Effect of the Solvent Temperatures on Dynamics of Serine Protease Proteinase K

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Peng Sang

2016-02-01

Full Text Available To obtain detailed information about the effect of the solvent temperatures on protein dynamics, multiple long molecular dynamics (MD simulations of serine protease proteinase K with the solute and solvent coupled to different temperatures (either 300 or 180 K have been performed. Comparative analyses demonstrate that the internal flexibility and mobility of proteinase K are strongly dependent on the solvent temperatures but weakly on the protein temperatures. The constructed free energy landscapes (FELs at the high solvent temperatures exhibit a more rugged surface, broader spanning range, and higher minimum free energy level than do those at the low solvent temperatures. Comparison between the dynamic hydrogen bond (HB numbers reveals that the high solvent temperatures intensify the competitive HB interactions between water molecules and protein surface atoms, and this in turn exacerbates the competitive HB interactions between protein internal atoms, thus enhancing the conformational flexibility and facilitating the collective motions of the protein. A refined FEL model was proposed to explain the role of the solvent mobility in facilitating the cascade amplification of microscopic motions of atoms and atomic groups into the global collective motions of the protein.

The control of neutrophil chemotaxis by inhibitors of cathepsin G and chymotrypsin.

Science.gov (United States)

Lomas, D A; Stone, S R; Llewellyn-Jones, C; Keogan, M T; Wang, Z M; Rubin, H; Carrell, R W; Stockley, R A

1995-10-06

Neutrophil chemotaxis plays an important role in the inflammatory response and when excessive or persistent may augment tissue damage. The effects of inhibitors indicated the involvement of one or more serine proteinases in human neutrophil migration and shape change in response to a chemoattractant. Monospecific antibodies, chloromethylketone inhibitors, and reactive-site mutants of alpha 1-antitrypsin and alpha 1-antichymotrypsin were used to probe the specificity of the proteinases involved in chemotaxis. Antibodies specific for cathepsin G inhibited chemotaxis. Moreover, rapid inhibitors of cathepsin G and alpha-chymotrypsin suppressed neutrophil chemotaxis to the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and zymosan-activated serum in multiple blind well assays and to fMLP in migration assays under agarose. The concentrations of antichymotrypsin mutants that reduced chemotaxis by 50% would inactivate free cathepsin G with a half-life of 1.5-3 s, whereas the concentrations of chloromethylketones required to produce a similar inhibition of chemotaxis would inactivate cathepsin G with a half-life of 345 s. These data suggest different modes of action for these two classes of inhibitors. Indeed the chloromethylketone inhibitors of cathepsin G (Z-Gly-Leu-Phe-CMK) and to a lesser extent of chymotrypsin (Cbz-Gly-Gly-Phe-CMK) mediated their effect by preventing a shape change in the purified neutrophils exposed to fMLP. Antichymotrypsin did not affect shape change in response to fMLP even at concentrations that were able to reduce neutrophil chemotaxis by 50%. These results support the involvement of cell surface proteinases in the control of cell migration and show that antichymotrypsin and chloromethylketones have differing modes of action. This opens the possibility for the rational design of anti-inflammatory agents targeted at neutrophil membrane enzymes.

Flexibility of cold- and heat-adapted subtilisin-like serine proteinases evaluated with fluorescence quenching and molecular dynamics

DEFF Research Database (Denmark)

Sigtryggsdóttir, Asta Rós; Papaleo, Elena; Thorbjarnardóttir, Sigríður H.

2014-01-01

activity of cold adapted enzymes when compared to homologues from thermophiles, reflects their higher molecular flexibility. To assess a potential difference in molecular flexibility between the two homologous proteinases, we have measured their Trp fluorescence quenching by acrylamide at different......The subtilisin-like serine proteinases, VPR, from a psychrotrophic Vibrio species and aqualysin I (AQUI) from the thermophile Thermus aquaticus, are structural homologues, but differ significantly with respect to stability and catalytic properties. It has been postulated that the higher catalytic...... to Trp (Y191W). A lower quenching effect of acrylamide on the intrinsic fluorescence of the thermophilic AQUI_Y191W was observed at all temperatures measured (10-55°C), suggesting that it possesses a more rigid structure than VPR. The MD analysis (Cα rmsf profiles) showed that even though VPR and AQUI...

Identification of a serine proteinase homolog (Sp-SPH) involved in immune defense in the mud crab Scylla paramamosain.

Science.gov (United States)

Zhang, Qiu-xia; Liu, Hai-peng; Chen, Rong-yuan; Shen, Kai-li; Wang, Ke-jian

2013-01-01

Clip domain serine proteinase homologs are involved in many biological processes including immune response. To identify the immune function of a serine proteinase homolog (Sp-SPH), originally isolated from hemocytes of the mud crab, Scylla paramamosain, the Sp-SPH was expressed recombinantly and purified for further studies. It was found that the Sp-SPH protein could bind to a number of bacteria (including Aeromonas hydrophila, Escherichia coli, Staphylococcus aureus, Vibrio fluvialis, Vibrio harveyi and Vibrio parahemolyticus), bacterial cell wall components such as lipopolysaccharide or peptidoglycan (PGN), and β-1, 3-glucan of fungus. But no direct antibacterial activity of Sp-SPH protein was shown by using minimum inhibitory concentration or minimum bactericidal concentration assays. Nevertheless, the Sp-SPH protein was found to significantly enhance the crab hemocyte adhesion activity (paired t-test, Pparahemolyticus which were both recognized by Sp-SPH protein, if pre-incubated with Sp-SPH protein, respectively. Whereas, the crabs died much faster when challenged with Vibrio alginolyiicus, a pathogenic bacterium not recognized by Sp-SPH protein, compared to those of crabs challenged with A. hydrophila or V. parahemolyticus when pre-coated with Sp-SPH protein. Taken together, these data suggested that Sp-SPH molecule might play an important role in immune defense against bacterial infection in the mud crab S. paramamosain.

In silico design, synthesis, and assays of specific substrates for proteinase 3: influence of fluorogenic and charged groups.

Science.gov (United States)

Narawane, Shailesh; Budnjo, Adnan; Grauffel, Cédric; Haug, Bengt Erik; Reuter, Nathalie

2014-02-13

Neutrophil serine proteases are specific regulators of the immune response, and proteinase 3 is a major target antigen in antineutrophil cytoplasmic antibody-associated vasculitis. FRET peptides containing 2-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) as fluorophore and quencher groups, respectively, have been widely used to probe proteases specificity. Using in silico design followed by enzymatic assays, we show that Abz and EDDnp significantly contribute to substrate hydrolysis by PR3. We also propose a new substrate specific for PR3.

Isolation and characterization of a serine proteinase with thrombin-like activity from the venom of the snake Bothrops asper

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A.V Pérez

2008-01-01

Full Text Available A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13% of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg and fibrinogen (minimum coagulant dose = 4.2 µg in vitro, and promotes defibrin(ogenation in vivo (minimum defibrin(ogenating dose = 1.0 µg. In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.

Application of Asian pumpkin (Cucurbita ficifolia) serine proteinase for production of biologically active peptides from casein.

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Dąbrowska, Anna; Szołtysik, Marek; Babij, Konrad; Pokora, Marta; Zambrowicz, Aleksandra; Chrzanowska, Józefa

2013-01-01

The main objective of this study was to determine potential application of a serine proteinase derived from Asian pumpkin for obtaining biologically active peptides from casein. The course of casein hydrolysis by three doses of the enzyme (50, 150, 300 U/mg of protein) was monitored for 24 hours by the determinations of: hydrolysis degree DH (%), free amino group content (μmole Gly/g), RP HPLC peptide profiles and by polyacrylamide gel electrophoresis. In all hydrolyzates analyzed antioxidant activities were determined using three tests: the ability to reduce iron ions in FRAP test, the ability to scavenge free radicals in DPPH test, and Fe(2+) chelating activity. The antimicrobial activity of obtained peptide fractions was determined as the ability to inhibit the growth of Escherichia coli, Bacillus cereus and Pseudomonas fluorescens in a diffusion plate test. The deepest degradation, expressed as the DH [%] and the free amino group content (67% and 7528 µmole Gly/mg, respectively), was noted in samples hydrolyzed with 300 U/ml of enzyme for 24 hours, while in other samples the determined values were about three and two times lower. The results were in agreement with the peptide profiles obtained by RP HPLC. The highest antioxidative activities determined in all tests were seen for the casein hydrolysate obtained with 300 U/mg protein of serine proteinase after 24 h of reaction (2.15 µM Trolox/mg, 96.15 µg Fe(3+)/mg, 814.97 µg Fe(2+)/mg). Antimicrobial activity was presented in three preparations. In other samples no antimicrobial activity was detected.

Identification of a Serine Proteinase Homolog (Sp-SPH) Involved in Immune Defense in the Mud Crab Scylla paramamosain

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Zhang, Qiu-xia; Liu, Hai-peng; Chen, Rong-yuan; Shen, Kai-li; Wang, Ke-jian

2013-01-01

Clip domain serine proteinase homologs are involved in many biological processes including immune response. To identify the immune function of a serine proteinase homolog (Sp-SPH), originally isolated from hemocytes of the mud crab, Scylla paramamosain, the Sp-SPH was expressed recombinantly and purified for further studies. It was found that the Sp-SPH protein could bind to a number of bacteria (including Aeromonas hydrophila, Escherichia coli, Staphylococcus aureus, Vibrio fluvialis, Vibrio harveyi and Vibrio parahemolyticus), bacterial cell wall components such as lipopolysaccharide or peptidoglycan (PGN), and β-1, 3-glucan of fungus. But no direct antibacterial activity of Sp-SPH protein was shown by using minimum inhibitory concentration or minimum bactericidal concentration assays. Nevertheless, the Sp-SPH protein was found to significantly enhance the crab hemocyte adhesion activity (paired t-test, Pparahemolyticus which were both recognized by Sp-SPH protein, if pre-incubated with Sp-SPH protein, respectively. Whereas, the crabs died much faster when challenged with Vibrio alginolyiicus, a pathogenic bacterium not recognized by Sp-SPH protein, compared to those of crabs challenged with A. hydrophila or V. parahemolyticus when pre-coated with Sp-SPH protein. Taken together, these data suggested that Sp-SPH molecule might play an important role in immune defense against bacterial infection in the mud crab S. paramamosain. PMID:23724001

Inhibition of growth hormone and prolactin secretion by a serine proteinase inhibitor

International Nuclear Information System (INIS)

Rappay, G.; Nagy, I.; Makara, G.B.; Horvath, G.; Karteszi, M.; Bacsy, E.; Stark, E.

1984-01-01

The action of the tripeptide aldehyde t-butyloxycarbonyl-DPhe-Pro-Arg-H (boc-fPR-H), belonging to a family of serine proteinase inhibitors, on the release of immunoreactive prolactin (iPRL) and growth hormone (iGH) has been studied. In rat anterior pituitary cell cultures and pituitary quarters 1 mM boc-fPR-H inhibited basal iPRL and iGH release. Thyroliberin-induced iPRL release by cultured cells was also markedly inhibited with a concomitant accumulation of intracellular iPRL. During the short- and long-term exposure of cells to boc-fPR-H there were no changes in total cell protein contents and in activities of some lysosomal marker enzymes. The marked inhibition of basal as well as stimulated hormone release in the presence of the enzyme inhibitor might suggest that at least a portion of the hormones is released via a proteolytic enzyme-dependent process

Anti-neutrophil cytoplasmic antibodies stimulate release of neutrophil microparticles.

LENUS (Irish Health Repository)

Hong, Ying

2012-01-01

The mechanisms by which anti-neutrophil cytoplasmic antibodies (ANCAs) may contribute to the pathogenesis of ANCA-associated vasculitis are not well understood. In this study, both polyclonal ANCAs isolated from patients and chimeric proteinase 3-ANCA induced the release of neutrophil microparticles from primed neutrophils. These microparticles expressed a variety of markers, including the ANCA autoantigens proteinase 3 and myeloperoxidase. They bound endothelial cells via a CD18-mediated mechanism and induced an increase in endothelial intercellular adhesion molecule-1 expression, production of endothelial reactive oxygen species, and release of endothelial IL-6 and IL-8. Removal of the neutrophil microparticles by filtration or inhibition of reactive oxygen species production with antioxidants abolished microparticle-mediated endothelial activation. In addition, these microparticles promoted the generation of thrombin. In vivo, we detected more neutrophil microparticles in the plasma of children with ANCA-associated vasculitis compared with that in healthy controls or those with inactive vasculitis. Taken together, these results support a role for neutrophil microparticles in the pathogenesis of ANCA-associated vasculitis, potentially providing a target for future therapeutics.

Plant Proteinase Inhibitor BbCI Modulates Lung Inflammatory Responses and Mechanic and Remodeling Alterations Induced by Elastase in Mice

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Rafael Almeida-Reis

2017-01-01

Full Text Available Background. Proteinases play a key role in emphysema. Bauhinia bauhinioides cruzipain inhibitor (BbCI is a serine-cysteine proteinase inhibitor. We evaluated BbCI treatment in elastase-induced pulmonary alterations. Methods.  C57BL/6 mice received intratracheal elastase (ELA group or saline (SAL group. One group of mice was treated with BbCI (days 1, 15, and 21 after elastase instillation, ELABC group. Controls received saline and BbCI (SALBC group. After 28 days, we evaluated respiratory mechanics, exhaled nitric oxide, and bronchoalveolar lavage fluid. In lung tissue we measured airspace enlargement, quantified neutrophils, TNFα-, MMP-9-, MMP-12-, TIMP-1-, iNOS-, and eNOS-positive cells, 8-iso-PGF2α, collagen, and elastic fibers in alveolar septa and airways. MUC-5-positive cells were quantified only in airways. Results. BbCI reduced elastase-induced changes in pulmonary mechanics, airspace enlargement and elastase-induced increases in total cells, and neutrophils in BALF. BbCI reduced macrophages and neutrophils positive cells in alveolar septa and neutrophils and TNFα-positive cells in airways. BbCI attenuated elastic and collagen fibers, MMP-9- and MMP-12-positive cells, and isoprostane and iNOS-positive cells in alveolar septa and airways. BbCI reduced MUC5ac-positive cells in airways. Conclusions. BbCI improved lung mechanics and reduced lung inflammation and airspace enlargement and increased oxidative stress levels induced by elastase. BbCI may have therapeutic potential in chronic obstructive pulmonary disease.

Identification of a serine proteinase homolog (Sp-SPH involved in immune defense in the mud crab Scylla paramamosain.

Directory of Open Access Journals (Sweden)

Qiu-xia Zhang

Full Text Available Clip domain serine proteinase homologs are involved in many biological processes including immune response. To identify the immune function of a serine proteinase homolog (Sp-SPH, originally isolated from hemocytes of the mud crab, Scylla paramamosain, the Sp-SPH was expressed recombinantly and purified for further studies. It was found that the Sp-SPH protein could bind to a number of bacteria (including Aeromonas hydrophila, Escherichia coli, Staphylococcus aureus, Vibrio fluvialis, Vibrio harveyi and Vibrio parahemolyticus, bacterial cell wall components such as lipopolysaccharide or peptidoglycan (PGN, and β-1, 3-glucan of fungus. But no direct antibacterial activity of Sp-SPH protein was shown by using minimum inhibitory concentration or minimum bactericidal concentration assays. Nevertheless, the Sp-SPH protein was found to significantly enhance the crab hemocyte adhesion activity (paired t-test, P<0.05, and increase phenoloxidase activity if triggered by PGN in vitro (paired t-test, P<0.05. Importantly, the Sp-SPH protein was demonstrated to promote the survival rate of the animals after challenge with A. hydrophila or V. parahemolyticus which were both recognized by Sp-SPH protein, if pre-incubated with Sp-SPH protein, respectively. Whereas, the crabs died much faster when challenged with Vibrio alginolyiicus, a pathogenic bacterium not recognized by Sp-SPH protein, compared to those of crabs challenged with A. hydrophila or V. parahemolyticus when pre-coated with Sp-SPH protein. Taken together, these data suggested that Sp-SPH molecule might play an important role in immune defense against bacterial infection in the mud crab S. paramamosain.

A case of proteinase 3 anti-neutrophil cytoplasmic antibody (PR3-ANCA positive/IgG4-related lung disease

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Hirokazu Touge

2017-01-01

Full Text Available IgG4-related lung disease (IgG4-RLD is a rare and chronic progressive autoimmune disease. We report a case of IgG4-related inflammatory pseudo-tumor of the lung that was seropositive for proteinase 3 anti-neutrophil cytoplasmic antibody (PR3-ANCA. A 61-year-old male had a mass lesion in the right lower lung field in chest X-ray. Transbronchial lung biopsy resulted in a pathological diagnosis of IgG4-RLD. The condition was improved by hormonal therapy.

Proteinases of human epidermis; a possible mechanism for polymorphonuclear leukocyte chemotaxis

Energy Technology Data Exchange (ETDEWEB)

Levine, N; Hatcher, V B; Lazarus, G S [Albert Einstein Coll. of Medicine, Bronx, N.Y. (USA); Montefiore Hospital, New York (USA); Duke Univ., Durham, N.C. (USA))

1976-12-08

Three neutral proteinases (EC 3.4.-,-) and cathepsin D have been identified in human epidermis utilizing a highly sensitive radioactive method. The proteinases were extracted in 1.0 M KCl and 0.1% Triton X-100 and separated by Sephadex G-75 chromatography. The neutral proteinase peaks were all inhibited by diisopropyl fluorophosphate and thus were serine proteinases. Incubation of the enzyme fractions with (/sup 3/H)diisopropyl fluorophosphate followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the two larger molecular weight proteinases were enzyme mixtures. The small molecular weight (/sup 3/H)diisopropyl fluorophosphate proteinase migrated as a single band. Injection of the small molecular weight neutral proteinase into rabbit skin produced a polymorphonuclear leukocyte infiltration and edema. The reaction was not observed with the diisopropul fluorophosphate-inhibited enzyme fraction. The release of neutral proteinases may be one of the signal events in the epidermal inflammatory response.

Neutrophil elastase processing of Gelatinase A is mediated by extracellular matrix

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Rice, A.; Banda, M.J. [Univ. of California, San Franciso, CA (United States)

1995-07-18

Gelatinase A (72-kDa type IV collagenase) is a metalloproteinase that is expressed by many cells in culture and is overexpressed by some tumor cells. It has been suggested that the serine proteinase neutrophil elastase might play a role iii the posttranslational processing of gelatinase A and that noncatalytic interactions between gelatinase A and components of the extracellular matrix might alter potential processing pathways. These questions were addressed with the use of gelatin substrate zymography, gelatinolytic activity assays, and amino acid sequence analysis. We found that neutrophil elastase does proteolytically modify gelatinase A by cleaving at a number of sites within gelatinase A. Sequential treatment of gelatinase A with 4-aminophenylmercuric acetate (APMA) and neutrophil elastase yielded an active gelatinase with a 4-fold increase in gelatinolytic activity. The increased gelatinolytic activity correlated with that of a 40-kDa fragment of gelatinase A. Matrix components altered the proteolytic modifications in gelatinase A that were mediated by neutrophil elastase. In the absence of gelatin, neutrophil elastase destructively degraded gelatinase A by hydrolyzing at least two bonds within the fibronectin-like gelatin-binding domain of gelatinase A. In the presence of gelatin, these two inactivating cleavage sites were protected, and cleavage at a site within the hemopexin-like carboxyl-terminal domain resulted in a truncated yet active gelatinase. The results suggest a regulatory role for extracellular matrix molecules in stabilizing gelatinase A fragments and in altering the availability of sites susceptible to destructive proteolysis by neutrophil elastase. 32 refs., 10 figs.

Proteinases in excretory-secretory products of Toxocara canis second-stage larvae: zymography and modeling insights.

Science.gov (United States)

González-Páez, Gonzalo Ernesto; Alba-Hurtado, Fernando; García-Tovar, Carlos Gerardo; Argüello-García, Raúl

2014-01-01

Components released in excretory-secretory products of Toxocara canis larvae (TES) include phosphatidylethanolamine-binding proteins (TES26), mucins (TES120, MUC2-5), and C-type lectins (TES32, TES70) and their biochemical, immunological, and diagnostic properties have been extensively studied albeit proteinase activities towards physiological substrates are almost unknown. Proteolytic activities in TES samples were first analyzed by gel electrophoresis with gelatin as substrate. Major activities of ~400, 120, and 32 kDa in TES were relatively similar over a broad pH range (5.5-9.0) and all these were of the serine-type as leupeptin abolished gelatinolysis. Further, the ~400 kDa component degraded all physiological substrates tested (laminin, fibronectin, albumin, and goat IgG) and the 120 kDa component degraded albumin and goat IgG while proteinases of lower MW (45, 32, and 26 kDa) only degraded laminin and fibronectin, preferentially at alkaline pH (9.0). By protein modeling approaches using the known sequences of TES components, only TES26 and MUC4 displayed folding patterns significantly related to reference serine proteinases. These data suggest that most of serine proteinase activities secreted in vitro by infective larvae of T. canis have intriguing nature but otherwise help the parasite to affect multiple components of somatic organs and bodily fluids within the infected host.

AbetaPP/APLP2 family of Kunitz serine proteinase inhibitors regulate cerebral thrombosis.

Science.gov (United States)

Xu, Feng; Previti, Mary Lou; Nieman, Marvin T; Davis, Judianne; Schmaier, Alvin H; Van Nostrand, William E

2009-04-29

The amyloid beta-protein precursor (AbetaPP) is best recognized as the precursor to the Abeta peptide that accumulates in the brains of patients with Alzheimer's disease, but less is known about its physiological functions. Isoforms of AbetaPP that contain a Kunitz-type serine proteinase inhibitor (KPI) domain are expressed in brain and, outside the CNS, in circulating blood platelets. Recently, we showed that KPI-containing forms of AbetaPP regulates cerebral thrombosis in vivo (Xu et al., 2005, 2007). Amyloid precursor like protein-2 (APLP2), a closely related homolog to AbetaPP, also possesses a highly conserved KPI domain. Virtually nothing is known of its function. Here, we show that APLP2 also regulates cerebral thrombosis risk. Recombinant purified KPI domains of AbetaPP and APLP2 both inhibit the plasma clotting in vitro. In a carotid artery thrombosis model, both AbetaPP(-/-) and APLP2(-/-) mice exhibit similar significantly shorter times to vessel occlusion compared with wild-type mice indicating a prothrombotic phenotype. Similarly, in an experimental model of intracerebral hemorrhage, both AbetaPP(-/-) and APLP2(-/-) mice produce significantly smaller hematomas with reduced brain hemoglobin content compared with wild-type mice. Together, these results indicate that AbetaPP and APLP2 share overlapping anticoagulant functions with regard to regulating thrombosis after cerebral vascular injury.

Purification and Characterization of an Extracellular Proteinase from Brevibacterium-Linens ATCC-9174

DEFF Research Database (Denmark)

Rattray, F P; Bockelmann, W; Fox, P F

1995-01-01

An extracellular serine proteinase from Brevibacterium linens ATCC 9174 was purified to homogeneity. pH and temperature optima were 8,5 and 50 degrees C, respectively. The results for the molecular mass of the proteinase were 56 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis...... and 126 kDa by gel filtration, indicating that the native enzyme exists as a dimer. Mg2+ and Ca2+ activated the proteinase, as did NaCl; however, Hg2+ Fe2+, and Zn2+ caused strong inhibition. The sequence of the first 20 N-terminal amino acids was NH2-Ala-Lys- Asn...

Neutrophil Protease Cleavage of Von Willebrand Factor in Glomeruli – An Anti-thrombotic Mechanism in the Kidney

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Ramesh Tati

2017-02-01

Full Text Available Adequate cleavage of von Willebrand factor (VWF prevents formation of thrombi. ADAMTS13 is the main VWF-cleaving protease and its deficiency results in development of thrombotic microangiopathy. Besides ADAMTS13 other proteases may also possess VWF-cleaving activity, but their physiological importance in preventing thrombus formation is unknown. This study investigated if, and which, proteases could cleave VWF in the glomerulus. The content of the glomerular basement membrane (GBM was studied as a reflection of processes occurring in the subendothelial glomerular space. VWF was incubated with human GBMs and VWF cleavage was assessed by multimer structure analysis, immunoblotting and mass spectrometry. VWF was cleaved into the smallest multimers by the GBM, which contained ADAMTS13 as well as neutrophil proteases, elastase, proteinase 3 (PR3, cathepsin-G and matrix-metalloproteinase 9. The most potent components of the GBM capable of VWF cleavage were in the serine protease or metalloprotease category, but not ADAMTS13. Neutralization of neutrophil serine proteases inhibited GBM-mediated VWF-cleaving activity, demonstrating a marked contribution of elastase and/or PR3. VWF-platelet strings formed on the surface of primary glomerular endothelial cells, in a perfusion system, were cleaved by both elastase and the GBM, a process blocked by elastase inhibitor. Ultramorphological studies of the human kidney demonstrated neutrophils releasing elastase into the GBM. Neutrophil proteases may contribute to VWF cleavage within the subendothelium, adjacent to the GBM, and thus regulate thrombus size. This anti-thrombotic mechanism would protect the normal kidney during inflammation and could also explain why most patients with ADAMTS13 deficiency do not develop severe kidney failure.

Redox regulation of peroxiredoxin and proteinases by ascorbate and thiols during pea root nodule senescence.

Science.gov (United States)

Groten, Karin; Dutilleul, Christelle; van Heerden, Philippus D R; Vanacker, Hélène; Bernard, Stéphanie; Finkemeier, Iris; Dietz, Karl-Josef; Foyer, Christine H

2006-02-20

Redox factors contributing to nodule senescence were studied in pea. The abundance of the nodule cytosolic peroxiredoxin but not the mitochondrial peroxiredoxin protein was modulated by ascorbate. In contrast to redox-active antioxidants such as ascorbate and cytosolic peroxiredoxin that decreased during nodule development, maximal extractable nodule proteinase activity increased progressively as the nodules aged. Cathepsin-like activities were constant throughout development but serine and cysteine proteinase activities increased during senescence. Senescence-induced cysteine proteinase activity was inhibited by cysteine, dithiotreitol, or E-64. Senescence-dependent decreases in redox-active factors, particularly ascorbate and peroxiredoxin favour decreased redox-mediated inactivation of cysteine proteinases.

A zymography analysis of proteinase activity present in Leptospira.

Science.gov (United States)

Madathiparambil, Madanan G; Cattavarayane, Sandhanakrishnan; Manickam, Gayathri D; Singh, Kavita; Perumana, Sudhakaran R; Sehgal, Subhash C

2011-03-01

Leptospirosis is a major public health problem caused by spirochete Leptospira which is an extracellular pathogen. During infection and invasion, the bacteria cross the physical barriers and later it encounter with the host defence mechanism. These processes may involve proteolytic degradation of the host tissue biomatrix. In an effort to understand the production and nature of Leptospiral proteinases, investigations were carried out using zymograpic methods. The results showed that the leptospires degrades different kind of protein substances such as gelatin, casein, and albumin. Gelatin zymography reveals that different serovars contain multiple gelatinases in the molecular weight range from 240 to 32 kDa. Studies using inhibitors suggested that the Leptospiral proteinases include metalloproteinases, serine or cysteine proteinases. The temperature sensitivity suggests that some of these proteinases are stable even at high temperatures. The presence of multiple gelatinases in Leptospira serovars suggests a critical role for these enzymes in Leptospiral invasion and pathogenesis.

PepJ is a new extracellular proteinase of Aspergillus nidulans.

Science.gov (United States)

Emri, T; Szilágyi, M; László, K; M-Hamvas, M; Pócsi, I

2009-01-01

Under carbon starvation, Aspergillus nidulans released a metallo-proteinase with activities comparable to those of PrtA, the major extracellular serine proteinase of the fungus. The relative molar mass of the enzyme was 19 kDa as determined with both denaturing and renaturing SDS PAGE, while its isoelectric point and pH and temperature optima were 8.6, 5.5 and 65 degrees C, respectively. The enzyme was stable at pH 3.5-10.5 and was still active at 95 degrees C in the presence of azocasein substrate. MALDI-TOF MS analysis demonstrated that the proteinase was encoded by the pepJ gene (locus ID AN7962.3), and showed high similarity to deuterolysin from Aspergillus oryzae. The size of the mature enzyme, its EDTA sensitivity and heat stability also supported the view that A. nidulans PepJ is a deuterolysin-type metallo-proteinase.

AβPP/APLP2 Family of Kunitz Serine Proteinase Inhibitors Regulate Cerebral Thrombosis

Science.gov (United States)

Xu, Feng; Previti, Mary Lou; Nieman, Marvin T.; Davis, Judianne; Schmaier, Alvin H.; Van Nostrand, William E.

2009-01-01

The amyloid β-protein precursor (AβPP) is best recognized as the precursor to the Aβ peptide that accumulates in the brains of patients with Alzheimer’s disease, but less is known about its physiological functions. Isoforms of AβPP that contain a Kunitz-type serine proteinase inhibitor (KPI) domain are expressed in brain and, outside the CNS, in circulating blood platelets. Recently, we showed that KPI-containing forms of AβPP regulates cerebral thrombosis in vivo (Xu et al., 2005 Proc. Natl. Acad. Sci. USA 102:18135–18140; Xu et al. 2007 Stroke 38:2598–2601). Amyloid precursor like protein-2 (APLP2), a closely related homolog to AβPP, also possesses a highly conserved KPI domain. Virtually nothing is known of its function. Here we show that APLP2 also regulates cerebral thrombosis risk. Recombinant purified KPI domains of AβPP and APLP2 both inhibit the plasma clotting in vitro. In a carotid artery thrombosis model both AβPP−/− and APLP2−/− mice exhibit similar significantly shorter times to vessel occlusion compared with wild-type mice indicating a pro-thrombotic phenotype. Similarly, in an experimental model of intracerebral hemorrhage both AβPP−/− and APLP2−/− mice produce significantly smaller hematomas with reduced brain hemoglobin content compared with wild-type mice. Together, these results indicate that AβPP and APLP2 share overlapping anticoagulant functions with regard to regulating thrombosis after cerebral vascular injury. PMID:19403832

The cell envelope subtilisin-like proteinase is a virulence determinant for Streptococcus suis

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Gottschalk Marcelo

2010-02-01

Full Text Available Abstract Background Streptococcus suis is a major swine pathogen and zoonotic agent that mainly causes septicemia, meningitis, and endocarditis. It has recently been suggested that proteinases produced by S. suis (serotype 2 are potential virulence determinants. In the present study, we screened a S. suis mutant library created by the insertion of Tn917 transposon in order to isolate a mutant deficient in a cell surface proteinase. We characterized the gene and assessed the proteinase for its potential as a virulence factor. Results Two mutants (G6G and M3G possessing a single Tn917 insertion were isolated. The affected gene coded for a protein (SSU0757 that shared a high degree of identity with Streptococccus thermophilus PrtS (95.9% and, to a lesser extent, with Streptococcus agalactiae CspA (49.5%, which are cell surface serine proteinases. The SSU0757 protein had a calculated molecular mass of 169.6 kDa and contained the catalytic triad characteristic of subtilisin family proteinases: motif I (Asp200, motif II (His239, and motif III (Ser568. SSU0757 also had the Gram-positive cell wall anchoring motif (Leu-Pro-X-Thr-Gly at the carboxy-terminus, which was followed by a hydrophobic domain. All the S. suis isolates tested, which belonged to different serotypes, possessed the gene encoding the SSU0757 protein. The two mutants devoid of subtilisin-like proteinase activity had longer generation times and were more susceptible to killing by whole blood than the wild-type parent strain P1/7. The virulence of the G6G and M3G mutants was compared to the wild-type strain in the CD1 mouse model. Significant differences in mortality rates were noted between the P1/7 group and the M3G and G6G groups (p Conclusion In summary, we identified a gene coding for a cell surface subtilisin-like serine proteinase that is widely distributed in S. suis. Evidences were brought for the involvement of this proteinase in S. suis virulence.

Cell-Wall-Bound Proteinase of Lactobacillus delbrueckii subsp. lactis ACA-DC 178: Characterization and Specificity for β-Casein

Science.gov (United States)

Tsakalidou, E.; Anastasiou, R.; Vandenberghe, I.; van Beeumen, J.; Kalantzopoulos, G.

1999-01-01

Lactobacillus delbrueckii subsp. lactis ACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca2+-free buffer. The crude proteinase extract shows its highest activity at pH 6.0 and 40°C. It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the enzyme is similar to the lactococcal PI-type proteinases, since it hydrolyzes β-casein mainly and α- and κ-caseins to a much lesser extent. The cell-wall-bound proteinase from L. delbrueckii subsp. lactis ACA-DC 178 liberates four main peptides from β-casein, which have been identified. PMID:10223997

CERTIFICATION REPORT The certification of the mass concentration of immunoglobulin G proteinase 3 anti-neutrophil cytoplasmic autoantibodies (IgG PR3 ANCA) in human serum: ERM® - DA483/IFCC

OpenAIRE

MONOGIOUDI EVANTHIA; HUTU DANA PETRONELA; CHAROUD-GOT JEAN; SHELDON JOANNA; SCHIMMEL HEINZ; TRAPMANN STEFANIE; MERONI PIERLUIGI; EMONS HENDRIK; ZEGERS INGRID

2017-01-01

This report describes the production and certification of ERM-DA483/IFCC, a serum protein reference material intended for the standardisation of measurements of immunoglobulin G proteinase 3 anti-neutrophil cytoplasmic autoantibodies (IgG PR3 ANCA). The material was produced according to ISO Guide 34:2009 [ ] and is certified in accordance with ISO Guide 35:2006. The raw material used to prepare ERM-DA483/IFCC was a plasmapheresis material containing a high concentration of IgG PR3 ANCA. A...

Kazal-type proteinase inhibitor from disk abalone (Haliotis discus discus): molecular characterization and transcriptional response upon immune stimulation.

Science.gov (United States)

Wickramaarachchi, W D Niroshana; De Zoysa, Mahanama; Whang, Ilson; Wan, Qiang; Lee, Jehee

2013-09-01

Proteinases and proteinase inhibitors are involved in several biological and physiological processes in all multicellular organisms. Proteinase inhibitors play a key role in regulating the activity of the respective proteinases. Among serine proteinase inhibitors, kazal-type proteinase inhibitors (KPIs) are widely found in mammals, avians, and a variety of invertebrates. In this study, we describe the identification of a kazal-type serine proteinase inhibitor (Ab-KPI) from the disk abalone, Haliotis discus discus, which is presumably involved in innate immunity. The full-length cDNA of Ab-KPI includes 600 bp nucleotides with an open reading frame (ORF) encoding a polypeptide of 143 amino acids. The deduced amino acid sequence of Ab-KPI contains a putative 17-amino acid signal peptide and two tandem kazal domains with high similarity to other kazal-type SPIs. Each kazal domain consists of reactive site (P1) residue containing a leucine (L), and a threonine (T) located in the second amino acid position after the second conserved cysteine of each domain. Temporal expression of Ab-KPI was assessed by real time quantitative PCR in hemocytes and mantle tissue following bacterial and viral hemorrhagic septicemia virus (VHSV) challenge, and tissue injury. At 6 h post-bacterial and -VHSV challenge, Ab-KPI expression in hemocytes was increased 14-fold and 4-fold, respectively, compared to control samples. The highest up-regulations upon tissue injury were shown at 9 h and 12 h in hemocytes and mantle, respectively. The transcriptional modulation of Ab-KPI following bacterial and viral challenges and tissue injury indicates that it might be involved in immune defense as well as wound healing process in abalone. Copyright © 2013 Elsevier Ltd. All rights reserved.

Expression of recombinant proteinase 3, the autoantigen in Wegener's granulomatosis, in insect cells

NARCIS (Netherlands)

Van der Geld, YM; Smook, MLF; Huitema, MG; Harmsen, MC; Limburg, PC; Kallenberg, CGM

2002-01-01

Proteinase 3 (PR3) is the major autoantigen for anti-neutrophil cytoplasmic antibodies (ANCA) in patients with Wegener's granulomatosis. Little is known about the major antigenic sites on PR3. To facilitate epitope mapping, PR3 was cloned in insect cells using a baculovirus expression system. Four

Contribution of neutrophils to acute lung injury.

Science.gov (United States)

Grommes, Jochen; Soehnlein, Oliver

2011-01-01

Treatment of acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS), remain unsolved problems of intensive care medicine. ALI/ARDS are characterized by lung edema due to increased permeability of the alveolar-capillary barrier and subsequent impairment of arterial oxygenation. Lung edema, endothelial and epithelial injury are accompanied by an influx of neutrophils into the interstitium and broncheoalveolar space. Hence, activation and recruitment of neutrophils are regarded to play a key role in progression of ALI/ARDS. Neutrophils are the first cells to be recruited to the site of inflammation and have a potent antimicrobial armour that includes oxidants, proteinases and cationic peptides. Under pathological circumstances, however, unregulated release of these microbicidal compounds into the extracellular space paradoxically can damage host tissues. This review focuses on the mechanisms of neutrophil recruitment into the lung and on the contribution of neutrophils to tissue damage in ALI.

Midgut proteinases of Sitotroga cerealella (Oliver) (Lepidoptera:Gelechiidae): Characterization and relationship to resistance in cereals

International Nuclear Information System (INIS)

Wu, Lan.

1989-01-01

Midgut proteinases are vital to the insects which digest ingested food in the midgut. Insect midgut proteinases, therefore, have been considered as possible targets for the control of insect pests. Proteinaceous proteinase inhibitors are very attractive for their potential use in developing insect resistant plant varieties via genetic engineering. Sitotroga cerealella is one of the major storage pests of cereals, and no antibiotic resistance in wheat against this insect has been identified to date. A series of diagnostic inhibitors, thiol-reducing agents and a metal-ion chelator were used in the identification of proteinases in crude extracts from S. cerealella larval midguts with both protein and ester substrates. The partial inhibition of proteolytic activity in crude midgut extract toward [ 3 H]-methemoglobin by pepstatin A suggested the presence of another proteinase which was sensitive to pepstatin A. The optimum pH range for the proteolytic activity, however, indicated that the major midgut proteinases were not carboxyl proteinases. Two proteinases were successfully purified by a combination of fractionation with ammonium sulfate, gel permeation and anion exchange chromatography. Characterization of the enzymes with the purified enzyme preparations confirmed that the two major proteinases were serine endoproteinases with trypsin-like and chymotrypsin-like specificities respectively. Bioassays were conducted using the artificial seeds to test naturally occurring proteinaceous proteinase inhibitors of potential value. Soybean trypsin inhibitor and the Bowman-Birk proteinase inhibitor had adverse effects on the development of the insect. A predictive model was constructed to evaluate effects of seed resistance in conjunction with other control methods on S. cerealella population dynamics

INCREASING THE THERMOSTABILITY OF THE NEUTRAL PROTEINASE OF BACILLUS-STEAROTHERMOPHILUS BY IMPROVEMENT OF INTERNAL HYDROGEN-BONDING

NARCIS (Netherlands)

EIJSINK, VGH; VRIEND, G; VANDERZEE, [No Value; VANDENBURG, B; VENEMA, G

1992-01-01

In an attempt to increase the thermostability of the neutral proteinase of Bacillus stearothermophilus the buried Ala-170 was replaced by serine. Molecular-dynamics simulations showed that Ser-170 stabilizes the enzyme by formation of an internal hydrogen bond. In addition, the hydroxy group of

Plant Proteinase Inhibitor BbCI Modulates Lung Inflammatory Responses and Mechanic and Remodeling Alterations Induced by Elastase in Mice

OpenAIRE

Almeida-Reis, Rafael; Theodoro-Junior, Osmar A.; Oliveira, Bruno T. M.; Oliva, Leandro V.; Toledo-Arruda, Alessandra C.; Bonturi, Camila R.; Brito, Marlon V.; Lopes, Fernanda D. T. Q. S.; Prado, Carla M.; Florencio, Ariana C.; Martins, Mílton A.; Owen, Caroline A.; Leick, Edna A.; Oliva, Maria L. V.; Tibério, Iolanda F. L. C.

2017-01-01

Background. Proteinases play a key role in emphysema. Bauhinia bauhinioides cruzipain inhibitor (BbCI) is a serine-cysteine proteinase inhibitor. We evaluated BbCI treatment in elastase-induced pulmonary alterations. Methods.??C57BL/6 mice received intratracheal elastase (ELA group) or saline (SAL group). One group of mice was treated with BbCI (days 1, 15, and 21 after elastase instillation, ELABC group). Controls received saline and BbCI (SALBC group). After 28 days, we evaluated respirator...

Heightened systemic levels of neutrophil and eosinophil granular proteins in pulmonary tuberculosis and reversal following treatment.

Science.gov (United States)

Moideen, Kadar; Kumar, Nathella Pavan; Nair, Dina; Banurekha, Vaithilingam V; Bethunaickan, Ramalingam; Babu, Subash

2018-04-09

Granulocytes are activated during tuberculosis (TB) infection and act as immune effector cells and granulocyte responses are implicated in TB pathogenesis. Plasma levels of neutrophil and eosinophil granular proteins provide an indirect measure of degranulation. In this study, we wanted to examine the levels of neutrophil and eosinophil granular proteins in individuals with pulmonary tuberculosis (PTB) and to compare them with the levels in latent TB (LTB) individuals. Hence, we measured the plasma levels of myeloperoxidase (MPO), neutrophil elastase, and proteinase-3; major basic protein (MBP), eosinophil derived neurotoxin (EDN), eosinophil cationic protein (ECP) and eosinophil peroxidase (EPX) in these individuals. Finally, we also measured the levels of all of these parameters in PTB individuals following anti-tuberculosis (ATT) treatment. Our data reveal that PTB individuals are characterized by significantly higher plasma levels of MPO, elastase, human proteinase 3 as well as MBP and EDN in comparison to LTB individuals. Our data also reveal that ATT resulted in reversal of all of these changes, indicating an association with TB disease. Finally, our data show that the systemic levels of MPO and proteinase-3 can significantly discriminate PTB from LTB individuals. Thus, our data suggest that neutrophil and eosinophil granular proteins could play a potential role in the innate immune response and therefore, the pathogenesis of pulmonary TB. Copyright © 2018 American Society for Microbiology.

A preliminary neutron crystallographic study of proteinase K at pD 6.5

Energy Technology Data Exchange (ETDEWEB)

Gardberg, Anna S [ORNL; Blakeley, Matthew P. [Institut Laue-Langevin (ILL); Myles, Dean A A [ORNL

2009-01-01

AbstractA preliminary neutron crystallographic study of the proteolytic enzyme proteinase K is presented. Large hydrogenated crystals were prepared in deuterated crystallization buffer using the vapour-diffusion method. Data were collected to a resolution of 2.3 on the LADI-III diffractometer at the Institut Laue Langevin (ILL) in 2.5 days. The results demonstrate the feasibility of a full neutron crystallographic analysis of this structure aimed at providing relevant information on the location of H atoms, particularly at the active site. This information will contribute to further understanding of the molecular mechanisms underlying proteinase K's catalytic activity and to an enriched understanding of the subtilisin clan of serine proteases.

Regulation of apoptosis and priming of neutrophil oxidative burst by diisopropyl fluorophosphate

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Tsang Jennifer LY

2010-07-01

Full Text Available Abstract Background Diisopropyl fluorophosphate (DFP is a serine protease inhibitor that is widely used as an inhibitor of endogenous proteases in in vitro neutrophil studies. Its effects on neutrophil function are unclear. We sought to determine the biological effects of DFP on human neutrophil apoptosis and oxidative burst. Methods We isolated neutrophils from healthy volunteers, incubated them with DFP (2.5 mM, and evaluated neutrophil elastase (NE activity, neutrophil degranulation, apoptosis as reflected in hypodiploid DNA formation and exteriorization of phosphatidylserine (PS, processing and activity of caspases-3 and -8, oxidative burst activity and hydrogen peroxide release. Results Consistent with its activity as a serine protease inhibitor, DFP significantly inhibited NE activity but not the degranulation of azurophilic granules. DFP inhibited constitutive neutrophil apoptosis as reflected in DNA fragmentation, and the processing and activity of caspases-3 and -8. DFP also inhibited priming of neutrophils for oxidative burst activity and hydrogen peroxide release. However, DFP enhanced the exteriorization of PS in a dose-dependent manner. Conclusion We conclude that DFP exerts significant effects on neutrophil inflammatory function that may confound the interpretation of studies that use it for its antiprotease activity. We further conclude that endogenous proteases play a role in the biology of constitutive neutrophil apoptosis.

Isolation, expression and characterization of a novel dual serine protease inhibitor, OH-TCI, from king cobra venom.

Science.gov (United States)

He, Ying-Ying; Liu, Shu-Bai; Lee, Wen-Hui; Qian, Jin-Qiao; Zhang, Yun

2008-10-01

Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibitor from Ophiophagus hannah) was isolated from king cobra venom by three chromatographic steps of gel filtration, trypsin affinity and reverse phase HPLC. OH-TCI is composed of 58 amino acid residues with a molecular mass of 6339Da. Successful expression of OH-TCI was performed as the maltose-binding fusion protein in E. coli DH5alpha. Much different from Oh11-1, the purified native and recombinant OH-TCI both had strong inhibitory activities against trypsin and chymotrypsin although the sequence identity (74.1%) between them is very high. The inhibitor constants (K(i)) of recombinant OH-TCI were 3.91 x 10(-7) and 8.46 x10(-8)M for trypsin and chymotrypsin, respectively. To our knowledge, it was the first report of Kunitz/BPTI serine proteinase inhibitor from snake venom that had equivalent trypsin and chymotrypsin inhibitory activities.

Interaction between tumor cell surface receptor RAGE and proteinase 3 mediates prostate cancer metastasis to bone

Science.gov (United States)

Kolonin, Mikhail G.; Sergeeva, Anna; Staquicini, Daniela I.; Smith, Tracey L.; Tarleton, Christy A.; Molldrem, Jeffrey J.; Sidman, Richard L.; Marchiò, Serena; Pasqualini, Renata; Arap, Wadih

2017-01-01

Human prostate cancer often metastasizes to bone, but the biological basis for such site-specific tropism remains largely unresolved. Recent work led us to hypothesize that this tropism may reflect pathogenic interactions between RAGE, a cell surface receptor expressed on malignant cells in advanced prostate cancer, and proteinase 3 (PR3), a serine protease present in inflammatory neutrophils and hematopoietic cells within the bone marrow microenvironment. In this study, we establish that RAGE-PR3 interaction mediates homing of prostate cancer cells to the bone marrow. PR3 bound to RAGE on the surface of prostate cancer cells in vitro, inducing tumor cell motility through a non-proteolytic signal transduction cascade involving activation and phosphorylation of ERK1/2 and JNK1. In preclinical models of experimental metastasis, ectopic expression of RAGE on human prostate cancer cells was sufficient to promote bone marrow homing within a short time frame. Our findings demonstrate how RAGE-PR3 interactions between human prostate cancer cells and the bone marrow microenvironment mediate bone metastasis during prostate cancer progression, with potential implications for prognosis and therapeutic intervention. PMID:28428279

Human neutrophils in auto-immunity.

Science.gov (United States)

Thieblemont, Nathalie; Wright, Helen L; Edwards, Steven W; Witko-Sarsat, Véronique

2016-04-01

Human neutrophils have great capacity to cause tissue damage in inflammatory diseases via their inappropriate activation to release reactive oxygen species (ROS), proteases and other tissue-damaging molecules. Furthermore, activated neutrophils can release a wide variety of cytokines and chemokines that can regulate almost every element of the immune system. In addition to these important immuno-regulatory processes, activated neutrophils can also release, expose or generate neoepitopes that have the potential to break immune tolerance and result in the generation of autoantibodies, that characterise a number of human auto-immune diseases. For example, in vasculitis, anti-neutrophil cytoplasmic antibodies (ANCA) that are directed against proteinase 3 or myeloperoxidase are neutrophil-derived autoantigens and activated neutrophils are the main effector cells of vascular damage. In other auto-immune diseases, these neutrophil-derived neoepitopes may arise from a number of processes that include release of granule enzymes and ROS, changes in the properties of components of their plasma membrane as a result of activation or apoptosis, and via the release of Neutrophil Extracellular Traps (NETs). NETs are extracellular structures that contain chromatin that is decorated with granule enzymes (including citrullinated proteins) that can act as neo-epitopes to generate auto-immunity. This review therefore describes the processes that can result in neutrophil-mediated auto-immunity, and the role of neutrophils in the molecular pathologies of auto-immune diseases such as vasculitis, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We discuss the potential role of NETs in these processes and some of the debate in the literature regarding the role of this phenomenon in microbial killing, cell death and auto-immunity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Localization and Functionality of the Inflammasome in Neutrophils

DEFF Research Database (Denmark)

Bakele, Martina; Joos, Melanie; Burdi, Sofia

2014-01-01

Neutrophils represent the major fraction of circulating immune cells and are rapidly recruited to sites of infection and inflammation. The inflammasome is a multiprotein complex that regulates the generation of IL-1 family proteins. The precise subcellular localization and functionality...... of the inflammasome in human neutrophils are poorly defined. Here we demonstrate that highly purified human neutrophils express key components of the NOD-like receptor family, pyrin domain containing 3 (NLRP3), and absent in melanoma 2 (AIM2) inflammasomes, particularly apoptosis-associated speck-like protein...... and released as protein, highly purified neutrophils neither expressed nor released IL-1α at baseline or upon stimulation. Upon inflammasome activation, highly purified neutrophils released substantially lower levels of IL-1β protein compared with partially purified neutrophils. Serine proteases and caspases...

Ixodes scapularis tick serine proteinase inhibitor (serpin gene family; annotation and transcriptional analysis

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Chalaire Katelyn C

2009-05-01

Full Text Available Abstract Background Serine proteinase inhibitors (Serpins are a large superfamily of structurally related, but functionally diverse proteins that control essential proteolytic pathways in most branches of life. Given their importance in the biology of many organisms, the concept that ticks might utilize serpins to evade host defenses and immunizing against or disrupting their functions as targets for tick control is an appealing option. Results A sequence homology search strategy has allowed us to identify at least 45 tick serpin genes in the Ixodes scapularis genome that are structurally segregated into 32 intronless and 13 intron-containing genes. Nine of the intron-containing serpins occur in a cluster of 11 genes that span 170 kb of DNA sequence. Based on consensus amino acid residues in the reactive center loop (RCL and signal peptide scanning, 93% are putatively inhibitory while 82% are putatively extracellular. Among the 11 different amino acid residues that are predicted at the P1 sites, 16 sequences possess basic amino acid (R/K residues. Temporal and spatial expression analyses revealed that 40 of the 45 serpins are differentially expressed in salivary glands (SG and/or midguts (MG of unfed and partially fed ticks. Ten of the 38 serpin genes were expressed from six to 24 hrs of feeding while six and fives genes each are predominantly or exclusively expressed in either MG and SG respectively. Conclusion Given the diversity among tick species, sizes of tick serpin families are likely to be variable. However this study provides insight on the potential sizes of serpin protein families in ticks. Ticks must overcome inflammation, complement activation and blood coagulation to complete feeding. Since these pathways are regulated by serpins that have basic residues at their P1 sites, we speculate that I. scapularis may utilize some of the serpins reported in this study to manipulate host defense. We have discussed our data in the context of

Purification and Characterization of a New Serine Protease (VLCII) Isolated from Vipera lebetina Venom: Its Role in Hemostasis.

Science.gov (United States)

Amel, Kadi-Saci; Fatima, Laraba-Djebari

2015-08-01

Snake venom serine proteinases (SVSPs) affect various physiological functions including blood coagulation, fibrinolysis, and platelet aggregation. Coagulant serine proteinase (VLCII) was purified from Vipera lebetina venom using three chromatographic steps: gel filtration on SephadexG-75, DEAE-Sephadex A-50, and reversed-phase high-performance liquid chromatography (RP-HPLC) on C8 column. VLCII appeared homogenous (60 kDa) when tested on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). VLCII as a thrombin-like enzyme was able to hydrolyze Nα-CBZ L-arginine-p-nitroanilide hydrochloride and could be a serine protease because it is inhibited by phenylmethylsulfonyl fluoride. The proteolytic activity of VLCII was not affected by ethylenediaminetetraacetic acid and 1.10-phenanthroline. It showed high coagulant activity against human plasma and cleaved both Aα chain and Bβ chain of bovine fibrinogen. The isolated VLCII displayed proaggregating effect on human platelet in a concentration-dependent manner with an absence of lag time. Clopidogrel P2Y12 adenosine diphosphate (ADP) receptor inhibitor reduced markedly the aggregating effect induced by VLCII than aspirin, indicating the involvement of ADP signaling pathway. © 2015 Wiley Periodicals, Inc.

Activated recombinant adenovirus proteinases

Science.gov (United States)

Anderson, Carl W.; Mangel, Walter F.

1999-08-10

This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

Papillon-Lefèvre syndrome patient reveals species-dependent requirements for neutrophil defenses

DEFF Research Database (Denmark)

Sørensen, Ole E.; Clemmensen, Stine N; Dahl, Sara L

2014-01-01

immunodeficiency. Here, we characterized a 24-year-old woman who had suffered from severe juvenile periodontal disease, but was otherwise healthy, and identified a homozygous missense mutation in CTSC indicative of PLS. Proteome analysis of patient neutrophil granules revealed that several proteins that normally......Papillon-Lefèvre syndrome (PLS) results from mutations that inactivate cysteine protease cathepsin C (CTSC), which processes a variety of serine proteases considered essential for antimicrobial defense. Despite serine protease-deficient immune cell populations, PLS patients do not exhibit marked......CAP-18 into the antibacterial peptide LL-37 in response to ionomycin. In immature myeloid cells from patient bone marrow, biosynthesis of CTSC and neutrophil serine proteases appeared normal along with initial processing and sorting to cellular storage. In contrast, these proteins were completely absent...

Genetic loci of Staphylococcus aureus associated with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides

NARCIS (Netherlands)

Glasner, Corinna; de Goffau, Marcus C; van Timmeren, Mirjan M; Schulze, Mirja L; Jansen, Benita; Tavakol, Mehri; van Wamel, Willem J B; Stegeman, Coen A; Kallenberg, Cees G M; Arends, Jan P; Rossen, John W; Heeringa, Peter; van Dijl, Jan Maarten

2017-01-01

The proteinase 3 (PR3)-positive anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) granulomatosis with polyangiitis (GPA) has been associated with chronic nasal S. aureus carriage, which is a risk factor for disease relapse. The present study was aimed at comparing the

Genetic loci of Staphylococcus aureus associated with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides

NARCIS (Netherlands)

C. Glasner (Corinna); M.C. De Goffau (Marcus C.); M.M. Van Timmeren (Mirjan M.); Schulze, M.L. (Mirja L.); Jansen, B. (Benita); M. Tavakol (Mehri); W.J.B. van Wamel (Willem); C.A. Stegeman; C.G.M. Kallenberg (Cees G. M.); J.P.A. Arends (Jan); J.W. Rossen (John); P. Heeringa (Peter); J.M. Dijl (Jan Maarten)

2017-01-01

textabstractThe proteinase 3 (PR3)-positive anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) granulomatosis with polyangiitis (GPA) has been associated with chronic nasal S. aureus carriage, which is a risk factor for disease relapse. The present study was aimed at

Anti-proteinase 3 anti-neutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system.

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Mark A Little

Full Text Available Evidence is lacking for direct pathogenicity of human anti-proteinase-3 (PR3 antibodies in development of systemic vasculitis and granulomatosis with polyangiitis (GPA, Wegener's granulomatosis. Progress in study of these antibodies in rodents has been hampered by lack of PR3 expression on murine neutrophils, and by different Fc-receptor affinities for IgG across species. Therefore, we tested whether human anti-PR3 antibodies can induce acute vasculitis in mice with a human immune system. Chimeric mice were generated by injecting human haematopoietic stem cells into irradiated NOD-scid-IL2Rγ⁻/⁻ mice. Matched chimera mice were treated with human IgG from patients with: anti-PR3 positive renal and lung vasculitis; patients with non-vasculitic renal disease; or healthy controls. Six-days later, 39% of anti-PR3 treated mice had haematuria, compared with none of controls. There was punctate bleeding on the surface of lungs of anti-PR3 treated animals, with histological evidence of vasculitis and haemorrhage. Anti-PR3 treated mice had mild pauci-immune proliferative glomerulonephritis, with infiltration of human and mouse leukocytes. In 3 mice (17% more severe glomerular injury was present. There were no glomerular changes in controls. Human IgG from patients with anti-PR3 autoantibodies is therefore pathogenic. This model of anti-PR3 antibody-mediated vasculitis may be useful in dissecting mechanisms of microvascular injury.

Anti-proteinase 3 anti-neutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system.

LENUS (Irish Health Repository)

Little, Mark A

2012-01-01

Evidence is lacking for direct pathogenicity of human anti-proteinase-3 (PR3) antibodies in development of systemic vasculitis and granulomatosis with polyangiitis (GPA, Wegener\\'s granulomatosis). Progress in study of these antibodies in rodents has been hampered by lack of PR3 expression on murine neutrophils, and by different Fc-receptor affinities for IgG across species. Therefore, we tested whether human anti-PR3 antibodies can induce acute vasculitis in mice with a human immune system. Chimeric mice were generated by injecting human haematopoietic stem cells into irradiated NOD-scid-IL2Rγ⁻\\/⁻ mice. Matched chimera mice were treated with human IgG from patients with: anti-PR3 positive renal and lung vasculitis; patients with non-vasculitic renal disease; or healthy controls. Six-days later, 39% of anti-PR3 treated mice had haematuria, compared with none of controls. There was punctate bleeding on the surface of lungs of anti-PR3 treated animals, with histological evidence of vasculitis and haemorrhage. Anti-PR3 treated mice had mild pauci-immune proliferative glomerulonephritis, with infiltration of human and mouse leukocytes. In 3 mice (17%) more severe glomerular injury was present. There were no glomerular changes in controls. Human IgG from patients with anti-PR3 autoantibodies is therefore pathogenic. This model of anti-PR3 antibody-mediated vasculitis may be useful in dissecting mechanisms of microvascular injury.

Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

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Araki Hiromasa

2007-04-01

Full Text Available Abstract Background Proteinase-activated receptors (PARs; PAR1–4 that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT which contributes to the increase in myofibroblast population. Methods EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells. Results Stimulation of PAR with thrombin (1 U/ml or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β. Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. Conclusion PAR4 stimulation of alveolar epithelial cells induced epithelial

Identification and Characterization of Roseltide, a Knottin-type Neutrophil Elastase Inhibitor Derived from Hibiscus sabdariffa

Science.gov (United States)

Loo, Shining; Kam, Antony; Xiao, Tianshu; Nguyen, Giang K. T.; Liu, Chuan Fa; Tam, James P.

2016-01-01

Plant knottins are of therapeutic interest due to their high metabolic stability and inhibitory activity against proteinases involved in human diseases. The only knottin-type proteinase inhibitor against porcine pancreatic elastase was first identified from the squash family in 1989. Here, we report the identification and characterization of a knottin-type human neutrophil elastase inhibitor from Hibiscus sabdariffa of the Malvaceae family. Combining proteomic and transcriptomic methods, we identified a panel of novel cysteine-rich peptides, roseltides (rT1-rT8), which range from 27 to 39 residues with six conserved cysteine residues. The 27-residue roseltide rT1 contains a cysteine spacing and amino acid sequence that is different from the squash knottin-type elastase inhibitor. NMR analysis demonstrated that roseltide rT1 adopts a cystine-knot fold. Transcriptome analyses suggested that roseltides are bioprocessed by asparagine endopeptidases from a three-domain precursor. The cystine-knot structure of roseltide rT1 confers its high resistance against degradation by endopeptidases, 0.2 N HCl, and human serum. Roseltide rT1 was shown to inhibit human neutrophil elastase using enzymatic and pull-down assays. Additionally, roseltide rT1 ameliorates neutrophil elastase-stimulated cAMP accumulation in vitro. Taken together, our findings demonstrate that roseltide rT1 is a novel knottin-type neutrophil elastase inhibitor with therapeutic potential for neutrophil elastase associated diseases. PMID:27991569

The vagina of women infected with Trichomonas vaginalis has numerous proteinases and antibody to trichomonad proteinases.

Science.gov (United States)

Alderete, J F; Newton, E; Dennis, C; Neale, K A

1991-12-01

Patients with trichomoniasis have serum antibody to numerous T. vaginalis cysteine proteinases, indicating that the proteinases are expressed in vivo. It was important, therefore, to examine for the presence of soluble trichomonad proteinases and/or antibody to the proteinases in the vagina of infected women. Vaginal washes (VWs) from 20 women were examined for the presence of proteinases by electrophoresis using acrylamide co-polymerised with gelatin as the indicator system. Antibody to proteinases in VWs was detected by an immunoprecipitation assay involving protein A-bearing Staphylococcus aureus first coated with anti-human immunoglobulin G (IgG) antibody, which was then added to VWs. For VWs having soluble proteinases, the bacteria were used to determine whether immune complexes between antibody and proteinases were present. VWs without soluble proteinases were incubated with the anti-human IgG treated bacteria before adding to detergent extracts of T. vaginalis. Individual isolates from the patients examined in this study were also analysed by one- and two-dimensional electrophoresis for their proteinase content. Finally, VWs were from patients without any history of other sexually transmitted diseases (STDs) as well as from individuals having numerous other STDs, including yeast, group B streptococcus, chlamydia, and syphilis. Approximately one-third of patients had soluble proteinases in the VWs; the remaining two-thirds (70%) of patients and normal women had no detectable proteinases in VWs. Half of the patients without soluble proteinases had IgG which, when bound to S. aureus, immunoprecipitated many proteinases from a detergent extract of T. vaginalis. All soluble proteinases and those precipitated from trichomonal extracts were inhibited by inhibitors of cysteine proteinases. Finally, patients having trichomoniasis in addition to numerous other STD agents, including yeast, group B streptococcus, chlamydia, and syphilis did not have soluble proteinases

Prolonged pharmacological inhibition of cathepsin C results in elimination of neutrophil serine proteases

DEFF Research Database (Denmark)

Guarino, Carla; Hamon, Yveline; Croix, Cécile

2017-01-01

cyclopropyl nitrile CatC inhibitor almost totally lack elastase. We confirmed the elimination of neutrophil elastase-like proteases by prolonged inhibition of CatC in a non-human primate. We also showed that neutrophils lacking elastase-like protease activities were still recruited to inflammatory sites....... These preclinical results demonstrate that the disappearance of neutrophil elastase-like proteases as observed in PLS patients can be achieved by pharmacological inhibition of bone marrow CatC. Such a transitory inhibition of CatC might thus help to rebalance the protease load during chronic inflammatory diseases...

Prospeção de inibidores de serinoproteinases em folhas de leguminosas arbóreas da floresta Amazônica Prospecting serine proteinase inhibitors in leaves from leguminous trees of the Amazon forest

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Larissa Ramos Chevreuil

2011-03-01

Full Text Available Os inibidores de proteinases são proteínas extensivamente investigadas nos tecidos de estocagem, mas pouco prospectadas em outros tecidos vegetais. O objetivo deste estudo foi detectar a presença de inibidores de serinoproteinases em extratos foliares de quinze espécies de leguminosas arbóreas da Amazônia. As espécies estudadas foram: Caesalpinia echinata, C. ferrea, Cedrelinga cateniformis, Copaifera multijuga, Dinizia excelsa, Enterolobium contortisiliquum, E. maximum, E. schomburgkii, Leucaena leucocephala, Ormosia paraensis, Parkia multijuga, P. pendula, P. platycephala, Swartzia corrugata e S. polyphylla. Folhas foram coletadas, secas a 30ºC durante 48 h, trituradas e submetidas à extração com NaCl (0,15 M, 10% p/v resultando no extrato total. Ensaios foram executados para determinar a concentração de proteínas e detectar a atividade inibitória contra a tripsina e quimotripsina bovina. Os teores de proteínas bruta e solúvel nos extratos foliares variaram de 7,9 a 31,2% e 1,3 a 14,8%, respectivamente. A atividade inibitória sobre a tripsina e quimotripsina foi observada em todos os extratos foliares. Contudo, nos extratos de E. maximum, L. leucocephala, P. pendula, S. corrugata e S. polyphylla a inibição foi maior sobre a tripsina, enquanto o extrato de P. multijuga foi mais efetivo contra a quimotripsina. Nós concluímos que nos extratos foliares de leguminosas arbóreas têm inibidores de serinoproteinases e exibem potencial aplicações biotecnológicas.The proteinase inhibitors are proteins extensively investigated in tissue storage, but few prospected in other plant tissues. The aim of this study was to detect the presence of serine proteinase inhibitors in leaf extracts from fifteen species of leguminous trees of the Amazon forest. The species studied were Caesalpinia echinata, C. ferrea, Cedrelinga cateniformis, Copaifera multijuga, Dinizia excelsa, Enterolobium contortisiliquum, E. maximum, E. schomburgkii

Potential role of recombinant secretory leucoprotease inhibitor in the prevention of neutrophil mediated matrix degradation.

Science.gov (United States)

Llewellyn-Jones, C G; Lomas, D A; Stockley, R A

1994-06-01

Neutrophil elastase is able to degrade connective tissue matrices and is thought to be involved in the pathogenesis of destructive lung diseases. The ability of recombinant secretory leucoprotease inhibitor (rSLPI) to inhibit neutrophil mediated degradation of fibronectin in vitro is demonstrated and its efficacy compared with native alpha-1-proteinase inhibitor (n alpha 1-PI), recombinant alpha-1-proteinase inhibitor (r alpha 1-PI), and the chemical elastase inhibitor ICI 200,355. When preincubated with neutrophils both rSLPI and r alpha 1-PI were effective inhibitors of fibronectin degradation although n alpha 1-PI and ICI 200,355 were less effective. Recombinant SLPI was the most effective inhibitor when the cells were allowed to adhere to fibronectin before the addition of the inhibitors. Preincubation of rSLPI (0.1 mumol/l) with the fibronectin plate resulted in almost total inhibition of fibronectin degradation (reduced to 3.3 (SE 0.9)% of control). Pretreating the fibronectin plate with 1 mumol/l rSLPI, r alpha 1-PI and ICI 200,355 followed by thorough washing before the addition of cells resulted in no inhibition of fibronectin degradation with r alpha 1-PI and the ICI inhibitor, but rSLPI retained its inhibitory effect. This effect could be reduced by adding rSLPI in high pH buffer or 2 mol/1 NaCl. It is postulated that rSLPI binds to fibronectin to form a protective layer which prevents its degradation by neutrophil elastase. It may prove to be the most useful therapeutic agent in the prevention of neutrophil mediated lung damage.

Co-factor activated recombinant adenovirus proteinases

Science.gov (United States)

Anderson, Carl W.; Mangel, Walter F.

1996-08-06

This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

Characterization of a New Cell Envelope Proteinase PrtP from Lactobacillus rhamnosus CGMCC11055.

Science.gov (United States)

Guo, Tingting; Ouyang, Xudong; Xin, Yongping; Wang, Yue; Zhang, Susu; Kong, Jian

2016-09-21

Cell envelope proteinases (CEPs) play essential roles in lactic acid bacteria growth in milk and health-promoting properties of fermented dairy products. The genome of Lactobacillus rhamnosus CGMCC11055 possesses two putative CEP genes prtP and prtR2, and the PrtP displays the distinctive domain organization from PrtR2 reported. The PrtP was purified and biochemically characterized. The results showed that the optimal activity occurred at 44 °C, pH 6.5. p-Amidinophenylmethylsulfonyl fluoride obviously inhibited enzymatic activity, suggesting PrtP was a member of serine proteinases. Under the optimal conditions, β-casein was a favorite substrate over αS1- and κ-casein, and 35 oligopeptides were identified in the β-casein hydrolysate, including the phosphoserine peptide and bioactive isoleucine-proline-proline. By analysis of the amino acid sequences of those oligopeptides, proline was the preferred residue at the breakdown site. Therefore, we speculated that PrtP was a new type of CEPs from Lb. rhamnosus.

Age is the work of art? Impact of neutrophil and organism age on neutrophil extracellular trap formation.

Science.gov (United States)

Ortmann, Weronika; Kolaczkowska, Elzbieta

2018-03-01

Neutrophil extracellular traps or NETs are released by highly activated neutrophils in response to infectious agents, sterile inflammation, autoimmune stimuli and cancer. In the cells, the nuclear envelop disintegrates and decondensation of chromatin occurs that depends on peptidylarginine deiminase 4 (PAD4) and neutrophil elastase (NE). Subsequently, proteins from neutrophil granules (e.g., NE, lactoferrin and myeloperoxidase) and the nucleus (histones) bind to decondensed DNA and the whole structure is ejected from the cell. The DNA decorated with potent antimicrobials and proteases can act to contain dissemination of infection and in sterile inflammation NETs were shown to degrade cytokines and chemokines via serine proteases. On the other hand, overproduction of NETs, or their inadequate removal and prolonged presence in vasculature or tissues, can lead to bystander damage or even initiation of diseases. Considering the pros and cons of NET formation, it is of relevance if the stage of neutrophil maturation (immature, mature and senescent cells) affects the capacity to produce NETs as the cells of different age-related phenotypes dominate in given (pathological) conditions. Moreover, the immune system of neonates and elderly individuals is weaker than in adulthood. Is the same pattern followed when it comes to NETs? The overall importance of individual and neutrophil age on the capacity to release NETs is reviewed in detail and the significance of these facts is discussed.

SwissProt search result: AK061101 [KOME

Lifescience Database Archive (English)

Full Text Available AK061101 006-207-A12 (P15156) Calcium-dependent serine proteinase precursor (EC 3.4....21.-) (CASP) [Contains: Calcium-dependent serine proteinase heavy chain; Calcium-dependent serine proteinase light chain] CASP_MESAU 4e-16 ...

SwissProt search result: AK112119 [KOME

Lifescience Database Archive (English)

Full Text Available AK112119 006-303-H09 (P15156) Calcium-dependent serine proteinase precursor (EC 3.4....21.-) (CASP) [Contains: Calcium-dependent serine proteinase heavy chain; Calcium-dependent serine proteinase light chain] CASP_MESAU 3e-12 ...

SwissProt search result: AK112005 [KOME

Lifescience Database Archive (English)

Full Text Available AK112005 006-202-F10 (P15156) Calcium-dependent serine proteinase precursor (EC 3.4....21.-) (CASP) [Contains: Calcium-dependent serine proteinase heavy chain; Calcium-dependent serine proteinase light chain] CASP_MESAU 5e-12 ...

Evidence for multiple modes of neutrophil serine protease recognition by the EAP family of Staphylococcal innate immune evasion proteins.

Science.gov (United States)

Stapels, Daphne A C; Woehl, Jordan L; Milder, Fin J; Tromp, Angelino T; van Batenburg, Aernoud A; de Graaf, Wilco C; Broll, Samuel C; White, Natalie M; Rooijakkers, Suzan H M; Geisbrecht, Brian V

2018-02-01

Neutrophils contain high levels of chymotrypsin-like serine proteases (NSPs) within their azurophilic granules that have a multitude of functions within the immune system. In response, the pathogen Staphylococcus aureus has evolved three potent inhibitors (Eap, EapH1, and EapH2) that protect the bacterium as well as several of its secreted virulence factors from the degradative action of NSPs. We previously showed that these so-called EAP domain proteins represent a novel class of NSP inhibitors characterized by a non-covalent inhibitory mechanism and a distinct target specificity profile. Based upon high levels of structural homology amongst the EAP proteins and the NSPs, as well as supporting biochemical data, we predicted that the inhibited complex would be similar for all EAP/NSP pairs. However, we present here evidence that EapH1 and EapH2 bind the canonical NSP, Neutrophil Elastase (NE), in distinct orientations. We discovered that alteration of EapH1 residues at the EapH1/NE interface caused a dramatic loss of affinity and inhibition of NE, while mutation of equivalent positions in EapH2 had no effect on NE binding or inhibition. Surprisingly, mutation of residues in an altogether different region of EapH2 severely impacted both the NE binding and inhibitory properties of EapH2. Even though EapH1 and EapH2 bind and inhibit NE and a second NSP, Cathepsin G, equally well, neither of these proteins interacts with the structurally related, but non-proteolytic granule protein, azurocidin. These studies expand our understanding of EAP/NSP interactions and suggest that members of this immune evasion protein family are capable of diverse target recognition modes. © 2017 The Protein Society.

Neutrophil-Derived Proteases Escalate Inflammation through Activation of IL-36 Family Cytokines

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Conor M. Henry

2016-02-01

Full Text Available Recent evidence has strongly implicated the IL-1 family cytokines IL-36α, IL-36β, and IL-36γ as key initiators of skin inflammation. Similar to the other members of the IL-1 family, IL-36 cytokines are expressed as inactive precursors and require proteolytic processing for activation; however, the responsible proteases are unknown. Here, we show that IL-36α, IL-36β, and IL-36γ are activated differentially by the neutrophil granule-derived proteases cathepsin G, elastase, and proteinase-3, increasing their biological activity ∼500-fold. Active IL-36 promoted a strong pro-inflammatory signature in primary keratinocytes and was sufficient to perturb skin differentiation in a reconstituted 3D human skin model, producing features resembling psoriasis. Furthermore, skin eluates from psoriasis patients displayed significantly elevated cathepsin G-like activity that was sufficient to activate IL-36β. These data identify neutrophil granule proteases as potent IL-36-activating enzymes, adding to our understanding of how neutrophils escalate inflammatory reactions. Inhibition of neutrophil-derived proteases may therefore have therapeutic benefits in psoriasis.

Gasdermin D Exerts Anti-inflammatory Effects by Promoting Neutrophil Death

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Hiroto Kambara

2018-03-01

Full Text Available Summary: Gasdermin D (GSDMD is considered a proinflammatory factor that mediates pyroptosis in macrophages to protect hosts from intracellular bacteria. Here, we reveal that GSDMD deficiency paradoxically augmented host responses to extracellular Escherichia coli, mainly by delaying neutrophil death, which established GSDMD as a negative regulator of innate immunity. In contrast to its activation in macrophages, in which activated inflammatory caspases cleave GSDMD to produce an N-terminal fragment (GSDMD-cNT to trigger pyroptosis, GSDMD cleavage and activation in neutrophils was caspase independent. It was mediated by a neutrophil-specific serine protease, neutrophil elastase (ELANE, released from cytoplasmic granules into the cytosol in aging neutrophils. ELANE-mediated GSDMD cleavage was upstream of the caspase cleavage site and produced a fully active ELANE-derived NT fragment (GSDMD-eNT that induced lytic cell death as efficiently as GSDMD-cNT. Thus, GSDMD is pleiotropic, exerting both pro- and anti-inflammatory effects that make it a potential target for antibacterial and anti-inflammatory therapies. : Kambara et al. find that GSDMD deficiency augments host responses to extracellular Escherichia coli, mainly by delaying neutrophil death, establishing GSDMD as a negative regulator of innate immunity. GSDMD cleavage and activation in neutrophils is mediated by ELANE, released from cytoplasmic granules into the cytosol in aging neutrophils. Keywords: GSDMD, neutrophil death, neutrophil elastase, innate immunity, host defense

Anti-neutrophil cytoplasmic antibodies in rheumatoid arthritis: two case reports and review of literature

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Spoerl David

2012-12-01

Full Text Available Abstract Background Anti-neutrophil cytoplasmic antibodies are typically detected in anti-neutrophil cytoplasmic antibody associated vasculitis, but are also present in a number of chronic inflammatory non-vasculitic conditions like rheumatoid arthritis. Rare cases of granulomatosis with polyangiitis (formerly known as Wegener’s granulomatosis, a vasculitic disorder frequently associated with the presence of anti-neutrophil cytoplasmic antibodies in patients with rheumatoid arthritis have been described in literature. Case presentation We report two middle-aged female patients with rheumatoid arthritis who developed anti-neutrophil cytoplasmic antibodies and symptoms reminiscent of granulomatosis with polyangiitis. Despite the lack of antibodies specific for proteinase 3 and the absence of a classical histology, we report a probable case of granulomatosis with polyangiitis in the first patient, and consider rheumatoid vasculitis in the second patient. Conclusion Taken together with previous reports, these cases highlight that anti-neutrophil cytoplasmic antibodies have to be evaluated very carefully in patients with rheumatoid arthritis. In this context, anti-neutrophil cytoplasmic antibodies detected by indirect immunofluorescence appear to have a low diagnostic value for granulomatosis with polyangiitis. Instead they may have prognostic value for assessing the course of rheumatoid arthritis.

HOMOLOGY MODELING AND PROTEIN ENGINEERING STRATEGY OF SUBTILASES, THE FAMILY OF SUBTILISIN-LIKE SERINE PROTEINASES

NARCIS (Netherlands)

SIEZEN, RJ; DEVOS, WM; LEUNISSEN, JAM

1991-01-01

Subtilases are members of the family of subtilisin-like serine proteases. Presently, > 50 subtilases are known, > 40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase

Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis

Science.gov (United States)

Millet, Arnaud; Martin, Katherine R.; Bonnefoy, Francis; Saas, Philippe; Mocek, Julie; Alkan, Manal; Terrier, Benjamin; Kerstein, Anja; Tamassia, Nicola; Satyanarayanan, Senthil Kumaran; Ariel, Amiram; Ribeil, Jean-Antoine; Guillevin, Loïc; Cassatella, Marco A.; Mueller, Antje; Thieblemont, Nathalie; Lamprecht, Peter; Mouthon, Luc; Perruche, Sylvain; Witko-Sarsat, Véronique

2015-01-01

Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating CD4+ T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology. PMID:26436651

Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases

NARCIS (Netherlands)

Siezen, Roland J.; Vos, Willem M. de; Leunissen, Jack A.M.; Dijkstra, Bauke W.

1991-01-01

Subtilases are members of the family of subtilisin-like serine proteases. Presently, >50 subtilases are known, >40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase and

CD177 modulates human neutrophil migration through activation-mediated integrin and chemoreceptor regulation.

Science.gov (United States)

Bai, Ming; Grieshaber-Bouyer, Ricardo; Wang, Junxia; Schmider, Angela B; Wilson, Zachary S; Zeng, Liling; Halyabar, Olha; Godin, Matthew D; Nguyen, Hung N; Levescot, Anaïs; Cunin, Pierre; Lefort, Craig T; Soberman, Roy J; Nigrovic, Peter A

2017-11-09

CD177 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed by a variable proportion of human neutrophils that mediates surface expression of the antineutrophil cytoplasmic antibody antigen proteinase 3. CD177 associates with β2 integrins and recognizes platelet endothelial cell adhesion molecule 1 (PECAM-1), suggesting a role in neutrophil migration. However, CD177 pos neutrophils exhibit no clear migratory advantage in vivo, despite interruption of in vitro transendothelial migration by CD177 ligation. We sought to understand this paradox. Using a PECAM-1-independent transwell system, we found that CD177 pos and CD177 neg neutrophils migrated comparably. CD177 ligation selectively impaired migration of CD177 pos neutrophils, an effect mediated through immobilization and cellular spreading on the transwell membrane. Correspondingly, CD177 ligation enhanced its interaction with β2 integrins, as revealed by fluorescence lifetime imaging microscopy, leading to integrin-mediated phosphorylation of Src and extracellular signal-regulated kinase (ERK). CD177-driven cell activation enhanced surface β2 integrin expression and affinity, impaired internalization of integrin attachments, and resulted in ERK-mediated attenuation of chemokine signaling. We conclude that CD177 signals in a β2 integrin-dependent manner to orchestrate a set of activation-mediated mechanisms that impair human neutrophil migration. © 2017 by The American Society of Hematology.

Processing of predicted substrates of fungal Kex2 proteinases from Candida albicans, C. glabrata, Saccharomyces cerevisiae and Pichia pastoris

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Bader Oliver

2008-07-01

Full Text Available Abstract Background Kexin-like proteinases are a subfamily of the subtilisin-like serine proteinases with multiple regulatory functions in eukaryotes. In the yeast Saccharomyces cerevisiae the Kex2 protein is biochemically well investigated, however, with the exception of a few well known proteins such as the α-pheromone precursors, killer toxin precursors and aspartic proteinase propeptides, very few substrates are known. Fungal kex2 deletion mutants display pleiotropic phenotypes that are thought to result from the failure to proteolytically activate such substrates. Results In this study we have aimed at providing an improved assembly of Kex2 target proteins to explain the phenotypes observed in fungal kex2 deletion mutants by in vitro digestion of recombinant substrates from Candida albicans and C. glabrata. We identified CaEce1, CA0365, one member of the Pry protein family and CaOps4-homolog proteins as novel Kex2 substrates. Conclusion Statistical analysis of the cleavage sites revealed extended subsite recognition of negatively charged residues in the P1', P2' and P4' positions, which is also reflected in construction of the respective binding pockets in the ScKex2 enzyme. Additionally, we provide evidence for the existence of structural constrains in potential substrates prohibiting proteolysis. Furthermore, by using purified Kex2 proteinases from S. cerevisiae, P. pastoris, C. albicans and C. glabrata, we show that while the substrate specificity is generally conserved between organisms, the proteinases are still distinct from each other and are likely to have additional unique substrate recognition.

The roles of serine protease, intracellular and extracellular phenoloxidase in activation of prophenoloxidase system, and characterization of phenoloxidase from shrimp haemocytes induced by lipopolysaccharide or dopamine

Science.gov (United States)

Xie, Peng; Pan, Luqing; Xu, Wujie; Yue, Feng

2013-09-01

We investigated the effects of lipopolysaccharide (LPS) and dopamine (DA) on the activation of the prophenoloxidase (proPO) system of Litopenaeus vannamei. LPS and DA were shown with a negative dose-dependent effect on hyalne cells (HC), semi-granular cells (SGC), large granular cells (LGC), and total haemocyte count (THC). When haemocytes were treated with LPS or DA, serine proteinase activity and intracellular phenoloxidase (PO) activity were significantly reduced, but extracellular PO activity increased significantly. These findings indicated that the reduction in haemocyte counts was mainly because of the degranulation and activation of the proPO system from semi-granule and large granule cells. The PKC inhibitor, chelerythrine, and the TPK inhibitor, genistein, had an inhibitory effect on extracellular PO activity, while serine proteinase and intracellular PO activity increased. This suggests that the LPS and DA induce the activation of proPO in haemocytes via PKC and TPK-related signaling pathways, but serine proteinase may be activated only by PKC, as the genistein effects were not statistically significant. Electrophoresis analysis revealed that POs induced by LPS or DA have the same molecular mass and high diphenolase activity. Two PO bands at 526 kDa and 272 kDa were observed in PAGE, while in the haemocyte lysate supernatant (HLS), only a 272-kDa band was observed. This band was resolved after SDS-PAGE under non-reducing and reducing conditions into two groups of POs, 166 kDa and 126 kDa, and 78.1 kDa and 73.6 kDa, respectively, suggesting that PO in L. vannamei is an oligomer, which may have different compositions intra- and extracellularly.

[Ulysses retrotransposon aspartate proteinase (Drosophila virilis)].

Science.gov (United States)

Volkov, D A; Savvateeva, L V; Dergousova, N I; Rumsh, L D

2002-01-01

Retrotransposones are mobile genetic elements occurring in genomes of bacteria, plants or animals. Retrotransposones were found to contain nucleotide sequences encoding proteins which are homological to retroviral aspartic proteinases. Our research has been focused on Ulysses which is mobile genetic element found in Drosophila virilis. We suggested a primary structure of Ulysses proteinase using comparative analysis of amino acid sequences of retroviral proteinases and proteinases from retrotransposones. The appropriate cDNA fragment has been cloned and expressed in E. coli. The purification of recombinant protein (12 kD) has been carried out by affinity chromatography using pepstatine-agarose. The obtained protein has proteolytic activity at optimum pH 5.5 like the majority of aspartic proteinases.

Protective effects of an aptamer inhibitor of neutrophil elastase in lung inflammatory injury

DEFF Research Database (Denmark)

Bless, N M; Smith, D; Charlton, J

1997-01-01

Neutrophils play an important part in the development of acute inflammatory injury. Human neutrophils contain high levels of the serine protease elastase, which is stored in azurophilic granules and is secreted in response to inflammatory stimuli. Elastase is capable of degrading many components...... of extracellular matrix [1-4] and has cytotoxic effects on endothelial cells [5-7] and airway epithelial cells. Three types of endogenous protease inhibitors control the activity of neutrophil elastase, including alpha-1 protease inhibitor (alpha-1PI), alpha-2 macroglobulin and secreted leukoproteinase inhibitor...... (SLPI) [8-10]. A disturbed balance between neutrophil elastase and these inhibitors has been found in various acute clinical conditions (such as adult respiratory syndrome and ischemia-reperfusion injury) and in chronic diseases. We investigated the effect of NX21909, a selected oligonucleotide (aptamer...

Neutrophil-Derived Proteases Escalate Inflammation through Activation of IL-36 Family Cytokines.

Science.gov (United States)

Henry, Conor M; Sullivan, Graeme P; Clancy, Danielle M; Afonina, Inna S; Kulms, Dagmar; Martin, Seamus J

2016-02-02

Recent evidence has strongly implicated the IL-1 family cytokines IL-36α, IL-36β, and IL-36γ as key initiators of skin inflammation. Similar to the other members of the IL-1 family, IL-36 cytokines are expressed as inactive precursors and require proteolytic processing for activation; however, the responsible proteases are unknown. Here, we show that IL-36α, IL-36β, and IL-36γ are activated differentially by the neutrophil granule-derived proteases cathepsin G, elastase, and proteinase-3, increasing their biological activity ~500-fold. Active IL-36 promoted a strong pro-inflammatory signature in primary keratinocytes and was sufficient to perturb skin differentiation in a reconstituted 3D human skin model, producing features resembling psoriasis. Furthermore, skin eluates from psoriasis patients displayed significantly elevated cathepsin G-like activity that was sufficient to activate IL-36β. These data identify neutrophil granule proteases as potent IL-36-activating enzymes, adding to our understanding of how neutrophils escalate inflammatory reactions. Inhibition of neutrophil-derived proteases may therefore have therapeutic benefits in psoriasis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Antibacterial activity of antileukoprotease.

Science.gov (United States)

Hiemstra, P S; Maassen, R J; Stolk, J; Heinzel-Wieland, R; Steffens, G J; Dijkman, J H

1996-01-01

Antileukoprotease (ALP), or secretory leukocyte proteinase inhibitor, is an endogenous inhibitor of serine proteinases that is present in various external secretions. ALP, one of the major inhibitors of serine proteinases present in the human lung, is a potent reversible inhibitor of elastase and, to a lesser extent, of cathepsin G. In equine neutrophils, an antimicrobial polypeptide that has some of the characteristics of ALP has been identified (M. A. Couto, S. S. L. Harwig, J. S. Cullor, J. P. Hughes, and R. I. Lehrer, Infect. Immun. 60:5042-5047, 1992). This report, together with the cationic nature of ALP, led us to investigate the antimicrobial activity of ALP. ALP was shown to display marked in vitro antibacterial activity against Escherichia coli and Staphylococcus aureus. On a molar basis, the activity of ALP was lower than that of two other cationic antimicrobial polypeptides, lysozyme and defensin. ALP comprises two homologous domains: its proteinase-inhibitory activities are known to be located in the second COOH-terminal domain, and the function of its first NH2-terminal domain is largely unknown. Incubation of intact ALP or its isolated first domain with E. coli or S. aureus resulted in killing of these bacteria, whereas its second domain displayed very little antibacterial activity. Together these data suggest a putative antimicrobial role for the first domain of ALP and indicate that its antimicrobial activity may equip ALP to contribute to host defense against infection. PMID:8890201

Potato type I and II proteinase inhibitors: modulating plant physiology and host resistance.

Science.gov (United States)

Turra, David; Lorito, Matteo

2011-08-01

Serine protease inhibitors (PIs) are a large and complex group of plant proteins. Members of the potato type I (Pin1) and II (Pin2) proteinase inhibitor families are among the first and most extensively characterized plant PIs. Many insects and phytopathogenic microorganisms use intracellular and extracellular serine proteases playing important roles in pathogenesis. Plants, however, are able to fight these pathogens through the activation of an intricate defence system that leads to the accumulation of various PIs, including Pin1 and Pin2. Several transgenic plants over-expressing members of the Pin1 and Pin2 families have been obtained in the last twenty years and their enhanced defensive capabilities demonstrated against insects, fungi and bacteria. Furthermore, Pin1 and Pin2 genetically engineered plants showed altered regulation of different plant physiological processes (e.g., dehydratation response, programmed cell death, plant growth, trichome density and branching), supporting an endogenous role in various plant species in addition to the well established defensive one. This review summarizes the current knowledge about Pin1 and Pin2 structure, the role of these proteins in plant defence and physiology, and their potential exploitation in biotechnology.

Autophagy Mediates Interleukin-1β Secretion in Human Neutrophils

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Leonardo Iula

2018-02-01

Full Text Available Interleukin-1β (IL-1β, a major pro-inflammatory cytokine, is a leaderless cytosolic protein whose secretion does not follow the classical endoplasmic reticulum-to-Golgi pathway, and for which a canonical mechanism of secretion remains to be established. Neutrophils are essential players against bacterial and fungi infections. These cells are rapidly and massively recruited from the circulation into infected tissues and, beyond of displaying an impressive arsenal of toxic weapons effective to kill pathogens, are also an important source of IL-1β in infectious conditions. Here, we analyzed if an unconventional secretory autophagy mechanism is involved in the exportation of IL-1β by these cells. Our findings indicated that inhibition of autophagy with 3-methyladenine and Wortmannin markedly reduced IL-1β secretion induced by LPS + ATP, as did the disruption of the autophagic flux with Bafilomycin A1 and E64d. These compounds did not noticeable affect neutrophil viability ruling out that the effects on IL-1β secretion were due to cell death. Furthermore, VPS34IN-1, a specific autophagy inhibitor, was still able to reduce IL-1β secretion when added after it was synthesized. Moreover, siRNA-mediated knockdown of ATG5 markedly reduced IL-1β secretion in neutrophil-differentiated PLB985 cells. Upon LPS + ATP stimulation, IL-1β was incorporated to an autophagic compartment, as was revealed by its colocalization with LC3B by confocal microscopy. Overlapping of IL-1β-LC3B in a vesicular compartment peaked before IL-1β increased in culture supernatants. On the other hand, stimulation of autophagy by cell starvation augmented the colocalization of IL-1β and LC3B and then promoted neutrophil IL-1β secretion. In addition, specific ELISAs indicated that although both IL-1β and pro-IL-1β are released to culture supernatants upon neutrophil stimulation, autophagy only promotes IL-1β secretion. Furthermore, the serine proteases inhibitor

In Vitro Activities against Cystic Fibrosis Pathogens of Synthetic Host Defence Propeptides Processed by Neutrophil Elastase.

LENUS (Irish Health Repository)

Desgranges, Stephane

2011-02-22

The antimicrobial and haemolytic activities of a host defence peptide can be controlled by modification as a propeptide of reduced net charge which can be processed by neutrophil elastase, a serine protease involved in chronic airway inflammation and infections associated with cystic fibrosis.

Isolation, cloning and structural characterisation of boophilin, a multifunctional Kunitz-type proteinase inhibitor from the cattle tick.

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Sandra Macedo-Ribeiro

Full Text Available Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine alpha-thrombin.boophilin complex, refined at 2.35 A resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S(1 pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9 degrees and is displaced by 6 A, while the C-terminal domain rotates almost 6 degrees accompanied by a 3 A displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P(1 residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin.boophilin.trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo.

Angiogenic activity of bFGF and VEGF suppressed by proteolytic cleavage by neutrophil elastase

International Nuclear Information System (INIS)

Ai, Shingo; Cheng Xianwu; Inoue, Aiko; Nakamura, Kae; Okumura, Kenji; Iguchi, Akihisa; Murohara, Toyoaki; Kuzuya, Masafumi

2007-01-01

Neutrophil elastase (NE), a serine protease released from the azurophil granules of activated neutrophil, proteolytically cleaves multiple cytokines, and cell surface proteins. In the present study, we examined whether NE affects the biological abilities of angiogenic growth factors such as basic-fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). NE degraded bFGF and VEGF in a time- and concentration-dependent manner, and these degradations were suppressed by sivelestat, a synthetic inhibitor of NE. The bFGF- or VEGF-mediated proliferative activity of human umbilical vein endothelial cells was inhibited by NE, and the activity was recovered by sivelestat. Furthermore, NE reduced the bFGF- or VEGF-induced tubulogenic response of the mice aortas, ex vivo angiogenesis assay, and these effects were also recovered by sivelestat. Neutrophil-derived NE degraded potent angiogenic factors, resulting in loss of their angiogenic activity. These findings provide additional insight into the role played by neutrophils in the angiogenesis process at sites of inflammation

Vasculites e ANCA – a propósito de um caso clínico

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Fernando Matos

1996-01-01

Full Text Available RESUMO: Apreseotamos o caso clínico de um doente de 68 anos de idade, com glomerulonefrite rapidamente progressiva e hemorragia pulmonar, presença de anticorpos anti-citoplasma dos neutrófilos (padrão perinuclear com especificidade anti-mieloperoxidase, e anticorpos anti-cardiolipina. A histologia renal confirmou o diagnóstico de poliarterite microscópica. A propósito deste caso, são feitas algumas considerações sobre a utilidade dos auto-anticorpos no diagnóstico das diferentes formas de vasculite. ABSRTRACT: We present the case report of a 68 year old man with rapidly progressive glomerulonephritis and pulmonary haemorrhage, with anti-neutrophil cytoplasmic auto-antibodies, perinuclear pattern with myeloperoxidase specifity, and anti-cardiolipin antibodies. Renal histology supported the diagnosis of microscopic polyarteritis. In reference to this case, some considerations regarding the usefullness of autoantibodies in different forms of vasculitis are made. Palavras-chave: Autoanticorpos, Antoantigénios, Endotélio Vascular, Mieloperoxidase, Neutrófilos, Serina proteinase, Vasculites, Key-Words: Autoantibodies, Autoantiges, Endothelium, vascular, Myelope-roxidase, Neutrophils, Serine proteinase, Vasculitis

A specific p47phox -serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites

DEFF Research Database (Denmark)

Dang, Pham My-Chan; Stensballe, Allan; Boussetta, Tarek

2006-01-01

mass spectrometry to show that GM-CSF and TNF-alpha induce phosphorylation of Ser345 on p47phox, a cytosolic component of NADPH oxidase, in human neutrophils. As Ser345 is located in the MAPK consensus sequence, we tested the effects of MAPK inhibitors. Inhibitors of the ERK1/2 pathway abrogated GM......Neutrophil NADPH oxidase plays a key role in host defense and in inflammation by releasing large amounts of superoxide and other ROSs. Proinflammatory cytokines such as GM-CSF and TNF-alpha prime ROS production by neutrophils through unknown mechanisms. Here we used peptide sequencing by tandem...

Cysteine proteinases and cystatins

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Adeliana S. Oliveira

2003-01-01

Full Text Available This review describeds the definition, localization, functions and examples of cysteine proteinases and their protein inhibitors in vertebrate, non-vertebrate animals and plants. These inhibitors are related with defense mechanisms of plant against pests. It also describes the factors involved in the specific cysteine proteinase-cystatin interaction and high degree of affinity and large specificity in this interaction which are not only represented by the compatibility between amino acid residues of the active site involved in catalysis, but also of all amino acid residues that participante in the enzyme-inhibitor interaction.Nesta revisão foram descritas definições, localizações, funções e exemplos de proteinases cisteínicas e suas proteinas inibidoras em animais vertebrados e invertebrados e plantas. Tratamos principalmente com aqueles inibidores que são relatados com o mecanismo de defesa da planta contra pestes. Em adição, comentamos sobre recentes trabalhos que contribuíram para uma melhor compreenção dos fatores envolvidos na interação específica proteinase cisteínica-cistatina. Por outro lado, chamamos atenção para o alto grau de afinidade e grande especificidade na interação que não são apenas representadas pela compatibilidade entre os residuos de aminoácidos do sítio ativo envolvidos na catalise, mas também de todos os resíduos de aminoácidos que participam da interação enzima-inibidor.

Detection and Characterization of Bacterial Proteinases Using Zymography.

Science.gov (United States)

Madanan, Madathiparambil G; Mechoor, Ambili

2017-01-01

Proteinases play a crucial role in invasion and pathogenesis of bacteria, especially the extracellular and membrane-bound forms. Analysis of these proteinases demands the isolation by retaining the enzymatic activity. The isolation procedures maintaining the native structure of the enzyme in its soluble form are also of extreme importance. The qualitative analyses of these proteinases are carried out by electrophoresis and zymography. Enzymatic characterization based on the effect of inhibitors and activators on gelatinase activity also can be assessed using this zymography. The membrane-bound proteinases can be isolated in their native and soluble form, still retaining the activity using 6-aminocaproic acid and sodium deoxycholate; the procedure of which is explained in this chapter.

Manipulation of Neutrophils by Porphyromonas gingivalis in the Development of Periodontitis.

Science.gov (United States)

Sochalska, Maja; Potempa, Jan

2017-01-01

The pathogenesis of the chronic periodontal disease is associated with a skewed host inflammatory response to periodontal pathogens, such as Porphyromonas gingivalis , that accounts for the majority of periodontal tissue damage. Neutrophils are the most abundant leukocytes in periodontal pockets and depending on the stage of the disease, also plentiful PMNs are present in the inflamed gingival tissue and the gingival crevice. They are the most efficient phagocytes and eliminate pathogens by a variety of means, which are either oxygen-dependent or -independent. However, these secretory lethal weapons do not strictly discriminate between pathogens and host tissue. Current studies describe conflicting findings about neutrophil involvement in periodontal disease. On one hand literature indicate that hyper-reactive neutrophils are the main immune cell type responsible for this observed tissue damage and disease progression. Deregulation of neutrophil survival and functions, such as chemotaxis, migration, secretion of antimicrobial peptides or enzymes, and production of reactive oxygen species, contribute to observed tissue injury and the clinical signs of periodontal disease. On the other hand neutrophils deficiencies in patients and mice also result in periodontal phenotype. Therefore, P. gingivalis represents a periodontal pathogen that manipulates the immune responses of PMNs, employing several virulence factors, such as gingipains, serine proteases, lipid phosphatases, or fimbriae. This review will sum up studies devoted to understanding different strategies utilized by P. gingivalis to manipulate PMNs survival and functions in order to inhibit killing by a granular content, prolong inflammation, and gain access to nutrient resources.

The role of secretory leukocyte proteinase inhibitor and elafin (elastase-specific inhibitor/skin-derived antileukoprotease as alarm antiproteinases in inflammatory lung disease

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Sallenave Jean-Michel

2000-08-01

Full Text Available Abstract Secretory leukocyte proteinase inhibitor and elafin are two low-molecular-mass elastase inhibitors that are mainly synthesized locally at mucosal sites. It is thought that their physicochemical properties allow them to efficiently inhibit target enzymes, such as neutrophil elastase, released into the interstitium. Historically, in the lung, these inhibitors were first purified from secretions of patients with chronic obstructive pulmonary disease and cystic fibrosis. This suggested that they might be important in controlling excessive neutrophil elastase release in these pathologies. They are upregulated by 'alarm signals' such as bacterial lipopolysaccharides, and cytokines such as interleukin-1 and tumor necrosis factor and have been shown to be active against Gram-positive and Gram-negative bacteria, so that they have joined the growing list of antimicrobial 'defensin-like' peptides produced by the lung. Their site of synthesis and presumed functions make them very attractive candidates as potential therapeutic agents under conditions in which the excessive release of elastase by neutrophils might be detrimental. Because of its natural tropism for the lung, the use of adenovirus-mediated gene transfer is extremely promising in such applications.

Autoactivation of proteinase A initiates activation of yeast vacuolar zymogens

DEFF Research Database (Denmark)

van den Hazel, H B; Kielland-Brandt, Morten; Winther, Jakob R.

1992-01-01

The Saccharomyces cerevisiae PEP4 gene encodes proteinase A, an aspartyl protease. pep4 mutants are defective in the activation of many vacuolar hydrolases, including proteinase B. We have expressed a pep4 mutation which directs the accumulation of pro-proteinase A with a defective active site. Co...

New aspartic proteinase of Ulysses retrotransposon from Drosophila virilis.

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Volkov, D A; Dergousova, N I; Rumsh, L D

2004-06-01

This work is focused on the investigation of a proteinase of Ulysses mobile genetic element from Drosophila virilis. The primary structure of this proteinase is suggested based on comparative analysis of amino acid sequences of aspartic proteinases from retroviruses and retrotransposons. The corresponding cDNA fragment has been cloned and expressed in E. coli. The protein accumulated in inclusion bodies. The recombinant protein (12 kD) was subjected to refolding and purified by affinity chromatography on pepstatin-agarose. Proteolytic activity of the protein was determined using oligopeptide substrates melittin and insulin B-chain. It was found that the maximum of the proteolytic activity is displayed at pH 5.5 as for the majority of aspartic proteinases. We observed that hydrolysis of B-chain of insulin was totally inhibited by pepstatin A in the micromolar concentration range. The molecular weight of the monomer of the Ulysses proteinase was determined by MALDI-TOF mass-spectrometry.

Neutrophils kill the parasite Trichomonas vaginalis using trogocytosis

Science.gov (United States)

Mercer, Frances; Ng, Shek Hang; Brown, Taylor M.; Boatman, Grace; Johnson, Patricia J.

2018-01-01

T. vaginalis, a human-infective parasite, causes the most common nonviral sexually transmitted infection (STI) worldwide and contributes to adverse inflammatory disorders. The immune response to T. vaginalis is poorly understood. Neutrophils (polymorphonuclear cells [PMNs]) are the major immune cell present at the T. vaginalis–host interface and are thought to clear T. vaginalis. However, the mechanism of PMN clearance of T. vaginalis has not been characterized. We demonstrate that human PMNs rapidly kill T. vaginalis in a dose-dependent, contact-dependent, and neutrophil extracellular trap (NET)-independent manner. In contrast to phagocytosis, we observed that PMN killing of T. vaginalis involves taking “bites” of T. vaginalis prior to parasite death, using trogocytosis to achieve pathogen killing. Both trogocytosis and parasite killing are dependent on the presence of PMN serine proteases and human serum factors. Our analyses provide the first demonstration, to our knowledge, of a mammalian phagocyte using trogocytosis for pathogen clearance and reveal a novel mechanism used by PMNs to kill a large, highly motile target. PMID:29408891

Elevated Systemic Levels of Eosinophil, Neutrophil, and Mast Cell Granular Proteins in Strongyloides Stercoralis Infection that Diminish following Treatment.

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Rajamanickam, Anuradha; Munisankar, Saravanan; Bhootra, Yukthi; Dolla, Chandra Kumar; Nutman, Thomas B; Babu, Subash

2018-01-01

Infection with the helminth parasite Strongyloides stercoralis ( Ss ) is commonly clinically asymptomatic that is often accompanied by peripheral eosinophilia. Granulocytes are activated during helminth infection and can act as immune effector cells. Plasma levels of eosinophil and neutrophil granular proteins convey an indirect measure of granulocyte degranulation and are prominently augmented in numerous helminth-infected patients. In this study, we sought to examine the levels of eosinophil, neutrophil, and mast cell activation-associated granule proteins in asymptomatic Ss infection and to understand their kinetics following anthelmintic therapy. To this end, we measured the plasma levels of eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase, eosinophil major basic protein, neutrophil elastase, myeloperoxidase, neutrophil proteinase-3, mast cell tryptase, leukotriene C4, and mast cell carboxypeptidase-A3 in individuals with asymptomatic Ss infection or without Ss infection [uninfected (UN)]. We also estimated the levels of all of these analytes in infected individuals following definitive treatment of Ss infection. We demonstrated that those infected individuals have significantly enhanced plasma levels of eosinophil cationic protein, eosinophil-derived neurotoxin, eosinophil peroxidase, eosinophil major basic protein, elastase, myeloperoxidase, mast cell tryptase, leukotriene C4, and carboxypeptidase-A3 compared to UN individuals. Following the treatment of Ss infection, each of these granulocyte-associated proteins drops significantly. Our data suggest that eosinophil, neutrophil, and mast cell activation may play a role in the response to Ss infection.

Functional specialization and evolution of leader proteinases in the family Closteroviridae.

Science.gov (United States)

Peng, C W; Peremyslov, V V; Mushegian, A R; Dawson, W O; Dolja, V V

2001-12-01

Members of the Closteroviridae and Potyviridae families of the plant positive-strand RNA viruses encode one or two papain-like leader proteinases. In addition to a C-terminal proteolytic domain, each of these proteinases possesses a nonproteolytic N-terminal domain. We compared functions of the several leader proteinases using a gene swapping approach. The leader proteinase (L-Pro) of Beet yellows virus (BYV; a closterovirus) was replaced with L1 or L2 proteinases of Citrus tristeza virus (CTV; another closterovirus), P-Pro proteinase of Lettuce infectious yellows virus (LIYV; a crinivirus), and HC-Pro proteinase of Tobacco etch virus (a potyvirus). Each foreign proteinase efficiently processed the chimeric BYV polyprotein in vitro. However, only L1 and P-Pro, not L2 and HC-Pro, were able to rescue the amplification of the chimeric BYV variants. The combined expression of L1 and L2 resulted in an increased RNA accumulation compared to that of the parental BYV. Remarkably, this L1-L2 chimera exhibited reduced invasiveness and inability to move from cell to cell. Similar analyses of the BYV hybrids, in which only the papain-like domain of L-Pro was replaced with those derived from L1, L2, P-Pro, and HC-Pro, also revealed functional specialization of these domains. In subcellular-localization experiments, distinct patterns were observed for the leader proteinases of BYV, CTV, and LIYV. Taken together, these results demonstrated that, in addition to a common proteolytic activity, the leader proteinases of closteroviruses possess specialized functions in virus RNA amplification, virus invasion, and cell-to-cell movement. The phylogenetic analysis suggested that functionally distinct L1 and L2 of CTV originated by a gene duplication event.

Cleavage preference distinguishes the two-component NS2B-NS3 serine proteinases of Dengue and West Nile viruses.

Science.gov (United States)

Shiryaev, Sergey A; Kozlov, Igor A; Ratnikov, Boris I; Smith, Jeffrey W; Lebl, Michal; Strongin, Alex Y

2007-02-01

Regulated proteolysis of the polyprotein precursor by the NS2B-NS3 protease is required for the propagation of infectious virions. Unless the structural and functional parameters of NS2B-NS3 are precisely determined, an understanding of its functional role and the design of flaviviral inhibitors will be exceedingly difficult. Our objectives were to define the substrate recognition pattern of the NS2B-NS3 protease of West Nile and Dengue virises (WNV and DV respectively). To accomplish our goals, we used an efficient, 96-well plate format, method for the synthesis of 9-mer peptide substrates with the general P4-P3-P2-P1-P1'-P2'-P3'-P4'-Gly structure. The N-terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag respectively. The synthesis was followed by the proteolytic cleavage of the synthesized, tagged peptides. Because of the strict requirement for the presence of basic amino acid residues at the P1 and the P2 substrate positions, the analysis of approx. 300 peptide sequences was sufficient for an adequate representation of the cleavage preferences of the WNV and DV proteinases. Our results disclosed the strict substrate specificity of the WNV protease for which the (K/R)(K/R)R/GG amino acid motifs was optimal. The DV protease was less selective and it tolerated well the presence of a number of amino acid residue types at either the P1' or the P2' site, as long as the other position was occupied by a glycine residue. We believe that our data represent a valuable biochemical resource and a solid foundation to support the design of selective substrates and synthetic inhibitors of flaviviral proteinases.

Phosphorylation of mouse serine racemase regulates D-serine synthesis

DEFF Research Database (Denmark)

Foltyn, Veronika N; Zehl, Martin; Dikopoltsev, Elena

2010-01-01

Serine racemase (SR) catalyses the synthesis of the transmitter/neuromodulator D-serine, which plays a major role in synaptic plasticity and N-methyl D-aspartate receptor neurotoxicity. We now report that SR is phosphorylated at Thr71 and Thr227 as revealed by mass spectrometric analysis and in v...... with a phosphorylation-deficient SR mutant indicate that Thr71 phosphorylation increases SR activity, suggesting a novel mechanism for regulating D-serine production....

Tumour-associated neutrophils and loss of epithelial PTEN can promote corticosteroid-insensitive MMP-9 expression in the chronically inflamed lung microenvironment.

Science.gov (United States)

Vannitamby, Amanda; Seow, Huei Jiunn; Anderson, Gary; Vlahos, Ross; Thompson, Michelle; Steinfort, Daniel; Irving, Louis B; Bozinovski, Steven

2017-12-01

Matrix metalloproteinase-9 (MMP-9) is increased in a number of pathological lung conditions, where the proteinase contributes to deleterious remodelling of the airways. While both lung cancer and COPD are associated with increased MMP-9 expression, the cellular and molecular drivers of MMP-9 remain unresolved. In this study, MMP-9 transcript measured within the tumour region from patients with non-small-cell lung cancer (NSCLC) and coexisting COPD was found to be uniformly increased relative to adjacent tumour-free tissue. MMP-9 gene expression and immunohistochemistry identified tumour-associated neutrophils, but not macrophages, as a predominant source of this proteinase. In addition, PTEN gene expression was significantly reduced in tumour and there was evidence of epithelial MMP-9 expression. To explore whether PTEN can regulate epithelial MMP-9 expression, a small interfering (si)RNA knockdown strategy was used in Beas-2B bronchial epithelial cells. PTEN knockdown by siRNA selectively increased MMP-9 expression in response to lipopolysaccharide in a corticosteroid-insensitive manner. In summary, tumour-associated neutrophils represent an important source of MMP-9 in NSCLC, and loss of epithelial PTEN may further augment steroid-insensitive expression. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Manipulation of Neutrophils by Porphyromonas gingivalis in the Development of Periodontitis

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Maja Sochalska

2017-05-01

Full Text Available The pathogenesis of the chronic periodontal disease is associated with a skewed host inflammatory response to periodontal pathogens, such as Porphyromonas gingivalis, that accounts for the majority of periodontal tissue damage. Neutrophils are the most abundant leukocytes in periodontal pockets and depending on the stage of the disease, also plentiful PMNs are present in the inflamed gingival tissue and the gingival crevice. They are the most efficient phagocytes and eliminate pathogens by a variety of means, which are either oxygen-dependent or -independent. However, these secretory lethal weapons do not strictly discriminate between pathogens and host tissue. Current studies describe conflicting findings about neutrophil involvement in periodontal disease. On one hand literature indicate that hyper-reactive neutrophils are the main immune cell type responsible for this observed tissue damage and disease progression. Deregulation of neutrophil survival and functions, such as chemotaxis, migration, secretion of antimicrobial peptides or enzymes, and production of reactive oxygen species, contribute to observed tissue injury and the clinical signs of periodontal disease. On the other hand neutrophils deficiencies in patients and mice also result in periodontal phenotype. Therefore, P. gingivalis represents a periodontal pathogen that manipulates the immune responses of PMNs, employing several virulence factors, such as gingipains, serine proteases, lipid phosphatases, or fimbriae. This review will sum up studies devoted to understanding different strategies utilized by P. gingivalis to manipulate PMNs survival and functions in order to inhibit killing by a granular content, prolong inflammation, and gain access to nutrient resources.

Propeptide-mediated inhibition of cognate gingipain proteinases.

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N Laila Huq

Full Text Available Porphyromonas gingivalis is a major pathogen associated with chronic periodontitis. The organism's cell-surface cysteine proteinases, the Arg-specific proteinases (RgpA, RgpB and the Lys-specific proteinase (Kgp, which are known as gingipains have been implicated as major virulence factors. All three gingipain precursors contain a propeptide of around 200 amino acids in length that is removed during maturation. The aim of this study was to characterize the inhibitory potential of the Kgp and RgpB propeptides against the mature cognate enzymes. Mature Kgp was obtained from P. gingivalis mutant ECR368, which produces a recombinant Kgp with an ABM1 motif deleted from the catalytic domain (rKgp that enables the otherwise membrane bound enzyme to dissociate from adhesins and be released. Mature RgpB was obtained from P. gingivalis HG66. Recombinant propeptides of Kgp and RgpB were produced in Escherichia coli and purified using nickel-affinity chromatography. The Kgp and RgpB propeptides displayed non-competitive inhibition kinetics with K(i values of 2.04 µM and 12 nM, respectively. Both propeptides exhibited selectivity towards their cognate proteinase. The specificity of both propeptides was demonstrated by their inability to inhibit caspase-3, a closely related cysteine protease, and papain that also has a relatively long propeptide. Both propeptides at 100 mg/L caused a 50% reduction of P. gingivalis growth in a protein-based medium. In summary, this study demonstrates that gingipain propeptides are capable of inhibiting their mature cognate proteinases.

Serine biosynthesis and transport defects.

Science.gov (United States)

El-Hattab, Ayman W

2016-07-01

l-serine is a non-essential amino acid that is biosynthesized via the enzymes phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). Besides its role in protein synthesis, l-serine is a potent neurotrophic factor and a precursor of a number of essential compounds including phosphatidylserine, sphingomyelin, glycine, and d-serine. Serine biosynthesis defects result from impairments of PGDH, PSAT, or PSP leading to systemic serine deficiency. Serine biosynthesis defects present in a broad phenotypic spectrum that includes, at the severe end, Neu-Laxova syndrome, a lethal multiple congenital anomaly disease, intermediately, infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end, the childhood disease with intellectual disability. A serine transport defect resulting from deficiency of the ASCT1, the main transporter for serine in the central nervous system, has been recently described in children with neurological manifestations that overlap with those observed in serine biosynthesis defects. l-serine therapy may be beneficial in preventing or ameliorating symptoms in serine biosynthesis and transport defects, if started before neurological damage occurs. Herein, we review serine metabolism and transport, the clinical, biochemical, and molecular aspects of serine biosynthesis and transport defects, the mechanisms of these diseases, and the potential role of serine therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF TWO SERINE PROTEASES AND THEIR POTENTIAL INVOLVEMENT IN PROPHENOLOXIDASE ACTIVATION IN Plutella xylostella.

Science.gov (United States)

Gao, Gang; Xu, Xiao-Xia; Yu, Jing; Li, Lin-Miao; Ju, Wen-Yan; Jin, Feng-Liang; Freed, Shoaib

2016-09-01

The proteolytic activation of prophenoloxidase (proPO) is a humoral defense mechanism in insects and crustaceans. Phenoloxidase (PO) is produced as an inactive precursor namely, proPO and is activated via specific proteolytic cleavage by proPO-activating proteinase. The current research reports two novel serine proteinase genes (PxSP1-768 bp and PxSP2-816 bp) from Plutella xylostella, encoding 255 and 271 amino acid residues, respectively. Tissue distribution analyses by semiquantitative reverse transcription-PCR (RT-PCR) revealed the resultant genes to be primarily expressed in the hemocytes, while quantitative-RT-PCR (qRT-PCR) assay showed that transcription level of PxSP1 and PxSP2 increased significantly after injection of the fungal pathogen Beauveria bassiana. Purified recombinant fusion proteins of PxSP2 and PxSP1 were injected to New Zealand white rabbits and polyclonal antibodies were generated with the titers of 1:12,800. After silencing the expression of PxSP2 by RNAi, the PO activity decreased significantly. The results show that PxSP2 is involved in prophenoloxidase activation in P. xylostella. © 2016 Wiley Periodicals, Inc.

Enhancement of catalytic activity of enzymes by heating in anhydrous organic solvents: 3D structure of a modified serine proteinase at high resolution.

Science.gov (United States)

Sharma, S; Tyagi, R; Gupta, M N; Singh, T P

2001-01-01

For the first time, it is demonstrated that exposure of an enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes, namely, proteinase K, wheat germ acid phosphatase, alpha-amylase, beta-glucosidase, chymotrypsin and trypsin were exposed to acetonitrile at 70 degrees C for three hr. The activities of these enzymes were found to be considerably enhanced. In order to understand the basis of this change in the activity of these enzymes, proteinase K was analyzed in detail using X-ray diffraction method. The overall structure of the enzyme was found to be similar to the native structure in aqueous environment. The hydrogen bonding system of the catalytic triad remained intact after the treatment. However, the water structure in the substrate binding site underwent some rearrangement as some of the water molecules were either displaced or completely absent. The most striking observation concerning the water structure was the complete deletion of the water molecule which occupied the position at the so-called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules were located in the recognition site. Interlinked through water molecules, the sites occupied by acetonitrile molecules were independent of water molecules. The acetonitrile molecules are involved in extensive interactions with the protein atoms. The methyl group of one of the acetonitrile molecules (CCN1) interacts simultaneously with the hydrophobic side chains of Leu 96, Ile 107 and Leu 133. The development of such a hydrophobic environment at the recognition site introduced a striking conformation change in Ile 107 by rotating its side chain about C alpha-C beta bond by 180 degrees to bring about the delta-methyl group within the range of attractive van der Waals interactions with the methyl group of CCN1. A similar

Role of Proteases in Chronic Obstructive Pulmonary Disease

Directory of Open Access Journals (Sweden)

Kailash C. Pandey

2017-08-01

Full Text Available Chronic obstructive pulmonary disease (COPD is generally associated with progressive destruction of airways and lung parenchyma. Various factors play an important role in the development and progression of COPD, like imbalance of proteases, environmental and genetic factors and oxidative stress. This review is specifically focused on the role of proteases and their imbalance in COPD. There are three classes (serine, mettalo, and cysteine of proteases involved in COPD. In serine proteases, neutrophil elastase, cathepsin G, and proteinase-3 are involved in destruction of alveolar tissue. Matrix-mettaloproteinase-9, 12, 13, plays an influential role in severity of COPD. Among cysteine proteases, caspase-3, caspases-8 and caspase-9 play an important role in controlling apoptosis. These proteases activities can be regulated by inhibitors like α-1-antitrypsin, neutrophil elastase inhibitor, and leukocyte protease inhibitor. Studies suggest that neutrophil elastase may be a therapeutic target for COPD, and specific inhibitor against this enzyme has potential role to control the disease. Current study suggests that Dipeptidyl Peptidase IV is a potential marker for COPD. Since the expression of proteases and its inhibitors play an important role in COPD pathogenesis, therefore, it is worth investigating the role of proteases and their regulation. Understanding the biochemical basis of COPD pathogenesis using advanced tools in protease biochemistry and aiming toward translational research from bench-to-bedside will have great impact to deal with this health problem.

Human seminal proteinase and prostate-specific antigen are the ...

Indian Academy of Sciences (India)

https://www.ias.ac.in/article/fulltext/jbsc/033/02/0195-0207. Keywords. Kallikrein; prostate cancer biomarker; proteinase activity; seminal plasma; tumour proliferation and metastasis; therapeutic target. Abstract. Human seminal proteinase and prostate-specific antigen (PSA) were each isolated from human seminal fluid and ...

Action of plant proteinase inhibitors on enzymes of physiopathological importance

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Maria Luiza V. Oliva

2009-09-01

Full Text Available Obtained from leguminous seeds, various plant proteins inhibit animal proteinases, including human, and can be considered for the development of compounds with biological activity. Inhibitors from the Bowman-Birk and plant Kunitz-type family have been characterized by proteinase specificity, primary structure and reactive site. Our group mostly studies the genus Bauhinia, mainly the species bauhinioides, rufa, ungulata and variegata. In some species, more than one inhibitor was characterized, exhibiting different properties. Although proteins from this group share high structural similarity, they present differences in proteinase inhibition, explored in studies using diverse biological models.Obtidas de sementes leguminosas, várias proteínas inibem proteinases de origem animal, incluindo humanas, e podem ser consideradas para o desenvolvimento de compostos com atividade biológica. Inibidores da família Bowman-Birk e da família Kunitz vegetal tem sido caracterizados em relação a especificidade para proteinase, estrutura primária e sitio reativo. O nosso grupo majoritariamente vem estudando o gênero Bauhinia, principalmente as espécies bauhinioides, rufa, ungulatae variegata. Em algumas espécies, mais de um inibidor com propriedades diferentes foi caracterizado. Embora tais proteínas apresentem alta similaridade estrutural, diferem quanto à inibição de proteinases, e foram exploradas em estudos utilizando diversos modelos biológicos.

Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways

Energy Technology Data Exchange (ETDEWEB)

Lund, Leif R; Romer, John; Thomasset, Nicole; Solberg, Helene; Pyke, Charles; Bissell, Mina J; Dano, Keld; Werb, Zena

1996-01-01

Postlactational involution of the mammary gland is characterized by two distinct physiological events: apoptosis of the secretory, epithelial cells undergoing programmed cell death, and proteolytic degradation of the mammary gland basement membrane. We examined the spatial and temporal patterns of apoptotic cells in relation to those of proteinases during involution of the BALB/c mouse mammary gland. Apoptosis was almost absent during lactation but became evident at day 2 of involution, when {beta}-casein gene expression was still high. Apoptotic cells were then seen at least up to day 8 of involution, when {beta}-casein gene expression was being extinguished. Expression of sulfated glycoprotein-2 (SGP-2), interleukin-1{beta} converting enzyme (ICE) and tissue inhibitor of metalloproteinases-1 was upregulated at day 2, when apoptotic cells were seen initially. Expression of the matrix metalloproteinases gelatinase A and stromelysin-1 and the serine proteinase urokinase-type plasminogen activator, which was low during lactation, was strongly upregulated in parallel starting at day 4 after weaning, coinciding with start of the collapse of the lobulo-alveolar structures and the intensive tissue remodeling in involution. The major sites of mRNA synthesis for these proteinases were fibroblast-like cells in the periductal stroma and stromal cells surrounding the collapsed alveoli, suggesting that the degradative phase of involution is due to a specialized mesenchymal-epithelial interaction. To elucidate the functional role of these proteinases during involution, at the onset of weaning we treated mice systemically with the glucocorticoid hydrocortisone, which is known to inhibit mammary gland involution. Although the initial wave of apoptotic cells appeared in the lumina of the gland, the dramatic regression and tissue remodeling usually evident by day 5 was substantially inhibited by systemic treatment with hydrocortisone. mRNA and protein for gelatinase A, stromelysin

Antimalarial effects of vinyl sulfone cysteine proteinase inhibitors.

OpenAIRE

Rosenthal, P J; Olson, J E; Lee, G K; Palmer, J T; Klaus, J L; Rasnick, D

1996-01-01

We evaluated the antimalarial effects of vinyl sulfone cysteine proteinase inhibitors. A number of vinyl sulfones strongly inhibited falcipain, a Plasmodium falciparum cysteine proteinase that is a critical hemoglobinase. In studies of cultured parasites, nanomolar concentrations of three vinyl sulfones inhibited parasite hemoglobin degradation, metabolic activity, and development. The antimalarial effects correlated with the inhibition of falcipain. Our results suggest that vinyl sulfones or...

The induction of proteinases in corn and soybean by anoxia

International Nuclear Information System (INIS)

VanToai, T.; Hwang, Shihying

1989-01-01

This study characterized the anaerobic changes in proteinase activities in corn and soybean roots and to investigate the possibility that these changes might contribute to the differential anaerobiosis tolerance of the two species. After 24 h of anoxia, crude protein extracts from H60 corn and Keller soybean root tips (10cm) were assayed for proteinase activities at pH range from 4.5 to 9.5. Turnover of aberrant proteins was studied in seedlings labelled with 3 H-leucine for 12 h under: (a) puromycin (0.64 mM) in air, (b) ethanol (1%) in air, (c) nitrogen and (d) air. After the treatment, the labelled proteins remaining in roots were determined every 2 h for 6 h. In both corn and soybean, activities of alkali proteinases increased, and activities of acid proteinases declined under anoxia. Neutral proteinases increase in anoxic corn roots, but decline in anoxic soybean roots. The protein turnover rate in corn treated with puromycin, ethanol and nitrogen was much higher than in control roots. The protein turnover rate in soybean roots treated with puromycin, ethanol was similar to the rate of the control. The results indicated that: (a) anoxic corn can degrade aberrant proteins, but anoxic soybean cannot, (b) the degradation of aberrant proteins in anoxic corn is accomplished by neutral proteinases, and (c) the accumulation of aberrant proteins in soybean might contribute to the susceptibility of this species to anoxia

Autophagy Primes Neutrophils for Neutrophil Extracellular Trap Formation during Sepsis.

Science.gov (United States)

Park, So Young; Shrestha, Sanjeeb; Youn, Young-Jin; Kim, Jun-Kyu; Kim, Shin-Yeong; Kim, Hyun Jung; Park, So-Hee; Ahn, Won-Gyun; Kim, Shin; Lee, Myung Goo; Jung, Ki-Suck; Park, Yong Bum; Mo, Eun-Kyung; Ko, Yousang; Lee, Suh-Young; Koh, Younsuck; Park, Myung Jae; Song, Dong-Keun; Hong, Chang-Won

2017-09-01

Neutrophils are key effectors in the host's immune response to sepsis. Excessive stimulation or dysregulated neutrophil functions are believed to be responsible for sepsis pathogenesis. However, the mechanisms regulating functional plasticity of neutrophils during sepsis have not been fully determined. We investigated the role of autophagy in neutrophil functions during sepsis in patients with community-acquired pneumonia. Neutrophils were isolated from patients with sepsis and stimulated with phorbol 12-myristate 13-acetate (PMA). The levels of reactive oxygen species generation, neutrophil extracellular trap (NET) formation, and granule release, and the autophagic status were evaluated. The effect of neutrophil autophagy augmentation was further evaluated in a mouse model of sepsis. Neutrophils isolated from patients who survived sepsis showed an increase in autophagy induction, and were primed for NET formation in response to subsequent PMA stimulation. In contrast, neutrophils isolated from patients who did not survive sepsis showed dysregulated autophagy and a decreased response to PMA stimulation. The induction of autophagy primed healthy neutrophils for NET formation and vice versa. In a mouse model of sepsis, the augmentation of autophagy improved survival via a NET-dependent mechanism. These results indicate that neutrophil autophagy primes neutrophils for increased NET formation, which is important for proper neutrophil effector functions during sepsis. Our study provides important insights into the role of autophagy in neutrophils during sepsis.

Coronavirus 3CL(pro) proteinase cleavage sites: Possible relevance to SARS virus pathology

DEFF Research Database (Denmark)

Kiemer, Lars; Lund, Ole; Brunak, Søren

2004-01-01

the picornaviruses it is known that pathology is related to proteolytic cleavage of host proteins by viral proteinases. Furthermore, several studies indicate that virus proliferation can be arrested using specific proteinase inhibitors supporting the belief that proteinases are indeed important during infection...

Perspectives of digestive pest control with proteinase inhibitors that mainly affect the trypsin-like activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae

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M.E. Pereira

2005-11-01

Full Text Available The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4ºC and the supernatants were used in enzymatic assays at 30ºC, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis was 1.14 ± 0.15 absorbance variation min-1 mg protein-1, at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis was 0.217 ± 0.02 mmol p-nitroaniline min-1 mg protein-1. The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50ºC, a temperature that reduces enzyme stability to 80 and 60% of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.

Human eosinophils constitutively express a unique serine protease, PRSS33.

Science.gov (United States)

Toyama, Sumika; Okada, Naoko; Matsuda, Akio; Morita, Hideaki; Saito, Hirohisa; Fujisawa, Takao; Nakae, Susumu; Karasuyama, Hajime; Matsumoto, Kenji

2017-07-01

Eosinophils play important roles in asthma, especially airway remodeling, by producing various granule proteins, chemical mediators, cytokines, chemokines and proteases. However, protease production by eosinophils is not fully understood. In the present study, we investigated the production of eosinophil-specific proteases/proteinases by transcriptome analysis. Human eosinophils and other cells were purified from peripheral blood by density gradient sedimentation and negative/positive selections using immunomagnetic beads. Protease/proteinase expression in eosinophils and release into the supernatant were evaluated by microarray analysis, qPCR, ELISA, flow cytometry and immunofluorescence staining before and after stimulation with eosinophil-activating cytokines and secretagogues. mRNAs for extracellular matrix proteins in human normal fibroblasts were measured by qPCR after exposure to recombinant protease serine 33 (PRSS33) protein (rPRSS33), created with a baculovirus system. Human eosinophils expressed relatively high levels of mRNA for metalloproteinase 25 (MMP25), a disintegrin and metalloprotease 8 (ADAM8), ADAM10, ADAM19 and PRSS33. Expression of PRSS33 was the highest and eosinophil-specific. PRSS33 mRNA expression was not affected by eosinophil-activating cytokines. Immunofluorescence staining showed that PRSS33 was co-localized with an eosinophil granule protein. PRSS33 was not detected in the culture supernatant of eosinophils even after stimulation with secretagogues, but its cell surface expression was increased. rPRSS33 stimulation of human fibroblasts increased expression of collagen and fibronectin mRNAs, at least in part via protease-activated receptor-2 activation. Activated eosinophils may induce fibroblast extracellular matrix protein synthesis via cell surface expression of PRSS33, which would at least partly explain eosinophils' role(s) in airway remodeling. Copyright © 2017 Japanese Society of Allergology. Production and hosting by Elsevier

Pathogenetic and Clinical Aspects of Anti-Neutrophil Cytoplasmic Autoantibody-Associated Vasculitides

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Peter Lamprecht

2018-04-01

Full Text Available Anti-neutrophil cytoplasmic autoantibodies (ANCA targeting proteinase 3 (PR3 and myeloperoxidase expressed by innate immune cells (neutrophils and monocytes are salient diagnostic and pathogenic features of small vessel vasculitis, comprising granulomatosis with polyangiitis (GPA, microscopic polyangiitis, and eosinophilic GPA. Genetic studies suggest that ANCA-associated vasculitides (AAV constitute separate diseases, which share common immunological and pathological features, but are otherwise heterogeneous. The successful therapeutic use of anti-CD20 antibodies emphasizes the prominent role of ANCA and possibly other autoantibodies in the pathogenesis of AAV. However, to elucidate causal effects in AAV, a better understanding of the complex interplay leading to the emergence of B lymphocytes that produce pathogenic ANCA remains a challenge. Different scenarios seem possible; e.g., the break of tolerance induced by a shift from non-pathogenic toward pathogenic autoantigen epitopes in inflamed tissue. This review gives a brief overview on current knowledge about genetic and epigenetic factors, barrier dysfunction and chronic non-resolving inflammation, necro-inflammatory auto-amplification of cellular death and inflammation, altered autoantigen presentation, alternative complement pathway activation, alterations within peripheral and inflamed tissue-residing T- and B-cell populations, ectopic lymphoid tissue neoformation, the characterization of PR3-specific T-cells, properties of ANCA, links between autoimmune disease and infection-triggered pathology, and animal models in AAV.

Serpins in arthropod biology

OpenAIRE

Meekins, David A.; Kanost, Michael R.; Michel, Kristin

2016-01-01

Serpins are the largest known family of serine proteinase inhibitors and perform a variety of physiological functions in arthropods. Herein, we review the field of serpins in arthropod biology, providing an overview of current knowledge and topics of interest. Serpins regulate insect innate immunity via inhibition of serine proteinase cascades that initiate immune responses such as melanization and antimicrobial peptide production. In addition, several serpins with anti-pathogen activity are ...

[Lactic acid bacteria proteinase and quality of fermented dairy products--A review].

Science.gov (United States)

Zhang, Shuang; Zhang, Lanwei; Han, Xue

2015-12-04

Lactic acid bacteria (LAB) could synthesize cell envelope proteinase with weak activity, which primarily degrades casein. In addition to its crucial role in the rapid growth of LAB in milk, LAB proteinases are also of industrial importance due to their contribution to the formation of texture and flavor of many fermented dairy products. The proteolytic system, properties of proteinase, the degradation product of casein and its effect on the quality of fermented dairy products were reviewed in this manuscript.

The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut.

Science.gov (United States)

Beton, Daniela; Guzzo, Cristiane R; Ribeiro, Alberto F; Farah, Chuck S; Terra, Walter R

2012-09-01

Cathepsin L-like proteinases (CAL) are major digestive proteinases in the beetle Tenebrio molitor. Procathepsin Ls 2 (pCAL2) and 3 (pCAL3) were expressed as recombinant proteins in Escherichia coli, purified and activated under acidic conditions. Immunoblot analyses of different T. molitor larval tissues demonstrated that a polyclonal antibody to pCAL3 recognized pCAL3 and cathepsin L 3 (CAL3) only in the anterior two-thirds of midgut tissue and midgut luminal contents of T. molitor larvae. Furthermore, immunocytolocalization data indicated that pCAL3 occurs in secretory vesicles and microvilli in anterior midgut. Therefore CAL3, like cathepsin L 2 (CAL2), is a digestive enzyme secreted by T. molitor anterior midgut. CAL3 hydrolyses Z-FR-MCA and Z-RR-MCA (typical cathepsin substrates), whereas CAL2 hydrolyses only Z-FR-MCA. Active site mutants (pCAL2C25S and pCAL3C26S) were constructed by replacing the catalytic cysteine with serine to prevent autocatalytic processing. Recombinant pCAL2 and pCAL3 mutants (pCAL2C25S and pCAL3C26S) were prepared, crystallized and their 3D structures determined at 1.85 and 2.1 Å, respectively. While the overall structure of these enzymes is similar to other members of the papain superfamily, structural differences in the S2 subsite explain their substrate specificities. The data also supported models for CAL trafficking to lysosomes and to secretory vesicles to be discharged into midgut contents. Copyright © 2012 Elsevier Ltd. All rights reserved.

Method for the production of l-serine using genetically engineered microorganisms deficient in serine degradation pathways

DEFF Research Database (Denmark)

2016-01-01

The present invention generally relates to the microbiological industry, and specifically to the production of L-serine using genetically modified bacteria. The present invention provides genetically modified microorganisms, such as bacteria, wherein the expression of genes encoding for enzymes...... concentrations of serine. The present invention also provides methods for the production of L-serine or L-serine derivative using such genetically modified microorganisms....

Characteristics of mutants designed to incorporate a new ion pair into the structure of a cold adapted subtilisin-like serine proteinase.

Science.gov (United States)

Sigurdardóttir, Anna Gudný; Arnórsdóttir, Jóhanna; Thorbjarnardóttir, Sigrídur H; Eggertsson, Gudmundur; Suhre, Karsten; Kristjánsson, Magnús M

2009-03-01

Structural comparisons of VPR, a subtilisin-like serine proteinase from a psychrotrophic Vibrio species and a thermophilic homologue, aqualysin I, have led us to hypothesize about the roles of different residues in the temperature adaptation of the enzymes. Some of these hypotheses are now being examined by analysis of mutants of the enzymes. The selected substitutions are believed to increase the stability of the cold adapted enzyme based on structural analysis of the thermostable structure. We report here on mutants, which were designed to incorporate an ion pair into the structure of VPR. The residues Asp17 and Arg259 are assumed to form an ion pair in aqualysin I. The cold adapted VPR contains Asn (Asn15) and Lys (Lys257) at corresponding sites in its structure. In VPR, Asn 15 is located on a surface loop with its side group pointing towards the side chain of Lys257. By substituting Asn15 by Asp (N15D) it was considered feasible that a salt bridge would form between the oppositely charged groups. To mimic further the putative salt bridge from the thermophile enzyme the corresponding double mutant (N15D/K257R) was also produced. The N15D mutation increased the thermal stability of VPR by approximately 3 degrees C, both in T(50%) and T(m). Addition of the K257R mutation did not however, increase the stability of the double mutant any further. Despite this stabilization of the VPR mutants the catalytic activity (k(cat)) against the substrate Suc-AAPF-NH-Np was increased in the mutants. Molecular dynamics simulations on wild type and the two mutant proteins suggested that indeed a salt bridge was formed in both cases. Furthermore, a truncated form of the N15D mutant (N15DDeltaC) was produced, lacking a 15 residue long C-terminal extended sequence not present in the thermophilic enzyme. In wild type VPR this supposedly moveable, negatively charged arm on the protein molecule might interfere with the new salt bridge introduced as a result of the N15D mutation

Phospholipase and proteinase activities of Candida spp. isolates from vulvovaginitis in Iran.

Science.gov (United States)

Shirkhani, S; Sepahvand, A; Mirzaee, M; Anbari, K

2016-09-01

This study aims to characterize phospholipase and proteinase activities of Candida isolates from 82 vulvovaginal candidiasis (VVC) and to study the relationship of these activities with vulvovaginitis. Totally 82 Candida isolates from vagina samples of VVC patients were randomly collected over the period between September and December 2014 from hospitalized patients at the general hospitals of Lorestan province, Iran. Isolates were previously identified by conventional mycological methods. The phospholipase and proteinase activities were evaluated by Egg yolk agar, Tween 80 opacity medium and agar plate methods. The most common Candida species was identified Candida albicans (n=34, 41.5%), followed by Candida famata (n=13, 15.8%), Candida tropicalis (n=11, 13.4%), and Candida parapsilosis (n=9, 11%). The most phospholipase activity was observed in Candida colliculosa (40%), followed by C. famata (38.5%), and Candida krusei (33.3%). The findings revealed that the correlation between phospholipase production by Candida spp. and the presence of VVC was not found to be statistically significant (P=0.91). All Candida spp. exhibited considerable proteinase activity; so that 100% of C. colliculosa, C. parapsilosis, Candida kefyr, and Candida intermedia isolates produced high proteinase activity with Pz 4+ scores. There was a significant correlation between proteinase production by Candida spp. and the presence of VVC (P=0.009). The obtained findings revealed that Candida spp. isolates may produce both virulence factors, phospholipase and proteinase. Although the phospholipase production was only observed in <40% of the isolates; however there was a significant association between proteinase production by Candida spp. and VVC. Copyright © 2016. Published by Elsevier Masson SAS.

Dynamics of postirradiation intracellular cysteine and aspartic proteinases profiles in proliferating and nonproliferating mammalian cells

International Nuclear Information System (INIS)

Korbelik, M.; Osmak, M.; Suhar, A.; Turk, V.; Skrk, J.

1990-01-01

Dynamics of postirradiation intracellular cysteine and aspartic proteinases profiles were examined in proliferating and nonproliferating Chinese hamster fibroblasts (V 79). The results show that there are significant alterations in cysteine and aspartic intracellular proteinases activity already in the early postirradiation period, which are different in proliferating and nonproliferating cells. Irradiation of the cells examined to low doses and up to 15 Gy induced an increase in cysteine proteinases activity in the early postexposure period, while at higher irradiation doses applied, the activity of these proteinases was decreased. These observations suggest that intracellular proteinases are actively participating in process involving recovery from radiation injury or cell killing. (orig.) [de

Neonatal disruption of serine racemase causes schizophrenia-like behavioral abnormalities in adulthood: clinical rescue by d-serine.

Directory of Open Access Journals (Sweden)

Hiroko Hagiwara

Full Text Available D-Serine, an endogenous co-agonist of the N-methyl-D-aspartate (NMDA receptor, is synthesized from L-serine by serine racemase (SRR. Given the role of D-serine in both neurodevelopment and the pathophysiology of schizophrenia, we examined whether neonatal disruption of D-serine synthesis by SRR inhibition could induce behavioral abnormalities relevant to schizophrenia, in later life.Neonatal mice (7-9 days were injected with vehicle or phenazine methosulfate (Met-Phen: 3 mg/kg/day, an SRR inhibitor. Behavioral evaluations, such as spontaneous locomotion, novel object recognition test (NORT, and prepulse inhibition (PPI were performed at juvenile (5-6 weeks old and adult (10-12 weeks old stages. In addition, we tested the effects of D-serine on PPI deficits in adult mice after neonatal Met-Phen exposure. Finally, we assessed whether D-serine could prevent the onset of schizophrenia-like behavior in these mice. Neonatal Met-Phen treatment reduced D-serine levels in the brain, 24 hours after the final dose. Additionally, this treatment caused behavioral abnormalities relevant to prodromal symptoms in juveniles and to schizophrenia in adults. A single dose of D-serine improved PPI deficits in adult mice. Interestingly, chronic administration of D-serine (900 mg/kg/day from P35 to P70 significantly prevented the onset of PPI deficits after neonatal Met-Phen exposure.This study shows that disruption of D-serine synthesis during developmental stages leads to behavioral abnormalities relevant to prodromal symptoms and schizophrenia, in later life. Furthermore, early pharmacological intervention with D-serine may prevent the onset of psychosis in adult.

Circumvention of normal constraints on granule protein gene expression in peripheral blood neutrophils and monocytes of patients with antineutrophil cytoplasmic autoantibody-associated glomerulonephritis.

Science.gov (United States)

Yang, Jia Jin; Pendergraft, William F; Alcorta, David A; Nachman, Patrick H; Hogan, Susan L; Thomas, Robin P; Sullivan, Pamela; Jennette, J Charles; Falk, Ronald J; Preston, Gloria A

2004-08-01

Granulopoiesis-related genes are distinctively upregulated in peripheral leukocytes of patients with antineutrophil cytoplasmic autoantibodies (ANCA)-associated glomerulonephritis. Affymetrix microarrays identified the upregulation of nine neutrophilic primary granule genes, including myeloperoxidase (MPO) and proteinase 3 (PR3), plus five secondary granule genes. Coordinate expression of granulocyte maturation marker CD35, measured by TaqMan PCR, and positive in situ staining for PR3 transcripts in polymorphic neutrophils and monocytes indicate that these genes are expressed in "mature" cells. Increased transcripts correlated with disease activity and absolute neutrophil values but not with "left shift," drug regimen, cytokine levels, hematuria, proteinuria, ANCA titer, serum creatinine, gender, or age. Upregulation of PR3 and MPO transcripts was specifically associated with ANCA disease (n = 56) as these changes were not detected in patients with ESRD (n = 25) or systemic lupus erythematosus (n = 17), as determined by TaqMan PCR. This is the first report of this phenomenon in nonneoplastic cells. The data raise the hypothesis that, in addition to the presence of anti-MPO or anti-PR3 autoantibodies, a second critical component in the cause of this disease is the reactivation of once-silenced genes leading to increased antigen availability.

Identification, classification and expression pattern analysis of sugarcane cysteine proteinases

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Gustavo Coelho Correa

2001-12-01

Full Text Available Cysteine proteases are peptidyl hydrolyses dependent on a cysteine residue at the active center. The physical and chemical properties of cysteine proteases have been extensively characterized, but their precise biological functions have not yet been completely understood, although it is known that they are involved in a number of events such as protein turnover, cancer, germination, programmed cell death and senescence. Protein sequences from different cysteine proteinases, classified as members of the E.C.3.4.22 sub-sub-class, were used to perform a T-BLAST-n search on the Brazilian Sugarcane Expressed Sequence Tags project (SUCEST data bank. Sequence homology was found with 76 cluster sequences that corresponded to possible cysteine proteinases. The alignments of these SUCEST clusters with the sequence of cysteine proteinases of known origins provided important information about the classification and possible function of these sugarcane enzymes. Inferences about the expression pattern of each gene were made by direct correlation with the SUCEST cDNA libraries from which each cluster was derived. Since no previous reports of sugarcane cysteine proteinases genes exists, this study represents a first step in the study of new biochemical, physiological and biotechnological aspects of sugarcane cysteine proteases.Proteinases cisteínicas são peptidil-hidrolases dependentes de um resíduo de cisteína em seu sítio ativo. As propriedades físico-químicas destas proteinases têm sido amplamente caracterizadas, entretanto suas funções biológicas ainda não foram completamente elucidadas. Elas estão envolvidas em um grande número de eventos, tais como: processamento e degradação protéica, câncer, germinação, morte celular programada e processos de senescência. Diferentes proteinases cisteínicas, classificadas pelo Comitê de Nomenclatura da União Internacional de Bioquímica e Biologia Molecular (IUBMB como pertencentes à sub

Applicability of Yeast Extracellular Proteinases in Brewing: Physiological and Biochemical Aspects

Science.gov (United States)

Bilinski, Carl A.; Russell, Inge; Stewart, Graham G.

1987-01-01

A general screening survey for expression of extracellular acid proteinase production was performed on over 100 cultures belonging to the genus Saccharomyces. Although two strains of Saccharomyces cerevisiae showed positive extracellular proteinase phenotypes in plate tests, it was not possible to demonstrate proteolytic activities in cell-free culture supernatants in assays performed at beer pH values. Of several yeasts from other genera examined, Saccharomycopsis fibuligera and Torulopsis magnoliae produced extracellular proteinases with desirable properties. Proteolytic activities were detected in assays performed at beer pH values and at lower temperature. Brewer's wort served as a highly inducing medium for extracellular proteinase production, with T. magnoliae yielding enzyme of highest specific activity. In fact, commencement of enzyme production was detected shortly after the onset of exponential growth in brewer's wort. Inclusion of crude enzyme preparations in brewer's wort inoculated simultaneously with brewer's yeast reduced final ethanol yields slightly and was found to be effective in reducing chill haze formation in bottled beer. PMID:16347298

Erythrocyte endogenous proteinase activity during blood bank storage.

Science.gov (United States)

de Angelis, V; de Matteis, M C; Orazi, B M; Santarossa, L; Della Toffola, L; Raineri, A; Vettore, L

1990-01-01

We studied proteolytic alterations of membrane proteins in ghosts derived from human red blood cells, preserved up to 35 days in the liquid state either as whole blood or with additive solution. The study was carried out by performing sodium dodecyl sulfate polyacrylamide gel electrophoresis of stromal proteins from erythrocytes, either previously treated with proteinase inhibitors or previously incubated in conditions promoting proteolysis. To differentiate the effect of erythrocyte from granulocyte proteinases, the investigation was also carried out in leukocyte-free red cell preparations. The results show: (1) the effects of endogenous proteinases on membrane proteins derived from red cells stored under blood bank conditions; (2) a decrease of proteolytic effects in ghosts derived from red cells which have been submitted to a longer storage; (3) a relevant influence of the red cell resuspending medium before lysis on the time-dependent onset and exhaustion of proteolysis in ghosts. The presence of increased proteolysis in ghosts could be regarded as a marker of molecular lesions induced in red cells by storage under blood bank conditions.

Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4).

Science.gov (United States)

Cunningham, Margaret R; McIntosh, Kathryn A; Pediani, John D; Robben, Joris; Cooke, Alexandra E; Nilsson, Mary; Gould, Gwyn W; Mundell, Stuart; Milligan, Graeme; Plevin, Robin

2012-05-11

Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.

Evolutionary patterns of proteinase activity in attine ant fungus gardens

DEFF Research Database (Denmark)

Semenova, Tatyana; Hughes, David Peter; Boomsma, Jacobus Jan

2011-01-01

hypothesized that fungal proteinase activity may have been under selection for efficiency and that different classes of proteinases might be involved. Results: We determined proteinase activity profiles across a wide pH range for fungus gardens of 14 Panamanian species of fungus-growing ants, representing...... classes. Remarkably, the single symbiont that is shared by species of the crown group of Atta and Acromyrmex leaf-cutting ants mostly showed metalloproteinase activity, suggesting that recurrent changes in enzyme production may have occurred throughout the domestication history of fungus-garden symbionts......Background: Attine ants live in symbiosis with a basidiomycetous fungus that they rear on a substrate of plant material. This indirect herbivory implies that the symbiosis is likely to be nitrogen deprived, so that specific mechanisms may have evolved to enhance protein availability. We therefore...

Trichoderma harzianum transformant has high extracellular alkaline proteinase expression during specific mycoparasitic interactions

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Goldman Maria Helena S.

1998-01-01

Full Text Available The mycoparasite Trichoderma harzianum produces an alkaline proteinase that may be specifically involved in mycoparasitism. We have constructed transformant strains of this fungus that overexpress this alkaline proteinase. Some of the transformants were assessed for alkaline proteinase activity, and those with higher activity than the wild type were selected for further studies. One of these transformant strains produced an elevated and constitutive pbr1 mRNA level during mycoparasitic interactions with Rhizoctonia solani.

Chronic neutrophilic leukemia.

Science.gov (United States)

Bredeweg, Arthur; Burch, Micah; Krause, John R

2018-01-01

Chronic neutrophilic leukemia is a rare myeloproliferative disorder characterized by a sustained peripheral blood neutrophilia, absence of the BCR/ABL oncoprotein, bone marrow hypercellularity with less than 5% myeloblasts and normal neutrophil maturation, and no dysplasia. This leukemia has been associated with mutations in the colony-stimulating factor 3 receptor (CSF3R) that may activate this receptor, leading to the proliferation of neutrophils that are the hallmark of chronic neutrophilic leukemia. We present a case of chronic neutrophilic leukemia and discuss the criteria for diagnosis and the significance of mutations found in this leukemia.

The Effect of Serine Protease Inhibitors on Airway Inflammation in a Chronic Allergen-Induced Asthma Mouse Model

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Chih-Che Lin

2014-01-01

Full Text Available Serine protease inhibitors reportedly attenuated airway inflammation and had antioxidant in multiorgan. However, the effects of the serine protease inhibitors nafamostat mesilate (FUT, gabexate mesilate (FOY, and ulinastatin (UTI on a long-term challenged mouse model of chronic asthma are unclear. BALB/c mice (6 mice/group were intratracheally inoculated with five doses of Dermatophagoides pteronyssinus (Der p; 50 μL, 1 mg/mL at one-week intervals. Therapeutic doses of FUT (0.0625 mg/kg, FOY (20 mg/kg, or UTI (10,000 U/kg were, respectively, injected intraperitoneally into these mice. Control mice received sterile PBS. At 3 days after the last challenge, mice were sacrificed to assess airway hyperresponsiveness (AHR, remodeling, and inflammation; lung histological features; and cytokine expression profiles. Compared with untreated controls, mice treated with FUT, FOY, and UTI had decreased AHR and goblet cell hyperplasia, decreased eosinophil and neutrophil infiltration, decreased Der p-induced IL-4 levels in serum and IL-5, IL-6, IL-13, and IL-17 levels in bronchoalveolar lavage fluid, and inhibited nuclear factor (NF-κB activity in lung tissues. The serine protease inhibitors FUT, FOY, and UTI have potential therapeutic benefits for treating asthma by downregulating Th2 cytokines and Th17 cell function and inhibiting NF-κB activation in lung tissue.

Elastase-induced emphysema: retention of instilled proteinase in the rat

International Nuclear Information System (INIS)

Sandhaus, R.A.; Janoff, A.

1982-01-01

Airway instillation of proteinases with the ability to degrade elastin has been used to produce disease in the rat analogous to human pulmonary emphysema. This study examined the retention, localization, and fate of endotracheally instilled elastase using 125 I labeled enzyme and immunoperoxidase histochemistry. Porcine pancreatic elastase labeled with 125 I was detected in rat lungs through 96 h after instillation; over half of the label was still present after 7 h. Similar results were obtained when elastase was reacted with a specific, catalytic site inactivator prior to instillation. Trypsin and denatured elastase, however, were cleared much more rapidly from the lung (less than half of the label present after 30 min). When lungs were homogenized after instillation of active elastase, the soluble fraction contained elastase bound to rat alpha1-antitrypsin. In addition, a small amount of label (less than 10%) appeared bound to insoluble components for extended periods of time. Using immunoperoxidase histochemistry, it was found that exogenous elastase was rapidly contained with pulmonary alveolar macrophages, as well as associated with alveolar septums and other parenchymal structures. Similar results were obtained with elastase from both porcine pancreas and human neutrophils. These results suggest that exogenous elastase in the rat, and perhaps endogenous elastolytic enzymes in humans, may have several fates in the lungs: complex formation with endogenous inhibitors, containment within the macrophage, and/or association with connective tissue targets

Effect of pH on the production of alkaline proteinase by alkalophilic Bacillus sp

International Nuclear Information System (INIS)

Kitada, Makio; Horikoshi, Koki

1976-01-01

The effect of the pH of the medium on the microbial growth and alkaline proteinase production, and on the uptake of various substances by alkalophilic Bacillus sp. No.8-1 were studied to investigate the physiological properties of alkalophilic bacteria. Both the microbial growth and alkaline proteinase production by replacement culture were maximum between pH 9 and 10. The alkaline proteinase production sources were also effective for the production. The uptake of various substances such as glucose, acetate, amino acids, and uracil, necessary for proteinase production by this strain, was maximum between pH 9 and 10. The uptake of α-aminoisobutyric acid, a nonmetabolizable amino acid analogue, was also maximum at pH 10. The pH-dependence of these substance was not due to their ionic forms being affected by extracellular pH. It was concluded from above results that good production of alkaline proteinase in alkaline media was due to the active uptake of various nutrients in this culture condition. (auth.)

Serpins of oat (Avena sativa) grain with distinct reactive centres and inhibitory specificity

DEFF Research Database (Denmark)

Hejgaard, Jørn; Hauge, S.

2002-01-01

Most proteinase inhibitors from plant seeds are assumed to contribute to broad-spectrum protection against pests and pathogens. In oat (Avena sativa L.) grain the main serine proteinase inhibitors were found to be serpins, which utilize a unique mechanism of irreversible inhibition. Four distinct...... inhibitors of the serpin superfamily were detected by native PAGE as major seed albumins and purified by thiophilic adsorption and anion exchange chromatography. The four serpins OSZa-d are the first proteinase inhibitors characterized from this cereal. An amino acid sequence close to the blocked N...... by chymotrypsin at the putative reactive centre bond P-1 -P-1 ' Tyrdown arrowSer, and no inhibition was detected. Together the oat grain serpins have a broader inhibitory specificity against digestive serine proteinases than represented by the major serpins of wheat, rye or barley grain. Presumably the serpins...

Production of a heterologous proteinase A by Saccharomyces kluyveri

DEFF Research Database (Denmark)

Møller, Kasper; Tidemand, L.D.; Winther, J.R.

2001-01-01

In order to evaluate the potential of Saccharomyces kluyveri for heterologous protein production, S. kluyveri Y159 was transformed with a S. cerevisiae-based multi-copy plasmid containing the S. cerevisiae PEP4 gene, which encodes proteinase A, under the control of its native promoter. As a refer......In order to evaluate the potential of Saccharomyces kluyveri for heterologous protein production, S. kluyveri Y159 was transformed with a S. cerevisiae-based multi-copy plasmid containing the S. cerevisiae PEP4 gene, which encodes proteinase A, under the control of its native promoter...

Coronavirus 3CLpro proteinase cleavage sites: Possible relevance to SARS virus pathology

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Blom Nikolaj

2004-06-01

Full Text Available Abstract Background Despite the passing of more than a year since the first outbreak of Severe Acute Respiratory Syndrome (SARS, efficient counter-measures are still few and many believe that reappearance of SARS, or a similar disease caused by a coronavirus, is not unlikely. For other virus families like the picornaviruses it is known that pathology is related to proteolytic cleavage of host proteins by viral proteinases. Furthermore, several studies indicate that virus proliferation can be arrested using specific proteinase inhibitors supporting the belief that proteinases are indeed important during infection. Prompted by this, we set out to analyse and predict cleavage by the coronavirus main proteinase using computational methods. Results We retrieved sequence data on seven fully sequenced coronaviruses and identified the main 3CL proteinase cleavage sites in polyproteins using alignments. A neural network was trained to recognise the cleavage sites in the genomes obtaining a sensitivity of 87.0% and a specificity of 99.0%. Several proteins known to be cleaved by other viruses were submitted to prediction as well as proteins suspected relevant in coronavirus pathology. Cleavage sites were predicted in proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR, transcription factors CREB-RP and OCT-1, and components of the ubiquitin pathway. Conclusions Our prediction method NetCorona predicts coronavirus cleavage sites with high specificity and several potential cleavage candidates were identified which might be important to elucidate coronavirus pathology. Furthermore, the method might assist in design of proteinase inhibitors for treatment of SARS and possible future diseases caused by coronaviruses. It is made available for public use at our website: http://www.cbs.dtu.dk/services/NetCorona/.

Neutrophils at work

DEFF Research Database (Denmark)

Nauseef, William M; Borregaard, Niels

2014-01-01

In this Review we discuss data demonstrating recently recognized aspects of neutrophil homeostasis in the steady state, granulopoiesis in 'emergency' conditions and interactions of neutrophils with the adaptive immune system. We explore in vivo observations of the recruitment of neutrophils from ...

Neutrophils in critical illness.

Science.gov (United States)

McDonald, Braedon

2018-03-01

During critical illness, dramatic alterations in neutrophil biology are observed including abnormalities of granulopoeisis and lifespan, cell trafficking and antimicrobial effector functions. As a result, neutrophils transition from powerful antimicrobial protectors into dangerous mediators of tissue injury and organ dysfunction. In this article, the role of neutrophils in the pathogenesis of critical illness (sepsis, trauma, burns and others) will be explored, including pathological changes to neutrophil function during critical illness and the utility of monitoring aspects of the neutrophil phenotype as biomarkers for diagnosis and prognostication. Lastly, we review findings from clinical trials of therapies that target the harmful effects of neutrophils, providing a bench-to-bedside perspective on neutrophils in critical illness.

Molecular cloning of a cysteine proteinase cDNA from the cotton boll weevil Anthonomus grandis (Coleoptera: Curculionidae).

Science.gov (United States)

De Oliveira Neto, Osmundo Brilhante; Batista, João Aguiar Nogueira; Rigden, Daniel John; Franco, Octávio Luiz; Fragoso, Rodrigo Rocha; Monteiro, Ana Carolina Santos; Monnerat, Rose Gomes; Grossi-De-Sa, Maria Fátima

2004-06-01

The cotton boll weevil (Anthonomus grandis) causes severe cotton crop losses in North and South America. This report describes the presence of cysteine proteinase activity in the cotton boll weevil. Cysteine proteinase inhibitors from different sources were assayed against total A. grandis proteinases but, unexpectedly, no inhibitor tested was particularly effective. In order to screen for active inhibitors against the boll weevil, a cysteine proteinase cDNA (Agcys1) was isolated from A. grandis larvae using degenerate primers and rapid amplification of cDNA ends (RACE) techniques. Sequence analysis showed significant homologies with other insect cysteine proteinases. Northern blot analysis indicated that the mRNA encoding the proteinase was transcribed mainly in the gut of larvae. No mRNA was detected in neonatal larvae, pupae, or in the gut of the adult insect, suggesting that Agcys1 is an important cysteine proteinase for larvae digestion. The isolated gene will facilitate the search for highly active inhibitors towards boll weevil larvae that may provide a new opportunity to control this important insect pest.

Action of trypsin on structural changes of collagen fibres from sea cucumber (Stichopus japonicus).

Science.gov (United States)

Liu, Zi-Qiang; Tuo, Feng-Yan; Song, Liang; Liu, Yu-Xin; Dong, Xiu-Ping; Li, Dong-Mei; Zhou, Da-Yong; Shahidi, Fereidoon

2018-08-01

Trypsin, a representative serine proteinase, was used to hydrolyse the collagen fibres from sea cucumber (Stichopus japonicus) to highlight the role of serine proteinase in the autolysis of sea cucumber. Partial disaggregation of collagen fibres into collagen fibrils upon trypsin treatment occurred. The trypsin treatment also caused a time-dependent release of water-soluble glycosaminoglycans and proteins. Therefore, the degradation of the proteoglycan bridges between collagen fibrils might account for the disaggregation of collagen fibrils. For trypsin-treated collagen fibres (72 h), the collagen fibrils still kept their structural integrity and showed characteristic D-banding pattern, and the dissolution rate of hydroxyproline was just 0.21%. Meanwhile, Fourier transform infrared analysis showed the collagen within trypsin-treated collagen fibres (72 h) still retaining their triple-helical conformation. These results suggested that serine proteinase participated in the autolysis of S. japonicus body wall by damaging the proteoglycan bridges between collagen fibrils and disintegrating the latter. Copyright © 2018 Elsevier Ltd. All rights reserved.

Determination of germ tube, phospholipase, and proteinase production by bloodstream isolates of Candida albicans

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Antonella Souza Mattei

2013-06-01

Full Text Available Introduction Candida albicans is a commensal and opportunistic agent that causes infection in immunocompromised individuals. Several attributes contribute to the virulence and pathogenicity of this yeast, including the production of germ tubes (GTs and extracellular hydrolytic enzymes, particularly phospholipase and proteinase. This study aimed to investigate GT production and phospholipase and proteinase activities in bloodstream isolates of C. albicans. Methods One hundred fifty-three C. albicans isolates were obtained from blood samples and analyzed for GT, phospholipase, and proteinase production. The assays were performed in duplicate in egg yolk medium containing bovine serum albumin and human serum. Results Detectable amounts of proteinase were produced by 97% of the isolates, and 78% of the isolates produced phospholipase. GTs were produced by 95% of the isolates. A majority of the isolates exhibited low levels of phospholipase production and high levels of proteinase production. Conclusions Bloodstream isolates of C. albicans produce virulence factors such as GT and hydrolytic enzymes that enable them to cause infection under favorable conditions.

Computational study of some benzamidine-based inhibitors of thrombin-like snake venom proteinases

Science.gov (United States)

Henriques, Elsa S.; Nascimento, Marco A. C.; Ramos, Maria João

Pit viper venoms contain a number of serine proteinases that, despite their observed coagulant thrombin-like action in vitro, exhibit a paradoxical benign defibrinogenating (anticoagulant) action in vivo, with clinical applications in preventing thrombi and improved blood circulation. Considering that several benzamidine-based inhibitors, some highly selective to thrombin, also inhibit the enzymatic activity of such venombins, the modeling of their enzyme-inhibitor interactions could provide valuable information on the topological factors that determine the divergences in activity. The first step, and the object of the present study, was to derive the necessary set of parameters, consistent with the CHARMM force field, and to perform molecular dynamics (MD) simulations on a few selected representatives of the inhibitors in question under physiological conditions. Bonding and van der Waals parameters were derived by analogy to similar ones in the existing force field. Net atomic charges were obtained with a restrained fitting to the molecular electrostatic potential generated at B3LYP/6-31G(d) level. The parameters were refined to reproduce the available experimental geometries and crystal data, and the MD simulations of the free inhibitors in aqueous solution at 298 K provided an insightful description of their available conformational space.

Neutrophil extracellular traps - the dark side of neutrophils

DEFF Research Database (Denmark)

Sørensen, Ole E.; Borregaard, Niels

2016-01-01

Neutrophil extracellular traps (NETs) were discovered as extracellular strands of decondensed DNA in complex with histones and granule proteins, which were expelled from dying neutrophils to ensnare and kill microbes. NETs are formed during infection in vivo by mechanisms different from those ori...

Glycine serine interconversion in the rooster

International Nuclear Information System (INIS)

Sugahara, Michihiro; Kandatsu, Makoto

1976-01-01

Serine was isolated by the column chromatography from the hydrolyzates of proteins of the serum, the liver and the pectoral muscle which were obtained from the roosters fed a diet containing 2- 14 C glycine for 16 - 17 days. The carbon chain of serine was cut off by treating with sodium periodate. The specific activity of each carbon (as barium carbonate) was estimated. Carboxyl carbon had little radioactivity. The specific activity of hydroxymethyl carbon was 10 - 19% of that of methylene carbon. Glycine isolated from the same hydrolyzates was degraded by ninhydrin oxidation. Formaldehyde produced from 2-C was oxidized to carbon dioxide by treating with mercuric chloride. Carboxyl carbon had little radioactivity. The specific activities of 2-C of glycine and 2-C of serine in the same tissue protein were compared. The ratio of serine 2-C/glycine 2-C was between 0.7 - 1.5. These results seem to indicate that glycine directly converts to serine in the rooster. The quantitative significance of the pathways of glycine (serine) biosynthesis is discussed. (auth.)

D-serine increases adult hippocampal neurogenesis

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Sebastien eSultan

2013-08-01

Full Text Available Adult hippocampal neurogenesis results in the continuous formation of new neurons and is a process of brain plasticity involved in learning and memory. The neurogenic niche regulates the stem cell proliferation and the differentiation and survival of new neurons and a major contributor to the neurogenic niche are astrocytes. Among the molecules secreted by astrocytes, D-serine is an important gliotransmitter and is a co-agonist of the glutamate, N-methyl-D-aspartate (NMDA receptor. D-serine has been shown to enhance the proliferation of neural stem cells in vitro, but its effect on adult neurogenesis in vivo is unknown. Here, we tested the effect of exogenous administration of D-serine on adult neurogenesis in the mouse dentate gyrus. We found that 1 week of treatment with D-serine increased cell proliferation in vivo and in vitro and increased the density of neural stem cells and transit amplifying progenitors. Furthermore, D-serine increased the survival of newborn neurons. Together, these results indicate that D-serine treatment resulted in the improvement of several steps of adult neurogenesis in vivo.

Proteinase-activated receptors - mediators of early and delayed normal tissue radiation responses

International Nuclear Information System (INIS)

Hauer-Jensen, M.

2003-01-01

Proteinase-activated receptors (PARs) are G-protein coupled receptors that are activated by proteolytic exposure of a receptor-tethered ligand. The discovery of this receptor family represents one of the most intriguing recent developments in signal transduction. PARs are involved in the regulation of many normal and pathophysiological processes, notably inflammatory and fibroproliferative responses to injury. Preclinical studies performed in our laboratory suggest that proteinase-activated receptor-1 (PAR-1) plays a critical role in the mechanism of chronicity of radiation fibrosis, while proteinase-activated receptor-2 (PAR-2) may mediate important fibroproliferative responses in irradiated intestine. Specifically, activation of PAR-1 by thrombin, and PAR-2 by pancreatic trypsin and mast cell proteinases, appears to be involved in acute radiation-induced inflammation, as well as in subsequent extracellular matrix deposition, leading to the development of intestinal wall fibrosis and clinical complications. Pharmacological modulators of PAR-1 or PAR-2 expression or activation would be potentially useful as preventive or therapeutic agents in patients who receive radiation therapy, especially if blockade could be targeted to specific tissues or cellular compartments

Targeting neutrophilic inflammation in severe neutrophilic asthma : can we target the disease-relevant neutrophil phenotype?

NARCIS (Netherlands)

Bruijnzeel, Piet L B; Uddin, Mohib; Koenderman, Leo

2015-01-01

In severe, neutrophilic asthma, neutrophils are thought to have an important role in both the maintenance of the disease and during exacerbations. These patients often display excessive, mucosal airway inflammation with unresolving neutrophilia. Because this variant of asthma is poorly controlled by

Biological roles of cysteine proteinases in the pathogenesis of Trichomonas vaginalis

Science.gov (United States)

Hernández, Hilda M.; Marcet, Ricardo; Sarracent, Jorge

2014-01-01

Human trichomonosis, infection with Trichomonas vaginalis, is the most common non-viral sexually transmitted disease in the world. The host-parasite interaction and pathophysiological processes of trichomonosis remain incompletely understood. This review focuses on the advancements reached in the area of the pathogenesis of T. vaginalis, especially in the role of the cysteine proteinases. It highlights various approaches made in this field and lists a group of trichomonad cysteine proteinases involved in diverse processes such as invasion of the mucous layer, cytoadherence, cytotoxicity, cytoskeleton disruption of red blood cells, hemolysis, and evasion of the host immune response. A better understanding of the biological roles of cysteine proteinases in the pathogenesis of this parasite could be used in the identification of new chemotherapeutic targets. An additional advantage could be the development of a vaccine in order to reduce transmission of T. vaginalis. PMID:25348828

The M358R variant of α_1-proteinase inhibitor inhibits coagulation factor VIIa

International Nuclear Information System (INIS)

Sheffield, William P.; Bhakta, Varsha

2016-01-01

The naturally occurring M358R mutation of the plasma serpin α_1-proteinase inhibitor (API) changes both its cleavable reactive centre bond to Arg–Ser and the efficacy with which it inhibits different proteases, reducing the rate of inhibition of neutrophil elastase, and enhancing that of thrombin, factor XIa, and kallikrein, by several orders of magnitude. Although another plasma serpin with an Arg–Ser reactive centre, antithrombin (AT), has been shown to inhibit factor VIIa (FVIIa), no published data are available with respect to FVIIa inhibition by API M358R. Recombinant bacterially-expressed API M358R and plasma-derived AT were therefore compared using gel-based and kinetic assays of FVIIa integrity and activity. Under pseudo-first order conditions of excess serpin over protease, both AT and API M358R formed denaturation-resistant inhibitory complexes with FVIIa in reactions accelerated by TF; AT, but not API M358R, also required heparin for maximal activity. The second order rate constant for heparin-independent API M358R-mediated FVIIa inhibition was determined to be 7.8 ± 0.8 × 10"2 M"−"1sec"−"1. We conclude that API M358R inhibits FVIIa by forming inhibitory complexes of the serpin type more rapidly than AT in the absence of heparin. The likely 20-fold excess of API M358R over AT in patient plasma during inflammation raises the possibility that it could contribute to the hemorrhagic tendencies manifested by rare individuals expressing this mutant serpin. - Highlights: • The inhibitory specificity of the serpin alpha-1-proteinase inhibitor (API) is sharply altered in the M358R variant. • API M358R forms denaturation-resistant complexes with coagulation factor VIIa at a rate accelerated by tissue factor but unaffected by heparin. • Complex formation was shown by gel-based assays and quantified kinetically by inhibition of FVIIa-dependent amidolysis.

Role of disulphide bonds in a thermophilic serine protease aqualysin I from Thermus aquaticus YT-1.

Science.gov (United States)

Sakaguchi, Masayoshi; Takezawa, Makoto; Nakazawa, Rie; Nozawa, Kazutaka; Kusakawa, Taro; Nagasawa, Takeshi; Sugahara, Yasusato; Kawakita, Masao

2008-05-01

A thermophilic serine protease, Aqualysin I, from Thermus aquaticus YT-1 has two disulphide bonds, which are also found in a psychrophilic serine protease from Vibrio sp. PA-44 and a proteinase K-like enzyme from Serratia sp. at corresponding positions. To understand the significance of these disulphide bonds in aqualysin I, we prepared mutants C99S, C194S and C99S/C194S (WSS), in which Cys69-Cys99, Cys163-Cys194 and both of these disulphide bonds, respectively, were disrupted by replacing Cys residues with Ser residues. All mutants were expressed stably in Escherichia coli. The C99S mutant was 68% as active as the wild-type enzyme at 40 degrees C in terms of k(cat) value, while C194S and WSS were only 6 and 3%, respectively, as active, indicating that disulphide bond Cys163-Cys194 is critically important for maintaining proper catalytic site conformation. Mutants C194S and WSS were less thermostable than wild-type enzyme, with a half-life at 90 degrees C of 10 min as compared to 45 min of the latter and with transition temperatures on differential scanning calorimetry of 86.7 degrees C and 86.9 degrees C, respectively. Mutant C99S was almost as stable as the wild-type aqualysin I. These results indicate that the disulphide bond Cys163-Cys194 is more important for catalytic activity and conformational stability of aqualysin I than Cys67-Cys99.

Proteinase activity in cell nuclei of rats exposed to γ-radiation and methyl nitrosourea

International Nuclear Information System (INIS)

Malakhova, L.V.; Surkenova, G.N.; Gaziev, A.I.

1990-01-01

Activity of nuclear proteinases in blood and liver cells of rats exposed to whole-body γ-irradiation (10 Gy) has been comparatively studied by the capacity of splitting the caseic substrate. Proteinase activity in nuclei of irradiated rat leukocytes was shown to increase by 2.5 times and to gradually decrease after 48 h reaching 150-160% as compared to the control. Two hours following a single injection of methyl nitrosourea the alteration in the activity of proteinases in nuclei of rat hepatocytes and leukocytes was different from the alteration of this index after γ-irradiation

Detection of Aspartic Proteinase Activities Using Gel Zymography.

Science.gov (United States)

Perera, Handunge Kumudu Irani

2017-01-01

Gel zymography is a two-stage process where the proteins from the test sample are first separated by electrophoresis followed by the detection of the activity of hydrolytic enzymes. Many zymography procedures use sodium dodecyl sulfate (SDS) polyacrylamide gels copolymerized with an appropriate substrate. The procedure described here uses native polyacrylamide gel electrophoresis (PAGE) in the absence of both SDS and substrate. In order to visualize aspartic proteinase activity, the gel is impregnated in bovine hemoglobin at pH 3.0 for 15 min after the electrophoresis procedure. Subsequently, the gel is incubated in a humid container in the absence of hemoglobin for 1 h at 37 °C. At the end, the gel is stained with amido black and destained. Clear areas against a dark background corresponding to aspartic proteinase activities can be detected.

The potency and specificity of the interaction between the IA3 inhibitor and its target aspartic proteinase from Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Phylip, L H; Lees, W E; Brownsey, B G

2001-01-01

The yeast IA3 polypeptide consists of only 68 residues, and the free inhibitor has little intrinsic secondary structure. IA3 showed subnanomolar potency toward its target, proteinase A from Saccharomyces cerevisiae, and did not inhibit any of a large number of aspartic proteinases with similar...... by the nontarget aspartic proteinases, it was not cleaved by proteinase A. The random coil IA3 polypeptide escapes cleavage by being stabilized in a helical conformation upon interaction with the active site of proteinase A. This results, paradoxically, in potent selective inhibition of the target enzyme....

Sphingoid bases and the serine catabolic enzyme CHA1 define a novel feedforward/feedback mechanism in the response to serine availability.

Science.gov (United States)

Montefusco, David J; Newcomb, Benjamin; Gandy, Jason L; Brice, Sarah E; Matmati, Nabil; Cowart, L Ashley; Hannun, Yusuf A

2012-03-16

Targets of bioactive sphingolipids in Saccharomyces cerevisiae were previously identified using microarray experiments focused on sphingolipid-dependent responses to heat stress. One of these heat-induced genes is the serine deamidase/dehydratase Cha1 known to be regulated by increased serine availability. This study investigated the hypothesis that sphingolipids may mediate the induction of Cha1 in response to serine availability. The results showed that inhibition of de novo synthesis of sphingolipids, pharmacologically or genetically, prevented the induction of Cha1 in response to increased serine availability. Additional studies implicated the sphingoid bases phytosphingosine and dihydrosphingosine as the likely mediators of Cha1 up-regulation. The yeast protein kinases Pkh1 and Pkh2, known sphingoid base effectors, were found to mediate CHA1 up-regulation via the transcription factor Cha4. Because the results disclosed a role for sphingolipids in negative feedback regulation of serine metabolism, we investigated the effects of disrupting this mechanism on sphingolipid levels and on cell growth. Intriguingly, exposure of the cha1Δ strain to high serine resulted in hyperaccumulation of endogenous serine and in turn a significant accumulation of sphingoid bases and ceramides. Under these conditions, the cha1Δ strain displayed a significant growth defect that was sphingolipid-dependent. Together, this work reveals a feedforward/feedback loop whereby the sphingoid bases serve as sensors of serine availability and mediate up-regulation of Cha1 in response to serine availability, which in turn regulates sphingolipid levels by limiting serine accumulation.

Understanding serine proteases implications on Leishmania spp lifecycle.

Science.gov (United States)

Alves, Carlos Roberto; Souza, Raquel Santos de; Charret, Karen Dos Santos; Côrtes, Luzia Monteiro de Castro; Sá-Silva, Matheus Pereira de; Barral-Veloso, Laura; Oliveira, Luiz Filipe Gonçalves; da Silva, Franklin Souza

2018-01-01

Serine proteases have significant functions over a broad range of relevant biological processes to the Leishmania spp lifecycle. Data gathered here present an update on the Leishmania spp serine proteases and the status of these enzymes as part of the parasite degradome. The serine protease genes (n = 26 to 28) in Leishmania spp, which encode proteins with a wide range of molecular masses (35 kDa-115 kDa), are described along with their degrees of chromosomal and allelic synteny. Amid 17 putative Leishmania spp serine proteases, only ∼18% were experimentally demonstrated, as: signal peptidases that remove the signal peptide from secretory pre-proteins, maturases of other proteins and with metacaspase-like activity. These enzymes include those of clans SB, SC and SF. Classical inhibitors of serine proteases are used as tools for the characterization and investigation of Leishmania spp. Endogenous serine protease inhibitors, which are ecotin-like, can act modulating host actions. However, crude or synthetic based-natural serine protease inhibitors, such as potato tuber extract, Stichodactyla helianthus protease inhibitor I, fukugetin and epoxy-α-lapachone act on parasitic serine proteases and are promising leishmanicidal agents. The functional interrelationship between serine proteases and other Leishmania spp proteins demonstrate essential functions of these enzymes in parasite physiology and therefore their value as targets for leishmaniasis treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

Increased tolerance towards serine obtained by adaptive laboratory evolution

DEFF Research Database (Denmark)

Mundhada, Hemanshu; Seoane, Jose Miguel; Koza, Anna

2014-01-01

The amino acid serine has previously been identified as one of the top 30 candidates of value added chemicals, making the production of serine from glucose attractive. Production of serine have previously been attempted in E. coli and C. glutamicum, however, titers sufficient for commercial...... by glyA), the conversion of serine to pyruvate (encoded by sdaA, sdaB and tdcG) was also deleted. As expected, the resulting strain turned out to be susceptible to even low concentrations of serine in the media. In order to improve the tolerance of the strain towards serine, adaptive laboratory evolution....... During the evolution experiment, the serine tolerance was increased substantially. Genome re-sequencing was subsequently used to analyze the genotype of a number of selected strains. These results reveal insights towards the adaptation process as well as the mechanism of serine tolerance....

Dental Enamel Development: Proteinases and Their Enamel Matrix Substrates

Science.gov (United States)

Bartlett, John D.

2013-01-01

This review focuses on recent discoveries and delves in detail about what is known about each of the proteins (amelogenin, ameloblastin, and enamelin) and proteinases (matrix metalloproteinase-20 and kallikrein-related peptidase-4) that are secreted into the enamel matrix. After an overview of enamel development, this review focuses on these enamel proteins by describing their nomenclature, tissue expression, functions, proteinase activation, and proteinase substrate specificity. These proteins and their respective null mice and human mutations are also evaluated to shed light on the mechanisms that cause nonsyndromic enamel malformations termed amelogenesis imperfecta. Pertinent controversies are addressed. For example, do any of these proteins have a critical function in addition to their role in enamel development? Does amelogenin initiate crystallite growth, does it inhibit crystallite growth in width and thickness, or does it do neither? Detailed examination of the null mouse literature provides unmistakable clues and/or answers to these questions, and this data is thoroughly analyzed. Striking conclusions from this analysis reveal that widely held paradigms of enamel formation are inadequate. The final section of this review weaves the recent data into a plausible new mechanism by which these enamel matrix proteins support and promote enamel development. PMID:24159389

Quantitative proteomics reveals differential biological processes in healthy neonatal cord neutrophils and adult neutrophils

KAUST Repository

Zhu, Jiang; Zhang, Huoming; Guo, Tiannan; Li, Wenying; Li, Huiyu; Zhu, Yi; Huang, Shiang

2014-01-01

Neonatal neutrophils are characterized by the immaturity of bactericidal mechanisms that contributes largely to neonatal mortality. However, underlying molecular mechanism associated with the immaturity remains incompletely understood. In this study, we performed comparative proteomic analysis on neonatal neutrophils derived from human cord blood and adult peripheral neutrophils. A total of 1332 proteins were identified and quantified, and 127 proteins were characterized as differentially expressed between adult and cord neutrophils. The differentially expressed proteins are mapped in KEGG pathways into five clusters and indicated impaired functions of neonatal neutrophils in proteasome, lysosome, phagosome, and leukocyte transendothelial migration. In particular, many proteins associated with NETosis, a critical mechanism for antimicrobial process and auto-clearance, were also found to be downregulated in cord neutrophils. This study represents a first comparative proteome profiling of neonatal and adult neutrophils, and provides a global view of differentially expressed proteome for enhancing our understanding of their various functional difference. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative proteomics reveals differential biological processes in healthy neonatal cord neutrophils and adult neutrophils

KAUST Repository

Zhu, Jiang

2014-06-11

Neonatal neutrophils are characterized by the immaturity of bactericidal mechanisms that contributes largely to neonatal mortality. However, underlying molecular mechanism associated with the immaturity remains incompletely understood. In this study, we performed comparative proteomic analysis on neonatal neutrophils derived from human cord blood and adult peripheral neutrophils. A total of 1332 proteins were identified and quantified, and 127 proteins were characterized as differentially expressed between adult and cord neutrophils. The differentially expressed proteins are mapped in KEGG pathways into five clusters and indicated impaired functions of neonatal neutrophils in proteasome, lysosome, phagosome, and leukocyte transendothelial migration. In particular, many proteins associated with NETosis, a critical mechanism for antimicrobial process and auto-clearance, were also found to be downregulated in cord neutrophils. This study represents a first comparative proteome profiling of neonatal and adult neutrophils, and provides a global view of differentially expressed proteome for enhancing our understanding of their various functional difference. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Oral neutrophil responses to acute prolonged exercise may not be representative of blood neutrophil responses.

Science.gov (United States)

Davison, Glen; Jones, Arwel Wyn

2015-03-01

Neutrophil numbers and function (oxidative burst) were assessed in peripheral blood and oral samples before and after prolonged exercise. Blood neutrophil count increased (∼3.5-fold, P < 0.001) and function decreased (30% ± 19% decrease, P = 0.005) postexercise. Oral neutrophil count (P = 0.392) and function (P = 0.334) were unchanged. Agreement between oral and blood neutrophil function responses to exercise was poor. These findings highlight the importance of studying neutrophils within various compartments/sample types.

The M358R variant of α{sub 1}-proteinase inhibitor inhibits coagulation factor VIIa

Energy Technology Data Exchange (ETDEWEB)

Sheffield, William P., E-mail: sheffiel@mcmaster.ca [Canadian Blood Services, Centre for Innovation, Hamilton, Ontario (Canada); Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario (Canada); Bhakta, Varsha [Canadian Blood Services, Centre for Innovation, Hamilton, Ontario (Canada)

2016-02-12

The naturally occurring M358R mutation of the plasma serpin α{sub 1}-proteinase inhibitor (API) changes both its cleavable reactive centre bond to Arg–Ser and the efficacy with which it inhibits different proteases, reducing the rate of inhibition of neutrophil elastase, and enhancing that of thrombin, factor XIa, and kallikrein, by several orders of magnitude. Although another plasma serpin with an Arg–Ser reactive centre, antithrombin (AT), has been shown to inhibit factor VIIa (FVIIa), no published data are available with respect to FVIIa inhibition by API M358R. Recombinant bacterially-expressed API M358R and plasma-derived AT were therefore compared using gel-based and kinetic assays of FVIIa integrity and activity. Under pseudo-first order conditions of excess serpin over protease, both AT and API M358R formed denaturation-resistant inhibitory complexes with FVIIa in reactions accelerated by TF; AT, but not API M358R, also required heparin for maximal activity. The second order rate constant for heparin-independent API M358R-mediated FVIIa inhibition was determined to be 7.8 ± 0.8 × 10{sup 2} M{sup −1}sec{sup −1}. We conclude that API M358R inhibits FVIIa by forming inhibitory complexes of the serpin type more rapidly than AT in the absence of heparin. The likely 20-fold excess of API M358R over AT in patient plasma during inflammation raises the possibility that it could contribute to the hemorrhagic tendencies manifested by rare individuals expressing this mutant serpin. - Highlights: • The inhibitory specificity of the serpin alpha-1-proteinase inhibitor (API) is sharply altered in the M358R variant. • API M358R forms denaturation-resistant complexes with coagulation factor VIIa at a rate accelerated by tissue factor but unaffected by heparin. • Complex formation was shown by gel-based assays and quantified kinetically by inhibition of FVIIa-dependent amidolysis.

Antinociceptive Effect of Rat D-Serine Racemase Inhibitors, L-Serine-O-Sulfate, and L-Erythro-3-Hydroxyaspartate in an Arthritic Pain Model

Directory of Open Access Journals (Sweden)

Claudio Laurido

2012-01-01

Full Text Available N-methyl-D-aspartic acid receptor (NMDAr activation requires the presence of D-serine, synthesized from L-serine by a pyridoxal 5′-phosphate-dependent serine racemase (SR. D-serine levels can be lowered by inhibiting the racemization of L-serine. L-serine-O-sulfate (LSOS and L-erythro-3-hydroxyaspartate (LEHA, among others, have proven to be effective in reducing the D-serine levels in culture cells. It is tempting then to try these compounds in their effectiveness to decrease nociceptive levels in rat arthritic pain. We measured the C-reflex paradigm and wind-up potentiation in the presence of intrathecally injected LSOS (100 μg/10 μL and LEHA (100 μg/10 μL in normal and monoarthritic rats. Both compounds decreased the wind-up activity in normal and monoarthritic rats. Accordingly, all the antinociceptive effects were abolished when 300 μg/10 μL of D-serine were injected intrathecally. Since no in vivo results have been presented so far, this constitutes the first evidence that SR inhibitions lower the D-serine levels, thus decreasing the NMDAr activity and the consequent development and maintenance of chronic pain.

The resistance of insects to plant proteinase inhibitors

NARCIS (Netherlands)

Jongsma, M.A.

1995-01-01

The research reported in this thesis describes the induction of proteinase inhibitor synthesis in solanaceous plants (tobacco and tomato), when lepidopteran larvae (Manduca sexta and Spodoptera exigua) are feeding on leaves. It is shown that the

Enhancement of catalytic efficiency of enzymes through exposure to anhydrous organic solvent at 70 degrees C. Three-dimensional structure of a treated serine proteinase at 2.2 A resolution.

Science.gov (United States)

Gupta, M N; Tyagi, R; Sharma, S; Karthikeyan, S; Singh, T P

2000-05-15

The enzyme behavior in anhydrous media has important applications in biotechnology. So far chemical modifications and protein engineering have been used to alter the catalytic power of the enzymes. For the first time, it is demonstrated that an exposure of enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes: proteinase K, wheat germ acid phosphatase, alpha-amylase, beta-glucosidase, chymotrypsin and trypsin have been exposed to acetonitrile at 70 degrees C for three hours. The activities of these enzymes were found to be considerably enhanced. In order to understand the basis of this change in the activity of these enzymes, the structure of one of these treated enzymes, proteinase K has been analyzed in detail using X-ray diffraction method. The overall structure of the enzyme is similar to the native structure in aqueous environment. The hydrogen bonding system of the catalytic triad is intact after the treatment. However, the water structure in the substrate binding site undergoes some rearrangement as some of the water molecules are either displaced or completely absent. The most striking observation concerning the water structure pertains to the complete deletion of the water molecule which occupied the position at the so-called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules are located in the recognition site. The sites occupied by acetonitrile molecules are independent of water molecules. The acetonitrile molecules are involved in extensive interactions with the protein atoms. All of them are interlinked through water molecules. The methyl group of one of the acetonitrile molecules (CCN1) interacts simultaneously with the hydrophobic side chains of Leu-96, Ile-107, and Leu-133. The development of such a hydrophobic environment at the recognition site

Complement Activation Induces Neutrophil Adhesion and Neutrophil-Platelet Aggregate Formation on Vascular Endothelial Cells

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Magdalena Riedl

2017-01-01

Discussion: Therefore, our findings of (i neutrophils adhering to complement-activated endothelial cells, (ii the formation of neutrophil-platelet aggregates on endothelial cells, and (iii the ability of aHUS serum to induce similar effects identify a possible role for neutrophils in aHUS manifestation.

Microbial Production of l-Serine from Renewable Feedstocks.

Science.gov (United States)

Zhang, Xiaomei; Xu, Guoqiang; Shi, Jinsong; Koffas, Mattheos A G; Xu, Zhenghong

2018-07-01

l-Serine is a non-essential amino acid that has wide and expanding applications in industry with a fast-growing market demand. Currently, extraction and enzymatic catalysis are the main processes for l-serine production. However, such approaches limit the industrial-scale applications of this important amino acid. Therefore, shifting to the direct fermentative production of l-serine from renewable feedstocks has attracted increasing attention. This review details the current status of microbial production of l-serine from renewable feedstocks. We also summarize the current trends in metabolic engineering strategies and techniques for the typical industrial organisms Corynebacterium glutamicum and Escherichia coli that have been developed to address and overcome major challenges in the l-serine production process. Copyright © 2018 Elsevier Ltd. All rights reserved.

pH-dependent processing of yeast procarboxypeptidase Y by proteinase A in vivo and in vitro

DEFF Research Database (Denmark)

Sørensen, S O; van den Hazel, H B; Kielland-Brandt, Morten

1994-01-01

Carboxypeptidase Y is a vacuolar enzyme from Saccharomyces cerevisiae. It enters the vacuole as a zymogen, procarboxypeptidase Y, which is immediately processed in a reaction involving two endoproteases, proteinase A and proteinase B. We have investigated the in vitro activation of purified proca...

Alterations in the Helicoverpa armigera midgut digestive physiology after ingestion of pigeon pea inducible leucine aminopeptidase.

Directory of Open Access Journals (Sweden)

Purushottam R Lomate

Full Text Available Jasmonate inducible plant leucine aminopeptidase (LAP is proposed to serve as direct defense in the insect midgut. However, exact functions of inducible plant LAPs in the insect midgut remain to be estimated. In the present investigation, we report the direct defensive role of pigeon pea inducible LAP in the midgut of Helicoverpa armigera (Lepidoptera: Noctuidae and responses of midgut soluble aminopeptidases and serine proteinases upon LAP ingestion. Larval growth and survival was significantly reduced on the diets supplemented with pigeon pea LAP. Aminopeptidase activities in larvae remain unaltered in presence or absence of inducible LAP in the diet. On the contrary, serine proteinase activities were significantly decreased in the larvae reared on pigeon pea LAP containing diet as compared to larvae fed on diet without LAP. Our data suggest that pigeon pea inducible LAP is responsible for the degradation of midgut serine proteinases upon ingestion. Reduction in the aminopeptidase activity with LpNA in the H. armigera larvae was compensated with an induction of aminopeptidase activity with ApNA. Our findings could be helpful to further dissect the roles of plant inducible LAPs in the direct plant defense against herbivory.

Serine proteinases and their inhibitors in fertilization

Czech Academy of Sciences Publication Activity Database

Jonáková, Věra

2000-01-01

Roč. 3, 3,4 (2000), s. 23 [ Proteolytic enzymes and their inhibitors in physiology and pathogenesis. 14.09.2000, Plzen] R&D Projects: GA ČR GV524/96/K162; GA ČR GA303/99/0357; GA MŠk VS96141 Grant - others:GA UK(CZ) 12/1998 Institutional research plan: CEZ:AV0Z5052915 Subject RIV: EB - Genetics ; Molecular Biology

Purification and characterization of cell-envelope proteinase from ...

African Journals Online (AJOL)

user

2012-10-18

Oct 18, 2012 ... phenylmethylsulfonyl fluoride;. ACE, angiotensin-I-converting enzyme. Poolman, 1998). Cell-envelope proteinase (CEP) play an important role in the lactobacillus proteolytic system. CEPs are the critical enzyme in the system (Kunji et al., 1996), since it is the only enzyme that can initiate the breakdown of.

Purification and characterization of a milk-clotting aspartic proteinase from globe artichoke (Cynara scolymus L.).

Science.gov (United States)

Llorente, Berta E; Brutti, Cristina B; Caffini, Néstor O

2004-12-29

The study of proteinase expression in crude extracts from different organs of the globe artichoke (Cynara scolymus L.) disclosed that enzymes with proteolytic and milk-clotting activity are mainly located in mature flowers. Maximum proteolytic activity was recorded at pH 5.0, and inhibition studies showed that only pepstatin, specific for aspartic proteinases, presented a significant inhibitory effect. Such properties, in addition to easy enzyme inactivation by moderate heating, make this crude protease extract potentially useful for cheese production. Adsorption with activated carbon, together with anion exchange and affinity chromatography, led to the isolation of a heterodimeric milk-clotting proteinase consisting of 30- and 15-kDa subunits. MALDI-TOF MS of the 15-kDa chain determined a 15.358-Da mass, and the terminal amino sequence presented 96% homology with the smaller cardosin A subunit. The amino terminal sequence of the 30-kDa chain proved to be identical to the larger cardosin A subunit. Electrophoresis evidenced proteinase self-processing that was confirmed by immunoblots presenting 62-, 30-, and 15-kDa bands.

Crystal Structure of Serine Racemase that Produces Neurotransmitter font-variant:small-caps">d-Serine for Stimulation of the NMDA Receptor

Science.gov (United States)

Goto, Masaru

font-variant:small-caps">d-Serine is an endogenous coagonist for the N-methyl-font-variant:small-caps">d-aspartate receptor and is involved in excitatory neurotransmission in the brain. Mammalian pyridoxal 5’-phosphate-dependent serine racemase, which is localized in the mammalian brain, catalyzes the racemization of font-variant:small-caps">l-serine to yield font-variant:small-caps">d-serine and vice versa. We have determined the structures of three forms of the mammalian enzyme homolog from Schizosaccharomyces pombe. Lys57 and Ser82 located on the protein and solvent sides, respectively, with respect to the cofactor plane, are acid-base catalysts that shuttle protons to the substrate. The modified enzyme, which has a unique lysino-font-variant:small-caps">d-alanyl residue at the active site, also binds the substrate serine in the active site, suggesting that the lysino-font-variant:small-caps">d-alanyl residue acts as a catalytic base in the same manner as Lys57 of the wild type enzyme.

Serine protease inhibitors of parasitic helminths.

Science.gov (United States)

Molehin, Adebayo J; Gobert, Geoffrey N; McManus, Donald P

2012-05-01

Serine protease inhibitors (serpins) are a superfamily of structurally conserved proteins that inhibit serine proteases and play key physiological roles in numerous biological systems such as blood coagulation, complement activation and inflammation. A number of serpins have now been identified in parasitic helminths with putative involvement in immune regulation and in parasite survival through interference with the host immune response. This review describes the serpins and smapins (small serine protease inhibitors) that have been identified in Ascaris spp., Brugia malayi, Ancylostoma caninum Onchocerca volvulus, Haemonchus contortus, Trichinella spiralis, Trichostrongylus vitrinus, Anisakis simplex, Trichuris suis, Schistosoma spp., Clonorchis sinensis, Paragonimus westermani and Echinococcus spp. and discusses their possible biological functions, including roles in host-parasite interplay and their evolutionary relationships.

The H1 histone-specific proteinase is associated with nuclear matrix and stimulated by DNA containing breaks of denatured sites

International Nuclear Information System (INIS)

Gaziev, A.I.; Kutsyj, M.P.

1988-01-01

Discovery of proteinase in nuclear matrix specific of H1 histone and dependent presence of breaks or denatured sites in DNA permits to assume that the given enzyme, obviously, participates in replication and DNA repair, in regulation of genes expression. Removal of H1 histone by proteinase is, probably, necessary for procedure of these processes, and, obviously, this proteinase suffers conformational changes in the composition of the DNA-histone complex. H1 histone disintegration in nucleohistone containing damaged sites of DNA by specific proteinase, probably, represents one of the mechanisms for providing DNA repair in cells of higher organisms

Pathogenic Bacterium Acinetobacter baumannii Inhibits the Formation of Neutrophil Extracellular Traps by Suppressing Neutrophil Adhesion

Science.gov (United States)

Kamoshida, Go; Kikuchi-Ueda, Takane; Nishida, Satoshi; Tansho-Nagakawa, Shigeru; Ubagai, Tsuneyuki; Ono, Yasuo

2018-01-01

Hospital-acquired infections caused by Acinetobacter baumannii have become problematic because of high rates of drug resistance. A. baumannii is usually harmless, but it may cause infectious diseases in an immunocompromised host. Although neutrophils are the key players of the initial immune response against bacterial infection, their interactions with A. baumannii remain largely unknown. A new biological defense mechanism, termed neutrophil extracellular traps (NETs), has been attracting attention. NETs play a critical role in bacterial killing by bacterial trapping and inactivation. Many pathogenic bacteria have been reported to induce NET formation, while an inhibitory effect on NET formation is rarely reported. In the present study, to assess the inhibition of NET formation by A. baumannii, bacteria and human neutrophils were cocultured in the presence of phorbol 12-myristate 13-acetate (PMA), and NET formation was evaluated. NETs were rarely observed during the coculture despite neutrophil PMA stimulation. Furthermore, A. baumannii prolonged the lifespan of neutrophils by inhibiting NET formation. The inhibition of NET formation by other bacteria was also investigated. The inhibitory effect was only apparent with live A. baumannii cells. Finally, to elucidate the mechanism of this inhibition, neutrophil adhesion was examined. A. baumannii suppressed the adhesion ability of neutrophils, thereby inhibiting PMA-induced NET formation. This suppression of cell adhesion was partly due to suppression of the surface expression of CD11a in neutrophils. The current study constitutes the first report on the inhibition of NET formation by a pathogenic bacterium, A. baumannii, and prolonging the neutrophil lifespan. This novel pathogenicity to inhibit NET formation, thereby escaping host immune responses might contribute to a development of new treatment strategies for A. baumannii infections. PMID:29467765

Pathogenic Bacterium Acinetobacter baumannii Inhibits the Formation of Neutrophil Extracellular Traps by Suppressing Neutrophil Adhesion

Directory of Open Access Journals (Sweden)

Go Kamoshida

2018-02-01

Full Text Available Hospital-acquired infections caused by Acinetobacter baumannii have become problematic because of high rates of drug resistance. A. baumannii is usually harmless, but it may cause infectious diseases in an immunocompromised host. Although neutrophils are the key players of the initial immune response against bacterial infection, their interactions with A. baumannii remain largely unknown. A new biological defense mechanism, termed neutrophil extracellular traps (NETs, has been attracting attention. NETs play a critical role in bacterial killing by bacterial trapping and inactivation. Many pathogenic bacteria have been reported to induce NET formation, while an inhibitory effect on NET formation is rarely reported. In the present study, to assess the inhibition of NET formation by A. baumannii, bacteria and human neutrophils were cocultured in the presence of phorbol 12-myristate 13-acetate (PMA, and NET formation was evaluated. NETs were rarely observed during the coculture despite neutrophil PMA stimulation. Furthermore, A. baumannii prolonged the lifespan of neutrophils by inhibiting NET formation. The inhibition of NET formation by other bacteria was also investigated. The inhibitory effect was only apparent with live A. baumannii cells. Finally, to elucidate the mechanism of this inhibition, neutrophil adhesion was examined. A. baumannii suppressed the adhesion ability of neutrophils, thereby inhibiting PMA-induced NET formation. This suppression of cell adhesion was partly due to suppression of the surface expression of CD11a in neutrophils. The current study constitutes the first report on the inhibition of NET formation by a pathogenic bacterium, A. baumannii, and prolonging the neutrophil lifespan. This novel pathogenicity to inhibit NET formation, thereby escaping host immune responses might contribute to a development of new treatment strategies for A. baumannii infections.

Heterogeneity of D-Serine Distribution in the Human Central Nervous System

Science.gov (United States)

Suzuki, Masataka; Imanishi, Nobuaki; Mita, Masashi; Hamase, Kenji; Aiso, Sadakazu

2017-01-01

D-serine is an endogenous ligand for N-methyl-D-aspartate glutamate receptors. Accumulating evidence including genetic associations of D-serine metabolism with neurological or psychiatric diseases suggest that D-serine is crucial in human neurophysiology. However, distribution and regulation of D-serine in humans are not well understood. Here, we found that D-serine is heterogeneously distributed in the human central nervous system (CNS). The cerebrum contains the highest level of D-serine among the areas in the CNS. There is heterogeneity in its distribution in the cerebrum and even within the cerebral neocortex. The neocortical heterogeneity is associated with Brodmann or functional areas but is unrelated to basic patterns of cortical layer structure or regional expressional variation of metabolic enzymes for D-serine. Such D-serine distribution may reflect functional diversity of glutamatergic neurons in the human CNS, which may serve as a basis for clinical and pharmacological studies on D-serine modulation. PMID:28604057

Neutrophil Extracellular Traps in Ulcerative Colitis

DEFF Research Database (Denmark)

Bjerg Bennike, Tue; Carlsen, Thomas Gelsing; Ellingsen, Torkell

2015-01-01

microscopy and confocal microscopy. RESULTS: We identified and quantified 5711 different proteins with proteomics. The abundance of the proteins calprotectin and lactotransferrin in the tissue correlated with the degree of tissue inflammation as determined by histology. However, fecal calprotectin did...... not correlate. Forty-six proteins were measured with a statistically significant differences in abundances between the UC colon tissue and controls. Eleven of the proteins with increased abundances in the UC biopsies were associated with neutrophils and neutrophil extracellular traps. The findings were...... validated by microscopy, where an increased abundance of neutrophils and the presence of neutrophil extracellular traps by extracellular DNA present in the UC colon tissue were confirmed. CONCLUSIONS: Neutrophils, induced neutrophil extracellular traps, and several proteins that play a part in innate...

Participation of intracellular cysteine proteinases, in particular cathepsin B, in degradation of collagen in periosteal tissue explants

NARCIS (Netherlands)

Creemers, L. B.; Hoeben, K. A.; Jansen, D. C.; Buttle, D. J.; Beertsen, W.; Everts, V.

1998-01-01

The involvement of cysteine proteinases in the degradation of soft connective tissue collagen was studied in cultured periosteal explants. Using cysteine proteinase inhibitors that were active intracellularly or extracellularly (Ep453 and Ep475, respectively), it was shown that over-all collagen

Multiple pathways for vacuolar sorting of yeast proteinase A

DEFF Research Database (Denmark)

Westphal, V; Marcusson, E G; Winther, Jakob R.

1996-01-01

The sorting of the yeast proteases proteinase A and carboxypeptidase Y to the vacuole is a saturable, receptor-mediated process. Information sufficient for vacuolar sorting of the normally secreted protein invertase has in fusion constructs previously been found to reside in the propeptide...

Expression of proteinase inhibitor II proteins during floral development in Solanum americanum.

Science.gov (United States)

Sin, Suk-Fong; Chye, Mee-Len

2004-10-01

The heterologous expression of serine proteinase inhibitor II (PIN2) proteins confers insect resistance in transgenic plants, but little is known of their endogenous roles. We have cloned two cDNAs encoding Solanum americanum PIN2 proteins, SaPIN2a and SaPIN2b. SaPIN2a is highly expressed in stem, particularly in the phloem, suggesting it could possibly regulate proteolysis in the sieve elements. When SaPIN2a was expressed in transgenic lettuce, we observed an inhibition of endogenous trypsin- and chymotrypsin-like activities. Here, we demonstrate that both SaPIN2a and SaPIN2b are expressed in floral tissues that are destined to undergo developmental programmed cell death (PCD), suggesting possible endogenous roles in inhibiting trypsin- and chymotrypsin-like activities during flower development. Northern and western blot analyses revealed that SaPIN2a and SaPIN2b mRNAs and proteins show highest expression early in floral development. In situ hybridization analysis and immunolocalization on floral sections, localized SaPIN2a and SaPIN2b mRNAs and their proteins to tissues that would apparently undergo PCD: the ovules, the stylar transmitting tissue, the stigma and the vascular bundles. Detection of PCD in floral sections was achieved using terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) analysis. Examination of the mid-style before, and 1 day after, pollination revealed that high expression of SaPIN2a and SaPIN2b in the style was inversely correlated with PCD.

Neutrophils: potential therapeutic targets in tularemia?

Directory of Open Access Journals (Sweden)

Lee-Ann H Allen

2013-12-01

Full Text Available The central role of neutrophils in innate immunity and host defense has long been recognized, and the ability of these cells to efficiently engulf and kill invading bacteria has been extensively studied, as has the role of neutrophil apoptosis in resolution of the inflammatory response. In the past few years additional immunoregulatory properties of neutrophils were discovered, and it is now clear that these cells play a much greater role in control of the immune response than was previously appreciated. In this regard, it is noteworthy that Francisella tularensis is one of relatively few pathogens that can successfully parasitize neutrophils as well as macrophages, DC and epithelial cells. Herein we will review the mechanisms used by F. tularensis to evade elimination by neutrophils. We will also reprise effects of this pathogen on neutrophil migration and lifespan as compared with other infectious and inflammatory disease states. In addition, we will discuss the evidence which suggests that neutrophils contribute to disease progression rather than effective defense during tularemia, and consider whether manipulation of neutrophil migration or turnover may be suitable adjunctive therapeutic strategies.

Effect of sevoflurane on human neutrophil apoptosis.

LENUS (Irish Health Repository)

Tyther, R

2012-02-03

BACKGROUND AND OBJECTIVE: Both chronic occupational exposure to volatile anaesthetic agents and acute in vitro exposure of neutrophils to isoflurane have been shown to inhibit the rate of apoptosis of human neutrophils. It is possible that inhibition of neutrophil apoptosis arises through delaying mitochondrial membrane potential collapse. We assessed mitochondrial depolarization and apoptosis in unexposed neutrophils and neutrophils exposed to sevoflurane in vivo. METHODS: A total of 20 mL venous blood was withdrawn pre- and postinduction of anaesthesia, the neutrophils isolated and maintained in culture. At 1, 12 and 24 h in culture, the percentage of neutrophil apoptosis was assessed by dual staining with annexin V-FITC and propidium iodide. Mitochondrial depolarization was measured using the dual emission styryl dye JC-1. RESULTS: Apoptosis was significantly inhibited in neutrophils exposed to sevoflurane in vivo at 24 (exposed: 38 (12)% versus control: 28 (11)%, P = 0.001), but not at 1 or 12 h, in culture. Mitochondrial depolarization was not delayed in neutrophils exposed to sevoflurane. CONCLUSIONS: The most important findings are that sevoflurane inhibits neutrophil apoptosis in vivo and that inhibition is not mediated primarily by an effect on mitochondrial depolarization.

Neutrophil programming dynamics and its disease relevance.

Science.gov (United States)

Ran, Taojing; Geng, Shuo; Li, Liwu

2017-11-01

Neutrophils are traditionally considered as first responders to infection and provide antimicrobial host defense. However, recent advances indicate that neutrophils are also critically involved in the modulation of host immune environments by dynamically adopting distinct functional states. Functionally diverse neutrophil subsets are increasingly recognized as critical components mediating host pathophysiology. Despite its emerging significance, molecular mechanisms as well as functional relevance of dynamically programmed neutrophils remain to be better defined. The increasing complexity of neutrophil functions may require integrative studies that address programming dynamics of neutrophils and their pathophysiological relevance. This review aims to provide an update on the emerging topics of neutrophil programming dynamics as well as their functional relevance in diseases.

Z α-1 antitrypsin deficiency and the endoplasmic reticulum stress response.

LENUS (Irish Health Repository)

Greene, Catherine M

2010-10-06

The serine proteinase inhibitor α-1 antitrypsin (AAT) is produced principally by the liver at the rate of 2 g\\/d. It is secreted into the circulation and provides an antiprotease protective screen throughout the body but most importantly in the lung, where it can neutralise the activity of the serine protease neutrophil elastase. Mutations leading to deficiency in AAT are associated with liver and lung disease. The most notable is the Z AAT mutation, which encodes a misfolded variant of the AAT protein in which the glutamic acid at position 342 is replaced by a lysine. More than 95% of all individuals with AAT deficiency carry at least one Z allele. ZAAT protein is not secreted effectively and accumulates intracellularly in the endoplasmic reticulum (ER) of hepatocytes and other AAT-producing cells. This results in a loss of function associated with decreased circulating and intrapulmonary levels of AAT. However, the misfolded protein acquires a toxic gain of function that impacts on the ER. A major function of the ER is to ensure correct protein folding. ZAAT interferes with this function and promotes ER stress responses and inflammation. Here the signalling pathways activated during ER stress in response to accumulation of ZAAT are described and therapeutic strategies that can potentially relieve ER stress are discussed.

Dynamic interactions of neutrophils and biofilms

Directory of Open Access Journals (Sweden)

Josefine Hirschfeld

2014-12-01

Full Text Available Background: The majority of microbial infections in humans are biofilm-associated and difficult to treat, as biofilms are highly resistant to antimicrobial agents and protect themselves from external threats in various ways. Biofilms are tenaciously attached to surfaces and impede the ability of host defense molecules and cells to penetrate them. On the other hand, some biofilms are beneficial for the host and contain protective microorganisms. Microbes in biofilms express pathogen-associated molecular patterns and epitopes that can be recognized by innate immune cells and opsonins, leading to activation of neutrophils and other leukocytes. Neutrophils are part of the first line of defense and have multiple antimicrobial strategies allowing them to attack pathogenic biofilms. Objective/design: In this paper, interaction modes of neutrophils with biofilms are reviewed. Antimicrobial strategies of neutrophils and the counteractions of the biofilm communities, with special attention to oral biofilms, are presented. Moreover, possible adverse effects of neutrophil activity and their biofilm-promoting side effects are discussed. Results/conclusion: Biofilms are partially, but not entirely, protected against neutrophil assault, which include the processes of phagocytosis, degranulation, and formation of neutrophil extracellular traps. However, virulence factors of microorganisms, microbial composition, and properties of the extracellular matrix determine whether a biofilm and subsequent microbial spread can be controlled by neutrophils and other host defense factors. Besides, neutrophils may inadvertently contribute to the physical and ecological stability of biofilms by promoting selection of more resistant strains. Moreover, neutrophil enzymes can degrade collagen and other proteins and, as a result, cause harm to the host tissues. These parameters could be crucial factors in the onset of periodontal inflammation and the subsequent tissue breakdown.

Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue.

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Busot, Grethel Yanet; McClure, Bruce; Ibarra-Sánchez, Claudia Patricia; Jiménez-Durán, Karina; Vázquez-Santana, Sonia; Cruz-García, Felipe

2008-01-01

After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen-stigma interactions that regulate pollen tube growth in Nicotiana.

Biomaterial associated impairment of local neutrophil function.

Science.gov (United States)

Kaplan, S S; Basford, R E; Kormos, R L; Hardesty, R L; Simmons, R L; Mora, E M; Cardona, M; Griffith, B L

1990-01-01

The effect of biomaterials on neutrophil function was studied in vitro to determine if these materials activated neutrophils and to determine the subsequent response of these neutrophils to further stimulation. Two biomaterials--polyurethane, a commonly used substance, and Velcro pile (used in the Jarvik 7 heart)--were evaluated. Two control substances, polyethylene and serum-coated polystyrene, were used for comparison. Neutrophil superoxide release was measured following incubation with these materials for 10, 30, and 120 min in the absence of additional stimulation and after stimulation with formylmethionylleucylphenylalanine (fMLP) or phorbol myristate acetate (PMA). The authors observed that the incubation of neutrophils on both polyurethane and Velcro resulted in substantially increased superoxide release that was greater after the 10 min than after the 30 or 120 min association. These activated neutrophils exhibited a poor additional response to fMLP but responded well to PMA. The effect of implantation of the Novacor left ventricular assist device on peripheral blood neutrophil function was also evaluated. The peripheral blood neutrophils exhibited normal superoxide release and chemotaxis. These studies suggest that biomaterials may have a profound local effect on neutrophils, which may predispose the patient to periprosthetic infection, but that the reactivity of circulating neutrophils is unimpaired.

Neutrophils Compromise Retinal Pigment Epithelial Barrier Integrity

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Jiehao Zhou

2010-01-01

Full Text Available We hypothesized that neutrophils and their secreted factors mediate breakdown of the integrity of the outer blood-retina-barrier by degrading the apical tight junctions of the retinal pigment epithelium (RPE. The effect of activated neutrophils or neutrophil cell lysate on apparent permeability of bovine RPE-Choroid explants was evaluated by measuring [H] mannitol flux in a modified Ussing chamber. The expression of matrix metalloproteinase- (MMP- 9 in murine peritoneal neutrophils, and the effects of neutrophils on RPE tight-junction protein expression were assessed by confocal microscopy and western blot. Our results revealed that basolateral incubation of explants with neutrophils decreased occludin and ZO-1 expression at 1 and 3 hours and increased the permeability of bovine RPE-Choroid explants by >3-fold (P<.05. Similarly, basolateral incubation of explants with neutrophil lysate decreased ZO-1 expression at 1 and 3 hours (P<.05 and increased permeability of explants by 75%. Further, we found that neutrophils prominently express MMP-9 and that incubation of explants with neutrophils in the presence of anti-MMP-9 antibody inhibited the increase in permeability. These data suggest that neutrophil-derived MMP-9 may play an important role in disrupting the integrity of the outer blood-retina barrier.

Visceral leishmaniasis patients display altered composition and maturity of neutrophils as well as impaired neutrophil effector functions

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Endalew Yizengaw

2016-11-01

Full Text Available Immunologically, active visceral leishmaniasis (VL is characterised by profound immunosuppression, severe systemic inflammatory responses and an impaired capacity to control parasite replication. Neutrophils are highly versatile cells, which play a crucial role in the induction as well as the resolution of inflammation, the control of pathogen replication and the regulation of immune responses. Neutrophil functions have been investigated in human cutaneous leishmaniasis, however, their role in human visceral leishmaniasis is poorly understood.In the present study we evaluated the activation status and effector functions of neutrophils in patients with active VL and after successful anti-leishmanial treatment. Our results show that neutrophils are highly activated and have degranulated; high levels of arginase, myeloperoxidase and elastase, all contained in neutrophils’ granules, were found in the plasma of VL patients. In addition, we show that a large proportion of these cells are immature. We also analysed effector functions of neutrophils that are essential for pathogen clearance and show that neutrophils have an impaired capacity to release neutrophil extracellular traps, produce reactive oxygen species and phagocytose bacterial particles, but not Leishmania parasites.Our results suggest that impaired effector functions, increased activation and immaturity of neutrophils play a key role in the pathogenesis of VL.

Proteases in Plasma and Kidney of db/db Mice as Markers of Diabetes-Induced Nephropathy

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Hadler-Olsen, E.; Winberg, J.-O.; Reinholt, F. P.; Larsen, T.; Uhlin-Hansen, L.; Jenssen, T.; Berg, E.; Kolset, S. O.

2011-01-01

Db/db mice are overweight, dyslipidemic and develop diabetic complications, relevant for similar complications in human type 2 diabetes. We have used db/db and db/+ control mice to investigate alterations in proteinase expression and activity in circulation and kidneys by SDS-PAGE zymography, electron microscopy, immunohistochemistry, Western blotting, and in situ zymography. Plasma from db/db mice contained larger amounts of serine proteinases compared to db/+ mice. Kidneys from the db/db mice had a significantly larger glomerular surface area and somewhat thicker glomerular basement membranes compared to the db/+ mice. Furthermore, kidney extracts from db/+ mice contained metalloproteinases with M r of approximately 92000, compatible with MMP-9, not observed in db/db mice. These results indicate that higher levels of serine proteinases in plasma may serve as potential markers for kidney changes in db/db mice, whereas a decrease in MMP-9 in the kidney may be related to the glomerular changes. PMID:22363890

Cytoplasmic lipid bodies of human neutrophilic leukocytes

International Nuclear Information System (INIS)

Weller, P.F.; Ackerman, S.J.; Nicholson-Weller, A.; Dvorak, A.M.

1989-01-01

The morphology and function of cytoplasmic lipid bodies in human neutrophils were evaluated. By transmission electron microscopy, neutrophil lipid bodies were cytoplasmic inclusions, usually several microns in diameter, that occasionally coalesced to attain a diameter up to 7 microM. Neutrophil lipid bodies were not enveloped by membrane but were often surrounded by a more electron-dense shell at their periphery. Normal peripheral blood neutrophils contained an average of approximately one lipid body per cell. Lipid bodies appeared in greater numbers in neutrophils from inflammatory lesions. Perturbation of neutrophils during conventional methods of cell isolation and purification modestly increased lipid body numbers in neutrophils, whereas incubation of neutrophils with 1 microM oleic acid rapidly induced lipid body formation over 30 to 60 minutes. After granulocytes were incubated for 2 hours with 3H-fatty acids, including arachidonic, oleic, and palmitic acids, electron microscopic autoradiography demonstrated that lipid bodies represented the predominant intracellular sites of localization of each of the three 3H-fatty acids. There was lesser labeling noted in the perinuclear cisterna, but not in cell membranes. Virtually all of each of the three 3H-fatty acids incorporated by the neutrophils were esterified into chromatographically resolved classes of neutral lipids or phospholipids. These findings indicate that cytoplasmic lipid bodies are more prominent in neutrophils in vivo engaged in inflammatory responses and that these organelles in human neutrophils function as sites of deposition of esterified, incorporated fatty acids

Functional study of elafin cleaved by Pseudomonas aeruginosa metalloproteinases.

LENUS (Irish Health Repository)

Guyot, Nicolas

2010-06-01

Elafin is a 6-kDa innate immune protein present at several epithelial surfaces including the pulmonary epithelium. It is a canonical protease inhibitor of two neutrophil serine proteases [neutrophil elastase (NE) and proteinase 3] with the capacity to covalently bind extracellular matrix proteins by transglutamination. In addition to these properties, elafin also possesses antimicrobial and immunomodulatory activities. The aim of the present study was to investigate the effect of Pseudomonas aeruginosa proteases on elafin function. We found that P. aeruginosa PAO1-conditioned medium and two purified Pseudomonas metalloproteases, pseudolysin (elastase) and aeruginolysin (alkaline protease), are able to cleave recombinant elafin. Pseudolysin was shown to inactivate the anti-NE activity of elafin by cleaving its protease-binding loop. Interestingly, antibacterial properties of elafin against PAO1 were found to be unaffected after pseudolysin treatment. In contrast to pseudolysin, aeruginolysin failed to inactivate the inhibitory properties of elafin against NE. Aeruginolysin cleaves elafin at the amino-terminal Lys6-Gly7 peptide bond, resulting in a decreased ability to covalently bind purified fibronectin following transglutaminase activity. In conclusion, this study provides evidence that elafin is susceptible to proteolytic cleavage at alternative sites by P. aeruginosa metalloproteinases, which can affect different biological functions of elafin.

D-serine : The right or wrong isoform?

NARCIS (Netherlands)

Fuchs, Sabine A; Berger, Ruud; de Koning, Tom J

2011-01-01

Only recently, d-amino acids have been identified in mammals. Of these, d-serine has been most extensively studied. d-Serine was found to play an important role as a neurotransmitter in the human central nervous system (CNS) by binding to the N-methyl-d-aspartate receptor (NMDAr), similar to

Leukotriene B4-Neutrophil Elastase Axis Drives Neutrophil Reverse Transendothelial Cell Migration In Vivo.

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Colom, Bartomeu; Bodkin, Jennifer V; Beyrau, Martina; Woodfin, Abigail; Ody, Christiane; Rourke, Claire; Chavakis, Triantafyllos; Brohi, Karim; Imhof, Beat A; Nourshargh, Sussan

2015-06-16

Breaching endothelial cells (ECs) is a decisive step in the migration of leukocytes from the vascular lumen to the extravascular tissue, but fundamental aspects of this response remain largely unknown. We have previously shown that neutrophils can exhibit abluminal-to-luminal migration through EC junctions within mouse cremasteric venules and that this response is elicited following reduced expression and/or functionality of the EC junctional adhesion molecule-C (JAM-C). Here we demonstrate that the lipid chemoattractant leukotriene B4 (LTB4) was efficacious at causing loss of venular JAM-C and promoting neutrophil reverse transendothelial cell migration (rTEM) in vivo. Local proteolytic cleavage of EC JAM-C by neutrophil elastase (NE) drove this cascade of events as supported by presentation of NE to JAM-C via the neutrophil adhesion molecule Mac-1. The results identify local LTB4-NE axis as a promoter of neutrophil rTEM and provide evidence that this pathway can propagate a local sterile inflammatory response to become systemic. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Tamoxifen induces apoptotic neutrophil efferocytosis in horses.

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Olave, C; Morales, N; Uberti, B; Henriquez, C; Sarmiento, J; Ortloff, A; Folch, H; Moran, G

2018-03-01

Macrophages and neutrophils are important cellular components in the process of acute inflammation and its subsequent resolution, and evidence increasingly suggests that they play important functions during the resolution of chronic, adaptive inflammatory processes. Exacerbated neutrophil activity can be harmful to surrounding tissues; this is important in a range of diseases, including allergic asthma and chronic obstructive pulmonary disease in humans, and equine asthma (also known as recurrent airway obstruction (RAO). Tamoxifen (TX) is a non-steroidal estrogen receptor modulator with effects on cell growth and survival. Previous studies showed that TX treatment in horses with induced acute pulmonary inflammation promoted early apoptosis of blood and BALF neutrophils, reduction of BALF neutrophils, and improvement in animals' clinical status. The aim of this study was to describe if TX induces in vitro efferocytosis of neutrophils by alveolar macrophages. Efferocytosis assay, myeloperoxidase (MPO) detection and translocation phosphatidylserine (PS) were performed on neutrophils isolated from peripheral blood samples from five healthy horses. In in vitro samples from heathy horses, TX treatment increases the phenomenon of efferocytosis of peripheral neutrophils by alveolar macrophages. Similar increases in supernatant MPO concentration and PS translocation were observed in TX-treated neutrophils, compared to control cells. In conclusion, these results confirm that tamoxifen has a direct effect on equine peripheral blood neutrophils, through stimulation of the engulfment of apoptotic neutrophils by alveolar macrophages.

Enhancement of L-Serine Production by Corynebacterium ...

African Journals Online (AJOL)

glutamicum SYPS-062 cultivation process for efficient production of L-serine on a large scale. ... central intermediate for a number of cellular .... impeller, oxygen and pH electrodes, under the ... equation. The yield of L-serine was regressed with respect to the medium ..... is not essential for activity but is required for inhibition.

Serine:glyoxylate aminotransferase mutant of barley

International Nuclear Information System (INIS)

Blackwell, R.; Murray, A.; Joy, K.; Lea, P.

1987-01-01

A photorespiratory mutant of barley (LaPr 85/84), deficient in both of the major peaks of serine:glyoxylate aminotransferase activity detected in the wild type, also lacks serine:pyruvate and asparagine:glyoxylate aminotransferase activities. Genetic analysis of the mutation demonstrated that these three activities are all carried on the same enzyme. The mutant, when placed in air, accumulated a large pool of serine, showed the expected rate (50%) of ammonia release during photorespiration but produced CO 2 at twice the wild type rate when it was fed [ 14 C] glyoxylate. Compared with the wild type, LaPr 85/84 exhibited abnormal transient changes in chlorophyll a fluorescence when the CO 2 concentration of the air was altered, indicating that the rates of the fluorescence quenching mechanisms were affected in vivo by the lack of this enzyme

Amperometric Self-Referencing Ceramic Based Microelectrode Arrays for D-Serine Detection.

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Campos-Beltrán, Diana; Konradsson-Geuken, Åsa; Quintero, Jorge E; Marshall, Lisa

2018-03-06

D-serine is the major D-amino acid in the mammalian central nervous system. As the dominant co-agonist of the endogenous synaptic NMDA receptor, D-serine plays a role in synaptic plasticity, learning, and memory. Alterations in D-serine are linked to neuropsychiatric disorders including schizophrenia. Thus, it is of increasing interest to monitor the concentration of D-serine in vivo as a relevant player in dynamic neuron-glia network activity. Here we present a procedure for amperometric detection of D-serine with self-referencing ceramic-based microelectrode arrays (MEAs) coated with D-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO). We demonstrate in vitro D-serine recordings with a mean sensitivity of 8.61 ± 0.83 pA/µM to D-serine, a limit of detection (LOD) of 0.17 ± 0.01 µM, and a selectivity ratio of 80:1 or greater for D-serine over ascorbic acid (mean ± SEM; n = 12) that can be used for freely moving studies.

Mitochondria in neutrophil apoptosis

NARCIS (Netherlands)

van Raam, B. J.; Verhoeven, A. J.; Kuijpers, T. W.

2006-01-01

Central in the regulation of the short life span of neutrophils are their mitochondria. These organelles hardly contribute to the energy status of neutrophils but play a vital role in the apoptotic process. Not only do the mitochondria contain cytotoxic proteins that are released during apoptosis

Neutrophil Responses to Sterile Implant Materials.

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Siddharth Jhunjhunwala

Full Text Available In vivo implantation of sterile materials and devices results in a foreign body immune response leading to fibrosis of implanted material. Neutrophils, one of the first immune cells to be recruited to implantation sites, have been suggested to contribute to the establishment of the inflammatory microenvironment that initiates the fibrotic response. However, the precise numbers and roles of neutrophils in response to implanted devices remains unclear. Using a mouse model of peritoneal microcapsule implantation, we show 30-500 fold increased neutrophil presence in the peritoneal exudates in response to implants. We demonstrate that these neutrophils secrete increased amounts of a variety of inflammatory cytokines and chemokines. Further, we observe that they participate in the foreign body response through the formation of neutrophil extracellular traps (NETs on implant surfaces. Our results provide new insight into neutrophil function during a foreign body response to peritoneal implants which has implications for the development of biologically compatible medical devices.

Neutrophil migration under normal and sepsis conditions.

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Lerman, Yelena V; Kim, Minsoo

2015-01-01

Neutrophil migration is critical for pathogen clearance and host survival during severe sepsis. Interaction of neutrophil adhesion receptors with ligands on endothelial cells results in firm adhesion of the circulating neutrophils, followed by neutrophil activation and directed migration to sites of infection through the basement membrane and interstitial extracellular matrix. Proteolytic enzymes and reactive oxygen species are produced and released by neutrophils in response to a variety of inflammatory stimuli. Although these mediators are important for host defense, they also promote tissue damage. Excessive neutrophil migration during the early stages of sepsis may lead to an exaggerated inflammatory response with associated tissue damage and subsequent organ dysfunction. On the other hand, dysregulation of migration and insufficient migratory response that occurs during the latter stages of severe sepsis contributes to neutrophils' inability to contain and control infection and impaired wound healing. This review discusses the major steps and associated molecules involved in the balance of neutrophil trafficking, the precise regulation of which during sepsis spells life or death for the host.

Fibrin(ogen)olytic activity of bumblebee venom serine protease

International Nuclear Information System (INIS)

Qiu Yuling; Choo, Young Moo; Yoon, Hyung Joo; Jia Jingming; Cui Zheng; Wang Dong; Kim, Doh Hoon; Sohn, Hung Dae; Jin, Byung Rae

2011-01-01

Bee venom is a rich source of pharmacologically active components; it has been used as an immunotherapy to treat bee venom hypersensitivity, and venom therapy has been applied as an alternative medicine. Here, we present evidence that the serine protease found in bumblebee venom exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a higher content of serine protease, which is one of its major components. Venom serine proteases from bumblebees did not cross-react with antibodies against the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. However, Bt-VSP is not a plasminogen activator, and its fibrinolytic activity is less than that of plasmin. Taken together, our results define roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings offer significant insight into the allergic reaction sequence that is initiated by bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis. - Graphical abstract: Display Omitted Highlights: → Bumblebee venom serine protease (Bt-VSP) is a fibrin(ogen)olytic enzyme. → Bt-VSP activates prothrombin. → Bt-VSP directly degrades fibrinogen into fibrin degradation products. → Bt-VSP is a hemostatically active protein that is a potent clinical agent.

Zymography in Multiwells for Quality Assessment of Proteinases.

Science.gov (United States)

Mechoor, Ambili; Madanan, Madathiparambil G

2017-01-01

Zymography is a well-standardized protocol for the qualitative assessment and analysis of proteinases under specified conditions. However, analysis of a large number of samples simultaneously becomes a challenge when the zymography is carried out by the usual protocol of electrophoresis. This can be overcome by assaying the matrix-degrading proteinases in substrate-impregnated gels in multiwells. Enzymes are copolymerized with 300 mL of 10% acrylamide impregnated with gelatin substrate and incubated for 16 h. The gels are then stained with Coomassie blue, destained with water, and visualized with the naked eye. The intensity; if needed can be measured with a densitometer or gel documentation system. This method has been tested for bacterial collagenases as well as some matrix-degrading metalloproteinases that were purified from rat mammary gland. It can also be used to characterize the enzymes with respect to the type and concentration of the cations required for activity and the role of other regulatory molecules that may affect the enzyme activity. The added advantage of this method is that the electrophoresis set up and electricity is not needed for the procedure.

Targeting Neutrophilic Inflammation Using Polymersome-Mediated Cellular Delivery.

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Robertson, James D; Ward, Jon R; Avila-Olias, Milagros; Battaglia, Giuseppe; Renshaw, Stephen A

2017-05-01

Neutrophils are key effector cells in inflammation and play an important role in neutralizing invading pathogens. During inflammation resolution, neutrophils undergo apoptosis before they are removed by macrophages, but if apoptosis is delayed, neutrophils can cause extensive tissue damage and chronic disease. Promotion of neutrophil apoptosis is a potential therapeutic approach for treating persistent inflammation, yet neutrophils have proven difficult cells to manipulate experimentally. In this study, we deliver therapeutic compounds to neutrophils using biocompatible, nanometer-sized synthetic vesicles, or polymersomes, which are internalized by binding to scavenger receptors and subsequently escape the early endosome through a pH-triggered disassembly mechanism. This allows polymersomes to deliver molecules into the cell cytosol of neutrophils without causing cellular activation. After optimizing polymersome size, we show that polymersomes can deliver the cyclin-dependent kinase inhibitor (R)-roscovitine into human neutrophils to promote apoptosis in vitro. Finally, using a transgenic zebrafish model, we show that encapsulated (R)-roscovitine can speed up inflammation resolution in vivo more efficiently than the free drug. These results show that polymersomes are effective intracellular carriers for drug delivery into neutrophils. This has important consequences for the study of neutrophil biology and the development of neutrophil-targeted therapeutics. Copyright © 2017 The Authors.

Neutrophil Reverse Migration Becomes Transparent with Zebrafish

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Taylor W. Starnes

2012-01-01

Full Text Available The precise control of neutrophil-mediated inflammation is critical for both host defense and the prevention of immunopathology. In vivo imaging studies in zebrafish, and more recently in mice, have made the novel observation that neutrophils leave a site of inflammation through a process called neutrophil reverse migration. The application of advanced imaging techniques to the genetically tractable, optically transparent zebrafish larvae was critical for these advances. Still, the mechanisms underlying neutrophil reverse migration and its effects on the resolution or priming of immune responses remain unclear. Here, we review the current knowledge of neutrophil reverse migration, its potential roles in host immunity, and the live imaging tools that make zebrafish a valuable model for increasing our knowledge of neutrophil behavior in vivo.

A 23-kDa protein as a substrate for protein kinase C in bovine neutrophils. Purification and partial characterization

International Nuclear Information System (INIS)

Stasia, M.J.; Dianoux, A.C.; Vignais, P.V.

1989-01-01

In 32 P i -loaded bovine neutrophils stimulated with phorbol myristate acetate (PMA), radioactivity was preferentially incorporated into a protein of low molecular mass, suggesting a PKC-dependent phosphorylation. This protein, termed 23-kDa protein, was predominantly localized in the cytosol. The apparent molecular mass of the purified protein range between 20 and 23 kDa. In the absence of mercaptoethanol, a dimer accumulated. Homogeneity of the 23-kDa protein was verified by 2D-PAGE analysis. Gel isoelectric focusing (IEF) of the purified 23-kDa protein followed by Coomassie blue staining allowed the visualization of our discrete protein bands with isoelectric points ranging between pH 6.3 and 6.7. Phosphorylation of the 23-kDa protein by [γ- 32 P]ATP in the presence of bovine neutrophil PKC supplemented with Ca 2+ , phosphatidylserine, and diacylglycerol or with PMA occurred on serine and required the presence of mercaptoethanol. IEF of the 32 P-labeled 23-kDa protein followed by autoradiography revealed for discrete bands with distinct isoelectric points similar to those of the bands stained by Coomassie blue after IEF on nonlabeled 23-kDa protein. The bands of the 23-kDa protein resolved by IEF and transfered to nitrocellulose showed ability to bind [ 35 S]GTP-γ-S. The immunoreactivity of antibodies raised in rabbits against the bovine neutrophil 23-kDa protein was demonstrated on immunoblots after SDS-PAGE. The 23-kDa protein differed also from several other proteins of similar molecular mass that have been identified in neutrophils, namely, calmodulin, the small subunit of the low-potential cytochrome b, and a low molecular weight protein which is ADP-ribosylated by the botulinum toxin

Soluble CD40 ligand stimulates CD40-dependent activation of the β2 integrin Mac-1 and protein kinase C zeda (PKCζ in neutrophils: implications for neutrophil-platelet interactions and neutrophil oxidative burst.

Directory of Open Access Journals (Sweden)

Rong Jin

Full Text Available Recent work has revealed an essential involvement of soluble CD40L (sCD40L in inflammation and vascular disease. Activated platelets are the major source of sCD40L, which has been implicated in platelet and leukocyte activation, although its exact functional impact on leukocyte-platelet interactions and the underlying mechanisms remain undefined. We aimed to determine the impact and the mechanisms of sCD40L on neutrophils. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte recruitment to the sites of injury. Our data show that CD40L contributes to neutrophil firm adhesion to and transmigration across activated surface-adherent platelets, possibly through two potential mechanisms. One involves the direct interaction of ligand-receptor (CD40L-CD40, i.e., platelet surface CD40L interaction with neutrophil CD40; another involves an indirect mechanism, i.e. soluble CD40L stimulates activation of the leukocyte-specific β2 integrin Mac-1 in neutrophils and thereby further promotes neutrophil adhesion and migration. Activation of the integrin Mac-1 is known to be critical for mediating neutrophil adhesion and migration. sCD40L activated Mac-1 in neutrophils and enhanced neutrophil-platelet interactions in wild-type neutrophils, but failed to elicit such responses in CD40-deficient neutrophils. Furthermore, our data show that the protein kinase C zeta (PKCζ is critically required for sCD40L-induced Mac-1 activation and neutrophil adhesive function. sCD40L strongly stimulated the focal clustering of Mac-1 (CD11b and the colocalization of Mac-1 with PKCζ in wild-type neutrophils, but had minimal effect in CD40-deficient neutrophils. Blocking PKCζ completely inhibited sCD40L-induced neutrophil firm adhesion. Moreover, sCD40L strongly stimulates neutrophil oxidative burst via CD40-dependent activation of PI3K/NF-KB, but independent of Mac-1 and PKCζ. These findings may contribute to a better

Targeting Neutrophilic Inflammation using Polymersome-Mediated Cellular Delivery

OpenAIRE

Robertson, J.D.; Ward, J.R.; Avila-Olias, M.; Battaglia, G.; Renshaw, S.A.

2017-01-01

Neutrophils are key effector cells in inflammation and play an important role in neutralizing invading pathogens. During inflammation resolution, neutrophils undergo apoptosis before they are removed by macrophages, but if apoptosis is delayed, neutrophils can cause extensive tissue damage and chronic disease. Promotion of neutrophil apoptosis is a potential therapeutic approach for treating persistent inflammation, yet neutrophils have proven difficult cells to manipulate experimentally. In ...

Leukotriene B4-Neutrophil Elastase Axis Drives Neutrophil Reverse Transendothelial Cell Migration In Vivo

Science.gov (United States)

Colom, Bartomeu; Bodkin, Jennifer V.; Beyrau, Martina; Woodfin, Abigail; Ody, Christiane; Rourke, Claire; Chavakis, Triantafyllos; Brohi, Karim; Imhof, Beat A.; Nourshargh, Sussan

2015-01-01

Summary Breaching endothelial cells (ECs) is a decisive step in the migration of leukocytes from the vascular lumen to the extravascular tissue, but fundamental aspects of this response remain largely unknown. We have previously shown that neutrophils can exhibit abluminal-to-luminal migration through EC junctions within mouse cremasteric venules and that this response is elicited following reduced expression and/or functionality of the EC junctional adhesion molecule-C (JAM-C). Here we demonstrate that the lipid chemoattractant leukotriene B4 (LTB4) was efficacious at causing loss of venular JAM-C and promoting neutrophil reverse transendothelial cell migration (rTEM) in vivo. Local proteolytic cleavage of EC JAM-C by neutrophil elastase (NE) drove this cascade of events as supported by presentation of NE to JAM-C via the neutrophil adhesion molecule Mac-1. The results identify local LTB4-NE axis as a promoter of neutrophil rTEM and provide evidence that this pathway can propagate a local sterile inflammatory response to become systemic. PMID:26047922

Analysis of green kiwi fruit (Actinidia deliciosa cv. Hayward) proteinases by two-dimensional zymography and direct identification of zymographic spots by mass spectrometry.

Science.gov (United States)

Larocca, Marilena; Rossano, Rocco; Riccio, Paolo

2010-11-01

Proteinases present in kiwi fruits are potentially allergenic enzymes belonging to the papain family of cysteine proteinases. Actinidin is a prominent kiwi enzyme. The study of kiwi proteinases is important for the follow-up of fruit maturation, a deeper insight in the allergenic properties of individual proteins, and the application of kiwi proteinases for meat tenderisation and other industrial purposes. Kiwi crude extracts were analysed by two-dimensional zymography on gelatin-containing gels. The digestion by the reactivated proteolytic enzymes after electrophoresis resulted in insights into kiwi proteinases. A mixture of several enzyme isotypes with the same pI but different molecular mass was observed. Clear spots, corresponding to the proteolytic activities, were excised, digested with trypsin, and submitted to MALDI-ToF mass spectrometry for protein identification. The most representative enzyme was actinidin. The innovative achievements of the present study are the: (1) two-dimensional zymographic map of kiwi gelatinases without the need for extensive purification; and (2) direct identification of proteinase isotypes by means of direct MALDI-ToF MS analysis of the zymographic spots. 2010 Society of Chemical Industry

GROUP B STREPTOCOCCUS CIRCUMVENTS NEUTROPHILS AND NEUTROPHIL EXTRACELLULAR TRAPS DURING AMNIOTIC CAVITY INVASION AND PRETERM LABOR

Science.gov (United States)

Boldenow, Erica; Gendrin, Claire; Ngo, Lisa; Bierle, Craig; Vornhagen, Jay; Coleman, Michelle; Merillat, Sean; Armistead, Blair; Whidbey, Christopher; Alishetti, Varchita; Santana-Ufret, Veronica; Ogle, Jason; Gough, Michael; Srinouanprachanh, Sengkeo; MacDonald, James W; Bammler, Theo K; Bansal, Aasthaa; Liggitt, H. Denny; Rajagopal, Lakshmi; Waldorf, Kristina M Adams

2016-01-01

Preterm birth is a leading cause of neonatal morbidity and mortality. Although microbial invasion of the amniotic cavity (MIAC) is associated with the majority of early preterm births, the temporal events that occur during MIAC and preterm labor are not known. Group B Streptococci (GBS) are β-hemolytic, gram-positive bacteria, which commonly colonize the vagina but have been recovered from the amniotic fluid in preterm birth cases. To understand temporal events that occur during MIAC, we utilized a unique chronically catheterized nonhuman primate model that closely emulates human pregnancy. This model allows monitoring of uterine contractions, timing of MIAC and immune responses during pregnancy-associated infections. Here, we show that adverse outcomes such as preterm labor, MIAC, and fetal sepsis were observed more frequently during infection with hemolytic GBS when compared to nonhemolytic GBS. Although MIAC was associated with systematic progression in chorioamnionitis beginning with chorionic vasculitis and progressing to neutrophilic infiltration, the ability of the GBS hemolytic pigment toxin to induce neutrophil cell death and subvert killing by neutrophil extracellular traps (NETs) in placental membranes in vivo facilitated MIAC and fetal injury. Furthermore, compared to maternal neutrophils, fetal neutrophils exhibit decreased neutrophil elastase activity and impaired phagocytic functions to GBS. Collectively, our studies demonstrate how a unique bacterial hemolytic lipid toxin enables GBS to circumvent neutrophils and NETs in placental membranes to induce fetal injury and preterm labor. PMID:27819066

Diisopropyl fluorophosphate labeling of sperm-associated proteinases

International Nuclear Information System (INIS)

Odem, R.R.; Willand, J.L.; Polakoski, K.L.

1990-01-01

Proteinase inhibitors have been shown to be capable of preventing various aspects of fertilization. Diisopropyl fluorophosphate (DFP) is an irreversible inhibitor of trypsin-like enzymes that is commercially available in a radiolabeled form. The experiments described herein were designed to determine if DFP would prevent sperm function in live, motile sperm and to identify the sperm proteins bound with DFP. DFP at 5 mM concentrations had no observable effect on sperm motility, but inhibited the penetration of zona-free hamster ova by human sperm (5.5%) compared to controls (33.5%). Acid extracts of motile sperm that had been incubated with radiolabeled DFP and collected by the swim-up procedure demonstrated the presence of radiolabeled DFP, and the autoradiography of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of these extracts localized the uptake of radiolabeled DFP to proteins in the molecular weight region of the proacrosin-acrosin system. Acid-extracted proteinases from semen samples incubated with DFP demonstrated a concentration-dependent inhibition of both esterolytic hydrolysis of benzoyl-arginine ethyl ester on spectrophotometric analysis and proteolytic activity on gelatin SDS-PAGE zymography. DFP-labeled proteins were precipitated by highly specific antibodies to proacrosin. These results demonstrated that DFP is capable of inhibiting sperm function, and that it associates with the proacrosin-acrosin system in live motile sperm

Diisopropyl fluorophosphate labeling of sperm-associated proteinases

Energy Technology Data Exchange (ETDEWEB)

Odem, R.R.; Willand, J.L.; Polakoski, K.L. (Washington Univ. School of Medicine, St. Louis, MO (USA))

1990-02-01

Proteinase inhibitors have been shown to be capable of preventing various aspects of fertilization. Diisopropyl fluorophosphate (DFP) is an irreversible inhibitor of trypsin-like enzymes that is commercially available in a radiolabeled form. The experiments described herein were designed to determine if DFP would prevent sperm function in live, motile sperm and to identify the sperm proteins bound with DFP. DFP at 5 mM concentrations had no observable effect on sperm motility, but inhibited the penetration of zona-free hamster ova by human sperm (5.5%) compared to controls (33.5%). Acid extracts of motile sperm that had been incubated with radiolabeled DFP and collected by the swim-up procedure demonstrated the presence of radiolabeled DFP, and the autoradiography of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of these extracts localized the uptake of radiolabeled DFP to proteins in the molecular weight region of the proacrosin-acrosin system. Acid-extracted proteinases from semen samples incubated with DFP demonstrated a concentration-dependent inhibition of both esterolytic hydrolysis of benzoyl-arginine ethyl ester on spectrophotometric analysis and proteolytic activity on gelatin SDS-PAGE zymography. DFP-labeled proteins were precipitated by highly specific antibodies to proacrosin. These results demonstrated that DFP is capable of inhibiting sperm function, and that it associates with the proacrosin-acrosin system in live motile sperm.

Neutrophil labeling with [99mTc]-technetium stannous colloid is complement receptor 3-mediated and increases the neutrophil priming response to lipopolysaccharide

International Nuclear Information System (INIS)

Gallagher, Hayley; Ramsay, Stuart C.; Barnes, Jodie; Maggs, Jacqueline; Cassidy, Nathan; Ketheesan, Natkunam

2006-01-01

Introduction: [ 99m Tc]-technetium stannous colloid (TcSnC)-labeled white cells are used to image inflammation. Neutrophil labeling with TcSnC is probably phagocytic, but the phagocytic receptor involved is not known. We hypothesised that complement receptor 3 (CR3) plays a key role. Phagocytic labeling could theoretically result in neutrophil activation or priming, affecting the behaviour of labeled cells. Fluorescence-activated cell sorter (FACS) analysis side scatter measurements can assess neutrophil activation and priming. Methods: We tested whether TcSnC neutrophil labeling is CR3-mediated by assessing if neutrophil uptake of TcSnC was inhibited by a monoclonal antibody (mAb) directed at the CD11b component of CR3. We tested if TcSnC-labeled neutrophils show altered activation or priming status, comparing FACS side scatter in labeled and unlabeled neutrophils and examining the effect of lipopolysaccharide (LPS), a known priming agent. Results: Anti-CD11b mAb reduced neutrophil uptake of TcSnC in a dose-dependent fashion. Labeled neutrophils did not show significantly increased side scatter compared to controls. LPS significantly increased side scatter in control cells and labeled neutrophils. However, the increase was significantly greater in labeled neutrophils than unlabeled cells. Conclusions: Neutrophil labeling with TcSnC is related to the function of CR3, a receptor which plays a central role in phagocytosis. TcSnC labeling did not significantly activate or prime neutrophils. However, labeled neutrophils showed a greater priming response to LPS. This could result in labeled neutrophils demonstrating increased adhesion on activated endothelium at sites of infection

Purification and characterization of a serine protease (CPM-2) with fibrinolytic activity from the dung beetles.

Science.gov (United States)

Ahn, Mi Young; Hahn, Bum-Soo; Ryu, Kang Sun; Hwang, Jae Sam; Kim, Yeong Shik

2005-07-01

Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gln Asp Phe Val Glu Glu Ile Leu. CPM-2 was inactivated by Cu2+ and Zn2+ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and alpha1-antitrypsin. However, EDTA, EGTA, cysteine, beta-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved Aalpha- and Bbeta-chains of fibrinogen and fibrin, and gamma-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.

Sexy again: the renaissance of neutrophils in psoriasis.

Science.gov (United States)

Schön, Michael P; Broekaert, Sigrid M C; Erpenbeck, Luise

2017-04-01

Notwithstanding their prominent presence in psoriatic skin, the functional role of neutrophilic granulocytes still remains somewhat enigmatic. Sparked by exciting scientific discoveries regarding neutrophil functions within the last years, the interest in these short-lived cells of the innate immune system has been boosted recently. While it had been known for some time that neutrophils produce and respond to a number of inflammatory mediators, recent research has linked neutrophils with the pathogenic functions of IL-17, possibly in conjunction with the formation of NETs (neutrophil extracellular traps). Antipsoriatic therapies exert their effects, at least in part, through interference with neutrophils. Neutrophils also appear to connect psoriasis with comorbid diseases. However, directly tampering with neutrophil functions is not trivial as evinced by the failure of therapeutic approaches targeting redundantly regulated cellular communication networks. It has also become apparent that neutrophils link important pathogenic functions of the innate and the adaptive immune system and that they are intricately involved in regulatory networks underlying the pathophysiology of psoriasis. In order to advocate intensified research into the role of this interesting cell population, we here highlight some features of neutrophils and put them into perspective with our current view of the pathophysiology of psoriasis. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Overlapping binding sites for trypsin and papain on a Kunitz-type proteinase inhibitor from Prosopis juliflora.

Science.gov (United States)

Franco, Octávio L; Grossi de Sá, Maria F; Sales, Maurício P; Mello, Luciane V; Oliveira, Adeliana S; Rigden, Daniel J

2002-11-15

Proteinase inhibitors are among the most promising candidates for expression by transgenic plants and consequent protection against insect predation. However, some insects can respond to the threat of the proteinase inhibitor by the production of enzymes insensitive to inhibition. Inhibitors combining more than one favorable activity are therefore strongly favored. Recently, a known small Kunitz trypsin inhibitor from Prosopis juliflora (PTPKI) has been shown to possess unexpected potent cysteine proteinase inhibitory activity. Here we show, by enzyme assay and gel filtration, that, unlike other Kunitz inhibitors with dual activities, this inhibitor is incapable of simultaneous inhibition of trypsin and papain. These data are most readily interpreted by proposing overlapping binding sites for the two enzymes. Molecular modeling and docking experiments favor an interaction mode in which the same inhibitor loop that interacts in a canonical fashion with trypsin can also bind into the papain catalytic site cleft. Unusual residue substitutions at the proposed interface can explain the relative rarity of twin trypsin/papain inhibition. Other changes seem responsible for the relative low affinity of PTPKI for trypsin. The predicted coincidence of trypsin and papain binding sites, once confirmed, would facilitate the search, by phage display for example, for mutants highly active against both proteinases. Copyright 2002 Wiley-Liss, Inc.

The helper component-proteinase of cowpea aphid-borne mosaic virus

NARCIS (Netherlands)

Mlotshwa, S.

2000-01-01

Cowpea aphid-borne mosaic potyvirus causes severe yield losses in cowpea, an important legume crop in semi-arid regions of Africa. We have elucidated the genomic sequence of the virus and subsequently focused our attention on the so-called helper component-proteinase (HC-Pro), a

Contribution of cutinase serine 42 side chain to the stabilization of the oxyanion transition state.

Science.gov (United States)

Nicolas, A; Egmond, M; Verrips, C T; de Vlieg, J; Longhi, S; Cambillau, C; Martinez, C

1996-01-16

Cutinase from the fungus Fusarium solani pisi is a lipolytic enzyme able to hydrolyze both aggregated and soluble substrates. It therefore provides a powerful tool for probing the mechanisms underlying lipid hydrolysis. Lipolytic enzymes have a catalytic machinery similar to those present in serine proteinases. It is characterized by the triad Ser, His, and Asp (Glu) residues, by an oxyanion binding site that stabilizes the transition state via hydrogen bonds with two main chain amide groups, and possibly by other determinants. It has been suggested on the basis of a covalently bond inhibitor that the cutinase oxyanion hole may consist not only of two main chain amide groups but also of the Ser42 O gamma side chain. Among the esterases and the serine and the cysteine proteases, only Streptomyces scabies esterase, subtilisin, and papain, respectively, have a side chain residue which is involved in the oxyanion hole formation. The position of the cutinase Ser42 side chain is structurally conserved in Rhizomucor miehei lipase with Ser82 O gamma, in Rhizopus delemar lipase with Thr83 O gamma 1, and in Candida antartica B lipase with Thr40 O gamma 1. To evaluate the increase in the tetrahedral intermediate stability provided by Ser42 O gamma, we mutated Ser42 into Ala. Furthermore, since the proper orientation of Ser42 O gamma is directed by Asn84, we mutated Asn84 into Ala, Leu, Asp, and Trp, respectively, to investigate the contribution of this indirect interaction to the stabilization of the oxyanion hole. The S42A mutation resulted in a drastic decrease in the activity (450-fold) without significantly perturbing the three-dimensional structure. The N84A and N84L mutations had milder kinetic effects and did not disrupt the structure of the active site, whereas the N84W and N84D mutations abolished the enzymatic activity due to drastic steric and electrostatic effects, respectively.

Neutrophils in Cancer: Two Sides of the Same Coin.

Science.gov (United States)

Uribe-Querol, Eileen; Rosales, Carlos

2015-01-01

Neutrophils are the most abundant leukocytes in blood and are considered to be the first line of defense during inflammation and infections. In addition, neutrophils are also found infiltrating many types of tumors. Tumor-associated neutrophils (TANs) have relevant roles in malignant disease. Indeed neutrophils may be potent antitumor effector cells. However, increasing clinical evidence shows TANs correlate with poor prognosis. The tumor microenvironment controls neutrophil recruitment and in turn TANs help tumor progression. Hence, TANs can be beneficial or detrimental to the host. It is the purpose of this review to highlight these two sides of the neutrophil coin in cancer and to describe recent studies that provide some light on the mechanisms for neutrophil recruitment to the tumor, for neutrophils supporting tumor progression, and for neutrophil activation to enhance their antitumor functions.

DNase I and proteinase K impair Listeria monocytogenes biofilm formation and induce dispersal of pre-existing biofilms.

Science.gov (United States)

Nguyen, Uyen T; Burrows, Lori L

2014-09-18

Current sanitation methods in the food industry are not always sufficient for prevention or dispersal of Listeria monocytogenes biofilms. Here, we determined if prevention of adherence or dispersal of existing biofilms could occur if biofilm matrix components were disrupted enzymatically. Addition of DNase during biofilm formation reduced attachment (biofilms with 100μg/ml of DNase for 24h induced incomplete biofilm dispersal, with biofilm remaining compared to control. In contrast, addition of proteinase K completely inhibited biofilm formation, and 72h biofilms-including those grown under stimulatory conditions-were completely dispersed with 100μg/ml proteinase K. Generally-regarded-as-safe proteases bromelain and papain were less effective dispersants than proteinase K. In a time course assay, complete dispersal of L. monocytogenes biofilms from both polystyrene and type 304H food-grade stainless steel occurred within 5min at proteinase K concentrations above 25μg/ml. These data confirm that both DNA and proteins are required for L. monocytogenes biofilm development and maintenance, and that these components of the biofilm matrix can be targeted for effective prevention and removal of biofilms. Copyright © 2014 Elsevier B.V. All rights reserved.

In vitro differential activity of phospholipases and acid proteinases of clinical isolates of Candida

Directory of Open Access Journals (Sweden)

Aurean D'Eça Júnior

2011-06-01

Full Text Available INTRODUCTION: Candida yeasts are commensals; however, if the balance of normal flora is disrupted or the immune defenses are compromised, Candida species can cause disease manifestations. Several attributes contribute to the virulence and pathogenicity of Candida, including the production of extracellular hydrolytic enzymes, particularly phospholipase and proteinase. This study aimed to investigate the in vitro activity of phospholipases and acid proteinases in clinical isolates of Candida spp. METHODS: Eighty-two isolates from hospitalized patients collected from various sites of origin were analyzed. Phospholipase production was performed in egg yolk medium and the production of proteinase was verified in a medium containing bovine serum albumin. The study was performed in triplicate. RESULTS: Fifty-six (68.3% of isolates tested were phospholipase positive and 16 (44.4% were positive for proteinase activity. C. tropicalis was the species with the highest number of positive isolates for phospholipase (91.7%. Statistically significant differences were observed in relation to production of phospholipases among species (p<0,0001 and among the strains from different sites of origin (p=0.014. Regarding the production of acid protease, the isolates of C. parapsilosis tested presented a larger number of producers (69.2%. Among the species analyzed, the percentage of protease producing isolates did not differ statistically (χ2=1.9 p=0.5901 (χ2=1.9 p=0.5901. CONCLUSIONS: The majority of C. non-albicans and all C. albicans isolates were great producers of hydrolytic enzymes and, consequently, might be able to cause infection under favorable conditions.

Regulation of neutrophil senescence by microRNAs.

Directory of Open Access Journals (Sweden)

Jon R Ward

2011-01-01

Full Text Available Neutrophils are rapidly recruited to sites of tissue injury or infection, where they protect against invading pathogens. Neutrophil functions are limited by a process of neutrophil senescence, which renders the cells unable to respond to chemoattractants, carry out respiratory burst, or degranulate. In parallel, aged neutrophils also undergo spontaneous apoptosis, which can be delayed by factors such as GMCSF. This is then followed by their subsequent removal by phagocytic cells such as macrophages, thereby preventing unwanted inflammation and tissue damage. Neutrophils translate mRNA to make new proteins that are important in maintaining functional longevity. We therefore hypothesised that neutrophil functions and lifespan might be regulated by microRNAs expressed within human neutrophils. Total RNA from highly purified neutrophils was prepared and subjected to microarray analysis using the Agilent human miRNA microarray V3. We found human neutrophils expressed a selected repertoire of 148 microRNAs and that 6 of these were significantly upregulated after a period of 4 hours in culture, at a time when the contribution of apoptosis is negligible. A list of predicted targets for these 6 microRNAs was generated from http://mirecords.biolead.org and compared to mRNA species downregulated over time, revealing 83 genes targeted by at least 2 out of the 6 regulated microRNAs. Pathway analysis of genes containing binding sites for these microRNAs identified the following pathways: chemokine and cytokine signalling, Ras pathway, and regulation of the actin cytoskeleton. Our data suggest that microRNAs may play a role in the regulation of neutrophil senescence and further suggest that manipulation of microRNAs might represent an area of future therapeutic interest for the treatment of inflammatory disease.

Neutrophils in Cancer: Two Sides of the Same Coin

Directory of Open Access Journals (Sweden)

Eileen Uribe-Querol

2015-01-01

Full Text Available Neutrophils are the most abundant leukocytes in blood and are considered to be the first line of defense during inflammation and infections. In addition, neutrophils are also found infiltrating many types of tumors. Tumor-associated neutrophils (TANs have relevant roles in malignant disease. Indeed neutrophils may be potent antitumor effector cells. However, increasing clinical evidence shows TANs correlate with poor prognosis. The tumor microenvironment controls neutrophil recruitment and in turn TANs help tumor progression. Hence, TANs can be beneficial or detrimental to the host. It is the purpose of this review to highlight these two sides of the neutrophil coin in cancer and to describe recent studies that provide some light on the mechanisms for neutrophil recruitment to the tumor, for neutrophils supporting tumor progression, and for neutrophil activation to enhance their antitumor functions.

Radiation-induced muscositis and neutrophil granulocytes in oral mucosa; Strahleninduzierte Mukositis und neutrophile Granulozyten in der Mundschleimhaut

Energy Technology Data Exchange (ETDEWEB)

Schmidberger, H.; Rave-Fraenk, M.; Kim, S.; Hille, A.; Pradier, O.; Hess, C.F. [Klinik fuer Strahlentherapie und Radioonkologie, Univ. Goettingen (Germany)

2003-10-01

Background: Chemotherapy-induced mucositis can be related to a decrease in oral neutrophils. We tested the relationship between radiation-induced mucositis and oral neutrophil counts. Patients and Methods: Oral neutrophil counts were obtained for ten patients with head and neck cancer who received radiotherapy of the pharynx and oral cavity. Four patients received additional chemotherapy (5-FU, Mitomycin). Counts were obtained before and during treatment; four healthy volunteers were included in the study as well. For evaluation, a quantitative mouth rinse assay, including neutrophil-staining with acridin-orange, was applied. Results: We observed large inter-individual variations with respect to neutrophil counts for patients and control persons (Table 1). During treatment (irradiation or chemoirradiation), large intra-individual variations were seen additionally (Figure 1). We found a correlation between neutrophil counts and clinical reaction grade. Neutrophil counts increased with increasing mucositis (Figure 2). This increase was more pronounced for patients treated with chemoirradiation compared to radiation alone. Treatment breaks at weekends had no clear influence on neutrophil counts. Conclusions: We observed a weak correlation between neutrophil counts and clinical reaction grade. However, the variations in neutrophil counts are too large to utilize this parameter as a surrogate for clinical mucositis grading. The assumption that a decrease in oral neutrophils is associated with radiation-induced mucositis was clearly negated. (orig.) [German] Hintergrund: Die chemotherapieinduzierte Mukositis kann mit einer Verarmung der Mundschleimhaut an neutrophilen Granulozyten vergesellschaftet sein. Wir ueberprueften den Zusammenhang zwischen der radiogenen Mukositis und der Anzahl neutrophiler Granulozyten. Patienten und Methoden: Bei zehn Patienten mit Tumoren der Kopf-Hals-Region, die sich einer Strahlentherapie unterzogen, wurde die Anzahl enoraler neutrophiler

Neutrophil labeling with [{sup 99m}Tc]-technetium stannous colloid is complement receptor 3-mediated and increases the neutrophil priming response to lipopolysaccharide

Energy Technology Data Exchange (ETDEWEB)

Gallagher, Hayley [School of Veterinary and Biomedical Sciences, James Cook University, Townsville, Queensland 4811 (Australia); Ramsay, Stuart C. [School of Medicine, James Cook University, Townsville, Queensland (Australia) and Townsville Nuclear Medicine, Mater Hospital, Townsville, Queensland 4812 (Australia)]. E-mail: stuart.ramsey@jcu.edu.au; Barnes, Jodie [School of Veterinary and Biomedical Sciences, James Cook University, Townsville, Queensland 4811 (Australia); Maggs, Jacqueline [Department of Nuclear Medicine, Townsville Hospital, Townsville, Queensland 4814 (Australia); Cassidy, Nathan [Townsville Nuclear Medicine, Mater Hospital, Townsville, Queensland 4812 (Australia); Ketheesan, Natkunam [School of Veterinary and Biomedical Sciences, James Cook University, Townsville, Queensland 4811 (Australia); School of Medicine, James Cook University, Townsville, Queensland (Australia)

2006-04-15

Introduction: [{sup 99m}Tc]-technetium stannous colloid (TcSnC)-labeled white cells are used to image inflammation. Neutrophil labeling with TcSnC is probably phagocytic, but the phagocytic receptor involved is not known. We hypothesised that complement receptor 3 (CR3) plays a key role. Phagocytic labeling could theoretically result in neutrophil activation or priming, affecting the behaviour of labeled cells. Fluorescence-activated cell sorter (FACS) analysis side scatter measurements can assess neutrophil activation and priming. Methods: We tested whether TcSnC neutrophil labeling is CR3-mediated by assessing if neutrophil uptake of TcSnC was inhibited by a monoclonal antibody (mAb) directed at the CD11b component of CR3. We tested if TcSnC-labeled neutrophils show altered activation or priming status, comparing FACS side scatter in labeled and unlabeled neutrophils and examining the effect of lipopolysaccharide (LPS), a known priming agent. Results: Anti-CD11b mAb reduced neutrophil uptake of TcSnC in a dose-dependent fashion. Labeled neutrophils did not show significantly increased side scatter compared to controls. LPS significantly increased side scatter in control cells and labeled neutrophils. However, the increase was significantly greater in labeled neutrophils than unlabeled cells. Conclusions: Neutrophil labeling with TcSnC is related to the function of CR3, a receptor which plays a central role in phagocytosis. TcSnC labeling did not significantly activate or prime neutrophils. However, labeled neutrophils showed a greater priming response to LPS. This could result in labeled neutrophils demonstrating increased adhesion on activated endothelium at sites of infection.

Serum proteinase inhibitors and other serum proteins in protein-energy malnutrition

NARCIS (Netherlands)

Schelp, F.P.; Migasena, P.; Pongpaew, P.; SCHREURS W.H.P

1977-01-01

1. The concentrations of serum protein albumin, prealbumin and transferrin were determined in twenty-eight cases of protein-energy malnutrition (PEM) with infection, together with the levels of serum proteinase inhibitors (PI), alpha1-antitrypsin (AT), alpha1-antichymotrypsin (Ach),

Neutrophils in Tuberculosis: Heterogeneity Shapes the Way?

Science.gov (United States)

2017-01-01

Infection with M. tuberculosis remains one of the most common infections in the world. The outcome of the infection depends on host ability to mount effective protection and balance inflammatory responses. Neutrophils are innate immune cells implicated in both processes. Accordingly, during M. tuberculosis infection, they play a dual role. Particularly, they contribute to the generation of effector T cells, participate in the formation of granuloma, and are directly involved in tissue necrosis, destruction, and infection dissemination. Neutrophils have a high bactericidal potential. However, data on their ability to eliminate M. tuberculosis are controversial, and the results of neutrophil depletion experiments are not uniform. Thus, the overall roles of neutrophils during M. tuberculosis infection and factors that determine these roles are not fully understood. This review analyzes data on neutrophil defensive and pathological functions during tuberculosis and considers hypotheses explaining the dualism of neutrophils during M. tuberculosis infection and tuberculosis disease. PMID:28626346

Effect of dopamine injection on the hemocyte count and prophenoloxidase system of the white shrimp Litopenaeus vannamei

Science.gov (United States)

Pan, Luqing; Hu, Fawen; Zheng, Debin

2011-09-01

Effects of dopamine injection on the hemocyte count, phenoloxidase activity, serine proteinase activity, proteinase inhibitor activity and α2-macroglobulin-like activity in L. vannamei were studied. Results showed that dopamine injection resulted in a significant effect on the parameters measured ( P < 0.05), while no significant difference was observed in the control group (0.85% NaCl). In the experimental groups, the hemocyte count reached the minimum in 3 h; granular and semi-granular cells became stable after 12 h and hyaline cells and the total hemocyte count became stable after 18 h. Phenoloxidase activity reached the minimum in 6 h, and then became stable after 9 h. Serine protease activity and proteinase inhibitor activity reached the minimum in 3 h, and α2-macroglobulin-like activity reached the maximum in 3 h, and all the three parameters became stable after 12 h. The results suggest that the activating mechanisms of the proPO system triggered by dopamine are different from those triggered by invasive agents or spontaneously activated under a normal physical condition.

Recombinant protein to analyze autoantibodies to proteinase 3 in systemic vasculitis

NARCIS (Netherlands)

Rarok, AA; Huitema, MG; van der Leij, MJ; van der Geld, YM; Berthold, H; Schmitt, J; Stegeman, CA; Limburg, PC; Kallenberg, CGM

2003-01-01

The presence of antineutrophil cytoplasmic autoantibodies with specificity for proteinase 3 (PR3-ANCA) usually is detected by enzyme-linked immunosorbent assay (ELISA) with purified PR3 as a substrate. We studied the technical performance of direct and capture ELISA using a recombinant

ACTIVATION OF A CRYPTIC D-SERINE DEAMINASE (DSD) GENE FROM PSEUDOMONAS CEPACIA 17616

Science.gov (United States)

D-serine inhibits growth of P. cepacia 17616; however, resistant mutants able to express an ordinarily cryptic D-serine deaminase (dsd) gene were isolated readily. The resistant strains formed high levels of a D-serine deaminase active on D-threonine as well as D-serine. IS eleme...

Activation of bovine neutrophils by Brucella spp.

Science.gov (United States)

Keleher, Lauren L; Skyberg, Jerod A

2016-09-01

Brucellosis is a globally important zoonotic infectious disease caused by gram negative bacteria of the genus Brucella. While many species of Brucella exist, Brucella melitensis, Brucella abortus, and Brucella suis are the most common pathogens of humans and livestock. The virulence of Brucella is largely influenced by its ability to evade host factors, including phagocytic killing mechanisms, which are critical for the host response to infection. The aim of this study was to characterize the bovine neutrophil response to virulent Brucella spp. Here, we found that virulent strains of smooth B. abortus, B. melitensis, B. suis, and virulent, rough, strains of Brucella canis possess similar abilities to resist killing by resting, or IFN-γ-activated, bovine neutrophils. Bovine neutrophils responded to infection with a time-dependent oxidative burst that varied little between Brucella spp. Inhibition of TAK1, or SYK kinase blunted the oxidative burst of neutrophils in response to Brucella infection. Interestingly, Brucella spp. did not induce robust death of bovine neutrophils. These results indicate that bovine neutrophils respond similarly to virulent Brucella spp. In addition, virulent Brucella spp., including naturally rough strains of B. canis, have a conserved ability to resist killing by bovine neutrophils. Copyright © 2016 Elsevier B.V. All rights reserved.

The complex interplay between neutrophils and cancer.

Science.gov (United States)

Rakic, Andrea; Beaudry, Paul; Mahoney, Douglas J

2018-03-01

Neutrophils are the most abundant type of white blood cell, and are an essential component of the innate immune system. They characteristically arrive rapidly at sites of infection and injury, and release a variety of cytokines and toxic molecules to eliminate pathogens and elicit an acute inflammatory response. Research into the function of neutrophils in cancer suggest they have divergent roles. Indeed, while most studies have found neutrophils to be associated with cancer progression, others have also documented anticancer effects. In this review, we describe the investigations into neutrophil populations that have been implicated in promoting tumor growth and metastasis as well those demonstrating antitumor functions. The collective research suggests a complex role for neutrophils in cancer biology, which raises the prospect of their targeting for the treatment of cancer.

Spontaneous neutrophil migration patterns during sepsis after major burns.

Science.gov (United States)

Jones, Caroline N; Moore, Molly; Dimisko, Laurie; Alexander, Andrew; Ibrahim, Amir; Hassell, Bryan A; Warren, H Shaw; Tompkins, Ronald G; Fagan, Shawn P; Irimia, Daniel

2014-01-01

Finely tuned to respond quickly to infections, neutrophils have amazing abilities to migrate fast and efficiently towards sites of infection and inflammation. Although neutrophils ability to migrate is perturbed in patients after major burns, no correlations have yet been demonstrated between altered migration and higher rate of infections and sepsis in these patients when compared to healthy individuals. To probe if such correlations exist, we designed microfluidic devices to quantify the neutrophil migration phenotype with high precision. Inside these devices, moving neutrophils are confined in channels smaller than the neutrophils and forced to make directional decisions at bifurcations and around posts. We employed these devices to quantify neutrophil migration across 18 independent parameters in 74 blood samples from 13 patients with major burns and 3 healthy subjects. Blinded, retrospective analysis of clinical data and neutrophil migration parameters revealed that neutrophils isolated from blood samples collected during sepsis migrate spontaneously inside the microfluidic channels. The spontaneous neutrophil migration is a unique phenotype, typical for patients with major burns during sepsis and often observed one or two days before the diagnosis of sepsis is confirmed. The spontaneous neutrophil migration phenotype is rare in patients with major burns in the absence of sepsis, and is not encountered in healthy individuals. Our findings warrant further studies of neutrophils and their utility for early diagnosing and monitoring sepsis in patients after major burns.

Inhibitory activity and conformational transition of alpha 1-proteinase inhibitor variants

NARCIS (Netherlands)

Schulze, A.J.; Huber, R.; Degryse, E.; Speck, D.; Bischoff, Rainer

1991-01-01

Several variants of alpha 1-proteinase inhibitor (alpha 1-PI) were investigated by spectroscopic methods and characterized according to their inhibitory activity. Replacement of Thr345 (P14) with Arg in alpha 1-PI containing an Arg residue in position 358 (yielding [Thr345----Arg,

Pathophysiology of neutrophil-mediated extracellular redox reactions.

Science.gov (United States)

Jaganjac, Morana; Cipak, Ana; Schaur, Rudolf Joerg; Zarkovic, Neven

2016-01-01

Neutrophil granulocyte leukocytes (neutrophils) play fundamental role in the innate immune response. In the presence of adequate stimuli, neutrophils release excessive amount of reactive oxygen species (ROS) that may induce cell and tissue injury. Oxidative burst of neutrophils acts as a double-edged sword. It may contribute to the pathology of atherosclerosis and brain injury but is also necessary in resolving infections. Moreover, neutrophil-derived ROS may also have both a tumor promoting and tumor suppressing role. ROS have a specific activities and diffusion distance, which is related to their short lifetime. Therefore, the manner in which ROS will act depends on the cells targeted and the intra- and extracellular levels of individual ROS, which can further cause production of reactive aldehydes like 4-hydroxynonenal (HNE) that act as a second messengers of ROS. In this review we discuss the influence of neutrophil mediated extracellular redox reactions in ischemia reperfusion injury, transplant rejection and chronic diseases (atherosclerosis, inflammatory bowel diseases and cancer). At the end a brief overview of cellular mechanisms to maintain ROS homeostasis is given.

Treatment with Rutin - A Therapeutic Strategy for Neutrophil-Mediated Inflammatory and Autoimmune Diseases - Anti-inflammatory Effects of Rutin on Neutrophils -

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Bahareh Abd Nikfarjam

2017-03-01

Full Text Available Objectives: Neutrophils represent the front line of human defense against infections. Immediately after stimulation, neutrophilic enzymes are activated and produce toxic mediators such as pro-inflammatory cytokines, nitric oxide (NO and myeloperoxidase (MPO. These mediators can be toxic not only to infectious agents but also to host tissues. Because flavonoids exhibit antioxidant and anti-inflammatory effects, they are subjects of interest for pharmacological modulation of inflammation. In the present study, the effects of rutin on stimulus-induced NO and tumor necrosis factor (TNF-α productions and MPO activity in human neutrophils were investigated. Methods: Human peripheral blood neutrophils were isolated using Ficoll-Hypaque density gradient centrifugation coupled with dextran T500 sedimentation. The cell preparations containing > 98% granulocytes were determined by morphological examination through Giemsa staining. Neutrophils were cultured in complete Roswell Park Memorial Institute (RPMI medium, pre-incubated with or without rutin (25 μM for 45 minutes, and stimulated with phorbol 12-myristate 13-acetate (PMA. Then, the TNF-α, NO and MPO productions were analyzed using enzyme-linked immunosorbent assay (ELISA, Griess Reagent, and MPO assay kits, respectively. Also, the viability of human neutrophils was assessed using tetrazolium salt 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT, and neutrophils were treated with various concentrations of rutin (1 - 100 μM, after which MTT was appended and incubated at 37ºC for 4 hour. Results: Rutin at concentrations up to 100 μM did not affect neutrophil viability during the 4-hour incubation period. Rutin significantly decreased the NO and TNF-α productions in human peripheral blood neutrophils compared to PMA-control cells (P < 0.001. Also, MPO activity was significantly reduced by rutin (P < 0.001. Conclusion: In this in vitro study, rutin had an anti-inflammatory effect

Thrombin Production and Human Neutrophil Elastase Sequestration by Modified Cellulosic Dressings and Their Electrokinetic Analysis

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Nicolette Prevost

2011-12-01

Full Text Available Wound healing is a complex series of biochemical and cellular events. Optimally, functional material design addresses the overlapping acute and inflammatory stages of wound healing based on molecular, cellular, and bio-compatibility issues. In this paper the issues addressed are uncontrolled hemostasis and inflammation which can interfere with the orderly flow of wound healing. In this regard, we review the serine proteases thrombin and elastase relative to dressing functionality that improves wound healing and examine the effects of charge in cotton/cellulosic dressing design on thrombin production and elastase sequestration (uptake by the wound dressing. Thrombin is central to the initiation and propagation of coagulation, and elastase is released from neutrophils that can function detrimentally in a stalled inflammatory phase characteristic of chronic wounds. Electrokinetic fiber surface properties of the biomaterials of this study were determined to correlate material charge and polarity with function relative to thrombin production and elastase sequestration. Human neutrophil elastase sequestration was assessed with an assay representative of chronic wound concentration with cotton gauze cross-linked with three types of polycarboxylic acids and one phosphorylation finish; thrombin production, which was assessed in a plasma-based assay via a fluorogenic peptide substrate, was determined for cotton, cotton-grafted chitosan, chitosan, rayon/polyester, and two kaolin-treated materials including a commercial hemorrhage control dressing (QuickClot Combat Gauze. A correlation in thrombin production to zeta potential was found. Two polycarboxylic acid cross linked and a phosphorylated cotton dressing gave high elastase sequestration.

Circadian rhythms of cysteine proteinases and cystatins, potential tumour markers, in normal sera

International Nuclear Information System (INIS)

Cimerman, N.; Krasovec, M.; Mesko-Brguljan, P.; Suskovic, S.; Kos, J.

2002-01-01

Circadian day/night variations have been evidenced in all major groups of organisms and at all levels of organisation of the organism. Circadian intra-individual variations are known for a number of analyses in serum including tumour-associated markers. It was suggested that the serum levels of cysteine proteinases and their inhibitors may be of clinical importance for prognosis and diagnosis in cancer. Since known circadian rhythms are important for choosing the best sampling time, interpretation of the results of a diagnostic test, patient monitoring, and timing of a therapy, our objective was to establish 24-h variations of cysteine proteinases, cathepsins B, H, L, and their low molecular weight inhibitors, stefin A, stefin B, and cystatin C, in sera from healthy subjects. (author)

Cerebrospinal fluid D-serine concentrations in major depressive disorder negatively correlate with depression severity.

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Ishiwata, Sayuri; Hattori, Kotaro; Sasayama, Daimei; Teraishi, Toshiya; Miyakawa, Tomoko; Yokota, Yuuki; Matsumura, Ryo; Nishikawa, Toru; Kunugi, Hiroshi

2018-01-15

D-serine is an endogenous co-agonist of N-methyl-D-aspartate receptor (NMDAR) and plays an important role in glutamate neurotransmission. Several studies suggested the possible involvement of D-serine related in the pathophysiology of psychiatric disorders including major depression disorders (MDD). We tried to examine whether cerebrospinal fluid (CSF) or plasma D-serine concentrations are altered in MDD and whether D-serine concentrations correlated with disease severity. 26 MDD patients and 27 healthy controls matched for age, sex and ethnicity were enrolled. We measured amino acids in these samples using by high-performance liquid chromatography with fluorometric detection. D-serine and L-serine, precursor of D-serine, levels in CSF or plasma were not significantly different in patients of MDD compared to controls. Furthermore, a significant correlation between D-serine levels in CSF and Hamilton Depression Rating Scale (HAMD)-17 score was observed (r = -0.65, p = 0.006). Furthermore, we found a positive correlation between CSF D-serine and HVA concentrations in MDD patients (r = 0.54, p = 0.007). CSF D-serine concentrations were correlated with those of plasma in MDD (r = 0.61, p = 0.01) but not in controls. In CSF, we also confirmed a significant correlation between D-serine and L-serine levels in MDD (r = 0.72, p depression severity and HVA concentrations and further investigation were required to reveal the effect of medication and disease heterogeneity. Copyright © 2017 Elsevier B.V. All rights reserved.

Pseudomonas aeruginosa ExoU augments neutrophil transepithelial migration.

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Pazos, Michael A; Lanter, Bernard B; Yonker, Lael M; Eaton, Alex D; Pirzai, Waheed; Gronert, Karsten; Bonventre, Joseph V; Hurley, Bryan P

2017-08-01

Excessive neutrophil infiltration of the lungs is a common contributor to immune-related pathology in many pulmonary disease states. In response to pathogenic infection, airway epithelial cells produce hepoxilin A3 (HXA3), initiating neutrophil transepithelial migration. Migrated neutrophils amplify this recruitment by producing a secondary gradient of leukotriene B4 (LTB4). We sought to determine whether this two-step eicosanoid chemoattractant mechanism could be exploited by the pathogen Pseudomonas aeruginosa. ExoU, a P. aeruginosa cytotoxin, exhibits phospholipase A2 (PLA2) activity in eukaryotic hosts, an enzyme critical for generation of certain eicosanoids. Using in vitro and in vivo models of neutrophil transepithelial migration, we evaluated the impact of ExoU expression on eicosanoid generation and function. We conclude that ExoU, by virtue of its PLA2 activity, augments and compensates for endogenous host neutrophil cPLA2α function, leading to enhanced transepithelial migration. This suggests that ExoU expression in P. aeruginosa can circumvent immune regulation at key signaling checkpoints in the neutrophil, resulting in exacerbated neutrophil recruitment.

Pseudomonas aeruginosa ExoU augments neutrophil transepithelial migration.

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Michael A Pazos

2017-08-01

Full Text Available Excessive neutrophil infiltration of the lungs is a common contributor to immune-related pathology in many pulmonary disease states. In response to pathogenic infection, airway epithelial cells produce hepoxilin A3 (HXA3, initiating neutrophil transepithelial migration. Migrated neutrophils amplify this recruitment by producing a secondary gradient of leukotriene B4 (LTB4. We sought to determine whether this two-step eicosanoid chemoattractant mechanism could be exploited by the pathogen Pseudomonas aeruginosa. ExoU, a P. aeruginosa cytotoxin, exhibits phospholipase A2 (PLA2 activity in eukaryotic hosts, an enzyme critical for generation of certain eicosanoids. Using in vitro and in vivo models of neutrophil transepithelial migration, we evaluated the impact of ExoU expression on eicosanoid generation and function. We conclude that ExoU, by virtue of its PLA2 activity, augments and compensates for endogenous host neutrophil cPLA2α function, leading to enhanced transepithelial migration. This suggests that ExoU expression in P. aeruginosa can circumvent immune regulation at key signaling checkpoints in the neutrophil, resulting in exacerbated neutrophil recruitment.

NETosis Delays Diabetic Wound Healing in Mice and Humans.

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Fadini, Gian Paolo; Menegazzo, Lisa; Rigato, Mauro; Scattolini, Valentina; Poncina, Nicol; Bruttocao, Andrea; Ciciliot, Stefano; Mammano, Fabio; Ciubotaru, Catalin Dacian; Brocco, Enrico; Marescotti, Maria Cristina; Cappellari, Roberta; Arrigoni, Giorgio; Millioni, Renato; Vigili de Kreutzenberg, Saula; Albiero, Mattia; Avogaro, Angelo

2016-04-01

Upon activation, neutrophils undergo histone citrullination by protein arginine deiminase (PAD)4, exocytosis of chromatin and enzymes as neutrophil extracellular traps (NETs), and death. In diabetes, neutrophils are primed to release NETs and die by NETosis. Although this process is a defense against infection, NETosis can damage tissue. Therefore, we examined the effect of NETosis on the healing of diabetic foot ulcers (DFUs). Using proteomics, we found that NET components were enriched in nonhealing human DFUs. In an independent validation cohort, a high concentration of neutrophil elastase in the wound was associated with infection and a subsequent worsening of the ulcer. NET components (elastase, histones, neutrophil gelatinase-associated lipocalin, and proteinase-3) were elevated in the blood of patients with DFUs. Circulating elastase and proteinase-3 were associated with infection, and serum elastase predicted delayed healing. Neutrophils isolated from the blood of DFU patients showed an increased spontaneous NETosis but an impaired inducible NETosis. In mice, skin PAD4 activity was increased by diabetes, and FACS detection of histone citrullination, together with intravital microscopy, showed that NETosis occurred in the bed of excisional wounds. PAD4 inhibition by Cl-amidine reduced NETting neutrophils and rescued wound healing in diabetic mice. Cumulatively, these data suggest that NETosis delays DFU healing. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Cross genome comparisons of serine proteases in Arabidopsis and rice

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Sowdhamini R

2006-08-01

Full Text Available Abstract Background Serine proteases are one of the largest groups of proteolytic enzymes found across all kingdoms of life and are associated with several essential physiological pathways. The availability of Arabidopsis thaliana and rice (Oryza sativa genome sequences has permitted the identification and comparison of the repertoire of serine protease-like proteins in the two plant species. Results Despite the differences in genome sizes between Arabidopsis and rice, we identified a very similar number of serine protease-like proteins in the two plant species (206 and 222, respectively. Nearly 40% of the above sequences were identified as potential orthologues. Atypical members could be identified in the plant genomes for Deg, Clp, Lon, rhomboid proteases and species-specific members were observed for the highly populated subtilisin and serine carboxypeptidase families suggesting multiple lateral gene transfers. DegP proteases, prolyl oligopeptidases, Clp proteases and rhomboids share a significantly higher percentage orthology between the two genomes indicating substantial evolutionary divergence was set prior to speciation. Single domain architectures and paralogues for several putative subtilisins, serine carboxypeptidases and rhomboids suggest they may have been recruited for additional roles in secondary metabolism with spatial and temporal regulation. The analysis reveals some domain architectures unique to either or both of the plant species and some inactive proteases, like in rhomboids and Clp proteases, which could be involved in chaperone function. Conclusion The systematic analysis of the serine protease-like proteins in the two plant species has provided some insight into the possible functional associations of previously uncharacterised serine protease-like proteins. Further investigation of these aspects may prove beneficial in our understanding of similar processes in commercially significant crop plant species.

Cryptococcus neoformans modulates extracellular killing by neutrophils

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Asfia eQureshi

2011-09-01

Full Text Available We recently established a key role for host sphingomyelin synthase (SMS in the regulation of the killing activity of neutrophils against Cryptococcus neoformans. In this work, we studied the effect of C. neoformans on the killing activity of neutrophils and whether SMS would still be a player against C. neoformans in immunocompromised mice lacking T and NK cells (Tgε26 mice. To this end, we analyzed whether C. neoformans would have any effect on neutrophil survival and killing in vitro and in vivo. We show that unlike C. albicans, neither the presence nor the capsule size of C. neoformans cells have any effect on neutrophil viability. Interestingly, melanized C. neoformans cells totally abrogated the killing activity of neutrophils. Next, we monitored how exposure of neutrophils to C. neoformans cells would interfere with any further killing activity of the medium and found that pre-incubation with live but not heat-killed fungal cells significantly inhibits further killing activity of the medium. We next studied whether activation of SMS at the site of C. neoformans infection is dependent on T and NK cells. Using matrix-assisted laser desorption-ionization (MALDI tissue imaging in infected lung we found that similarly to previous observations in the isogenic wild type CBA/J mice, SM 16:0 levels are significantly elevated at the site of infection in mice lacking T and NK cells but only at early time points. This study highlights that C. neoformans may negatively regulate the killing activity of neutrophils and that SMS activation in neutrophils appears to be partially independent of T and/or NK cells.

Phenotypic Diversity and Plasticity in Circulating Neutrophil Subpopulations in Cancer

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Jitka Y. Sagiv

2015-02-01

Full Text Available Controversy surrounds neutrophil function in cancer because neutrophils were shown to provide both pro- and antitumor functions. We identified a heterogeneous subset of low-density neutrophils (LDNs that appear transiently in self-resolving inflammation but accumulate continuously with cancer progression. LDNs display impaired neutrophil function and immunosuppressive properties, characteristics that are in stark contrast to those of mature, high-density neutrophils (HDNs. LDNs consist of both immature myeloid-derived suppressor cells (MDSCs and mature cells that are derived from HDNs in a TGF-β-dependent mechanism. Our findings identify three distinct populations of circulating neutrophils and challenge the concept that mature neutrophils have limited plasticity. Furthermore, our findings provide a mechanistic explanation to mitigate the controversy surrounding neutrophil function in cancer.

[1-14C]Glycolate metabolism and serine biosynthesis in soybean plants

International Nuclear Information System (INIS)

Calmes, J.; Viala, G.; Latche, J.C.; Cavalie, G.

1977-01-01

[1- 14 C]Glycolate metabolism was examined in leafy shoots of soybean plants (Glycine max (L.) Merr., var. Adepta). Only small amounts of 14 C were incorporated into evolved carbon dioxide and glucidic compounds. Free and protein glycine was labelled but higher levels of radioactivity were found in free serine. Changes in the distribution of 14 C with time showed that metabolic conversion glycollate → glycine → serine occurred very early and serine biosynthesis was more important in the shoot than in the leaves. Carbon dioxide labelling was always slight compared to serine labelling. These data suggest strong relations between glycollate and nitrogen metabolism

The VA, VCD, Raman and ROA spectra of tri-L-serine in aqueous solution

DEFF Research Database (Denmark)

Jürgensen, Vibeke Würtz; Jalkanen, Karl J.

2006-01-01

The structures of one conformer of the nonionic neutral and zwitterionic species of L-serinyl L-serinyl L-serine (SSS or tri-L-serine), together with its cationic and anionic species and the capped N-acetyl tri-L-serine N'-methylamide analog were optimized with density functional theory with the ......The structures of one conformer of the nonionic neutral and zwitterionic species of L-serinyl L-serinyl L-serine (SSS or tri-L-serine), together with its cationic and anionic species and the capped N-acetyl tri-L-serine N'-methylamide analog were optimized with density functional theory...

Hidden truth of circulating neutrophils (polymorphonuclear neutrophil function in periodontally healthy smoker subjects

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Chitra Agarwal

2016-01-01

Full Text Available Context: Tobacco smoking is considered to be a major risk factor associated with periodontal disease. Smoking exerts a major effect on the protective elements of the immune response, resulting in an increase in the extent and severity of periodontal destruction. Aims: The aim of the present study was to assess viability and phagocytic function of neutrophils in circulating blood of the smokers and nonsmokers who are periodontally healthy. Settings and Design: Two hundred subjects in the mean range of 20–30 years of age were included in the study population. It was a retrospective study carried out for 6 months. Materials and Methods: Two hundred subjects were divided into four groups: 50 nonsmokers, 50 light smokers (15 cigarettes/day. Full mouth plaque index, sulcus bleeding index, and probing depths were measured. Percentage viability of circulating neutrophils and average number of phagocytosed Candida albicans were recorded. Statistical Analysis Used: Means and standard deviations were calculated from data obtained within the groups. Comparison between the smokers and nonsmokers was performed by Kruskal–Wallis ANOVA analysis. Comparison between smoker groups was performed using Mann–Whitney–Wilcoxon test. Results: Percentage viability of neutrophils was significantly less in heavy smokers (66.9 ± 4.0, moderate (76.6 ± 4.2, light smokers (83.1 ± 2.5 as compared to nonsmokers (92.3 ± 2.6 (P < 0.01. The ability of neutrophils to phagocytose, i.e., mean particle number was significantly less in light smokers (3.5 ± 0.5, moderate smokers (2.3 ± 0.5, and heavy smokers (1.4 ± 0.5 compared to nonsmokers (4.9 ± 0.7 (P < 0.01 with evidence of dose-response effect. Conclusions: Smoking significantly affects neutrophils viability and phagocytic function in periodontally healthy population.

Biomaterial-induced alterations of neutrophil superoxide production.

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Kaplan, S S; Basford, R E; Mora, E; Jeong, M H; Simmons, R L

1992-08-01

Because periprosthetic infection remains a vexing problem for patients receiving implanted devices, we evaluated the effect of several materials on neutrophil free radical production. Human peripheral blood neutrophils were incubated with several sterile, lipopolysaccharide (LPS)-free biomaterials used in surgically implantable prosthetic devices: polyurethane, woven dacron, and velcro. Free radical formation as the superoxide (O2-) anion was evaluated by cytochrome c reduction in neutrophils that were exposed to the materials and then removed and in neutrophils allowed to remain in association with the materials. Neutrophils exposed to polyurethane or woven dacron for 30 or 60 min and then removed consistently exhibited an enhanced release of O2- after simulation via receptor engagement with formyl methionyl-leucyl-phenylalanine. Enhanced reactivity to stimulation via protein kinase C with phorbol myristate acetate, however, was not consistently observed. The cells evaluated for O2- release during continuous association with the biomaterials showed enhanced metabolic activity during short periods of association (especially with polyurethane and woven dacron). Although O2- release by neutrophils in association with these materials decreased with longer periods of incubation, it was not obliterated. These studies, therefore, show that several commonly used biomaterials activate neutrophils soon after exposure and that this activated state diminishes with prolonged exposure but nevertheless remains measurable. The diminishing level of activity with prolonged exposure, however, suggests that ultimately a depletion of reactivity may occur and may result in increased susceptibility to periprosthetic infection.

Superoxide anion production by human neutrophils activated by Trichomonas vaginalis.

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Song, Hyun-Ouk; Ryu, Jae-Sook

2013-08-01

Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O2 (.-)) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis.

Advanced Role of Neutrophils in Common Respiratory Diseases

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Jinping Liu

2017-01-01

Full Text Available Respiratory diseases, always being a threat towards the health of people all over the world, are most tightly associated with immune system. Neutrophils serve as an important component of immune defense barrier linking innate and adaptive immunity. They participate in the clearance of exogenous pathogens and endogenous cell debris and play an essential role in the pathogenesis of many respiratory diseases. However, the pathological mechanism of neutrophils remains complex and obscure. The traditional roles of neutrophils in severe asthma, chronic obstructive pulmonary diseases (COPD, pneumonia, lung cancer, pulmonary fibrosis, bronchitis, and bronchiolitis had already been reviewed. With the development of scientific research, the involvement of neutrophils in respiratory diseases is being brought to light with emerging data on neutrophil subsets, trafficking, and cell death mechanism (e.g., NETosis, apoptosis in diseases. We reviewed all these recent studies here to provide you with the latest advances about the role of neutrophils in respiratory diseases.

Characterization of Yersinia pestis Interactions with Human Neutrophils In vitro

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Sophia C. Dudte

2017-08-01

Full Text Available Yersinia pestis is a gram-negative, zoonotic, bacterial pathogen, and the causative agent of plague. The bubonic form of plague occurs subsequent to deposition of bacteria in the skin by the bite of an infected flea. Neutrophils are recruited to the site of infection within the first few hours and interactions between neutrophils and Y. pestis have been demonstrated in vivo. In contrast to macrophages, neutrophils have been considered non-permissive to Y. pestis intracellular survival. Several studies have shown killing of the vast majority of Y. pestis ingested by human neutrophils. However, survival of 10–15% of Y. pestis after phagocytosis by neutrophils is consistently observed. Furthermore, these surviving bacteria eventually replicate within and escape from the neutrophils. We set out to further characterize the interactions between Y. pestis and human neutrophils by (1 determining the effects of known Y. pestis virulence factors on bacterial survival after uptake by neutrophils, (2 examining the mechanisms employed by the neutrophil to kill the majority of intracellular Y. pestis, (3 determining the activation phenotype of Y. pestis-infected neutrophils, and (4 characterizing the Y. pestis-containing phagosome in neutrophils. We infected human neutrophils in vitro with Y. pestis and assayed bacterial survival and uptake. Deletion of the caf1 gene responsible for F1 capsule production resulted in significantly increased uptake of Y. pestis. Surprisingly, while the two-component regulator PhoPQ system is important for survival of Y. pestis within neutrophils, pre-induction of this system prior to infection did not increase bacterial survival. We used an IPTG-inducible mCherry construct to distinguish viable from non-viable intracellular bacteria and determined the association of the Y. pestis-containing phagosome with neutrophil NADPH-oxidase and markers of primary, secondary and tertiary granules. Additionally, we show that inhibition of

Neutrophil extracellular trap formation in supragingival biofilms.

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Hirschfeld, Josefine; Dommisch, Henrik; Skora, Philipp; Horvath, Gabor; Latz, Eicke; Hoerauf, Achim; Waller, Tobias; Kawai, Toshihisa; Jepsen, Søren; Deschner, James; Bekeredjian-Ding, Isabelle

2015-01-01

Oral biofilms are the causative agents of the highly prevalent oral diseases periodontitis and caries. Additionally, the host immune response is thought to play a critical role in disease onset. Neutrophils are known to be a key host response factor to bacterial challenge on host surfaces. Release of neutrophil extracellular traps (NETs) as a novel antimicrobial defense strategy has gained increasing attention in the past years. Here, we investigated the influx of neutrophils into the dental plaque and the ability of oral bacteria to trigger intra-biofilm release of NETs and intracellular proteins. Supragingival biofilms and whole saliva were sampled from systemically healthy subjects participating in an experimental gingivitis study. Biofilms were analysed by immunofluorescence followed by confocal and fluorescence microscopy. Moreover, concentrations of cytokines and immune-associated proteins in biofilm suspensions and saliva were assessed by ELISA. Neutrophils obtained from blood were stimulated with twelve bacterial species isolated from cultured biofilms or with lipopolysaccharide to monitor NET formation. Neutrophils, NETs, neutrophil-associated proteins (myeloperoxidase, elastase-2, cathepsin G, cathelicidin LL-37), interleukin-8, interleukin-1β and tumor necrosis factor were detected within plaque samples and saliva. All tested bacterial species as well as the polymicrobial samples isolated from the plaque of each donor induced release of NETs and interleukin-8. The degree of NET formation varied among different subjects and did not correlate with plaque scores or clinical signs of local inflammation. Our findings indicate that neutrophils are attracted towards dental biofilms, in which they become incorporated and where they are stimulated by microbes to release NETs and immunostimulatory proteins. Thus, neutrophils and NETs may be involved in host biofilm control, although their specific role needs to be further elucidated. Moreover, inter

Gβ1 is required for neutrophil migration in zebrafish.

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Ke, Wenfan; Ye, Ding; Mersch, Kacey; Xu, Hui; Chen, Songhai; Lin, Fang

2017-08-01

Signaling mediated by G protein-coupled receptors (GPCRs) is essential for the migration of cells toward chemoattractants. The recruitment of neutrophils to injured tissues in zebrafish larvae is a useful model for studying neutrophil migration and trafficking in vivo. Indeed, the study of this process led to the discovery that PI3Kγ is required for the polarity and motility of neutrophils, features that are necessary for the directed migration of these cells to wounds. However, the mechanism by which PI3Kγ is activated remains to be determined. Here we show that signaling by specifically the heterotrimeric G protein subunit Gβ1 is critical for neutrophil migration in response to wounding. In embryos treated with small-molecule inhibitors of Gβγ signaling, neutrophils failed to migrate to wound sites. Although both the Gβ1 and Gβ4 isoforms are expressed in migrating neutrophils, only deficiency for the former (morpholino-based knockdown) interfered with the directed migration of neutrophils towards wounds. The Gβ1 deficiency also impaired the ability of cells to change cell shape and reduced their general motility, defects that are similar to those in neutrophils deficient for PI3Kγ. Transplantation assays showed that the requirement for Gβ1 in neutrophil migration is cell autonomous. Finally, live imaging revealed that Gβ1 is required for polarized activation of PI3K, and for the actin dynamics that enable neutrophil migration. Collectively, our data indicate that Gβ1 signaling controls proper neutrophil migration by activating PI3K and modulating actin dynamics. Moreover, they illustrate a role for a specific Gβ isoform in chemotaxis in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Convergent synthesis of a deuterium-labeled serine dipeptide lipid for analysis of biological samples.

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Dietz, Christopher; Clark, Robert B; Nichols, Frank C; Smith, Michael B

2017-05-30

Bacterial serine dipeptide lipids are known to promote inflammatory processes and are detected in human tissues associated with periodontal disease or atherosclerosis. Accurate quantification of bacterial serine lipid, specifically lipid 654 [((S)-15-methyl-3-((13-methyltetradecanoyl)oxy)hexadecanoyl)glycyl-l-serine, (3S)-l-serine] isolated from Porphyromonas gingivalis, in biological samples requires the preparation of a stable isotope internal standard for sample supplementation and subsequent mass spectrometric analysis. This report describes the convergent synthesis of a deuterium-substituted serine dipeptide lipid, which is an isotopically labeled homologue that represents a dominant form of serine dipeptide lipid recovered in bacteria. Copyright © 2017 John Wiley & Sons, Ltd.

Prevention of vascular inflammation by nanoparticle targeting of adherent neutrophils

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Wang, Zhenjia; Li, Jing; Cho, Jaehyung; Malik, Asrar B.

2014-03-01

Inflammatory diseases such as acute lung injury and ischaemic tissue injury are caused by the adhesion of a type of white blood cell--polymorphonuclear neutrophils--to the lining of the circulatory system or vascular endothelium and unchecked neutrophil transmigration. Nanoparticle-mediated targeting of activated neutrophils on vascular endothelial cells at the site of injury may be a useful means of directly inactivating neutrophil transmigration and hence mitigating vascular inflammation. Here, we report a method employing drug-loaded albumin nanoparticles, which efficiently deliver drugs into neutrophils adherent to the surface of the inflamed endothelium. Using intravital microscopy of tumour necrosis factor-α-challenged mouse cremaster post-capillary venules, we demonstrate that fluorescently tagged albumin nanoparticles are largely internalized by neutrophils adherent to the activated endothelium via cell surface Fcɣ receptors. Administration of albumin nanoparticles loaded with the spleen tyrosine kinase inhibitor, piceatannol, which blocks `outside-in' β2 integrin signalling in leukocytes, detached the adherent neutrophils and elicited their release into the circulation. Thus, internalization of drug-loaded albumin nanoparticles into neutrophils inactivates the pro-inflammatory function of activated neutrophils, thereby offering a promising approach for treating inflammatory diseases resulting from inappropriate neutrophil sequestration and activation.

L-Serine overproduction with minimization of by-product synthesis by engineered Corynebacterium glutamicum.

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Zhu, Qinjian; Zhang, Xiaomei; Luo, Yuchang; Guo, Wen; Xu, Guoqiang; Shi, Jinsong; Xu, Zhenghong

2015-02-01

The direct fermentative production of L-serine by Corynebacterium glutamicum from sugars is attractive. However, superfluous by-product accumulation and low L-serine productivity limit its industrial production on large scale. This study aimed to investigate metabolic and bioprocess engineering strategies towards eliminating by-products as well as increasing L-serine productivity. Deletion of alaT and avtA encoding the transaminases and introduction of an attenuated mutant of acetohydroxyacid synthase (AHAS) increased both L-serine production level (26.23 g/L) and its productivity (0.27 g/L/h). Compared to the parent strain, the by-products L-alanine and L-valine accumulation in the resulting strain were reduced by 87 % (from 9.80 to 1.23 g/L) and 60 % (from 6.54 to 2.63 g/L), respectively. The modification decreased the metabolic flow towards the branched-chain amino acids (BCAAs) and induced to shift it towards L-serine production. Meanwhile, it was found that corn steep liquor (CSL) could stimulate cell growth and increase sucrose consumption rate as well as L-serine productivity. With addition of 2 g/L CSL, the resulting strain showed a significant improvement in the sucrose consumption rate (72 %) and the L-serine productivity (67 %). In fed-batch fermentation, 42.62 g/L of L-serine accumulation was achieved with a productivity of 0.44 g/L/h and yield of 0.21 g/g sucrose, which was the highest production of L-serine from sugars to date. The results demonstrated that combined metabolic and bioprocess engineering strategies could minimize by-product accumulation and improve L-serine productivity.

Candida albicans escapes from mouse neutrophils

DEFF Research Database (Denmark)

Ermert, David; Niemiec, Maria J; Röhm, Marc

2013-01-01

is the most widely used model organism. Neutrophils are essential immune cells to prevent opportunistic mycoses. To explore potential differences between the rodent infection model and the human host, we compared the interactions of C. albicans with neutrophil granulocytes from mice and humans. We revealed...

Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation.

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Alemán, Omar Rafael; Mora, Nancy; Cortes-Vieyra, Ricarda; Uribe-Querol, Eileen; Rosales, Carlos

2016-01-01

Neutrophils (PMN) are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA) are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil.

Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation

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Omar Rafael Alemán

2016-01-01

Full Text Available Neutrophils (PMN are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil.

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

Science.gov (United States)

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.

Hypoxia upregulates neutrophil degranulation and potential for tissue injury

Science.gov (United States)

Hoenderdos, Kim; Lodge, Katharine M; Hirst, Robert A; Chen, Cheng; Palazzo, Stefano G C; Emerenciana, Annette; Summers, Charlotte; Angyal, Adri; Porter, Linsey; Juss, Jatinder K; O'Callaghan, Christopher; Chilvers, Edwin R

2016-01-01

Background The inflamed bronchial mucosal surface is a profoundly hypoxic environment. Neutrophilic airway inflammation and neutrophil-derived proteases have been linked to disease progression in conditions such as COPD and cystic fibrosis, but the effects of hypoxia on potentially harmful neutrophil functional responses such as degranulation are unknown. Methods and results Following exposure to hypoxia (0.8% oxygen, 3 kPa for 4 h), neutrophils stimulated with inflammatory agonists (granulocyte-macrophage colony stimulating factor or platelet-activating factor and formylated peptide) displayed a markedly augmented (twofold to sixfold) release of azurophilic (neutrophil elastase, myeloperoxidase), specific (lactoferrin) and gelatinase (matrix metalloproteinase-9) granule contents. Neutrophil supernatants derived under hypoxic but not normoxic conditions induced extensive airway epithelial cell detachment and death, which was prevented by coincubation with the antiprotease α-1 antitrypsin; both normoxic and hypoxic supernatants impaired ciliary function. Surprisingly, the hypoxic upregulation of neutrophil degranulation was not dependent on hypoxia-inducible factor (HIF), nor was it fully reversed by inhibition of phospholipase C signalling. Hypoxia augmented the resting and cytokine-stimulated phosphorylation of AKT, and inhibition of phosphoinositide 3-kinase (PI3K)γ (but not other PI3K isoforms) prevented the hypoxic upregulation of neutrophil elastase release. Conclusion Hypoxia augments neutrophil degranulation and confers enhanced potential for damage to respiratory airway epithelial cells in a HIF-independent but PI3Kγ-dependent fashion. PMID:27581620

Novel Aggregation Properties of Candida albicans Secreted Aspartyl Proteinase Sap6 Mediate Virulence in Oral Candidiasis.

Science.gov (United States)

Kumar, Rohitashw; Saraswat, Darpan; Tati, Swetha; Edgerton, Mira

2015-07-01

Candida albicans, a commensal fungus of the oral microbiome, causes oral candidiasis in humans with localized or systemic immune deficiencies. Secreted aspartic proteinases (Saps) are a family of 10 related proteases and are virulence factors due to their proteolytic activity, as well as their roles in adherence and colonization of host tissues. We found that mice infected sublingually with C. albicans cells overexpressing Sap6 (SAP6 OE and a Δsap8 strain) had thicker fungal plaques and more severe oral infection, while infection with the Δsap6 strain was attenuated. These hypervirulent strains had highly aggregative colony structure in vitro and higher secreted proteinase activity; however, the levels of proteinase activity of C. albicans Saps did not uniformly match their abilities to damage cultured oral epithelial cells (SCC-15 cells). Hyphal induction in cells overexpressing Sap6 (SAP6 OE and Δsap8 cells) resulted in formation of large cell-cell aggregates. These aggregates could be produced in germinated wild-type cells by addition of native or heat-inactivated Sap6. Sap6 bound only to germinated cells and increased C. albicans adhesion to oral epithelial cells. The adhesion properties of Sap6 were lost upon deletion of its integrin-binding motif (RGD) and could be inhibited by addition of RGD peptide or anti-integrin antibodies. Thus, Sap6 (but not Sap5) has an alternative novel function in cell-cell aggregation, independent of its proteinase activity, to promote infection and virulence in oral candidiasis.

Neutrophilic dermatoses in a patient with collagenous colitis

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Didac Barco

2010-01-01

Full Text Available We report the case of a 75-year old woman with collagenous colitis who presented with erythematous and edematous plaques on the periorbital and eyelid regions, accompanied by oral ulcers. Histopathology showed a dermal neutrophilic infiltrate plus mild septal and lobular panniculitis with lymphocytes, neutrophils and eosinophils. Five years earlier she had presented a flare of papules and vesicles on the trunk, together with oral ulcers; a skin biopsy revealed a neutrophilic dermal infiltrate and Sweet’s syndrome was diagnosed. Both the neutrophilic panniculitis and the Sweet’s syndrome were accompanied by fever, malaise and diarrhea. Cutaneous and intestinal symptoms disappeared with corticoid therapy. The two types of neutrophilic dermatoses that appeared in periods of colitis activity suggest that intestinal and cutaneous manifestations may be related.

Neutrophil adhesion and chemotaxis depend on substrate mechanics

International Nuclear Information System (INIS)

Jannat, Risat A; Hammer, Daniel A; Robbins, Gregory P; Ricart, Brendon G; Dembo, Micah

2010-01-01

Neutrophil adhesion to the vasculature and chemotaxis within tissues play critical roles in the inflammatory response to injury and pathogens. Unregulated neutrophil activity has been implicated in the progression of numerous chronic and acute diseases such as rheumatoid arthritis, asthma and sepsis. Cell migration of anchorage-dependent cells is known to depend on both chemical and mechanical interactions. Although neutrophil responses to chemical cues have been well characterized, little is known about the effect of underlying tissue mechanics on neutrophil adhesion and migration. To address this question, we quantified neutrophil migration and traction stresses on compliant hydrogel substrates with varying elasticity in a micromachined gradient chamber in which we could apply either a uniform concentration or a precise gradient of the bacterial chemoattractant fMLP. Neutrophils spread more extensively on substrates of greater stiffness. In addition, increasing the stiffness of the substrate leads to a significant increase in the chemotactic index for each fMLP gradient tested. As the substrate becomes stiffer, neutrophils generate higher traction forces without significant changes in cell speed. These forces are often displayed in pairs and focused in the uropod. Increases in the mean fMLP concentration beyond the K D of the receptor lead to a decrease in chemotactic index on all surfaces. Blocking with an antibody against β 2 -integrins leads to a significant reduction, but not an elimination, of directed motility on stiff materials, but no change in motility on soft materials, suggesting neutrophils can display both integrin-dependent and integrin-independent motility. These findings are critical for understanding how neutrophil migration may change in different mechanical environments in vivo and can be used to guide the design of migration inhibitors that more efficiently target inflammation.

Neutrophil adhesion and chemotaxis depend on substrate mechanics

Energy Technology Data Exchange (ETDEWEB)

Jannat, Risat A; Hammer, Daniel A [Department of Bioengineering, University of Pennsylvania, 240 Skirkanich Hall, 210 South 33rd Street, Philadelphia, PA 19104 (United States); Robbins, Gregory P; Ricart, Brendon G [Department of Chemical and Biomolecular Engineering, University of Pennsylvania, 311A Towne Building, 220 South 33rd Street, Philadelphia, PA 19104 (United States); Dembo, Micah, E-mail: hammer@seas.upenn.ed [Department of Biomedical Engineering, Boston University, 44 Cummington Street, Boston, MA 02215 (United States)

2010-05-19

Neutrophil adhesion to the vasculature and chemotaxis within tissues play critical roles in the inflammatory response to injury and pathogens. Unregulated neutrophil activity has been implicated in the progression of numerous chronic and acute diseases such as rheumatoid arthritis, asthma and sepsis. Cell migration of anchorage-dependent cells is known to depend on both chemical and mechanical interactions. Although neutrophil responses to chemical cues have been well characterized, little is known about the effect of underlying tissue mechanics on neutrophil adhesion and migration. To address this question, we quantified neutrophil migration and traction stresses on compliant hydrogel substrates with varying elasticity in a micromachined gradient chamber in which we could apply either a uniform concentration or a precise gradient of the bacterial chemoattractant fMLP. Neutrophils spread more extensively on substrates of greater stiffness. In addition, increasing the stiffness of the substrate leads to a significant increase in the chemotactic index for each fMLP gradient tested. As the substrate becomes stiffer, neutrophils generate higher traction forces without significant changes in cell speed. These forces are often displayed in pairs and focused in the uropod. Increases in the mean fMLP concentration beyond the K{sub D} of the receptor lead to a decrease in chemotactic index on all surfaces. Blocking with an antibody against {beta}{sub 2}-integrins leads to a significant reduction, but not an elimination, of directed motility on stiff materials, but no change in motility on soft materials, suggesting neutrophils can display both integrin-dependent and integrin-independent motility. These findings are critical for understanding how neutrophil migration may change in different mechanical environments in vivo and can be used to guide the design of migration inhibitors that more efficiently target inflammation.

Chemotactic Activity on Human Neutrophils to Streptococcus mutans

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Tetiana Haniastuti

2013-07-01

Full Text Available Objective: The aim of this study was to evaluate chemotactic activity o neutrophil to S. mutans. Chemotaxis assay was performed in blind well chambers. Materials and Methods: Hanks balanced salt solution (HBSS containing 106 S. mutans,  108 S. mutans, 10-8 M fMLP, or HBSS alone were placed in the lower wells of the chamber and covered with polycorbonate membrane filter. Neutrophils suspension (2x105 cells was then placed in the upper compartment. After incubation for 60 mins at 37ºC in a humidified atmosphere with 5% CO2, the filters were removed and stained with Giemsa. Result: ANOVA revealed statistically significant differences among groups (p<0.05, indicating that S. mutans induced neutrophils chemotaxis. The number of neutrophils migration in response to 108 S. mutans and 106 S. mutans were signifiantly greater compared to fMLP (p<0.05. Conclusion: S. mutans may activate human neutrophils, resulting in the chemotaxis of the neutrophils.DOI: 10.14693/jdi.v16i2.99

Effect of Isolation Techniques on Viability of Bovine Blood Neutrophils

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P. Sláma

2006-01-01

Full Text Available The effect of selected isolation methods on the viability of neutrophil granulocytes (neutrophils from the blood of healthy Holstein x Bohemian Red Pied crossbred heifers was evaluated. Two methods of neutrophil isolation were used: a neutrophil isolation on the basis of hypotonic erythrocyte lysis (in two variants: after the erythrocyte lysis proper, the cells were centrifuged at either 200 g or 1000 g, and b neutrophil isolation with FACS Lysing Solution as the lysing agent. The viability of the isolated neutrophils was evaluated on the basis of apoptosis and necrosis. The results obtained with flow cytometry (FCM suggest that, from the isolation techniques used, the method based on FACS Lysing Solution impaired the neutrophil viability least. After the application of this method, 5.36 ± 2.15% of neutrophils were apoptotic and 0.51 ± 0.12% were necrotic. In contrast, when the hypotonic erythrocyte lysis was used, the proportion of apoptotic neutrophils amounted to 42.14 ± 7.12% and 49.00 ± 14.70%, respectively, and 41.12 ± 5.55% and 36.91 ± 24.38% respectively of necrotic neutrophils (P < 0.01. This was also confirmed by the light microscopy. After the isolation with FASC Lysing Solution, 1.92 ± 1.74% of neutrophils were apoptotic and 1.05 ± 0.76% were necrotic, as distinct from after the hypotonic erythrocyte lysis where 9.43 ± 3.69% of neutrophils were apoptotic and 12.67 ± 4.74% of necrotic after centrifugation at 200 g, while 12.60 ± 4.35 were apoptotic and 14.96 ± 12.64% were necrotic after centrifugation at 1000 g. It follows from the above-mentioned data that hypotonic lysis is not a suitable method for the isolation of neutrophils, as the method itself markedly affects cell viability.

Site-specific DNA Inversion by Serine Recombinases

Science.gov (United States)

2015-01-01

Reversible site-specific DNA inversion reactions are widely distributed in bacteria and their viruses. They control a range of biological reactions that most often involve alterations of molecules on the surface of cells or phage. These programmed DNA rearrangements usually occur at a low frequency, thereby preadapting a small subset of the population to a change in environmental conditions, or in the case of phages, an expanded host range. A dedicated recombinase, sometimes with the aid of additional regulatory or DNA architectural proteins, catalyzes the inversion of DNA. RecA or other components of the general recombination-repair machinery are not involved. This chapter discusses site-specific DNA inversion reactions mediated by the serine recombinase family of enzymes and focuses on the extensively studied serine DNA invertases that are stringently controlled by the Fis-bound enhancer regulatory system. The first section summarizes biological features and general properties of inversion reactions by the Fis/enhancer-dependent serine invertases and the recently described serine DNA invertases in Bacteroides. Mechanistic studies of reactions catalyzed by the Hin and Gin invertases are then discussed in more depth, particularly with regards to recent advances in our understanding of the function of the Fis/enhancer regulatory system, the assembly of the active recombination complex (invertasome) containing the Fis/enhancer, and the process of DNA strand exchange by rotation of synapsed subunit pairs within the invertasome. The role of DNA topological forces that function in concert with the Fis/enhancer controlling element in specifying the overwhelming bias for DNA inversion over deletion and intermolecular recombination is emphasized. PMID:25844275

New L-Serine Derivative Ligands as Cocatalysts for Diels-Alder Reaction

Science.gov (United States)

Sousa, Carlos A. D.; Rodríguez-Borges, José E.; Freire, Cristina

2013-01-01

New L-serine derivative ligands were prepared and tested as cocatalyst in the Diels-Alder reactions between cyclopentadiene (CPD) and methyl acrylate, in the presence of several Lewis acids. The catalytic potential of the in situ formed complexes was evaluated based on the reaction yield. Bidentate serine ligands showed good ability to coordinate medium strength Lewis acids, thus boosting their catalytic activity. The synthesis of the L-serine ligands proved to be highly efficient and straightforward. PMID:24383009

Serine integrase chimeras with activity in E. coli and HeLa cells

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Alfonso P. Farruggio

2014-09-01

Full Text Available In recent years, application of serine integrases for genomic engineering has increased in popularity. The factor-independence and unidirectionality of these large serine recombinases makes them well suited for reactions such as site-directed vector integration and cassette exchange in a wide variety of organisms. In order to generate information that might be useful for altering the specificity of serine integrases and to improve their efficiency, we tested a hybridization strategy that has been successful with several small serine recombinases. We created chimeras derived from three characterized members of the serine integrase family, phiC31, phiBT1, and TG1 integrases, by joining their amino- and carboxy-terminal portions. We found that several phiBT1-phiC31 (BC and phiC31-TG1 (CT hybrid integrases are active in E. coli. BC chimeras function on native att-sites and on att-sites that are hybrids between those of the two donor enzymes, while CT chimeras only act on the latter att-sites. A BC hybrid, BC{−1}, was also active in human HeLa cells. Our work is the first to demonstrate chimeric serine integrase activity. This analysis sheds light on integrase structure and function, and establishes a potentially tractable means to probe the specificity of the thousands of putative large serine recombinases that have been revealed by bioinformatics studies.

d(− Lactic Acid-Induced Adhesion of Bovine Neutrophils onto Endothelial Cells Is Dependent on Neutrophils Extracellular Traps Formation and CD11b Expression

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Pablo Alarcón

2017-08-01

Full Text Available Bovine ruminal acidosis is of economic importance as it contributes to reduced milk and meat production. This phenomenon is mainly attributed to an overload of highly fermentable carbohydrate, resulting in increased d(− lactic acid levels in serum and plasma. Ruminal acidosis correlates with elevated acute phase proteins in blood, along with neutrophil activation and infiltration into various tissues leading to laminitis and aseptic polysynovitis. Previous studies in bovine neutrophils indicated that d(− lactic acid decreased expression of L-selectin and increased expression of CD11b to concentrations higher than 6 mM, suggesting a potential role in neutrophil adhesion onto endothelia. The two aims of this study were to evaluate whether d(− lactic acid influenced neutrophil and endothelial adhesion and to trigger neutrophil extracellular trap (NET production (NETosis in exposed neutrophils. Exposure of bovine neutrophils to 5 mM d(− lactic acid elevated NET release compared to unstimulated neutrophil negative controls. Moreover, this NET contains CD11b and histone H4 citrullinated, the latter was dependent on PAD4 activation, a critical enzyme in DNA decondensation and NETosis. Furthermore, NET formation was dependent on d(− lactic acid plasma membrane transport through monocarboxylate transporter 1 (MCT1. d(− lactic acid enhanced neutrophil adhesion onto endothelial sheets as demonstrated by in vitro neutrophil adhesion assays under continuous physiological flow conditions, indicating that cell adhesion was a NET- and a CD11b/ICAM-1-dependent process. Finally, d(− lactic acid was demonstrated for the first time to trigger NETosis in a PAD4- and MCT1-dependent manner. Thus, d(− lactic acid-mediated neutrophil activation may contribute to neutrophil-derived pro-inflammatory processes, such as aseptic laminitis and/or polysynovitis in animals suffering acute ruminal acidosis.

The cystic fibrosis neutrophil: a specialized yet potentially defective cell.

LENUS (Irish Health Repository)

Hayes, Elaine

2012-02-01

Cystic fibrosis (CF) is one of the commonest genetically inherited diseases in the world. It is characterized by recurrent respiratory tract infections eventually leading to respiratory failure. One of the hallmarks of this disease is a persistent and predominantly neutrophil driven inflammation. Neutrophils provide the first line of defence by killing and digesting phagocytosed bacteria and fungi, yet despite advances in our understanding of the molecular and cellular basis of CF, there remains a paradox of why recruited CF neutrophils fail to eradicate bacterial infections in the lung. This review describes mechanisms involved in neutrophil migration, microbial killing and apoptosis leading to inflammatory resolution. We discuss dysregulated neutrophil activity and consider genetic versus inflammatory neutrophil reprogramming in CF and ultimately pharmacological modulation of the CF neutrophil for therapeutic intervention.

The cystic fibrosis neutrophil: a specialized yet potentially defective cell.

LENUS (Irish Health Repository)

Hayes, Elaine

2011-04-01

Cystic fibrosis (CF) is one of the commonest genetically inherited diseases in the world. It is characterized by recurrent respiratory tract infections eventually leading to respiratory failure. One of the hallmarks of this disease is a persistent and predominantly neutrophil driven inflammation. Neutrophils provide the first line of defence by killing and digesting phagocytosed bacteria and fungi, yet despite advances in our understanding of the molecular and cellular basis of CF, there remains a paradox of why recruited CF neutrophils fail to eradicate bacterial infections in the lung. This review describes mechanisms involved in neutrophil migration, microbial killing and apoptosis leading to inflammatory resolution. We discuss dysregulated neutrophil activity and consider genetic versus inflammatory neutrophil reprogramming in CF and ultimately pharmacological modulation of the CF neutrophil for therapeutic intervention.

A group-specific inhibitor of lysosomal cysteine proteinases selectively inhibits both proteolytic degradation and presentation of the antigen dinitrophenyl-poly-L-lysine by guinea pig accessory cells to T cells

DEFF Research Database (Denmark)

Buus, S; Werdelin, O

1986-01-01

of antigens by guinea pig accessory cells. The proteinase inhibitor benzyloxycarbonyl-phenylalanylalanine-diazomethyl-ketone, which selectively inhibits cysteine proteinases, was used to block this set of enzymes in cultured cells. We demonstrate that the selective inhibition of the cysteine proteinases...

Different innate neutrophil responses in controlled and uncontrolled asthma

NARCIS (Netherlands)

Tang, Francesca; Foxley, Gloria; Gibson, Peter; Burgess, Janette; Baines, Katherine; Oliver, Brian

2015-01-01

Introduction: Respiratory viruses are a major cause of asthma exacerbations. Neutrophilic inflammation occurs during infections and is associated with difficult to treat asthma. The role of neutrophils in viral infections and whether neutrophil dysfunction contributes to exacerbation pathogenesis

Molecular cloning of Kazal-type proteinase inhibitor of the shrimp Fenneropenaeus chinensis.

Science.gov (United States)

Kong, Hee Jeong; Cho, Hyun Kook; Park, Eun-Mi; Hong, Gyeong-Eun; Kim, Young-Ok; Nam, Bo-Hye; Kim, Woo-Jin; Lee, Sang-Jun; Han, Hyon Sob; Jang, In-Kwon; Lee, Chang Hoon; Cheong, Jaehun; Choi, Tae-Jin

2009-01-01

Proteinase inhibitors play important roles in host defence systems involving blood coagulation and pathogen digestion. We isolated and characterized a cDNA clone for a Kazal-type proteinase inhibitor (KPI) from a hemocyte cDNA library of the oriental white shrimp Fenneropenaeus chinensis. The KPI gene consists of three exons and two introns. KPI cDNA contains an open reading frame of 396 bp, a polyadenylation signal sequence AATAAA, and a poly (A) tail. KPI cDNA encodes a polypeptide of 131 amino acids with a putative signal peptide of 21 amino acids. The deduced amino acid sequence of KPI contains two homologous Kazal domains, each with six conserved cysteine residues. The mRNA of KPI is expressed in the hemocytes of healthy shrimp, and the higher expression of KPI transcript is observed in shrimp infected with the white spot syndrome virus (WSSV), suggesting a potential role for KPI in host defence mechanisms.

Characterization of the mature cell surface proteinase of Lactobacillus delbrueckii subsp. lactis CRL 581.

Science.gov (United States)

Villegas, Josefina M; Brown, Lucía; Savoy de Giori, Graciela; Hebert, Elvira M

2015-05-01

The cell envelope-associated proteinase (CEP) of Lactobacillus delbrueckii subsp. lactis CRL 581 (PrtL) has an essential role in bacterial growth, contributes to the flavor and texture development of fermented products, and can release bioactive health-beneficial peptides during milk fermentation. The genome of L. delbrueckii subsp. lactis CRL 581 possesses only one gene that encodes PrtL, which consists of 1924 amino acids and is a multidomain protein anchored to the cell via its W domain. PrtL was extracted from the cell under high ionic strength conditions using NaCl, suggesting an electrostatic interaction between the proteinase and the cell envelope. The released PrtL was purified and biochemically characterized; its activity was maximal at temperatures between 37 and 40 °C and at pH between 7 and 8. Under optimal conditions, PrtL exhibited higher affinity for succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide than for succinyl-alanyl-glutamyl-prolyl-phenylalanine-p-nitroanilide, while methoxy-succinyl-arginyl-prolyl-tyrosyl-p-nitroanilide was not degraded. A similar α- and β-casein degradation pattern was observed with the purified and the cell envelope-bound proteinase. Finally, on the basis of its specificity towards caseins and the unique combination of amino acids at residues thought to be involved in substrate specificity, PrtL can be classified as a representative of a new group of CEP.

Neutrophils, a candidate biomarker and target for radiation therapy?

Science.gov (United States)

Schernberg, Antoine; Blanchard, Pierre; Chargari, Cyrus; Deutsch, Eric

2017-11-01

Neutrophils are the most abundant blood-circulating white blood cells, continuously generated in the bone marrow. Growing evidence suggests they regulate the innate and adaptive immune system during tumor evolution. This review will first summarize the recent findings on neutrophils as a key player in cancer evolution, then as a potential biomarker, and finally as therapeutic targets, with respective focuses on the interplay with radiation therapy. A complex interplay: Neutrophils have been associated with tumor progression through multiple pathways. Ionizing radiation has cytotoxic effects on cancer cells, but the sensitivity to radiation therapy in vivo differ from isolated cancer cells in vitro, partially due to the tumor microenvironment. Different microenvironmental states, whether baseline or induced, can modulate or even attenuate the effects of radiation, with consequences for therapeutic efficacy. Inflammatory biomarkers: Inflammation-based scores have been widely studied as prognostic biomarkers in cancer patients. We have performed a large retrospective cohort of patients undergoing radiation therapy (1233 patients), with robust relationship between baseline blood neutrophil count and 3-year's patient's overall survival in patients with different cancer histologies. (Pearson's correlation test: p = .001, r = -.93). Therapeutic approaches: Neutrophil-targeting agents are being developed for the treatment of inflammatory and autoimmune diseases. Neutrophils either can exert antitumoral (N1 phenotype) or protumoral (N2 phenotype) activity, depending on the Tumor Micro Environment. Tumor associated N2 neutrophils are characterized by high expression of CXCR4, VEGF, and gelatinase B/MMP9. TGF-β within the tumor microenvironment induces a population of TAN with a protumor N2 phenotype. TGF-β blockade slows tumor growth through activation of CD8 + T cells, macrophages, and tumor associated neutrophils with an antitumor N1 phenotype. This supports

Tumor-Associated Macrophages and Neutrophils in Tumor Microenvironment

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Jaehong Kim

2016-01-01

Full Text Available Distinct tumor microenvironment forms in each progression step of cancer and has diverse capacities to induce both adverse and beneficial consequences for tumorigenesis. It is now known that immune cells can be activated to favor tumor growth and progression, most probably influenced by the tumor microenvironment. Tumor-associated macrophages and tumor-associated neutrophils can exert protumoral functions, enhancing tumor cell invasion and metastasis, angiogenesis, and extracellular matrix remodeling, while inhibiting the antitumoral immune surveillance. Considering that neutrophils in inflammatory environments recruit macrophages and that recruited macrophages affect neutrophil functions, there may be various degrees of interaction between tumor-associated macrophages and tumor-associated neutrophils. Platelets also play an important role in the recruitment and regulation of monocytic and granulocytic cells in the tumor tissues, suggesting that platelet function may be essential for generation of tumor-associated macrophages and tumor-associated neutrophils. In this review, we will explore the biology of tumor-associated macrophages and tumor-associated neutrophils and their possible interactions in the tumor microenvironment. Special attention will be given to the recruitment and activation of these tumor-associated cells and to the roles they play in maintenance of the tumor microenvironment and progression of tumors.

Granule protein processing and regulated secretion in neutrophils

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Avinash eSheshechalam

2014-09-01

Full Text Available Neutrophils are part of a family of granulocytes that, together with eosinophils and basophils, play an essential role in innate immunity. Neutrophils are the most abundant circulating leukocytes and are vital for rapid immune responses, being recruited to sites of injury or infection within minutes, where they can act as specialized phagocytic cells. However, another prominent function of neutrophils is the release of pro-inflammatory compounds, including cytokines, chemokines and digestive enzymes, which are stored in intracellular compartments and released through regulated exocytosis. Hence, an important feature that contributes to rapid immune responses is capacity of neutrophils to synthesize and store pre-formed pro-inflammatory mediators in specialized intracellular vesicles and thus no new synthesis is required. This review will focus on advancement in three topics relevant to neutrophil secretion. First we will examine what is known about basal level pro-inflammatory mediator synthesis, trafficking and storage in secretory compartments. Second, we will review recent advancements in the mechanisms that control vesicle mobilization and the release of pre-formed mediators. Third, we will examine the upregulation and de novo synthesis of pro-inflammatory mediators by neutrophils engaged at sites of infection.

Swell activated chloride channel function in human neutrophils

Energy Technology Data Exchange (ETDEWEB)

Salmon, Michael D. [Leukocyte and Ion Channel Research Laboratory, School of Health and Biosciences, University of East London, Stratford Campus, London E15 4LZ (United Kingdom); Ahluwalia, Jatinder, E-mail: j.ahluwalia@uel.ac.uk [Leukocyte and Ion Channel Research Laboratory, School of Health and Biosciences, University of East London, Stratford Campus, London E15 4LZ (United Kingdom)

2009-04-17

Non-excitable cells such as neutrophil granulocytes are the archetypal inflammatory immune cell involved in critical functions of the innate immune system. The electron current generated (I{sub e}) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential. For continuous function of the NADPH oxidase, I{sub e} has to be balanced to preserve electroneutrality, if not; sufficient depolarisation would prevent electrons from leaving the cell and neutrophil function would be abrogated. Subsequently, the depolarisation generated by the neutrophil NADPH oxidase I{sub e} must be counteracted by ion transport. The finding that depolarisation required counter-ions to compensate electron transport was followed by the observation that chloride channels activated by swell can counteract the NADPH oxidase membrane depolarisation. In this mini review, we discuss the research findings that revealed the essential role of swell activated chloride channels in human neutrophil function.

Effect of moderate exercise on peritoneal neutrophils from juvenile rats.

Science.gov (United States)

Braz, Glauber Ruda; Ferreira, Diorginis Soares; Pedroza, Anderson Apolonio; da Silva, Aline Isabel; Sousa, Shirley Maria; Pithon-Curi, Tania Cristina; Lagranha, Claudia

2015-09-01

Previous studies showed that moderate exercise in adult rats enhances neutrophil function, although no studies were performed in juvenile rats. We evaluated the effects of moderate exercise on the neutrophil function in juvenile rats. Viability and neutrophils function were evaluated. Moderate exercise did not impair the viability and mitochondrial transmembrane potential of neutrophils, whereas there was greater reactive oxygen species production (164%; p < 0.001) and phagocytic capacity (29%; p < 0.05). Our results suggest that moderate exercise in juvenile rats improves neutrophil function, similar to adults.

Mosaic serine proteases in the mammalian central nervous system.

Science.gov (United States)

Mitsui, Shinichi; Watanabe, Yoshihisa; Yamaguchi, Tatsuyuki; Yamaguchi, Nozomi

2008-01-01

We review the structure and function of three kinds of mosaic serine proteases expressed in the mammalian central nervous system (CNS). Mosaic serine proteases have several domains in the proenzyme fragment, which modulate proteolytic function, and a protease domain at the C-terminus. Spinesin/TMPRSS5 is a transmembrane serine protease whose presynaptic distribution on motor neurons in the spinal cord suggests that it is significant for neuronal plasticity. Cell type-specific alternative splicing gives this protease diverse functions by modulating its intracellular localization. Motopsin/PRSS12 is a mosaic protease, and loss of its function causes mental retardation. Recent reports indicate the significance of this protease for cognitive function. We mention the fibrinolytic protease, tissue plasminogen activator (tPA), which has physiological and pathological functions in the CNS.

Demodex-associated bacterial proteins induce neutrophil activation.

LENUS (Irish Health Repository)

2012-02-01

Background: Patients with rosacea demonstrate a higher density of Demodex mites in their skin than controls. A bacterium isolated from a Demodex mite from a patient with papulopustular rosacea (PPR) was previously shown to provoke an immune response in patients with PPR or ocular rosacea thus suggesting a possible role for bacterial proteins in the etiology of this condition. Objectives: To examine the response of neutrophils to proteins derived from a bacterium isolated from a Demodex mite. Methods: Bacterial cells were lysed and proteins were partially purified by AKTA-FPLC. Isolated neutrophils were exposed to bacterial proteins and monitored for alterations in migration, degranulation and cytokine production. Results: Neutrophils exposed to proteins from Bacillus cells demonstrated increased levels of migration and elevated release of MMP-9, an enzyme known to degrade collagen and cathelicidin, an antimicrobial peptide. In addition neutrophils exposed to the bacterial proteins demonstrated elevated rates of Il-8 and TNF-alpha production. Conclusions: Proteins produced by a bacterium isolated from a Demodex mite have the ability to increase the migration, degranulation and cytokine production abilities of neutrophils. These results suggest that bacteria may play a role in the inflammatory erythema associated with rosacea.

Identification and characterization of fusolisin, the Fusobacterium nucleatum autotransporter serine protease.

Directory of Open Access Journals (Sweden)

Lior Doron

Full Text Available Fusobacterium nucleatum is an oral anaerobe associated with periodontal disease, adverse pregnancy outcomes and colorectal carcinoma. A serine endopeptidase of 61-65 kDa capable of damaging host tissue and of inactivating immune effectors was detected previously in F. nucleatum. Here we describe the identification of this serine protease, named fusolisin, in three oral F. nucleatum sub-species. Gel zymogram revealed fusobacterial proteolytic activity with molecular masses ranging from 55-101 kDa. All of the detected proteases were inhibited by the serine protease inhibitor PMSF. analysis revealed that all of the detected proteases are encoded by genes encoding an open reading frame (ORF with a calculated mass of approximately 115 kDa. Bioinformatics analysis of the identified ORFs demonstrated that they consist of three domains characteristic of autotransporters of the type Va secretion system. Our results suggest that the F. nucleatum fusolisins are derived from a precursor of approximately 115 kDa. After crossing the cytoplasmic membrane and cleavage of the leader sequence, the C-terminal autotransporter domain of the remaining 96-113 kDa protein is embedded in the outer membrane and delivers the N-terminal S8 serine protease passenger domain to the outer cell surface. In most strains the N-terminal catalytic 55-65 kDa domain self cleaves and liberates itself from the autotransporter domain after its transfer across the outer cell membrane. In F. nucleatum ATCC 25586 this autocatalytic activity is less efficient resulting in a full length membrane-anchored serine protease. The mature serine protease was found to cleave after Thr, Gly, Ala and Leu residues at the P1 position. Growth of F. nucleatum in complex medium was inhibited when serine protease inhibitors were used. Additional experiments are needed to determine whether fusolisin might be used as a target for controlling fusobacterial infections.

Ketamine Metabolites Enantioselectively Decrease Intracellular D-Serine Concentrations in PC-12 Cells.

Directory of Open Access Journals (Sweden)

Nagendra S Singh

Full Text Available D-Serine is an endogenous NMDA receptor co-agonist that activates synaptic NMDA receptors modulating neuronal networks in the cerebral cortex and plays a key role in long-term potentiation of synaptic transmission. D-serine is associated with NMDA receptor neurotoxicity and neurodegeneration and elevated D-serine concentrations have been associated with Alzheimer's and Parkinsons' diseases and amyotrophic lateral sclerosis. Previous studies have demonstrated that the ketamine metabolites (rac-dehydronorketamine and (2S,6S-hydroxynorketamine decrease intracellular D-serine concentrations in a concentration dependent manner in PC-12 cells. In the current study, PC-12 cells were incubated with a series of ketamine metabolites and the IC50 values associated with attenuated intracellular D-serine concentrations were determined. The results demonstrate that structural and stereochemical features of the studied compounds contribute to the magnitude of the inhibitory effect with (2S,6S-hydroxynorketamine and (2R,6R-hydroxynorketamine displaying the most potent inhibition with IC50 values of 0.18 ± 0.04 nM and 0.68 ± 0.09 nM. The data was utilized to construct a preliminary 3D-QSAR/pharmacophore model for use in the design of new and more efficient modulators of D-serine.

Protective role of purified cysteine proteinases against Fasciola gigantica infection in experimental animals.

Science.gov (United States)

El-Ahwany, Eman; Rabia, Ibrahim; Nagy, Faten; Zoheiry, Mona; Diab, Tarek; Zada, Suher

2012-03-01

Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freund's adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 F. gigantica metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, IgG(1), and IgG(2) (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-γ, and TNF-α, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-β, and IL-6, showed significant increases (P<0.05). In conclusion, it has been found that CP released by F. gigantica are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships.

A secreted aspartic proteinase from Glomerella cingulata: purification of the enzyme and molecular cloning of the cDNA.

Science.gov (United States)

Clark, S J; Templeton, M D; Sullivan, P A

1997-04-01

A secreted aspartic proteinase from Glomerella cingulata (GcSAP) was purified to homogeneity by ion exchange chromatography. The enzyme has an M, of 36000 as estimated by SDS-PAGE, optimal activity from pH 3.5 to pH 4.0 and is inhibited by pepstatin. The N-terminal sequence, 23 residues long, was used to design a gene-specific primer. This was used in 3' RACE (rapid amplification of cDNA ends) PCR to amplify a 1.2 kb fragment of the gcsap cDNA. A second gene-specific primer was designed and used in 5' RACE PCR to clone the 5' region. This yielded a 600 bp DNA fragment and completed the open reading frame. The gcsap open reading frame encodes a protein with a 78 residue prepro-sequence typical of other fungal secreted aspartic proteinases. Based on the deduced sequence, the mature enzyme contains 329 amino acids and shows approximately 40% identity to other fungal aspartic proteinases. Subsequent cloning and sequencing of gcsap fragments obtained from PCR with genomic DNA revealed a 73 bp intron beginning at nt 728. Southern analyses at medium and high stringency indicated that G. cingulata possesses one gene for the secreted aspartic proteinase, and Northern blots indicated that gene expression was induced by exogenous protein and repressed by ammonium salts. GcSAP is a putative pathogenicity factor of G. cingulata, and it will now be possible to create SAP-mutants and assess the role GcSAP plays in pathogenicity.

Protein C Inhibitor-A Novel Antimicrobial Agent

NARCIS (Netherlands)

Malmström, E.; Mörgelin, M.; Malmsten, M.; Johansson, L.; Norrby-Teglund, A.; Shannon, O.; Schmidtchen, A.; Meijers, J.C.M.; Herwald, H.

2009-01-01

Protein C inhibitor (PCI) is a heparin-binding serine proteinase inhibitor belonging to the family of serpin proteins. Here we describe that PCI exerts broad antimicrobial activity against bacterial pathogens. This ability is mediated by the interaction of PCI with lipid membranes, which

Understanding binding affinity : A combined isothermal titration calorimetry/molecular dynamics study of the binding of a series of hydrophobically modified benzamidinium chloride inhibitors to trypsin

NARCIS (Netherlands)

Talhout, Reinskje; Villa, Alessandra; Mark, AE; Engberts, JBFN

2003-01-01

The binding of a series of p-alkylbenzamidinium chloride inhibitors to the serine proteinase trypsin over a range of temperatures has been studied using isothermal titration (micro)calorimetry and molecular dynamics simulation techniques. The inhibitors have small structural variations at the para

Organization of the gene coding for human protein C inhibitor (plasminogen activator inhibitor-3). Assignment of the gene to chromosome 14

NARCIS (Netherlands)

Meijers, J. C.; Chung, D. W.

1991-01-01

Protein C inhibitor (plasminogen activator inhibitor-3) is a plasma glycoprotein and a member of the serine proteinase inhibitor superfamily. In the present study, the human gene for protein C inhibitor was isolated and characterized from three independent phage that contained overlapping inserts

Interval and continuous exercise regimens suppress neutrophil-derived microparticle formation and neutrophil-promoted thrombin generation under hypoxic stress.

Science.gov (United States)

Chen, Yi-Ching; Ho, Ching-Wen; Tsai, Hsing-Hua; Wang, Jong-Shyan

2015-04-01

Acute hypoxic exposure increases vascular thrombotic risk. The release of procoagulant-rich microparticles from neutrophils accelerates the pathogenesis of inflammatory thrombosis. The present study explicates the manner in which interval and continuous exercise regimens affect neutrophil-derived microparticle (NDMP) formation and neutrophil/NDMP-mediated thrombin generation (TG) under hypoxic condition. A total of 60 sedentary males were randomized to perform either aerobic interval training [AIT; 3-min intervals at 40% and 80% V̇O2max (maximal O2 consumption)] or moderate continuous training (MCT; sustained 60% V̇O2max) for 30 min/day, 5 days/week for 5 weeks, or to a control (CTL) group who did not receive any form of training. At rest and immediately after hypoxic exercise test (HE, 100 W under 12% O2 for 30 min), the NDMP characteristics and dynamic TG were measured by flow cytometry and thrombinography respectively. Before the intervention, HE (i) elevated coagulant factor VIII/fibrinogen concentrations and shortened activated partial thromboplastin time (aPTT), (ii) increased total and tissue factor (TF)-rich/phosphatidylserine (PS)-exposed NDMP counts and (iii) enhanced the peak height and rate of TG promoted by neutrophils/NDMPs. Following the 5-week intervention, AIT exhibited higher enhancement of V̇O2max than did MCT. Notably, both MCT and AIT attenuated the extents of HE-induced coagulant factor VIII/fibrinogen elevations and aPTT shortening. Furthermore, the two exercise regimens significantly decreased TF-rich/PS-exposed NDMP formation and depressed neutrophil/NDMP-mediated dynamic TG at rest and following HE. Hence, we conclude that AIT is superior to MCT for enhancing aerobic capacity. Moreover, either AIT or MCT effectively ameliorates neutrophil/NDMP-promoted TG by down-regulating expression of procoagulant factors during HE, which may reduce thrombotic risk evoked by hypoxia. Moreover, either AIT or MCT effectively ameliorates neutrophil

Engineering of High Yield Production of L-serine in Escherichia coli

DEFF Research Database (Denmark)

Mundhada, Hemanshu; Schneider, Konstantin; Christensen, Hanne Bjerre

2016-01-01

by deletion of three L-serine deaminases sdaA, sdaB, and tdcG, as well as serine hydroxyl methyl transferase (SHMT) encoded by glyA. Upon overexpression of the serine production pathway, consisting of a feedback resistant version of serA along with serB and serC, this quadruple deletion strain showed a very...

Proteinase K processing of rabbit muscle creatine kinase

DEFF Research Database (Denmark)

Leydier, C; Andersen, Jens S.; Couthon, F

1997-01-01

Proteinase K cleaves selectively both cytosolic and mitochondrial isoforms of creatine kinase leading to the appearance of two fragments, a large N-terminal one (K1) and a small C-terminal peptide (K2) which remain associated together. The loss of enzymatic activity correlates with the extent...... of monomer cleavage. N-terminal sequencing of the K2 fragments from rabbit cytosolic and pig mitochondrial creatine kinase shows that these peptides begin with A328 and A324, respectively. Electrospray ionization mass spectrometry demonstrates that K2 peptide is composed of 53 residues (A328-K380). However...

Neutrophils, dendritic cells and Toxoplasma.

Science.gov (United States)

Denkers, Eric Y; Butcher, Barbara A; Del Rio, Laura; Bennouna, Soumaya

2004-03-09

Toxoplasma gondii rapidly elicits strong Type 1 cytokine-based immunity. The necessity for this response is well illustrated by the example of IFN-gamma and IL-12 gene knockout mice that rapidly succumb to the effects of acute infection. The parasite itself is skilled at sparking complex interactions in the innate immune system that lead to protective immunity. Neutrophils are one of the first cell types to arrive at the site of infection, and the cells release several proinflammatory cytokines and chemokines in response to Toxoplasma. Dendritic cells are an important source of IL-12 during infection with T. gondii and other microbial pathogens, and they are also specialized for high-level antigen presentation to T lymphocytes. Tachyzoites express at least two types of molecules that trigger innate immune cell cytokine production. One of these involves Toll-like receptor/MyD88 pathways common to many microbial pathogens. The second pathway is less conventional and involves molecular mimicry between a parasite cyclophilin and host CC chemokine receptor 5-binding ligands. Neutrophils, dendritic cells and Toxoplasma work together to elicit the immune response required for host survival. Cytokine and chemokine cross-talk between parasite-triggered neutrophils and dendritic cells results in recruitment, maturation and activation of the latter. Neutrophil-empowered dendritic cells possess properties expected of highly potent antigen presenting cells that drive T helper 1 generation.

Secreted aspartate proteinases, a virulence factor of Candida spp.: Occurrence among clinical isolates

Czech Academy of Sciences Publication Activity Database

Hamal, P.; Dostál, Jiří; Raclavský, V.; Krylová, M.; Pichová, Iva; Hrušková-Heidingsfeldová, Olga

2004-01-01

Roč. 49, č. 4 (2004), s. 491-496 ISSN 0015-5632 R&D Projects: GA MZd NI6485 Institutional research plan: CEZ:AV0Z4055905 Keywords : Candida spp. * aspartate proteinases * RAPD typing Subject RIV: CE - Biochemistry Impact factor: 1.034, year: 2004

Neutrophil heterogeneity: implications for homeostasis and pathogenesis

NARCIS (Netherlands)

Silvestre-Roig, Carlos; Hidalgo, Andres; Soehnlein, Oliver

2016-01-01

Neutrophils are polymorphonuclear leukocytes of the phagocytic system that act as first line of host defense against invading pathogens but are also important mediators of inflammation-induced injury. In contrast to other members of the innate immune system, neutrophils are classically considered a

Tissue-transglutaminase contributes to neutrophil granulocyte differentiation and functions.

Science.gov (United States)

Balajthy, Zoltán; Csomós, Krisztián; Vámosi, György; Szántó, Attila; Lanotte, Michel; Fésüs, László

2006-09-15

Promyelocytic NB4 leukemia cells undergo differentiation to granulocytes following retinoic acid treatment. Here we report that tissue transglutaminase (TG2), a protein cross-linking enzyme, was induced, then partially translocated into the nucleus, and became strongly associated with the chromatin during the differentiation process. The transglutaminase-catalyzed cross-link content of both the cytosolic and the nuclear protein fractions increased while NB4 cells underwent cellular maturation. Inhibition of cross-linking activity of TG2 by monodansylcadaverin in these cells led to diminished nitroblue tetrazolium (NBT) positivity, production of less superoxide anion, and decreased expression of GP91PHOX, the membrane-associated subunit of NADPH oxidase. Neutrophils isolated from TG2(-/-) mice showed diminished NBT reduction capacity, reduced superoxide anion formation, and down-regulation of the gp91phox subunit of NADPH oxidase, compared with wild-type cells. It was also observed that TG2(-/-) mice exhibited increased neutrophil phagocytic activity, but had attenuated neutrophil chemotaxis and impaired neutrophil extravasation with higher neutrophil counts in their circulation during yeast extract-induced peritonitis. These results clearly suggest that TG2 may modulate the expression of genes related to neutrophil functions and is involved in several intracellular and extracellular functions of extravasating neutrophil.

Phenotypic changes in neutrophils related to anti-inflammatory therapy.

Science.gov (United States)

Barton, A E; Bayley, D L; Mikami, M; Llewellyn-Jones, C G; Stockley, R A

2000-01-03

Previous work from the group has shown that non-steroidal anti-inflammatory agents given to volunteers and patients inhibit PMN function possibly by affecting the developing neutrophil during the differentiation process. In this study indomethacin treatment in vivo reduced neutrophil chemotaxis and proteolytic degradation of fibronectin, with a maximal effect after 14 days. Stimulated neutrophil adherence to fibronectin was also reduced but this was not due to quantitative changes in beta(2) integrin expression or function. L-Selectin expression on resting and stimulated neutrophils was increased after 14 days and there was a small decrease in plasma levels of soluble L-selectin. These effects, however, could not be reproduced by treatment of neutrophils with indomethacin in vitro, suggesting they are due to effects on differentiating/maturing PMNs. In an attempt to interpret these changes, studies were performed with dexamethasone, which is known to alter neutrophil function and kinetics. Dexamethasone treatment reduced chemotaxis and increased superoxide generation after 1 day and was associated with increased expression of activated beta(2) integrins and reduced L-selectin expression on resting neutrophils. This suggests the appearance of mainly 'activated' cells as a result of demargination and indicates that the effects of indomethacin are distinctive and not related to changes in compartmentalisation.

Neutrophils in Homeostasis, Immunity, and Cancer.

Science.gov (United States)

Nicolás-Ávila, José Ángel; Adrover, José M; Hidalgo, Andrés

2017-01-17

Neutrophils were among the first leukocytes described and visualized by early immunologists. Prominent effector functions during infection and sterile inflammation classically placed them low in the immune tree as rapid, mindless aggressors with poor regulatory functions. This view is currently under reassessment as we uncover new aspects of their life cycle and identify transcriptional and phenotypic diversity that endows them with regulatory properties that extend beyond their lifetime in the circulation. These properties are revealing unanticipated roles for neutrophils in supporting homeostasis, as well as complex disease states such as cancer. We focus this review on these emerging functions in order to define the true roles of neutrophils in homeostasis, immunity, and disease. Copyright © 2017 Elsevier Inc. All rights reserved.

Neutrophilic dermatoses in a patient with collagenous colitis

OpenAIRE

Didac Barco; Maria A. Barnadas; Esther Roé; Francisco J. Sancho; Elena Ricart; Agustín Alomar

2010-01-01

We report the case of a 75-year old woman with collagenous colitis who presented with erythematous and edematous plaques on the periorbital and eyelid regions, accompanied by oral ulcers. Histopathology showed a dermal neutrophilic infiltrate plus mild septal and lobular panniculitis with lymphocytes, neutrophils and eosinophils. Five years earlier she had presented a flare of papules and vesicles on the trunk, together with oral ulcers; a skin biopsy revealed a neutrophilic dermal infiltrate...

Vasculitis and antineutrophil cytoplasmic autoantibodies associated with propylthiouracil therapy

NARCIS (Netherlands)

Dolman, K. M.; Gans, R. O.; Vervaat, T. J.; Zevenbergen, G.; Maingay, D.; Nikkels, R. E.; Donker, A. J.; von dem Borne, A. E.; Goldschmeding, R.

1993-01-01

Vasculitis is a rare complication of propylthiouracil therapy. Antineutrophil cytoplasmic antibodies (ANCA) have been described in association with several vasculitic disorders. We report detection of ANCA against human neutrophil elastase, proteinase 3, and myeloperoxidase in serum from six

Neutrophil Leukocyte: Combustive Microbicidal Action and Chemiluminescence

Directory of Open Access Journals (Sweden)

Robert C. Allen

2015-01-01

Full Text Available Neutrophil leukocytes protect against a varied and complex array of microbes by providing microbicidal action that is simple, potent, and focused. Neutrophils provide such action via redox reactions that change the frontier orbitals of oxygen (O2 facilitating combustion. The spin conservation rules define the symmetry barrier that prevents direct reaction of diradical O2 with nonradical molecules, explaining why combustion is not spontaneous. In burning, the spin barrier is overcome when energy causes homolytic bond cleavage producing radicals capable of reacting with diradical O2 to yield oxygenated radical products that further participate in reactive propagation. Neutrophil mediated combustion is by a different pathway. Changing the spin quantum state of O2 removes the symmetry restriction to reaction. Electronically excited singlet molecular oxygen (O2*1 is a potent electrophilic reactant with a finite lifetime that restricts its radius of reactivity and focuses combustive action on the target microbe. The resulting exergonic dioxygenation reactions produce electronically excited carbonyls that relax by light emission, that is, chemiluminescence. This overview of neutrophil combustive microbicidal action takes the perspectives of spin conservation and bosonic-fermionic frontier orbital considerations. The necessary principles of particle physics and quantum mechanics are developed and integrated into a fundamental explanation of neutrophil microbicidal metabolism.

The aspartic proteinase from Saccharomyces cerevisiae folds its own inhibitor into a helix

DEFF Research Database (Denmark)

Li, M; Phylip, L H; Lees, W E

2000-01-01

Aspartic proteinase A from yeast is specifically and potently inhibited by a small protein called IA3 from Saccharomyces cerevisiae. Although this inhibitor consists of 68 residues, we show that the inhibitory activity resides within the N-terminal half of the molecule. Structures solved at 2...

Systems biology of neutrophil differentiation and immune response

DEFF Research Database (Denmark)

Theilgaard-Mönch, Kim; Porse, Bo T; Borregaard, Niels

2005-01-01

Systems biology has emerged as a new scientific field, which aims at investigating biological processes at the genomic and proteomic levels. Recent studies have unravelled aspects of neutrophil differentiation and immune responses at the systems level using high-throughput technologies. These stu......Systems biology has emerged as a new scientific field, which aims at investigating biological processes at the genomic and proteomic levels. Recent studies have unravelled aspects of neutrophil differentiation and immune responses at the systems level using high-throughput technologies....... These studies have identified a plethora of novel effector proteins stored in the granules of neutrophils. In addition, these studies provide evidence that neutrophil differentiation and immune response are governed by a highly coordinated transcriptional programme that regulates cellular fate and function...

Diverse novel functions of neutrophils in immunity, inflammation, and beyond.

Science.gov (United States)

Mócsai, Attila

2013-07-01

Neutrophils have long been considered simple suicide killers at the bottom of the hierarchy of the immune response. That view began to change 10-20 yr ago, when the sophisticated mechanisms behind how neutrophils locate and eliminate pathogens and regulate immunity and inflammation were discovered. The last few years witnessed a new wave of discoveries about additional novel and unexpected functions of these cells. Neutrophils have been proposed to participate in protection against intracellular pathogens such as viruses and mycobacteria. They have been shown to intimately shape the adaptive immune response at various levels, including marginal zone B cells, plasmacytoid dendritic cells and T cell populations, and even to control NK cell homeostasis. Neutrophils have been shown to mediate an alternative pathway of systemic anaphylaxis and to participate in allergic skin reactions. Finally, neutrophils were found to be involved in physiological and pathological processes beyond the immune system, such as diabetes, atherosclerosis, and thrombus formation. Many of those functions appear to be related to their unique ability to release neutrophil extracellular traps even in the absence of pathogens. This review summarizes those novel findings on versatile functions of neutrophils and how they change our view of neutrophil biology in health and disease.

Serpins in plants and green algae

DEFF Research Database (Denmark)

Roberts, Thomas Hugh; Hejgaard, Jørn

2008-01-01

. Serpins have been found in diverse species of the plant kingdom and represent a distinct clade among serpins in multicellular organisms. Serpins are also found in green algae, but the evolutionary relationship between these serpins and those of plants remains unknown. Plant serpins are potent inhibitors...... of mammalian serine proteinases of the chymotrypsin family in vitro but, intriguingly, plants and green algae lack endogenous members of this proteinase family, the most common targets for animal serpins. An Arabidopsis serpin with a conserved reactive centre is now known to be capable of inhibiting...

Imaging neutrophil migration dynamics using micro-optical coherence tomography (Conference Presentation)

Science.gov (United States)

Chu, Kengyeh K.; Yonker, Lael; Som, Avira; Pazos, Michael; Kusek, Mark E.; Hurley, Bryan P.; Tearney, Guillermo J.

2016-03-01

Neutrophils are immune cells that undergo chemotaxis, detecting and migrating towards a chemical signal gradient. Neutrophils actively migrate across epithelial boundaries, interacting with the epithelium to selectively permit passage without compromising the epithelial barrier. In many inflammatory disorders, excessive neutrophil migration can cause damage to the epithelium itself. The signaling pathways and mechanisms that facilitate trans-epithelial migration are not fully characterized. Our laboratory has developed micro-optical coherence tomography (μOCT), which has 2 μm lateral resolution and 1 μm axial resolution. As a high-resolution native contrast modality, μOCT can directly visualize individual neutrophils as they interact with a cell layer cultured on a transwell filter. A chemoattractant can be applied to the apical side of inverted monolayer, and human neutrophils placed in the basolateral compartment, while μOCT captures 3D images of the chemotaxis. μOCT images can also generate quantitative metrics of migration volume to study the dependence of chemotaxis on monolayer cell type, chemoattractant type, and disease state of the neutrophils. For example, a disease known as leukocyte adhesion deficiency (LAD) can be simulated by treating neutrophils with antibodies that interfere with the CD18 receptor, a facilitator of trans-epithelial migration. We conducted a migration study of anti-CD18 treated and control neutrophils using T84 intestinal epithelium as a barrier. After one hour, μOCT time-lapse imaging indicated a strong difference in the fraction of neutrophils that remain attached to the epithelium after migration (0.67 +/- 0.12 attached anti-CD18 neutrophils, 0.23 +/- 0.08 attached control neutrophils, n = 6, p < 0.05), as well as a modest but non-significant decrease in total migration volume for treated neutrophils. We can now integrate μOCT-derived migration metrics with simultaneously acquired measurements of transepithelial electrical

Roles of the Picornaviral 3C Proteinase in the Viral Life Cycle and Host Cells

Directory of Open Access Journals (Sweden)

Di Sun

2016-03-01

Full Text Available The Picornaviridae family comprises a large group of non-enveloped viruses that have a major impact on human and veterinary health. The viral genome contains one open reading frame encoding a single polyprotein that can be processed by viral proteinases. The crucial 3C proteinases (3Cpros of picornaviruses share similar spatial structures and it is becoming apparent that 3Cpro plays a significant role in the viral life cycle and virus host interaction. Importantly, the proteinase and RNA-binding activity of 3Cpro are involved in viral polyprotein processing and the initiation of viral RNA synthesis. In addition, 3Cpro can induce the cleavage of certain cellular factors required for transcription, translation and nucleocytoplasmic trafficking to modulate cell physiology for viral replication. Due to interactions between 3Cpro and these essential factors, 3Cpro is also involved in viral pathogenesis to support efficient infection. Furthermore, based on the structural conservation, the development of irreversible inhibitors and discovery of non-covalent inhibitors for 3Cpro are ongoing and a better understanding of the roles played by 3Cpro may provide insights into the development of potential antiviral treatments. In this review, the current knowledge regarding the structural features, multiple functions in the viral life cycle, pathogen host interaction, and development of antiviral compounds for 3Cpro is summarized.

[The significances of peripheral neutrophils CD(55) and myeloperoxidase expression in patients with myeloperoxidase-specific anti-neutrophil cytoplasmic antibody associated vasculitis].

Science.gov (United States)

Zhou, X L; Zheng, M J; Shuai, Z W; Zhang, L; Zhang, M M; Chen, S Y

2017-06-01

Objective: To investigate the expression of CD(55) and myeloperoxidase (MPO) on neutrophils in patients with MPO-specific anti-neutrophil cytoplasmic antibody associated vasculitis(MPO-AAV), and analyze the relationship between the expression and clinical manifestation. Methods: Forty untreated patients with active MPO-AAV (patient group) and 30 healthy volunteers (control group) were enrolled in this study. The CD(55) on neutrophils and both membrane and cytoplasmic MPO were detected by flow cytometry. Serum fragment-from the activated complement factor B(Ba) and MPO were measured by ELISA. The clinical activity of vasculitis was valued by Birmingham vasculitis activity score-version 3(BVAS-V3). The significance of laboratory data was evaluated by Spearman correlation test and multivariate linear regression analysis. Results: (1)The mean fluorescence intensity(MFI) of CD(55) expressed on neutrophils was significantly higher than that in control group[4 068.6±2 306.0 vs 2 999.5±1 504.9, P =0.033]. Similar results of serum MPO and Ba in patient group were found compared to controls [500.0(381.0, 612.7) IU/L vs 286.9(225.5, 329.1) IU/L, P <0.001; 35.2(25.2, 79.5) ng/L vs 18.0(15.0, 28.0) ng/L, P <0.001], respectively. However, MIF of cytoplasmic MPO in patients was significantly lower than that of control group(1 577.1±1 175.9 vs 3 105.3±2 323.0, P =0.003) . (2) In patient group, cytoplasmic intensity of MPO was negatively associated with the serum levels of MPO( r =-0.710, P <0.001) and Ba ( r =-0.589, P =0.001). Moreover, serum MPO was positively associated with serum Ba( r =0.691, P <0.001). Membrane intensity of CD(55) on neutrophils was positively correlated with patient age ( r =0.514, P =0.001), C reactive protein ( r =0.376, P =0.018), peripheral neutrophils count ( r =0.485, P =0.001) and BVAS-V3 ( r =0.484, P =0.002), whereas negative correlation between membrane CD(55) and disease duration was seen ( r =-0.403, P =0.01). (3) The result of multiple

Neutrophils in traumatic brain injury (TBI): friend or foe?

Science.gov (United States)

Liu, Yang-Wuyue; Li, Song; Dai, Shuang-Shuang

2018-05-17

Our knowledge of the pathophysiology about traumatic brain injury (TBI) is still limited. Neutrophils, as the most abundant leukocytes in circulation and the first-line transmigrated immune cells at the sites of injury, are highly involved in the initiation, development, and recovery of TBI. Nonetheless, our understanding about neutrophils in TBI is obsolete, and mounting evidences from recent studies have challenged the conventional views. This review summarizes what is known about the relationships between neutrophils and pathophysiology of TBI. In addition, discussions are made on the complex roles as well as the controversial views of neutrophils in TBI.

The binding mechanism of a peptidic cyclic serine protease inhibitor

DEFF Research Database (Denmark)

Jiang, Longguang; Svane, Anna Sigrid P.; Sørensen, Hans Peter

2011-01-01

Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries......, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical...... inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding...

N-Terminomics TAILS Identifies Host Cell Substrates of Poliovirus and Coxsackievirus B3 3C Proteinases That Modulate Virus Infection

Science.gov (United States)

Jagdeo, Julienne M.; Dufour, Antoine; Klein, Theo; Solis, Nestor; Kleifeld, Oded; Kizhakkedathu, Jayachandran; Luo, Honglin; Overall, Christopher M.

2018-01-01

ABSTRACT Enteroviruses encode proteinases that are essential for processing of the translated viral polyprotein. In addition, viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. Although some host protein substrates of enterovirus proteinases have been identified, the full repertoire of targets remains unknown. We used a novel quantitative in vitro proteomics-based approach, termed terminal amine isotopic labeling of substrates (TAILS), to identify with high confidence 72 and 34 new host protein targets of poliovirus and coxsackievirus B3 (CVB3) 3C proteinases (3Cpros) in HeLa cell and cardiomyocyte HL-1 cell lysates, respectively. We validated a subset of candidate substrates that are targets of poliovirus 3Cpro in vitro including three common protein targets, phosphoribosylformylglycinamidine synthetase (PFAS), hnRNP K, and hnRNP M, of both proteinases. 3Cpro-targeted substrates were also cleaved in virus-infected cells but not noncleavable mutant proteins designed from the TAILS-identified cleavage sites. Knockdown of TAILS-identified target proteins modulated infection both negatively and positively, suggesting that cleavage by 3Cpro promotes infection. Indeed, expression of a cleavage-resistant mutant form of the endoplasmic reticulum (ER)-Golgi vesicle-tethering protein p115 decreased viral replication and yield. As the first comprehensive study to identify and validate functional enterovirus 3Cpro substrates in vivo, we conclude that N-terminomics by TAILS is an effective strategy to identify host targets of viral proteinases in a nonbiased manner. IMPORTANCE Enteroviruses are positive-strand RNA viruses that encode proteases that cleave the viral polyprotein into the individual mature viral proteins. In addition, viral proteases target host proteins in order to modulate cellular pathways and block antiviral responses in order to facilitate virus infection

Inhibition of neutrophil migration by aggregated immunoglobulin attached to micropore membranes.

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Kemp, A S; Brown, S

1980-01-01

The effect of substrate-bound immunoglobulin on neutrophil migration was examined. Immunoglobulin aggregates bound to micropore membranes inhibited the neutrophil response to a chemotactic stimulus. This inhibition was reversed by the presence of aggregates in suspension suggesting competition between substrate-bound and free aggregates for neutrophil surface binding sites. The immobilization of neutrophils by substrate-bound aggregated immunoglobulin suggests a mechanism for the accumulation of neutrophils at sites of immune complex deposition and tissue-bound antibodies in vivo. PMID:7380477

Distinct Trypanosoma cruzi isolates induce activation and apoptosis of human neutrophils.

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Luísa M D Magalhães

Full Text Available Neutrophils are critical players in the first line of defense against pathogens and in the activation of subsequent cellular responses. We aimed to determine the effects of the interaction of Trypanosoma cruzi with human neutrophils, using isolates of the two major discrete type units (DTUs associated with Chagas' disease in Latin America (clone Col1.7G2 and Y strain, DTU I and II, respectively. Thus, we used CFSE-stained trypomastigotes to measure neutrophil-T. cruzi interaction, neutrophil activation, cytokine expression and death, after infection with Col1.7G2 and Y strain. Our results show that the frequency of CFSE+ neutrophils, indicative of interaction, and CFSE intensity on a cell-per-cell basis were similar when comparing Col1.7G2 and Y strains. Interaction with T. cruzi increased neutrophil activation, as measured by CD282, CD284, TNF and IL-12 expression, although at different levels between the two strains. No change in IL-10 expression was observed after interaction of neutrophils with either strain. We observed that exposure to Y and Col1.7G2 caused marked neutrophil death. This was specific to neutrophils, since interaction of either strain with monocytes did not cause death. Our further analysis showed that neutrophil death was a result of apoptosis, which was associated with an upregulation of TNF-receptor, TNF and FasLigand, but not of Fas. Induction of TNF-associated neutrophil apoptosis by the different T. cruzi isolates may act as an effective common mechanism to decrease the host's immune response and favor parasite survival.

The effect of midazolam on neutrophil mitogen-activated protein kinase.

LENUS (Irish Health Repository)

Ghori, Kamran

2010-06-01

Neutrophil p38 mitogen-activated protein kinase (MAPK) is a key enzyme in the intracellular signalling pathway that is responsible for many neutrophil functions, which are important in neutrophil-endothelial interaction. The imidazole compounds are inhibitors of this enzyme system. The objectives of this in-vitro investigation were to examine the effect of midazolam on neutrophil p38 MAPK activation (phosphorylation) following in-vitro ischaemia-reperfusion injury, and the expression of adhesion molecule CD11b\\/CD18.

Effect of oral antiseptic agents on phospholipase and proteinase enzymes of Candida albicans.

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Uygun-Can, Banu; Kadir, Tanju; Gumru, Birsay

2016-02-01

Candida-associated denture stomatitis is the most prevalent form of oral candida infections among the denture wearers. Generally, antiseptic oral rinses used in the treatment of these infections are considered as an adjunct or alternative antifungal treatment. Studies have suggested that the intraoral concentrations of antiseptics decrease substantially to the sub-therapeutic levels on account of the dynamics of the oral cavity. This condition yields the question about the minimum antiseptic concentration that effect the character or pathogenesis of Candida during treatment. The extracellular phospholipase and proteinase enzymes of Candida albicans are regarded to have a crucial role in the pathogenesis of human fungal infections. Therefore, the aim of this study was to investigate the effect of different sub-therapeutic concentrations of chlorhexidine gluconate, hexetidine and triclosan on the production of these enzymes by C. albicans strains isolated from 20 patients with denture stomatitis. Phospholipase test was done by using Sabouraud dextrose agar with egg yolk, proteinase test was done by using bovine serum albumin agar. Phospholipase test was done by using Sabouraud dextrose agar with egg yolk, proteinase test was done by using bovine serum albumin agar. Exoenzyme production of 20 strains which were brief exposured to sub-therapeutic concentrations of three antiseptic agents decreased significantly compared with the strains that were not exposured with antiseptic values (pantiseptics (pantiseptic was compared, there were no significant differences between enzymatic activities (p>0.05). The results of this study show that sub-therapeutic levels of each antiseptic may modulate candidal exoenzyme production, consequently suppressing pathogenicity of C. albicans. Copyright © 2015 Elsevier Ltd. All rights reserved.

Exposure to tobacco-derived materials induces overproduction of secreted proteinases in mast cells

International Nuclear Information System (INIS)

Small-Howard, Andrea; Turner, Helen

2005-01-01

Mast cells reside at interfaces with the environment, including the mucosa of the respiratory and gastrointestinal tracts. This localization exposes mast cells to inhaled, or ingested, environmental challenges. In the airways of smokers, resident immune cells will be in contact with the condensed components of cigarette smoke. Mast cells are of particular interest due to their ability to promote airway remodeling and mucus hypersecretion. Clinical data show increased levels of mast cell-secreted tryptase and increased numbers of degranulated mast cells in the lavage and bronchial tissue of smokers. Since mast cell-secreted proteinases (MCPTs), including tryptases, contribute to pathological airway remodeling, we investigated the relationship between mast cell proteinases and smoke exposure. We exposed a mast cell line to cigarette smoke condensate (CSC). We show that CSC exposure increases MCPT levels in mast cells using an assay for tryptase-type MCPT activity. We hypothesized that this increase in MCPT activity reflects a CSC-induced increase in the cytosolic pool of proteinase molecules, via stimulation of MCPT transcription. Transcript array data suggested that mRNA changes in response to CSC were limited in number and peaked after 3 h of CSC exposure. However, we noted marked transcriptional regulation of several MCPT genes. CSC-induced changes in the mRNA levels for MCPTs were confirmed using quantitative RT-PCR. Taken together, our data suggest that chronic exposure to cigarette smoke up-regulates MCPT levels in mast cells at both the protein and the mRNA level. We suggest that the pathological airway remodeling that has been described in clinical studies of smoke inhalation may be attributable to MCPT overproduction in vivo

Cell-type specific mechanisms of D-serine uptake and release in the brain

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Magalie eMartineau

2014-05-01

Full Text Available Accumulating evidence during the last decade established that D-serine is a key signaling molecule utilized by neurons and astroglia in the mammalian central nervous system. D-serine is increasingly appreciated as the main physiological endogenous coagonist for synaptic NMDA receptors at central excitatory synapses; it is mandatory for long-term changes in synaptic strength, memory, learning, and social interactions. Alterations in the extracellular levels of D-serine leading to disrupted cell-cell signaling are a trademark of many chronic or acute neurological (i.e. Alzheimer disease, epilepsy, stroke and psychiatric (i.e. schizophrenia disorders, and are associated with addictive behavior (i.e. cocaine addiction. Indeed, fine tuning of the extracellular levels of D-serine, achieved by various molecular machineries and signaling pathways, is necessary for maintenance of accurate NMDA receptor functions. Here, we review the experimental data supporting the notion that astroglia and neurons use different pathways to regulate levels of extracellular D-serine.

Effects of Wharton's jelly-derived mesenchymal stem cells on neonatal neutrophils

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Khan I

2014-12-01

Full Text Available Imteyaz Khan,1 Liying Zhang,2 Moiz Mohammed,1 Faith E Archer,1 Jehan Abukharmah,1 Zengrong Yuan,2 S Saif Rizvi,1 Michael G Melek,1 Arnold B Rabson,1,2 Yufang Shi,2 Barry Weinberger,1 Anna M Vetrano1,21Department of Pediatrics, Division of Neonatology, Rutgers Robert Wood Johnson Medical School, 2Rutgers Child Health Institute of New Jersey, New Brunswick, NJ, USABackground: Mesenchymal stem cells (MSCs have been proposed as autologous therapy for inflammatory diseases in neonates. MSCs from umbilical cord Wharton's jelly (WJ-MSCs are accessible, with high proliferative capacity. The effects of WJ-MSCs on neutrophil activity in neonates are not known. We compared the effects of WJ-MSCs on apoptosis and the expression of inflammatory, oxidant, and antioxidant mediators in adult and neonatal neutrophils.Methods: WJ-MSCs were isolated, and their purity and function were confirmed by flow cytometry. Neutrophils were isolated from cord and adult blood by density centrifugation. The effects of neutrophil/WJ-MSC co-culture on apoptosis and gene and protein expression were measured.Results: WJ-MSCs suppressed neutrophil apoptosis in a dose-dependent manner. WJ-MSCs decreased gene expression of NADPH oxidase-1 in both adult and neonatal neutrophils, but decreased heme oxygenase-1 and vascular endothelial growth factor and increased catalase and cyclooxygenase-2 in the presence of lipopolysaccharide only in adult cells. Similarly, generation of interleukin-8 was suppressed in adult but not neonatal neutrophils. Thus, WJ-MSCs dampened oxidative, vascular, and inflammatory activity by adult neutrophils, but neonatal neutrophils were less responsive. Conversely, Toll-like receptor-4, and cyclooxygenase-2 were upregulated in WJ-MSCs only in the presence of adult neutrophils, suggesting an inflammatory MSC phenotype that is not induced by neonatal neutrophils.Conclusion: Whereas WJ-MSCs altered gene expression in adult neutrophils in ways suggesting anti

Epitope specificity determines pathogenicity and detectability in ANCA-associated vasculitis

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ABSTRACT BACKGROUND Anti-neutrophil cytoplasmic autoantibodies (ANCA) specific for myeloperoxidase (MPO) or proteinase 3 (PR3) are detectable in >90% of patients with ANCA-associated vasculitis (AAV). ANCA titers do not correlate well with disease activity. In vivo and in vi...

Suppressed neutrophil function in children with acute lymphoblastic leukemia.

Science.gov (United States)

Tanaka, Fumiko; Goto, Hiroaki; Yokosuka, Tomoko; Yanagimachi, Masakatsu; Kajiwara, Ryosuke; Naruto, Takuya; Nishimaki, Shigeru; Yokota, Shumpei

2009-10-01

Infection is a major obstacle in cancer chemotherapy. Neutropenia has been considered to be the most important risk factor for severe infection; however, other factors, such as impaired neutrophil function, may be involved in susceptibility to infection in patients undergoing chemotherapy. In this study, we analyzed neutrophil function in children with acute lymphoblastic leukemia (ALL). Whole blood samples were obtained from 16 children with ALL at diagnosis, after induction chemotherapy, and after consolidation chemotherapy. Oxidative burst and phagocytic activity of neutrophils were analyzed by flow cytometry. Oxidative burst of neutrophils was impaired in ALL patients. The percentage of neutrophils with normal oxidative burst after PMA stimulation was 59.0 +/- 13.2 or 70.0 +/- 21.0% at diagnosis or after induction chemotherapy, respectively, which was significantly lower compared with 93.8 +/- 6.1% in healthy control subjects (P = 0.00004, or 0.002, respectively); however, this value was normal after consolidation chemotherapy. No significant differences were noted in phagocytic activity in children with ALL compared with healthy control subjects. Impaired oxidative burst of neutrophils may be one risk factor for infections in children with ALL, especially in the initial periods of treatment.

l-Serine Enhances Light-Induced Circadian Phase Resetting in Mice and Humans.

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Yasuo, Shinobu; Iwamoto, Ayaka; Lee, Sang-Il; Ochiai, Shotaro; Hitachi, Rina; Shibata, Satomi; Uotsu, Nobuo; Tarumizu, Chie; Matsuoka, Sayuri; Furuse, Mitsuhiro; Higuchi, Shigekazu

2017-12-01

Background: The circadian clock is modulated by the timing of ingestion or food composition, but the effects of specific nutrients are poorly understood. Objective: We aimed to identify the amino acids that modulate the circadian clock and reset the light-induced circadian phase in mice and humans. Methods: Male CBA/N mice were orally administered 1 of 20 l-amino acids, and the circadian and light-induced phase shifts of wheel-running activity were analyzed. Antagonists of several neurotransmitter pathways were injected before l-serine administration, and light-induced phase shifts were analyzed. In addition, the effect of l-serine on the light-induced phase advance was investigated in healthy male students (mean ± SD age 22.2 ± 1.8 y) by using dim-light melatonin onset (DLMO) determined by saliva samples as an index of the circadian phase. Results: l-Serine administration enhanced light-induced phase shifts in mice (1.86-fold; P light-dark cycle by 6 h, l-serine administration slightly accelerated re-entrainment to the shifted cycle. In humans, l-serine ingestion before bedtime induced significantly larger phase advances of DLMO after bright-light exposure during the morning (means ± SEMs-l-serine: 25.9 ± 6.6 min; placebo: 12.1 ± 7.0 min; P light-induced phase resetting in mice and humans, and it may be useful for treating circadian disturbances. © 2017 American Society for Nutrition.

Granule Associated Serine Proteases of Hematopoietic Cells - An Analysis of Their Appearance and Diversification during Vertebrate Evolution.

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Srinivas Akula

Full Text Available Serine proteases are among the most abundant granule constituents of several hematopoietic cell lineages including mast cells, neutrophils, cytotoxic T cells and NK cells. These proteases are stored in their active form in the cytoplasmic granules and in mammals are encoded from four different chromosomal loci: the chymase locus, the met-ase locus, the T cell tryptase and the mast cell tryptase locus. In order to study their appearance during vertebrate evolution we have performed a bioinformatic analysis of related genes and gene loci from a large panel of metazoan animals from sea urchins to placental mammals for three of these loci: the chymase, met-ase and granzyme A/K loci. Genes related to mammalian granzymes A and K were the most well conserved and could be traced as far back to cartilaginous fish. Here, the granzyme A and K genes were found in essentially the same chromosomal location from sharks to humans. However in sharks, no genes clearly identifiable as members of the chymase or met-ase loci were found. A selection of these genes seemed to appear with bony fish, but sometimes in other loci. Genes related to mammalian met-ase locus genes were found in bony fish. Here, the most well conserved member was complement factor D. However, genes distantly related to the neutrophil proteases were also identified in this locus in several bony fish species, indicating that this locus is also old and appeared at the base of bony fish. In fish, a few of the chymase locus-related genes were found in a locus with bordering genes other than the mammalian chymase locus and some were found in the fish met-ase locus. This indicates that a convergent evolution rather than divergent evolution has resulted in chymase locus-related genes in bony fish.

Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant.

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Duan, X; Li, X; Xue, Q; Abo-el-Saad, M; Xu, D; Wu, R

1996-04-01

We introduced the potato proteinase inhibitor II (PINII) gene (pin2) into several Japonica rice varieties, and regenerated a large number of transgenic rice plants. Wound-inducible expression of the pin2 gene driven by its own promoter, together with the first intron of the rice actin 1 gene (act1), resulted in high-level accumulation of the PINII protein in the transgenic plants. The introduced pin2 gene was stably inherited in the second, third, and fourth generations, as shown by molecular analyses. Based on data from the molecular analyses, several homozygous transgenic lines were obtained. Bioassay for insect resistance with the fifth-generation transgenic rice plants showed that transgenic rice plants had increased resistance to a major rice insect pest, pink stem borer (Sesamia inferens). Thus, introduction of an insecticidal proteinase inhibitor gene into cereal plants can be used as a general strategy for control of insect pests.

Changes in Neutrophil Functions in Astronauts

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Kaur, Indreshpal; Simons, Elizabeth R.; Castro, Victoria; Pierson, Duane L.

2002-01-01

Neutrophil functions (phagocytosis, oxidative burst, degranulation) and expression of surface markers involved in these functions were studied in 25 astronauts before and after 4 space shuttle missions. Space flight duration ranged from 5 to 11 days. Blood specimens were obtained 10 days before launch (preflight or L-10), immediately after landing (landing or R+0), and again at 3 days after landing (postflight or R+3). Blood samples were also collected from 9 healthy low-stressed subjects at 3 time points simulating a 10-day shuttle mission. The number of neutrophils increased at landing by 85 percent when compared to the preflight numbers. Neutrophil functions were studied in whole blood using flow cytometric methods. Phagocytosis of E.coli-FITC and oxidative burst capacity of the neutrophils following the 9 to 11 day missions were lower at all three sampling points than the mean values for control subjects. Phagocytosis and oxidative burst capacity of the astronauts was decreased even 10-days before space flight. Mission duration appears to be a factor in phagocytic and oxidative functions. In contrast, following the short-duration (5-days) mission, these functions were unchanged from control values. No consistent changes in degranulation were observed following either short or medium length space missions. The expression of CD16, CD32, CD11a, CD11b, CD11c, L-selectin and CD36 was measured and found to be variable. Specifically, CD16 and CD32 did not correlate with the changes in oxidative burst and phagocytosis. We can conclude from this study that the stresses associated with space flight can alter the important functions of neutrophils.

Dosage compensation of serine-4 transfer RNA in Drosophila melanogaster

International Nuclear Information System (INIS)

Birchler, J.A.; Owenby, R.K.; Jacobson, K.B.

1982-01-01

A dosage series of the X chromosome site for serine-4 transfer RNA consisting of one of three copies in females and one to two in males was constructed to test whether transfer RNA expression is governed by dosage compensation. A dosage effect on the level of the serine-4 isoacceptor was observed in both females and males when the structural locus was varied. However, in males, each dose had a relatively greater expression so the normal one dose was slightly greater than the total female value and the duplicated male had the highest relative expression of all the types examined. Serine-4 levels in males and females from an isogenic Oregon-R stock were similar. Thus the transfer RNA levels conform to the expectations of dosage compensation

Neutrophilic nodules in the intestinal walls of Japanese monkeys associated with the neutrophil chemotactic activity of larval extracts and secretions of Oesophagostomum aculeatum.

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Horii, Y; Ishii, A; Owhashi, M; Miyoshi, M; Usui, M

1985-01-01

High neutrophil chemotactic activity was detected in the culture medium from Oesophagostomum aculeatum larvae in vitro using blind-well chambers with Millipore filters, and guinea pig leucocytes as indicator cells. Neutrophil chemotactic activity was also detected in the extract from larval worms in a dose dependent fashion. This activity was detected in the low molecular weight fractions adjacent to a sodium chloride marker by gel filtration on Sephadex G200. These results were further confirmed with monkey neutrophils. The possible role of this activity in the formation of granulomatous lesions rich in neutrophils found in O aculeatum infections in the Japanese monkey is discussed.

Heterogeneity in Neutrophil Microparticles Reveals Distinct Proteome and Functional Properties*

Science.gov (United States)

Dalli, Jesmond; Montero-Melendez, Trinidad; Norling, Lucy V; Yin, Xiaoke; Hinds, Charles; Haskard, Dorian; Mayr, Manuel; Perretti, Mauro

2013-01-01

Altered plasma neutrophil microparticle levels have recently been implicated in a number of vascular and inflammatory diseases, yet our understanding of their actions is very limited. Herein, we investigate the proteome of neutrophil microparticles in order to shed light on their biological actions. Stimulation of human neutrophils, either in suspension or adherent to an endothelial monolayer, led to the production of microparticles containing >400 distinct proteins with only 223 being shared by the two subsets. For instance, postadherent microparticles were enriched in alpha-2 macroglobulin and ceruloplasmin, whereas microparticles produced by neutrophils in suspension were abundant in heat shock 70 kDa protein 1. Annexin A1 and lactotransferrin were expressed in both microparticle subsets. We next determined relative abundance of these proteins in three types of human microparticle samples: healthy volunteer plasma, plasma of septic patients and skin blister exudates finding that these proteins were differentially expressed on neutrophil microparticles from these samples reflecting in part the expression profiles we found in vitro. Functional assessment of the neutrophil microparticles subsets demonstrated that in response to direct stimulation neutrophil microparticles produced reactive oxygen species and leukotriene B4 as well as locomoted toward a chemotactic gradient. Finally, we investigated the actions of the two neutrophil microparticles subsets described herein on target cell responses. Microarray analysis with human primary endothelial cells incubated with either microparticle subset revealed a discrete modulation of endothelial cell gene expression profile. These findings demonstrate that neutrophil microparticles are heterogenous and can deliver packaged information propagating the activation status of the parent cell, potentially exerting novel and fundamental roles both under homeostatic and disease conditions. PMID:23660474

Evaluation of oxidative stress in D-serine induced nephrotoxicity

International Nuclear Information System (INIS)

Orozco-Ibarra, Marisol; Medina-Campos, Omar Noel; Sanchez-Gonzalez, Dolores Javier; Martinez-Martinez, Claudia Maria; Floriano-Sanchez, Esau; Santamaria, Abel; Ramirez, Victoria; Bobadilla, Norma A.; Pedraza-Chaverri, Jose

2007-01-01

It has been suggested that oxidative stress is involved in D-serine-induced nephrotoxicity. The purpose of this study was to assess if oxidative stress is involved in this experimental model using several approaches including (a) the determination of several markers of oxidative stress and the activity of some antioxidant enzymes in kidney and (b) the use of compounds with antioxidant or prooxidant effects. Rats were sacrificed at several periods of time (from 3 to 24 h) after a single i.p. injection of D-serine (400 mg/kg). Control rats were injected with L-serine (400 mg/kg) and sacrificed 24 h after. The following markers were used to assess the temporal aspects of renal damage: (a) urea nitrogen (BUN) and creatinine in blood serum, (b) kidney injury molecule (KIM-1) mRNA levels, and (c) tubular necrotic damage. In addition, creatinine clearance, proteinuria, and urinary excretion of N-acetyl-β-D-glucosaminidase (NAG) were measured 24 h after D-serine injection. Protein carbonyl content, malondialdehyde (MDA), 4-hydroxy-2-nonenal (4-HNE), fluorescent products of lipid peroxidation, reactive oxygen species (ROS), glutathione (GSH) content, and heme oxygenase-1 (HO-1) expression were measured as markers of oxidative stress in the kidney. Additional experiments were performed using the following compounds with antioxidant or pro-oxidant effects before D-serine injection: (a) α-phenyl-tert-butyl-nitrone (PBN), a spin trapping agent; (b) 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron(III) (FeTPPS), a soluble complex able to metabolize peroxynitrite; (c) aminotriazole (ATZ), a catalase (CAT) inhibitor; (d) stannous chloride (SnCl 2 ), an HO-1 inductor; (e) tin mesoporphyrin (SnMP), an HO inhibitor. In the time-course study, serum creatinine and BUN increased significantly on 15-24 and 20-24 h, respectively, and KIM-1 mRNA levels increased significantly on 6-24 h. Histological analyses revealed tubular necrosis at 12 h. The activity of antioxidant enzymes

Ly6G-mediated depletion of neutrophils is dependent on macrophages.

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Bruhn, Kevin W; Dekitani, Ken; Nielsen, Travis B; Pantapalangkoor, Paul; Spellberg, Brad

2016-01-01

Antibody-mediated depletion of neutrophils is commonly used to study neutropenia. However, the mechanisms by which antibodies deplete neutrophils have not been well defined. We noticed that mice deficient in complement and macrophages had blunted neutrophil depletion in response to anti-Ly6G monoclonal antibody (MAb) treatment. In vitro, exposure of murine neutrophils to anti-Ly6G MAb in the presence of plasma did not result in significant depletion of cells, either in the presence or absence of complement. In vivo, anti-Ly6G-mediated neutrophil depletion was abrogated following macrophage depletion, but not complement depletion, indicating a requirement for macrophages to induce neutropenia by this method. These results inform the use and limitations of anti-Ly6G antibody as an experimental tool for depleting neutrophils in various immunological settings.

[Experimental approach to the prophylaxis and treatment of acute lung injury syndrome with proteinase inhibitors and corvitin].

Science.gov (United States)

Moĭbenko, O O; Kubyshkin, A V; Kharchenko, V Z; Horokhova, N Iu; Semenets', P F

2003-01-01

The results of a combined study of the proteolysis on a model of post-ischemic toxemia in rats showed a decrease in antiproteinase potential and an activation of proteolysis. The activation of proteolysis and inhibition of antiproteinases was observed not only in the blood, but also in the bronchoalveolar secretion. Those changes were accompanied with the changes in the morphological structure of the lungs. The data obtained have shown a high effectiveness of proteinase inhibitor (contrical) and an antioxidant of flavonoid group (corvetine). Those drugs decreased the morphological changes in the lungs and prevented the development of imbalance in proteinase-inhibitor system. The prophylactic effect was more considerable when both drugs were used in a combined way.

Neutrophils in oral paracoccidioidomycosis and the involvement of Nrf2.

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Vera Cavalcanti Araújo

Full Text Available Neutrophils have been implicated in granuloma formation in several infectious diseases, in addition to their main phagocytic and pathogen destruction role. It has been demonstrated that Nrf2 regulates antioxidant protection in neutrophils, attenuating inflammation without compromising the hosts bacterial defense. In this study, we analyzed the presence of neutrophils in Paracoccidioides brasiliensis mycosis (PCM, as well as the immunoexpression of Nrf2. Thirty-nine cases of oral PCM were classified according to quantity of fungi and to the presence of loose or well-organized granulomas and microabscesses. An Nrf2 antibody was used for immunohistochemical analysis. The results showed that neutrophils are present in microabscesses and loose granulomas, but were absent in structured granulomas. A greater quantity of fungi was shown in cases with only loose granulomas when compared to loose and well organized granulomas. Nrf2 was observed in the nuclei of neutrophils of loose granulomas and abscesses, with its expression in loose granulomas maintained despite the additional presence of well organized granulomas in the same specimen. This study suggests that neutrophils participate in P. brasiliensis granuloma formation and that Nrf2 has a possible role in neutrophil survival, via modulation of the inflammatory response.

Comparison of self-processing of foot-and-mouth disease virus leader proteinase and porcine reproductive and respiratory syndrome virus leader proteinase nsp1α

International Nuclear Information System (INIS)

Steinberger, Jutta; Kontaxis, Georg; Rancan, Chiara; Skern, Tim

2013-01-01

The foot-and-mouth disease virus leader proteinase (Lb pro ) cleaves itself off the nascent viral polyprotein. NMR studies on the monomeric variant Lb pro L200F provide structural evidence for intramolecular self-processing. 15 N-HSQC measurements of Lb pro L200F showed specifically shifted backbone signals in the active and substrate binding sites compared to the monomeric variant sLb pro , lacking six C-terminal residues. This indicates transient intramolecular interactions between the C-terminal extension (CTE) of one molecule and its own active site. Contrastingly, the porcine reproductive and respiratory syndrome virus (PRRSV) leader proteinase nsp1α, with a papain-like fold like Lb pro , stably binds its own CTE. Parts of the β-sheet domains but none of the α-helical domains of Lb pro and nsp1α superimpose; consequently, the α-helical domain of nsp1α is oriented differently relative to its β-sheet domain. This provides a large interaction surface for the CTE with the globular domain, stabilising the intramolecular complex. Consequently, self-processing inactivates nsp1α but not Lb pro . - Highlights: • We examine self-processing of the leader protease of foot-and-mouth disease virus. • NMR analysis strongly supports intramolecular self-processing. • Self-processing is a dynamic process with no stable complex. • Structural comparison with nsp1α of PRRSV which forms stable intramolecular complex. • Subdomain orientation explains differences in stability of intramolecular complexes

In vivo activation of equine eosinophils and neutrophils by experimental Strongylus vulgaris infections.

Science.gov (United States)

Dennis, V A; Klei, T R; Chapman, M R; Jeffers, G W

1988-12-01

Eosinophils and neutrophils from ponies with Strongylus vulgaris-induced eosinophilia (eosinophilic ponies; activated eosinophils and neutrophils) were assayed in vitro for chemotactic and chemokinetic responses to zymosan-activated serum (ZAS) using the filter system in Boyden chambers, for Fc and complement (C) receptors using the EA and EAC-rosette assays, respectively, and for phagocytic and bactericidal activities using opsonized Escherichia coli and the acridine orange method. The responses of activated eosinophils and neutrophils in the above assays were compared with those of eosinophils and neutrophils from S. vulgaris-naive ponies without eosinophilia (noneosinophilic ponies; nonactivated eosinophils and neutrophils). Differences in cell density following centrifugation in a continuous Percoll gradient were used to further characterize the heterogeneity of activated eosinophils and neutrophils. Activated and nonactivated eosinophils demonstrated similar chemotactic responses to ZAS while activated and nonactivated neutrophils demonstrated similar chemokinetic responses to ZAS. A higher percentage of activated eosinophils and neutrophils expressed Fc and C receptors compared with nonactivated cells (P less than 0.05). Generally, higher percentages of eosinophils and neutrophils expressed C than Fc receptors. However, the percentage of neutrophils with both receptors was higher than that of eosinophils. Phagocytosis and killing of E. coli by either type of eosinophil were not consistently observed. Both activated and nonactivated neutrophils phagocytized E. coli and significant differences between the two cell types were not observed. The bacterial activity, however, of activated neutrophils was significantly greater than that obtained using nonactivated neutrophils (P less than 0.05). Activated eosinophils and neutrophils were both separated into two distinct fractions based on differences in cell densities. A higher percentage of band 2 eosinophils

Contribution of the D-Serine-dependent pathway to the cellular mechanisms underlying cognitive aging

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Emilie Rouaud

2010-02-01

Full Text Available An association between age-related memory impairments and changes in functional plasticity in the aging brain has been under intense study within the last decade. In this article, we show that an impaired activation of the strychnine-insensitive glycine site of N-Methyl-D-Aspartate receptors (NMDA-R by its agonist D-serine contributes to deficits of synaptic plasticity in the hippocampus of memory-impaired aged rats. Supplementation with exogenous D-serine prevents the age-related deficits of isolated NMDA-R-dependent synaptic potentials as well as those of theta-burst-induced long-term potentiation and synaptic depotentiation. Endogenous levels of D-serine are reduced in the hippocampus with aging, that correlates with a weaker expression of serine racemase synthesizing the amino acid. On the contrary, the affinity of D-serine binding to NMDA-R is not affected by aging. These results point to a critical role for the D-serine-dependent pathway in the functional alterations of the brain underlying memory impairment and provide key information in the search for new therapeutic strategies for the treatment of memory deficits in the elderly.

File list: Unc.Bld.50.AllAg.Neutrophils [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Unc.Bld.50.AllAg.Neutrophils hg19 Unclassified Blood Neutrophils SRX956546,SRX95655...2,SRX956549 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Bld.50.AllAg.Neutrophils.bed ...

File list: Unc.Bld.05.AllAg.Neutrophils [Chip-atlas[Archive

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Full Text Available Unc.Bld.05.AllAg.Neutrophils hg19 Unclassified Blood Neutrophils SRX956546,SRX95655...2,SRX956549 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Bld.05.AllAg.Neutrophils.bed ...

File list: Unc.Bld.10.AllAg.Neutrophils [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Unc.Bld.10.AllAg.Neutrophils hg19 Unclassified Blood Neutrophils SRX956552,SRX95654...9,SRX956546 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Unc.Bld.10.AllAg.Neutrophils.bed ...

Characterization of proteinases from the midgut of Rhipicephalus (Boophilus microplus involved in the generation of antimicrobial peptides

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Craik Charles S

2010-07-01

Full Text Available Abstract Background Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins. A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins. Results An aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1'. Conclusions BmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora.

Purification, characterization and cloning of an aspartic proteinase inhibitor from squash phloem exudate.

Science.gov (United States)

Christeller, J T; Farley, P C; Ramsay, R J; Sullivan, P A; Laing, W A

1998-05-15

Phloem exudate from squash fruit contains heat-inactivated material which inhibits pepsin activity. This inhibitory activity was purified by mild acid treatment, chromatography on trypsin-agarose, Sephadex G-75 and reverse-phase HPLC, resulting in the elution of three peaks with pepsin-inhibitory activity. N-terminal sequencing indicated a common sequence of MGPGPAIGEVIG and the presence of minor species with seven- or two-amino-acid N-terminal extensions beyond this point. Microheterogeneity in this end sequence was exhibited within and between two preparations. Internal sequencing of a major peak after a trypsin digestion gave the sequence FYNVVVLEK. The common N-terminal sequence was used to design a degenerate primer for 3' rapid amplification of cDNA ends and cDNA clones encoding two isoforms of the inhibitor were obtained. The open reading frames of both cDNAs encoded proteins (96% identical) which contained the experimentally determined internal sequence. The amino acid content calculated from the predicted amino acid sequence was very similar to that measured by amino acid analysis of the purified inhibitor. The two predicted amino acid sequences (96 residues) had neither similarity to any other aspartic proteinase inhibitor nor similarity to any other protein. The inhibitors have a molecular mass of 10,552 Da, measured by matrix-assisted laser-desorption ionisation time-of-flight mass spectrometry and approximately 10,000 Da by SDS/PAGE, and behave as dimers of approximately 21,000 Da during chromatography on Superdex G-75 gel-filtration medium. The calculated molecular masses from the predicted amino acid sequences were 10,551 Da and 10,527 Da. The inhibitor was capable of inhibiting pepsin (Ki = 2 nM) and a secreted aspartic proteinase from the fungus Glomerella cingulata (Ki = 20 nM). The inhibitor, which is stable over acid and neutral pH, has been named squash aspartic proteinase inhibitor (SQAPI).

The uropathogenic species Staphylococcus saprophyticus tolerates a high concentration of D-serine.

Science.gov (United States)

Sakinç, Türkân; Michalski, Nadine; Kleine, Britta; Gatermann, Sören G

2009-10-01

Human urine contains a relatively high concentration of d-serine, which is toxic to several nonuropathogenic bacteria, but can be utilized or detoxified by uropathogenic Escherichia coli (UPEC). The sequenced genome of uropathogenic Staphylococcus saprophyticus contains a gene with homology to the d-serine deaminase gene (dsdA) of UPEC. We found the gene in several clinical isolates of S. saprophyticus; however, the gene was absent in Staphylococcus xylosus and Staphylococcus cohnii, phylogenetically close relatives of S. saprophyticus, and could also not be detected in isolates of Staphylococcus aureus, Staphylococcus epidermidis and 13 other staphylococcal species. In addition, the genomes of other sequenced staphylococci do not harbor homologues of this operon. Interestingly, S. saprophyticus could grow in media supplemented with relatively high concentrations of d-serine, whereas S. aureus, S. epidermidis and other staphylococcal species could not. The association of the dsdA gene with growth in media including d-serine was proved by introducing the gene into S. aureus Newman. Given the fact that UPEC and S. saprophyticus tolerate this compound, d-serine utilization and detoxification may be a general property of uropathogenic bacteria. © 2009 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

D-Serine and Glycine Differentially Control Neurotransmission during Visual Cortex Critical Period.

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Claire N J Meunier

Full Text Available N-methyl-D-aspartate receptors (NMDARs play a central role in synaptic plasticity. Their activation requires the binding of both glutamate and d-serine or glycine as co-agonist. The prevalence of either co-agonist on NMDA-receptor function differs between brain regions and remains undetermined in the visual cortex (VC at the critical period of postnatal development. Here, we therefore investigated the regulatory role that d-serine and/or glycine may exert on NMDARs function and on synaptic plasticity in the rat VC layer 5 pyramidal neurons of young rats. Using selective enzymatic depletion of d-serine or glycine, we demonstrate that d-serine and not glycine is the endogenous co-agonist of synaptic NMDARs required for the induction and expression of Long Term Potentiation (LTP at both excitatory and inhibitory synapses. Glycine on the other hand is not involved in synaptic efficacy per se but regulates excitatory and inhibitory neurotransmission by activating strychnine-sensitive glycine receptors, then producing a shunting inhibition that controls neuronal gain and results in a depression of synaptic inputs at the somatic level after dendritic integration. In conclusion, we describe for the first time that in the VC both D-serine and glycine differentially regulate somatic depolarization through the activation of distinct synaptic and extrasynaptic receptors.

Spontaneous neutrophil activation in HTLV-1 infected patients

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Jaqueline B. Guerreiro

Full Text Available Human T cell lymphotropic Virus type-1 (HTLV-1 induces lymphocyte activation and proliferation, but little is known about the innate immune response due to HTLV-1 infection. We evaluated the percentage of neutrophils that metabolize Nitroblue tetrazolium (NBT to formazan in HTLV-1 infected subjects and the association between neutrophil activation and IFN-gamma and TNF-alpha levels. Blood was collected from 35 HTLV-1 carriers, from 8 patients with HAM/TSP (HTLV-1- associated myelopathy; 22 healthy individuals were evaluated for spontaneous and lipopolysaccharide (LPS-stimulated neutrophil activity (reduction of NBT to formazan. The production of IFN-gamma and TNF-alpha by unstimulated mononuclear cells was determined by ELISA. Spontaneous NBT levels, as well as spontaneous IFN-gamma and TNF-alpha production, were significantly higher (p<0.001 in HTLV-1 infected subjects than in healthy individuals. A trend towards a positive correlation was noted, with increasing percentage of NBT positive neutrophils and levels of IFN-gamma. The high IFN-gamma producing HTLV-1 patient group had significantly greater NBT than healthy controls, 43±24% and 17±4.8% respectively (p< 0.001, while no significant difference was observed between healthy controls and the low IFN-gamma-producing HTLV-1 patient group (30±20%. Spontaneous neutrophil activation is another marker of immune perturbation resulting from HTLV-1 infection. In vivo activation of neutrophils observed in HTLV-1 infected subjects is likely to be the same process that causes spontaneous IFN-gamma production, or it may partially result from direct IFN-gamma stimulation.

SiMA: A simplified migration assay for analyzing neutrophil migration.

Science.gov (United States)

Weckmann, Markus; Becker, Tim; Nissen, Gyde; Pech, Martin; Kopp, Matthias V

2017-07-01

In lung inflammation, neutrophils are the first leukocytes migrating to an inflammatory site, eliminating pathogens by multiple mechanisms. The term "migration" describes several stages of neutrophil movement to reach the site of inflammation, of which the passage of the interstitium and basal membrane of the airway are necessary to reach the site of bronchial inflammation. Currently, several methods exist (e.g., Boyden Chamber, under-agarose assay, or microfluidic systems) to assess neutrophil mobility. However, these methods do not allow for parameterization on single cell level, that is, the individual neutrophil pathway analysis is still considered challenging. This study sought to develop a simplified yet flexible method to monitor and quantify neutrophil chemotaxis by utilizing commercially available tissue culture hardware, simple video microscopic equipment and highly standardized tracking. A chemotaxis 3D µ-slide (IBIDI) was used with different chemoattractants [interleukin-8 (IL-8), fMLP, and Leukotriene B4 (LTB 4 )] to attract neutrophils in different matrices like Fibronectin (FN) or human placental matrix. Migration was recorded for 60 min using phase contrast microscopy with an EVOS ® FL Cell Imaging System. The images were normalized and texture based image segmentation was used to generate neutrophil trajectories. Based on these spatio-temporal information a comprehensive parameter set is extracted from each time series describing the neutrophils motility, including velocity and directness and neutrophil chemotaxis. To characterize the latter one, a sector analysis was employed enabling the quantification of the neutrophils response to the chemoattractant. Using this hard- and software framework we were able to identify typical migration profiles of the chemoattractants IL-8, fMLP, and LTB 4 , the effect of the matrices FN versus HEM as well as the response to different medications (Prednisolone). Additionally, a comparison of four asthmatic and

Improvement in regional CBF by L-serine contributes to its neuroprotective effect in rats after focal cerebral ischemia.

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Tao-Jie Ren

Full Text Available To investigate the mechanisms underlying the neuroprotective effect of L-serine, permanent focal cerebral ischemia was induced by occlusion of the middle cerebral artery while monitoring cerebral blood flow (CBF. Rats were divided into control and L-serine-treated groups after middle cerebral artery occlusion. The neurological deficit score and brain infarct volume were assessed. Nissl staining was used to quantify the cortical injury. L-serine and D-serine levels in the ischemic cortex were analyzed with high performance liquid chromatography. We found that L-serine treatment: 1 reduced the neurological deficit score, infarct volume and cortical neuron loss in a dose-dependent manner; 2 improved CBF in the cortex, and this effect was inhibited in the presence of apamin plus charybdotoxin while the alleviation of both neurological deficit score and infarct volume was blocked; and 3 increased the amount of L-serine and D-serine in the cortex, and inhibition of the conversion of L-serine into D-serine by aminooxyacetic acid did not affect the reduction of neurological deficit score and infarct volume by L-serine. In conclusion, improvement in regional CBF by L-serine may contribute to its neuroprotective effect on the ischemic brain, potentially through vasodilation which is mediated by the small- and intermediate-conductance Ca(2+-activated K(+ channels on the cerebral blood vessel endothelium.

Formation of neutrophil extracellular traps under low oxygen level

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Katja Branitzki-Heinemann

2016-11-01

Full Text Available Since their discovery, neutrophil extracellular traps (NETs have been characterized as a fundamental host innate immune defense mechanism. Conversely, excessive NET release may have a variety of detrimental consequences for the host. A fine balance between NET formation and elimination is necessary to sustain a protective effect during an infectious challenge. Our own recently published data revealed that stabilization of hypoxia inducible factor 1α (HIF-1α by the iron chelating HIF-1α-agonist desferoxamine or AKB-4924 enhanced the release of phagocyte extracellular traps. Since HIF-1α is a global regulator of the cellular response to low oxygen, we hypothesized that NET formation may be similarly increased under low oxygen conditions. Hypoxia occurs in tissues during infection or inflammation, mostly due to overconsumption of oxygen by pathogens and recruited immune cells. Therefore, experiments were performed to characterize the formation of NETs under hypoxic oxygen conditions compared to normoxia. Human blood-derived neutrophils were isolated and incubated under normoxic (21% oxygen level and compared to hypoxic (1% conditions. Dissolved oxygen levels were monitored in the primary cell culture using a Fibox4-PSt3 measurement system. The formation of NETs was quantified by fluorescence microscopy in response to the known NET-inducer phorbol 12-myristate 13-acetate (PMA or S. aureus wildtype and a nuclease-deficient mutant. In contrast to our hypothesis, spontaneous NET formation of neutrophils incubated under hypoxia was distinctly reduced compared to control neutrophils incubated under normoxia. Furthermore, neutrophils incubated under hypoxia showed significantly reduced formation of NETs in response to PMA. Gene expression analysis revealed that mRNA level of hif-1α as well as hif-1α target genes was not altered. However, in good correlation to the decreased NET formation under hypoxia, the cholesterol content of the neutrophils was

File list: His.Bld.50.AllAg.Neutrophils [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: His.Bld.20.AllAg.Neutrophils [Chip-atlas[Archive

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File list: His.Bld.05.AllAg.Neutrophils [Chip-atlas[Archive

Lifescience Database Archive (English)

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RhoA determines disease progression by controlling neutrophil motility and restricting hyperresponsiveness

DEFF Research Database (Denmark)

Jennings, Richard T; Strengert, Monika; Hayes, Patti

2014-01-01

Neutrophil responses are central to host protection and inflammation. Neutrophil activation follows a two-step process where priming amplifies responses to activating stimuli. Priming is essential for life span extension, chemotaxis and respiratory burst activity. Here we show that the cytoskeletal...... organizer RhoA suppresses neutrophil priming via formins. Premature granule exocytosis in Rho-deficient neutrophils activated numerous signaling pathways and amplified superoxide generation. Deletion of Rho altered front-to-back coordination by simultaneously increasing uropod elongation, leading edge...... neutrophils exacerbated LPS-mediated lung injury, deleting Rho in innate immune cells was highly protective in Influenza A virus infection. Hence, Rho is a key regulator of disease progression by maintaining neutrophil quiescence and suppressing hyperresponsiveness....

Activated signature of antiphospholipid syndrome neutrophils reveals potential therapeutic target

Science.gov (United States)

Knight, Jason S.; Meng, He; Coit, Patrick; Yalavarthi, Srilakshmi; Sule, Gautam; Gandhi, Alex A.; Grenn, Robert C.; Mazza, Levi F.; Ali, Ramadan A.; Renauer, Paul; Wren, Jonathan D.; Bockenstedt, Paula L.; Wang, Hui; Eitzman, Daniel T.; Sawalha, Amr H.

2017-01-01

Antiphospholipid antibodies, present in one-third of lupus patients, increase the risk of thrombosis. We recently reported a key role for neutrophils — neutrophil extracellular traps (NETs), in particular — in the thrombotic events that define antiphospholipid syndrome (APS). To further elucidate the role of neutrophils in APS, we performed a comprehensive transcriptome analysis of neutrophils isolated from patients with primary APS. Moreover, APS-associated venous thrombosis was modeled by treating mice with IgG prepared from APS patients, followed by partial restriction of blood flow through the inferior vena cava. In patients, APS neutrophils demonstrated a proinflammatory signature with overexpression of genes relevant to IFN signaling, cellular defense, and intercellular adhesion. For in vivo studies, we focused on P-selectin glycoprotein ligand-1 (PSGL-1), a key adhesion molecule overexpressed in APS neutrophils. The introduction of APS IgG (as compared with control IgG) markedly potentiated thrombosis in WT mice, but not PSGL-1–KOs. PSGL-1 deficiency was also associated with reduced leukocyte vessel wall adhesion and NET formation. The thrombosis phenotype was restored in PSGL-1–deficient mice by infusion of WT neutrophils, while an anti–PSGL-1 monoclonal antibody inhibited APS IgG–mediated thrombosis in WT mice. PSGL-1 represents a potential therapeutic target in APS. PMID:28931754

File list: ALL.Bld.20.AllAg.Neutrophils [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: His.Bld.05.AllAg.Neutrophils [Chip-atlas[Archive

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File list: ALL.Bld.50.AllAg.Neutrophils [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: His.Bld.10.AllAg.Neutrophils [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: His.Bld.50.AllAg.Neutrophils [Chip-atlas[Archive

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Evidence for a self-enforcing inflammation in neutrophil-mediated chronic diseases

NARCIS (Netherlands)

Overbeek, S.A.

2011-01-01

In summary, this thesis provides evidence for the self-sustaining role of neutrophils in the inflammatory state in the pathogenesis of COPD and CD. In active disease, neutrophils release proteolytic enzymes that breakdown collagen. One of the collagen fragments can be neutrophilic chemoattractant

File list: His.Bld.20.AllAg.Neutrophils [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available His.Bld.20.AllAg.Neutrophils hg19 Histone Blood Neutrophils SRX956551,SRX956548,SRX...956545,SRX956544,SRX956547,SRX956550 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Bld.20.AllAg.Neutrophils.bed ...

Effects of ghrelin on the apoptosis of human neutrophils in vitro

Science.gov (United States)

Li, Bin; Zeng, Mian; Zheng, Haichong; Huang, Chunrong; He, Wanmei; Lu, Guifang; Li, Xia; Chen, Yanzhu; Xie, Ruijie

2016-01-01

Acute respiratory distress syndrome (ARDS) is characterized by lung inflammation and the diffuse infiltration of neutrophils into the alveolar space. Neutrophils are abundant, short-lived leukocytes that play a key role in immune defense against microbial infections. These cells die via apoptosis following the activation and uptake of microbes, and will also enter apoptosis spontaneously at the end of their lifespan if they do not encounter pathogens. Apoptosis is essential for the removal of neutrophils from inflamed tissues and for the timely resolution of neutrophilic inflammation. Ghrelin is an endogenous ligand for the growth hormone (GH) secretagogue receptor, produced and secreted mainly from the stomach. Previous studies have reported that ghrelin exerts anti-inflammatory effects in lung injury through the regulation of the apoptosis of different cell types; however, the ability of ghrelin to regulate alveolar neutrophil apoptosis remains largely undefined. We hypothesized that ghrelin may have the ability to modulate neutrophil apoptosis. In this study, to examine this hypothesis, we investigated the effects of ghrelin on freshly isolated neutrophils in vitro. Our findings demonstrated a decrease in the apoptotic ratio (as shown by flow cytometry), as well as in the percentage of cells with decreased mitochondrial membrane potential (ΔΨm) and in the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling-positive rate, accompanied by an increased B-cell lymphoma 2/Bax ratio and the downregulation of cleaved caspase-3 in neutrophils following exposure to lipopolysaccharide (100 ng/ml). However, pre-treatment with ghrelin at a physiological level (100 nM) did not have a notable influence on the neutrophils in all the aforementioned tests. Our findings suggest that ghrelin may not possess the ability to modulate the neutrophil lifespan in vitro. PMID:27431014

Matrix metalloproteinase-8 overexpression prevents proper tissue repair

DEFF Research Database (Denmark)

Danielsen, Patricia L; Holst, Anders V; Maltesen, Henrik R

2011-01-01

The collagenolytic matrix metalloproteinase-8 (MMP-8) is essential for normal tissue repair but is often overexpressed in wounds with disrupted healing. Our aim was to study the impact of a local excess of this neutrophil-derived proteinase on wound healing using recombinant adenovirus...

The digestion of phagocytosed collagen is inhibited by the proteinase inhibitors leupeptin and E-64

NARCIS (Netherlands)

Everts, V.; Beertsen, W.; Tigchelaar-Gutter, W.

1985-01-01

Using morphometric methods the effects of the thiol-proteinase inhibitors leupeptin and E-64 on the digestion of intracytoplasmic collagen fibrils were studied in cultured mouse bone explants. Both drugs caused a dose-dependent increase of lysosomal structures containing cross-banded collagen

A Neutrophil Phenotype Model for Extracorporeal Treatment of Sepsis.

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Alexander D Malkin

2015-10-01

Full Text Available Neutrophils play a central role in eliminating bacterial pathogens, but may also contribute to end-organ damage in sepsis. Interleukin-8 (IL-8, a key modulator of neutrophil function, signals through neutrophil specific surface receptors CXCR-1 and CXCR-2. In this study a mechanistic computational model was used to evaluate and deploy an extracorporeal sepsis treatment which modulates CXCR-1/2 levels. First, a simplified mechanistic computational model of IL-8 mediated activation of CXCR-1/2 receptors was developed, containing 16 ODEs and 43 parameters. Receptor level dynamics and systemic parameters were coupled with multiple neutrophil phenotypes to generate dynamic populations of activated neutrophils which reduce pathogen load, and/or primed neutrophils which cause adverse tissue damage when misdirected. The mathematical model was calibrated using experimental data from baboons administered a two-hour infusion of E coli and followed for a maximum of 28 days. Ensembles of parameters were generated using a Bayesian parallel tempering approach to produce model fits that could recreate experimental outcomes. Stepwise logistic regression identified seven model parameters as key determinants of mortality. Sensitivity analysis showed that parameters controlling the level of killer cell neutrophils affected the overall systemic damage of individuals. To evaluate rescue strategies and provide probabilistic predictions of their impact on mortality, time of onset, duration, and capture efficacy of an extracorporeal device that modulated neutrophil phenotype were explored. Our findings suggest that interventions aiming to modulate phenotypic composition are time sensitive. When introduced between 3-6 hours of infection for a 72 hour duration, the survivor population increased from 31% to 40-80%. Treatment efficacy quickly diminishes if not introduced within 15 hours of infection. Significant harm is possible with treatment durations ranging from 5

Reprogramming One-Carbon Metabolic Pathways To Decouple l-Serine Catabolism from Cell Growth in Corynebacterium glutamicum.

Science.gov (United States)

Zhang, Yun; Shang, Xiuling; Lai, Shujuan; Zhang, Yu; Hu, Qitiao; Chai, Xin; Wang, Bo; Liu, Shuwen; Wen, Tingyi

2018-02-16

l-Serine, the principal one-carbon source for DNA biosynthesis, is difficult for microorganisms to accumulate due to the coupling of l-serine catabolism and microbial growth. Here, we reprogrammed the one-carbon unit metabolic pathways in Corynebacterium glutamicum to decouple l-serine catabolism from cell growth. In silico model-based simulation showed a negative influence on glyA-encoding serine hydroxymethyltransferase flux with l-serine productivity. Attenuation of glyA transcription resulted in increased l-serine accumulation, and a decrease in purine pools, poor growth and longer cell shapes. The gcvTHP-encoded glycine cleavage (Gcv) system from Escherichia coli was introduced into C. glutamicum, allowing glycine-derived 13 CH 2 to be assimilated into intracellular purine synthesis, which resulted in an increased amount of one-carbon units. Gcv introduction not only restored cell viability and morphology but also increased l-serine accumulation. Moreover, comparative proteomic analysis indicated that abundance changes of the enzymes involved in one-carbon unit cycles might be responsible for maintaining one-carbon unit homeostasis. Reprogramming of the one-carbon metabolic pathways allowed cells to reach a comparable growth rate to accumulate 13.21 g/L l-serine by fed-batch fermentation in minimal medium. This novel strategy provides new insights into the regulation of cellular properties and essential metabolite accumulation by introducing an extrinsic pathway.

Integration of ARTP mutagenesis with biosensor-mediated high-throughput screening to improve L-serine yield in Corynebacterium glutamicum.

Science.gov (United States)

Zhang, Xin; Zhang, Xiaomei; Xu, Guoqiang; Zhang, Xiaojuan; Shi, Jinsong; Xu, Zhenghong

2018-05-03

L-Serine is widely used in the pharmaceutical, food, and cosmetics industries. Although direct fermentative production of L-serine from sugar in Corynebacterium glutamicum has been achieved, the L-serine yield remains relatively low. In this study, atmospheric and room temperature plasma (ARTP) mutagenesis was used to improve the L-serine yield based on engineered C. glutamicum ΔSSAAI strain. Subsequently, we developed a novel high-throughput screening method using a biosensor constructed based on NCgl0581, a transcriptional factor specifically responsive to L-serine, so that L-serine concentration within single cell of C. glutamicum can be monitored via fluorescence-activated cell sorting (FACS). Novel L-serine-producing mutants were isolated from a large library of mutagenized cells. The mutant strain A36-pDser was screened from 1.2 × 10 5 cells, and the magnesium ion concentration in the medium was optimized specifically for this mutant. C. glutamicum A36-pDser accumulated 34.78 g/L L-serine with a yield of 0.35 g/g sucrose, which were 35.9 and 66.7% higher than those of the parent C. glutamicum ΔSSAAI-pDser strain, respectively. The L-serine yield achieved in this mutant was the highest of all reported L-serine-producing strains of C. glutamicum. Moreover, the whole-genome sequencing identified 11 non-synonymous mutations of genes associated with metabolic and transport pathways, which might be responsible for the higher L-serine production and better cell growth in C. glutamicum A36-pDser. This study explored an effective mutagenesis strategy and reported a novel high-throughput screening method for the development of L-serine-producing strains.

A novel nonsense mutation in cathepsin C gene in an Egyptian ...

African Journals Online (AJOL)

Background: Cathepsin C gene (CTSC) (MIM#602365) is a lysosomal cysteine proteinase coding gene which encodes for CTSC protein that plays a major role in the activation of granule serine proteases, particularly leukocyte elastase and granzymes A and B. This activity was proposed to play a role in epithelial ...

Regulation of calcium homeostasis in activated human neutrophils ...

African Journals Online (AJOL)

Objectives. The objectives of the current study were to: (i) present an integrated model for the restoration of calcium homeostasis in activated human neutrophils based on current knowledge and recent research; and (ii) identify potential targets for the modulation of calcium fluxes in activated neutrophils based on this model ...

Fasciola gigantica cathepsin L proteinase-based synthetic peptide for immunodiagnosis and prevention of sheep fasciolosis

Czech Academy of Sciences Publication Activity Database

Ježek, Jan; El Ridi, R.; Salah, M.; Wagih, A.; Aziz, H. W.; Tallima, H.; El Shafie, M. H.; Khalek, T. A.; Ammou, F. F. A.; Strongylis, C.; Moussis, V.; Tsikaris, V.

2008-01-01

Roč. 90, č. 3 (2008), s. 349-357 ISSN 0006-3525 Institutional research plan: CEZ:AV0Z40550506 Keywords : cathepsin L proteinase * peptides * sequential oligopeptide carriers * synthetic peptide vaccine * Fasciiola gigantica Subject RIV: CC - Organic Chemistry Impact factor: 2.823, year: 2008

Obesity is associated with more activated neutrophils in African American male youth.

Science.gov (United States)

Xu, X; Su, S; Wang, X; Barnes, V; De Miguel, C; Ownby, D; Pollock, J; Snieder, H; Chen, W; Wang, X

2015-01-01

There is emerging evidence suggesting the role of peripheral blood leukocytes in the pathogenesis of obesity and related diseases. However, few studies have taken a genome-wide approach to investigating gene expression profiles in peripheral leukocytes between obese and lean individuals with the consideration of obesity-related shifts in leukocyte types. We conducted this study in 95 African Americans (AAs) of both genders (age 14-20 years, 46 lean and 49 obese). Complete blood count with differential test (CBC) was performed in whole blood. Genome-wide gene expression analysis was obtained using the Illumina HumanHT-12 V4 Beadchip with RNA extracted from peripheral leukocytes. Out of the 95 participants, 64 had neutrophils stored. The validation study was based on real-time PCR with RNA extracted from purified neutrophils. CBC test suggested that, in males, obesity was associated with increased neutrophil percentage (P=0.03). Genome-wide gene expression analysis showed that, in males, the majority of the most differentially expressed genes were related to neutrophil activation. Validation of the gene expression levels of ELANE (neutrophil elastase) and MPO (myeloperoxidase) in purified neutrophils demonstrated that the expression of these two genes--important biomarkers of neutrophils activation--were significantly elevated in obese males (P=0.01 and P=0.02, respectively). The identification of increased neutrophil percentage and activation in obese AA males suggests that neutrophils have an essential role in the pathogenesis of obesity-related disease. Further functional and mechanistic studies on neutrophils may contribute to the development of novel intervention strategies reducing the burden associated with obesity-related health problems.

Comparison of self-processing of foot-and-mouth disease virus leader proteinase and porcine reproductive and respiratory syndrome virus leader proteinase nsp1α

Energy Technology Data Exchange (ETDEWEB)

Steinberger, Jutta [Max F. Perutz Laboratories, Medical University of Vienna, Department of Medical Biochemistry, Dr. Bohr-Gasse 9/3, A-1030 Vienna (Austria); Kontaxis, Georg [Max F. Perutz Laboratories, University of Vienna, Department of Structural and Computational Biology, Campus Vienna Biocenter 5, A-1030 Vienna (Austria); Rancan, Chiara [Helmholtz Zentrum München, Department of Gene Vectors, Haematologikum, Marchioninistrasse 25, D-81377 Munich (Germany); Skern, Tim, E-mail: timothy.skern@meduniwien.ac.at [Max F. Perutz Laboratories, Medical University of Vienna, Department of Medical Biochemistry, Dr. Bohr-Gasse 9/3, A-1030 Vienna (Austria)

2013-09-01

The foot-and-mouth disease virus leader proteinase (Lb{sup pro}) cleaves itself off the nascent viral polyprotein. NMR studies on the monomeric variant Lb{sup pro} L200F provide structural evidence for intramolecular self-processing. {sup 15}N-HSQC measurements of Lb{sup pro} L200F showed specifically shifted backbone signals in the active and substrate binding sites compared to the monomeric variant sLb{sup pro}, lacking six C-terminal residues. This indicates transient intramolecular interactions between the C-terminal extension (CTE) of one molecule and its own active site. Contrastingly, the porcine reproductive and respiratory syndrome virus (PRRSV) leader proteinase nsp1α, with a papain-like fold like Lb{sup pro}, stably binds its own CTE. Parts of the β-sheet domains but none of the α-helical domains of Lb{sup pro} and nsp1α superimpose; consequently, the α-helical domain of nsp1α is oriented differently relative to its β-sheet domain. This provides a large interaction surface for the CTE with the globular domain, stabilising the intramolecular complex. Consequently, self-processing inactivates nsp1α but not Lb{sup pro}. - Highlights: • We examine self-processing of the leader protease of foot-and-mouth disease virus. • NMR analysis strongly supports intramolecular self-processing. • Self-processing is a dynamic process with no stable complex. • Structural comparison with nsp1α of PRRSV which forms stable intramolecular complex. • Subdomain orientation explains differences in stability of intramolecular complexes.

File list: ALL.Bld.10.AllAg.Neutrophils [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available ALL.Bld.10.AllAg.Neutrophils hg19 All antigens Blood Neutrophils SRX956551,SRX95654...8,SRX956545,SRX956544,SRX956550,SRX956552,SRX956549,SRX956546,SRX956547 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/ALL.Bld.10.AllAg.Neutrophils.bed ...

Functional Properties of a Cysteine Proteinase from Pineapple Fruit with Improved Resistance to Fungal Pathogens in Arabidopsis thaliana

Directory of Open Access Journals (Sweden)

Wei Wang

2014-02-01

Full Text Available In plant cells, many cysteine proteinases (CPs are synthesized as precursors in the endoplasmic reticulum, and then are subject to post-translational modifications to form the active mature proteinases. They participate in various cellular and physiological functions. Here, AcCP2, a CP from pineapple fruit (Ananas comosus L. belonging to the C1A subfamily is analyzed based on the molecular modeling and homology alignment. Transcripts of AcCP2 can be detected in the different parts of fruits (particularly outer sarcocarps, and gradually increased during fruit development until maturity. To analyze the substrate specificity of AcCP2, the recombinant protein was overexpressed and purified from Pichia pastoris. The precursor of purified AcCP2 can be processed to a 25 kDa active form after acid treatment (pH 4.3. Its optimum proteolytic activity to Bz-Phe-Val-Arg-NH-Mec is at neutral pH. In addition, the overexpression of AcCP2 gene in Arabidopsis thaliana can improve the resistance to fungal pathogen of Botrytis cinerea. These data indicate that AcCP2 is a multifunctional proteinase, and its expression could cause fruit developmental characteristics of pineapple and resistance responses in transgenic Arabidopsis plants.

Disentangling the effects of tocilizumab on neutrophil survival and function.

Science.gov (United States)

Gaber, Timo; Hahne, Martin; Strehl, Cindy; Hoff, Paula; Dörffel, Yvonne; Feist, Eugen; Burmester, Gerd-Rüdiger; Buttgereit, Frank

2016-06-01

The synovial tissue in rheumatoid arthritis (RA) represents a hypoxic environment with up-regulated pro-inflammatory cytokines and cellular infiltrates including neutrophils. Although inhibition of the interleukin (IL)6 receptor pathway by tocilizumab is a potent treatment option for RA, it may also cause adverse effects such as an occasionally high-grade neutropenia. We analysed the impact of tocilizumab on survival, mediator secretion, oxidative burst, phagocytosis and energy availability of high-dose toll-like receptor (TLR)2/4-stimulated neutrophils (to mimic an arthritis flare) under normoxic versus hypoxic conditions. Human neutrophils were purified, pre-treated with varying doses of tocilizumab, dexamethasone or human IgG1 and high-dose-stimulated with lipopolysaccharide (LPS) alone-triggering TLR2/4-, LPS plus IL6, or left unstimulated. Cells were then incubated under normoxic (18 % O2) or hypoxic (1 % O2) conditions and subsequently analysed. Neutrophil survival and energy availability were significantly decreased by tocilizumab in a dose-dependent manner in high-dose TLR2/4-stimulated cells, but to a greater extent under normoxia as compared to hypoxia. We also found high-dose LPS-stimulated oxidative burst and phagocytosis of neutrophils to be higher under hypoxic versus normoxic conditions, but this difference was reduced by tocilizumab. Finally, we observed that tocilizumab affected neutrophil mediator secretion as a function of oxygen availability. Tocilizumab is known for both beneficial effects and a higher incidence of neutropenia when treating RA patients. Our results suggest that both effects can at least in part be explained by a reduction in neutrophil survival, a dose-dependent inhibition of hypoxia-induced NADPH oxidase-mediated oxidative burst and phagocytosis of infiltrating hypoxic neutrophils and an alteration of mediator secretion.

Yersinia pestis targets neutrophils via complement receptor 3

Science.gov (United States)

Merritt, Peter M.; Nero, Thomas; Bohman, Lesley; Felek, Suleyman; Krukonis, Eric S.; Marketon, Melanie M.

2015-01-01

Yersinia species display a tropism for lymphoid tissues during infection, and the bacteria select innate immune cells for delivery of cytotoxic effectors by the type III secretion system. Yet the mechanism for target cell selection remains a mystery. Here we investigate the interaction of Yersinia pestis with murine splenocytes to identify factors that participate in the targeting process. We find that interactions with primary immune cells rely on multiple factors. First, the bacterial adhesin Ail is required for efficient targeting of neutrophils in vivo. However, Ail does not appear to directly mediate binding to a specific cell type. Instead, we find that host serum factors direct Y. pestis to specific innate immune cells, particularly neutrophils. Importantly, specificity towards neutrophils was increased in the absence of bacterial adhesins due to reduced targeting of other cell types, but this phenotype was only visible in the presence of mouse serum. Addition of antibodies against complement receptor 3 and CD14 blocked target cell selection, suggesting that a combination of host factors participate in steering bacteria toward neutrophils during plague infection. PMID:25359083

Failure of rabbit neutrophils to secrete endogenous pyrogen when stimulated with staphylococci.

Science.gov (United States)

Hanson, D F; Murphy, P A; Windle, B E

1980-06-01

Cells obtained from acute peritoneal exudates in rabbits were separated into neutrophil and mononuclear populations by centrifugation on colloidal silica gradients. When these populations were separately incubated in tissue culture medium in the presence of opsonized Staphylococcus epidermidis, endogenous pyrogen was secreted only by the adherent cells of the mononuclear population. Pyrogen production by neutrophils could not have amounted to as much as 1% of the pyrogen produced by macrophages. When mononuclear cells were added back to purified neutrophils, no pyrogen was produced that could not be accounted for by the number of macrophages added. Rabbit blood cells were similarly fractionated on colloidal silica gradients. Again, endogenous pyrogen was made only by the adherent mononuclear population. The neutrophils isolated on these gradients appeared to be morphologically normal and were 85% viable as judged by dye exclusion. They showed normal random motility. Both blood and exudate neutrophils responded chemotactically to N-formyl Met-Leu-Phe, and blood neutrophils responded chemotactically to zymosan-activated serum. Both kinds of neutrophils phagocytosed zymosan particles and both killed opsonized S. epidermidis in a roller tube system. Both blood and exudate neutrophils showed normal superoxide production when stimulated with opsonized zymosan particles. This evidence suggests that macrophages are the only source of endogenous pyrogens, and that pyrogens secreted by cell populations that are rich in neutrophils are to be attributed to the monocytes or macrophages that they contain.

Heterogeneity of neutrophil antibodies in patients with primary Sjögren's syndrome.

Science.gov (United States)

Lamour, A; Le Corre, R; Pennec, Y L; Cartron, J; Youinou, P

1995-11-01

Our aims were to determine the prevalence of neutrophil antibodies in patients with primary Sjögren's syndrome (pSS), identify their target antigen(s), and evaluate their functional significance. Neutrophil antibodies were detected using an indirect immunofluorescence (IIIF) test and an enzyme-linked immunosorbent assay (ELISA), using recombinant human Fc-gamma receptor (Fc gamma RIIIb) as a capture agent. Luminol-dependent chemiluminescence was then measured by an established technique. Antibodies to neutrophils were detected in 30 of 66 patients (45%) and categorized on the basis of positivity for the two assays: IIF+/ELISA+ (group A: five patients), IIF+/ELISA- (group B: five patients), and IFF-/ELISA+ (group C: 20 patients). All positive sera contained antibodies directed to the neutrophil specific Fc gamma RIIIb, and none of them bound to NAnull neutrophils. The titer of neutrophil-reactive antibodies (groups A and B) showed no correlation with the neutrophil count, but these autoantibodies did reduce the cell ability to generate a respiratory burst. Thus, neutrophil antibodies are common in patients with pSS. Their main target appears to be Fc gamma RIII, and this may partly account for the dysfunction in Fc gamma R-mediated clearance by the reticuloendothelial system reported in these patients.

LOCALIZATION OF POLYSOME-BOUND ALBUMIN AND SERINE DEHYDRATASE IN RAT LIVER CELL FRACTIONS

Science.gov (United States)

Ikehara, Yukio; Pitot, Henry C.

1973-01-01

The polysomes involved in albumin and serine dehydratase synthesis were identified and localized by the binding to rat liver polysomes of anti-rat serum albumin and anti-serine dehydratase [125I]Fab dimer and monomer. Techniques were developed for the isolation of undegraded free and membrane-bound polysomes and for the preparation of [125I]Fab monomers and dimers from the IgG obtained from the antisera to the two proteins, rat serum albumin and serine dehydratase. The distribution of anti-rat serum albumin [125I]Fab dimer in the polysome profile is in accordance with the size of polysomes that are expected to be synthesizing albumin. By direct precipitation, it has been demonstrated that nascent chains isolated from the membrane-bound polysomes by puromycin were precipitated by anti-rat serum albumin-IgG at a level of 5–6 times those released from free polysomes. Anti-rat serum albumin-[125I]Fab dimer reacted with membrane-bound polysomes almost exclusively compared to the binding of nonimmune, control [125I]Fab dimer; a significant degree of binding of anti-rat serum albumin-[125I]Fab to free polysomes was also obtained. The [125I]Fab dimer made from normal control rabbit serum does not react with polysomes from liver at all and this preparation will not interact with polysomes extracted from tissues that do not synthesize rat serum albumin. Both anti-serine dehydratase-[125I]Fab monomer and dimer react with free and bound polysomes from livers of animals fed a chow diet or those fed a high 90% protein diet and given glucagon. In the latter instance, however, it is clear that the majority of the binding occurs to the bound polysomes. Furthermore, the specificity of this reaction may be further shown by the use of kidney polysomes that do not normally synthesize serine dehydratase. When these latter polysomes are isolated, even after the addition of crude and purified serine dehydratase, no reaction with anti-serine dehydratase-Fab fragments could be

Neutrophil CD64 has a high negative predictive value for exclusion ...

African Journals Online (AJOL)

[13] Multiple studies agree that neutrophil CD64 has high diagnostic speci city and sensitivity.[13,18-20] In the light of the above information, we prospectively evaluated the usefulness of neutrophil CD64 expression in diagnosing neonatal infection. e main objective of the study was quantitation of neutrophil. CD64 by ow ...

Neutrophil targeted nano-drug delivery system for chronic obstructive lung diseases.

Science.gov (United States)

Vij, Neeraj; Min, Taehong; Bodas, Manish; Gorde, Aakruti; Roy, Indrajit

2016-11-01

The success of drug delivery to target airway cell(s) remains a significant challenge due to the limited ability of nanoparticle (NP) systems to circumvent protective airway-defense mechanisms. The size, density, surface and physical-chemical properties of nanoparticles are the key features that determine their ability to navigate across the airway-barrier. We evaluated here the efficacy of a PEGylated immuno-conjugated PLGA-nanoparticle (PINP) to overcome this challenge and selectively deliver drug to specific inflammatory cells (neutrophils). We first characterized the size, shape, surface-properties and neutrophil targeting using dynamic laser scattering, transmission electron microscopy and flow cytometry. Next, we assessed the efficacy of neutrophil-targeted PINPs in transporting through the airway followed by specific binding and release of drug to neutrophils. Finally, our results demonstrate the efficacy of PINP mediated non-steroidal anti-inflammatory drug-(ibuprofen) delivery to neutrophils in murine models of obstructive lung diseases, based on its ability to control neutrophilic-inflammation and resulting lung disease. Copyright © 2016 Elsevier Inc. All rights reserved.

The role of phagocytosis, oxidative burst and neutrophil extracellular traps in the interaction between neutrophils and the periodontal pathogen Porphyromonas gingivalis.

Science.gov (United States)

Jayaprakash, K; Demirel, I; Khalaf, H; Bengtsson, T

2015-10-01

Neutrophils are regarded as the sentinel cells of innate immunity and are found in abundance within the gingival crevice. Discovery of neutrophil extracellular traps (NETs) within the gingival pockets prompted us to probe the nature of the interactions of neutrophils with the prominent periopathogen Porphyromonas gingivalis. Some of the noted virulence factors of this Gram-negative anaerobe are gingipains: arginine gingipains (RgpA/B) and lysine gingipain (Kgp). The aim of this study was to evaluate the role of gingipains in phagocytosis, formation of reactive oxygen species, NETs and CXCL8 modulation by using wild-type strains and isogenic gingipain mutants. Confocal imaging showed that gingipain mutants K1A (Kgp) and E8 (RgpA/B) induced extracellular traps in neutrophils, whereas ATCC33277 and W50 were phagocytosed. The viability of both ATCC33277 and W50 dwindled as the result of phagocytosis and could be salvaged by cytochalasin D, and the bacteria released high levels of lipopolysaccharide in the culture supernatant. Porphyromonas gingivalis induced reactive oxygen species and CXCL8 with the most prominent effect being that of the wild-type strain ATCC33277, whereas the other wild-type strain W50 was less effective. Quantitative real-time polymerase chain reaction revealed a significant CXCL8 expression by E8. All the tested P. gingivalis strains increased cytosolic free calcium. In conclusion, phagocytosis is the primary neutrophil response to P. gingivalis, although NETs could play an accessory role in infection control. Although gingipains do not seem to directly regulate phagocytosis, NETs or oxidative burst in neutrophils, their proteolytic properties could modulate the subsequent outcomes such as nutrition acquisition and survival by the bacteria. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Diverse novel functions of neutrophils in immunity, inflammation, and beyond

OpenAIRE

Mocsai, A.

2013-01-01

Neutrophils have long been considered simple suicide killers at the bottom of the hierarchy of the immune response. That view began to change 10–20 yr ago, when the sophisticated mechanisms behind how neutrophils locate and eliminate pathogens and regulate immunity and inflammation were discovered. The last few years witnessed a new wave of discoveries about additional novel and unexpected functions of these cells. Neutrophils have been proposed to participate in protection against intracellu...

High-affinity binding of two molecules of cysteine proteinases to low-molecular-weight kininogen.

Science.gov (United States)

Turk, B.; Stoka, V.; Björk, I.; Boudier, C.; Johansson, G.; Dolenc, I.; Colic, A.; Bieth, J. G.; Turk, V.

1995-01-01

Human low-molecular-weight kininogen (LK) was shown by fluorescence titration to bind two molecules of cathepsins L and S and papain with high affinity. By contrast, binding of a second molecule of cathepsin H was much weaker. The 2:1 binding stoichiometry was confirmed by titration monitored by loss of enzyme activity and by sedimentation velocity experiments. The kinetics of binding of cathepsins L and S and papain showed the two proteinase binding sites to have association rate constants kass,1 = 10.7-24.5 x 10(6) M-1 s-1 and kass,2 = 0.83-1.4 x 10(6) M-1 s-1. Comparison of these kinetic constants with previous data for intact LK and its separated domains indicate that the faster-binding site is also the tighter-binding site and is present on domain 3, whereas the slower-binding, lower-affinity site is on domain 2. These results also indicate that there is no appreciable steric hindrance for the binding of proteinases between the two binding sites or from the kininogen light chain. PMID:8528085

Antibacterial activity of silver nanoparticles synthesized from serine

Energy Technology Data Exchange (ETDEWEB)

Jayaprakash, N. [Catalysis and Nanomaterials Research Laboratory, Department of Chemistry, Loyola College, Chennai 600 034 (India); SRM Valliammai Engineering College, Department of Chemistry, Chennai 603 203 (India); Judith Vijaya, J., E-mail: jjvijayaloyola@yahoo.co.in [Catalysis and Nanomaterials Research Laboratory, Department of Chemistry, Loyola College, Chennai 600 034 (India); John Kennedy, L. [Materials Division, School of Advanced Sciences, VIT University, Chennai Campus, Chennai 600 048 (India); Priadharsini, K.; Palani, P. [Department of Center for Advanced Study in Botany, University of Madras, Guindy Campus, Chennai 600 025 (India)

2015-04-01

Silver nanoparticles (Ag NPs) were synthesized by a simple microwave irradiation method using polyvinyl pyrrolidone (PVP) as a capping agent and serine as a reducing agent. UV–Visible spectra were used to confirm the formation of Ag NPs by observing the surface plasmon resonance (SPR) band at 443 nm. The emission spectrum of Ag NPs showed an emission band at 484 nm. In the presence of microwave radiation, serine acts as a reducing agent, which was confirmed by Fourier transformed infrared (FT-IR) spectrum. High-resolution transmission electron microscopy (HR-TEM) and high-resolution scanning electron microscopy (HR-SEM) were used to investigate the morphology of the synthesized sample. These images showed the sphere-like morphology. The elemental composition of the sample was determined by the energy dispersive X-ray analysis (EDX). Selected area electron diffraction (SAED) was used to find the crystalline nature of the Ag NPs. The electrochemical behavior of the synthesized Ag NPs was analyzed by the cyclic voltammetry (CV). Antibacterial experiments showed that the prepared Ag NPs showed relatively similar antibacterial activities, when compared with AgNO{sub 3} against Gram-positive and Gram-negative bacteria. - Highlights: • Microwave irradiation method is used to synthesize silver nanoparticles. • Highly stable silver nanoparticles are produced from serine. • A detailed study of antibacterial activities is discussed. • Formation mechanism of silver microspheres has been proposed.

Random mutagenesis of human serine racemase reveals residues important for the enzymatic activity

Czech Academy of Sciences Publication Activity Database

Hoffman, Hillary Elizabeth; Jirásková, Jana; Zvelebil, M.; Konvalinka, Jan

2010-01-01

Roč. 75, č. 1 (2010), s. 59-79 ISSN 0010-0765 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : D-serine * serine racemase * random mutagenesis Subject RIV: CE - Biochemistry Impact factor: 0.853, year: 2010

Anti-proteinase 3 antibodies in diffuse systemic sclerosis (SSc with normotensive renal impairment: is it suggestive for an overlapping between SSc and idiopathic vasculitis?

Directory of Open Access Journals (Sweden)

V. Campanella

2011-09-01

Full Text Available Objective. To test the prevalence of anti-neutrophil cytoplasmic antibodies (ANCA in systemic sclerosis (SSc and to verify a possible association of ANCA with normotensive renal involvement in SSc. Patients and methods: 51 patients affected by SSc, 35 with diffuse scleroderma (dSSc and 16 with limited scleroderma (lSSc, were tested for ANCA by indirect immunofluorescence (IIF on human ethanol and formalin-acetone-fixed granulocytes (before and after DNase treatment, by conventional enzyme linked immuno-sorbent assay (ELISA and by capture-ELISA. Results. Six out of 51 selected SSc patients had ANCA by IIF (11.7% and five presented a perinuclear/nuclear atypical ANCA pattern. In all cases we only found anti-proteinase3 (aPR3 antibodies. All ANCA positive patients had diffuse form of SSc (17.1%, all were anti-Scl70 positive (aScl70, five patients had proteinuria, three had microscopic haematuria. All ANCA positive patients were normotensive with normal renin plasma levels, the mean erythrocyte sedimentation rate (ESR was higher in this group compared to the other SSc patients. Conclusions. Our study shows that aPR3 is not rare in dSSc. According to the clinical and serological findings and to the recent literature, we can hypothesise that when ANCA are found in SSc, an overlapping of scleroderma with systemic necrotizing vasculitis should be suspected.

Evasion of Human Neutrophil-Mediated Host Defense during Toxoplasma gondii Infection.

Science.gov (United States)

Lima, Tatiane S; Gov, Lanny; Lodoen, Melissa B

2018-02-13

Neutrophils are a major player in host immunity to infection; however, the mechanisms by which human neutrophils respond to the intracellular protozoan parasite Toxoplasma gondii are still poorly understood. In the current study, we found that, whereas primary human monocytes produced interleukin-1beta (IL-1β) in response to T. gondii infection, human neutrophils from the same blood donors did not. Moreover, T. gondii inhibited lipopolysaccharide (LPS)-induced IL-1β synthesis in human peripheral blood neutrophils. IL-1β suppression required active parasite invasion, since heat-killed or mycalolide B-treated parasites did not inhibit IL-1β release. By investigating the mechanisms involved in this process, we found that T. gondii infection of neutrophils treated with LPS resulted in reduced transcript levels of IL-1β and NLRP3 and reduced protein levels of pro-IL-1β, mature IL-1β, and the inflammasome sensor NLRP3. In T. gondii -infected neutrophils stimulated with LPS, the levels of MyD88, TRAF6, IKKα, IKKβ, and phosphorylated IKKα/β were not affected. However, LPS-induced IκBα degradation and p65 phosphorylation were reduced in T. gondii- infected neutrophils, and degradation of IκBα was reversed by treatment with the proteasome inhibitor MG-132. Finally, we observed that T. gondii inhibited the cleavage and activity of caspase-1 in human neutrophils. These results indicate that T. gondii suppression of IL-1β involves a two-pronged strategy whereby T. gondii inhibits both NF-κB signaling and activation of the NLRP3 inflammasome. These findings represent a novel mechanism of T. gondii evasion of human neutrophil-mediated host defense by targeting the production of IL-1β. IMPORTANCE Toxoplasma gondii is an obligate intracellular parasite that infects approximately one-third of humans worldwide and can invade virtually any nucleated cell in the human body. Although it is well documented that neutrophils infiltrate the site of acute T

Cationic ferritin uptake by cultured anterior pituitary cells treated with the proteinase inhibitor, BOC-DPhe-Phe-Lys-H.

Science.gov (United States)

Gaál, G; Bácsy, E; Rappay, G

1988-01-01

Cultured cells from the anterior pituitary glands of adult rats were treated with the tripeptide aldehyde proteinase inhibitor, BOC-DPhe-Phe-Lys-H. The addition of this tripeptide aldehyde decreased the in vitro release of prolactin to 25% of the control value, while the release of growth hormone in the same cultures decreased to 33% of the control value. Prolactin immunostaining was stronger in semithin sections of proteinase-inhibitor-treated cultures than in control sections. After 2 h treatment with the inhibitor, prolactin- and growth hormone-containing secretory granules were numerous, and the number of crinophagic vacuoles had increased. In the presence of the inhibitor, the overall cytoarchitecture of parenchymal cells was well preserved, and the pathway of the uptake of cationic ferritin appeared to be unaffected.

Induction of hyperresponsiveness in human airway tissue by neutrophils--mechanism of action.

Science.gov (United States)

Anticevich, S Z; Hughes, J M; Black, J L; Armour, C L

1996-05-01

The two main features of asthma are bronchial hyperresponsiveness and inflammation. The inflammatory response in asthma consists of infiltration and activation of a variety of inflammatory cells including neutrophils. Our previous studies have shown that stimulated neutrophil supernatants cause hyperresponsiveness of human bronchial tissue in vitro. To investigate the effect of the sensitization status of the tissue and the albumin concentration used to prepare supernatants on the response of human bronchial tissue to stimulated neutrophil supernatants. Neutrophil supernatants were prepared from human isolated blood in the presence of varying concentrations of albumin (0%, 0.1% and 4%). Neutrophil supernatants were added to sensitized and non-sensitized human isolated bronchial tissue which was stimulated with electrical field stimulation (EFS) (20 s every 4 min). Receptor antagonists specific for the prostaglandin and thromboxane (10(-7) M GR32191), platelet activating factor (10(-6) M WEB 2086), leukotriene D4 (10(-6) M MK-679) and neurokinin A (10(-7) M SR48968) receptors were used to identify neutrophil products responsible for the effects observed in the bronchial tissue. In non-sensitized human bronchial tissue, stimulated neutrophil supernatants induced a direct contraction in the presence of 0% and 0.1% but not 4% albumin. This contraction was due to leukotriene D4 as MK-679 completely inhibited the contraction. In contrast, stimulated neutrophil supernatants increased responsiveness of sensitized human bronchial tissue to EFS. The increased responsiveness was observed only in the presence of 0.1% albumin, with the site of modulation likely to be prejunctional on the parasympathetic nerve. The increased responsiveness was not inhibited by any of the antagonists tested. Sensitization status of the tissue and albumin concentration effect the responsiveness of human bronchial tissue to stimulated neutrophil supernatant. Our results suggest a possible role for

Intervention with Serine Protease Activity with Small Peptides

DEFF Research Database (Denmark)

Xu, Peng

2015-01-01

Serine proteases perform proteolytic reactions in many physiological and metabolic processes and have been certified as targets for therapeutics. Small peptides can be used as potent antagonists to target serine proteases and intervene with their activities. Urokinase-type plasminogen activator (u......PA) plays an important role in plasminogen activation system, which has many physiological and pathological functions and is closely associated with the metastasis of tumor cells. Based on a mono-cyclic peptidic inhibitor of murine uPA (muPA), mupain-1, which was screened out from a phage-display library...... before, we elucidated the binding and inhibitory mechanism by using multiple techniques, like X-ray crystallography, site-directed mutagenesis, isothermal titration calorimetry and surface plasmon resonance analysis. By studying the peptide-enzyme interaction, we discovered an unusual inhibitor...

Crystal structure and characterization of a novel L-serine ammonia-lyase from Rhizomucor miehei

Energy Technology Data Exchange (ETDEWEB)

Qin, Zhen [College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center of Food Nutrition and Human Health, China Agricultural University, Beijing 100083 (China); Yan, Qiaojuan [College of Engineering, China Agricultural University, Beijing 100083 (China); Ma, Qingjun [Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071 (China); Jiang, Zhengqiang, E-mail: zhqjiang@cau.edu.cn [College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center of Food Nutrition and Human Health, China Agricultural University, Beijing 100083 (China)

2015-10-23

L-serine ammonia-lyase, as a member of the β-family of pyridoxal-5′-phosphate (PLP) dependent enzymes, catalyzes the conversion of L-serine (L-threonine) to pyruvate (α-ketobutyrate) and ammonia. The crystal structure of L-serine ammonia-lyase from Rhizomucor miehei (RmSDH) was solved at 1.76 Å resolution by X-ray diffraction method. The overall structure of RmSDH had the characteristic β-family PLP dependent enzyme fold. It consisted of two distinct domains, both of which show the typical open twisted α/β structure. A PLP cofactor was located in the crevice between the two domains, which was attached to Lys52 by a Schiff-base linkage. Unique residue substitutions (Gly78, Pro79, Ser146, Ser147 and Thr312) were discovered at the catalytic site of RmSDH by comparison of structures of RmSDH and other reported eukaryotic L-serine ammonia-lyases. Optimal pH and temperature of the purified RmSDH were 7.5 and 40 °C, respectively. It was stable in the pH range of 7.0–9.0 and at temperatures below 40 °C. This is the first crystal structure of a fungal L-serine ammonia-lyase. It will be useful to study the catalytic mechanism of β-elimination enzymes and will provide a basis for further enzyme engineering. - Highlights: • The crystal structure of a fungal L-serine ammonia-lyase (RmSDH) was solved. • Five unique residue substitutions are found at the catalytic site of RmSDH. • RmSDH was expressed in Pichia. pastoris and biochemically characterized. • RmSDH has potential application in splitting D/L-serine.

Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions

NARCIS (Netherlands)

Glogauer, Michael; Marchal, Christophe C.; Zhu, Fei; Worku, Aelaf; Clausen, Björn E.; Foerster, Irmgard; Marks, Peter; Downey, Gregory P.; Dinauer, Mary; Kwiatkowski, David J.

2003-01-01

Defects in myeloid cell function in Rac2 knockout mice underline the importance of this isoform in activation of NADPH oxidase and cell motility. However, the specific role of Rac1 in neutrophil function has been difficult to assess since deletion of Rac1 results in embryonic lethality in mice. To

Serine esterase and hemolytic activity in human cloned cytotoxic T lymphocytes

OpenAIRE

1988-01-01

Target cell lysis by most murine cytotoxic T lymphocytes appears to be mediated by a complement (C9)-like protein called perforin, contained in high-density cytoplasmic granules. These granules also contain high levels of serine esterase activity, which may also play a role in cytolysis. Analysis of 17 cloned human cytotoxic T lymphocytes revealed the presence of serine esterase that is very similar to its murine counterpart in substrate and inhibitor specificities, pH optimum, and molecular ...

Neutrophil evasion strategies by Streptococcus pneumoniae and Staphylococcus aureus.

Science.gov (United States)

Lewis, Megan L; Surewaard, Bas G J

2018-03-01

Humans are well equipped to defend themselves against bacteria. The innate immune system employs diverse mechanisms to recognize, control and initiate a response that can destroy millions of different microbes. Microbes that evade the sophisticated innate immune system are able to escape detection and could become pathogens. The pathogens Streptococcus pneumoniae and Staphylococcus aureus are particularly successful due to the development of a wide variety of virulence strategies for bacterial pathogenesis and they invest significant efforts towards mechanisms that allow for neutrophil evasion. Neutrophils are a primary cellular defense and can rapidly kill invading microbes, which is an indispensable function for maintaining host health. This review compares the key features of Streptococcus pneumoniae and Staphylococcus aureus in epidemiology, with a specific focus on virulence mechanisms utilized to evade neutrophils in bacterial pathogenesis. It is important to understand the complex interactions between pathogenic bacteria and neutrophils so that we can disrupt the ability of pathogens to cause disease.

Pnserpin: A Novel Serine Protease Inhibitor from Extremophile Pyrobaculum neutrophilum

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Huan Zhang

2017-01-01

Full Text Available Serine protease inhibitors (serpins are native inhibitors of serine proteases, constituting a large protein family with members spread over eukaryotes and prokaryotes. However, only very few prokaryotic serpins, especially from extremophiles, have been characterized to date. In this study, Pnserpin, a putative serine protease inhibitor from the thermophile Pyrobaculum neutrophilum, was overexpressed in Escherichia coli for purification and characterization. It irreversibly inhibits chymotrypsin-, trypsin-, elastase-, and subtilisin-like proteases in a temperature range from 20 to 100 °C in a concentration-dependent manner. The stoichiometry of inhibition (SI of Pnserpin for proteases decreases as the temperature increases, indicating that the inhibitory activity of Pnserpin increases with the temperature. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that Pnserpin inhibits proteases by forming a SDS-resistant covalent complex. Homology modeling and molecular dynamic simulations predicted that Pnserpin can form a stable common serpin fold. Results of the present work will help in understanding the structural and functional characteristics of thermophilic serpin and will broaden the current knowledge about serpins from extremophiles.

Effects of acrolein on leukotriene biosynthesis in human neutrophils.

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Berry, Karin A Zemski; Henson, Peter M; Murphy, Robert C

2008-12-01

Acrolein is a toxic, highly reactive alpha,beta-unsaturated aldehyde that is present in high concentrations in cigarette smoke. In the current study, the effect of acrolein on eicosanoid synthesis in stimulated human neutrophils was examined. Eicosanoid synthesis in neutrophils was initiated by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) and subsequent stimulation with formyl-methionyl-leucyl-phenylalanine (fMLP) and 5-lipoxygenase (5-LO) products in addition to small amounts of cyclooxygenase (COX) products were detected using LC/MS/MS. A dose-dependent decrease in the formation of 5-LO products was observed in GM-CSF/fMLP-stimulated neutrophils when acrolein (0-50 microM) was present with almost complete inhibition at > or = 25 microM acrolein. The production of COX products was not affected by acrolein in these cells. The effect of acrolein was examined on key parts of the eicosanoid pathway, such as arachidonic acid release, intracellular calcium ion concentration, and adenosine production. In addition, the direct effect of acrolein on 5-LO enzymatic activity was probed using a recombinant enzyme. Some of these factors were affected by acrolein but did not completely explain the almost complete inhibition of 5-LO product formation in GM-CSF/fMLP-treated cells with acrolein. In addition, the effect of acrolein on different stimuli that initiate the 5-LO pathway [platelet-activating factor (PAF)/fMLP, GM-CSF/PAF, opsonized zymosan, and A23187] was examined. Acrolein had no significant effect on the leukotriene production in neutrophils stimulated with PAF/fMLP, GM-CSF/ PAF, or OPZ. Additionally, 50% inhibition of the 5-LO pathway was observed in A23187-stimulated neutrophils. Our results suggest that acrolein has a profound effect on the 5-LO pathway in neutrophils, which may have implications in disease states, such as chronic obstructive pulmonary disease and other pulmonary disease, where both activated neutrophils and acrolein are

Neutrophil-Derived MMP-8 Drives AMPK-Dependent Matrix Destruction in Human Pulmonary Tuberculosis

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Ong, Catherine W. M.; Elkington, Paul T.; Brilha, Sara; Ugarte-Gil, Cesar; Tome-Esteban, Maite T.; Tezera, Liku B.; Pabisiak, Przemyslaw J.; Moores, Rachel C.; Sathyamoorthy, Tarangini; Patel, Vimal; Gilman, Robert H.; Porter, Joanna C.; Friedland, Jon S.

2015-01-01

Pulmonary cavities, the hallmark of tuberculosis (TB), are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8) secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs) contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease. PMID:25996154

Neutrophil-Derived MMP-8 Drives AMPK-Dependent Matrix Destruction in Human Pulmonary Tuberculosis.

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Ong, Catherine W M; Elkington, Paul T; Brilha, Sara; Ugarte-Gil, Cesar; Tome-Esteban, Maite T; Tezera, Liku B; Pabisiak, Przemyslaw J; Moores, Rachel C; Sathyamoorthy, Tarangini; Patel, Vimal; Gilman, Robert H; Porter, Joanna C; Friedland, Jon S

2015-05-01

Pulmonary cavities, the hallmark of tuberculosis (TB), are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8) secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs) contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease.

Epigenetic Activation of ASCT2 in the Hippocampus Contributes to Depression-Like Behavior by Regulating D-Serine in Mice

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Jiesi Wang

2017-05-01

Full Text Available The roles of D-serine in depression are raised concerned recently as an intrinsic co-agonist for the NMDA receptor. However, the mechanisms underlying its regulation are not fully elucidated. ASCT2 is a Na+-dependent D-serine transporter. We found that decreased D-serine and increased hippocampal ASCT2 levels correlated with chronic social defeat stress (CSDS in mice. Lentivirus-mediated shRNA-mediated knockdown of ASCT2 and the administration of exogenous D-serine in the hippocampus alleviated CSDS-induced social avoidance and immobility. In vivo and in vitro experiments revealed that upregulation of ASCT2 expression in CSDS was regulated through histone hyper-acetylation, not DNA methylation in its promoter region. Immunohistochemistry demonstrated the co-localization of ASCT2 and D-serine. Uptake of D-serine by ASCT2 was demonstrated by in vivo and in vitro experiments. Our results indicate that CSDS induces ASCT2 expression through epigenetic activation and decreases hippocampal D-serine levels, leading to social avoidance, and immobility. Thus, targeting D-serine transport represents an attractive new strategy for treating depression.

Intracellular localization of Treponema denticola chymotrypsin-like proteinase in chronic periodontitis

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Emilia Marttila

2014-07-01

Full Text Available Treponema denticola is an important periodontal pathogen capable of tissue invasion. Its chymotrypsin-like proteinase (CTLP can degrade a number of basement membrane components in vitro, thus suggesting a contribution to tissue invasion by the spirochete. The aim of this study was to analyze the localization of CTLP in chronic periodontitis tissues ex vivo. A polyclonal antibody specific to T. denticola cell-bound CTLP was used to detect the spirochetes in the gingival tissues of patients with moderate to severe chronic periodontitis (n=25 by immunohistochemistry and periodic acid-Schiff staining (PAS. The presence of T. denticola in the periodontal tissue samples was analyzed by PCR. Periodontal tissue samples of 12 of the 25 patients were found to be positive for T. denticola by PCR. Moreover, CTLP could be detected in the periodontal tissues of all these patients by immunohistochemistry. In the epithelium, the CTLP was mostly intracellular. Typically, the positive staining could be seen throughout the whole depth of the epithelium. When detected extracellularly, CTLP was localized mainly as granular deposits. The connective tissue stained diffusely positive in four cases. The positive staining co-localized with the PAS stain in nine cases. T. denticola and its CTLP could be detected in diseased human periodontium both intra- and extracellularly. The granular staining pattern was suggestive of the presence of T. denticola bacteria, whereas the more diffused staining pattern was indicative of the recent presence of the bacterium and shedding of the cell-bound proteinase.

The emerging role of neutrophils in thrombosis – The journey of TF through NETs

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Konstantinos eKambas

2012-12-01

Full Text Available The production of TF by neutrophils and their contribution in thrombosis was until recently a matter of scientific debate. Experimental data suggested the de novo TF production by neutrophils under inflammatory stimuli, while others proposed that these cells acquired microparticle-derived TF. Recent experimental evidence revealed the critical role of neutrophils in thrombotic events. Neutrophil derived TF has been implicated in this process in several human and animal models. Additionally, neutrophil extracellular trap (NET release has emerged as a major contributor in neutrophil-driven thrombogenicity in disease models including sepsis, deep venous thrombosis and malignancy. It is suggested that NETs provide the scaffold for fibrin deposition and platelet entrapment and subsequent activation. The recently reported autophagy-dependent extracellular delivery of TF in NETs further supports the involvement of neutrophils in thrombosis. Herein, we seek to review novel data regarding the role of neutrophils in thrombosis, emphasizing the implication of TF and NETs.

A thermolabile aspartic proteinase from Mucor mucedo DSM 809: gene identification, cloning, and functional expression in Pichia pastoris.

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Yegin, Sirma; Fernandez-Lahore, Marcelo

2013-06-01

In this study, the cDNA encoding the aspartic proteinase of Mucor mucedo DSM 809 has been identified by RNA ligased-mediated and oligo-capping rapid amplification of cDNA ends (RACE) technique. The gene contained an open reading frame of 1,200 bp and encoded for a signal peptide of 21 amino acid residues. Two N-glycosylation sites were observed within the identified sequence. The proteinase gene was cloned into the vector pGAPZαA and expressed in Pichia pastoris X-33 for the first time. The protein has been secreted in functionally active form into the culture medium. The expression system does not require any acid activation process. The factors affecting the expression level were optimized in shaking flask cultures. Maximum enzyme production was observed with an initial medium pH of 3.5 at 20 °C and 220 rpm shaking speed utilizing 4 % glucose as a carbon and energy source. The enzyme was purified with cation exchange chromatography and further studies revealed that the enzyme was secreted in glycosylated form. The purified enzyme exhibited remarkable sensitivity to thermal treatment and became completely inactivated after incubation at 55 °C for 10 min. These results indicated that the recombinant proteinase could be considered as a potential rennet candidate for the cheese-making industry.

Iodinated contrast media induce neutrophil apoptosis through a mitochondrial and caspase mediated pathway.

LENUS (Irish Health Repository)

Fanning, N F

2012-02-03

Iodinated contrast media (ICM) can induce apoptosis (programmed cell death) in renal, myocardial and endothelial cells. Following intravascular injection, circulating immune cells are exposed to high concentrations of ICM. As neutrophils constitutively undergo apoptosis we hypothesized that ICM may adversely affect neutrophil survival. Our aim was to investigate the effect of ICM on neutrophil apoptosis. Neutrophils were isolated from healthy subjects and cultured in vitro with ionic (diatrizoate and ioxaglate) and non-ionic (iohexol and iotrolan) ICM. The effect of ICM on neutrophil apoptosis in both unstimulated and lipopolysaccharide-stimulated neutrophils was determined by annexin V flow cytometry. The influence of physicochemical properties of the different ICM on apoptosis of neutrophils was also studied. We further investigated the effects of ICM on key intracellular signal pathways, including p38 mitogen-activated protein kinase (MAPK) by Western blotting, and mitochondrial depolarization and caspase activity by flow cytometry. Isoiodine concentrations (20 mg ml(-1)) of ionic (diatrizoate 69.6+\\/-2.9%; ioxaglate 58.9+\\/-2.0%) and non-ionic (iohexol 57.3+\\/-2.9%; iotrolan 57.1+\\/-2.6%) ICM significantly induced neutrophil apoptosis over control levels (47.7+\\/-1.4%). The apoptotic effect of ICM was influenced by their chemical structure, with ionic ICM having a more significant (p<0.01) apoptotic effect than non-ionic ICM (p<0.05). Furthermore, ICM reversed the anti-apoptotic effect of lipopolysaccharide (1000 ng ml(-1)) treated neutrophils to control levels (23.0+\\/-3.5% to 61.2+\\/-5.3%; n=4; p<0.05). These agents induce apoptosis through a p38 MAPK independent pathway that results in mitochondrial depolarization, and is dependent on caspase activation. As neutrophils play a central role in host response to infection and injury, ICM, through induction of neutrophil apoptosis, could have a significant deleterious effect on host immune defence and

Serpins in arthropod biology.

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Meekins, David A; Kanost, Michael R; Michel, Kristin

2017-02-01

Serpins are the largest known family of serine proteinase inhibitors and perform a variety of physiological functions in arthropods. Herein, we review the field of serpins in arthropod biology, providing an overview of current knowledge and topics of interest. Serpins regulate insect innate immunity via inhibition of serine proteinase cascades that initiate immune responses such as melanization and antimicrobial peptide production. In addition, several serpins with anti-pathogen activity are expressed as acute-phase serpins in insects upon infection. Parasitoid wasps can downregulate host serpin expression to modulate the host immune system. In addition, examples of serpin activity in development and reproduction in Drosophila have also been discovered. Serpins also function in host-pathogen interactions beyond immunity as constituents of venom in parasitoid wasps and saliva of blood-feeding ticks and mosquitoes. These serpins have distinct effects on immunosuppression and anticoagulation and are of interest for vaccine development. Lastly, the known structures of arthropod serpins are discussed, which represent the serpin inhibitory mechanism and provide a detailed overview of the process. Copyright © 2016 Elsevier Ltd. All rights reserved.

Neutrophil activation during acetaminophen hepatotoxicity and repair in mice and humans

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Williams, C. David; Bajt, Mary Lynn [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Sharpe, Matthew R. [Department of Internal Medicine, University of Kansas Hospital, Kansas City, KS (United States); McGill, Mitchell R. [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Farhood, Anwar [Department of Pathology, St. David' s North Austin Medical Center, Austin, TX 78756 (United States); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States)

2014-03-01

Following acetaminophen (APAP) overdose there is an inflammatory response triggered by the release of cellular contents from necrotic hepatocytes into the systemic circulation which initiates the recruitment of neutrophils into the liver. It has been demonstrated that neutrophils do not contribute to APAP-induced liver injury, but their role and the role of NADPH oxidase in injury resolution are controversial. C57BL/6 mice were subjected to APAP overdose and neutrophil activation status was determined during liver injury and liver regeneration. Additionally, human APAP overdose patients (ALT: > 800 U/L) had serial blood draws during the injury and recovery phases for the determination of neutrophil activation. Neutrophils in the peripheral blood of mice showed an increasing activation status (CD11b expression and ROS priming) during and after the peak of injury but returned to baseline levels prior to complete injury resolution. Hepatic sequestered neutrophils showed an increased and sustained CD11b expression, but no ROS priming was observed. Confirming that NADPH oxidase is not critical to injury resolution, gp91{sup phox}−/− mice following APAP overdose displayed no alteration in injury resolution. Peripheral blood from APAP overdose patients also showed increased neutrophil activation status after the peak of liver injury and remained elevated until discharge from the hospital. In mice and humans, markers of activation, like ROS priming, were increased and sustained well after active liver injury had subsided. The similar findings between surviving patients and mice indicate that neutrophil activation may be a critical event for host defense or injury resolution following APAP overdose, but not a contributing factor to APAP-induced injury. - Highlights: • Neutrophil (PMN) function increases during liver repair after acetaminophen overdose. • Liver repair after acetaminophen (APAP)-overdose is not dependent on NADPH oxidase. • Human PMNs do not appear

FBXO22 Protein Is Required for Optimal Synthesis of the N-Methyl-d-Aspartate (NMDA) Receptor Coagonist d-Serine

DEFF Research Database (Denmark)

Dikopoltsev, Elena; Foltyn, Veronika N; Zehl, Martin

2014-01-01

d-Serine is a physiological activator of NMDA receptors (NMDARs) in the nervous system that mediates several NMDAR-mediated processes ranging from normal neurotransmission to neurodegeneration. d-Serine is synthesized from l-serine by serine racemase (SR), a brain-enriched enzyme. However, little......, SR interacts preferentially with free FBXO22 species. In vivo ubiquitination and SR half-life determination indicate that FBXO22 does not target SR to the proteasome system. FBXO22 primarily affects SR subcellular localization and seems to increase d-serine synthesis by preventing the association...... is known about the regulation of d-serine synthesis. We now demonstrate that the F-box only protein 22 (FBXO22) interacts with SR and is required for optimal d-serine synthesis in cells. Although FBXO22 is classically associated with the ubiquitin system and is recruited to the Skip1-Cul1-F-box E3 complex...

Protein kinase A phosphorylates serine 267 in the homeodomain of engrailed-2 leading to decreased DNA binding

DEFF Research Database (Denmark)

Hjerrild, Majbrit; Stensballe, Allan; Jensen, Ole N

2004-01-01

Engrailed-2 (En-2) belongs to an evolutionarily conserved family of DNA binding homeodomain-containing proteins that are expressed in mammalian brain during development. Here, we demonstrate that serine 267 in the homeodomain of En-2 is phosphorylated by protein kinase A (PKA) in forskolin......-treated COS-7 cells. Furthermore, we analyze the physiological function of En-2 phosphorylation by PKA. The nuclear localization of En-2 is not influenced by the phosphorylation of serine 267. However, substitution of serine 267 with alanine resulted in increased binding of En-2 to DNA, while replacing serine...

A novel bacterial transport mechanism of Acinetobacter baumannii via activated human neutrophils through interleukin-8.

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Kamoshida, Go; Tansho-Nagakawa, Shigeru; Kikuchi-Ueda, Takane; Nakano, Ryuichi; Hikosaka, Kenji; Nishida, Satoshi; Ubagai, Tsuneyuki; Higashi, Shouichi; Ono, Yasuo

2016-12-01

Hospital-acquired infections as a result of Acinetobacter baumannii have become problematic because of high rates of drug resistance. Although neutrophils play a critical role in early protection against bacterial infection, their interactions with A. baumannii remain largely unknown. To elucidate the interactions between A. baumannii and human neutrophils, we cocultured these cells and analyzed them by microscopy and flow cytometry. We found that A. baumannii adhered to neutrophils. We next examined neutrophil and A. baumannii infiltration into Matrigel basement membranes by an in vitro transmigration assay. Neutrophils were activated by A. baumannii, and invasion was enhanced. More interestingly, A. baumannii was transported together by infiltrating neutrophils. Furthermore, we observed by live cell imaging that A. baumannii and neutrophils moved together. In addition, A. baumannii-activated neutrophils showed increased IL-8 production. The transport of A. baumannii was suppressed by inhibiting neutrophil infiltration by blocking the effect of IL-8. A. baumannii appears to use neutrophils for transport by activating these cells via IL-8. In this study, we revealed a novel bacterial transport mechanism that A. baumannii exploits human neutrophils by adhering to and inducing IL-8 release for bacterial portage. This mechanism might be a new treatment target. © Society for Leukocyte Biology.

Cervico-vaginal immunoglobulin G levels increase post-ovulation independently of neutrophils.

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Katrein Schaefer

Full Text Available The prevalence of sexually transmitted infections (STIs is often higher in females than in males. Although the reproductive cycle profoundly modulates local immunity in the female reproductive tract (FRT system, significant gaps in our knowledge of the immunobiology of the FRT still exist. An intriguing and frequently observed characteristic of the FRT is the predominant presence of immunoglobulin (Ig G in cervico-vaginal secretions. We show here that in the mouse, IgG accumulation was enhanced approximately 5-fold post-ovulation, and was accompanied by an influx of neutrophils into the FRT. To determine whether these two events were causally related, we performed short-term neutrophil depletion experiments at individual stages throughout the estrous cycle. Our results demonstrate that neutrophils were not necessary for cycle-dependent tissue remodeling and cycle progression and that cycle-dependent IgG accumulation occurred independent of neutrophils. We thus conclude that neutrophil influx and IgG accumulation are independent events that occur in the FRT during the reproductive cycle.

Identification of Placental Aspartic Proteinase in the Eurasian Beaver (Castor fiber L.).

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Lipka, Aleksandra; Panasiewicz, Grzegorz; Majewska, Marta; Paukszto, Lukasz; Bieniek-Kobuszewska, Martyna; Szafranska, Bozena

2018-04-18

Aspartic proteinases (AP) form a multigenic group widely distributed in various organisms and includes pepsins (pep), cathepsins D and E, pregnancy associated glycoproteins (PAGs) as well as plant, fungal, and retroviral proteinases. This study describes the transcript identification and expression localization of the AP within the discoid placenta of the Castor fiber . We identified 1257 bp of the AP cDNA sequence, encoding 391 amino acids (aa) of the polypeptide precursor composed of 16 aa signal peptide, 46 aa pro-piece, and 329 aa of the mature protein. Within the AP precursor, one site of potential N -glycosylation (NPS 119–121 ) and two Asp residues (D) specific for the catalytic cleft of AP were identified (VLFDTGSSNLWV 91–102 and GIVDTGTSLLTV 277–288 ). The highest homology of the identified placental AP nucleotide and aa sequence was to mouse pepsinogen C (75.8% and 70.1%, respectively). Identified AP also shared high homology with other superfamily members: PAGs, cathepsins, and napsins. The AP identified in this study was named as pepsinogen/PAG-Like (pep/PAG-L). Diversified pep/PAG-L protein profiles with a dominant 58 kDa isoform were identified. Immune reactive signals of the pep/PAG-L were localized within the trophectodermal cells of the beaver placenta. This is the first report describing the placental AP (pep/PAG-L) in the C. fiber .

Computer-assisted image analysis assay of human neutrophil chemotaxis in vitro

DEFF Research Database (Denmark)

Jensen, P; Kharazmi, A

1991-01-01

We have developed a computer-based image analysis system to measure in-filter migration of human neutrophils in the Boyden chamber. This method is compared with the conventional manual counting techniques. Neutrophils from healthy individuals and from patients with reduced chemotactic activity were....... Another advantage of the assay is that it can be used to show the migration pattern of different populations of neutrophils from both healthy individuals and patients....

Neutrophils reduce the parasite burden in Leishmania (Leishmania amazonensis-infected macrophages.

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Erico Vinícius de Souza Carmo

2010-11-01

Full Text Available Studies on the role of neutrophils in Leishmania infection were mainly performed with L. (L major, whereas less information is available for L. (L amazonensis. Previous results from our laboratory showed a large infiltrate of neutrophils in the site of infection in a mouse strain resistant to L. (L. amazonensis (C3H/HePas. In contrast, the susceptible strain (BALB/c displayed a predominance of macrophages harboring a high number of amastigotes and very few neutrophils. These findings led us to investigate the interaction of inflammatory neutrophils with L. (L. amazonensis-infected macrophages in vitro.Mouse peritoneal macrophages infected with L. (L. amazonensis were co-cultured with inflammatory neutrophils, and after four days, the infection was quantified microscopically. Data are representative of three experiments with similar results. The main findings were 1 intracellular parasites were efficiently destroyed in the co-cultures; 2 the leishmanicidal effect was similar when cells were obtained from mouse strains resistant (C3H/HePas or susceptible (BALB/c to L. (L. amazonensis; 3 parasite destruction did not require contact between infected macrophages and neutrophils; 4 tumor necrosis factor alpha (TNF-α, neutrophil elastase and platelet activating factor (PAF were involved with the leishmanicidal activity, and 5 destruction of the parasites did not depend on generation of oxygen or nitrogen radicals, indicating that parasite clearance did not involve the classical pathway of macrophage activation by TNF-α, as reported for other Leishmania species.The present results provide evidence that neutrophils in concert with macrophages play a previously unrecognized leishmanicidal effect on L. (L. amazonensis. We believe these findings may help to understand the mechanisms involved in innate immunity in cutaneous infection by this Leishmania species.

Serum and glucocorticoid-regulated kinase 1 regulates neutrophil clearance during inflammation resolution.

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Burgon, Joseph; Robertson, Anne L; Sadiku, Pranvera; Wang, Xingang; Hooper-Greenhill, Edward; Prince, Lynne R; Walker, Paul; Hoggett, Emily E; Ward, Jonathan R; Farrow, Stuart N; Zuercher, William J; Jeffrey, Philip; Savage, Caroline O; Ingham, Philip W; Hurlstone, Adam F; Whyte, Moira K B; Renshaw, Stephen A

2014-02-15

The inflammatory response is integral to maintaining health by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralize invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein serum and glucocorticoid-regulated kinase 1 (SGK1). We have characterized the expression patterns and regulation of SGK family members in human neutrophils and shown that inhibition of SGK activity completely abrogates the antiapoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signaling and thus may prove a valuable therapeutic target for the treatment of inflammatory disease.

Sulforaphane restores cellular glutathione levels and reduces chronic periodontitis neutrophil hyperactivity in vitro.

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Irundika H K Dias

Full Text Available The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2 (. - by the nicotinamide adenine dinucleotide (NADPH oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2, a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients' neutrophils exhibit a low reduced glutathione (GSH/oxidised glutathione (GSSG ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC, and modifier (GCLM subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2 (. - production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis.

Sulforaphane restores cellular glutathione levels and reduces chronic periodontitis neutrophil hyperactivity in vitro.

Science.gov (United States)

Dias, Irundika H K; Chapple, Ian L C; Milward, Mike; Grant, Melissa M; Hill, Eric; Brown, James; Griffiths, Helen R

2013-01-01

The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN) to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2 (. -) by the nicotinamide adenine dinucleotide (NADPH) oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients' neutrophils exhibit a low reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC), and modifier (GCLM) subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2 (. -) production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis.

New strategy for sepsis: Targeting a key role of platelet-neutrophil interaction

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Xu Wang

2014-07-01

Full Text Available Neutrophil and platelet are essential arms of the innate immune response. In sepsis, platelet abnormal activation as well as neutrophil paralysis are well recognized. For platelet, it is characterized by the contribution to disseminated intravascular coagulation (DIC and the enhanced inflammation response. In terms of neutrophil, its dysfunction is manifested by the impaired recruitment and migration to the infectious foci, abnormal sequestration in the remote organs, and the delayed clearance. More recently, it has been apparent that together platelet-neutrophil interaction can induce a faster and harder response during sepsis. This article focuses on the activation of platelet, dysfunction of neutrophil, and the interaction between them during sepsis and profiles some of the molecular mechanisms and outcomes in these cellular dialogues, providing a novel strategy for treatment of sepsis.

Role of β1 Integrin in Tissue Homing of Neutrophils During Sepsis

Science.gov (United States)

Sarangi, Pranita P.; Hyun, Young-Min; Lerman, Yelena V.; Pietropaoli, Anthony P.; Kim, Minsoo

2012-01-01

Aberrant activation of neutrophils during sepsis results in the widespread release of pro-inflammatory mediators, leading to multi-organ system failure and death. However, aberrant activation of neutrophils during sepsis results in the widespread release of harmful inflammatory mediators causing host tissue injuries that can lead to multi organ system failure and death. One of the pivotal components of neutrophil migration during inflammation is the expression of surface integrins. In this study, we show that administration of a cyclic analog of RGD peptide (Arg-Gly-Asp) significantly reduced the number of tissue-invading neutrophils and the degree of sepsis-induced lethality in mice as compared to control peptide. Secondly, β1 integrin (CD29) was highly up-regulated on the neutrophils isolated from both septic patients and animals. Finally, conditional genetic ablation of β1 integrin from granulocytes also improved survival and bacterial clearance in septic animals Thus, our results indicate that expression of β1 integrin is important for modulating neutrophil trafficking during sepsis, and that therapeutics designed against β1 integrins may be beneficial. PMID:22683734

Neutrophils are not less sensitive than other blood leukocytes to the genomic effects of glucocorticoids.

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Gaelle Hirsch

Full Text Available Neutrophils are generally considered less responsive to glucocorticoids compared to other inflammatory cells. The reported increase in human neutrophil survival mediated by these drugs partly supports this assertion. However, it was recently shown that dexamethasone exerts potent anti-inflammatory effects in equine peripheral blood neutrophils. Few comparative studies of glucocorticoid effects in neutrophils and other leukocytes have been reported and a relative insensitivity of neutrophils to these drugs could not be ruled out.We assessed glucocorticoid-responsiveness in equine and human peripheral blood neutrophils and neutrophil-depleted leukocytes.Blood neutrophils and neutrophil-depleted leukocytes were isolated from 6 healthy horses and 4 human healthy subjects. Cells were incubated for 5 h with or without LPS (100 ng/mL alone or combined with hydrocortisone, prednisolone or dexamethasone (10(-8 M and 10(-6 M. IL-1β, TNF-α, IL-8, glutamine synthetase and GR-α mRNA expression was quantified by qPCR. Equine neutrophils were also incubated for 20 h with or without the three glucocorticoids and cell survival was assessed by flow cytometry and light microscopy on cytospin preparations.We found that glucocorticoids down-regulated LPS-induced pro-inflammatory mRNA expression in both cell populations and species. These drugs also significantly increased glutamine synthetase gene expression in both equine cell populations. The magnitude of glucocorticoid response between cell populations was generally similar in both species. We also showed that dexamethasone had a comparable inhibitory effect on pro-inflammatory gene expression in both human and equine neutrophils. As reported in other species, glucocorticoids significantly increase the survival in equine neutrophils.Glucocorticoids exert genomic effects of similar magnitude on neutrophils and on other blood leukocytes. We speculate that the poor response to glucocorticoids observed in some

Neutrophils Are Not Less Sensitive Than Other Blood Leukocytes to the Genomic Effects of Glucocorticoids

Science.gov (United States)

Hirsch, Gaelle; Lavoie-Lamoureux, Anouk; Beauchamp, Guy; Lavoie, Jean-Pierre

2012-01-01

Background Neutrophils are generally considered less responsive to glucocorticoids compared to other inflammatory cells. The reported increase in human neutrophil survival mediated by these drugs partly supports this assertion. However, it was recently shown that dexamethasone exerts potent anti-inflammatory effects in equine peripheral blood neutrophils. Few comparative studies of glucocorticoid effects in neutrophils and other leukocytes have been reported and a relative insensitivity of neutrophils to these drugs could not be ruled out. Objective We assessed glucocorticoid-responsiveness in equine and human peripheral blood neutrophils and neutrophil-depleted leukocytes. Methods Blood neutrophils and neutrophil-depleted leukocytes were isolated from 6 healthy horses and 4 human healthy subjects. Cells were incubated for 5 h with or without LPS (100 ng/mL) alone or combined with hydrocortisone, prednisolone or dexamethasone (10−8 M and 10−6 M). IL-1β, TNF-α, IL-8, glutamine synthetase and GR-α mRNA expression was quantified by qPCR. Equine neutrophils were also incubated for 20 h with or without the three glucocorticoids and cell survival was assessed by flow cytometry and light microscopy on cytospin preparations. Results We found that glucocorticoids down-regulated LPS-induced pro-inflammatory mRNA expression in both cell populations and species. These drugs also significantly increased glutamine synthetase gene expression in both equine cell populations. The magnitude of glucocorticoid response between cell populations was generally similar in both species. We also showed that dexamethasone had a comparable inhibitory effect on pro-inflammatory gene expression in both human and equine neutrophils. As reported in other species, glucocorticoids significantly increase the survival in equine neutrophils. Conclusions Glucocorticoids exert genomic effects of similar magnitude on neutrophils and on other blood leukocytes. We speculate that the poor response to

Dihydroxyoctadecamonoenoate esters inhibit the neutrophil ...

Indian Academy of Sciences (India)

PRAKASH

that observed with arachidonic acid treatment (Li et al 1996). ..... An alternative possibility is that the methyl DiHOMEs .... nitric oxide-derived reactive species in vascular cells; Circ. ... necrosis factor 1-alpha-initiated neutrophil responses and.

p21-Activated kinase (PAK regulates cytoskeletal reorganization and directional migration in human neutrophils.

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Asako Itakura

Full Text Available Neutrophils serve as a first line of defense in innate immunity owing in part to their ability to rapidly migrate towards chemotactic factors derived from invading pathogens. As a migratory function, neutrophil chemotaxis is regulated by the Rho family of small GTPases. However, the mechanisms by which Rho GTPases orchestrate cytoskeletal dynamics in migrating neutrophils remain ill-defined. In this study, we characterized the role of p21-activated kinase (PAK downstream of Rho GTPases in cytoskeletal remodeling and chemotactic processes of human neutrophils. We found that PAK activation occurred upon stimulation of neutrophils with f-Met-Leu-Phe (fMLP, and PAK accumulated at the actin-rich leading edge of stimulated neutrophils, suggesting a role for PAK in Rac-dependent actin remodeling. Treatment with the pharmacological PAK inhibitor, PF3758309, abrogated the integrity of RhoA-mediated actomyosin contractility and surface adhesion. Moreover, inhibition of PAK activity impaired neutrophil morphological polarization and directional migration under a gradient of fMLP, and was associated with dysregulated Ca(2+ signaling. These results suggest that PAK serves as an important effector of Rho-family GTPases in neutrophil cytoskeletal reorganization, and plays a key role in driving efficient directional migration of human neutrophils.

The Contribution of Proteinase-Activated Receptors to Intracellular Signaling, Transcellular Transport and Autophagy in Alzheimer´s Disease

Czech Academy of Sciences Publication Activity Database

Matěj, R.; Rohan, Z.; Holada, K.; Olejár, Tomáš

2015-01-01

Roč. 12, č. 1 (2015), s. 2-12 ISSN 1567-2050 Institutional support: RVO:67985823 Keywords : Alzheimer ´s Disease * autophagy * proteinase-activated receptors Subject RIV: EA - Cell Biology Impact factor: 3.145, year: 2015

G-CSF maintains controlled neutrophil mobilization during acute inflammation by negatively regulating CXCR2 signaling

Science.gov (United States)

Bajrami, Besnik; Zhu, Haiyan; Zhang, Yu C.

2016-01-01

Cytokine-induced neutrophil mobilization from the bone marrow to circulation is a critical event in acute inflammation, but how it is accurately controlled remains poorly understood. In this study, we report that CXCR2 ligands are responsible for rapid neutrophil mobilization during early-stage acute inflammation. Nevertheless, although serum CXCR2 ligand concentrations increased during inflammation, neutrophil mobilization slowed after an initial acute fast phase, suggesting a suppression of neutrophil response to CXCR2 ligands after the acute phase. We demonstrate that granulocyte colony-stimulating factor (G-CSF), usually considered a prototypical neutrophil-mobilizing cytokine, was expressed later in the acute inflammatory response and unexpectedly impeded CXCR2-induced neutrophil mobilization by negatively regulating CXCR2-mediated intracellular signaling. Blocking G-CSF in vivo paradoxically elevated peripheral blood neutrophil counts in mice injected intraperitoneally with Escherichia coli and sequestered large numbers of neutrophils in the lungs, leading to sterile pulmonary inflammation. In a lipopolysaccharide-induced acute lung injury model, the homeostatic imbalance caused by G-CSF blockade enhanced neutrophil accumulation, edema, and inflammation in the lungs and ultimately led to significant lung damage. Thus, physiologically produced G-CSF not only acts as a neutrophil mobilizer at the relatively late stage of acute inflammation, but also prevents exaggerated neutrophil mobilization and the associated inflammation-induced tissue damage during early-phase infection and inflammation. PMID:27551153

Identification and purification of O-acetyl-L-serine sulphhydrylase in Penicillium chrysogenum

DEFF Research Database (Denmark)

østergaard, Simon; Theilgaard, Hanne Birgitte; Nielsen, Jens Bredal

1998-01-01

We have demonstrated that Penicillium chrysogenum possesses the L-cysteine biosynthetic enzyme O-acetyI-L-serine sulphhydrylase (EC 4.2.99.8) of the direct sulphhydrylation pathway. The finding of this enzyme, and thus the presence of the direct sulphhydrylation pathway in P. chrysogenum, creates...... the potential for increasing the overall yield in penicillin production by enhancing the enzymatic activity of this microorganism. Only O-acetyl-L-serine sulphhydrylase and O-acetyl-L-homoserine sulphhydrylase (EC 4.2.99.10) have been demonstrated to use O-acetyl-L-serine as substrate for the formation of L-cysteine....... The purified enzyme did not catalyse the formation of L-homocysteine from O-acetyl-L-homoserine and sulphide, excluding the possibility that the purified enzyme was O-acetyI-L-homoserine sulphhydrylase with multiple substrate specificity. The purification enhanced the enzymatic specific activity 93-fold...

Accumulation of 111In-neutrophils in rabbit skin in allergic and non-allergic inflammatory reactions in vivo. Inhibition by neutrophil pretreatment in vitro with a monoclonal antibody recognizing the CD18 antigen

International Nuclear Information System (INIS)

Nourshargh, S.; Rampart, M.; Hellewell, P.G.; Jose, P.J.; Harlan, J.M.; Edwards, A.J.; Williams, T.J.

1989-01-01

The mAb 60.3 recognizes the neutrophil CD18 Ag. We have investigated the effect of in vitro pretreatment of radiolabeled neutrophils with mAb 60.3 on their accumulation in vivo. Further, we have compared the in vivo effects of mAb 60.3 with its effects on neutrophil adherence in vitro. Neutrophil accumulation in vivo was measured in response to: (1) exogenous mediators FMLP, C5a des Arg, LTB4 and IL-1; (2) endogenous mediators generated in a non-allergic inflammatory reaction induced by zymosan; and (3) endogenous mediators generated in two allergic inflammatory reactions, a passive cutaneous anaphylactic reaction and a reversed passive Arthus reaction in rabbit skin. Pretreatment of neutrophils with mAb 60.3 inhibited their accumulation in all the responses. The results demonstrate that there is a common mechanism mediating neutrophil accumulation in these inflammatory reactions. Neutrophils pretreated with mAb 60.3 were also unresponsive to chemoattractants in in vitro adherence assays. However, the antibody-treated neutrophils responded normally to FMLP and C5a with respect to granular enzyme release. These results suggest that the basal expression of CD18 Ag is important for the adherence of neutrophils to microvascular endothelial cells stimulated by the local generation, or administration, of chemical mediators in vivo. Despite the fact that mediators such as FMLP can increase CD18 expression in vitro, it appears more likely that such mediators act in vivo by inducing a conformational change in the basally expressed neutrophil adhesive molecules

Hexokinase 2 from Saccharomyces cerevisiae: regulation of oligomeric structure by in vivo phosphorylation at serine-14.

Science.gov (United States)

Behlke, J; Heidrich, K; Naumann, M; Müller, E C; Otto, A; Reuter, R; Kriegel, T

1998-08-25

Homodimeric hexokinase 2 from Saccharomyces cerevisiae is known to have two sites of phosphorylation: for serine-14 the modification in vivo increases with glucose exhaustion [Kriegel et al. (1994) Biochemistry 33, 148-152], while for serine-157 it occurs in vitro with ATP in the presence of nonphosphorylateable five-carbon analogues of glucose [Heidrich et al. (1997) Biochemistry 36, 1960-1964]. We show now by site-directed mutagenesis and sedimentation analysis that serine-14 phosphorylation affects the oligomeric state of hexokinase, its substitution by glutamate causing complete dissociation; glutamate exchange for serine-157 does not. Phosphorylation of wild-type hexokinase at serine-14 likewise causes dissociation in vitro. In view of the higher glucose affinity of monomeric hexokinase and the high hexokinase concentration in yeast [Womack, F., and Colowick, S. P. (1978) Arch. Biochem. Biophys. 191, 742-747; Mayes, E. L., Hoggett, J. G., and Kellett, G. L. (1983) Eur. J. Biochem. 133, 127-134], we speculate that the in vivo phosphorylation at serine-14 as transiently occurring in glucose derepression might provide a mechanism to improve glucose utilization from low level and/or that nuclear localization of the monomer might be involved in the signal transduction whereby glucose causes catabolite repression.

Development of cathepsin-L cysteine proteinase based Dot-enzyme-linked immunosorbent assay for the diagnosis of Fasciola gigantica infection in buffaloes.

Science.gov (United States)

Varghese, Anju; Raina, O K; Nagar, Gaurav; Garg, Rajat; Banerjee, P S; Maharana, B R; Kollannur, Justin D

2012-02-10

Native cathepsin-L cysteine proteinase (28 kDa) was purified from the excretory secretory products of Fasciola gigantica and was used for sero-diagnosis of F. gigantica infection in buffaloes by Dot-enzyme-linked immunosorbent assay (Dot-ELISA). The test detected F. gigantica field infection in these animals with a sensitivity of ∼ 90%. No specific IgG antibody binding was displayed by sera obtained from 76 buffaloes considered to be Fasciola and other parasite-free by microscopic examination of faeces and necropsy examination of liver, rumen and intestine. Additionally, sera from 156 Fasciola-free buffaloes, yet infected with Gigantocotyle explanatum, Paramphistomum epiclitum, Gastrothylax spp., Strongyloides papillosus and hydatid cyst were all negative, indicating that F. gigantica cathepsin-L cysteine proteinase does not cross-react with these helminth parasites in natural infection of the host. The data indicated that cathepsin-L cysteine proteinase based Dot-ELISA reached ∼ 90% sensitivity and 100% specificity with relation to above parasites in the detection of bubaline fasciolosis. The present Dot-ELISA diagnostic assay is relevant to the field diagnosis of F. gigantica infection in buffaloes. Copyright © 2011 Elsevier B.V. All rights reserved.

Stanniocalcin-1 Potently Inhibits the Proteolytic Activity of the Metalloproteinase Pregnancy-associated Plasma Protein-A

DEFF Research Database (Denmark)

Kløverpris, Søren; Mikkelsen, Jakob Hauge; Pedersen, Josefine Hvidkjær

2015-01-01

regulation in these species. Several physiological functions of STC1 have been reported, although many molecular details are still lacking. We here demonstrate that STC1 is an inhibitor of the metzincin metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A), which modulates insulin-like growth...... that the homologous STC2 inhibits PAPP-A proteolytic activity, and that this depends on the formation of a covalent complex between the inhibitor and the proteinase, mediated by Cys-120 of STC2. We find that STC1 is unable to bind covalently to PAPP-A, in agreement with the absence of a corresponding cysteine residue....... It rather binds to PAPP-A with high affinity (KD = 75 pm). We further demonstrate that both STC1 and STC2 show inhibitory activity toward PAPP-A2, but not selected serine proteinases and metalloproteinases. We therefore conclude that the STCs are proteinase inhibitors, probably restricted in specificity...

Brewer’s spent grain and corn steep liquor as alternative culture medium substrates for proteinase production by Streptomyces malaysiensis AMT-3

Science.gov (United States)

do Nascimento, Rodrigo Pires; Junior, Nelson Alves; Coelho, Rosalie Reed Rodrigues

2011-01-01

Brewer’s spent grain and corn steep liquor or yeast extract were used as the sole organic forms for proteinase production by Streptomyces malaysiensis in submerged fermentation. The influence of the C and N concentrations, as well as the incubation periods, were assessed. Eight proteolytic bands were detected through gelatin-gel-electrophoresis in the various extracts obtained from the different media and after different incubation periods, with apparent molecular masses of 20, 35, 43, 50, 70, 100, 116 and 212 kDa. The results obtained suggest an opportunity for exploring this alternative strategy for proteinases production by actinomycetes, using BSG and CSL as economically feasible substrates. PMID:24031767

111Indium-labeled neutrophil migration into the lungs of bleomycin-treated rabbits assessed noninvasively by external scintigraphy

International Nuclear Information System (INIS)

Haslett, C.; Shen, A.S.; Feldsien, D.C.; Allen, D.; Henson, P.M.; Cherniack, R.M.

1989-01-01

Factors controlling neutrophil migration into the lung are poorly understood, but their identification is important for our understanding of the pathogenesis of inflammatory lung diseases. Pulmonary inflammation is difficult to quantify, and neutrophils in tissues and BAL may not accurately represent cell migration. In this study, intravenously delivered pulses of rabbit neutrophils labeled with Indium-111 (111In-neutrophils) were used to monitor neutrophil migration into the lungs. Radioactivity quantified in the lung region of interest (ROI) of external gamma camera scintigrams recorded 24 h after intravenous 111In-neutrophil injection accurately reflected the actual neutrophil-associated lung tissue radioactivity. ROI radioactivity at 24 h also correlated closely with the percent of 111In-neutrophils that had migrated into lavageable air spaces, and this parameter therefore provided an index of total lung 111In-neutrophil migration. Using 24-h ROI radioactivity and percent of injected 111In-neutrophils recovered in BAL at 24 h as indices of neutrophil migration into the lung, it was found that intratracheal saline caused only a transient neutrophil migration, whereas 10 U/kg intratracheal bleomycin induced migration that persisted for as long as 3 wk. 111In-neutrophil migration into the lung, assessed by external scintigraphy, correlated with total neutrophils quantified in histologic sections (r = 0.71, p = 0.006). The data suggest that this approach will be valuable in investigating mechanisms controlling neutrophil migration in lung inflammation, and that 111In-neutrophil scintigraphy may provide a noninvasive index of total lung neutrophil load that might be useful in staging inflammation in patchy diseases such as idiopathic pulmonary fibrosis

Kunitz Proteinase Inhibitors Limit Water Stress Responses in White Clover (Trifolium repens L.) Plants.

Science.gov (United States)

Islam, Afsana; Leung, Susanna; Nikmatullah, Aluh; Dijkwel, Paul P; McManus, Michael T

2017-01-01

The response of plants to water deficiency or drought is a complex process, the perception of which is triggered at the molecular level before any visible morphological responses are detected. It was found that different groups of plant proteinase inhibitors (PIs) are induced and play an active role during abiotic stress conditions such as drought. Our previous work with the white clover ( Trifolium repens L.) Kunitz Proteinase Inhibitor ( Tr-KPI ) gene family showed that Tr-KPIs are differentially regulated to ontogenetic and biotic stress associated cues and that, at least some members of this gene family may be required to maintain cellular homeostasis. Altered cellular homeostasis may also affect abiotic stress responses and therefore, we aimed to understand if distinct Tr-PKI members function during drought stress. First, the expression level of three Tr-KPI genes, Tr-KPI1 , Tr-KPI2 , and Tr-KPI5 , was measured in two cultivars and one white clover ecotype with differing capacity to tolerate drought. The expression of Tr-KPI1 and Tr-KPI5 increased in response to water deficiency and this was exaggerated when the plants were treated with a previous period of water deficiency. In contrast, proline accumulation and increased expression of Tr-NCED1 , a gene encoding a protein involved in ABA biosynthesis, was delayed in plants that experienced a previous drought period. RNAi knock-down of Tr-KPI1 and Tr-KPI5 resulted in increased proline accumulation in leaf tissue of plants grown under both well-watered and water-deficit conditions. In addition, increased expression of genes involved in ethylene biosynthesis was found. The data suggests that Tr-KPIs , particularly Tr-KPI5 , have an explicit function during water limitation. The results also imply that the Tr-KPI family has different in planta proteinase targets and that the functions of this protein family are not solely restricted to one of storage proteins or in response to biotic stress.

Kunitz Proteinase Inhibitors Limit Water Stress Responses in White Clover (Trifolium repens L. Plants

Directory of Open Access Journals (Sweden)

Afsana Islam

2017-10-01

Full Text Available The response of plants to water deficiency or drought is a complex process, the perception of which is triggered at the molecular level before any visible morphological responses are detected. It was found that different groups of plant proteinase inhibitors (PIs are induced and play an active role during abiotic stress conditions such as drought. Our previous work with the white clover (Trifolium repens L. Kunitz Proteinase Inhibitor (Tr-KPI gene family showed that Tr-KPIs are differentially regulated to ontogenetic and biotic stress associated cues and that, at least some members of this gene family may be required to maintain cellular homeostasis. Altered cellular homeostasis may also affect abiotic stress responses and therefore, we aimed to understand if distinct Tr-PKI members function during drought stress. First, the expression level of three Tr-KPI genes, Tr-KPI1, Tr-KPI2, and Tr-KPI5, was measured in two cultivars and one white clover ecotype with differing capacity to tolerate drought. The expression of Tr-KPI1 and Tr-KPI5 increased in response to water deficiency and this was exaggerated when the plants were treated with a previous period of water deficiency. In contrast, proline accumulation and increased expression of Tr-NCED1, a gene encoding a protein involved in ABA biosynthesis, was delayed in plants that experienced a previous drought period. RNAi knock-down of Tr-KPI1 and Tr-KPI5 resulted in increased proline accumulation in leaf tissue of plants grown under both well-watered and water-deficit conditions. In addition, increased expression of genes involved in ethylene biosynthesis was found. The data suggests that Tr-KPIs, particularly Tr-KPI5, have an explicit function during water limitation. The results also imply that the Tr-KPI family has different in planta proteinase targets and that the functions of this protein family are not solely restricted to one of storage proteins or in response to biotic stress.

Enhanced neutrophil chemotactic activity after bronchial challenge in subjects with grain dust-induced asthma.

Science.gov (United States)

Park, H S; Jung, K S

1998-03-01

There have been few reports suggesting involvement of neutrophils in induction of bronchoconstriction after inhalation of grain dust. To understand the role of neutrophils in pathogenesis of grain dust-induced asthma. We observed serum neutrophil chemotactic activity during grain dust-bronchoprovocation tests in six asthmatic subjects with positive bronchial challenges (group I). They were compared with those of six symptomatic subjects from the same workplace with negative bronchial challenges (group II). After grain dust inhalation, serum neutrophil chemotactic activity significantly increased at 30 minutes (P = .028), and then decreased to baseline level at 240 minutes (P = .028) in five subjects of group I having isolated early asthmatic responses. Enhanced neutrophil chemotactic activity was persistent for up to 240 minutes in one asthmatic subject having both early and late asthmatic responses. There was, however, no significant change in serum neutrophil chemotactic activity during bronchial challenges in subjects of group II. Pre-incubation of sera with anti-interleukin-8 (IL-8) antibody did not affect the neutrophil chemotactic activity results of group I subjects. These results suggest that enhanced neutrophil chemotactic activity distinct from IL-8 may contribute to significant bronchoconstriction induced by grain dust.

ATM-mediated Snail Serine 100 phosphorylation regulates cellular radiosensitivity

International Nuclear Information System (INIS)

Boohaker, Rebecca J.; Cui, Xiaoli; Stackhouse, Murray; Xu, Bo

2013-01-01

Purpose: Activation of the DNA damage responsive protein kinase ATM is a critical step for cellular survival in response to ionizing irradiation (IR). Direct targets of ATM regulating radiosensitivity remain to be fully investigated. We have recently reported that ATM phosphorylates the transcriptional repressor Snail on Serine 100. We aimed to further study the functional significance of ATM-mediated Snail phosphorylation in response to IR. Material and methods: We transfected vector-only, wild-type, the Serine 100 to alanine (S100A) or to glutamic acid (S100E) substitution of Snail into various cell lines. We assessed colony formation, γ-H2AX focus formation and the invasion index in the cells treated with or without IR. Results: We found that over-expression of the S100A mutant Snail in HeLa cells significantly increased radiosensitivity. Meanwhile the expression of S100E, a phospho-mimicking mutation, resulted in enhanced radio-resistance. Interestingly, S100E could rescue the radiosensitive phenotype in ATM-deficient cells. We also found that expression of S100E increased γ-H2AX focus formation and compromised inhibition of invasion in response to IR independent of cell survival. Conclusion: ATM-mediated Snail Serine 100 phosphorylation in response to IR plays an important part in the regulation of radiosensitivity

Burn injury reduces neutrophil directional migration speed in microfluidic devices.

Directory of Open Access Journals (Sweden)

Kathryn L Butler

2010-07-01

Full Text Available Thermal injury triggers a fulminant inflammatory cascade that heralds shock, end-organ failure, and ultimately sepsis and death. Emerging evidence points to a critical role for the innate immune system, and several studies had documented concurrent impairment in neutrophil chemotaxis with these post-burn inflammatory changes. While a few studies suggest that a link between neutrophil motility and patient mortality might exist, so far, cumbersome assays have prohibited exploration of the prognostic and diagnostic significance of chemotaxis after burn injury. To address this need, we developed a microfluidic device that is simple to operate and allows for precise and robust measurements of chemotaxis speed and persistence characteristics at single-cell resolution. Using this assay, we established a reference set of migration speed values for neutrophils from healthy subjects. Comparisons with samples from burn patients revealed impaired directional migration speed starting as early as 24 hours after burn injury, reaching a minimum at 72-120 hours, correlated to the size of the burn injury and potentially serving as an early indicator for concurrent infections. Further characterization of neutrophil chemotaxis using this new assay may have important diagnostic implications not only for burn patients but also for patients afflicted by other diseases that compromise neutrophil functions.

Impaired neutrophil function in intestinal lymphangiectasia.

OpenAIRE

Bolton, R P; Cotter, K L; Losowsky, M S

1986-01-01

Impaired neutrophil chemotaxis and phagocytosis were shown in three patients with intestinal lymphangiectasia. Abnormalities in cell associated and serum derived activity occurred, and possible mechanisms are suggested.

With Friends Like These: The Complex Role of Neutrophils in the Progression of Severe Pneumonia

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Roger D. Pechous

2017-05-01

Full Text Available Pneumonia is a leading cause of death from infection in the United States and across the globe. During pulmonary infection, clear resolution of host inflammatory responses occurs in the absence of appreciable lung damage. Neutrophils are the first wave of leukocytes to arrive in the lung upon infection. After activation, neutrophils traffic from the vasculature via transendothelial migration through the lung interstitium and into the alveolar space. Successful pulmonary immunity requires neutrophil-mediated killing of invading pathogens by phagocytosis and release of a myriad of antimicrobial molecules, followed by resolution of inflammation, neutrophil apoptosis, and clearing of dead or dying neutrophils by macrophages. In addition to their antimicrobial role, it is becoming clear that neutrophils are also important modulators of innate and adaptive immune responses, primarily through the release of cytokines and recruitment of additional waves of neutrophils into the airways. Though typically essential to combating severe pneumonia, neutrophil influx into the airways is a double-edged sword: Overzealous neutrophil activation can cause severe tissue damage as a result of the release of toxic agents including proteases, cationic polypeptides, cytokines, and reactive oxygen species (ROS aimed at killing invading microbes. In extreme cases, the damage caused by neutrophils and other innate immune mediators become the primary source of morbidity and mortality. Here, we review the complex role of neutrophils during severe pneumonia by highlighting specific molecules and processes that contribute to pulmonary immunity, but can also drive progression of severe disease. Depending on the identity of the infectious agent, enhancing or suppressing neutrophil-mediated responses may be key to effectively treating severe and typically lethal pneumonia.

Streptococcus sanguinis induces neutrophil cell death by production of hydrogen peroxide.

Science.gov (United States)

Sumioka, Ryuichi; Nakata, Masanobu; Okahashi, Nobuo; Li, Yixuan; Wada, Satoshi; Yamaguchi, Masaya; Sumitomo, Tomoko; Hayashi, Mikako; Kawabata, Shigetada

2017-01-01

Streptococcus is the dominant bacterial genus in the human oral cavity and a leading cause of infective endocarditis. Streptococcus sanguinis belongs to the mitis group of streptococci and produces hydrogen peroxide (H2O2) by the action of SpxB, a pyruvate oxidase. In this study, we investigated the involvement of SpxB in survival of S. sanguinis in human blood and whether bacterial H2O2 exhibits cytotoxicity against human neutrophils. Results of a bactericidal test with human whole blood revealed that the spxB mutation in S. sanguinis is detrimental to its survival in blood. When S. sanguinis strains were exposed to isolated neutrophils, the bacterial survival rate was significantly decreased by spxB deletion. Furthermore, human neutrophils exposed to the S. sanguinis wild-type strain, in contrast to those exposed to an spxB mutant strain, underwent cell death with chromatin de-condensation and release of web-like extracellular DNA, reflecting induction of neutrophil extracellular traps (NETs). Since reactive oxygen species-mediated NET induction requires citrullination of arginine residues in histone proteins and subsequent chromatin de-condensation, we examined citrullination levels of histone in infected neutrophils. It is important to note that the citrullinated histone H3 was readily detected in neutrophils infected with the wild-type strain, as compared to infection with the spxB mutant strain. Moreover, decomposition of streptococcal H2O2 with catalase reduced NET induction. These results suggest that H2O2 produced by S. sanguinis provokes cell death of neutrophils and NET formation, thus potentially affecting bacterial survival in the bloodstream.

Streptococcus sanguinis induces neutrophil cell death by production of hydrogen peroxide.

Directory of Open Access Journals (Sweden)

Ryuichi Sumioka

Full Text Available Streptococcus is the dominant bacterial genus in the human oral cavity and a leading cause of infective endocarditis. Streptococcus sanguinis belongs to the mitis group of streptococci and produces hydrogen peroxide (H2O2 by the action of SpxB, a pyruvate oxidase. In this study, we investigated the involvement of SpxB in survival of S. sanguinis in human blood and whether bacterial H2O2 exhibits cytotoxicity against human neutrophils. Results of a bactericidal test with human whole blood revealed that the spxB mutation in S. sanguinis is detrimental to its survival in blood. When S. sanguinis strains were exposed to isolated neutrophils, the bacterial survival rate was significantly decreased by spxB deletion. Furthermore, human neutrophils exposed to the S. sanguinis wild-type strain, in contrast to those exposed to an spxB mutant strain, underwent cell death with chromatin de-condensation and release of web-like extracellular DNA, reflecting induction of neutrophil extracellular traps (NETs. Since reactive oxygen species-mediated NET induction requires citrullination of arginine residues in histone proteins and subsequent chromatin de-condensation, we examined citrullination levels of histone in infected neutrophils. It is important to note that the citrullinated histone H3 was readily detected in neutrophils infected with the wild-type strain, as compared to infection with the spxB mutant strain. Moreover, decomposition of streptococcal H2O2 with catalase reduced NET induction. These results suggest that H2O2 produced by S. sanguinis provokes cell death of neutrophils and NET formation, thus potentially affecting bacterial survival in the bloodstream.

Toxicity to cotton boll weevil Anthonomus grandis of a trypsin inhibitor from chickpea seeds.

Science.gov (United States)

de P G Gomes, Angélica; Dias, Simoni C; Bloch, Carlos; Melo, Francislete R; Furtado, José R; Monnerat, Rose G; Grossi-de-Sá, Maria F; Franco, Octávio L

2005-02-01

Cotton (Gossypium hirsutum L.) is an important agricultural commodity, which is attacked by several pests such as the cotton boll weevil Anthonomus grandis. Adult A. grandis feed on fruits and leaf petioles, reducing drastically the crop production. The predominance of boll weevil digestive serine proteinases has motivated inhibitor screenings in order to discover new ones with the capability to reduce the digestion process. The present study describes a novel proteinase inhibitor from chickpea seeds (Cicer arietinum L.) and its effects against A. grandis. This inhibitor, named CaTI, was purified by using affinity Red-Sepharose Cl-6B chromatography, followed by reversed-phase HPLC (Vydac C18-TP). SDS-PAGE and MALDI-TOF analyses, showed a unique monomeric protein with a mass of 12,877 Da. Purified CaTI showed significant inhibitory activity against larval cotton boll weevil serine proteinases (78%) and against bovine pancreatic trypsin (73%), when analyzed by fluorimetric assays. Although the molecular mass of CaTI corresponded to alpha-amylase/trypsin bifunctional inhibitors masses, no inhibitory activity against insect and mammalian alpha-amylases was observed. In order to observe CaTI in vivo effects, an inhibitor rich fraction was added to an artificial diet at different concentrations. At 1.5% (w/w), CaTI caused severe development delay, several deformities and a mortality rate of approximately 45%. These results suggested that CaTI could be useful in the production of transgenic cotton plants with enhanced resistance toward cotton boll weevil.

The effect of cigarette smoking on neutrophil kinetics in human lungs [see comments

International Nuclear Information System (INIS)

MacNee, W.; Wiggs, B.; Belzberg, A.S.; Hogg, J.C.

1989-01-01

Neutrophils may play a part in the pathogenesis of the centrilobular emphysema associated with cigarette smoking. The capillary bed of the lungs concentrates neutrophils approximately 100-fold with respect to erythrocytes, producing a large pool of marginated cells. We examined the effect of cigarette smoking on the kinetics of this pool of cells, using 99mTc-labeled erythrocytes to measure regional blood velocity and 111In-labeled neutrophils to measure the removal of neutrophils during the first passage through the pulmonary circulation, their subsequent washout from the lungs, and the effect of local blood velocity on the number of neutrophils retained in each lung region. We observed no difference in these measurements between subjects who had never smoked (n = 6) and smokers who did not smoke during the study (n = 12). However, subjects who did smoke during the study (n = 12) had a significantly slower rate of washout of radiolabeled neutrophils from the lung (0.08 +/- 0.04 of the total per minute, as compared with 0.13 +/- 0.06 in smokers who did not smoke during the experiment and 0.14 +/- 0.08 in non-smokers) (P = 0.02). We also observed an increase in the regional retention of labeled neutrophils with respect to blood velocity in 5 of the 12 subjects who smoked during the study, but in none of the other subjects. We conclude that the presence of cigarette smoke in the lungs of some subjects increases the local concentration of neutrophils, and suggest that the lesions that characterize emphysema may be a result of the destruction of lung tissue by neutrophils that remain within pulmonary microvessels