in animal disease models. Three of the new analogs are analyzed in more detail as they possess strong and novel binding characteristics and are promising candidates for further optimization. This work paves the way for the development of an improved therapeutic for age-related macular degeneration, and other complement system-mediated diseases, compared to known compstatin variants. © 2012 John Wiley & Sons A/S.

Purification and characterization of complement-activating acidic polysaccharides from the fruits of Capsicum annuum.

PubMed

Paik, Soon-Young; Ra, Kyung Soo; Chang, In Seop; Park, Yoon Chang; Park, Hee Sung; Baik, Hyung Suk; Yun, Jong Won; Choi, Jang Won

2003-03-31

Hot water-soluble crude polysaccharide (HCAP-0) that was obtained from the fruits of Capsicum annuum showed potent anti-complementary activity. The activity was unchanged by pronase digestion, but decreased by periodate oxidation. The HCAP-0 was fractionated by DEAE ion-exchange chromatography to give two major fractions, HCAP-II and III. These two fractions were finally purified by gel filtration to give HCAP-IIa, HCAPIIIa1, and IIIa2 fractions that had high anti-complementary activities. The HCAP-IIIa1 and IIIa2 consisted of homogeneous polysaccharides. The anti-complementary activities were unaffected by treatment with polymyxin B, indicating that the modes of complement activation were not due to preexisting lipopolysaccharide. The molecular weight and sugar content of HCAP-IIIa2 had potent anti-complementary activity. The highest yields were 55 kDa and 75.9%, and the molar ratio of galactose (Ara:Gal, 1.0:4.6) was higher than other sugars. The crossed immuno-electrophoresis showed that both classical and alternative pathways were activated by HCAP-IIIa2.

Use of Functionalized Carbon Nanotubes for Covalent Attachment of Nanotubes to Silicon

NASA Technical Reports Server (NTRS)

Tour, James M.; Dyke, Christopher A.; Maya, Francisco; Stewart, Michael P.; Chen, Bo; Flatt, Austen K.

2012-01-01

The purpose of the invention is to covalently attach functionalized carbon nanotubes to silicon. This step allows for the introduction of carbon nanotubes onto all manner of silicon surfaces, and thereby introduction of carbon nano - tubes covalently into silicon-based devices, onto silicon particles, and onto silicon surfaces. Single-walled carbon nanotubes (SWNTs) dispersed as individuals in surfactant were functionalized. The nano - tube was first treated with 4-t-butylbenzenediazonium tetrafluoroborate to give increased solubility to the carbon nanotube; the second group attached to the sidewall of the nanotube has a silyl-protected terminal alkyne that is de-protected in situ. This gives a soluble carbon nanotube that has functional groups appended to the sidewall that can be attached covalently to silicon. This reaction was monitored by UV/vis/NJR to assure direct covalent functionalization.

Advances in Antibody Design

PubMed Central

Tiller, Kathryn E.; Tessier, Peter M.

2017-01-01

The use of monoclonal antibodies as therapeutics requires optimizing several of their key attributes. These include binding affinity and specificity, folding stability, solubility, pharmacokinetics, effector functions, and compatibility with the attachment of additional antibody domains (bispecific antibodies) and cytotoxic drugs (antibody–drug conjugates). Addressing these and other challenges requires the use of systematic design methods that complement powerful immunization and in vitro screening methods. We review advances in designing the binding loops, scaffolds, domain interfaces, constant regions, post-translational and chemical modifications, and bispecific architectures of antibodies and fragments thereof to improve their bioactivity. We also highlight unmet challenges in antibody design that must be overcome to generate potent antibody therapeutics. PMID:26274600

Bulk functionalization of graphene using diazonium compounds and amide reaction

NASA Astrophysics Data System (ADS)

Peng, Chang; Xiong, Yuzi; Liu, Zhibo; Zhang, Fan; Ou, Encai; Qian, Jiangtao; Xiong, Yuanqin; Xu, Weijian

2013-09-01

An efficient and convenient method is applied to introduce varieties of simple functionalities onto the graphene surface for the bulk preparation, which begins with pristine graphite that does not require initial oxidative damage of the graphene basal planes. Diazonium compounds functionalized reaction is demonstrated and it successfully prevented the aggregation of graphene for which providing solubility in high polar organic media or even in volatile solvents such as ethanol and acetone. This approach is complemented by the phenyl carboxylic diazonium salt functionalized graphene (PCFG) attachment of a symmetrically substituted zinc phthalocyanine (PCFG-Pc) using the amide reaction, which is used for the covalent introduction of a complex phthalocyanine molecule.

Proteolysis of complement factors iC3b and C5 by the serine protease prostate-specific antigen in prostatic fluid and seminal plasma.

PubMed

Manning, Michael L; Williams, Simon A; Jelinek, Christine A; Kostova, Maya B; Denmeade, Samuel R

2013-03-15

Prostate-specific Ag (PSA) is a serine protease that is expressed exclusively by normal and malignant prostate epithelial cells. The continued high-level expression of PSA by the majority of men with both high- and low-grade prostate cancer throughout the course of disease progression, even in the androgen-ablated state, suggests that PSA has a role in the pathogenesis of disease. Current experimental and clinical evidence suggests that chronic inflammation, regardless of the cause, may predispose men to prostate cancer. The responsibility of the immune system in immune surveillance and eventually tumor progression is well appreciated but not completely understood. In this study, we used a mass spectrometry-based evaluation of prostatic fluid obtained from diseased prostates after removal by radical prostatectomy to identify potential immunoregulatory proteins. This analysis revealed the presence of Igs and the complement system proteins C3, factor B, and clusterin. Verification of these findings by Western blot confirmed the high-level expression of C3 in the prostatic fluid and the presence of a previously uncharacterized C-terminal C3 cleavage product. Biochemical analysis of this C3 cleavage fragment revealed a putative PSA cleavage site after tyrosine-1348. Purified PSA was able to cleave iC3b and the related complement protein C5. These results suggest a previously uncharacterized function of PSA as an immunoregulatory protease that could help to create an environment hospitable to malignancy through proteolysis of the complement system.

Novel Mutations Causing C5 Deficiency in Three North-African Families.

PubMed

Colobran, Roger; Franco-Jarava, Clara; Martín-Nalda, Andrea; Baena, Neus; Gabau, Elisabeth; Padilla, Natàlia; de la Cruz, Xavier; Pujol-Borrell, Ricardo; Comas, David; Soler-Palacín, Pere; Hernández-González, Manuel

2016-05-01

The complement system plays a central role in defense to encapsulated bacteria through opsonization and membrane attack complex (MAC) dependent lysis. The three activation pathways (classical, lectin, and alternative) converge in the cleavage of C5, which initiates MAC formation and target lysis. C5 deficiency is associated to recurrent infections by Neisseria spp. In the present study, complement deficiency was suspected in three families of North-African origin after one episode of invasive meningitis due to a non-groupable and two uncommon Meningococcal serotypes (E29, Y). Activity of alternative and classical pathways of complement were markedly reduced and the measurement of terminal complement components revealed total C5 absence. C5 gene analysis revealed two novel mutations as causative of the deficiency: Family A propositus carried a homozygous deletion of two adenines in the exon 21 of C5 gene, resulting in a frameshift and a truncated protein (c.2607_2608del/p.Ser870ProfsX3 mutation). Families B and C probands carried the same homozygous deletion of three consecutive nucleotides (CAA) in exon 9 of the C5 gene, leading to the deletion of asparagine 320 (c.960_962del/p.Asn320del mutation). Family studies confirmed an autosomal recessive inheritance pattern. Although sharing the same geographical origin, families B and C were unrelated. This prompted us to investigate this mutation prevalence in a cohort of 768 North-African healthy individuals. We identified one heterozygous carrier of the p.Asn320del mutation (allelic frequency = 0.065 %), indicating that this mutation is present at low frequency in North-African population.

Purification and cDNA cloning of a protein derived from Flammulina velutipes that increases the permeability of the intestinal Caco-2 cell monolayer.

PubMed

Watanabe, H; Narai, A; Shimizu, M

1999-06-01

A new protein that decreases transepithelial electrical resistance (TEER) in the human intestinal Caco-2 cell monolayer was found in a water-soluble fraction of the mushroom Flammulina velutipes. This protein, termed TEER-decreasing protein (TDP), is not cytotoxic and does not induce cell detachment, but rapidly increases the tight junctional permeability for water-soluble marker substances such as Lucifer Yellow CH (Mr 457) through the paracellular pathway. TDP was isolated and purified from the aqueous extract of F. velutipes by chromatographic means. Purified TDP was found to be a simple, nonglycosylated protein without intermolecular disulfide bonds, and the apparent molecular mass as estimated by SDS/PAGE and gel filtration is 30 kDa. It was revealed that the N-terminal amino-acid sequence of purified TDP is identical to the recently reported N-terminal sequence of flammutoxin, a membrane-perturbing hemolytic protein, for which the complete primary structure has not yet been reported [Tomita, T., Ishikawa, D., Noguchi, T., Katayama, E., and Hashimoto, Y. (1998) Biochem. J. 333, 24794-24799]. The cDNA coding for TDP was cloned by 5' and 3' rapid amplification of cDNA ends. The ORF encodes a protein with 272 amino-acid residues showing no homology to known proteins. Relevant studies using TDP cDNA will provide insight into the structure-function relationships of membrane pore-forming toxins.

C-terminal oligomerization of podocin mediates interallelic interactions.

PubMed

Stráner, Pál; Balogh, Eszter; Schay, Gusztáv; Arrondel, Christelle; Mikó, Ágnes; L'Auné, Gerda; Benmerah, Alexandre; Perczel, András; K Menyhárd, Dóra; Antignac, Corinne; Mollet, Géraldine; Tory, Kálmán

2018-07-01

Interallelic interactions of membrane proteins are not taken into account while evaluating the pathogenicity of sequence variants in autosomal recessive disorders. Podocin, a membrane-anchored component of the slit diaphragm, is encoded by NPHS2, the major gene mutated in hereditary podocytopathies. We formerly showed that its R229Q variant is only pathogenic when trans-associated to specific 3' mutations and suggested the causal role of an abnormal C-terminal dimerization. Here we show by FRET analysis and size exclusion chromatography that podocin oligomerization occurs exclusively through the C-terminal tail (residues 283-382): principally through the first C-terminal helical region (H1, 283-313), which forms a coiled coil as shown by circular dichroism spectroscopy, and through the 332-348 region. We show the principal role of the oligomerization sites in mediating interallelic interactions: while the monomer-forming R286Tfs*17 podocin remains membranous irrespective of the coexpressed podocin variant identity, podocin variants with an intact H1 significantly influence each other's localization (r 2  = 0.68, P = 9.2 × 10 -32 ). The dominant negative effect resulting in intracellular retention of the pathogenic F344Lfs*4-R229Q heterooligomer occurs in parallel with a reduction in the FRET efficiency, suggesting the causal role of a conformational rearrangement. On the other hand, oligomerization can also promote the membrane localization: it can prevent the endocytosis of F344Lfs*4 or F344* podocin mutants induced by C-terminal truncation. In conclusion, C-terminal oligomerization of podocin can mediate both a dominant negative effect and interallelic complementation. Interallelic interactions of NPHS2 are not restricted to the R229Q variant and have to be considered in compound heterozygous individuals. Copyright © 2018 Elsevier B.V. All rights reserved.

In Vitro Iron Availability from Insects and Sirloin Beef.

PubMed

Latunde-Dada, Gladys O; Yang, Wenge; Vera Aviles, Mayra

2016-11-09

Interest in the consumption of insects (entomophagy) as an alternative environmentally sustainable source of protein in the diet of humans has recently witnessed a surge. Knowledge of the nutrient composition and, in particular, the bioavailability of minerals from insects is currently sparse. This study evaluated the availability of Fe, Ca, Cu, Mg, Mn, and Zn from four commonly eaten insects and compared these to sirloin beef. Soluble iron from the samples was measured by inductively coupled plasma optical emission spectrometry (ICP-OES). Iron bioavailability was determined using an in vitro simulated peptic-pancreatic digestion, followed by measurement of ferritin (a surrogate marker for iron absorption) in Caco-2 cells. Cricket and sirloin beef had comparably higher levels of Fe, Ca, and Mn than grasshopper, meal, and buffalo worms. However, iron solubility was significantly higher from the insect samples than from beef. The complementation of whole-wheat flour with insect or beef protein resulted in overall decreases in mineral content and iron solubility in the composite mixtures. Collectively, the data show that grasshopper, cricket, and mealworms contain significantly higher chemically available Ca, Cu, Mg, Mn, and Zn than sirloin. However, buffalo worms and sirloin exhibited higher iron bioavailability comparable to that of FeSO 4 . Commonly consumed insect species could be excellent sources of bioavailable iron and could provide the platform for an alternative strategy for increased mineral intake in the diets of humans.

Isolation and initial characterisation of complement components C3 and C4 of the nurse shark and the channel catfish.

PubMed

Dodds, A W; Smith, S L; Levine, R P; Willis, A C

1998-01-01

Complement components C3 and C4 have been isolated from the serum of the nurse shark (Ginglymostoma cirratum) and of the channel catfish (Ictalurus punctatus). As in the higher vertebrates, the fish C4 proteins have three-chain structures while the C3 proteins have two-chain structures. All four proteins have intra-chain thioesters located within their highest molecular mass polypeptides. N-terminal sequence analysis of the polypeptides has confirmed the identity of the proteins. In all cases except the catfish C3 alpha-chain, which appears to have a blocked N-terminus, sequence similarities are apparent in comparisons with the chains of C3 and C4 from higher vertebrates. We have confirmed that the activity/protein previously designated C2n is the nurse shark analogue of mammalian C4. This is the first report of structural evidence for C4 in both the bony and cartilaginous fish.

Homology-based modeling of the Erwinia amylovora type III secretion chaperone DspF used to identify amino acids required for virulence and interaction with the effector DspE.

PubMed

Triplett, Lindsay R; Wedemeyer, William J; Sundin, George W

2010-09-01

The structure of DspF, a type III secretion system (T3SS) chaperone required for virulence of the fruit tree pathogen Erwinia amylovora, was modeled based on predicted structural homology to characterized T3SS chaperones. This model guided the selection of 11 amino acid residues that were individually mutated to alanine via site-directed mutagenesis. Each mutant was assessed for its effect on virulence complementation, dimerization and interaction with the N-terminal chaperone-binding site of DspE. Four amino acid residues were identified that did not complement the virulence defect of a dspF knockout mutant, and three of these residues were required for interaction with the N-terminus of DspE. This study supports the significance of the predicted beta-sheet helix-binding groove in DspF chaperone function. Copyright 2010 Elsevier Masson SAS. All rights reserved.

Molecular defects leading to human complement component C6 deficiency in an African-American family

PubMed Central

Zhu, Z-B; Totemchokchyakarn, K; Atkinson, T P; Volanakis, J E

1998-01-01

Complement component C6 deficiency (C6D) was diagnosed in a 16-year-old African-American male with meningococcal meningitis. The patient's father and two brothers also had C6D, but gave no history of meningitis or other neisserial infection. By using exon-specific polymerase chain reaction (PCR)/single-strand conformation polymorphism as a screening step and nucleotide sequencing of target exons, we determined that the proband was a compound heterozygote for two C6 gene mutations. The first, 1195delC located in exon 7, is a novel mutation, while the second, 1936delG in exon 12, has been described before to cause C6D in an unrelated African-American individual. Both mutations result in premature termination codons and C6 null alleles. Allele-specific PCR indicated that the proband's two brothers also inherited the 1195delC mutation from their heterozygous mother and the 1936delG mutation from their homozygous father. PMID:9472666

Banded karyotype of the Konya wild sheep (Ovis orientalis anatolica Valenciennes, 1856) from Turkey

PubMed Central

Arslan, Atilla; Zima, Jan

2011-01-01

Abstract Thekaryotype, C-banding, and nucleoar organizer regions (NORs) of eight specimens ofKonya wild sheepfrom Turkey were examined. The complement included six large metacentric autosomes, 46 acrocentric autosomes of decreasing size, a medium-sized acrocentric X chromosome, and a small bi-armed Y chromosome (the diploid chromosome number 2n=54, the number of autosomal arms NFa=58, the number of chromosome arms NF=61). G-banding allowed reliable identification of all the chromosome pairs and the pairing of homologous elements. All the autosomes possessed distinct centromeric or pericentromeric C-positive bands. The X chromosome had a pericentromeric C-positive band, and the Y chromosome was entirely C-heterochromatic. The NORs were located in the terminal regions of the long arms of three metacentric and two acrocentric autosomes. The karyotype of the Konya wild sheep and its banding patterns are quite similar to chromosome complement reported in domestic sheep and European mouflon. PMID:24260621

C-terminal peptides of tissue factor pathway inhibitor are novel host defense molecules.

PubMed

Papareddy, Praveen; Kalle, Martina; Kasetty, Gopinath; Mörgelin, Matthias; Rydengård, Victoria; Albiger, Barbara; Lundqvist, Katarina; Malmsten, Martin; Schmidtchen, Artur

2010-09-03

Tissue factor pathway inhibitor (TFPI) inhibits tissue factor-induced coagulation, but may, via its C terminus, also modulate cell surface, heparin, and lipopolysaccharide interactions as well as participate in growth inhibition. Here we show that C-terminal TFPI peptide sequences are antimicrobial against the gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, gram-positive Bacillus subtilis and Staphylococcus aureus, as well as the fungi Candida albicans and Candida parapsilosis. Fluorescence studies of peptide-treated bacteria, paired with analysis of peptide effects on liposomes, showed that the peptides exerted membrane-breaking effects similar to those seen for the "classic" human antimicrobial peptide LL-37. The killing of E. coli, but not P. aeruginosa, by the C-terminal peptide GGLIKTKRKRKKQRVKIAYEEIFVKNM (GGL27), was enhanced in human plasma and largely abolished in heat-inactivated plasma, a phenomenon linked to generation of antimicrobial C3a and activation of the classic pathway of complement activation. Furthermore, GGL27 displayed anti-endotoxic effects in vitro and in vivo in a mouse model of LPS shock. Importantly, TFPI was found to be expressed in the basal layers of normal epidermis, and was markedly up-regulated in acute skin wounds as well as wound edges of chronic leg ulcers. Furthermore, C-terminal fragments of TFPI were associated with bacteria present in human chronic leg ulcers. These findings suggest a new role for TFPI in cutaneous defense against infections.

Short term effects of milrinone on biomarkers of necrosis, apoptosis, and inflammation in patients with severe heart failure

PubMed Central

Lanfear, David E; Hasan, Reema; Gupta, Ramesh C; Williams, Celeste; Czerska, Barbara; Tita, Cristina; Bazari, Rasha; Sabbah, Hani N

2009-01-01

Introduction Inotropes are associated with adverse outcomes in heart failure (HF), raising concern they may accelerate myocardial injury. Whether biomarkers of myocardial necrosis, inflammation and apoptosis change in response to acute milrinone administration is not well established. Methods Ten patients with severe HF and reduced cardiac output who were to receive milrinone were studied. Blood samples were taken just before initiation of milrinone and after 24 hours of infusion. Dosing was at the discretion of the patient's attending physician (range 0.25–0.5 mcg/kg/min). Plasma measurements of troponin, myoglobin, N-terminal-pro-BNP, interleukin-6, tumor necrosis factor-α, soluble Fas, and soluble Fas-ligand were performed at both time points. Results Troponin was elevated at baseline in all patients (mean 0.1259 ± 0.17 ng/ml), but there was no significant change after 24 hours of milrinone (mean 0.1345 ± 0.16 ng/ml, p = 0.44). There were significant improvements in interleukin-6, tumor necrosis factor-α, soluble Fas, and soluble Fas-ligand (all p < 0.05) indicative of reduced inflammatory and apoptotic signaling compared to baseline. Conclusion In conclusion, among patients with severe HF and low cardiac output, ongoing myocardial injury is common, and initiation of milrinone did not result in exacerbation of myocardial injury but instead was associated with salutary effects on other biomarkers. PMID:19640280

FZD4S, a splicing variant of frizzled-4, encodes a soluble-type positive regulator of the WNT signaling pathway.

PubMed

Sagara, N; Kirikoshi, H; Terasaki, H; Yasuhiko, Y; Toda, G; Shiokawa, K; Katoh, M

2001-04-06

Frizzled-1 (FZD1)-FZD10 are seven-transmembrane-type WNT receptors, and SFRP1-SFRP5 are soluble-type WNT antagonists. These molecules are encoded by mutually distinct genes. We have previously isolated and characterized the 7.7-kb FZD4 mRNA, encoding a seven-transmembrane receptor with the extracellular cysteine-rich domain (CRD). Here, we have cloned and characterized FZD4S, a splicing variant of the FZD4 gene. FZD4S, corresponding to the 10.0-kb FZD4 mRNA, consisted of exon 1, intron 1, and exon 2 of the FZD4 gene. FZD4S encoded a soluble-type polypeptide with the N-terminal part of CRD, and was expressed in human fetal kidney. Injection of synthetic FZD4S mRNA into the ventral marginal zone of Xenopus embryos at the 4-cell stage did not induce axis duplication by itself, but augmented the axis duplication potential of coinjected Xwnt-8 mRNA. These results indicate that the FZD4 gene gives rise to soluble-type FZD4S as well as seven-transmembrane-type FZD4 due to alternative splicing, and strongly suggest that FZD4S plays a role as a positive regulator of the WNT signaling pathway. Copyright 2001 Academic Press.

Efficacy and safety of Meriva®, a curcumin-phosphatidylcholine complex, during extended administration in osteoarthritis patients.

PubMed

Belcaro, Gianni; Cesarone, Maria Rosaria; Dugall, Mark; Pellegrini, Luciano; Ledda, Andrea; Grossi, Maria Giovanna; Togni, Stefano; Appendino, Giovanni

2010-12-01

In a previous three-month study of Meriva, a proprietary curcumin-phosphatidylcholine phytosome complex, decreased joint pain and improvement in joint function were observed in 50 osteoarthritis (OA) patients. Since OA is a chronic condition requiring prolonged treatment, the long-term efficacy and safety of Meriva were investigated in a longer (eight months) study involving 100 OA patients. The clinical end points (Western Ontario and McMaster Universities [WOMAC] score, Karnofsky Performance Scale Index, and treadmill walking performance) were complemented by the evaluation of a series of inflammatory markers (interleukin [IL]-1beta, IL-6, soluble CD40 ligand [sCD40L], soluble vascular cell adhesion molecule (sVCAM)-1, and erythrocyte sedimentation rate [ESR]). This represents the most ambitious attempt, to date, to evaluate the clinical efficacy and safety of curcumin as an anti-inflammatory agent. Significant improvements of both the clinical and biochemical end points were observed for Meriva compared to the control group. This, coupled with an excellent tolerability, suggests that Meriva is worth considering for the long-term complementary management of osteoarthritis.

The C-terminal region of Trypanosoma cruzi MASPs is antigenic and secreted via exovesicles

PubMed Central

De Pablos, Luis Miguel; Díaz Lozano, Isabel María; Jercic, Maria Isabel; Quinzada, Markela; Giménez, Maria José; Calabuig, Eva; Espino, Ana Margarita; Schijman, Alejandro Gabriel; Zulantay, Inés; Apt, Werner; Osuna, Antonio

2016-01-01

Trypanosoma cruzi is the etiological agent of Chagas disease, a neglected and emerging tropical disease, endemic to South America and present in non-endemic regions due to human migration. The MASP multigene family is specific to T. cruzi, accounting for 6% of the parasite’s genome and plays a key role in immune evasion. A common feature of MASPs is the presence of two conserved regions: an N-terminal region codifying for signal peptide and a C-terminal (C-term) region, which potentially acts as GPI-addition signal peptide. Our aim was the analysis of the presence of an immune response against the MASP C-term region. We found that this region is highly conserved, released via exovesicles (EVs) and has an associated immune response as revealed by epitope affinity mapping, IFA and inhibition of the complement lysis assays. We also demonstrate the presence of a fast IgM response in Balb/c mice infected with T. cruzi. Our results reveal the presence of non-canonical secreted peptides in EVs, which can subsequently be exposed to the immune system with a potential role in evading immune system targets in the parasite. PMID:27270330

COP9 signalosome subunit 7 from Arabidopsis interacts with and regulates the small subunit of ribonucleotide reductase (RNR2).

PubMed

Halimi, Yair; Dessau, Moshe; Pollak, Shaul; Ast, Tslil; Erez, Tamir; Livnat-Levanon, Nurit; Karniol, Baruch; Hirsch, Joel A; Chamovitz, Daniel A

2011-09-01

The COP9 Signalosome protein complex (CSN) is a pleiotropic regulator of plant development and contains eight-subunits. Six of these subunits contain the PCI motif which mediates specific protein interactions necessary for the integrity of the complex. COP9 complex subunit 7 (CSN7) contains an N-terminal PCI motif followed by a C-terminal extension which is also necessary for CSN function. A yeast-interaction trap assay identified the small subunit of ribonucelotide reductase (RNR2) from Arabidopsis as interacting with the C-terminal section of CSN7. This interaction was confirmed in planta by both bimolecular fluorescence complementation and immuoprecipitation assays with endogenous proteins. The subcellular localization of RNR2 was primarily nuclear in meristematic regions, and cytoplasmic in adult cells. RNR2 was constitutively nuclear in csn7 mutant seedlings, and was also primarily nuclear in wild type seedlings following exposure to UV-C. These two results correlate with constitutive expression of several DNA-damage response genes in csn7 mutants, and to increased tolerance of csn7 seedlings to UV-C treatment. We propose that the CSN is a negative regulator of RNR activity in Arabidopsis.

The N-terminal region of the Plantago asiatica mosaic virus coat protein is required for cell-to-cell movement but is dispensable for virion assembly.

PubMed

Ozeki, Johji; Hashimoto, Masayoshi; Komatsu, Ken; Maejima, Kensaku; Himeno, Misako; Senshu, Hiroko; Kawanishi, Takeshi; Kagiwada, Satoshi; Yamaji, Yasuyuki; Namba, Shigetou

2009-06-01

Potexvirus cell-to-cell movement requires coat protein (CP) and movement proteins. In this study, mutations in two conserved in-frame AUG codons in the 5' region of the CP open reading frame of Plantago asiatica mosaic virus (PlAMV) were introduced, and virus accumulation of these mutants was analyzed in inoculated and upper noninoculated leaves. When CP was translated only from the second AUG codon, virus accumulation in inoculated leaves was lower than that of wild-type PlAMV, and the viral spread was impaired. Trans-complementation analysis showed that the leucine residue at the third position (Leu-3) of CP is important for cell-to-cell movement of PlAMV. The 14-amino-acid N-terminal region of CP was dispensable for virion formation. Immunoprecipitation assays conducted with an anti-TGBp1 antibody indicated that PlAMV CP interacts with TGBp1 in vivo and that this interaction is not affected by alanine substitution at Leu-3. These results support the concept that the N-terminal region of potexvirus CP can be separated into two distinct functional domains.

Enhanced neuroinvasion by smaller, soluble prions.

PubMed

Bett, Cyrus; Lawrence, Jessica; Kurt, Timothy D; Orru, Christina; Aguilar-Calvo, Patricia; Kincaid, Anthony E; Surewicz, Witold K; Caughey, Byron; Wu, Chengbiao; Sigurdson, Christina J

2017-04-21

Infectious prion aggregates can propagate from extraneural sites into the brain with remarkable efficiency, likely transported via peripheral nerves. Yet not all prions spread into the brain, and the physical properties of a prion that is capable of transit within neurons remain unclear. We hypothesized that small, diffusible aggregates spread into the CNS via peripheral nerves. Here we used a structurally diverse panel of prion strains to analyze how the prion conformation impacts transit into the brain. Two prion strains form fibrils visible ultrastructurally in the brain in situ, whereas three strains form diffuse, subfibrillar prion deposits and no visible fibrils. The subfibrillar strains had significantly higher levels of soluble prion aggregates than the fibrillar strains. Primary neurons internalized both the subfibrillar and fibril-forming prion strains by macropinocytosis, and both strain types were transported from the axon terminal to the cell body in vitro. However in mice, only the predominantly soluble, subfibrillar prions, and not the fibrillar prions, were efficiently transported from the tongue to the brain. Sonicating a fibrillar prion strain increased the solubility and enabled prions to spread into the brain in mice, as evident by a 40% increase in the attack rate, indicating that an increase in smaller particles enhances prion neuroinvasion. Our data suggest that the small, highly soluble prion particles have a higher capacity for transport via nerves. These findings help explain how prions that predominantly assemble into subfibrillar states can more effectively traverse into and out of the CNS, and suggest that promoting fibril assembly may slow the neuron-to-neuron spread of protein aggregates.

Four subunits that are shared by the three classes of RNA polymerase are functionally interchangeable between Homo sapiens and Saccharomyces cerevisiae.

PubMed Central

Shpakovski, G V; Acker, J; Wintzerith, M; Lacroix, J F; Thuriaux, P; Vigneron, M

1995-01-01

Four cDNAs encoding human polypeptides hRPB7.0, hRPB7.6, hRPB17, and hRPB14.4 (referred to as Hs10 alpha, Hs10 beta, Hs8, and Hs6, respectively), homologous to the ABC10 alpha, ABC10 beta, ABC14.5, and ABC23 RNA polymerase subunits (referred to as Sc10 alpha, Sc10 beta, Sc8, and Sc6, respectively) of Saccharomyces cerevisiae, were cloned and characterized for their ability to complement defective yeast mutants. Hs10 alpha and the corresponding Sp10 alpha of Schizosaccharomyces pombe can complement an S. cerevisiae mutant (rpc10-delta::HIS3) defective in Sc10 alpha. The peptide sequences are highly conserved in their carboxy-terminal halves, with an invariant motif CX2CX12RCX2CGXR corresponding to a canonical zinc-binding domain. Hs10 beta, Sc10 beta, and the N subunit of archaeal RNA polymerase are homologous. An invariant CX2CGXnCCR motif presumably forms an atypical zinc-binding domain. Hs10 beta, but not the archaeal subunit, complemented an S. cerevisiae mutant (rpb10-delta 1::HIS3) lacking Sc10 beta. Hs8 complemented a yeast mutant (rpb8-delta 1::LYS2) defective in the corresponding Sc8 subunit, although with a strong thermosensitive phenotype. Interspecific complementation also occurred with Hs6 and with the corresponding Dm6 cDNA of Drosophila melanogaster. Hs6 cDNA and the Sp6 cDNA of S. pombe are dosage-dependent suppressors of rpo21-4, a mutation generating a slowly growing yeast defective in the largest subunit of RNA polymerase II. Finally, a doubly chimeric S. cerevisiae strain bearing the Sp6 cDNA and the human Hs10 beta cDNA was also viable. No interspecific complementation was observed for the human hRPB25 (Hs5) homolog of the yeast ABC27 (Sc5) subunit. PMID:7651387

Structural features and complement-fixing activity of pectin from three Brassica oleracea varieties: white cabbage, kale, and red kale.

PubMed

Samuelsen, Anne Berit; Westereng, Bjørge; Yousif, Osman; Holtekjølen, Ann Katrin; Michaelsen, Terje E; Knutsen, Svein H

2007-02-01

Leaves of different cabbage species are used both as food and as wound healing remedies in traditional medicine. This supposed wound healing activity might be connected to presence of immunomodulating water soluble polysaccharides. To study this, three different cabbage varieties, white cabbage (W), kale (K), and red kale (RK), were pretreated with 80% ethanol and then extracted with water at 50 degrees C and 100 degrees C for isolation of polysaccharide-containing fractions. The fractions were analyzed for monosaccharide composition, glycosidic linkages, Mw distribution, protein content, and phenolic compounds and then tested for complement-fixing activity. All fractions contained pectin type polysaccharides with linkages corresponding to homogalacturonan and hairy regions. Those extracted at 50 degrees C contained higher amounts of neutral side chains and were more active in the complement-fixation test than those extracted at 100 degrees C. The fractions can be ranged by decreasing activity: K-50 > RK-50 > W-50 approximately = K-100 > RK100 approximately = W-100. Studies on structure-activity relationships (SAR) employing multivariate statistical analysis strongly suggest that the magnitude of the measured activity is influenced by the content of certain side chains in the polymers. High activity correlates to large neutral side chains with high amounts of (1-->6)- and (1-->3,6)-linked Gal and low amounts of (1-->4)-linked GalA but not on molecular weight distribution of the polymers.

Sequestration of Sup35 by aggregates of huntingtin fragments causes toxicity of [PSI+] yeast.

PubMed

Zhao, Xiaohong; Park, Yang-Nim; Todor, Horia; Moomau, Christine; Masison, Daniel; Eisenberg, Evan; Greene, Lois E

2012-07-06

Expression of huntingtin fragments with 103 glutamines (HttQ103) is toxic in yeast containing either the [PIN(+)] prion, which is the amyloid form of Rnq1, or [PSI(+)] prion, which is the amyloid form of Sup35. We find that HttQP103, which has a polyproline region at the C-terminal end of the polyQ repeat region, is significantly more toxic in [PSI(+)] yeast than in [PIN(+)], even though HttQP103 formed multiple aggregates in both [PSI(+)] and [PIN(+)] yeast. This toxicity was only observed in the strong [PSI(+)] variant, not the weak [PSI(+)] variant, which has more soluble Sup35 present than the strong variant. Furthermore, expression of the MC domains of Sup35, which retains the C-terminal domain of Sup35, but lacks the N-terminal prion domain, almost completely rescued HttQP103 toxicity, but was less effective in rescuing HttQ103 toxicity. Therefore, the toxicity of HttQP103 in yeast containing the [PSI(+)] prion is primarily due to sequestration of the essential protein, Sup35.

The Ribosome-Bound Chaperones RAC and Ssb1/2p Are Required for Accurate Translation in Saccharomyces cerevisiae

PubMed Central

Rakwalska, Magdalena; Rospert, Sabine

2004-01-01

The chaperone homologs RAC (ribosome-associated complex) and Ssb1/2p are anchored to ribosomes; Ssb1/2p directly interacts with nascent polypeptides. The absence of RAC or Ssb1/2p results in a similar set of phenotypes, including hypersensitivity against the aminoglycoside paromomycin, which binds to the small ribosomal subunit and compromises the fidelity of translation. In order to understand this phenomenon we measured the frequency of translation termination and misincorporation in vivo and in vitro with a novel reporter system. Translational fidelity was impaired in the absence of functional RAC or Ssb1/2p, and the effect was further enhanced by paromomycin. The mutant strains suffered primarily from a defect in translation termination, while misincorporation was compromised to a lesser extent. Consistently, a low level of soluble translation termination factor Sup35p enhanced growth defects in the mutant strains. Based on the combined data we conclude that RAC and Ssb1/2p are crucial in maintaining translational fidelity beyond their postulated role as chaperones for nascent polypeptides. PMID:15456889

The ribosome-bound chaperones RAC and Ssb1/2p are required for accurate translation in Saccharomyces cerevisiae.

PubMed

Rakwalska, Magdalena; Rospert, Sabine

2004-10-01

The chaperone homologs RAC (ribosome-associated complex) and Ssb1/2p are anchored to ribosomes; Ssb1/2p directly interacts with nascent polypeptides. The absence of RAC or Ssb1/2p results in a similar set of phenotypes, including hypersensitivity against the aminoglycoside paromomycin, which binds to the small ribosomal subunit and compromises the fidelity of translation. In order to understand this phenomenon we measured the frequency of translation termination and misincorporation in vivo and in vitro with a novel reporter system. Translational fidelity was impaired in the absence of functional RAC or Ssb1/2p, and the effect was further enhanced by paromomycin. The mutant strains suffered primarily from a defect in translation termination, while misincorporation was compromised to a lesser extent. Consistently, a low level of soluble translation termination factor Sup35p enhanced growth defects in the mutant strains. Based on the combined data we conclude that RAC and Ssb1/2p are crucial in maintaining translational fidelity beyond their postulated role as chaperones for nascent polypeptides.

Solution structure analysis of the HPV16 E6 oncoprotein reveals a self-association mechanism required for E6-mediated degradation of p53

PubMed Central

Zanier, Katia; Sidi, Abdellahi ould M’hamed ould; Boulade-Ladame, Charlotte; Rybin, Vladimir; Chappelle, Anne; Atkinson, Andrew; Kieffer, Bruno; Travé, Gilles

2012-01-01

The viral oncoprotein E6 is an essential factor for cervical cancers induced by “high-risk” mucosal HPV. Among other oncogenic activities, E6 recruits the ubiquitin ligase E6AP to promote the ubiquitination and subsequent proteasomal degradation of p53. E6 is prone to self-association, which long precluded its structural analysis. Here we found that E6 specifically dimerizes through its N-terminal domain and that disruption of the dimer interface strongly increases E6 solubility. This allowed us to raise the first structural data covering the entire HPV16 E6 protein, including the high-resolution NMR structures of the two zinc-binding domains of E6 and a robust data-driven model structure of the N-terminal domain homodimer. Interestingly, homodimer interface mutations that disrupt E6 self-association also inactivate E6-mediated p53 degradation. These data suggest that E6 needs to self-associate via its N-terminal domain to promote the poly-ubiquitination of p53 by E6AP. PMID:22483108

Structure of a shear-thickening polysaccharide extracted from the New Zealand black tree fern, Cyathea medullaris.

PubMed

Wee, May S M; Matia-Merino, Lara; Carnachan, Susan M; Sims, Ian M; Goh, Kelvin K T

2014-09-01

A shear-thickening water-soluble polysaccharide was purified from mucilage extracted from the fronds of the New Zealand black tree fern (Cyathea medullaris or 'mamaku' in Māori) and its structure characterised. Constituent sugar analysis by three complementary methods, combined with linkage analysis (of carboxyl reduced samples) and 1H and 13C nuclear magnetic resonance spectroscopy (NMR) revealed a glucuronomannan comprising a backbone of 4-linked methylesterified glucopyranosyl uronic acid and 2-linked mannopyranosyl residues, branched at O-3 of 45% and at both O-3 and O-4 of 53% of the mannopyranosyl residues with side chains likely comprising terminal xylopyranosyl, terminal galactopyranosyl, non-methylesterified terminal glucopyranosyl uronic acid and 3-linked glucopyranosyl uronic acid residues. The weight-average molecular weight of the purified polysaccharide was ∼1.9×10(6) Da as determined by size-exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS). The distinctive rheological properties of this polysaccharide are discussed in relation to its structure. Copyright © 2014 Elsevier B.V. All rights reserved.

Relationship of Circulating C5a and Complement Factor H Levels With Disease Control in Pregnant Women With Asthma.

PubMed

Bohács, Anikó; Bikov, András; Ivancsó, István; Czaller, Ibolya; Böcskei, Renáta; Müller, Veronika; Rigó, János; Losonczy, György; Tamási, Lilla

2016-04-01

Asthma often complicates pregnancy and represents a risk of serious pregnancy complications. The complement system contributes to asthma pathogenesis and is up-regulated in healthy gestation as well. The anaphylatoxin C5a has a major pro-inflammatory role, and the complement factor H is a main soluble regulator protein both in asthma and during pregnancy; however, peripheral levels of these complement factors and their relationship to disease control have not yet been evaluated in pregnant subjects with asthma. The present study aimed to investigate circulating C5a and complement factor H levels in asthma (non-pregnant subjects with asthma; n = 19) and in pregnancy with asthma (pregnant subjects with asthma; n = 22), compared with healthy non-pregnant (n = 21) and healthy pregnant women (n = 13) and to test their relationship to clinical parameters of asthma (lung function, airway inflammation, and symptoms). Circulating C5a levels were higher in the pregnant asthma subject group compared with the healthy non-pregnant, healthy pregnant, and non-pregnant asthma groups: median 2.629 (interquartile range [IQR] 2.257-3.052) ng/mL versus 1.84 (IQR 1.576-2.563), 1.783 (IQR 0.6064-2.786), and 2.024 (IQR 1.232-2.615) ng/mL, respectively (P = .02 in all cases). C5a correlated negatively with FEV1 (r = -0.44, P = .039) and FVC values (r = -0.64, P = .001) in the pregnant asthma group and positively with fraction of exhaled nitric oxide levels in the non-pregnant asthma group (n = 12, r = 0.78, P = .004). Complement factor H levels were elevated in both the healthy pregnant and pregnant asthma subject groups compared with the healthy non-pregnant group (median 1,082 [IQR 734.9-1,224] and 910.7 [IQR 614.5-1076] μg/mL vs. 559.7 [IQR 388.7-783.1] μg/mL, P = .002 and P = .004, respectively) but not in the pregnant asthma group compared with the non-pregnant asthma group (median 687.4 [IQR 441.6-947.6] μg/mL, P = .10). Asthma during pregnancy increases the circulating level of pro-inflammatory C5a, which is accompanied by impaired lung function and partly counteracted by the gestation-specific elevation of regulatory complement factor H level (detected in pregnancy both in healthy and subjects with asthma). Copyright © 2016 by Daedalus Enterprises.

A New Solution Route to Hydrogen Terminated Silicon Nanoparticles: Synthesis, Functionalization, and Water Stability

PubMed Central

Zhang, Xiaoming; Neiner, Doinita; Wang, Shizhong; Louie, Angelique Y.; Kauzlarich, Susan M.

2010-01-01

Hydrogen capped silicon nanoparticles with strong blue photoluminescence were synthesized by the metathesis reaction of sodium silicide, NaSi, with NH4Br. The hydrogen capped Si nanoparticles were further terminated with octyl groups and then coated with a polymer to render them water soluble. The nanoparticles were characterized by TEM, FT-IR, UV-VIS absorption, and photoluminescence. The Si nanoparticles were shown to have an average diameter of 3.9 ±1.3 nm and exhibited room-temperature photoluminescence with a peak maximum at 438 nm with a quantum efficiency of 32% in hexane and 18% in water; the emission was stable in ambient air for up to 2 months. These nanoparticles could hold great potential as a non-heavy element containing quantum dot for applications in biology. PMID:25170189

Receptor-mediated protein kinase activation and the mechanism of transmembrane signaling in bacterial chemotaxis.

PubMed Central

Liu, Y; Levit, M; Lurz, R; Surette, M G; Stock, J B

1997-01-01

Chemotaxis responses of Escherichia coli and Salmonella are mediated by type I membrane receptors with N-terminal extracytoplasmic sensing domains connected by transmembrane helices to C-terminal signaling domains in the cytoplasm. Receptor signaling involves regulation of an associated protein kinase, CheA. Here we show that kinase activation by a soluble signaling domain construct involves the formation of a large complex, with approximately 14 receptor signaling domains per CheA dimer. Electron microscopic examination of these active complexes indicates a well defined bundle composed of numerous receptor filaments. Our findings suggest a mechanism for transmembrane signaling whereby stimulus-induced changes in lateral packing interactions within an array of receptor-sensing domains at the cell surface perturb an equilibrium between active and inactive receptor-kinase complexes within the cytoplasm. PMID:9405352

Palisade endings: cholinergic sensory organs or effector organs?

PubMed

Blumer, Roland; Konakci, Kadriye Zeynep; Pomikal, Christine; Wieczorek, Grazyna; Lukas, Julius-Robert; Streicher, Johannes

2009-03-01

This study aims to complement the authors' prior findings on palisade endings in extraocular muscles (EOMs) of monkeys, and to clarify whether palisade endings are cholinergic motor or cholinergic sensory. Macaque monkeys (Macaca fascicularis, n = 10) of both sexes were analyzed using three-dimensional (3D) reconstructions, confocal laser scanning microscopy (CLSM), and conventional/immuno transmission electron microscopy (TEM). For CLSM, we used three combinations of triple fluorescent labeling. EOM wholemounts were labeled with cholinergic markers, including choline acetyltransferase (ChAT), choline transporter (ChT), vesicular acetylcholine transporter (VAChT), and a classical postsynaptic marker for motor terminals, namely alpha-bungarotoxin. Muscle fibers were counterstained with phalloidin. 3D reconstructions were done of triple-labeled palisade endings. For immuno TEM, tissue was labeled with antibody against ChAT. Concordant with prior findings, the authors demonstrated that palisade endings at the muscle fiber tips arose from nerve fibers that are ChAT-positive. In 25% of the cases, axons forming palisade endings established multiple neuromuscular contacts outside the palisade complex. Such additional neuromuscular contacts were motor terminals, as demonstrated by alpha-bungarotoxin binding. All palisade endings established nerve terminals on the tendon. In 40% of the palisade endings, nerve terminals were observed on the muscle fiber as well. Neurotendinous contacts and neuromuscular contacts in palisade endings were ChT/ChAT/VAChT-immunoreactive. Neuromuscular contacts exhibited structural features of motor terminals and were also alpha-bungarotoxin positive. The present study ascertained that palisade endings are cholinergic motor organs. Therefore, it was concluded that palisade endings are not candidates to provide eye-position signals.

Strains Responsible for Invasive Meningococcal Disease in Patients With Terminal Complement Pathway Deficiencies.

PubMed

Rosain, Jérémie; Hong, Eva; Fieschi, Claire; Martins, Paula Vieira; El Sissy, Carine; Deghmane, Ala-Eddine; Ouachée, Marie; Thomas, Caroline; Launay, David; de Pontual, Loïc; Suarez, Felipe; Moshous, Despina; Picard, Capucine; Taha, Muhamed-Kheir; Frémeaux-Bacchi, Véronique

2017-04-15

Patients with terminal complement pathway deficiency (TPD) are susceptible to recurrent invasive meningococcal disease (IMD). Neisseria meningitidis (Nm) strains infecting these patients are poorly documented in the literature. We identified patients with TPD and available Nm strains isolated during IMD. We investigated the genetic basis of the different TPDs and the characteristics of the Nm strains. We included 56 patients with C5 (n = 8), C6 (n = 20), C7 (n = 18), C8 (n = 9), or C9 (n = 1) deficiency. Genetic study was performed in 47 patients and 30 pathogenic variants were identified in the genes coding for C5 (n = 4), C6 (n = 5), C7 (n = 12), C8 (n = 7), and C9 (n = 2). We characterized 61 Nm strains responsible for IMD in the 56 patients with TPD. The most frequent strains belonged to groups Y (n = 27 [44%]), B (n = 18 [30%]), and W (n = 8 [13%]). Hyperinvasive clonal complexes (CC11, CC32, CC41/44, and CC269) were responsible for 21% of IMD cases. The CC23 predominates and represented 26% of all invasive isolates. Eleven of the 15 clonal complexes identified fit to 12 different clonal complexes belonging to carriage strains. Unusual meningococcal strains with low level of virulence similar to carriage strains are most frequently responsible for IMD in patients with TPD. © The Author 217. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Age-related macular degeneration and modification of systemic complement factor H production through liver transplantation.

PubMed

Khandhadia, Samir; Hakobyan, Svetlana; Heng, Ling Z; Gibson, Jane; Adams, David H; Alexander, Graeme J; Gibson, Jonathan M; Martin, Keith R; Menon, Geeta; Nash, Kathryn; Sivaprasad, Sobha; Ennis, Sarah; Cree, Angela J; Morgan, B Paul; Lotery, Andrew J

2013-08-01

To investigate whether modification of liver complement factor H (CFH) production, by alteration of liver CFH Y402H genotype through liver transplantation (LT), influences the development of age-related macular degeneration (AMD). Multicenter, cross-sectional study. We recruited 223 Western European patients ≥ 55 years old who had undergone LT ≥ 5 years previously. We determined AMD status using a standard grading system. Recipient CFH Y402H genotype was obtained from DNA extracted from recipient blood samples. Donor CFH Y402H genotype was inferred from recipient plasma CFH Y402H protein allotype, measured using enzyme-linked immunosorbent assays. This approach was verified by genotyping donor tissue from a subgroup of patients. Systemic complement activity was ascertained by measuring levels of plasma complement proteins using an enzyme-linked immunosorbent assay, including substrates (C3, C4), activation products (C3a, C4a, and terminal complement complex), and regulators (total CFH, C1 inhibitor). We evaluated AMD status and recipient and donor CFH Y402H genotype. In LT patients, AMD was associated with recipient CFH Y402H genotype (P = 0.036; odds ratio [OR], 1.6; 95% confidence interval [CI], 1.0-2.4) but not with donor CFH Y402H genotype (P = 0.626), after controlling for age, sex, smoking status, and body mass index. Recipient plasma CFH Y402H protein allotype predicted donor CFH Y402H genotype with 100% accuracy (n = 49). Plasma complement protein or activation product levels were similar in LT patients with and without AMD. Compared with previously reported prevalence figures (Rotterdam Study), LT patients demonstrated a high prevalence of both AMD (64.6% vs 37.1%; OR, 3.09; P<0.001) and the CFH Y402H sequence variation (41.9% vs 36.2%; OR, 1.27; P = 0.014). Presence of AMD is not associated with modification of hepatic CFH production. In addition, AMD is not associated with systemic complement activity in LT patients. These findings suggest that local intraocular complement activity is of greater importance in AMD pathogenesis. The high AMD prevalence observed in LT patients may be associated with the increased frequency of the CFH Y402H sequence variation. The authors have no proprietary or commercial interest in any materials discussed in this article. Copyright © 2013 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

Soluble and Membrane-Bound β-Glucosidases Are Involved in Trimming the Xyloglucan Backbone.

PubMed

Sampedro, Javier; Valdivia, Elene R; Fraga, Patricia; Iglesias, Natalia; Revilla, Gloria; Zarra, Ignacio

2017-02-01

In many flowering plants, xyloglucan is a major component of primary cell walls, where it plays an important role in growth regulation. Xyloglucan can be degraded by a suite of exoglycosidases that remove specific sugars. In this work, we show that the xyloglucan backbone, formed by (1→4)-linked β-d-glucopyranosyl residues, can be attacked by two different Arabidopsis (Arabidopsis thaliana) β-glucosidases from glycoside hydrolase family 3. While BGLC1 (At5g20950; for β-glucosidase active against xyloglucan 1) is responsible for all or most of the soluble activity, BGLC3 (At5g04885) is usually a membrane-anchored protein. Mutations in these two genes, whether on their own or combined with mutations in other exoglycosidase genes, resulted in the accumulation of partially digested xyloglucan subunits, such as GXXG, GXLG, or GXFG. While a mutation in BGLC1 had significant effects on its own, lack of BGLC3 had only minor effects. On the other hand, double bglc1 bglc3 mutants revealed a synergistic interaction that supports a role for membrane-bound BGLC3 in xyloglucan metabolism. In addition, bglc1 bglc3 was complemented by overexpression of either BGLC1 or BGLC3 In overexpression lines, BGLC3 activity was concentrated in a microsome-enriched fraction but also was present in soluble form. Finally, both genes were generally expressed in the same cell types, although, in some cases, BGLC3 was expressed at earlier stages than BGLC1 We propose that functional specialization could explain the separate localization of both enzymes, as a membrane-bound β-glucosidase could specifically digest soluble xyloglucan without affecting the wall-bound polymer. © 2017 American Society of Plant Biologists. All Rights Reserved.

Rituximab fails where eculizumab restores renal function in C3nef-related DDD.

PubMed

Rousset-Rouvière, Caroline; Cailliez, Mathilde; Garaix, Florentine; Bruno, Daniele; Laurent, Daniel; Tsimaratos, Michel

2014-06-01

Dense deposit disease (DDD), a C3 glomerulopathy (C3G), is a rare disease with unfavorable progression towards end-stage kidney disease. The pathogenesis of DDD is due to cytotoxic effects related to acquired or genetic dysregulation of the complement alternative pathway, which is at times accompanied by the production of C3 nephritic factor (C3NeF), an auto-antibody directed against the alternative C3 convertase. Available treatments include plasma exchange, CD20-targeted antibodies, and a terminal complement blockade via the anti-C5 monoclonal antibody eculizumab. We report here the case of an 8-year-old child with C3NeF and refractory DDD who presented with a nephritic syndrome. She tested positive for C3NeF activity; C3 was undetectable. Genetic analyses of the alternative complement pathway were normal. Methylprednisolone pulses and mycophenolate mofetil treatment resulted in complete recovery of renal function and a reduction in proteinuria. Corticosteroids were tapered and then withdrawn. Four months after corticosteroid discontinuation, hematuria and proteinuria recurred, and a renal biopsy confirmed an active DDD with a majority of extracapillary crescents. Despite an increase in immunosuppressive drugs, including methylprednisolone pulses and rituximab therapy, the patient suffered acute renal failure within 3 weeks, requiring dialysis. Eculizumab treatment resulted in a quick and impressive response. Hematuria very quickly resolved, kidney function improved, and no further dialysis was required. The patient received bimonthly eculizumab injections of 600 mg, allowing for normalization of renal function and reduction of proteinuria to <0.5 g per day. Since then, she continues to receive eculizumab. Complement regulation pathway-targeted therapy may be a specific and useful treatment for rapidly progressing DDD prior to the development of glomerulosclerosis. Our data provide evidence supporting the pivotal role of complement alternative pathway abnormalities in C3G with DDD.

Shark complement: an assessment.

PubMed

Smith, S L

1998-12-01

The classical (CCP) and alternative (ACP) pathways of complement activation have been established for the nurse shark (Ginglymostoma cirratum). The isolation of a cDNA clone encoding a mannan-binding protein-associated serine protease (MASP)-1-like protein from the Japanese dogfish (Triakis scyllia) suggests the presence of a lectin pathway. The CCP consists of six functionally distinct components: C1n, C2n, C3n, C4n, C8n and C9n, and is activated by immune complexes in the presence of Ca++ and Mg++ ions. The ACP is antibody independent, requiring Mg++ ions and a heat-labile 90 kDa factor B-like protein for activity. Proteins considered homologues of C1q, C3 and C4 (C2n) of the mammalian complement system have been isolated from nurse shark serum. Shark C1q is composed of at least two chain types each showing 50% identity to human C1q chains A and B. Partial sequence of the globular domain of one of the chains shows it to be C1q-like rather than like mannan-binding protein. N-terminal amino acid sequences of the alpha and beta chain of shark C3 and C4 molecules show significant identity with corresponding human C3 and C4 chains. A sequence representing shark C4 gamma chain, shows little similarity to human C4 gamma chain. The terminal shark components C8n and C9n are functional analogues of mammalian C8 and C9. Anaphylatoxin activity has been demonstrated in activated shark serum, and porcine C5a desArg induces shark leucocyte chemotaxis. The deduced amino acid sequence of a partial C3 cDNA clone from the nurse shark shows 50%, 30% and 24% homology with the corresponding region of mammalian C3, C4 and alpha 2-macroglobulin. Deduced amino acid sequence data from partial Bf/C2 cDNA clones, two from the nurse shark and one from the Japanese dogfish, suggest that at least one species of elasmobranch has two distinct Bf/C2 genes.

The guanidinium group as a key part of water-soluble polymer carriers for siRNA complexation and protection against degradation.

PubMed

Tabujew, Ilja; Freidel, Christoph; Krieg, Bettina; Helm, Mark; Koynov, Kaloian; Müllen, Klaus; Peneva, Kalina

2014-07-01

Here, the preparation of a novel block copolymer consisting of a statistical copolymer N-(2-hydroxypropyl) methacrylamide-s-N-(3-aminopropyl) methacrylamide and a short terminal 3-guanidinopropyl methacrylamide block is reported. This polymer structure forms neutral but water-soluble nanosized complexes with siRNA. The siRNA block copolymer complexes are first analyzed using agarose gel electrophoresis and their size is determined with fluorescence correlation spectroscopy. The protective properties of the polymer against RNA degradation are investigated by treating the siRNA block copolymer complexes with RNase V1. Heparin competition assays confirm the efficient release of the cargo in vitro. In addition, the utilization of microscale thermophoresis is demonstrated for the determination of the binding strength between a fluorescently labeled polyanion and a polymer molecule. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Importance of interlayer H bonding structure to the stability of layered minerals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Conroy, Michele; Soltis, Jennifer A.; Wittman, Rick S.

2017-10-16

The exact atomic structures of layered minerals have been difficult to characterize because the layers often possess out-of-plane hydrogen atoms that cannot be detected by many analytical techniques. However, the ordering of these bonds are thought to play a fundamental role in the structural stability and solubility of layered minerals. We report a new strategy of using the intense radiation field of a focused electron beam to probe the effect of differences in hydrogen bonding networks on mineral solubility while simultaneously imaging the dissolution behavior in real time via liquid cell electron microscopy. We show the loss in hydrogens frommore » interlayers of boehmite (γ-AlOOH) resulted in 2D nanosheets exfoliating from the bulk that subsequently and rapidly dissolved. However gibbsite (γ-Al(OH)3), with its higher concentration of OH terminating groups, was more accommodating to the deprotonation and stable under the beam.« less

Isolation, purification and some structural features of the mucilaginous exudate from Musa paradisiaca.

PubMed

Mondal, S K; Ray, B; Thakur, S; Ghosal, P K

2001-03-01

The water-soluble polysaccharides isolated from the vascular gel of Musa paradisiaca, were fractionated via anion exchange chromatography into four fractions. Fractionated polymers contained arabinose, xylose and galacturonic acid as major sugars, together with traces of galactose, rhamnose, mannose and glucose residues. Methylation analysis revealed the presence of a highly branched arabinoxylan with a significant amount of terminal arabinopyranosyl units and an arabinogalactan type I pectin. Periodate oxidation studies supported the results of methylation analysis.

Copper(II) carboxylate promoted intramolecular diamination of terminal alkenes: improved reaction conditions and expanded substrate scope.

PubMed

Zabawa, Thomas P; Chemler, Sherry R

2007-05-10

The copper(II) carboxylate promoted diamination reaction has been improved by the use of the organic soluble copper(II) neodecanoate [Cu(ND)2]. Cu(ND)2 allowed the less-polar solvent dichloroethane (DCE) to be used, and as a consequence, decomposition of less-reactive substrates could be avoided. High diastereoselectivity was observed in the synthesis of 2,5-disubstituted pyrrolidines. Ureas, bis(anilines), and alpha-amido pyrroles derived from 2-allylaniline could also participate in the diamination reaction.

Silk Spinning in Silkworms and Spiders

PubMed Central

Andersson, Marlene; Johansson, Jan; Rising, Anna

2016-01-01

Spiders and silkworms spin silks that outcompete the toughness of all natural and manmade fibers. Herein, we compare and contrast the spinning of silk in silkworms and spiders, with the aim of identifying features that are important for fiber formation. Although spiders and silkworms are very distantly related, some features of spinning silk seem to be universal. Both spiders and silkworms produce large silk proteins that are highly repetitive and extremely soluble at high pH, likely due to the globular terminal domains that flank an intermediate repetitive region. The silk proteins are produced and stored at a very high concentration in glands, and then transported along a narrowing tube in which they change conformation in response primarily to a pH gradient generated by carbonic anhydrase and proton pumps, as well as to ions and shear forces. The silk proteins thereby convert from random coil and alpha helical soluble conformations to beta sheet fibers. We suggest that factors that need to be optimized for successful production of artificial silk proteins capable of forming tough fibers include protein solubility, pH sensitivity, and preservation of natively folded proteins throughout the purification and initial spinning processes. PMID:27517908

Definition of the HLA-A29 peptide ligand motif allows prediction of potential T-cell epitopes from the retinal soluble antigen, a candidate autoantigen in birdshot retinopathy.

PubMed Central

Boisgerault, F; Khalil, I; Tieng, V; Connan, F; Tabary, T; Cohen, J H; Choppin, J; Charron, D; Toubert, A

1996-01-01

The peptide-binding motif of HLA-A29, the predisposing allele for birdshot retinopathy, was determined after acid-elution of endogenous peptides from purified HLA-A29 molecules. Individual and pooled HPLC fractions were sequenced by Edman degradation. Major anchor residues could be defined as glutamate at the second position of the peptide and as tyrosine at the carboxyl terminus. In vitro binding of polyglycine synthetic peptides to purified HLA-A29 molecules also revealed the need for an auxiliary anchor residue at the third position, preferably phenylalanine. By using this motif, we synthesized six peptides from the retinal soluble antigen, a candidate autoantigen in autoimmune uveoretinitis. Their in vitro binding was tested on HLA-A29 and also on HLA-B44 and HLA-B61, two alleles sharing close peptide-binding motifs. Two peptides derived from the carboxyl-terminal sequence of the human retinal soluble antigen bound efficiently to HLA-A29. This study could contribute to the prediction of T-cell epitopes from retinal autoantigens implicated in birdshot retinopathy. PMID:8622959

Cytoplasmic expression of a thermostable invertase from Thermotoga maritima in Lactococcus lactis.

PubMed

Pek, Han Bin; Lim, Pei Yu; Liu, Chengcheng; Lee, Dong-Yup; Bi, Xuezhi; Wong, Fong Tian; Ow, Dave Siak-Wei

2017-05-01

To evaluate the secretory and cytoplasmic expression of a thermostable Thermogata maritima invertase in Lactococcus lactis. The thermostable invertase from T. maritima was cloned with and without the USP45 secretory peptide into the pNZ8148 vector for nisin-inducible expression in L. lactis. The introduction of an USP45 secretion peptide at the N-terminal of the enzyme led to a loss of protein solubility. Computational homology modeling and hydrophobicity studies indicated that the USP45 peptide exposes a stretch of hydrophobic amino acids on the protein surface resulting in lower solubility. Removal of the USP45 secretion peptide allowed a soluble and functional invertase to be expressed intracellularly in L. lactis. Immobilized metal affinity chromatography purification of the cell lysate with nickel-NTA gave a single protein band on SDS-PAGE, while E. coli-expressed invertase consistently co-purified with an additional band. The yields of the purified invertase from E. coli and L. lactis were 14.1 and 6.3 mg/l respectively. Invertase can be expressed in L. lactis and purified in a functional form. L. lactis is a suitable host for the production of food-grade invertase for use in the food and biotechnology industries.

Silk Spinning in Silkworms and Spiders.

PubMed

Andersson, Marlene; Johansson, Jan; Rising, Anna

2016-08-09

Spiders and silkworms spin silks that outcompete the toughness of all natural and manmade fibers. Herein, we compare and contrast the spinning of silk in silkworms and spiders, with the aim of identifying features that are important for fiber formation. Although spiders and silkworms are very distantly related, some features of spinning silk seem to be universal. Both spiders and silkworms produce large silk proteins that are highly repetitive and extremely soluble at high pH, likely due to the globular terminal domains that flank an intermediate repetitive region. The silk proteins are produced and stored at a very high concentration in glands, and then transported along a narrowing tube in which they change conformation in response primarily to a pH gradient generated by carbonic anhydrase and proton pumps, as well as to ions and shear forces. The silk proteins thereby convert from random coil and alpha helical soluble conformations to beta sheet fibers. We suggest that factors that need to be optimized for successful production of artificial silk proteins capable of forming tough fibers include protein solubility, pH sensitivity, and preservation of natively folded proteins throughout the purification and initial spinning processes.

Thermodynamic modeling of the Ge-La binary system

NASA Astrophysics Data System (ADS)

Liu, Miao; Li, Chang-rong; Du, Zhen-min; Guo, Cui-ping; Niu, Chun-ju

2012-08-01

The Ge-La binary system was critically assessed by means of the calculation of phase diagram (CALPHAD) technique. The associate model was used for the liquid phase containing the constituent species Ge, La, Ge3La5, and Ge1.7La. The terminal solid solution diamond-(Ge) with a small solubility of La was described using the substitutional model, in which the excess Gibbs energy was formulated with the Redlich-Kister equation. The compounds with homogeneity ranges, α(Ge1.7La), β(Ge1.7La), and (GeLa), were modeled using two sublattices as α(Ge,La)1.7La, β(Ge,La)1.7La, and (Ge,La)(Ge,La), respectively. The intermediate phases with no solubility ranges, Ge4La5, Ge3La4, Ge3La5, and GeLa3, were treated as stoichiometric compounds. The three allotropic modifications of La, dhcp-La, fcc-La, and bcc-La, were kept as pure element phases since no solubility of Ge in La was reported. A set of self-consistent thermodynamic parameters of the Ge-La binary system was obtained. The calculation results agree well with the available experimental data from literatures.

Disease-linked mutations in factor H reveal pivotal role of cofactor activity in self-surface-selective regulation of complement activation.

PubMed

Kerr, Heather; Wong, Edwin; Makou, Elisavet; Yang, Yi; Marchbank, Kevin; Kavanagh, David; Richards, Anna; Herbert, Andrew P; Barlow, Paul N

2017-08-11

Spontaneous activation enables the complement system to respond very rapidly to diverse threats. This activation is efficiently suppressed by complement factor H (CFH) on self-surfaces but not on foreign surfaces. The surface selectivity of CFH, a soluble protein containing 20 complement-control protein modules (CCPs 1-20), may be compromised by disease-linked mutations. However, which of the several functions of CFH drives this self-surface selectivity remains unknown. To address this, we expressed human CFH mutants in Pichia pastoris We found that recombinant I62-CFH (protective against age-related macular degeneration) and V62-CFH functioned equivalently, matching or outperforming plasma-derived CFH, whereas R53H-CFH, linked to atypical hemolytic uremic syndrome (aHUS), was defective in C3bBb decay-accelerating activity (DAA) and factor I cofactor activity (CA). The aHUS-linked CCP 19 mutant D1119G-CFH had virtually no CA on (self-like) sheep erythrocytes ( E S ) but retained DAA. The aHUS-linked CCP 20 mutant S1191L/V1197A-CFH (LA-CFH) had dramatically reduced CA on E S but was less compromised in DAA. D1119G-CFH and LA-CFH both performed poorly at preventing complement-mediated hemolysis of E S PspCN, a CFH-binding Streptococcus pneumoniae protein domain, binds CFH tightly and increases accessibility of CCPs 19 and 20. PspCN did not improve the DAA of any CFH variant on E S Conversely, PspCN boosted the CA, on E S , of I62-CFH, R53H-CFH, and LA-CFH and also enhanced hemolysis protection by I62-CFH and LA-CFH. We conclude that CCPs 19 and 20 are critical for efficient CA on self-surfaces but less important for DAA. Exposing CCPs 19 and 20 with PspCN and thus enhancing CA on self-surfaces may reverse deficiencies of some CFH variants. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Fanconi Anemia Complementation Group A (FANCA) Protein Has Intrinsic Affinity for Nucleic Acids with Preference for Single-stranded Forms*

PubMed Central

Yuan, Fenghua; Qian, Liangyue; Zhao, Xinliang; Liu, Jesse Y.; Song, Limin; D'Urso, Gennaro; Jain, Chaitanya; Zhang, Yanbin

2012-01-01

The Fanconi anemia complementation group A (FANCA) gene is one of 15 disease-causing genes and has been found to be mutated in ∼60% of Fanconi anemia patients. Using purified protein, we report that human FANCA has intrinsic affinity for nucleic acids. FANCA binds to both single-stranded (ssDNA) and double-stranded (dsDNA) DNAs; however, its affinity for ssDNA is significantly higher than for dsDNA in an electrophoretic mobility shift assay. FANCA also binds to RNA with an intriguingly higher affinity than its DNA counterpart. FANCA requires a certain length of nucleic acids for optimal binding. Using DNA and RNA ladders, we determined that the minimum number of nucleotides required for FANCA recognition is ∼30 for both DNA and RNA. By testing the affinity between FANCA and a variety of DNA structures, we found that a 5′-flap or 5′-tail on DNA facilitates its interaction with FANCA. A patient-derived FANCA truncation mutant (Q772X) has diminished affinity for both DNA and RNA. In contrast, the complementing C-terminal fragment of Q772X, C772–1455, retains the differentiated nucleic acid-binding activity (RNA > ssDNA > dsDNA), indicating that the nucleic acid-binding domain of FANCA is located primarily at its C terminus, where most disease-causing mutations are found. PMID:22194614

Fanconi anemia complementation group A (FANCA) protein has intrinsic affinity for nucleic acids with preference for single-stranded forms.

PubMed

Yuan, Fenghua; Qian, Liangyue; Zhao, Xinliang; Liu, Jesse Y; Song, Limin; D'Urso, Gennaro; Jain, Chaitanya; Zhang, Yanbin

2012-02-10

The Fanconi anemia complementation group A (FANCA) gene is one of 15 disease-causing genes and has been found to be mutated in ∼60% of Fanconi anemia patients. Using purified protein, we report that human FANCA has intrinsic affinity for nucleic acids. FANCA binds to both single-stranded (ssDNA) and double-stranded (dsDNA) DNAs; however, its affinity for ssDNA is significantly higher than for dsDNA in an electrophoretic mobility shift assay. FANCA also binds to RNA with an intriguingly higher affinity than its DNA counterpart. FANCA requires a certain length of nucleic acids for optimal binding. Using DNA and RNA ladders, we determined that the minimum number of nucleotides required for FANCA recognition is ∼30 for both DNA and RNA. By testing the affinity between FANCA and a variety of DNA structures, we found that a 5'-flap or 5'-tail on DNA facilitates its interaction with FANCA. A patient-derived FANCA truncation mutant (Q772X) has diminished affinity for both DNA and RNA. In contrast, the complementing C-terminal fragment of Q772X, C772-1455, retains the differentiated nucleic acid-binding activity (RNA > ssDNA > dsDNA), indicating that the nucleic acid-binding domain of FANCA is located primarily at its C terminus, where most disease-causing mutations are found.

Hiding in plain sight: immune evasion by the staphylococcal protein SdrE.

PubMed

Herr, Andrew B; Thorman, Alexander W

2017-05-10

The human immune system is responsible for identification and destruction of invader cells, such as the bacterial pathogen Staphylococcus aureus In response, S. aureus brings to the fight a large number of virulence factors, including several that allow it to evade the host immune response. The staphylococcal surface protein SdrE was recently reported to bind to complement Factor H, an important regulator of complement activation. Factor H attaches to the surface of host cells to inhibit complement activation and amplification, preventing the destruction of the host cell. SdrE binding to Factor H allows S. aureus to mimic a host cell and reduces bacterial killing by granulocytes. In a new study published in Biochemical Journal , Zhang et al. describe crystal structures of SdrE and its complex with the C-terminal portion of Factor H. The structure of SdrE and its interaction with the Factor H peptide closely resemble a family of surface proteins that recognize extracellular matrix components such as fibrinogen. However, unbound SdrE forms a novel 'Closed' conformation with an occluded peptide-binding groove. These structures reveal a fascinating mechanism for immune evasion and provide a potential avenue for the development of novel antimicrobial agents to target SdrE. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

FANCJ Helicase Operates in the Fanconi Anemia DNA Repair Pathway and the Response to Replicational Stress

PubMed Central

Wu, Yuliang; Brosh, Robert M.

2009-01-01

Fanconi anemia (FA) is an autosomal recessive disorder characterized by multiple congenital anomalies, progressive bone marrow failure, and high cancer risk. Cells from FA patients exhibit spontaneous chromosomal instability and hypersensitivity to DNA interstrand cross-linking (ICL) agents. Although the precise mechanistic details of the FA/BRCA pathway of ICL-repair are not well understood, progress has been made in the identification of the FA proteins that are required for the pathway. Among the 13 FA complementation groups from which all the FA genes have been cloned, only a few of the FA proteins are predicted to have direct roles in DNA metabolism. One of the more recently identified FA proteins, shown to be responsible for complementation of the FA complementation group J, is the BRCA1 Associated C-terminal Helicase (BACH1, designated FANCJ), originally identified as a protein associated with breast cancer. FANCJ has been proposed to function downstream of FANCD2 monoubiquitination, a critical event in the FA pathway. Evidence supports a role for FANCJ in a homologous recombination (HR) pathway of double strand break (DSB) repair. In this review, we will summarize the current knowledge in terms of FANCJ functions through its enzymatic activities and protein interactions. The molecular roles of FANCJ in DNA repair and the response to replicational stress will be discussed. PMID:19519404

C1 inhibitor-mediated myocardial protection from chronic intermittent hypoxia-induced injury

PubMed Central

Fu, Jinrong; Guo, Furong; Chen, Cheng; Yu, Xiaoman; Hu, Ke; Li, Mingjiang

2016-01-01

The optimal treatment for chronic intermittent hypoxia (CIH)-induced cardiovascular injuries has yet to be determined. The aim of the current study was to explore the potential protective effect and mechanism of a C1 inhibitor in CIH in the myocardium. The present study used a rat model of CIH in which complement regulatory protein, known as C1 inhibitor (C1INH), was administered to the rats in the intervention groups. Cardiomyocyte apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. The expression of proteins associated with the apoptotic pathway, such as B-cell lymphoma 2 (Bcl-2), Bax and caspase-3 were detected by western blot analysis. The expression of complement C3 protein and RNA were also analyzed. C1INH was observed to improve the cardiac function in rats with CIH. Myocardial myeloperoxidase activity, a marker of neutrophil infiltration, was significantly decreased in the C1INH intervention group compared with the CIH control group, and cardiomyocyte apoptosis was significantly attenuated (P<0.05). Western blotting and reverse transcription-polymerase chain reaction analysis indicated that the protein expression levels of Bcl-2 were decreased and those of Bax were increased in the CIH group compared with the normal control group, but the protein expression levels of Bcl-2 were increased and those of Bax were decreased in the C1INH intervention group, as compared with the CIH group. Furthermore, the CIH-induced expression and synthesis of complement C3 in the myocardium were also reduced in the C1INH intervention group. C1INH, in addition to inhibiting complement activation and inflammation, preserved cardiac function in CIH-mediated myocardial cell injury through an anti-apoptotic mechanism. PMID:27698713

Eculizumab treatment and impaired opsonophagocytic killing of meningococci by whole blood from immunized adults.

PubMed

Konar, Monica; Granoff, Dan M

2017-08-17

Eculizumab, a humanized anti-complement C5 monoclonal antibody (mAb) for treatment of paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome, blocks the terminal complement pathway required for serum bactericidal activity (SBA). Because treated patients are at >1000-fold increased risk of meningococcal disease, vaccination is recommended; whether vaccination can protect by opsonophagocytic activity in the absence of SBA is not known. Meningococci were added to anticoagulated blood from 12 healthy adults vaccinated with meningococcal serogroup B and serogroup A, C, W, Y vaccines. Bacterial survival was measured after 3-hour incubation in the presence of eculizumab or control complement factor D inhibitor ACH-4471, which blocks the complement alternative pathway (AP) and is in phase 2 development for treatment of PNH. In the absence of inhibitors, colony formation units (CFUs) per milliliter in blood from all 12 immunized subjects decreased from ∼4000 at time 0 to sterile cultures at 3 hours. In the presence of eculizumab, there was a >22-fold increase in geometric mean CFUs per milliliter (90 596 and 114 683 CFU/mL for serogroup B and C strains, respectively; P < .0001 compared with time 0). In the presence of ACH-4471, there was a >12-fold decrease (23 and 331 CFU/mL, respectively; P < .0001). The lack of meningococci killing by blood containing eculizumab resulted from inhibition of release of C5a, a C5 split product needed for upregulation of phagocytosis. The results provide an explanation for the large number of cases of meningococcal disease in immunized patients being treated with eculizumab and suggest that vaccination may provide better protection against meningococcal disease in patients treated with an AP-specific inhibitor. © 2017 by The American Society of Hematology.

Transcriptional profiles of pulmonary innate immune responses to isogenic antibiotic-susceptible and multidrug-resistant Pseudomonas aeruginosa.

PubMed

Tam, Vincent H; Pérez, Cynthia; Ledesma, Kimberly R; Lewis, Russell E

2018-04-01

The virulence of an isogenic pair of Pseudomonas aeruginosa strains was studied under similar experimental conditions in two animal infection models. The time to death was significantly longer for the multidrug resistant (MDR) than the wild-type strain. The transcriptional profiles of 84 innate immune response genes in the lungs of immune competent Balb/C mice were further compared. Significantly weaker expression of genes involved in production of soluble pattern recognition receptor and complement were observed in animals infected with the MDR strain. Altered patterns of innate immune system activation may explain the attenuated virulence in MDR bacteria. © 2018 The Societies and John Wiley & Sons Australia, Ltd.

Structural and functional aspects of C1-inhibitor.

PubMed

Bos, Ineke G A; Hack, C Erik; Abrahams, Jan Pieter

2002-09-01

C1-Inh is a serpin that inhibits serine proteases from the complement and the coagulation pathway. C1-Inh consists of a serpin domain and a unique N-terminal domain and is heavily glycosylated. Non-functional mutants of C1-Inh can give insight into the inhibitory mechanism of C1-Inh. This review describes a novel 3D model of C1-Inh, based on a newly developed homology modelling method. This model gives insight into a possible potentiation mechanism of C1-Inh and based on this model the essential residues for efficient inhibition by C1-Inh are discussed.

Water-soluble Coenzyme Q10 formulation (Q-ter) promotes outer hair cell survival in a guinea pig model of noise induced hearing loss (NIHL).

PubMed

Fetoni, Anna Rita; Piacentini, Roberto; Fiorita, Antonella; Paludetti, Gaetano; Troiani, Diana

2009-02-27

The mitochondrial respiratory chain is a powerful source of reactive oxygen species (ROS) also in noise induced hearing loss (NIHL) and anti-oxidants and free-radicals scavengers have been shown to attenuate the damage. Coenzyme Q(10) (CoQ(10)) or ubiquinone has a bioenergetic role as a component of the mithocondrial respiratory chain, it inhibits mitochondrial lipid peroxidation, inducing ATP production and it is involved in ROS removal and prevention of oxidative stress-induced apoptosis. However the therapeutic application of CoQ(10) is limited by the lack of solubility and poor bio- availability, therefore it is a challenge to improve its water solubility in order to ameliorate the efficacy in tissues and fluids. This study was conducted in a model of acoustic trauma in the guinea pig where the effectiveness of CoQ(10) was compared with a soluble formulation of CoQ(10) (multicomposite CoQ(10) Terclatrate, Q-ter) given intraperitoneally 1 h before and once daily for 3 days after pure tone noise exposure (6 kHz for 1 h at 120 dB SPL). Functional and morphological studies were carried out by measuring auditory brainstem responses, scanning electron microscopy for hair cell loss count, active caspase 3 staining and terminal deoxynucleotidyl transferase-mediated dUTP labelling assay in order to identify initial signs of apoptosis. Treatments decreased active caspase 3 expression and the number of apoptotic cells, but animals injected with Q-ter showed a greater degree of activity in preventing apoptosis and thus in improving hearing. These data confirm that solubility of Coenzyme Q(10) improves the ability of CoQ(10) in preventing oxidative injuries that result from mitochondrial dysfunction.

Automated microfluidic platform for studies of carbon dioxide dissolution and solubility in physical solvents.

PubMed

Abolhasani, Milad; Singh, Mayank; Kumacheva, Eugenia; Günther, Axel

2012-05-07

We present an automated microfluidic (MF) approach for the systematic and rapid investigation of carbon dioxide (CO(2)) mass transfer and solubility in physical solvents. Uniformly sized bubbles of CO(2) with lengths exceeding the width of the microchannel (plugs) were isothermally generated in a co-flowing physical solvent within a gas-impermeable, silicon-based MF platform that is compatible with a wide range of solvents, temperatures and pressures. We dynamically determined the volume reduction of the plugs from images that were accommodated within a single field of view, six different downstream locations of the microchannel at any given flow condition. Evaluating plug sizes in real time allowed our automated strategy to suitably select inlet pressures and solvent flow rates such that otherwise dynamically self-selecting parameters (e.g., the plug size, the solvent segment size, and the plug velocity) could be either kept constant or systematically altered. Specifically, if a constant slug length was imposed, the volumetric dissolution rate of CO(2) could be deduced from the measured rate of plug shrinkage. The solubility of CO(2) in the physical solvent was obtained from a comparison between the terminal and the initial plug sizes. Solubility data were acquired every 5 min and were within 2-5% accuracy as compared to literature data. A parameter space consisting of the plug length, solvent slug length and plug velocity at the microchannel inlet was established for different CO(2)-solvent pairs with high and low gas solubilities. In a case study, we selected the gas-liquid pair CO(2)-dimethyl carbonate (DMC) and volumetric mass transfer coefficients 4-30 s(-1) (translating into mass transfer times between 0.25 s and 0.03 s), and Henry's constants, within the range of 6-12 MPa.

Separation and characterization of acetyl and non-acetyl hemicelluloses of Arundo donax by ammonium sulfate precipitation.

PubMed

Peng, Feng; Bian, Jing; Peng, Pai; Xiao, Huan; Ren, Jun-Li; Xu, Feng; Sun, Run-Cang

2012-04-25

Delignified Arundo donax was sequentially extracted with DMSO, saturated barium hydroxide, and 1.0 M aqueous NaOH solution. The yields of the soluble fractions were 10.2, 6.7, and 10.0% (w/w), respectively, of the dry Arundo donax materials. The DMSO-, Ba(OH)(2)- and NaOH-soluble hemicellulosic fractions were further fractionated into two subfractions by gradient 50% and 80% saturation ammonium sulfate precipitation, respectively. Monosaccharide, molecular weight, FT-IR, and 1D ((1)H and (13)C) and 2D (HSQC) NMR analysis revealed the differences in structural characteristics and physicochemical properties among the subfractions. The subfractions precipitated with 50% saturation ammonium sulfate had lower arabinose/xylose and glucuronic acid/xylose ratios but had higher molecular weight than those of the subfractions precipitated by 80% saturation ammonium sulfate. FT-IR and NMR analysis revealed that the highly acetylated DMSO-soluble hemicellulosic subfraction (H(D50)) could be precipitated with a relatively lower concentration of 50% saturated ammonium sulfate, and thus the gradient ammonium sulfate precipitation technique could discriminate acetyl and non-acetyl hemicelluloses. It was found that the DMSO-soluble subfraction H(D50) precipitated by 50% saturated ammonium sulfate mainly consisted of poorly substituted O-acetyl arabino-4-O-methylglucurono xylan with terminal units of arabinose linked on position 3 of xylose, 4-O-methylglucuronic acid residues linked on position 2 of the xylan bone, and the acetyl groups (degree of acetylation, 37%) linked on position 2 or 3. The DMSO-soluble subfraction H(D80) precipitated by 80% saturated ammonium sulfate was mainly composed of highly substituted arabino-4-O-methylglucurono xylan and β-d-glucan.

Carbon Dots as Versatile Photosensitizers for Solar-Driven Catalysis with Redox Enzymes.

PubMed

Hutton, Georgina A M; Reuillard, Bertrand; Martindale, Benjamin C M; Caputo, Christine A; Lockwood, Colin W J; Butt, Julea N; Reisner, Erwin

2016-12-28

Light-driven enzymatic catalysis is enabled by the productive coupling of a protein to a photosensitizer. Photosensitizers used in such hybrid systems are typically costly, toxic, and/or fragile, with limited chemical versatility. Carbon dots (CDs) are low-cost, nanosized light-harvesters that are attractive photosensitizers for biological systems as they are water-soluble, photostable, nontoxic, and their surface chemistry can be easily modified. We demonstrate here that CDs act as excellent light-absorbers in two semibiological photosynthetic systems utilizing either a fumarate reductase (FccA) for the solar-driven hydrogenation of fumarate to succinate or a hydrogenase (H 2 ase) for reduction of protons to H 2 . The tunable surface chemistry of the CDs was exploited to synthesize positively charged ammonium-terminated CDs (CD-NHMe 2 + ), which were capable of transferring photoexcited electrons directly to the negatively charged enzymes with high efficiency and stability. Enzyme-based turnover numbers of 6000 mol succinate (mol FccA) -1 and 43,000 mol H 2 (mol H 2 ase) -1 were reached after 24 h. Negatively charged carboxylate-terminated CDs (CD-CO 2 - ) displayed little or no activity, and the electrostatic interactions at the CD-enzyme interface were determined to be essential to the high photocatalytic activity observed with CD-NHMe 2 + . The modular surface chemistry of CDs together with their photostability and aqueous solubility make CDs versatile photosensitizers for redox enzymes with great scope for their utilization in photobiocatalysis.

RAGE splicing variants in mammals.

PubMed

Sterenczak, Katharina Anna; Nolte, Ingo; Murua Escobar, Hugo

2013-01-01

The receptor for advanced glycation end products (RAGE) is a multiligand receptor of environmental stressors which plays key roles in pathophysiological processes, including immune/inflammatory disorders, Alzheimer's disease, diabetic arteriosclerosis, tumorigenesis, and metastasis. Besides the full-length RAGE protein in humans nearly 20 natural occurring RAGE splicing variants were described on mRNA and protein level. These naturally occurring isoforms are characterized by either N-terminally or C-terminally truncations and are discussed as possible regulators of the full-length RAGE receptor either by competitive ligand binding or by displacing the full-length protein in the membrane. Accordingly, expression deregulations of the naturally occurring isoforms were supposed to have significant effect on RAGE-mediated disorders. Thereby the soluble C-truncated RAGE isoforms present in plasma and tissues are the mostly focused isoforms in research and clinics. Deregulations of the circulating levels of soluble RAGE forms were reported in several RAGE-associated pathological disorders including for example atherosclerosis, diabetes, renal failure, Alzheimer's disease, and several cancer types. Regarding other mammalian species, the canine RAGE gene showed high similarities to the corresponding human structures indicating RAGE to be evolutionary highly conserved between both species. Similar to humans the canine RAGE showed a complex and extensive splicing activity leading to a manifold pattern of RAGE isoforms. Due to the similarities seen in several canine and human diseases-including cancer-comparative structural and functional analyses allow the development of RAGE and ligand-specific therapeutic approaches beneficial for human and veterinary medicine.

Acyclic cucurbit[n]uril-type molecular containers: influence of glycoluril oligomer length on their function as solubilizing agents.

PubMed

Gilberg, Laura; Zhang, Ben; Zavalij, Peter Y; Sindelar, Vladimir; Isaacs, Lyle

2015-04-07

We present the synthesis of a series of six new glycoluril derived molecular clips and acyclic CB[n]-type molecular containers (1–3) that all feature SO3(−) solubilizing groups but differ in the number of glycoluril rings between the two terminal dialkoxyaromatic sidewalls. We report the X-ray crystal structure of 3b which shows that its dialkoxynaphthalene sidewalls actively define a hydrophobic cavity with high potential to engage in π–π interactions with insoluble aromatic guests. Compounds 1–3 possess very good solubility characteristics (≥38 mM) and undergo only very weak self-association (Ks < 92 M(−1)) in water. The weak self-association is attributed to unfavorable SO3(−)···SO3(−) electrostatic interactions in the putative dimers 12–42. Accordingly, we created phase solubility diagrams to study their ability to act as solubilizing agents for four water insoluble drugs (PBS-1086, camptothecin, β-estradiol, and ziprasidone). We find that the containers 3a and 3b which feature three glycoluril rings between the terminal dialkoxy-o-xylylene and dialkoxynaphthalene sidewalls are less efficient solubilizing agents than 4a and 4b because of their smaller hydrophobic cavities. Containers 1 and 2 behave as molecular clip type receptors and therefore possess the ability to bind to and thereby solubilize aromatic drugs like camptothecin, ziprasidone, and PBS-1086.

Acyclic Cucurbit[n]uril-Type Molecular Containers: Influence of Glycoluril Oligomer Length on their Function as Solubilizing Agents

PubMed Central

Gilberg, Laura; Zhang, Ben; Zavalij, Peter Y.; Sindelar, Vladimir; Isaacs, Lyle

2015-01-01

We present the synthesis of a series of six new glycoluril derived molecular clips and acyclic CB[n]-type molecular containers (1 – 3) that all feature SO3− solubilizing groups but differ in the number of glycoluril rings between the two terminal dialkoxyaromatic sidewalls. We report the x-ray crystal structure of 3b which shows that its dialkoxynaphthalene sidewalls actively define a hydrophobic cavity with high potential to engage in π–π interactions with insoluble aromatic guests. Compounds 1 – 3 possess very good solubility characteristics (≥ 38 mM) and undergo only very weak self-association (Ks < 92 M−1) in water. The weak self-association is attributed to unfavorable SO3−•••SO3− electrostatic interactions in the putative dimers 12 – 42. Accordingly, we created phase solubility diagrams to study their ability to act as solubilizing agents for four water insoluble drugs (PBS-1086, camptothecin, β-estradiol, and ziprasidone). We find that the containers 3a and 3b which feature three glycoluril rings between the terminal dialkoxy-o-xylylene and dialkoxynaphthalene sidewalls are less efficient solubilizing agents than 4a and 4b because of their smaller hydrophobic cavities. Containers 1 and 2 behave as molecular clip type receptors and therefore possess the ability to bind to and thereby solubilize aromatic drugs like camptothecin and ziprasidone, and PBS-1086. PMID:25731639

Soluble ST2 and N-terminal pro-brain natriuretic peptide combination. Useful biomarker for predicting outcome of childhoodpulmonary arterial hypertension.

PubMed

Chida, Ayako; Sato, Hiroki; Shintani, Masaki; Nakayama, Tomotaka; Kawamura, Yoichi; Furutani, Yoshiyuki; Inai, Kei; Saji, Tsutomu; Matsuoka, Rumiko; Nonoyama, Shigeaki; Nakanishi, Toshio

2014-01-01

Some potential biomarkers have been reported recently in patients with pulmonary arterial hypertension (PAH), but the most clinically useful among these potential biomarkers, especially in childhood PAH, has not been identified. Therefore, this study investigated which biomarker is useful in assessing severity of and patient prognosis in childhood idiopathic PAH (IPAH)/heritable PAH (HPAH). Fifty-nine patients who were younger than 16 years at onset of IPAH/HPAH were selected. The following 10 biomarker candidates were quantified: high-sensitivity troponin T, human heart fatty acid-binding protein, N-terminal pro-brain natriuretic peptide (NT-proBNP), pentraxin-3, soluble ST2 (sST2), angiopoietin-2 (Ang-2), matrix metalloproteinase 2, tenascin C, endostatin (ES), and thymidine kinase. Functional characteristics and clinical outcomes were analyzed retrospectively. NT-proBNP, sST2, Ang-2, and ES correlated well with New York Heart Association class. On area under the receiver operating characteristic curve analysis, sST2 had a significantly good relationship with prognosis. On Kaplan-Meier curve and univariate Cox regression analyses, elevated sST2 and NT-proBNP level predicted poor outcome of the present patients with childhood IPAH/HPAH. Furthermore, patients with elevated sST2 had significantly worse prognosis among those with high NT-proBNP. The sST2 and NT-proBNP combination is a useful biomarker to predict clinical condition and outcome in patients with childhood IPAH/HPAH.  

Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization*

PubMed Central

Wang, Weiyi; Jiang, Qiyang; Argentini, Manuela; Cornu, David; Gigant, Benoît; Knossow, Marcel; Wang, Chunguang

2012-01-01

The kinesin-13 Kif2C hydrolyzes ATP and uses the energy released to disassemble microtubules. The mechanism by which this is achieved remains elusive. Here we show that Kif2C-(sN+M), a monomeric construct consisting of the motor domain with the proximal part of the N-terminal Neck extension but devoid of its more distal, unstructured, and highly basic part, has a robust depolymerase activity. When detached from microtubules, the Kif2C-(sN+M) nucleotide-binding site is occupied by ATP at physiological concentrations of adenine nucleotides. As a consequence, Kif2C-(sN+M) starts its interaction with microtubules in that state, which differentiates kinesin-13s from motile kinesins. Moreover, in this ATP-bound conformational state, Kif2C-(sN+M) has a higher affinity for soluble tubulin compared with microtubules. We propose a mechanism in which, in the first step, the specificity of ATP-bound Kif2C for soluble tubulin causes it to stabilize a curved conformation of tubulin heterodimers at the ends of microtubules. Data from an ATPase-deficient Kif2C mutant suggest that, then, ATP hydrolysis precedes and is required for tubulin release to take place. Finally, comparison with Kif2C-Motor indicates that the binding specificity for curved tubulin and, accordingly, the microtubule depolymerase activity are conferred to the motor domain by its N-terminal Neck extension. PMID:22403406

Kidney lesions in baboons infected with Schistosoma mansoni.

PubMed Central

Houba, V; Sturrock, R F; Butterworth, A E

1977-01-01

Glomerular lesions in baboons (Papio anubis) infected with different dosage regimes of Schistosoma mansoni were studied by immunofluorescence and light microscopy on kidney sections and by countercurrent immunoelectrophoresis on kidney homogenates and tissue eluates. Mild lesions, characterized by focal and segmental deposits of immune complexes, developed in sixty-two out of 103 baboons, irrespective of the intensity and duration of the infection. Severe, diffuse lesions developed in six baboons after prolonged and heavy infections. Adult worm and soluble egg antigens, together with IgM, IgG and C3, were detected in most of the severe lesions and in some of the mild lesions. In some animals, antigens were detected in most of the severe lesions and in some of the mild lesions. In some animals, antigens were detected in acid homogenates and eluates of kidneys which showed no deposits of immunoglobulins or complement. These observations indicate that renal lesions in S. mansoni infections may be attributable to the deposition of immune complexes pre-formed in the circulation. However, the demonstration of antigens alone in some animals may suggest an alternative possibility, namely that antigens are deposited first with a subsequent binding of antibody and complement. PMID:414868

Ab initio structure search and in situ 7Li NMR studies of discharge products in the Li–S battery system

DOE PAGES

See, Kimberly A.; Leskes, Michal; Griffin, John M.; ...

2014-11-10

The high theoretical gravimetric capacity of the Li–S battery system makes it an attractive candidate for numerous energy storage applications. In practice, cell performance is plagued by low practical capacity and poor cycling. In an effort to explore the mechanism of the discharge with the goal of better understanding performance, we examine the Li–S phase diagram using computational techniques and complement this with an in situ 7Li NMR study of the cell during discharge. Both the computational and experimental studies are consistent with the suggestion that the only solid product formed in the cell is Li 2S, formed soon aftermore » cell discharge is initiated. In situ NMR spectroscopy also allows the direct observation of soluble Li +-species during cell discharge; species that are known to be highly detrimental to capacity retention. We suggest that during the first discharge plateau, S is reduced to soluble polysulfide species concurrently with the formation of a solid component (Li 2S) which forms near the beginning of the first plateau, in the cell configuration studied here. The NMR data suggest that the second plateau is defined by the reduction of the residual soluble species to solid product (Li 2S). Lastly, a ternary diagram is presented to rationalize the phases observed with NMR during the discharge pathway and provide thermodynamic underpinnings for the shape of the discharge profile as a function of cell composition.« less

Modification and restriction of T-even bacteriophages. In vitro degradation of deoxyribonucleic acid containing 5-hydroxymethylctosine.

PubMed

Fleischman, R A; Cambell, J L; Richardson, C C

1976-03-25

Using the single-stranded circular DNA of bacteriophage fd as template, double-stranded circular DNA has been prepared in vitro with either 5-hydroxymethylcytosine ([hmdC]DNA) or cytosine ([dC]DNA) in the product strand. Extracts prepared from Escherichia coli cells restrictive to T-even phage containing nonglucosylated DNA degrade [hmdC]DNA to acid-soluble material in vitro, but do not degrade [dC]dna. In contrast, extracts prepared from E. coli K12 rglA- rglB-, a strain permissive to T-even phage containing nonglucosylated DNA, do not degrade [hmdC]DNA or [dC]DNA. In addition, glucosylation of the [hmdC]DNA renders it resistant to degradation by extracts from restrictive strains. The conversion of [hmdC]DNA to acid-soluble material in vitro consists of an HmCyt-specific endonucleolytic cleavage requiring the presence of the RglB gene product to form a linear molecule, followed by a non-HmCyt-specific hydrolysis of the linear DNA to acid-soluble fragments, catalyzed in part by exonuclease V. The RglB protein present in extracts of E. coli K12 rglA- rglB+ has been purified 200-fold by complementation with extracts from E. coli K12 rglA- rglB-. The purified RglB protein does not contain detectable HmCyt-specific endonuclease or exonuclease activity. In vitro endonucleolytic cleavage of [hmdC]DNA thus requires additional factors present in cell extracts.

Mutants of Saccharomyces cerevisiae defective in the farnesylation of Ras proteins.

PubMed Central

Goodman, L E; Judd, S R; Farnsworth, C C; Powers, S; Gelb, M H; Glomset, J A; Tamanoi, F

1990-01-01

Ras proteins are post-translationally modified by farnesylation. In the present investigation, we identified an activity in crude soluble extracts of yeast cells that catalyzes the transfer of a farnesyl moiety from farnesyl pyrophosphate to yeast RAS2 protein. RAS2 proteins having a C-terminal Cys-Ali-Ali-Xaa sequence (where Ali is an aliphatic amino acid and Xaa is the unspecified C-terminal amino acid) served as substrates for this reaction, whereas RAS2 proteins with an altered or deleted Cys-Ali-Ali-Xaa sequence did not. A yeast mutant, dpr1/ram1, originally isolated as a Ras-processing mutant was shown to be defective in farnesyltransferase activity. In addition, another mutant, ram2, also was defective in the transferase activity. These results demonstrate that at least two genes, DPR1/RAM1 and RAM2, are required for the farnesyltransferase activity in yeast. Images PMID:2124698

Two Variants of Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) with Additional Protein Domains: Synthesis in an Escherichia coli Heterologous Expression System.

PubMed

Karyagina, A S; Boksha, I S; Grunina, T M; Demidenko, A V; Poponova, M S; Sergienko, O V; Lyashchuk, A M; Galushkina, Z M; Soboleva, L A; Osidak, E O; Bartov, M S; Gromov, A V; Lunin, V G

2017-05-01

Two variants of recombinant human bone morphogenetic protein-2 (rhBMP-2) with additional N-terminal protein domains were obtained by expression in E. coli. The N-terminal domains were s-tag (15-a.a. oligopeptide from bovine pancreatic ribonuclease A) and lz (leucine zipper dimerization domain from yeast transcription factor GCN4). The s-tag-BMP-2 and lz-BMP-2 were purified by a procedure that excluded a long refolding stage. The resulting dimeric proteins displayed higher solubility compared to rhBMP-2 without additional protein domains. Biological activity of both proteins was demonstrated in vitro by induction of alkaline phosphatase in C2C12 cells, and the activity of s-tag-BMP-2 in vivo was shown in various experimental animal models.

Functionalization of multi-walled carbon nanotubes with thermo-responsive azide-terminated poly(N-isopropylacrylamide) via click reactions.

PubMed

Su, Xin; Shuai, Ya; Guo, Zanru; Feng, Yujun

2013-04-18

Covalently functionalized multi-walled carbon nanotubes (MWNTs) were prepared by grafting well-defined thermo-responsive poly(N-isopropylacrylamide) (PNIPAM) via click reactions. First, azide-terminated poly(N-isopropylacrylamide) (N3-PNIPAM) was synthesized by reversible addition fragmentation chain-transfer (RAFT) polymerization, and then the N₃-PNIPAM moiety was connected onto MWNTs by click chemistry. The products were characterized by means of FT-IR, TGA and TEM. The results show that the modification of MWNTs is very successful and MWNTs functionalized by N₃-PNIPAM (MWNTs-PNIPAM) have good solubility and stability in water. TEM images show the functionalized MWNTs are dispersed individually, indicating that the bundles of original MWNTs are separated into individual tubes by surface modification with polymer chains. These MWNTs modified with PNIPAM represent a potential nano-material for preparation of hydrophilic composite materials.

Functional Implications of Domain Organization Within Prokaryotic Rhomboid Proteases.

PubMed

Panigrahi, Rashmi; Lemieux, M Joanne

2015-01-01

Intramembrane proteases are membrane embedded enzymes that cleave transmembrane substrates. This interesting class of enzyme and its water mediated substrate cleavage mechanism occurring within the hydrophobic lipid bilayer has drawn the attention of researchers. Rhomboids are a family of ubiquitous serine intramembrane proteases. Bacterial forms of rhomboid proteases are mainly composed of six transmembrane helices that are preceded by a soluble N-terminal domain. Several crystal structures of the membrane domain of the E. coli rhomboid protease ecGlpG have been solved. Independently, the ecGlpG N-terminal cytoplasmic domain structure was solved using both NMR and protein crystallography. Despite these structures, we still do not know the structure of the full-length protein, nor do we know the functional role of these domains in the cell. This chapter will review the structural and functional roles of the different domains associated with prokaryotic rhomboid proteases. Lastly, we will address questions remaining in the field.

Isolation and characterization of beta- and gamma-caseins from horse milk.

PubMed Central

Visser, S; Jenness, R; Mullin, R J

1982-01-01

Three groups of casein components were isolated from horse milk. Group I is almost insoluble at acid and neutral pH, and is rather heterogeneous on alkaline gels with or without sodium dodecyl sulphate. Group II shows strong similarity to beta-casein from other species, as concluded from its amino acid composition and its N- and C-terminal sequences. This group consists of five electrophoretically distinguishable forms, all containing ester phosphate groups but no carbohydrate. Group III is composed of C-terminal fragments of the beta-like (group II) fraction and probably arises from the action of a plasmin-like enzyme present in horse milk. It does not contain phosphate or carbohydrate. Homology of this group with bovine gamma-caseins is demonstrated. Both beta- and gamma-like caseins are more soluble at 4 degrees C than at room temperature. Images Fig. 1. Fig. 3. Fig. 5. PMID:6213224

Analysis of the Heterogeneity of the 40,000 Molecular Weight Tuber Glycoprotein of Potatoes by Immunological Methods and by NH2-Terminal Sequence Analysis 1

PubMed Central

Park, William D.; Blackwood, Cheri; Mignery, Greg A.; Hermodson, Mark A.; Lister, Richard M.

1983-01-01

Among the major soluble tuber proteins of potato (Solanum tuberosum L.) is a group of glycoproteins having apparent molecular weights of approximately 40,000. This group of proteins as purified by ion-exchange and affinity chromatography has been given the trivial name `patatin.' Patatin exists in a number of charge forms which differ between potato cultivars and in some cases can also be resolved into a number of bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, by immunodiffusion and immunoelectrophoresis, it was found that the isoforms of patatin are immunologically identical both within a cultivar as well as between cultivars. A high degree of homology between the isoforms of patatin is also indicated by NH2-terminal amino acid sequence analysis. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:16662777

Biophysical Analysis of Anopheles gambiae Leucine-Rich Repeat Proteins APL1A1, APL1B and APL1C and Their Interaction with LRIM1

DOE PAGES

Williams, Marni; Summers, Brady J.; Baxter, Richard H. G.; ...

2015-03-16

Natural infection of Anopheles gambiae by malaria-causing Plasmodium parasites is significantly influenced by the APL1 genetic locus. The locus contains three closely related leucine-rich repeat (LRR) genes, APL1A, APL1B and APL1C. Multiple studies have reported the participation of APL1A—C in the immune response of A. gambiae to invasion by both rodent and human Plasmodium isolates. APL1C forms a heterodimer with the related LRR protein LRIM1 via a C-terminal coiled-coil domain that is also present in APL1A and APL1B. The LRIM1/APL1C heterodimer protects A. gambiae from infection by binding the complement-like protein TEP1 to form a stable and active immune complex.more » We report solution x-ray scatting data for the LRIM1/APL1C heterodimer, the oligomeric state of LRIM1/APL1 LRR domains in solution and the crystal structure of the APL1B LRR domain. The LRIM1/APL1C heterodimeric complex has a flexible and extended structure in solution. In contrast to the APL1A, APL1C and LRIM1 LRR domains, the APL1B LRR domain is a homodimer. The crystal structure of APL1B-LRR shows that the homodimer is formed by an N-terminal helix that complements for the absence of an N-terminal capping motif in APL1B, which is a unique distinction within the LRIM1/APL1 protein family. Full-length APL1A 1 and APL1B form a stable complex with LRIM1. Our results support a model in which APL1A 1, APL1B and APL1C can all form an extended, flexible heterodimer with LRIM1, providing a repertoire of functional innate immune complexes to protect A. gambiae from a diverse array of pathogens.« less

Biophysical Analysis of Anopheles gambiae Leucine-Rich Repeat Proteins APL1A1, APL1B and APL1C and Their Interaction with LRIM1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Williams, Marni; Summers, Brady J.; Baxter, Richard H. G.

Natural infection of Anopheles gambiae by malaria-causing Plasmodium parasites is significantly influenced by the APL1 genetic locus. The locus contains three closely related leucine-rich repeat (LRR) genes, APL1A, APL1B and APL1C. Multiple studies have reported the participation of APL1A—C in the immune response of A. gambiae to invasion by both rodent and human Plasmodium isolates. APL1C forms a heterodimer with the related LRR protein LRIM1 via a C-terminal coiled-coil domain that is also present in APL1A and APL1B. The LRIM1/APL1C heterodimer protects A. gambiae from infection by binding the complement-like protein TEP1 to form a stable and active immune complex.more » We report solution x-ray scatting data for the LRIM1/APL1C heterodimer, the oligomeric state of LRIM1/APL1 LRR domains in solution and the crystal structure of the APL1B LRR domain. The LRIM1/APL1C heterodimeric complex has a flexible and extended structure in solution. In contrast to the APL1A, APL1C and LRIM1 LRR domains, the APL1B LRR domain is a homodimer. The crystal structure of APL1B-LRR shows that the homodimer is formed by an N-terminal helix that complements for the absence of an N-terminal capping motif in APL1B, which is a unique distinction within the LRIM1/APL1 protein family. Full-length APL1A 1 and APL1B form a stable complex with LRIM1. Our results support a model in which APL1A 1, APL1B and APL1C can all form an extended, flexible heterodimer with LRIM1, providing a repertoire of functional innate immune complexes to protect A. gambiae from a diverse array of pathogens.« less

Docking simulation analysis of range data requirements for the orbital maneuvering vehicle

NASA Technical Reports Server (NTRS)

Micheal, J. D.; Vinz, F. L.

1985-01-01

The results of an initial study are reported assess the controllability of the Orbital Maneuvering Vehicle (OMV) for terminal closure and docking are reported. The vehicle characteristics used in this study are those of the Marshall Space Flight Center (MSFC) baseline OMV which were published with the request for proposals for preliminary design of this vehicle. This simulation was conducted at MSFC using the Target Motion Simulator. The study focused on the OMV manual mode capability to accommodate both stabilized and tumbling target engagements with varying complements of range and range rate data displayed to the OMV operator. Four trained test subjects performed over 400 simulated orbital dockings during this study. A firm requirement for radar during the terminal closure and dock phase of the OMV mission was not established by these simulations. Fifteen pound thrusters recommended in the MSFC baseline design were found to be advantageous for initial rate matching maneuvers with unstabilized targets; however, lower thrust levels were desirable for making the final docking maneuvers.

G5 PAMAM dendrimer versus liposome: a comparison study on the in vitro transepithelial transport and in vivo oral absorption of simvastatin.

PubMed

Qi, Rong; Zhang, Heran; Xu, Lu; Shen, Wenwen; Chen, Cong; Wang, Chao; Cao, Yini; Wang, Yunan; van Dongen, Mallory A; He, Bing; Wang, Siling; Liu, George; Banaszak Holl, Mark M; Zhang, Qiang

2015-07-01

This study compared formulation effects of a dendrimer and a liposome preparation on the water solubility, transepithelial transport, and oral bioavailability of simvastatin (SMV). Amine-terminated G5 PAMAM dendrimer (G5-NH2) was chosen to form SMV/G5-NH2 molecular complexes, and SMV-liposomes were prepared by using a thin film dispersion method. The effects of these preparations on the transepithelial transport were investigated in vitro using Caco-2 cell monolayers. Results indicated that the solubility and transepithelial transport of SMV were significantly improved by both formulations. Pharmacokinetic studies in rats also revealed that both the SMV/G5-NH2 molecular complexes and the SMV-liposomes significantly improved the oral bioavailability of SMV with the liposomes being more effective than the G5-NH2. The overall better oral absorption of SMV-liposomes as compared to SMV/G5-NH2 molecular complexes appeared to arise from better liposomal solubilization and encapsulation of SMV and more efficient intracellular SMV delivery. Various carrier systems have been designed to enhance drug delivery via the oral route. In this study, the authors compared G5 PAMAM dendrimers to liposome preparations in terms of solubility, transepithelial transport, and oral bioavailability of this poorly water-soluble drug. This understanding has improved our knowledge in the further development of drug carrier systems. Copyright © 2015 Elsevier Inc. All rights reserved.

Soluble extracellular matrix metalloproteinase inducer (EMMPRIN, EMN) regulates cancer-related cellular functions by homotypic interactions with surface CD147.

PubMed

Knutti, Nadine; Kuepper, Michael; Friedrich, Karlheinz

2015-11-01

EMMPRIN (extracellular matrix metalloproteinase inducer) is a widely expressed glycoprotein and a member of the immunoglobulin superfamily which exists in both a membrane-spanning and a soluble form. Homotypic interactions of EMMPRIN underlie its multiple roles in normal development and pathological situations such as viral infections, Alzheimer's disease and cancer. This study employed a recombinant soluble, fully glycosylated EMMPRIN domain (rhsEMN) as a tool to characterize the structural basis of EMMPRIN-EMMPRIN receptor (EMNR) contacts and their functional effects on MCF-7 breast carcinoma cells. rhsEMN did not form dimers in solution but bound to surface EMMPRIN (EMN) on MCF-7 cells with high affinity and was readily internalized. The interaction interface for the homotypic contact was localized to the N-terminal Ig domain. rhsEMN exerted a stimulatory effect on proliferation of MCF-7 cells whereas it reduced cell migration in a dose-dependent manner. These effects were accompanied by an upregulation of endogenous EMMPRIN as well as of matrix metalloproteinase-14 (MMP-14), a membrane-bound protease involved in the extracellular release of soluble EMMPRIN, indicating a regulatory feedback mechanism. The proliferation-promoting activity of rhsEMN was mimicked by a novel functional antibody directed to EMMPRIN, underscoring that crosslinking of cell surface EMMPRIN (EMNR) is crucial for eliciting intracellular signalling. Addressing malignancy-related signal transduction in HEK-293 cells, we could show that rhsEMN triggers the oncogenic Wnt pathway. © 2015 FEBS.

Dysfibrinogen Kagoshima with the amino acid substitution gammaThr-314 to Ile: analyses of molecular abnormalities and thrombophilic nature of this abnormal molecule.

PubMed

Niwa, Kazuki; Mimuro, Jun; Miyata, Masaaki; Sugo, Teruko; Ohmori, Tsukasa; Madoiwa, Seiji; Tei, Chuwa; Sakata, Yoichi

2008-01-01

Emerging lines of evidence have suggested that certain dysfibrinogens present a significant risk of thrombosis. The thrombophilic nature of a new-type of dysfibrinogen Kagoshima identified in a 36-year-old female with deep vein thrombosis during the postpartum period was studied. Based on the analyses of the patient fibrinogen and the fibrinogen genes, fibrinogen Kagoshima was shown to have the amino acid substitution of gammaThr-314 to Ile that resulted in impaired function and hypofibrinogenemia. Polymerization of fibrin monomers derived from patient fibrinogen was severely impaired with a partial correction in the presence of calcium ions, causing very low clottability and delayed cross-linking of patient fibrin catalyzed by activated factor XIII. Because of the low clottability, a large amount of soluble fibrin was formed upon thrombin treatment, resulting in an increase of thrombin in the soluble fraction. Additionally, tPA-mediated plasmin generation on fibrin was impaired and calcium-ion-dependent integrity of the gamma-chain D domain of Kagoshima fibrinogen was perturbed. The presence of many tapered-fiber ends inside the tangled fibrin networks, observed by scanning electron microscopy, suggested early termination of fibrin polymerization and the structural alteration. These data suggest that fibrinogen Kagoshima is dysfunctional, giving rise to formation of fibrinolysis-resistant soluble fibrin polymers and entrance of soluble fibrin associating with thrombin to the circulation, partly accounting for the thrombophilic nature of the affected fibrinogen and fibrin molecules.

Novel Biomarkers to Improve the Prediction of Cardiovascular Event Risk in Type 2 Diabetes Mellitus.

PubMed

van der Leeuw, Joep; Beulens, Joline W J; van Dieren, Susan; Schalkwijk, Casper G; Glatz, Jan F C; Hofker, Marten H; Verschuren, W M Monique; Boer, Jolanda M A; van der Graaf, Yolanda; Visseren, Frank L J; Peelen, Linda M; van der Schouw, Yvonne T

2016-05-31

We evaluated the ability of 23 novel biomarkers representing several pathophysiological pathways to improve the prediction of cardiovascular event (CVE) risk in patients with type 2 diabetes mellitus beyond traditional risk factors. We used data from 1002 patients with type 2 diabetes mellitus from the Second Manifestations of ARTertial disease (SMART) study and 288 patients from the European Prospective Investigation into Cancer and Nutrition-NL (EPIC-NL). The associations of 23 biomarkers (adiponectin, C-reactive protein, epidermal-type fatty acid binding protein, heart-type fatty acid binding protein, basic fibroblast growth factor, soluble FMS-like tyrosine kinase-1, soluble intercellular adhesion molecule-1 and -3, matrix metalloproteinase [MMP]-1, MMP-3, MMP-9, N-terminal prohormone of B-type natriuretic peptide, osteopontin, osteonectin, osteocalcin, placental growth factor, serum amyloid A, E-selectin, P-selectin, tissue inhibitor of MMP-1, thrombomodulin, soluble vascular cell adhesion molecule-1, and vascular endothelial growth factor) with CVE risk were evaluated by using Cox proportional hazards analysis adjusting for traditional risk factors. The incremental predictive performance was assessed with use of the c-statistic and net reclassification index (NRI; continuous and based on 10-year risk strata 0-10%, 10-20%, 20-30%, >30%). A multimarker model was constructed comprising those biomarkers that improved predictive performance in both cohorts. N-terminal prohormone of B-type natriuretic peptide, osteopontin, and MMP-3 were the only biomarkers significantly associated with an increased risk of CVE and improved predictive performance in both cohorts. In SMART, the combination of these biomarkers increased the c-statistic with 0.03 (95% CI 0.01-0.05), and the continuous NRI was 0.37 (95% CI 0.21-0.52). In EPIC-NL, the multimarker model increased the c-statistic with 0.03 (95% CI 0.00-0.03), and the continuous NRI was 0.44 (95% CI 0.23-0.66). Based on risk strata, the NRI was 0.12 (95% CI 0.03-0.21) in SMART and 0.07 (95% CI -0.04-0.17) in EPIC-NL. Of the 23 evaluated biomarkers from different pathophysiological pathways, N-terminal prohormone of B-type natriuretic peptide, osteopontin, MMP-3, and their combination improved CVE risk prediction in 2 separate cohorts of patients with type 2 diabetes mellitus beyond traditional risk factors. However, the number of patients reclassified to a different risk stratum was limited. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Terminal Differentiation of Adult Hippocampal Progenitor Cells Is a Step Functionally Dissociable from Proliferation and Is Controlled by Tis21, Id3 and NeuroD2

PubMed Central

Micheli, Laura; Ceccarelli, Manuela; Gioia, Roberta; D’Andrea, Giorgio; Farioli-Vecchioli, Stefano; Costanzi, Marco; Saraulli, Daniele; Cestari, Vincenzo; Tirone, Felice

2017-01-01

Cell proliferation and differentiation are interdependent processes. Here, we have asked to what extent the two processes of neural progenitor cell amplification and differentiation are functionally separated. Thus, we analyzed whether it is possible to rescue a defect of terminal differentiation in progenitor cells of the dentate gyrus, where new neurons are generated throughout life, by inducing their proliferation and/or their differentiation with different stimuli appropriately timed. As a model we used the Tis21 knockout mouse, whose dentate gyrus neurons, as demonstrated by us and others, have an intrinsic defect of terminal differentiation. We first tested the effect of two proliferative as well as differentiative neurogenic stimuli, one pharmacological (fluoxetine), the other cognitive (the Morris water maze (MWM) training). Both effectively enhanced the number of new dentate gyrus neurons produced, and fluoxetine also reduced the S-phase length of Tis21 knockout dentate gyrus progenitor cells and increased the rate of differentiation of control cells, but neither factor enhanced the defective rate of differentiation. In contrast, the defect of terminal differentiation was fully rescued by in vivo infection of proliferating dentate gyrus progenitor cells with retroviruses either silencing Id3, an inhibitor of neural differentiation, or expressing NeuroD2, a proneural gene expressed in terminally differentiated dentate gyrus neurons. This is the first demonstration that NeuroD2 or the silencing of Id3 can activate the differentiation of dentate gyrus neurons, complementing a defect of differentiation. It also highlights how the rate of differentiation of dentate gyrus neurons is regulated genetically at several levels and that a neurogenic stimulus for amplification of neural stem/progenitor cells may not be sufficient in itself to modify this rate. PMID:28740463

Immune complexes in chronic Chagas disease patients are formed by exovesicles from Trypanosoma cruzi carrying the conserved MASP N-terminal region

PubMed Central

Díaz Lozano, Isabel María; De Pablos, Luis Miguel; Longhi, Silvia Andrea; Zago, María Paola; Schijman, Alejandro Gabriel; Osuna, Antonio

2017-01-01

The exovesicles (EVs) are involved in pathologic host-parasite immune associations and have been recently used as biomarkers for diagnosis of infectious diseases. The release of EVs by Trypanosoma cruzi, the causative agent of Chagas disease, has recently been described, with different protein cargoes including the MASP multigene family of proteins MASPs are specific to this parasite and characterized by a conserved C-terminal (C-term) region and an N-terminal codifying for a signal peptide (SP). In this investigation, we identified immature MASP proteins containing the MASP SP in EVs secreted by the infective forms of the parasite. Those EVs are responsible for the formation of immune complexes (ICs) containing anti-MASP SP IgGs in patients with different (cardiac, digestive and asymptomatic) chronic Chagas disease manifestations. Moreover, purified EVs as well as the MASP SP inhibit the action of the complement system and also show a significant association with the humoral response in patients with digestive pathologies. These findings reveal a new route for the secretion of MASP proteins in T. cruzi, which uses EVs as vehicles for immature and misfolded proteins, forming circulating immune complexes. Such complexes could be used in the prognosis of digestive pathologies of clinical forms of Chagas disease. PMID:28294160

Immune complexes in chronic Chagas disease patients are formed by exovesicles from Trypanosoma cruzi carrying the conserved MASP N-terminal region

NASA Astrophysics Data System (ADS)

Díaz Lozano, Isabel María; de Pablos, Luis Miguel; Longhi, Silvia Andrea; Zago, María Paola; Schijman, Alejandro Gabriel; Osuna, Antonio

2017-03-01

The exovesicles (EVs) are involved in pathologic host-parasite immune associations and have been recently used as biomarkers for diagnosis of infectious diseases. The release of EVs by Trypanosoma cruzi, the causative agent of Chagas disease, has recently been described, with different protein cargoes including the MASP multigene family of proteins MASPs are specific to this parasite and characterized by a conserved C-terminal (C-term) region and an N-terminal codifying for a signal peptide (SP). In this investigation, we identified immature MASP proteins containing the MASP SP in EVs secreted by the infective forms of the parasite. Those EVs are responsible for the formation of immune complexes (ICs) containing anti-MASP SP IgGs in patients with different (cardiac, digestive and asymptomatic) chronic Chagas disease manifestations. Moreover, purified EVs as well as the MASP SP inhibit the action of the complement system and also show a significant association with the humoral response in patients with digestive pathologies. These findings reveal a new route for the secretion of MASP proteins in T. cruzi, which uses EVs as vehicles for immature and misfolded proteins, forming circulating immune complexes. Such complexes could be used in the prognosis of digestive pathologies of clinical forms of Chagas disease.

[Diagnostic Significance of BAT in Anaphylaxis to Non-ionic Contrast Media].

PubMed

Zhang, Hao-yue; Xu, Su-jun; Tang, Xiao-xian; Niu, Ji-jun; Guo, Xiang-jie; Gao, Cai-rong

2015-06-01

To investigate the diagnostic significance of basophil activation test (BAT) in anaphylaxis to non-ionic contrast media through testing the content of CD63, mast cell-carboxypeptidase A3 (MC-CPA3), and terminal complement complex SC5b-9 of the individuals by testing their levels in the normal immune group and the anaphylaxis groups to β-lactam drugs and non -ionic contrast media. The CD63 expression of basophilic granulocyte in blood was detected by flow cytometry. The levels of MC-CPA3 in blood serum and SC5b-9 in blood plasma were detected by ELISA. The CD63 expression of basophilic granulocyte in blood, the levels of MC-CPA3 and SC5b-9 of anaphylaxis to non-ionic contrast media and β-lactam drugs were significantly higher than that in normal immune group (P < 0.05). There is activation of basophilic granulocytes, mast cells and complement system in anaphylaxis to non-ionic contrast media. BAT can be used to diagnose the anaphylaxis to non-ionic contrast media.

Zinc-induced Self-association of Complement C3b and Factor H

PubMed Central

Nan, Ruodan; Tetchner, Stuart; Rodriguez, Elizabeth; Pao, Po-Jung; Gor, Jayesh; Lengyel, Imre; Perkins, Stephen J.

2013-01-01

The sub-retinal pigment epithelial deposits that are a hallmark of age-related macular degeneration contain both C3b and millimolar levels of zinc. C3 is the central protein of complement, whereas C3u is formed by the spontaneous hydrolysis of the thioester bridge in C3. During activation, C3 is cleaved to form active C3b, then C3b is inactivated by Factor I and Factor H to form the C3c and C3d fragments. The interaction of zinc with C3 was quantified using analytical ultracentrifugation and x-ray scattering. C3, C3u, and C3b associated strongly in >100 μm zinc, whereas C3c and C3d showed weak association. With zinc, C3 forms soluble oligomers, whereas C3u and C3b precipitate. We conclude that the C3, C3u, and C3b association with zinc depended on the relative positions of C3d and C3c in each protein. Computational predictions showed that putative weak zinc binding sites with different capacities exist in all five proteins, in agreement with experiments. Factor H forms large oligomers in >10 μm zinc. In contrast to C3b or Factor H alone, the solubility of the central C3b-Factor H complex was much reduced at 60 μm zinc and even more so at >100 μm zinc. The removal of the C3b-Factor H complex by zinc explains the reduced C3u/C3b inactivation rates by zinc. Zinc-induced precipitation may contribute to the initial development of sub-retinal pigment epithelial deposits in the retina as well as reducing the progression to advanced age-related macular degeneration in higher risk patients. PMID:23661701

Lectin-dependent enhancement of Ebola virus infection via soluble and transmembrane C-type lectin receptors.

PubMed

Brudner, Matthew; Karpel, Marshall; Lear, Calli; Chen, Li; Yantosca, L Michael; Scully, Corinne; Sarraju, Ashish; Sokolovska, Anna; Zariffard, M Reza; Eisen, Damon P; Mungall, Bruce A; Kotton, Darrell N; Omari, Amel; Huang, I-Chueh; Farzan, Michael; Takahashi, Kazue; Stuart, Lynda; Stahl, Gregory L; Ezekowitz, Alan B; Spear, Gregory T; Olinger, Gene G; Schmidt, Emmett V; Michelow, Ian C

2013-01-01

Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes.

Lectin-Dependent Enhancement of Ebola Virus Infection via Soluble and Transmembrane C-type Lectin Receptors

PubMed Central

Lear, Calli; Chen, Li; Yantosca, L. Michael; Scully, Corinne; Sarraju, Ashish; Sokolovska, Anna; Zariffard, M. Reza; Eisen, Damon P.; Mungall, Bruce A.; Kotton, Darrell N.; Omari, Amel; Huang, I-Chueh; Farzan, Michael; Takahashi, Kazue; Stuart, Lynda; Stahl, Gregory L.; Ezekowitz, Alan B.; Spear, Gregory T.; Olinger, Gene G.; Schmidt, Emmett V.; Michelow, Ian C.

2013-01-01

Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes. PMID:23573288

The Arabidopsis COP9 signalosome subunit 7 is a model PCI domain protein with subdomains involved in COP9 signalosome assembly.

PubMed

Dessau, Moshe; Halimi, Yair; Erez, Tamir; Chomsky-Hecht, Orna; Chamovitz, Daniel A; Hirsch, Joel A

2008-10-01

The COP9 Signalosome (CSN) is a multiprotein complex that was originally identified in Arabidopsis thaliana as a negative regulator of photomorphogenesis and subsequently shown to be a general eukaryotic regulator of developmental signaling. The CSN plays various roles, but it has been most often implicated in regulating protein degradation pathways. Six of eight CSN subunits bear a sequence motif called PCI. Here, we report studies of subunit 7 (CSN7) from Arabidopsis, which contains such a motif. Our in vitro and structural results, based on 1.5 A crystallographic data, enable a definition of a PCI domain, built from helical bundle and winged helix subdomains. Using functional binding assays, we demonstrate that the PCI domain (residues 1 to 169) interacts with two other PCI proteins, CSN8 and CSN1. CSN7 interactions with CSN8 use both PCI subdomains. Furthermore, we show that a C-terminal tail outside of this PCI domain is responsible for association with the non-PCI subunit, CSN6. In vivo studies of transgenic plants revealed that the overexpressed CSN7 PCI domain does not assemble into the CSN, nor can it complement a null mutation of CSN7. However, a CSN7 clone that contains the PCI domain plus part of the CSN6 binding domain can complement the null mutation in terms of seedling viability and photomorphogenesis. These transgenic plants, though, are defective in adult growth, suggesting that the CSN7 C-terminal tail plays additional functional roles. Together, the findings have implications for CSN assembly and function, highlighting necessary interactions between subunits.

Yersinia adhesin A (YadA)--beauty & beast.

PubMed

Mühlenkamp, Melanie; Oberhettinger, Philipp; Leo, Jack C; Linke, Dirk; Schütz, Monika S

2015-02-01

The trimeric autotransporter adhesin Yersinia adhesin A is the prototype of the type Vc secretion systems. It is expressed by enteropathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis strains, but not by Yersinia pestis. A characteristic trait of YadA is its modular composition and trimeric nature. YadA consists of an N-terminal passenger domain which is exposed on the bacterial cell surface. The translocation of this passenger onto the surface is facilitated by a C-terminal β-barrel domain which concomitantly anchors YadA into the outer membrane with three YadA monomers contributing to the formation of a single β-barrel. In Y. enterocolitica, but not Y. pseudotuberculosis, YadA is a decisive virulence factor and its deletion renders the bacteria virtually avirulent in mouse models of infection. This striking importance of YadA in infection may derive from its manifold functions in host cell interaction. Presumably the most important function of YadA is that it mediates adhesion to extracellular matrix components of eukaryotic host cells. Only tight adhesion allows for the injection of "anti-host" effector proteins via a type III secretion system into the host cell cytosol. These effector proteins enable Yersinia to subvert the host immune system in order to replicate and establish infection. YadA is also essential for the survival of Y. enterocolitica upon contact with serum, an important immune-evasion mechanism called serum resistance. To this end, YadA interacts with several components of the host complement system, the first line of immune defense. This review will summarize recent findings about the structure and biogenesis of YadA and its interactions with the host complement system. Copyright © 2015 Elsevier GmbH. All rights reserved.

Human Lipooligosaccharide IGG That Prevents Endemic Meningococcal Disease Recognizes an Internal Lacto-N-neotetraose Structure*

PubMed Central

Cheng, Hui; Yang, Zhijie; Estabrook, Michele M.; John, Constance M.; Jarvis, Gary A.; McLaughlin, Stephanie; Griffiss, J. McLeod

2011-01-01

Antibodies that initiate complement-mediated killing of Neisseria meningitidis as they enter the bloodstream from the oropharynx protect against disseminated disease. Human IgGs that bind the neisserial L7 lipooligosaccharide (LOS) are bactericidal for L3,7 and L2,4 meningococci in the presence of human complement. These strains share a lacto-N-neotetraose (nLc4) LOS α chain. We used a set of mutants that have successive saccharide deletions from the nLc4 α chain to characterize further the binding and bactericidal activity of nLc4 LOS IgG. We found that the nLc4 α chain conforms at least four different antigens. We separately purified IgG that required the nLc4 (non-reducing) terminal galactose (Gal) for binding and IgG that bound the truncated nLc3 α chain that lacks this Gal residue. IgG that bound the internal nLc3 α chain killed both L3,7 and L2,4 strains, whereas IgG that required the nLc4 terminal Gal residue for binding killed L2,4 stains but not L3,7 strains. These results show that the diversity of LOS antibodies in human serum is as much a function of the conformation of multiple antigens by a single glycoform as of the production of multiple glycoforms. Differences in sensitivity to killing by human nLc4 LOS IgG may account for the fact that fully two-thirds of endemic group B meningococcal disease in infants and children is caused by L3,7 strains, but only 20% is caused by L2,4 stains. PMID:22027827

Biomarkers and mortality after transient ischemic attack and minor ischemic stroke: population-based study.

PubMed

Greisenegger, Stefan; Segal, Helen C; Burgess, Annette I; Poole, Debbie L; Mehta, Ziyah; Rothwell, Peter M

2015-03-01

Premature death after transient ischemic attack or stroke is more often because of heart disease or cancer than stroke. Previous studies found blood biomarkers not usefully predictive of nonfatal stroke but possibly of all-cause death. This association might be explained by potentially treatable occult cardiac disease or cancer. We therefore aimed to validate the association of a panel of biomarkers with all-cause death, particularly cardiac death and cancer death, despite the absence of associations with risk of nonfatal vascular events. Fifteen biomarkers were measured in 929 consecutive patients in a population-based study (Oxford Vascular Study), recruited from 2002 and followed up to 2013. Associations were determined by Cox regression. Model discrimination was assessed by c-statistic and the integrated discrimination improvement. During 5560 patient-years of follow-up, none of the biomarkers predicted risk of nonfatal vascular events. However, soluble tumor necrosis factor α receptor-1, von Willebrand factor, heart-type fatty-acid-binding protein, and N-terminal pro-B-type natriuretic peptide were independently predictive of all-cause death (n=361; adjusted hazard ratio per SD, 95% confidence interval: heart-type fatty-acid-binding protein: 1.31, 1.12-1.56, P=0.002; N-terminal pro-B-type natriuretic peptide: 1.34, 1.11-1.62, P=0.002; soluble tumor necrosis factor α receptor-1: 1.45, 1.26-1.66, P=0.02; von Willebrand factor: 1.19, 1.04-1.36, P=0.01). The independent contribution of the four biomarkers taken together added prognostic information and improved model discrimination (integrated discrimination improvement=0.028, P=0.0001). N-terminal pro-B-type natriuretic peptide was most predictive of vascular death (adjusted hazard ratio=1.80, 95% confidence interval, 1.34-2.41, P<0.0001), whereas heart-type fatty-acid-binding protein predicted cancer deaths (1.64, 1.26-2.12, P=0.0002). Associations were strongest in patients without known prior cardiac disease or cancer. Several biomarkers predicted death of any cause after transient ischemic attack and minor stroke. N-terminal pro-B-type natriuretic peptide and heart-type fatty-acid-binding protein might improve patient selection for additional screening for occult cardiac disease or cancer, respectively. However, our results require validation in future studies. © 2015 American Heart Association, Inc.

New RTM/RI Resins for the HSCT

NASA Technical Reports Server (NTRS)

Harris, Frank W.

1999-01-01

In the first portion of this work, 1,2,3,3,4,4-hexafluoro-1,2-bis[4-(dimethylhydroxysilyl)phenoxy]cyclobutane and 1,2,3,3,4,4-hexafluoro-1,2-bis[3-(dimethylhydroxysilyl)phenoxy]cyclobutane were prepared and homopolymerized to afford polymers with excellent thermal stability and Tgs of 27 C and -12 C, respectively. Despite the moderately high wt% of fluorin in the polymer structure (23.8%), these polymers had poor fuel resistance. In fact, swelling measurements indicate that these polymers had apparent solubility parameters of about 18.2 J (exp 1/2) m (exp -3/2) (toluene). Copolymerization of the disilanol monomers with fluorosilicone monomers afforded copolymers containing 20-30 wt% of the perfluorocyclobutane-containing structure displayed adequate fuel resistance, enhanced thermal stability, and a Tg low enough to meet the requirements of a High Speed Civil Transport (HSCT) fuel tank sealant. In the second part of this work, trifluorovinylether-terminated oligomers were prepared and polymerized via cyclodimerization. Initially, an alpha, omega-silanol-terminated fluorosilicone was endcapped with trifluorovinylether end groups via a two-step synthetic sequence. The oligomer was thermally cyclodimerized to a polymer that displayed thermal stability similar to that of a fluorosilicone homopolymer. Second, 1,3-bis[4-trifluorovinyl(oxy)phenyl]-1,3-(3,3,3-trifluoropropyl)dimethyldisiloxane and 1,3-bis{3-trifluorovinyl(oxy)phenyl]-1,3-(3,3,3-trifluoropropyl)dimethyldisiloxane were prepared and cyclodimerized to afford polymers that contained pendant trifluoropropyl groups. The pendant trifluoropropyl groups did enhance solvent resistance in aliphatic hydrocarbon solvents, however, no improvement was observed in aromatic hydrocarbon solvents. These polymers also displayed excellent thermal stability. In the last part of this work, a series of monomers was prepared by the DCC-promoted esterification of 4-[trifluorovinyl(oxy)benzoic acid with alpha, omega-functionalized hydrogenated and partially fluorinated alcohols. The monomers were cyclodimerized to the corresponding polymers. The polymers that did not contain beta hydrogens displayed significantly higher thermal stability than the fully hydrogenated polymers. A commercially-available alpha,omega-hydroxy-terminated perfluoropolyether was then functionalized with 4-[trifluorovinyl(oxy)benzoylchloride. An attempt was made to polymerize the resulting oligomer via the cyclodimerization of the terminal trifluorovinylether moieties. Although the viscosity of the oligomer increased significantly during polymerization, Gel Permeation Chromatography (GPC) analysis revealed that the Tetrahydrofuran (THF) soluble portion of the polymer did not have high molecular weight.

Structural characterization and rheological behavior of a heteroxylan extracted from Plantago notata Lagasca (Plantaginaceae) seeds.

PubMed

Benaoun, Fatima; Delattre, Cédric; Boual, Zakaria; Ursu, Alina V; Vial, Christophe; Gardarin, Christine; Wadouachi, Anne; Le Cerf, Didier; Varacavoudin, Tony; Ould El-Hadj, Mohamed Didi; Michaud, Philippe; Pierre, Guillaume

2017-11-01

Plantago notata (Plantaginaceae) is a spontaneous plant from Septentrional Algerian Sahara currently used by traditional healers to treat stomach disorders, inflammations or wound healing. A water-soluble polysaccharide, called PSPN (PolySaccharide fraction from Plantago Notata), was extracted and purified from the seeds of this semi-arid plant. The structural features of this mucilage were evaluated by colorimetric assays, Fourier transformed infrared spectroscopy (FT-IR), gas chromatography coupled to mass spectrometry (GC/MS) and 1 H/ 13 C Nuclear Magnetic Resonance (NMR) spectroscopy. PSPN is a heteroxylan with a backbone composed of β-(1,3)-d-Xylp and β-(1,4)-d-Xylp highly branched, through (O)-2 and (O)-3 positions of β-(1,4)-d-Xylp by various side chains and terminal monosaccharides such as α-l-Araf-(1,3)-β-d-Xylp, β-d-Xylp-(1,2)-β-d-Xylp, terminal Xylp or terminal Araf. The physico-chemical and rheological analysis of this polysaccharide in dilute and semi diluted regimes showed that PSPN exhibites a molecular weight of 2.3×10 6 g/mol and a pseudoplastic behavior. Copyright © 2017 Elsevier Ltd. All rights reserved.

Crystallographic studies of the anthrax lethal toxin. Final report, 1 July 1994-31 December 1996

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frederick, C.A.

1997-01-01

Protective Antigen (PA) is the central component of the three-part protein toxin secreted by Bacillus anthraces, the organism responsible for anthrax. Following proteolytic activation on the host cell surface, PA forms a membrane-inserting heptamer that translocates the toxic enzymes into the cytosol. We have solved the crystal structure of monomeric PA at 2.1 A resolution and the water-soluble heptamer at 4.5 A resolution. The monomer is organized mainly into antiparallel b-sheets and has four domains: an N-terminal domain containing two calcium ions; a heptamerization domain containing a large flexible loop implicated in membrane insertion; a small domain of unknown function;more » and a C-terminal receptor-binding domain. Removal of a 20 kDa fragment from the N-terminal domain permits assembly of the heptamer, a ring-shaped structure with a negatively charged lumen, and exposes a large hydrophobic surface for binding the toxic enzymes. We present a model of pH-dependent membrane insertion involving formation of a porin-like membrane-spanning b barrel. These studies greatly enhance current understanding of the mechanism of anthrax intoxication, and will be useful in the design of recombinant anthrax vaccines.« less

Synaptic Vesicle Mobility and Presynaptic F-Actin Are Disrupted in a N-ethylmaleimide–sensitive Factor Allele of Drosophila

PubMed Central

Nunes, Paula; Haines, Nicola; Kuppuswamy, Venkat; Fleet, David J.

2006-01-01

N-ethylmaleimide sensitive factor (NSF) can dissociate the soluble NSF attachment receptor (SNARE) complex, but NSF also participates in other intracellular trafficking functions by virtue of SNARE-independent activity. Drosophila that express a neural transgene encoding a dominant-negative form of NSF2 show an 80% reduction in the size of releasable synaptic vesicle pool, but no change in the number of vesicles in nerve terminal boutons. Here we tested the hypothesis that vesicles in the NSF2 mutant terminal are less mobile. Using a combination of genetics, pharmacology, and imaging we find a substantial reduction in vesicle mobility within the nerve terminal boutons of Drosophila NSF2 mutant larvae. Subsequent analysis revealed a decrease of filamentous actin in both NSF2 dominant-negative and loss-of-function mutants. Lastly, actin-filament disrupting drugs also decrease vesicle movement. We conclude that a factor contributing to the NSF mutant phenotype is a reduction in vesicle mobility, which is associated with decreased presynaptic F-actin. Our data are consistent with a model in which actin filaments promote vesicle mobility and suggest that NSF participates in establishing or maintaining this population of actin. PMID:16914524

Mechanistic Basis Of Calmodulin Mediated Estrogen Receptor Alpha Activation and Antiestrogen Resistance

DTIC Science & Technology

2010-06-01

for solubility (Figure 5). We call this protein Trx -ERA241-320. We also produced a similar protein construct, but with only residues 241-273 of...ERa, as a “control” (Figure 5). We call this protein Trx -ERA241-273. Because CaM binds tightly to the N-terminal extended ligand binding domain of...ERa (residues 286- 552, see above), we hypothesized that Trx - ERA241-320 would bind tightly to CaM, but that Trx -ERA241-273 would not. The genetic

Galactic chemical evolution and nucleocosmochronology - Standard model with terminated infall

NASA Technical Reports Server (NTRS)

Clayton, D. D.

1984-01-01

Some exactly soluble families of models for the chemical evolution of the Galaxy are presented. The parameters considered include gas mass, the age-metallicity relation, the star mass vs. metallicity, the age distribution, and the mean age of dwarfs. A short BASIC program for calculating these parameters is given. The calculation of metallicity gradients, nuclear cosmochronology, and extinct radioactivities is addressed. An especially simple, mathematically linear model is recommended as a standard model of galaxies with truncated infall due to its internal consistency and compact display of the physical effects of the parameters.

Proof of Principle: Immobilisation of Robust CuII3 TbIII -Macrocycles on Small, Suitably Pre-functionalised Gold Nanoparticles.

PubMed

Feltham, Humphrey L C; Dumas, Christophe; Mannini, Matteo; Otero, Edwige; Sainctavit, Philippe; Sessoli, Roberta; Meledandri, Carla J; Brooker, Sally

2017-02-21

In a proof-of-principle study, a soluble macrocyclic single-molecule magnet (SMM) containing a Cu II 3 Tb III magnetic core was covalently grafted onto small gold nanoparticles pre-functionalised with carboxylate-terminated tethers. A modified microemulsion method allowed production of the small and monodisperse nanoparticles (approximately 3.5 nm in diameter) for the chemisorption of a large amount of intact macrocyclic complexes in the hybrid system. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Copper(II) Carboxylate Promoted Intramolecular Diamination of Terminal Alkenes: Improved Reaction Conditions and Expanded Substrate Scope

PubMed Central

Zabawa, Thomas P.

2008-01-01

The copper(II) carboxylate promoted diamination reaction has been improved by the use of the organic soluble copper(II) neodecanoate [Cu(ND)2]. Cu(ND)2 allowed the less polar solvent, dichloroethane (DCE) to be used, and as a consequence, decomposition of less reactive substrates could be avoided. High diastereoselectivity was observed in the synthesis of 2,5-disubstituted pyrrolidines. Ureas, bis(anilines) and α-amido pyrroles derived from 2-allylaniline could also participate in the diamination reaction. PMID:17447781

Study on the fluorescence characteristics of carbon dots

NASA Astrophysics Data System (ADS)

Mao, Xiao-Jiao; Zheng, Hu-Zhi; Long, Yi-Juan; Du, Juan; Hao, Jian-Yu; Wang, Ling-Ling; Zhou, Dong-Bo

2010-02-01

Herein, we prepared water-soluble fluorescent carbon dots with diameter about 1.5 nm from cheap commercial lampblack. These fluorescent carbon nanoparticles are stable toward photobleaching and stable in water for more than half a year without fluorescence decrease. In order to improve its fluorescence properties, we passivated these nanoparticles with bisamino-terminated polyethylene glycol (PEG 1500N). Therefore, both fluorescence quantum yield and lifetime increased after this progress. In addition, the passivated carbon dots were more inert to solvent than the bare one and showed different responses to pH change.

Proteolytic processing of the vitellogenin precursor in the boll weevil, Anthonomus grandis.

PubMed

Heilmann, L J; Trewitt, P M; Kumaran, A K

1993-01-01

The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M(r)s of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M(r) vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M(r) honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M(r) boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10-15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein.

Mocr: A novel fusion tag for enhancing solubility that is compatible with structural biology applications

PubMed Central

DelProposto, James; Majmudar, Chinmay Y.; Smith, Janet L.; Brown, William Clay

2010-01-01

A persistent problem in heterologous protein production is insolubility of the target protein when expressed to high level in the host cell. A widely employed strategy for overcoming this problem is the use of fusion tags. The best fusion tags promote solubility, may function as purification handles and either do not interfere with downstream applications or may be removed from the passenger protein preparation. A novel fusion tag is identified that meets these criteria. This fusion tag is a monomeric mutant of the Ocr protein (0.3 gene product) of bacteriophage T7. This fusion tag displays solubilizing activity with a variety of different passenger proteins. We show that it may be used as a purification handle similar to other fusion tags. Its small size and compact structure are compatible with its use in downstream applications of the passenger protein or it may be removed and purified away from the passenger protein. The use of monomeric Ocr (Mocr) as a complement to other fusion tags such as maltose-binding protein will provide greater flexibility in protein production and processing for a wide variety of protein applications. PMID:18824232

Staphylococcus aureus Phenol-Soluble Modulins Impair Interleukin Expression in Bovine Mammary Epithelial Cells

PubMed Central

Deplanche, Martine; Alekseeva, Ludmila; Semenovskaya, Ksenia; Fu, Chih-Lung; Dessauge, Frederic; Finot, Laurence; Petzl, Wolfram; Zerbe, Holm; Le Loir, Yves; Rainard, Pascal; Smith, David G. E.; Germon, Pierre; Otto, Michael

2016-01-01

The role of the recently described interleukin-32 (IL-32) in Staphylococcus aureus-induced mastitis, an inflammation of the mammary gland, is unclear. We determined expression of IL-32, IL-6, and IL-8 in S. aureus- and Escherichia coli-infected bovine mammary gland epithelial cells. Using live bacteria, we found that in S. aureus-infected cells, induction of IL-6 and IL-8 expression was less pronounced than in E. coli-infected cells. Notably, IL-32 expression was decreased in S. aureus-infected cells, while it was increased in E. coli-infected cells. We identified the staphylococcal phenol-soluble modulin (PSM) peptides as key contributors to these effects, as IL-32, IL-6, and IL-8 expression by epithelial cells exposed to psm mutant strains was significantly increased compared to that in cells exposed to the isogenic S. aureus wild-type strain, indicating that PSMs inhibit the production of these interleukins. The use of genetically complemented strains confirmed this observation. Inasmuch as the decreased expression of IL-32, which is involved in dendritic cell maturation, impairs immune responses, our results support a PSM-dependent mechanism that allows for the development of chronic S. aureus-related mastitis. PMID:27001539

Mocr: a novel fusion tag for enhancing solubility that is compatible with structural biology applications.

PubMed

DelProposto, James; Majmudar, Chinmay Y; Smith, Janet L; Brown, William Clay

2009-01-01

A persistent problem in heterologous protein production is insolubility of the target protein when expressed to high level in the host cell. A widely employed strategy for overcoming this problem is the use of fusion tags. The best fusion tags promote solubility, may function as purification handles and either do not interfere with downstream applications or may be removed from the passenger protein preparation. A novel fusion tag is identified that meets these criteria. This fusion tag is a monomeric mutant of the Ocr protein (0.3 gene product) of bacteriophage T7. This fusion tag displays solubilizing activity with a variety of different passenger proteins. We show that it may be used as a purification handle similar to other fusion tags. Its small size and compact structure are compatible with its use in downstream applications of the passenger protein or it may be removed and purified away from the passenger protein. The use of monomeric Ocr (Mocr) as a complement to other fusion tags such as maltose-binding protein will provide greater flexibility in protein production and processing for a wide variety of protein applications.

The Surface Chemistry of Metal Chalcogenide Nanocrystals

NASA Astrophysics Data System (ADS)

Anderson, Nicholas Charles

The surface chemistry of metal chalcogenide nanocrystals is explored through several interrelated analytical investigations. After a brief discussion of the nanocrystal history and applications, molecular orbital theory is used to describe the electronic properties of semiconductors, and how these materials behave on the nanoscale. Quantum confinement plays a major role in dictating the optical properties of metal chalcogenide nanocrystals, however surface states also have an equally significant contribution to the electronic properties of nanocrystals due to the high surface area to volume ratio of nanoscale semiconductors. Controlling surface chemistry is essential to functionalizing these materials for biological imaging and photovoltaic device applications. To better understand the surface chemistry of semiconducting nanocrystals, three competing surface chemistry models are presented: 1.) The TOPO model, 2.) the Non-stoichiometric model, and 3.) the Neutral Fragment model. Both the non-stoichiometric and neutral fragment models accurately describe the behavior of metal chalcogenide nanocrystals. These models rely on the covalent bond classification system, which divides ligands into three classes: 1.) X-type, 1-electron donating ligands that balance charge with excess metal at the nanocrystal surface, 2.) L-type, 2-electron donors that bind metal sites, and 3.) Z-type, 2-electron acceptors that bind chalcogenide sites. Each of these ligand classes is explored in detail to better understand the surface chemistry of metal chalcogenide nanocrystals. First, chloride-terminated, tri-n-butylphosphine (Bu 3P) bound CdSe nanocrystals were prepared by cleaving carboxylate ligands from CdSe nanocrystals with chlorotrimethylsilane in Bu3P solution. 1H and 31P{1H} nuclear magnetic resonance spectra of the isolated nanocrystals allowed assignment of distinct signals from several free and bound species, including surface-bound Bu3P and [Bu3P-H]+[Cl]- ligands as well as a Bu3P complex of cadmium chloride. Nuclear magnetic resonance spectroscopy supports complete cleavage of the X-type carboxylate ligands. Combined with measurements of the Se:Cd:Cl ratio using Rutherford backscattering spectrometry, these studies support a structural model of nanocrystals where chloride ligands terminate the crystal lattice by balancing the charges of excess Cd2+ ions. The adsorption of dative phosphine ligands leads to nanocrystals who's solubility is afforded by reversibly bound and readily exchanged L-type ligands, e.g. primary amines and phosphines. The other halides (Br and I) can also be used to prepare Bu 3P-bound, halide-terminated CdSe nanocrystals, however these nanocrystals are not soluble after exchange. The change in binding affinity of Bu 3P over the halide series is briefly discussed. Next, we report a series of L-type ligand exchanges using Bu3P-bound, chloride-terminated CdSe nanocrystals with several Lewis bases, including aromatic, cyclic, and non-cyclic sulfides, and ethers; primary, secondary, and tertiary amines and phosphines; tertiary phosphine chalcogenides; primary alcohols, isocyanides, and isothiocyanides. Using 31P nuclear magnetic resonance spectroscopy, we establish a relative binding affinity for these ligands that reflects electronic considerations but is dominated primarily by steric interactions, as determined by comparing binding affinity to Tolmann cone angles. We also used chloride-terminated CdSe nanocrystals to explore the reactivity of ionic salts at nanocrystal surfaces. These salts, particularly [Bu3P-H]+[Cl]-, bind nanocrystals surfaces as L-type ligands, making them soluble in polar solvents such as acetonitrile. This information should provide insight for rational ligand design for future applications involving metal chalcogenide nanocrystals. The strongest ligand, primary n-alkylamine, rapidly displace the Bu3P from halide-terminated CdSe nanocrystals, leading to amine-bound nanocrystals with higher dative ligand coverages and greatly increased photoluminescence quantum yields. The importance of ligand coverage to both the UV-visible absorption and photoluminescence spectra are discussed. (Abstract shortened by UMI.).

Retention of a Bean Phaseolin/Maize γ-Zein Fusion in the Endoplasmic Reticulum Depends on Disulfide Bond Formation[W

PubMed Central

Pompa, Andrea; Vitale, Alessandro

2006-01-01

Most seed storage proteins of the prolamin class accumulate in the endoplasmic reticulum (ER) as large insoluble polymers termed protein bodies (PBs), through mechanisms that are still poorly understood. We previously showed that a fusion between the Phaseolus vulgaris vacuolar storage protein phaseolin and the N-terminal half of the Zea mays prolamin γ-zein forms ER-located PBs. Zeolin has 6 Cys residues and, like γ-zein with 15 residues, is insoluble unless reduced. The contribution of disulfide bonds to zeolin destiny was determined by studying in vivo the effects of 2-mercaptoethanol (2-ME) and by zeolin mutagenesis. We show that in tobacco (Nicotiana tabacum) protoplasts, 2-ME enhances interactions of newly synthesized proteins with the ER chaperone BiP and inhibits the secretory traffic of soluble proteins with or without disulfide bonds. In spite of this general inhibition, 2-ME enhances the solubility of zeolin and relieves its retention in the ER, resulting in increased zeolin traffic. Consistently, mutated zeolin unable to form disulfide bonds is soluble and efficiently enters the secretory traffic without 2-ME treatment. We conclude that disulfide bonds that lead to insolubilization are a determinant for PB-mediated protein accumulation in the ER. PMID:17041149

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

PubMed Central

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

2016-01-01

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

Chemical characterization of heteropolysaccharides from green and black teas (Camellia sinensis) and their anti-ulcer effect.

PubMed

Scoparo, Camila T; Souza, Lauro M; Dartora, Nessana; Sassaki, Guilherme L; Santana-Filho, Arquimedes P; Werner, Maria Fernanda P; Borato, Débora G; Baggio, Cristiane H; Iacomini, Marcello

2016-05-01

In order to obtain polysaccharides from green and black teas (Camellia sinensis), commercial leaves were submitted to infusion and then to alkaline extraction. The extracts were fractionated by freeze-thawing process, giving insoluble and soluble fractions. Complex arabinogalactan protein from the soluble fractions of both teas (GTPS and BTPS) were determined by methylation analysis and (1)H/(13)C-HSQC spectroscopy, showing a main chain of (1→3)-β-Galp, substituted at O-6 by (1→6)-linked β-Galp with side chains of α-Araf and terminal units of α-Araf, α-Fucp and α-Rhap. A highly branched heteroxylan from the insoluble fractions (GTPI and BTPI) showed in methylation analysis and (1)H/(13)C-HSQC spectroscopy the main chain of (1→4)-β-Xylp, substituted in O-3 by α-Araf, β-Galp and α-Glcp units. Evaluating their gastroprotective activity, the fractions containing the soluble heteropolysaccharides from green (GTPS) and black teas (BTPS) reduced the gastric lesions induced by ethanol. Furthermore, the fraction of insoluble heteropolysaccharides of green (GTPI) and black (BTPI) teas also protected the gastric mucosa. In addition, the maintenance of gastric mucus and reduced glutathione (GSH) levels was involved in the polysaccharides gastroprotection. Copyright © 2016 Elsevier B.V. All rights reserved.

The soluble extracellular fragment of neuroligin-1 targets Aβ oligomers to the postsynaptic region of excitatory synapses.

PubMed

Dinamarca, Margarita C; Di Luca, Monica; Godoy, Juan A; Inestrosa, Nibaldo C

2015-10-09

Amyloid-β oligomers (Aβo) play a major role in the synaptic dysfunction of Alzheimer's disease (AD). Neuroligins are postsynaptic cell-adhesion molecules, that share an extracellular domain with high degree of similarity to acetylcholinesterase (AChE), one of the first putative Aβo receptors. We recently found that Aβo interact with the soluble N-terminal fragment of neuroligin-1 (NL-1). We report here that Aβo associate with NL-1 at excitatory hippocampal synapses, whereas almost no association was observed with neuroligin-2, an isoform present at inhibitory synapses. Studies using purified hippocampal postsynaptic densities indicate that NL-1 interacts with Aβo in a complex with GluN2B-containing NMDA receptors. Additionally, the soluble fragment of NL-1 was used as a scavenger for Aβo. Field excitatory postsynaptic potentials indicate that fragments of NL-1 protect hippocampal neurons from the impairment induced by Aβo. To our knowledge, this is the first report of the interaction between this extracellular fragment of NL-1 and Aβo, strongly suggest that NL-1 facilitates the targeting of Aβo to the postsynaptic regions of excitatory synapses. Copyright © 2015 Elsevier Inc. All rights reserved.

Functionalization of ZnO nanoparticles by 3-mercaptopropionic acid for aqueous curcumin delivery: Synthesis, characterization, and anticancer assessment.

PubMed

Ghaffari, Seyed-Behnam; Sarrafzadeh, Mohammad-Hossein; Fakhroueian, Zahra; Shahriari, Shadab; Khorramizadeh, M Reza

2017-10-01

Inherent biocompatibility and stability of zinc oxide nanoparticles (ZnO-NPs) and their biomedical potentials make them an emerging candidate for drug delivery. The aim of this study was to develop and assess a simple procedure for surface functionalization of ZnO-NPs by 3-mercaptopropionic acid (MPA) for water-soluble curcumin delivery. Carboxyl-terminated ZnO nanoparticles were successfully made using ZnCl 2 and NaOH in the presence of MPA. The functional groups were activated by 1,1'-carbonyldiimidazole (CDI) and the curcumin bonding was carried out at room temperature for 24h. The core-shell nanocomposite had a significant better solubility versus free curcumin, as characterized by XRD, FTIR, UV-Vis spectrophotometry, DLS, and TEM, p<0.005. In addition, MTT cytotoxicity assessment on MDA-MB-231 breast cancer cells revealed a drop of IC 50 values from 5μg/mL to 3.3μg/mL for free curcumin and ZnO-MPA-curcumin complex, respectively. This result showed an augmented cancer-inhibitory effect of nanoconjugate complex. In conclusion, the presented improved solubility and elevated functionality of novel ZnO-MPA-curcumin nanoformula is promising, and could be considered for new therapeutic endeavors. Copyright © 2017 Elsevier B.V. All rights reserved.

Follicular Dendritic Cells Retain Infectious HIV in Cycling Endosomes

PubMed Central

Heesters, Balthasar A.; Lindqvist, Madelene; Vagefi, Parsia A.; Scully, Eileen P.; Schildberg, Frank A.; Altfeld, Marcus; Walker, Bruce D.; Kaufmann, Daniel E.; Carroll, Michael C.

2015-01-01

Despite the success of antiretroviral therapy (ART), it does not cure Human Immunodeficiency Virus (HIV) and discontinuation results in viral rebound. Follicular dendritic cells (FDC) are in direct contact with CD4+ T cells and they retain intact antigen for prolonged periods. We found that human FDC isolated from patients on ART retain infectious HIV within a non-degradative cycling compartment and transmit infectious virus to uninfected CD4 T cells in vitro. Importantly, treatment of the HIV+ FDC with a soluble complement receptor 2 purges the FDC of HIV virions and prevents viral transmission in vitro. Our results provide an explanation for how FDC can retain infectious HIV for extended periods and suggest a therapeutic strategy to achieve cure in HIV-infected humans. PMID:26623655

Research of home energy management system based on technology of PLC and ZigBee

NASA Astrophysics Data System (ADS)

Wei, Qi; Shen, Jiaojiao

2015-12-01

In view of the problem of saving effectively energy and energy management in home, this paper designs a home energy intelligent control system based on power line carrier communication and wireless ZigBee sensor networks. The system is based on ARM controller, power line carrier communication and wireless ZigBee sensor network as the terminal communication mode, and realizes the centralized and intelligent control of home appliances. Through the combination of these two technologies, the advantages of the two technologies complement each other, and provide a feasible plan for the construction of energy-efficient, intelligent home energy management system.

Cloning, expression and functional characterization of Schizosaccharomyces pombe TFIIB.

PubMed

Tamayo, Evelyn; Maldonado, Edio

2002-09-27

The transcription factor TFIIB has been identified and cloned from the yeast Schizosaccharomyces pombe. The cloned polypeptide is highly homologous to human TFIIB and to Saccharomyces cerevisiae TFIIB. S. pombe TFIIB is a 340-amino-acid-long protein and it possesses a repeated motif of 75 amino acids near the carboxy-terminal region. The purified recombinant protein is able to bind to the TBP-DNA promoter complex in gel retardation experiments. Recombinant S. pombe TFIIB is active in in vitro transcription assays, since it can complement the transcription activity of a S. pombe cell extract in which TFIIB was depleted by using antibodies.

DNA Sequence Analysis of a Complementary DNA for Cold-Regulated Arabidopsis Gene cor15 and Characterization of the COR 15 Polypeptide 1

PubMed Central

Lin, Chentao; Thomashow, Michael F.

1992-01-01

Previous studies have indicated that changes in gene expression occur in Arabidopsis thaliana L. (Heyn) during cold acclimation and that certain of the cor (cold-regulated) genes encode polypeptides that share the unusual property of remaining soluble upon boiling in aqueous solution. Here, we identify a cDNA clone for a cold-regulated gene encoding one of the “boiling-stable” polypeptides, COR15. DNA sequence analysis indicated that the gene, designated cor15, encodes a 14.7-kilodalton hydrophilic polypeptide having an N-terminal amino acid sequence that closely resembles transit peptides that target proteins to the stromal compartment of chloroplasts. Immunological studies indicated that COR15 is processed in vivo and that the mature polypeptide, COR 15m, is present in the soluble fraction of chloroplasts. Possible functions of COR 15m are discussed. ImagesFigure 1Figure 4Figure 5Figure 6Figure 7 PMID:16668917

A Major Binding Protein for Leukemia Inhibitory Factor in Normal Mouse Serum: Identification as a Soluble Form of the Cellular Receptor

NASA Astrophysics Data System (ADS)

Layton, Meredith J.; Cross, Bronwyn A.; Metcalf, Donald; Ward, Larry D.; Simpson, Richard J.; Nicola, Nicos A.

1992-09-01

A protein that specifically binds leukemia inhibitory factor (LIF) has been isolated from normal mouse serum by using four successive fractionation steps: chromatography on a LIF affinity matrix, anion-exchange chromatography, size-exclusion chromatography, and preparative native gel electrophoresis. The purified LIF-binding protein (LBP) is a glycoprotein with an apparent molecular mass of 90 kDa that specifically binds 125I-labeled murine LIF with an affinity comparable to that of the low-affinity cellular LIF receptor (K_d = 600 pM). N-terminal sequencing has identified this protein as a soluble truncated form of the α chain of the cellular LIF receptor. LBP is present in normal mouse serum at high levels (1 μg/ml) and these levels are elevated in pregnant mice and reduced in neonatal mice. Since normal serum concentrations of LBP can block the biological actions of LIF in culture, LBP may serve as an inhibitor of the systemic effects of locally produced LIF.

Gum and deposit formation in diesel fuels. Final report, 1984-1988

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mayo, F.R.; Mill, T.

1988-05-15

The authors examined two aspects of the stability of diesel fuels in storage: the formation of sediments in suspension, which subsequently clog filters, and the formation of soluble gum, which passes the filters but then forms hard deposits on hot engine parts. Research on fuel stability at SRI during the last 6 years has shown that soluble gum appears first on storage, and then part of it grows into sediment. If the oxidation mixture is agitated gently, the precipitating gum grows on the surface gum, and no loose sediment is formed. Three mechanisms of gum formation were distinguished: (1) amore » process intimately associated with chain propagation and termination during oxidation, (2) a coupling of fuel molecules by decomposing peroxides in the absence of oxygen, and (3) a condensation of naphthols and aldehydes from the oxidation of alkylnaphthalenes. The polymeric oxidation products from a JP-8 fuel are shown to be largely responsible for deposits in the Jet Fuel Thermal Oxidation Tester (JFTOT).« less

Structure-Based Discovery of Nonpeptide Allatostatin Analogues for Pest Control.

PubMed

Huang, Shan-Shan; Chen, Shan-Shan; Zhang, Hong-Ling; Yang, Han; Yang, Hui-Juan; Ren, Yu-Jie; Kai, Zhen-Peng

2018-04-11

FGLamide allatostatins (ASTs) are regarded as possible insecticide candidates, although their lack of in vivo effects, rapid degradation, poor water solubility, and high production costs preclude their practical use in pest control. In contrast to previous research, the C-terminal tripeptide (FGLa) was selected as the lead compound in this study. Five nonpeptide AST analogues (2-amino-1-[3-oxo-3-(substituted-anilino)propyl]pyridinium nitrate derivatives) were designed on the basis of the structure-activity relationship and docking results of FGLa. All of the nonpeptide analogues (S1-S5) were more potent against juvenile-hormone (JH) biosynthesis than the lead compound. They significantly inhibited the biosynthesis of JH in vivo following injection. A pest-control application demonstrated that S1 and S3 have larvicidal effects following oral administration (the IC 50 values were 0.020 and 0.0016 mg/g, respectively). The good oral toxicities and excellent water solubilities of S1 and S3 suggest that they have considerable potential as insecticides for pest management.

Microbial expression of alkaloid biosynthetic enzymes for characterization of their properties.

PubMed

Minami, Hiromichi; Ikezawa, Nobuhiro; Sato, Fumihiko

2010-01-01

A wide variety of secondary metabolites are produced in higher plants. These metabolites are synthesized in specific organs/cells at certain developmental stages and/or under specific environmental conditions. Since these biosynthetic activities are rather restricted and difficult to detect, the biochemical characterization of biosynthetic enzymes involved in secondary metabolism has been limited compared to those involved in primary metabolism. Recently, however, progress in tissue culture and molecular biology has made it easier to study biosynthetic enzymes. Here we describe protocols for expressing some biosynthetic enzymes in Escherichia coli expression systems, since this system is both efficient and cost-effective. First, we describe a standard system for expressing biosynthetic enzymes as a soluble protein under the T7 promoter of the pET expression system in E. coli. In addition, the successful expression of cytochrome P450 in E. coli in an active soluble form with N-terminal modification is discussed, since P450 is the critical enzyme in secondary metabolite biosynthesis.

Molecular cloning and sequence analysis of the gene coding for the 57kDa soluble antigen of the salmonid fish pathogen Renibacterium salmoninarum

USGS Publications Warehouse

Chien, Maw-Sheng; Gilbert , Teresa L.; Huang, Chienjin; Landolt, Marsha L.; O'Hara, Patrick J.; Winton, James R.

1992-01-01

The complete sequence coding for the 57-kDa major soluble antigen of the salmonid fish pathogen, Renibacterium salmoninarum, was determined. The gene contained an opening reading frame of 1671 nucleotides coding for a protein of 557 amino acids with a calculated Mr value of 57190. The first 26 amino acids constituted a signal peptide. The deduced sequence for amino acid residues 27–61 was in agreement with the 35 N-terminal amino acid residues determined by microsequencing, suggesting the protein in synthesized as a 557-amino acid precursor and processed to produce a mature protein of Mr 54505. Two regions of the protein contained imperfect direct repeats. The first region contained two copies of an 81-residue repeat, the second contained five copies of an unrelated 25-residue repeat. Also, a perfect inverted repeat (including three in-frame UAA stop codons) was observed at the carboxyl-terminus of the gene.

A switch in disulfide linkage during minicollagen assembly in Hydra nematocysts.

PubMed

Engel, U; Pertz, O; Fauser, C; Engel, J; David, C N; Holstein, T W

2001-06-15

The smallest known collagens with only 14 Gly-X-Y repeats referred to as minicollagens are the main constituents of the capsule wall of nematocysts. These are explosive organelles found in Hydra, jellyfish, corals and other Cnidaria. Minicollagen-1 of Hydra recombinantly expressed in mammalian 293 cells contains disulfide bonds within its N- and C-terminal Cys-rich domains but no interchain cross-links. It is soluble and self-associates through non-covalent interactions to form 25-nm-long trimeric helical rod-like molecules. We have used a polyclonal antibody prepared against the recombinant protein to follow the maturation of minicollagens from soluble precursors present in the endoplasmic reticulum and post-Golgi vacuoles to the disulfide-linked insoluble assembly form of the wall. The switch from intra- to intermolecular disulfide bonds is associated with 'hardening' of the capsule wall and provides an explanation for its high tensile strength and elasticity. The process is comparable to disulfide reshuffling between the NC1 domains of collagen IV in mammalian basement membranes.

Reduced graphene oxide composites with water soluble copolymers having tailored lower critical solution temperatures and unique tube-like structure

NASA Astrophysics Data System (ADS)

Namvari, Mina; Biswas, Chandra S.; Galluzzi, Massimiliano; Wang, Qiao; Du, Bing; Stadler, Florian J.

2017-03-01

Nanohybrids of graphene with water soluble polymer were synthesized using ‘grafting from’ method. GO, prepared by modified Hummers’ method, was first reacted with sodium azide. Alkyne-terminated RAFT-CTA was synthesized by reaction of propargyl alcohol and S-1-dodecyl-S’-(α,α‘-dimethyl-α”-acetic acid) trithiocarbonate. RAFT-CTA was grafted onto the GO sheets by facile click-reaction and subsequently, N-isopropylacrylamide (NIPAM) and N-ethyleacrylamide (NEAM) were polymerized on graphene sheets via RAFT polymerization method. The respective copolymers with different ratios were also prepared. The nanohybrids were characterized by FTIR, XRD, TGA, Raman, SEM, and AFM. Both SEM and AFM clearly showed rod-like structures for rGO-PNEAM. XRD showed a small peak at 2θ = 19.21°, corresponding to d-spacing ≈ 4.6 Å. In addition, the nanohybrids showed a very broad temperature range for the LCST in water between ca. 30 and 70 °C.

Role of Plant-Specific N-Terminal Domain of Maize CK2β1 Subunit in CK2β Functions and Holoenzyme Regulation

PubMed Central

Vélez-Bermúdez, Isabel C.; Carretero-Paulet, Lorenzo; Lumbreras, Victoria; Pagès, Montserrat

2011-01-01

Protein kinase CK2 is a highly pleiotropic Ser/Thr kinase ubiquituous in eukaryotic organisms. CK2 is organized as a heterotetrameric enzyme composed of two types of subunits: the catalytic (CK2α) and the regulatory (CK2β). The CK2β subunits enhance the stability, activity and specificity of the holoenzyme, but they can also perform functions independently of the CK2 tetramer. CK2β regulatory subunits in plants differ from their animal or yeast counterparts, since they present an additional specific N-terminal extension of about 90 aminoacids that shares no homology with any previously characterized functional domain. Sequence analysis of the N-terminal domain of land plant CK2β subunit sequences reveals its arrangement through short, conserved motifs, some of them including CK2 autophosphorylation sites. By using maize CK2β1 and a deleted version (ΔNCK2β1) lacking the N-terminal domain, we have demonstrated that CK2β1 is autophosphorylated within the N-terminal domain. Moreover, the holoenzyme composed with CK2α1/ΔNCK2β1 is able to phosphorylate different substrates more efficiently than CK2α1/CK2β1 or CK2α alone. Transient overexpression of CK2β1 and ΔNCK2β1 fused to GFP in different plant systems show that the presence of N-terminal domain enhances aggregation in nuclear speckles and stabilizes the protein against proteasome degradation. Finally, bimolecular fluorescence complementation (BiFC) assays show the nuclear and cytoplasmic location of the plant CK2 holoenzyme, in contrast to the individual CK2α/β subunits mainly observed in the nucleus. All together, our results support the hypothesis that the plant-specific N-terminal domain of CK2β subunits is involved in the down-regulation of the CK2 holoenzyme activity and in the stabilization of CK2β1 protein. In summary, the whole amount of data shown in this work suggests that this domain was acquired by plants for regulatory purposes. PMID:21789193

The EspF N-Terminal of Enterohemorrhagic Escherichia coli O157:H7 EDL933w Imparts Stronger Toxicity Effects on HT-29 Cells than the C-Terminal

PubMed Central

Wang, Xiangyu; Du, Yanli; Hua, Ying; Fu, Muqing; Niu, Cong; Zhang, Bao; Zhao, Wei; Zhang, Qiwei; Wan, Chengsong

2017-01-01

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 EspF is an important multifunctional protein that destroys the tight junctions of intestinal epithelial cells and promotes host cell apoptosis. However, its molecular mechanism remains elusive. We knocked out the espF sequence (747 bp, ΔespF), N-terminal sequence (219 bp, ΔespFN), and C-terminal sequence (528 bp, ΔespFC) separately using the pKD46-mediated λ Red homologous recombination system. Then, we built the corresponding complementation strains, namely, ΔespF/pespF, ΔespFN/pespFN, and ΔespFC/pespFC by overlap PCR, which were used in infecting HT-29 cells and BALB/C mice. The level of reactive oxygen species, cell apoptosis, mitochondrial trans-membrane potential, inflammatory factors, transepithelial electrical resistance (TER), and animal mortality were evaluated by DCFH-DA, double staining of Annexin V-FITC/PI, JC-1 staining, ELISA kit, and a mouse assay. The wild-type (WT), ΔespF, ΔespF/pespF, ΔespFC, ΔespFC/pespFC, ΔespFN, and ΔespFN/pespFN groups exhibited apoptotic rates of 68.3, 27.9, 64.9, 65.7, 73.4, 41.3, and 35.3% respectively, and mean TNF-α expression levels of 428 pg/mL, 342, 466, 446, 381, 383, and 374 pg/mL, respectively. In addition, the apoptotic rates and TNF-α levels of the WT, ΔespF/pespF, and ΔespFC were significantly higher than that of ΔespF, ΔespFN, ΔespFC/pespFC, and ΔespFN/pespFN group (p < 0.05). The N-terminal of EspF resulted in an increase in the number of apoptotic cells, TNF-α secretion, ROS generation, mitochondria apoptosis, and pathogenicity in BalB/c mice. In conclusion, the N-terminal domain of the Enterohemorrhagic E. coli O157:H7 EspF more strongly promotes apoptosis and inflammation than the C-terminal domain. PMID:28983470

The Role of Chaperone-subunit Usher Domain Interactions in the Mechanism of Bacterial Pilus Biogenesis Revealed by ESI-MS*

PubMed Central

Morrissey, Bethny; Leney, Aneika C.; Toste Rêgo, Ana; Phan, Gilles; Allen, William J.; Verger, Denis; Waksman, Gabriel; Ashcroft, Alison E.; Radford, Sheena E.

2012-01-01

The PapC usher is a β-barrel outer membrane protein essential for assembly and secretion of P pili that are required for adhesion of pathogenic E. coli, which cause the development of pyelonephritis. Multiple protein subunits form the P pilus, the highly specific assembly of which is coordinated by the usher. Despite a wealth of structural knowledge, how the usher catalyzes subunit polymerization and orchestrates a correct and functional order of subunit assembly remain unclear. Here, the ability of the soluble N-terminal (UsherN), C-terminal (UsherC2), and Plug (UsherP) domains of the usher to bind different chaperone-subunit (PapDPapX) complexes is investigated using noncovalent electrospray ionization mass spectrometry. The results reveal that each usher domain is able to bind all six PapDPapX complexes, consistent with an active role of all three usher domains in pilus biogenesis. Using collision induced dissociation, combined with competition binding experiments and dissection of the adhesin subunit, PapG, into separate pilin and adhesin domains, the results reveal why PapG has a uniquely high affinity for the usher, which is consistent with this subunit always being displayed at the pilus tip. In addition, we show how the different soluble usher domains cooperate to coordinate and control efficient pilus assembly at the usher platform. As well as providing new information about the protein-protein interactions that determine pilus biogenesis, the results highlight the power of noncovalent MS to interrogate biological mechanisms, especially in complex mixtures of species. PMID:22371487

The role of chaperone-subunit usher domain interactions in the mechanism of bacterial pilus biogenesis revealed by ESI-MS.

PubMed

Morrissey, Bethny; Leney, Aneika C; Toste Rêgo, Ana; Phan, Gilles; Allen, William J; Verger, Denis; Waksman, Gabriel; Ashcroft, Alison E; Radford, Sheena E

2012-07-01

The PapC usher is a β-barrel outer membrane protein essential for assembly and secretion of P pili that are required for adhesion of pathogenic E. coli, which cause the development of pyelonephritis. Multiple protein subunits form the P pilus, the highly specific assembly of which is coordinated by the usher. Despite a wealth of structural knowledge, how the usher catalyzes subunit polymerization and orchestrates a correct and functional order of subunit assembly remain unclear. Here, the ability of the soluble N-terminal (UsherN), C-terminal (UsherC2), and Plug (UsherP) domains of the usher to bind different chaperone-subunit (PapDPapX) complexes is investigated using noncovalent electrospray ionization mass spectrometry. The results reveal that each usher domain is able to bind all six PapDPapX complexes, consistent with an active role of all three usher domains in pilus biogenesis. Using collision induced dissociation, combined with competition binding experiments and dissection of the adhesin subunit, PapG, into separate pilin and adhesin domains, the results reveal why PapG has a uniquely high affinity for the usher, which is consistent with this subunit always being displayed at the pilus tip. In addition, we show how the different soluble usher domains cooperate to coordinate and control efficient pilus assembly at the usher platform. As well as providing new information about the protein-protein interactions that determine pilus biogenesis, the results highlight the power of noncovalent MS to interrogate biological mechanisms, especially in complex mixtures of species.

The monoclonal antibody that recognizes an epitope in the C-terminal region of the fibrinogen alpha-chain reacts with soluble fibrin and fibrin monomer generated by thrombin but not with those formed as plasmin degradation products.

PubMed

Suzuki, Akiko; Ebinuma, Hiroyuki; Matsuo, Masanao; Miyazaki, Osamu; Yago, Hirokazu

2007-01-01

The presence of soluble fibrin (SF) provides evidence of thrombin activation in the blood; therefore, SF is a useful marker for diagnosing blood coagulation diseases such as disseminated intravascular coagulation (DIC). The antibody that specifically detects SF could be a useful tool for diagnosing thrombotic diseases. By using an acid-solubilized desAA-FM (fibrin monomer) as an immunogen, we developed a monoclonal antibody, namely J2-23, which specifically reacts with SF and FM. We examined the specificity of J2-23 by ELISA and immunoblotting and confirmed the reactivity of J2-23 with SF and FM by gel filtration. J2-23 specifically reacted with SF, but not with fibrinogen or plasmic fibrinogen-derived Fbg-X, Fbg-Y, Fbg-E, and D; thrombin-treated Fbn-X, Fbn-Y, and Fbn-E; and plasmic cross-linked fibrin (DD, XDP). The epitope recognized by J2-23 was located within the Aalpha 502-521 region on the C-terminal of the fibrinogen alpha-chain. The reactivity of J2-23 disappeared following the action of the fibrinolytic enzyme plasmin. Furthermore, J2-23 reacted not only with SF but also with FM in plasma from DIC patients. This indicated that J2-23 specifically detected coagulation without reflecting the plasmin action. We demonstrated the potential of J2-23 as a useful antibody for detecting SF for diagnosing blood coagulation.

Amino Acid Substitutions of Coiled-Coil Protein Tpr Abrogate Anchorage to the Nuclear Pore Complex but Not Parallel, In-Register Homodimerization

PubMed Central

Hase, Manuela E.; Kuznetsov, Nikolai V.; Cordes, Volker C.

2001-01-01

Tpr is a protein component of nuclear pore complex (NPC)-attached intranuclear filaments. Secondary structure predictions suggest a bipartite structure, with a large N-terminal domain dominated by heptad repeats (HRs) typical for coiled-coil–forming proteins. Proposed functions for Tpr have included roles as a homo- or heteropolymeric architectural element of the nuclear interior. To gain insight into Tpr's ultrastructural properties, we have studied recombinant Tpr segments by circular dichroism spectroscopy, chemical cross-linking, and rotary shadowing electron microscopy. We show that polypeptides of the N-terminal domain homodimerize in vitro and represent α-helical molecules of extended rod-like shape. With the use of a yeast two-hybrid approach, arrangement of the coiled-coil is found to be in parallel and in register. To clarify whether Tpr can self-assemble further into homopolymeric filaments, the full-length protein and deletion mutants were overexpressed in human cells and then analyzed by confocal immunofluorescence microscopy, cell fractionation, and immuno-electron microscopy. Surplus Tpr, which does not bind to the NPC, remains in a soluble state of ∼7.5 S and occasionally forms aggregates of entangled molecules but neither self-assembles into extended linear filaments nor stably binds to other intranuclear structures. Binding to the NPC is shown to depend on the integrity of individual HRs; amino acid substitutions within these HRs abrogate NPC binding and render the protein soluble but do not abolish Tpr's general ability to homodimerize. Possible contributions of Tpr to the structural organization of the nuclear periphery in somatic cells are discussed. PMID:11514627

Protein trans-splicing on an M13 bacteriophage: towards directed evolution of a semisynthetic split intein by phage display.

PubMed

Garbe, Daniel; Thiel, Ilka V; Mootz, Henning D

2010-10-01

Split inteins link their fused peptide or protein sequences with a peptide bond in an autocatalytic reaction called protein trans-splicing. This reaction is becoming increasingly important for a variety of applications in protein semisynthesis, polypeptide circularisation, construction of biosensors, or segmental isotopic labelling of proteins. However, split inteins exhibit greatly varying solubility, efficiency and tolerance towards the nature of the fused sequences as well as reaction conditions. We envisioned that phage display as an in vitro selection technique would provide a powerful tool for the directed evolution of split inteins with improved properties. As a first step towards this goal, we show that presentation of active split inteins on an M13 bacteriophage is feasible. Two different C-terminal intein fragments of the Ssp DnaB intein, artificially split at amino acid positions 104 and 11, were encoded in a phagemid vector in fusion to a truncated gpIII protein. For efficient production of hybrid phages, the presence of a soluble domain tag at their N-termini was necessary. Immunoblot analysis revealed that the hybrid phages supported protein trans-splicing with a protein or a synthetic peptide, respectively, containing the complementary intein fragment. Incorporation of biotin or desthiobiotin by this reaction provides a straightforward strategy for future enrichment of desired mutants from randomised libraries of the C-terminal intein fragments on streptavidin beads. Protein semisynthesis on a phage could also be exploited for the selection of chemically modified proteins with unique properties. © 2010 European Peptide Society and John Wiley & Sons, Ltd.

Constitutive Endocytosis of VEGFR2 Protects the Receptor against Shedding.

PubMed

Basagiannis, Dimitris; Christoforidis, Savvas

2016-08-05

VEGFR2 plays a fundamental role in blood vessel formation and in life threatening diseases, such as cancer angiogenesis and cardiovascular disorders. Although inactive growth factor receptors are mainly localized at the plasma membrane, VEGFR2 undergoes constitutive endocytosis (in the absence of ligand) and recycling. Intriguingly, the significance of these futile transport cycles of VEGFR2 remains unclear. Here we found that, unexpectedly, the function of constitutive endocytosis of VEGFR2 is to protect the receptor against plasma membrane cleavage (shedding), thereby preserving the functional state of the receptor until the time of activation by VEGF. Inhibition of constitutive endocytosis of VEGFR2, by interference with the function of clathrin, dynamin, or Rab5, increases dramatically the cleavage/shedding of VEGFR2. Shedding of VEGFR2 produces an N-terminal soluble fragment (100 kDa, s100), which is released in the extracellular space, and a residual C-terminal part (130 kDa, p130) that remains integrated at the plasma membrane. The released soluble fragment (s100) co-immunoprecipitates with VEGF, in line with the topology of the VEGF-binding domain at the N terminus of VEGFR2. Increased shedding of VEGFR2 (via inhibition of constitutive endocytosis) results in reduced response to VEGF, consistently with the loss of the VEGF-binding domain from the membrane remnant of VEGFR2. These data suggest that constitutive internalization of VEGFR2 protects the receptor against shedding and provides evidence for an unprecedented mechanism via which endocytosis can regulate the fate and activity of growth factor receptors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

PAMAM dendrimers as a carbamazepine delivery system for neurodegenerative diseases: A biophysical and nanotoxicological characterization.

PubMed

Igartúa, Daniela E; Martinez, Carolina S; Temprana, C Facundo; Alonso, Silvia Del V; Prieto, M Jimena

2018-06-10

Carbamazepine (CBZ) is an antiepileptic drug, which also could be used in the treatment of neurodegenerative diseases, such as the Alzheimer's disease. However, its use has been limited due to its low solubility, inefficient pharmacokinetic profiles, and multiple side effects. PAMAM dendrimers, ethylenediamine core, generation 4.0 (amine terminal groups) and 4.5 (carboxylate terminal groups) (DG4.0 and DG4.5 respectively) are polymers that can increase drug solubility through complexation. Thus, the aim of this work was to obtain and characterize complexes between CBZ and dendrimers. Both DG4.0 and DG4.5 allowed the incorporation of ∼20 molecules of CBZ per dendrimer, into their hydrophobic pockets. DG4.0-CBZ and DG4.5-CBZ complexes were found to be stable for 90 days at 37 °C and resistant to a lyophilization process, presenting controlled drug release. Also, the complexes nanotoxicity was tested ex vivo (human red blood cells), in vitro (N2a cell line), and in vivo (zebrafish). No hemolytic effect was observed in the ex vivo model. As regards in vitro toxicity, the DG4.5-CBZ complexes significantly reduced the toxicity caused by the free drug. Moreover, the DG4.5-CBZ did not cause neurotoxicity or cardiotoxicity in zebrafish larvae. In conclusion, a stable and biocompatible drug delivery system based on the DG4.5 capable of complex the CBZ has been developed. This achievement highlights the advantages of using negatively charged dendrimers for nanomedicine. Copyright © 2018 Elsevier B.V. All rights reserved.

Constitutive Endocytosis of VEGFR2 Protects the Receptor against Shedding*

PubMed Central

Basagiannis, Dimitris; Christoforidis, Savvas

2016-01-01

VEGFR2 plays a fundamental role in blood vessel formation and in life threatening diseases, such as cancer angiogenesis and cardiovascular disorders. Although inactive growth factor receptors are mainly localized at the plasma membrane, VEGFR2 undergoes constitutive endocytosis (in the absence of ligand) and recycling. Intriguingly, the significance of these futile transport cycles of VEGFR2 remains unclear. Here we found that, unexpectedly, the function of constitutive endocytosis of VEGFR2 is to protect the receptor against plasma membrane cleavage (shedding), thereby preserving the functional state of the receptor until the time of activation by VEGF. Inhibition of constitutive endocytosis of VEGFR2, by interference with the function of clathrin, dynamin, or Rab5, increases dramatically the cleavage/shedding of VEGFR2. Shedding of VEGFR2 produces an N-terminal soluble fragment (100 kDa, s100), which is released in the extracellular space, and a residual C-terminal part (130 kDa, p130) that remains integrated at the plasma membrane. The released soluble fragment (s100) co-immunoprecipitates with VEGF, in line with the topology of the VEGF-binding domain at the N terminus of VEGFR2. Increased shedding of VEGFR2 (via inhibition of constitutive endocytosis) results in reduced response to VEGF, consistently with the loss of the VEGF-binding domain from the membrane remnant of VEGFR2. These data suggest that constitutive internalization of VEGFR2 protects the receptor against shedding and provides evidence for an unprecedented mechanism via which endocytosis can regulate the fate and activity of growth factor receptors. PMID:27298320

The intrinsically disordered C-terminal region of Arabidopsis thaliana TCP8 transcription factor acts both as a transactivation and self-assembly domain.

PubMed

Valsecchi, Isabel; Guittard-Crilat, Emilie; Maldiney, Régis; Habricot, Yvette; Lignon, Sabrina; Lebrun, Régine; Miginiac, Emile; Ruelland, Eric; Jeannette, Emmanuelle; Lebreton, Sandrine

2013-09-01

TCPs are plant specific transcription factors with non-canonical basic helix-loop-helix domains. While Arabidopsis thaliana has 24 TCPs involved in cell proliferation and differentiation, their mode of action has not been fully elucidated. Using bioinformatic tools, we demonstrate that TCP transcription factors belong to the intrinsically disordered proteins (IDP) family and that disorder is higher in class I TCPs than in class II TCPs. In particular, using bioinformatic and biochemical approaches, we have characterized TCP8, a class I TCP. TCP8 exhibits three intrinsically disordered regions (IDR) made of more than 50 consecutive residues, in which phosphorylable Ser residues are mainly clustered. Phosphorylation of Ser-211 that belongs to the central IDR was confirmed by mass spectrometry. Yeast two-hybrid assays also showed that the C-terminal IDR corresponds to a transactivation domain. Moreover, biochemical experiments demonstrated that TCP8 tends to oligomerize in dimers, trimers and higher-order multimers. Bimolecular fluorescence complementation (BiFC) experiments carried out on a truncated form of TCP8 lacking the C-terminal IDR indicated that it is effectively required for the pronounced self-assembly of TCP8. These data were reinforced by the prediction of a coiled coil domain in this IDR. The C-terminal IDR acts thus as an oligomerization domain and also a transactivation domain. Moreover, many Molecular Recognition Features (MoRFs) were predicted, indicating that TCP8 could interact with several partners to fulfill a fine regulation of transcription in response to various stimuli.

Can direct extracellular electron transfer occur in the absence of outer membrane cytochromes in Desulfovibrio vulgaris?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Elias, Dwayne A; Zane, Mr. Grant M.; Auer, Dr. Manfred

2010-01-01

Extracellular electron transfer has been investigated over several decades via forms of soluble electron transfer proteins that are exported for extracellular reoxidation. More recently, several organisms have been shown to reduce extracellular metals via the direct transfer of electron through appendages; also known as nanowires. They have been reported most predominantly in Shewanella and Geobacter. While the relevancy and composition of these structures in each genus has been debated, both possess outer membrane cytochrome complexes that could theoretically come into direct contact with solid phase oxidized metals. Members of the genus Desulfovibrio apparently have no such cytochromes although similar appendagesmore » are present, are electrically conductive, and are different from flagella. Upon U(VI)-reduction, the structures in Desulfovibrio become coated with U(IV). Deletion of flagellar genes did not alter soluble or amorphous Fe(III) or U(VI) reduction, or appendage appearance. Removal of the chromosomal pilA gene hampered amorphous Fe(III)-reduction by ca. 25%, but cells lacking the native plasmid, pDV1, reduced soluble Fe(III) and U(VI) at ca. 50% of the wild type rate while amorphous Fe(III)-reduction slowed to ca. 20% of the wild type rate. Appendages were present in all deletions as well as pDV1, except pilA. Gene complementation restored all activities and morphologies to wild type levels. This suggests that pilA encodes the structural component, whereas genes within pDV1 may provide the reactive members. How such appendages function without outer membrane cytochromes is under investigation.« less

Comparative c-type cytochrome expression analysis in Shewanella oneidensis strain MR-1 and Anaeromyxobacter dehalogenans strain 2CP-C grown with soluble and insoluble oxidised metal electron acceptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nissen, Silke; Liu, Xiaoxin; Chourey, Karuna

2012-01-01

The genomes of Shewanella oneidensis strain MR-1 and Anaeromyxobacter dehalogenans strain 2CP-C encode 40 and 69 putative c-type cytochrome genes, respectively. Deletion mutant and biochemical studies have assigned specific functions to a few c-type cytochromes involved in electron transfer to oxidised metals in Shewanella oneidensis strain MR-1. Although promising, the genetic approach is limited to gene deletions that produce a distinct phenotype, and organism for which a genetic system is available. To more comprehensively investigate and compare c-type cytochrome expression in Shewanella oneidensis strain MR-1 and Anaeromyxobacter dehalogenans strain 2CP-C, proteomic measurements were used to characterise lysates of cells grownmore » with soluble Fe(III) (as ferric citrate) and insoluble Mn(IV) (as MnO2) as electron acceptors. Strain MR-1 expressed 19 and 20, and strain 2CP-C expressed 27 and 25 c-type cytochromes when grown with Fe(III) and Mn(IV), respectively. The majority of c-type cytochromes (77% for strain MR-1 and 63% for strain 2CP-C) were expressed under both growth conditions; however, the analysis also revealed unique c-type cytochromes that were specifically expressed in cells grown with soluble Fe(III) or insoluble Mn(IV). Proteomic characterisation proved to be a promising approach for determining the c-type cytochrome complement expressed under different growth conditions, and will help elucidating the specific functions of more c-type cytochromes that are the basis for Shewanella and Anaeromyxobacter respiratory versatility.« less

1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose Binds to the N-terminal Metal Binding Region to Inhibit Amyloid β-protein Oligomer and Fibril Formation.

PubMed

de Almeida, Natália E C; Do, Thanh D; LaPointe, Nichole E; Tro, Michael; Feinstein, Stuart C; Shea, Joan-Emma; Bowers, Michael T

2017-09-01

The early oligomerization of amyloid β -protein (A β ) is a crucial step in the etiology of Alzheimer's disease (AD), in which soluble and highly neurotoxic oligomers are produced and accumulated inside neurons. In search of therapeutic solutions for AD treatment and prevention, potent inhibitors that remodel A β assembly and prevent neurotoxic oligomer formation offer a promising approach. In particular, several polyphenolic compounds have shown anti-aggregation properties and good efficacy on inhibiting oligomeric amyloid formation. 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose is a large polyphenol that has been shown to be effective at inhibiting aggregation of full-length A β 1-40 and A β 1-42 , but has the opposite effect on the C-terminal fragment A β 25-35 . Here, we use a combination of ion mobility coupled to mass spectrometry (IMS-MS), transmission electron microscopy (TEM) and molecular dynamics (MD) simulations to elucidate the inhibitory effect of PGG on aggregation of full-length A β 1-40 and A β 1-42 . We show that PGG interacts strongly with these two peptides, especially in their N-terminal metal binding regions, and suppresses the formation of A β 1-40 tetramer and A β 1-42 dodecamer. By exploring multiple facets of polyphenol-amyloid interactions, we provide a molecular basis for the opposing effects of PGG on full-length A β and its C-terminal fragments.

Paralogs of the C-Terminal Domain of the Cyanobacterial Orange Carotenoid Protein Are Carotenoid Donors to Helical Carotenoid Proteins1[OPEN

PubMed Central

Muzzopappa, Fernando; Wilson, Adjélé; Yogarajah, Vinosa; Cot, Sandrine; Perreau, François; Montigny, Cédric; Bourcier de Carbon, Céline

2017-01-01

The photoactive Orange Carotenoid Protein (OCP) photoprotects cyanobacteria cells by quenching singlet oxygen and excess excitation energy. Its N-terminal domain is the active part of the protein, and the C-terminal domain regulates the activity. Recently, the characteristics of a family of soluble carotenoid-binding proteins (Helical Carotenoid Proteins [HCPs]), paralogs of the N-terminal domain of OCP, were described. Bioinformatics studies also revealed the existence of genes coding for homologs of CTD. Here, we show that the latter genes encode carotenoid proteins (CTDHs). This family of proteins contains two subgroups with distinct characteristics. One CTDH of each clade was further characterized, and they proved to be very good singlet oxygen quenchers. When synthesized in Escherichia coli or Synechocystis PCC 6803, CTDHs formed dimers that share a carotenoid molecule and are able to transfer their carotenoid to apo-HCPs and apo-OCP. The CTDHs from clade 2 have a cysteine in position 103. A disulfide bond is easily formed between the monomers of the dimer preventing carotenoid transfer. This suggests that the transfer of the carotenoid could be redox regulated in clade 2 CTDH. We also demonstrate here that apo-OCPs and apo-CTDHs are able to take the carotenoid directly from membranes, while HCPs are unable to do so. HCPs need the presence of CTDH to become holo-proteins. We propose that, in cyanobacteria, the CTDHs are carotenoid donors to HCPs. PMID:28935842

Celecoxib restores angiogenic factor expression at the maternal-fetal interface in the BPH/5 mouse model of preeclampsia.

PubMed

Reijnders, Dorien; Liu, Chin-Chi; Xu, Xinjing; Zhao, Anna M; Olson, Kelsey N; Butler, Scott D; Douglas, Nataki C; Sones, Jenny L

2018-05-01

Preeclampsia (PE), a hypertensive disease of pregnancy, is a leading cause of fetal and maternal morbidity/mortality. Early angiogenic and inflammatory disturbances within the placenta are thought to underlie the development of the maternal PE syndrome and poor pregnancy outcomes. However, the exact etiology remains largely unknown. Here, we use the BPH/5 mouse model of PE to elucidate the way in which inflammation early in pregnancy contributes to abnormal expression of angiogenic factors at the maternal-fetal interface. We have previously described improvement in maternal hypertension and fetal growth restriction in this model after treatment with the anti-inflammatory cyclooxygenase-2 (Cox2) specific inhibitor celecoxib. To further characterize the mechanisms by which celecoxib improves poor pregnancy outcomes in BPH/5 mice, we determined expression of angiogenic factors and complement pathway components after celecoxib. In BPH/5 implantation sites there was increased hypoxia inducible factor-1α ( Hif1α), heme oxygenase-1 ( Ho-1), and stem cell factor ( Scf) mRNA concomitant with elevated prostaglandin synthase 2 ( Ptgs2), encoding Cox2, and elevated VEGF protein. Angiopoietin 1 ( Ang1), tunica interna endothelial cell kinase-2 receptor ( Tie2), complement factor 3 ( C3), and complement factor B ( CfB) were increased in midgestation BPH/5 placentae. Whereas BPH/5 expression levels of VEGF, Ang1, and Tie2 normalized after celecoxib, placental C3 and CfB mRNA remained unchanged. However, celecoxib did reduce the pregnancy-specific circulating soluble fms-like tyrosine kinase-1 (sFlt-1) rise in BPH/5 mice at midgestation. These data show that elevated Cox2 during implantation contributes to placental angiogenic factor imbalances in the BPH/5 mouse model of PE.

Hemocompatibility evaluation in vitro of methoxy polyethyleneglycol-polycaprolactone copolymer solutions.

PubMed

Hu, Qian; Zhang, Yi; Wang, Changyong; Xu, Jiake; Wu, Jianping; Liu, Zonghua; Xue, Wei

2016-03-01

Amphiphilic block copolymer methoxy polyethyleneglycol-polycaprolactone (mPEG-PCL) has attracted interest in the biomedical field, due to its water solubility and biodegradability. Nevertheless, the blood safety of mPEG-PCL copolymers has not been investigated in detail. Because mPEG-PCL copolymers introduced in vivo would inevitably interact with blood tissue, an investigation of possible interactions of mPEG-PCL with key blood components is crucial. We studied the effects of two mPEG-PCL copolymer solutions on blood coagulation, the morphology and lysis of human red blood cells (RBCs), the structure of plasma fibrinogen, complement activation, and platelet aggregation. We found that higher concentrations of the mPEG-PCL copolymers impaired blood clotting, and the copolymers had little impact on the morphology or lysis of RBCs. From the spectroscopy results, the copolymers affected the local microstructure of fibrinogen. The copolymers significantly activated the complement system in a concentration-dependent way. At higher concentrations, the copolymers impaired platelet aggregation, which may have been mediated by an inhibition of the arachidonic acid pathway. These findings provide important information that may be useful for the molecular design and biomedical applications of mPEG-PCL copolymers. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 802-812, 2016. © 2016 Wiley Periodicals, Inc.

Therapeutic inhibition of the complement system. Y2K update.

PubMed

Asghar, S S; Pasch, M C

2000-09-01

Activation of complement is an essential part of the mechanism of pathogenesis of a large number of human diseases; its inhibition by pharmacological means is likely to suppress disease processes in complement mediated diseases. From this point of view low molecular weight synthetic inhibitors of complement are being developed and high molecular weight natural inhibitors of human origin present in plasma or embedded in cell membrane are being purified or produced in their recombinant forms. This review is concerned with high molecular weight inhibitors, some of which are already in clinical use but may be efficacious in many other diseases in which they have not yet been tried. C1-esterase inhibitor (C1-INH) concentrate prepared from human plasma is being successfully used for the treatment of hereditary angioneurotic edema. Recently, C1-INH has been found to be consumed in severe inflammation and has been shown to exert beneficial effects in several inflammatory conditions such as human sepsis, post-operative myocardial dysfunction due to reperfusion injury, severe capillary leakage syndrome after bone marrow transplantation, reperfusion injury after lung transplantation, burn, and cytotoxicity caused by IL-2 therapy in cancer. Factor I has been used for the treatment of factor I deficiency. Recombinant soluble forms of membrane cofactor protein (MCP), and decay accelerating factor (DAF) have not yet been tried in humans but have been shown to be effective in immune complex mediate inflammation in animals. Organs of pigs transgenic for one or more of human membrane regulators of complement namely membrane cofactor protein (MCP), decay accelerating factor (DAF) or CD59, are being produced for transplantation into humans. They have been shown to be resistant to hyperacute rejection in non-human primates; acute vascular rejection is still a problem in their clinical use. It is hoped that these observations together with future developments will make xeno-transplantation in clinical practice a reality. Several recombinant variants of complement receptor 1 (CR1) have been produced. The most effective of these appears to be sCR1-SLe x, sCR1 part of which inhibits complement and carbohydrate Sle x moiety inhibits selectin mediated interactions of neutrophils and lymphocytes with endothelium. Although clinical trials of sCR1 in humans is eagerly awaited, several of the recombinant versions of sCR1 have been shown to suppress ischemia/reperfusion injury, thermal trauma, and immune complex mediated inflammation. They have also been shown to be effective in experimental models of systemic sclerosis, arthritis, myasthenia gravis, Guillain Barré syndrome and glomerulonephritis. Intravenous immunoglobulin, three of the most prominent properties of which are neutralization of autoantibody activity, suppression of autoantibody production and inhibition of complement activity, is being used in several diseases. These include autoimmune thrombocyopenic purpura, Kawasaki disease and several neurological diseases such as myasthenia gravis and Guillain Barre syndrome. In many uncontrolled small scale studies intravenous immunoglobulin has been shown to be effective in many immunological including dermatological diseases; controlled clinical trials in a large number of patients with these diseases is needed to establish the efficacy. It is hoped that in future therapeutic inhibition of complement will be one of the major approaches to combat many human diseases.

Premature terminator analysis sheds light on a hidden world of bacterial transcriptional attenuation.

PubMed

Naville, Magali; Gautheret, Daniel

2010-01-01

Bacterial transcription attenuation occurs through a variety of cis-regulatory elements that control gene expression in response to a wide range of signals. The signal-sensing structures in attenuators are so diverse and rapidly evolving that only a small fraction have been properly annotated and characterized to date. Here we apply a broad-spectrum detection tool in order to achieve a more complete view of the transcriptional attenuation complement of key bacterial species. Our protocol seeks gene families with an unusual frequency of 5' terminators found across multiple species. Many of the detected attenuators are part of annotated elements, such as riboswitches or T-boxes, which often operate through transcriptional attenuation. However, a significant fraction of candidates were not previously characterized in spite of their unmistakable footprint. We further characterized some of these new elements using sequence and secondary structure analysis. We also present elements that may control the expression of several non-homologous genes, suggesting co-transcription and response to common signals. An important class of such elements, which we called mobile attenuators, is provided by 3' terminators of insertion sequences or prophages that may be exapted as 5' regulators when inserted directly upstream of a cellular gene. We show here that attenuators involve a complex landscape of signal-detection structures spanning the entire bacterial domain. We discuss possible scenarios through which these diverse 5' regulatory structures may arise or evolve.

Ama1p-activated anaphase-promoting complex regulates the destruction of Cdc20p during meiosis II

PubMed Central

Tan, Grace S.; Magurno, Jennifer; Cooper, Katrina F.

2011-01-01

The execution of meiotic divisions in Saccharomyces cerevisiae is regulated by anaphase-promoting complex/cyclosome (APC/C)–mediated protein degradation. During meiosis, the APC/C is activated by association with Cdc20p or the meiosis-specific activator Ama1p. We present evidence that, as cells exit from meiosis II, APC/CAma1 mediates Cdc20p destruction. APC/CAma1 recognizes two degrons on Cdc20p, the destruction box and destruction degron, with either domain being sufficient to mediate Cdc20p destruction. Cdc20p does not need to associate with the APC/C to bind Ama1p or be destroyed. Coimmunoprecipitation analyses showed that the diverged amino-terminal region of Ama1p recognizes both Cdc20p and Clb1p, a previously identified substrate of APC/CAma1. Domain swap experiments revealed that the C-terminal WD region of Cdh1p, when fused to the N-terminal region of Ama1p, could direct most of Ama1p functions, although at a reduced level. In addition, this fusion protein cannot complement the spore wall defect in ama1Δ strains, indicating that substrate specificity is also derived from the WD repeat domain. These findings provide a mechanism to temporally down-regulate APC/CCdc20 activity as the cells complete meiosis II and form spores. PMID:21118994

Isolation and Characterization of a TERMINAL FLOWER Homolog and Its Correlation with Juvenility in Citrus

PubMed Central

Pillitteri, Lynn Jo; Lovatt, Carol J.; Walling, Linda L.

2004-01-01

TERMINAL FLOWER is a key regulator of floral timing in Arabidopsis and other herbaceous species. A homolog of this gene, CsTFL, was isolated from the hybrid perennial tree crop Washington navel orange (Citrus sinensis L. Osbeck). The deduced amino acid sequence of CsTFL was 65% identical to the Arabidopsis TFL1 protein. Wild-type Arabidopsis plants ectopically expressing CsTFL showed late-flowering phenotypes similar to those described for overexpression of Arabidopsis TFL1. In addition, the 35S:CsTFL transgene complemented the tfl1-2 mutant. The severity of the overexpression phenotypes correlated with the amount of CsTFL transcript that accumulated. Unlike many model systems that have been studied, C. sinensis maintains two distinguishable CsTFL alleles. CsTFL transcripts from either allele were not detected in adult vegetative tissues using reverse transcription-PCR, but CsTFL RNAs were detected in all floral organs. In addition, real-time PCR determined that juvenility in citrus was positively correlated with CsTFL transcript accumulation and negatively correlated with the floral-regulatory genes, LEAFY and APETALA1, RNA levels. PMID:15235113

Isoprenylation of the plant molecular chaperone ANJ1 facilitates membrane association and function at high temperature.

PubMed

Zhu, J K; Bressan, R A; Hasegawa, P M

1993-09-15

We demonstrate that ANJ1, a higher plant homolog of the bacterial molecular chaperone DnaJ, is a substrate in vitro for protein farnesyl- and geranylgeranyl-transferase activities present in cell extracts of the plant Atriplex nummularia and yeast Saccharomyces cerevisiae. Isoprenylation did not occur when cysteine was replaced by serine in the CAQQ motif at the carboxyl terminus of ANJ1, indicating that this sequence functions as a CaaX consensus sequence for polyisoprenylation (where C is cysteine, a is an aliphatic residue, and X is any amino acid residue). Substitution of leucine for the terminal glutamine did not result in the expected geranylgeranylation as occurs with mammalian proteins containing a carboxyl-terminal leucine. Unlike the wild-type ANJ1, neither of the proteins containing these amino acid substitutions could functionally complement the yeast temperature-sensitive mutant mas5. Farnesylation enhanced the association of ANJ1 with A. nummularia microsomal membranes. Electrophoretic mobility of ANJ1 from the plant indicated that the protein is isoprenylated in vivo.

Isoprenylation of the plant molecular chaperone ANJ1 facilitates membrane association and function at high temperature.

PubMed Central

Zhu, J K; Bressan, R A; Hasegawa, P M

1993-01-01

We demonstrate that ANJ1, a higher plant homolog of the bacterial molecular chaperone DnaJ, is a substrate in vitro for protein farnesyl- and geranylgeranyl-transferase activities present in cell extracts of the plant Atriplex nummularia and yeast Saccharomyces cerevisiae. Isoprenylation did not occur when cysteine was replaced by serine in the CAQQ motif at the carboxyl terminus of ANJ1, indicating that this sequence functions as a CaaX consensus sequence for polyisoprenylation (where C is cysteine, a is an aliphatic residue, and X is any amino acid residue). Substitution of leucine for the terminal glutamine did not result in the expected geranylgeranylation as occurs with mammalian proteins containing a carboxyl-terminal leucine. Unlike the wild-type ANJ1, neither of the proteins containing these amino acid substitutions could functionally complement the yeast temperature-sensitive mutant mas5. Farnesylation enhanced the association of ANJ1 with A. nummularia microsomal membranes. Electrophoretic mobility of ANJ1 from the plant indicated that the protein is isoprenylated in vivo. Images Fig. 1 Fig. 2 Fig. 3 Fig. 5 Fig. 6 Fig. 7 PMID:8378331

Crystal structure of the N-terminal domain of the secretin GspD from ETEC determined with the assistance of a nanobody

PubMed Central

Korotkov, Konstantin V.; Pardon, Els

2009-01-01

Summary Secretins are among the largest bacterial outer membrane proteins known. Here we report the crystal structure of the periplasmic N-terminal domain of GspD (peri-GspD) from the type 2 secretion system (T2SS) secretin in complex with a “nanobody”, the VHH domain of a “heavy-chain” camelid antibody. Two different crystal forms contained the same compact peri-GspD:nanobody heterotetramer. The nanobody contacts peri-GspD mainly via CDR3 and framework residues. The peri-GspD structure reveals three subdomains with the second and third subdomains exhibiting the KH-fold which also occurs in ring-forming proteins of the type 3 secretion system. The first subdomain of GspD is related to domains in phage tail proteins and outer membrane TonB-dependent receptors. A dodecameric peri-GspD model is proposed in which a solvent-accessible β-strand of the first subdomain interacts with secreted proteins and/or T2SS partner proteins by β-strand complementation. PMID:19217396

Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8)

PubMed Central

Russo, James J.; Bohenzky, Roy A.; Chien, Ming-Cheng; Chen, Jing; Yan, Ming; Maddalena, Dawn; Parry, J. Preston; Peruzzi, Daniela; Edelman, Isidore S.; Chang, Yuan; Moore, Patrick S.

1996-01-01

The genome of the Kaposi sarcoma-associated herpesvirus (KSHV or HHV8) was mapped with cosmid and phage genomic libraries from the BC-1 cell line. Its nucleotide sequence was determined except for a 3-kb region at the right end of the genome that was refractory to cloning. The BC-1 KSHV genome consists of a 140.5-kb-long unique coding region flanked by multiple G+C-rich 801-bp terminal repeat sequences. A genomic duplication that apparently arose in the parental tumor is present in this cell culture-derived strain. At least 81 ORFs, including 66 with homology to herpesvirus saimiri ORFs, and 5 internal repeat regions are present in the long unique region. The virus encodes homologs to complement-binding proteins, three cytokines (two macrophage inflammatory proteins and interleukin 6), dihydrofolate reductase, bcl-2, interferon regulatory factors, interleukin 8 receptor, neural cell adhesion molecule-like adhesin, and a D-type cyclin, as well as viral structural and metabolic proteins. Terminal repeat analysis of virus DNA from a KS lesion suggests a monoclonal expansion of KSHV in the KS tumor. PMID:8962146

The crystal structure of the Hsp90 co-chaperone Cpr7 from Saccharomyces cerevisiae.

PubMed

Qiu, Yu; Ge, Qiangqiang; Wang, Mingxing; Lv, Hui; Ebrahimi, Mohammad; Niu, Liwen; Teng, Maikun; Li, Xu

2017-03-01

The versatility of Hsp90 can be attributed to the variety of co-chaperone proteins that modulate the role of Hsp90 in many cellular processes. As a co-chaperone of Hsp90, Cpr7 is essential for accelerating the cell growth in an Hsp90-containing trimeric complex. Here, we report the crystal structure of Cpr7 at a resolution of 1.8Å. It consists of an N-terminal PPI domain and a C-terminal TPR domain, and exhibits a U-shape conformation. Our studies revealed the aggregation state of Cpr7 in solution and the interaction properties between Cpr7 and the MEEVD sequence from the C-terminus of Hsp90. In addition, the structure and sequence analysis between Cpr7 and homologues revealed the structure basis both for the function differences between Cpr6 and Cpr7 and the functional complements between Cns1 and Cpr7. Our studies facilitate the understanding of Cpr7 and provide decent insights into the molecular mechanisms of the Hsp90 co-chaperone pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

Bassoon-disruption slows vesicle replenishment and induces homeostatic plasticity at a CNS synapse

PubMed Central

Mendoza Schulz, Alejandro; Jing, Zhizi; María Sánchez Caro, Juan; Wetzel, Friederike; Dresbach, Thomas; Strenzke, Nicola; Wichmann, Carolin; Moser, Tobias

2014-01-01

Endbulb of Held terminals of auditory nerve fibers (ANF) transmit auditory information at hundreds per second to bushy cells (BCs) in the anteroventral cochlear nucleus (AVCN). Here, we studied the structure and function of endbulb synapses in mice that lack the presynaptic scaffold bassoon and exhibit reduced ANF input into the AVCN. Endbulb terminals and active zones were normal in number and vesicle complement. Postsynaptic densities, quantal size and vesicular release probability were increased while vesicle replenishment and the standing pool of readily releasable vesicles were reduced. These opposing effects canceled each other out for the first evoked EPSC, which showed unaltered amplitude. We propose that ANF activity deprivation drives homeostatic plasticity in the AVCN involving synaptic upscaling and increased intrinsic BC excitability. In vivo recordings from individual mutant BCs demonstrated a slightly improved response at sound onset compared to ANF, likely reflecting the combined effects of ANF convergence and homeostatic plasticity. Further, we conclude that bassoon promotes vesicular replenishment and, consequently, a large standing pool of readily releasable synaptic vesicles at the endbulb synapse. PMID:24442636

Role of a disulfide-bonded peptide loop within human complement C9 in the species-selectivity of complement inhibitor CD59.

PubMed

Husler, T; Lockert, D H; Sims, P J

1996-03-12

CD59 antigen is a membrane glycoprotein that inhibits the activity of the C9 component of the C5b-9 membrane attack complex (MAC), thereby protecting human cells from lysis by human complement. The complement-inhibitory activity of CD59 is species-selective, and is most effective toward C9 derived from human or other primate plasma. The species-selective activity of CD59 was recently used to map the segment of human C9 that is recognized by this MAC inhibitor, using recombinant rabbit/human C9 chimeras that retain lytic function within the MAC [Husler, T., Lockert, D. H., Kaufman, K. M., Sodetz, J. M., & Sims, P. J. (1995) J. Biol. Chem. 270,3483-3486]. These experiments suggested that the CD59 recognition domain was contained between residues 334 and 415 in human C9. By analyzing the species-selective lytic activity of recombinant C9 with chimeric substitutions internal to this segment, we now demonstrate that the site in human C9 uniquely recognized by CD59 is centered on those residues contained between C9 Cys359/Cys384, with an additional contribution by residues C-terminal to this segment. Consistent with its role as a CD59 recognition domain, CD59 specifically bound a human C9-derived peptide corresponding to residues 359-384, and antibody (Fab) raised against this C9-derived peptide inhibited the lytic activity of human MAC. Mutant human C9 in which Ala was substituted for Cys359/384 was found to express normal lytic activity and to be fully inhibited by CD59. This suggests that the intrachain Cys359/Cys384 disulfide bond within C9 is not required to maintain the conformation of this segment of C9 for interaction with CD59.

A Triblock Copolymer Design Leads to Robust Hybrid Hydrogels for High-Performance Flexible Supercapacitors.

PubMed

Zhang, Guangzhao; Chen, Yunhua; Deng, Yonghong; Wang, Chaoyang

2017-10-18

We report here an intriguing hybrid conductive hydrogel as electrode for high-performance flexible supercapacitor. The key is using a rationally designed water-soluble ABA triblock copolymer (termed as IAOAI) containing a central poly(ethylene oxide) block (A) and terminal poly(acrylamide) (PAAm) block with aniline moieties randomly incorporated (B), which was synthesized by reversible additional fragment transfer polymerization. The subsequent copolymerization of aniline monomers with the terminated aniline moieties on the IAOAI polymer generates a three-dimensional cross-linking hybrid network. The hybrid hydrogel electrode demonstrates robust mechanical flexibility, remarkable electrochemical capacitance (919 F/g), and cyclic stability (90% capacitance retention after 1000 cycles). Moreover, the flexible supercapacitor based on this hybrid hydrogel electrode presents a large specific capacitance (187 F/g), superior to most reported conductive hydrogel-based supercapacitors. With the demonstrated additional favorable cyclic stability and excellent capacitive and rate performance, this hybrid hydrogel-based supercapacitor holds great promise for flexible energy-storage device.

N-terminal dual lipidation-coupled molecular targeting into the primary cilium.

PubMed

Kumeta, Masahiro; Panina, Yulia; Yamazaki, Hiroya; Takeyasu, Kunio; Yoshimura, Shige H

2018-06-13

The primary cilium functions as an "antenna" for cell signaling, studded with characteristic transmembrane receptors and soluble protein factors, raised above the cell surface. In contrast to the transmembrane proteins, targeting mechanisms of nontransmembrane ciliary proteins are poorly understood. We focused on a pathogenic mutation that abolishes ciliary localization of retinitis pigmentosa 2 protein and revealed a dual acylation-dependent ciliary targeting pathway. Short N-terminal sequences which contain myristoylation and palmitoylation sites are sufficient to target a marker protein into the cilium in a palmitoylation-dependent manner. A Golgi-localized palmitoyltransferase DHHC-21 was identified as the key enzyme controlling this targeting pathway. Rapid turnover of the targeted protein was ensured by cholesterol-dependent membrane fluidity, which balances highly and less-mobile populations of the molecules within the cilium. This targeting signal was found in a set of signal transduction molecules, suggesting a general role of this pathway in proper ciliary organization, and dysfunction in ciliary disorders. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

A dual affinity-tag strategy for the expression and purification of human linker histone H1.4 in Escherichia coli.

PubMed

Ryan, Daniel P; Tremethick, David J

2016-04-01

Linker histones are an abundant and critical component of the eukaryotic chromatin landscape. They play key roles in regulating the higher order structure of chromatin and many genetic processes. Higher eukaryotes possess a number of different linker histone subtypes and new data are consistently emerging that indicate these subtypes are functionally distinct. We were interested in studying one of the most abundant human linker histone subtypes, H1.4. We have produced recombinant full-length H1.4 in Escherichia coli. An N-terminal Glutathione-S-Transferase tag was used to promote soluble expression and was combined with a C-terminal hexahistidine tag to facilitate a simple non-denaturing two-step affinity chromatography procedure that results in highly pure full-length H1.4. The purified H1.4 was shown to be functional via in vitro chromatin assembly experiments and remains active after extended storage at -80 °C. Copyright © 2015 Elsevier Inc. All rights reserved.

Structural investigation of a novel heteropolysaccharide from the fruiting bodies of Boletus edulis.

PubMed

Zhang, An-qiang; Liu, Ye; Xiao, Nan-nan; Zhang, Yang; Sun, Pei-long

2014-03-01

A novel water-soluble heteropolysaccharide, BEPF1, was isolated from the fruiting bodies of Boletus edulis with boiling water extraction and purified by Sephacryl S-300, with a molecular weight of 1.08×10(4)Da. Sugar composition of BEPF1 showed that it was composed of l-fucose, d-mannose, d-glucose and d-galactose in the ratio of 0.21:0.23:1.17:1.00. Methylation analysis together with (1)H, (13)C and 2D NMR spectroscopy established that BEPF1 was consisted of α-d-(1→6)-galactopyranan backbone with a terminal of α-l-fucosyl unit on O-2 of the 2-d-(2→6)-galactosyl units, β-d-(1→6)-4-O-Me-glucopyranan and β-d-(1→6)-glucopyranan backbone with a terminal β-d-glucosyl unit and it also contained a minor of 2,6-β-d-Mannopyranan residues. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

Functional characterisation of ganglioside-induced differentiation-associated protein 1 as a glutathione transferase.

PubMed

Shield, Alison J; Murray, Tracy P; Board, Philip G

2006-09-08

Mutations in the ganglioside-induced differentiation-associated protein 1 (GDAP1) gene have been linked with Charcot-Marie-Tooth (CMT) disease. This protein, and its paralogue GDAP1L1, appear to be structurally related to the cytosolic glutathione S-transferases (GST) including an N-terminal thioredoxin fold domain with conserved active site residues. The specific function, of GDAP1 remains unknown. To further characterise their structure and function we purified recombinant human GDAP1 and GDAP1L1 proteins using bacterial expression and immobilised metal affinity chromatography. Like other cytosolic GSTs, GDAP1 protein has a dimeric structure. Although the full-length proteins were largely insoluble, the deletion of a proposed C-terminal transmembrane domain allowed the preparation of soluble protein. The purified proteins were assayed for glutathione-dependent activity against a library of 'prototypic' GST substrates. No evidence of glutathione-dependent activity or an ability to bind glutathione immobilised on agarose was found.

Purification and characterization of Phaseolus vulgaris alpha-D-galactosidase isozymes.

PubMed

Dhar, M; Mitra, M; Hata, J; Butnariu, O; Smith, D

1994-11-01

A highly purified preparation of alpha-D-galactosidase [E.C. 3.2.1.22] isozymes was obtained from Phaseolus vulgaris (pinto bean) seeds by extraction, salt precipitation, ion exchange, and affinity chromatography. The final preparation was homogeneous by SDS-PAGE but revealed isozymes of relative mass of 38.3 and 39.6 kDa. The N-terminal sequence for both isozymes was identical, LANGLAKT (one letter code for amino acids). Relative native molecular mass was estimated at 149.3 kDa by Sephacryl S-200 chromatography. Activity was unaffected by ionic strength at high enzyme concentrations, and was specific for alpha-D-galactoside conjugates. No protease or hemagglutinin activity was detected, and activity was stable at 4 degrees C. Studies with soluble oligosaccharides demonstrated high activity against the selected straight and branched-chain substrates. The enzyme was active against terminal alpha 1-3 galactosyl residues on human and rabbit erythrocyte membranes. Because of its activity against membrane glycoconjugates, these isozymes may have potential utility for modifying membrane epitopes on native erythrocytes.

Study on the fluorescence characteristics of carbon dots.

PubMed

Mao, Xiao-Jiao; Zheng, Hu-Zhi; Long, Yi-Juan; Du, Juan; Hao, Jian-Yu; Wang, Ling-Ling; Zhou, Dong-Bo

2010-02-01

Herein, we prepared water-soluble fluorescent carbon dots with diameter about 1.5 nm from cheap commercial lampblack. These fluorescent carbon nanoparticles are stable toward photobleaching and stable in water for more than half a year without fluorescence decrease. In order to improve its fluorescence properties, we passivated these nanoparticles with bisamino-terminated polyethylene glycol (PEG(1500 N)). Therefore, both fluorescence quantum yield and lifetime increased after this progress. In addition, the passivated carbon dots were more inert to solvent than the bare one and showed different responses to pH change. Copyright (c) 2009 Elsevier B.V. All rights reserved.

Isolation and Characterization of a Soluble NADPH-Dependent Fe(III) Reductase from Geobacter sulfurreducens

PubMed Central

Kaufmann, Franz; Lovley, Derek R.

2001-01-01

NADPH is an intermediate in the oxidation of organic compounds coupled to Fe(III) reduction in Geobacter species, but Fe(III) reduction with NADPH as the electron donor has not been studied in these organisms. Crude extracts of Geobacter sulfurreducens catalyzed the NADPH-dependent reduction of Fe(III)-nitrilotriacetic acid (NTA). The responsible enzyme, which was recovered in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. Its specific activity for Fe(III) reduction was 65 μmol · min−1 · mg−1. The soluble Fe(III) reductase was specific for NADPH and did not utilize NADH as an electron donor. Although the enzyme reduced several forms of Fe(III), Fe(III)-NTA was the preferred electron acceptor. The protein possessed methyl viologen:NADP+ oxidoreductase activity and catalyzed the reduction of NADP+ with reduced methyl viologen as electron donor at a rate of 385 U/mg. The enzyme consisted of two subunits with molecular masses of 87 and 78 kDa and had a native molecular mass of 320 kDa, as determined by gel filtration. The purified enzyme contained 28.9 mol of Fe, 17.4 mol of acid-labile sulfur, and 0.7 mol of flavin adenine dinucleotide per mol of protein. The genes encoding the two subunits were identified in the complete sequence of the G. sulfurreducens genome from the N-terminal amino acid sequences derived from the subunits of the purified protein. The sequences of the two subunits had about 30% amino acid identity to the respective subunits of the formate dehydrogenase from Moorella thermoacetica, but the soluble Fe(III) reductase did not possess formate dehydrogenase activity. This soluble Fe(III) reductase differs significantly from previously characterized dissimilatory and assimilatory Fe(III) reductases in its molecular composition and cofactor content. PMID:11443080

Impact of protein domains on PE_PGRS30 polar localization in Mycobacteria.

PubMed

De Maio, Flavio; Maulucci, Giuseppe; Minerva, Mariachiara; Anoosheh, Saber; Palucci, Ivana; Iantomasi, Raffaella; Palmieri, Valentina; Camassa, Serena; Sali, Michela; Sanguinetti, Maurizio; Bitter, Wilbert; Manganelli, Riccardo; De Spirito, Marco; Delogu, Giovanni

2014-01-01

PE_PGRS proteins are unique to the Mycobacterium tuberculosis complex and a number of other pathogenic mycobacteria. PE_PGRS30, which is required for the full virulence of M. tuberculosis (Mtb), has three main domains, i.e. an N-terminal PE domain, repetitive PGRS domain and the unique C-terminal domain. To investigate the role of these domains, we expressed a GFP-tagged PE_PGRS30 protein and a series of its functional deletion mutants in different mycobacterial species (Mtb, Mycobacterium bovis BCG and Mycobacterium smegmatis) and analysed protein localization by confocal microscopy. We show that PE_PGRS30 localizes at the mycobacterial cell poles in Mtb and M. bovis BCG but not in M. smegmatis and that the PGRS domain of the protein strongly contributes to protein cellular localization in Mtb. Immunofluorescence studies further showed that the unique C-terminal domain of PE_PGRS30 is not available on the surface, except when the PGRS domain is missing. Immunoblot demonstrated that the PGRS domain is required to maintain the protein strongly associated with the non-soluble cellular fraction. These results suggest that the repetitive GGA-GGN repeats of the PGRS domain contain specific sequences that contribute to protein cellular localization and that polar localization might be a key step in the PE_PGRS30-dependent virulence mechanism.

The use of plasma-activated covalent attachment of early domains of tropoelastin to enhance vascular compatibility of surfaces

PubMed Central

Hiob, Matti A.; Wise, Steven G.; Kondyurin, Alexey; Waterhouse, Anna; Bilek, Marcela M.; Ng, Martin K. C.; Weiss, Anthony S.

2013-01-01

All current metallic vascular prostheses, including stents, exhibit suboptimal biocompatibility. Improving the re-endothelialization and reducing the thrombogenicity of these devices would substantially improve their clinical efficacy. Tropoelastin (TE), the soluble precursor of elastin, mediates favorable endothelial cell interactions while having low thrombogenicity. Here we show that constructs of TE corresponding to the first 10 (“N10”) and first 18 (“N18”) N-terminal domains of the molecule facilitate endothelial cell attachment and proliferation equivalent to the performance of full-length TE. This N-terminal ability contrasts with the known role of the C-terminus of TE in facilitating cell attachment, particularly of fibroblasts. When immobilized on a plasma-activated coating (“PAC”), N10 and N18 retained their bioactivity and endothelial cell interactive properties, demonstrating attachment and proliferation equivalent to full-length TE. In whole blood assays, both N10 and N18 maintained the low thrombogenicity of PAC. Furthermore, these N-terminal constructs displayed far greater resistance to protease degradation by blood serine proteases kallikrein and thrombin than did full-length TE. When immobilized onto a PAC surface, these shorter constructs form a modified metal interface to establish a platform technology for biologically compatible, implantable cardiovascular devices. PMID:23863453

Macrophage matrix metalloproteinase-12 dampens inflammation and neutrophil influx in arthritis.

PubMed

Bellac, Caroline L; Dufour, Antoine; Krisinger, Michael J; Loonchanta, Anantasak; Starr, Amanda E; Auf dem Keller, Ulrich; Lange, Philipp F; Goebeler, Verena; Kappelhoff, Reinhild; Butler, Georgina S; Burtnick, Leslie D; Conway, Edward M; Roberts, Clive R; Overall, Christopher M

2014-10-23

Resolution of inflammation reduces pathological tissue destruction and restores tissue homeostasis. Here, we used a proteomic protease substrate discovery approach, terminal amine isotopic labeling of substrates (TAILS), to analyze the role of the macrophage-specific matrix metalloproteinase-12 (MMP12) in inflammation. In murine peritonitis, MMP12 inactivates antithrombin and activates prothrombin, prolonging the activated partial thromboplastin time. Furthermore, MMP12 inactivates complement C3 to reduce complement activation and inactivates the chemoattractant anaphylatoxins C3a and C5a, whereas iC3b and C3b opsonin cleavage increases phagocytosis. Loss of these anti-inflammatory activities in collagen-induced arthritis in Mmp12(-/-) mice leads to unresolved synovitis and extensive articular inflammation. Deep articular cartilage loss is associated with massive neutrophil infiltration and abnormal DNA neutrophil extracellular traps (NETs). The NETs are rich in fibrin and extracellular actin, which TAILS identified as MMP12 substrates. Thus, macrophage MMP12 in arthritis has multiple protective roles in countering neutrophil infiltration, clearing NETs, and dampening inflammatory pathways to prepare for the resolution of inflammation. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.