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Sample records for phosphorylation-dependent neutrophil adherence

  1. Formyl Met-Leu-Phe-Stimulated FPR1 Phosphorylation in Plate-Adherent Human Neutrophils: Enhanced Proteolysis but Lack of Inhibition by Platelet-Activating Factor

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    Algirdas J. Jesaitis

    2018-01-01

    Full Text Available N-formyl-Met-Leu-Phe (fMLF is a model PAMP/DAMP driving human PMN to sites of injury/infection utilizing the GPCR, FPR1. We examined a microtiter plate format for measurement of FPR1 phosphorylation in adherent PMN at high densities and found that a new phosphosensitive FPR1 fragment, 25K-FPR1, accumulates in SDS-PAGE extracts. 25K-FPR1 is fully inhibited by diisopropylfluorophosphate PMN pretreatment but is not physiologic, as its formation failed to be significantly perturbed by ATP depletion, time and temperature of adherence, or adherence mechanism. 25K-FPR1 was minimized by extracting fMLF-exposed PMN in lithium dodecylsulfate at 4°C prior to reduction/alkylation. After exposure of adherent PMN to a 5 log range of PAF before or after fMLF, unlike in suspension PMN, no inhibition of fMLF-induced FPR1 phosphorylation was observed. However, PAF induced the release of 40% of PMN lactate dehydrogenase, implying significant cell lysis. We infer that PAF-induced inhibition of fMLF-dependent FPR1 phosphorylation observed in suspension PMN does not occur in the unlysed adherent PMN. We speculate that although the conditions of the assay may induce PAF-stimulated necrosis, the cell densities on the plates may approach levels observed in inflamed tissues and provide for an explanation of PAF’s divergent effects on FPR1 phosphorylation as well as PMN function.

  2. 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.

  3. A stable aspirin-triggered lipoxin A4 analog blocks phosphorylation of leukocyte-specific protein 1 in human neutrophils.

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    Ohira, Taisuke; Bannenberg, Gerard; Arita, Makoto; Takahashi, Minoru; Ge, Qingyuan; Van Dyke, Thomas E; Stahl, Gregory L; Serhan, Charles N; Badwey, John A

    2004-08-01

    Lipoxins and their aspirin-triggered 15-epimers are endogenous anti-inflammatory agents that block neutrophil chemotaxis in vitro and inhibit neutrophil influx in several models of acute inflammation. In this study, we examined the effects of 15-epi-16-(p-fluoro)-phenoxy-lipoxin A(4) methyl ester, an aspirin-triggered lipoxin A(4)-stable analog (ATLa), on the protein phosphorylation pattern of human neutrophils. Neutrophils stimulated with the chemoattractant fMLP were found to exhibit intense phosphorylation of a 55-kDa protein that was blocked by ATLa (10-50 nM). This 55-kDa protein was identified as leukocyte-specific protein 1, a downstream component of the p38-MAPK cascade in neutrophils, by mass spectrometry, Western blotting, and immunoprecipitation experiments. ATLa (50 nM) also reduced phosphorylation/activation of several components of the p38-MAPK pathway in these cells (MAPK kinase 3/MAPK kinase 6, p38-MAPK, MAPK-activated protein kinase-2). These results indicate that ATLa exerts its anti-inflammatory effects, at least in part, by blocking activation of the p38-MAPK cascade in neutrophils, which is known to promote chemotaxis and other proinflammatory responses by these cells.

  4. fMLP-Induced IL-8 Release Is Dependent on NADPH Oxidase in Human Neutrophils

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    María A. Hidalgo

    2015-01-01

    Full Text Available N-Formyl-methionyl-leucyl-phenylalanine (fMLP and platelet-activating factor (PAF induce similar intracellular signalling profiles; but only fMLP induces interleukin-8 (IL-8 release and nicotinamide adenine dinucleotide phosphate reduced (NADPH oxidase activity in neutrophils. Because the role of ROS on IL-8 release in neutrophils is until now controversial, we assessed if NADPH oxidase is involved in the IL-8 secretions and PI3K/Akt, MAPK, and NF-κB pathways activity induced by fMLP. Neutrophils were obtained from healthy volunteers. IL-8 was measured by ELISA, IL-8 mRNA by qPCR, and ROS production by luminol-amplified chemiluminescence, reduction of ferricytochrome c, and FACS. Intracellular pH changes were detected by spectrofluorescence. ERK1/2, p38 MAPK, and Akt phosphorylation were analysed by immunoblotting and NF-κB was analysed by immunocytochemistry. Hydroxy-3-methoxyaceto-phenone (HMAP, diphenyleneiodonium (DPI, and siRNA Nox2 reduced the ROS and IL-8 release in neutrophils treated with fMLP. HMAP, DPI, and amiloride (a Na+/H+ exchanger inhibitor inhibited the Akt phosphorylation and did not affect the p38 MAPK and ERK1/2 activity. DPI and HMAP reduced NF-κB translocation induced by fMLP. We showed that IL-8 release induced by fMLP is dependent on NADPH oxidase, and ROS could play a redundant role in cell signalling, ultimately activating the PI3K/Akt and NF-κB pathways in neutrophils.

  5. 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.

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    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

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

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    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...

  7. GSK3β-dependent inhibition of AMPK potentiates activation of neutrophils and macrophages and enhances severity of acute lung injury

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    Park, Dae Won; Jiang, Shaoning; Liu, Yanping; Siegal, Gene P.; Inoki, Ken; Abraham, Edward

    2014-01-01

    Although AMP-activated protein kinase (AMPK) is involved in regulating carbohydrate and lipid metabolism, activated AMPK also plays an anti-inflammatory role in many cell populations. However, despite the ability of AMPK activation to diminish the severity of inflammatory responses, previous studies have found that AMPK activity is diminished in LPS-treated neutrophils and also in lungs of mice with LPS-induced acute lung injury (ALI). Since GSK3β participates in regulating AMPK activity, we examined potential roles for GSK3β in modulating LPS-induced activation of neutrophils and macrophages and in influencing severity of ALI. We found that GSK3β-dependent phosphorylation of T479-AMPK was associated with pT172 dephosphorylation and inactivation of AMPK following TLR4 engagement. GSK3β inhibitors BIO (6-bromoindirubin-3′-oxime), SB216763, or siRNA knockdown of GSK3β, but not the PI3K/AKT inhibitor LY294002, prevented Thr172-AMPK dephosphorylation. Exposure to LPS resulted in rapid binding between IKKβ and AMPKα, and phosphorylation of S485-AMPK by IKKβ. These results suggest that IKKβ-dependent phosphorylation of S485-AMPK was an essential step in subsequent phosphorylation and inactivation AMPK by GSK3β. Inhibition of GSK3β activity delayed IκBα degradation and diminished expression of the proinflammatory TNF-α in LPS-stimulated neutrophils and macrophages. In vivo, inhibition of GSK3β decreased the severity of LPS-induced lung injury as assessed by development of pulmonary edema, production of TNF-α and MIP-2, and release of the alarmins HMGB1 and histone 3 in the lungs. These results show that inhibition of AMPK by GSK3β plays an important contributory role in enhancing LPS-induced inflammatory responses, including worsening the severity of ALI. PMID:25239914

  8. Neutrophil adhesion and chemotaxis depend on substrate mechanics

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    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.

  9. Neutrophil adhesion and chemotaxis depend on substrate mechanics

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    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.

  10. Anti-Inflammatory benefits of antibiotic-induced neutrophil apoptosis: tulathromycin induces caspase-3-dependent neutrophil programmed cell death and inhibits NF-kappaB signaling and CXCL8 transcription.

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    Fischer, Carrie D; Beatty, Jennifer K; Zvaigzne, Cheryl G; Morck, Douglas W; Lucas, Merlyn J; Buret, A G

    2011-01-01

    Clearance of apoptotic neutrophils is a central feature of the resolution of inflammation. Findings indicate that immuno-modulation and induction of neutrophil apoptosis by macrolide antibiotics generate anti-inflammatory benefits via mechanisms that remain obscure. Tulathromycin (TUL), a new antimicrobial agent for bovine respiratory disease, offers superior clinical efficacy for reasons not fully understood. The aim of this study was to identify the immuno-modulating effects of tulathromycin and, in this process, to establish tulathromycin as a new model for characterizing the novel anti-inflammatory properties of antibiotics. Bronchoalveolar lavage specimens were collected from Holstein calves 3 and 24 h postinfection, challenged intratracheally with live Mannheimia haemolytica (2 × 10(7) CFU), and treated with vehicle or tulathromycin (2.5 mg/kg body weight). Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and enzyme-linked immunosorbent assay (ELISA) revealed that tulathromycin treatment significantly increased leukocyte apoptosis and reduced levels of proinflammatory leukotriene B(4) in M. haemolytica-challenged calves. In vitro, tulathromycin concentration dependently induced apoptosis in freshly isolated bovine neutrophils from healthy steers in a capase-3-dependent manner but failed to induce apoptosis in bovine fibroblasts, epithelial cells, and endothelial cells, as well as freshly isolated bovine blood monocytes and monocyte-derived macrophages. The proapoptotic effects of TUL were also, in part, drug specific; equimolar concentrations of penicillin G, oxytetracycline, and ceftiofur failed to cause apoptosis in bovine neutrophils. In addition, tulathromycin significantly reduced levels of phosphorylated IκBα, nuclear translocation of NF-κB p65, and mRNA levels of proinflammatory interleukin-8 in lipopolysaccharide (LPS)-stimulated bovine neutrophils. The findings illustrate novel mechanisms through which

  11. 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.

  12. Hypoxia upregulates neutrophil degranulation and potential for tissue injury

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    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

  13. MAC-1 Glycoprotein Family mediates adherence of neutrophils to endothelial cells stimulated by leukotriene B/sub 4/ and platelet activating factor

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    Tonnesen, M.G.; Anderson, D.C.; Springer, T.A.; Knedler, A.; Avdi, N.; Henson, P.M.

    1986-03-01

    The process of neutrophil (N) adhesion to and migration through endothelium (EC), an early event in the induction of the acute inflammatory response, has been attributed to the generation of extravascular chemoattractant peptides and lipids. Although both leukotriene B/sub 4/ (LTB/sub 4/) and platelet activating factor (PAF) enhance N adherence to EC, the mechanisms involved in this interaction are still not completely understood. Since the MAC-1 Glycoprotein (GP) Family has recently been shown to be required for a variety of adherence-dependent functions of stimulated N, the authors questioned whether these adherence-associated GP might be involved in N adherence to EC stimulated by LTB/sub 4/ or PAF. Using a microtiter adherence assay with /sup 111/In labeled N, they assessed the ability of N from patients with MAC-1, LFA-1 Deficiency to adhere to monolayers of human omental microvascular or umbilical vein EC as well as to serum-coated plastic. Patient N exhibited markedly diminished adherence in response to LTB/sub 4/ or PAF compared to normal controls. LTB/sub 4/ and PAF enhanced expression of the MAC-1 GP Family on the surface of normal N as determined by flow cytofluorimetry using a monoclonal antibody (TS1/18) to the GP common beta subunit. In addition TS1/18 (20 ..mu..g/ml) completely inhibited N adherence stimulated by either LTB/sub 4/ (10/sup -8/M) or PAF(10/sup -11/M). Thus, the MAC-1 GP Family appears to be important in chemotactic factor regulation of N adherence to EC.

  14. 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.

  15. Sulfur mustard primes human neutrophils for increased degranulation and stimulates cytokine release via TRPM2/p38 MAPK signaling

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    Ham, Hwa-Yong [Department of Pharmacology, Infectious Diseases Medical Research Center, College of Medicine, Hallym University, Chuncheon (Korea, Republic of); Hong, Chang-Won, E-mail: chyj7983@hallym.ac.kr [Department of Chemical and Biological Warfare Research, The Armed Forces Medical Research Institute, Daejeon (Korea, Republic of); Lee, Si-Nae [Department of Pharmacology, Infectious Diseases Medical Research Center, College of Medicine, Hallym University, Chuncheon (Korea, Republic of); Kwon, Min-Soo [Department of Pharmacology, School of Medicine, CHA University, Seongnam (Korea, Republic of); Kim, Yeon-Ja [Department of Pharmacology, Infectious Diseases Medical Research Center, College of Medicine, Hallym University, Chuncheon (Korea, Republic of); Song, Dong-Keun, E-mail: dksong@hallym.ac.kr [Department of Pharmacology, Infectious Diseases Medical Research Center, College of Medicine, Hallym University, Chuncheon (Korea, Republic of)

    2012-01-01

    Sulfur mustard (2,2′-bis-chloroethyl-sulfide; SM) has been a military threat since the World War I. The emerging threat of bioterrorism makes SM a major threat not only to military but also to civilian world. SM injury elicits an inflammatory response characterized by infiltration of neutrophils. Although SM was reported to prime neutrophils, the mechanism has not been identified yet. In the present study, we investigated the mechanism of SM-induced priming in human neutrophils. SM increased [Ca{sup 2+}]{sub i} in human neutrophils in a concentration-dependent fashion. Transient receptor potential melastatin (TRPM) 2 inhibitors (clotrimazole, econazole and flufenamic acid) and silencing of TRPM2 by shRNA attenuated SM-induced [Ca{sup 2+}]{sub i} increase. SM primed degranulation of azurophil and specific granules in response to activation by fMLP as previously reported. SB203580, an inhibitor of p38 MAPK, inhibited SM-induced priming. Neither PD98057, an ERK inhibitor, nor SP600215, a JNK inhibitor, inhibited SM-induced priming. In addition, SM enhanced phosphorylation of NF-kB p65 and release of TNF-α, interleukin (IL)-6 and IL-8. SB203580 inhibited SM-induced NF-kB phosphorylation and cytokine release. These results suggest the involvement of TRPM2/p38 MAPK pathway in SM-induced priming and cytokines release in neutrophils. -- Highlights: ► SM increased [Ca{sup 2+}]{sub i} in human neutrophils through TPRM2-mediated calcium influx. ► SM primed degranulation of azurophil and specific granules. ► SM enhanced p38 MAPK and NF-κB p65 phosphorylation in human neutrophils. ► SM enhanced release of TNF-α, interleukin (IL)-6 and IL-8 from human neutrophils. ► SB203580 inhibited SM-induced priming, NF-κB p65 phosphorylation and cytokine release.

  16. 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

  17. 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.

  18. Sphingosine 1-phosphate mediates hyperalgesia via a neutrophil-dependent mechanism.

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    Amanda Finley

    Full Text Available Novel classes of pain-relieving molecules are needed to fill the void between non-steroidal anti-inflammatory agents and narcotics. We have recently shown that intraplantar administration of sphingosine 1-phosphate (S1P in rats causes peripheral sensitization and hyperalgesia through the S1P(1 receptor subtype (S1PR(1: the mechanism(s involved are largely unknown and were thus explored in the present study. Intraplantar injection of carrageenan in rats led to a time-dependent development of thermal hyperalgesia that was associated with pronounced edema and infiltration of neutrophils in paw tissues. Inhibition of 1 S1P formation with SK-I, a sphingosine kinase inhibitor, 2 S1P bioavailability with the S1P blocking antibody Sphingomab, LT1002 (but not its negative control, LT1017 or 3 S1P actions through S1PR(1 with the selective S1PR(1 antagonist, W146 (but not its inactive enantiomer, W140 blocked thermal hyperalgesia and infiltration of neutrophils. Taken together, these findings identify S1P as an important contributor to inflammatory pain acting through S1PR(1 to elicit hyperalgesia in a neutrophil-dependant manner. In addition and in further support, we demonstrate that the development of thermal hyperalgesia following intraplantar injection of S1P or SEW2871 (an S1PR(1 agonist was also associated with neutrophilic infiltration in paw tissues as these events were attenuated by fucoidan, an inhibitor of neutrophilic infiltration. Importantly, FTY720, an FDA-approved S1P receptor modulator known to block S1P-S1PR(1 signaling, attenuated carrageenan-induced thermal hyperalgesia and associated neutrophil infiltration. Targeting the S1P/S1PR(1 axis opens a therapeutic strategy for the development of novel non-narcotic anti-hyperalgesic agents.

  19. 5-Lipoxygenase-Dependent Recruitment of Neutrophils and Macrophages by Eotaxin-Stimulated Murine Eosinophils

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    Ricardo Alves Luz

    2014-01-01

    Full Text Available The roles of eosinophils in antimicrobial defense remain incompletely understood. In ovalbumin-sensitized mice, eosinophils are selectively recruited to the peritoneal cavity by antigen, eotaxin, or leukotriene(LTB4, a 5-lipoxygenase (5-LO metabolite. 5-LO blockade prevents responses to both antigen and eotaxin. We examined responses to eotaxin in the absence of sensitization and their dependence on 5-LO. BALB/c or PAS mice and their mutants (5-LO-deficient ALOX; eosinophil-deficient GATA-1 were injected i.p. with eotaxin, eosinophils, or both, and leukocyte accumulation was quantified up to 24 h. Significant recruitment of eosinophils by eotaxin in BALB/c, up to 24 h, was accompanied by much larger numbers of recruited neutrophils and monocytes/macrophages. These effects were abolished by eotaxin neutralization and 5-LO-activating protein inhibitor MK886. In ALOX (but not PAS mice, eotaxin recruitment was abolished for eosinophils and halved for neutrophils. In GATA-1 mutants, eotaxin recruited neither neutrophils nor macrophages. Transfer of eosinophils cultured from bone-marrow of BALB/c donors, or from ALOX donors, into GATA-1 mutant recipients, i.p., restored eotaxin recruitment of neutrophils and showed that the critical step dependent on 5-LO is the initial recruitment of eosinophils by eotaxin, not the secondary neutrophil accumulation. Eosinophil-dependent recruitment of neutrophils in naive BALB/c mice was associated with increased binding of bacteria.

  20. Hypertonic Saline Suppresses NADPH Oxidase-Dependent Neutrophil Extracellular Trap Formation and Promotes Apoptosis

    Directory of Open Access Journals (Sweden)

    Ajantha Nadesalingam

    2018-03-01

    Full Text Available Tonicity of saline (NaCl is important in regulating cellular functions and homeostasis. Hypertonic saline is administered to treat many inflammatory diseases, including cystic fibrosis. Excess neutrophil extracellular trap (NET formation, or NETosis, is associated with many pathological conditions including chronic inflammation. Despite the known therapeutic benefits of hypertonic saline, its underlying mechanisms are not clearly understood. Therefore, we aimed to elucidate the effects of hypertonic saline in modulating NETosis. For this purpose, we purified human neutrophils and induced NETosis using agonists such as diacylglycerol mimetic phorbol myristate acetate (PMA, Gram-negative bacterial cell wall component lipopolysaccharide (LPS, calcium ionophores (A23187 and ionomycin from Streptomyces conglobatus, and bacteria (Pseudomonas aeruginosa and Staphylococcus aureus. We then analyzed neutrophils and NETs using Sytox green assay, immunostaining of NET components and apoptosis markers, confocal microscopy, and pH sensing reagents. This study found that hypertonic NaCl suppresses nicotinamide adenine dinucleotide phosphate oxidase (NADPH2 or NOX2-dependent NETosis induced by agonists PMA, Escherichia coli LPS (0111:B4 and O128:B12, and P. aeruginosa. Hypertonic saline also suppresses LPS- and PMA- induced reactive oxygen species production. It was determined that supplementing H2O2 reverses the suppressive effect of hypertonic saline on NOX2-dependent NETosis. Many of the aforementioned suppressive effects were observed in the presence of equimolar concentrations of choline chloride and osmolytes (d-mannitol and d-sorbitol. This suggests that the mechanism by which hypertonic saline suppresses NOX2-dependent NETosis is via neutrophil dehydration. Hypertonic NaCl does not significantly alter the intracellular pH of neutrophils. We found that hypertonic NaCl induces apoptosis while suppressing NOX2-dependent NETosis. In contrast, hypertonic

  1. Cathepsin G-dependent modulation of platelet thrombus formation in vivo by blood neutrophils.

    Directory of Open Access Journals (Sweden)

    Nauder Faraday

    Full Text Available Neutrophils are consistently associated with arterial thrombotic morbidity in human clinical studies but the causal basis for this association is unclear. We tested the hypothesis that neutrophils modulate platelet activation and thrombus formation in vivo in a cathepsin G-dependent manner. Neutrophils enhanced aggregation of human platelets in vitro in dose-dependent fashion and this effect was diminished by pharmacologic inhibition of cathepsin G activity and knockdown of cathepsin G expression. Tail bleeding time in the mouse was prolonged by a cathepsin G inhibitor and in cathepsin G knockout mice, and formation of neutrophil-platelet conjugates in blood that was shed from transected tails was reduced in the absence of cathepsin G. Bleeding time was highly correlated with blood neutrophil count in wildtype but not cathepsin G deficient mice. In the presence of elevated blood neutrophil counts, the anti-thrombotic effect of cathepsin G inhibition was greater than that of aspirin and additive to it when administered in combination. Both pharmacologic inhibition of cathepsin G and its congenital absence prolonged the time for platelet thrombus to form in ferric chloride-injured mouse mesenteric arterioles. In a vaso-occlusive model of ischemic stroke, inhibition of cathepsin G and its congenital absence improved cerebral blood flow, reduced histologic brain injury, and improved neurobehavioral outcome. These experiments demonstrate that neutrophil cathepsin G is a physiologic modulator of platelet thrombus formation in vivo and has potential as a target for novel anti-thrombotic therapies.

  2. Selective inhibition of extracellular oxidants liberated from human neutrophils--A new mechanism potentially involved in the anti-inflammatory activity of hydroxychloroquine.

    Science.gov (United States)

    Jančinová, Viera; Pažoureková, Silvia; Lucová, Marianna; Perečko, Tomáš; Mihalová, Danica; Bauerová, Katarína; Nosáľ, Radomír; Drábiková, Katarína

    2015-09-01

    Hydroxychloroquine is used in the therapy of rheumatoid arthritis or lupus erythematosus. Although these diseases are often accompanied by activation of neutrophils, there are still few data relating to the impact of hydroxychloroquine on these cells. We investigated the effect of orally administered hydroxychloroquine on neutrophil oxidative burst in rats with adjuvant arthritis. In human neutrophils, extra- and intracellular formation of oxidants, mobilisation of intracellular calcium and the phosphorylation of proteins regulating NADPH oxidase assembly were analysed. Administration of hydroxychloroquine decreased the concentration of oxidants in blood of arthritic rats. The inhibition was comparable with the reference drug methotrexate, yet it was not accompanied by a reduction in neutrophil count. When both drugs were co-applied, the effect became more pronounced. In isolated human neutrophils, treatment with hydroxychloroquine resulted in reduced mobilisation of intracellular calcium, diminished concentration of external oxidants and in decreased phosphorylation of Ca(2+)-dependent protein kinase C isoforms PKCα and PKCβII, which regulate activation of NADPH oxidase on plasma membrane. On the other hand, no reduction was observed in intracellular oxidants or in the phosphorylation of p40(phox) and PKCδ, two proteins directing the oxidase assembly to intracellular membranes. Hydroxychloroquine reduced neutrophil-derived oxidants potentially involved in tissue damage and protected those capable to suppress inflammation. The observed effects may represent a new mechanism involved in the anti-inflammatory activity of this drug. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. 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.

  4. СHANGES IN PARAMETERS OF LUMINOL-DEPENDENT AND LUCIGENIN-DEPENDENT CHEMILUMINESCENCE OF PERIPHERAL BLOOD NEUTROPHILS IN PATIENTS WITH BLADDER CANCER IN THE DISEASE DYNAMICS

    Directory of Open Access Journals (Sweden)

    L. M. Kurtasova

    2015-01-01

    Full Text Available The study deals with parameters of luminol-dependent and lucigenin-dependent chemiluminescence (CL of peripheral blood neutrophils from patients with bladder cancer (BC prior to surgical treatment. We examined sixty patients (45 to 55 years old with advanced bladder cancer (TNM prior to the operation, and forty-six patients at 10 days after surgical treatment. A control group consisted of 56 healthy donors. Luminol-dependent and lucigenin-dependent chemiluminescence of blood neutrophils was assessed according to De Sole et al. (1983. Chemiluminescence assays of peripheral blood neutrophils from the patients with bladder cancer revealed changes in production of reactive oxygen species (ROS, both for initial stage of oxidation reaction, and total level of active oxygen radicals. We have found disturbed values of primary-to-secondary ROS ratio in the cells. In the patients with bladder cancer, some changes in oxidative metabolism of the blood neutrophils have been registered. These alterations may play an important role in promotion of potential effector cell functions, thus, probably, affecting the whole-scale development of a cytopathic effect exerted by neutrophilic granulocytes. 

  5. Proteomic Analysis of Calcium- and Phosphorylation-dependentCalmodulin Complexes in Mammalian Cells

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Deok-Jin; Wang, Daojing

    2006-05-26

    Protein conformational changes due to cofactor binding (e.g. metal ions, heme) and/or posttranslational modifications (e.g. phosphorylation) modulate dynamic protein complexes. Calmodulin (CaM) plays an essential role in regulating calcium (Ca{sup 2+}) signaling and homeostasis. No systematic approach on the identification of phosphorylation-dependent Ca{sup 2+}/CaM binding proteins has been published. Herein, we report a proteome-wide study of phosphorylation-dependent CaM binding proteins from mammalian cells. This method, termed 'Dynamic Phosphoprotein Complex Trapping', 'DPPC Trapping' for short, utilizes a combination of in vivo and in vitro assays. The basic strategy is to drastically shift the equilibrium towards endogenous phosphorylation of Ser, Thr, and Tyr at the global scale by inhibiting corresponding phosphatases in vivo. The phosphorylation-dependent calmodulin-binding proteins are then trapped in vitro in a Ca{sup 2+}-dependent manner by CaM-Sepharose chromatography. Finally, the isolated calmodulin-binding proteins are separated by SDS-PAGE and identified by LC/MS/MS. In parallel, the phosphorylation-dependent binding is visualized by silver staining and/or Western blotting. Using this method, we selectively identified over 120 CaM-associated proteins including many previously uncharacterized. We verified ubiquitin-protein ligase EDD1, inositol 1, 4, 5-triphosphate receptor type 1 (IP{sub 3}R1), and ATP-dependent RNA helicase DEAD box protein 3 (DDX3), as phosphorylation-dependent CaM binding proteins. To demonstrate the utilities of our method in understanding biological pathways, we showed that pSer/Thr of IP{sub 3}R1 in vivo by staurosporine-sensitive kinase(s), but not by PKA/PKG/PKC, significantly reduced the affinity of its Ca{sup 2+}-dependent CaM binding. However, pSer/Thr of IP{sub 3}R1 did not substantially affect its Ca{sup 2+}-independent CaM binding. We further showed that phosphatase PP1, but not PP2A or PP2B

  6. CARD9-Dependent Neutrophil Recruitment Protects against Fungal Invasion of the Central Nervous System.

    Directory of Open Access Journals (Sweden)

    Rebecca A Drummond

    2015-12-01

    Full Text Available Candida is the most common human fungal pathogen and causes systemic infections that require neutrophils for effective host defense. Humans deficient in the C-type lectin pathway adaptor protein CARD9 develop spontaneous fungal disease that targets the central nervous system (CNS. However, how CARD9 promotes protective antifungal immunity in the CNS remains unclear. Here, we show that a patient with CARD9 deficiency had impaired neutrophil accumulation and induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid despite uncontrolled CNS Candida infection. We phenocopied the human susceptibility in Card9-/- mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected Card9-/- brains, from both myeloid and resident glial cellular sources, whereas cell-intrinsic neutrophil chemotaxis is Card9-independent. Taken together, our data highlight the critical role of CARD9-dependent neutrophil trafficking into the CNS and provide novel insight into the CNS fungal susceptibility of CARD9-deficient humans.

  7. 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

  8. Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

    KAUST Repository

    Bigeard, Jean; Rayapuram, Naganand; Pflieger, Delphine; Hirt, Heribert

    2014-01-01

    In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

  9. Phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins

    KAUST Repository

    Bigeard, Jean

    2014-07-10

    In eukaryotes, most of the DNA is located in the nucleus where it is organized with histone proteins in a higher order structure as chromatin. Chromatin and chromatin-associated proteins contribute to DNA-related processes such as replication and transcription as well as epigenetic regulation. Protein functions are often regulated by PTMs among which phosphorylation is one of the most abundant PTM. Phosphorylation of proteins affects important properties, such as enzyme activity, protein stability, or subcellular localization. We here describe the main specificities of protein phosphorylation in plants and review the current knowledge on phosphorylation-dependent regulation of plant chromatin and chromatin-associated proteins. We also outline some future challenges to further elucidate protein phosphorylation and chromatin regulation.

  10. 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.

  11. Phosphorylation of purified mitochondrial Voltage-Dependent Anion Channel by c-Jun N-terminal Kinase-3 modifies channel voltage-dependence

    Directory of Open Access Journals (Sweden)

    Rajeev Gupta

    2017-06-01

    Full Text Available Voltage-Dependent Anion Channel (VDAC phosphorylated by c-Jun N-terminal Kinase-3 (JNK3 was incorporated into the bilayer lipid membrane. Single-channel electrophysiological properties of the native and the phosphorylated VDAC were compared. The open probability versus voltage curve of the native VDAC displayed symmetry around the voltage axis, whereas that of the phosphorylated VDAC showed asymmetry. This result indicates that phosphorylation by JNK3 modifies voltage-dependence of VDAC.

  12. Phosphorylation of purified mitochondrial Voltage-Dependent Anion Channel by c-Jun N-terminal Kinase-3 modifies channel voltage-dependence.

    Science.gov (United States)

    Gupta, Rajeev; Ghosh, Subhendu

    2017-06-01

    Voltage-Dependent Anion Channel (VDAC) phosphorylated by c-Jun N-terminal Kinase-3 (JNK3) was incorporated into the bilayer lipid membrane. Single-channel electrophysiological properties of the native and the phosphorylated VDAC were compared. The open probability versus voltage curve of the native VDAC displayed symmetry around the voltage axis, whereas that of the phosphorylated VDAC showed asymmetry. This result indicates that phosphorylation by JNK3 modifies voltage-dependence of VDAC.

  13. Modulation of oxygen-dependent and oxygen-independent metabolism of neutrophilic granulocytes by quantum points.

    Science.gov (United States)

    Pleskova, S N; Mikheeva, E R

    2011-08-01

    Inhibition of neutrophilic granulocyte metabolism under the effect of semiconductor quantum points was demonstrated. The status of the oxidative system was evaluated by the NBT test, nonoxidative status by the lysosomal cationic test. It was found that quantum points in a dose of 0.1 mg/ml irrespective of their core and composition of coating significantly inhibited oxygen-dependent and oxygen-independent metabolism of neutrophilic granulocytes.

  14. Priming of the neutrophil NADPH oxidase activation: role of p47phox phosphorylation and NOX2 mobilization to the plasma membrane.

    Science.gov (United States)

    El-Benna, Jamel; Dang, Pham My-Chan; Gougerot-Pocidalo, Marie-Anne

    2008-07-01

    Neutrophils play an essential role in host defense against microbial pathogens and in the inflammatory reaction. Upon activation, neutrophils produce superoxide anion (O*2), which generates other reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical (OH*) and hypochlorous acid (HOCl), together with microbicidal peptides and proteases. The enzyme responsible for O2* production is called the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of two trans-membrane proteins (p22phox and gp91phox/NOX2, which form the cytochrome b558), three cytosolic proteins (p47phox, p67phox, p40phox) and a GTPase (Rac1 or Rac2), which assemble at membrane sites upon cell activation. NADPH oxidase activation in phagocytes can be induced by a large number of soluble and particulate factors. Three major events accompany NAPDH oxidase activation: (1) protein phosphorylation, (2) GTPase activation, and (3) translocation of cytosolic components to the plasma membrane to form the active enzyme. Actually, the neutrophil NADPH oxidase exists in different states: resting, primed, activated, or inactivated. The resting state is found in circulating blood neutrophils. The primed state can be induced by neutrophil adhesion, pro-inflammatory cytokines, lipopolysaccharide, and other agents and has been characterized as a "ready to go" state, which results in a faster and higher response upon exposure to a second stimulus. The active state is found at the inflammatory or infection site. Activation is induced by the pathogen itself or by pathogen-derived formylated peptides and other agents. Finally, inactivation of NADPH oxidase is induced by anti-inflammatory agents to limit inflammation. Priming is a "double-edged sword" process as it contributes to a rapid and efficient elimination of the pathogens but can also induce the generation of large quantities of toxic ROS by hyperactivation of

  15. Th-17 regulatory cytokines IL-21, IL-23, and IL-6 enhance neutrophil production of IL-17 cytokines during asthma.

    Science.gov (United States)

    Halwani, Rabih; Sultana, Asma; Vazquez-Tello, Alejandro; Jamhawi, Amer; Al-Masri, Abeer A; Al-Muhsen, Saleh

    2017-11-01

    In a subset of severe asthma patients, chronic airway inflammation is associated with infiltration of neutrophils, Th-17 cells and elevated expression of Th-17-derived cytokines (e.g., interleukin [IL]-17, IL-21, IL-22). Peripheral neutrophils from allergic asthmatics are known to express higher IL-17 cytokine levels than those from healthy subjects, but the regulatory mechanisms involved are not well understood. We hypothesize that Th-17 regulatory cytokines could modulate IL-17 expression in neutrophils. Peripheral blood neutrophils isolated from asthmatics were stimulated with IL-21, IL-23, and IL-6 cytokines and their ability to produce IL-17A and IL-17F was determined relative to healthy controls. Signal transducer and activator of transcription 3 (STAT3) phosphorylation levels were measured in stimulated neutrophil using flow cytometry. The requirement for STAT3 phosphorylation was determined by blocking its activation using a specific chemical inhibitor. Stimulating asthmatic neutrophils with IL-21, 23, and 6 enhanced the production of IL-17A and IL-17F at significantly higher levels comparatively to healthy controls. Stimulating neutrophils with IL-21, IL-23, and IL-6 cytokines enhanced STAT3 phosphorylation, in all cases. Interestingly, inhibiting STAT3 phosphorylation using a specific chemical inhibitor dramatically blocked the ability of neutrophils to produce IL-17, demonstrating that STAT3 activation is the major factor mediating IL-17 gene expression. These findings suggest that neutrophil infiltration in lungs of severe asthmatics may represent an important source of pro-inflammatory IL-17A and -F cytokines, a production enhanced by Th-17 regulatory cytokines, and thus providing a feedback mechanism that sustains inflammation. Our results suggest that STAT3 pathway could be a potential target for regulating neutrophilic inflammation during severe asthma.

  16. A novel bacterial transport mechanism of Acinetobacter baumannii via activated human neutrophils through interleukin-8.

    Science.gov (United States)

    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.

  17. The Extracellular Matrix of Candida albicans Biofilms Impairs Formation of Neutrophil Extracellular Traps.

    Science.gov (United States)

    Johnson, Chad J; Cabezas-Olcoz, Jonathan; Kernien, John F; Wang, Steven X; Beebe, David J; Huttenlocher, Anna; Ansari, Hamayail; Nett, Jeniel E

    2016-09-01

    Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix.

  18. Linear motif atlas for phosphorylation-dependent signaling

    DEFF Research Database (Denmark)

    Miller, Martin Lee; Jensen, LJ; Diella, F

    2008-01-01

    bind to them remains a challenge. NetPhorest is an atlas of consensus sequence motifs that covers 179 kinases and 104 phosphorylation-dependent binding domains [Src homology 2 (SH2), phosphotyrosine binding (PTB), BRCA1 C-terminal (BRCT), WW, and 14-3-3]. The atlas reveals new aspects of signaling...

  19. 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.

  20. 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.

  1. Ca(2+)-calmodulin-dependent phosphorylation of islet secretory granule proteins

    International Nuclear Information System (INIS)

    Watkins, D.T.

    1991-01-01

    The effect of Ca2+ and calmodulin on phosphorylation of islet secretory granule proteins was studied. Secretory granules were incubated in a phosphorylation reaction mixture containing [32P]ATP and test reagents. The 32P-labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 32P content was visualized by autoradiography, and the relative intensities of specific bands were quantitated. When the reaction mixture contained EGTA and no added Ca2+, 32P was incorporated into two proteins with molecular weights of 45,000 and 13,000. When 10(-4) M Ca2+ was added without EGTA, two additional proteins (58,000 and 48,000 Mr) were phosphorylated, and the 13,000-Mr protein was absent. The addition of 2.4 microM calmodulin markedly enhanced the phosphorylation of the 58,000- and 48,000-Mr proteins and resulted in the phosphorylation of a major protein whose molecular weight (64,000 Mr) is identical to that of one of the calmodulin binding proteins located on the granule surface. Calmodulin had no effect on phosphorylation in the absence of Ca2+ but was effective in the presence of calcium between 10 nM and 50 microM. Trifluoperazine and calmidazolium, calmodulin antagonists, produced a dose-dependent inhibition of the calmodulin effect. 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester that activates protein kinase C, produced no increase in phosphorylation, and 1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride, an inhibitor of protein kinase C, had no effect. These results indicate that Ca(2+)-calmodulin-dependent protein kinases and endogenous substrates are present in islet secretory granules

  2. Neutrophil transmigration mediated by the neutrophil-specific antigen CD177 is influenced by the endothelial S536N dimorphism of platelet endothelial cell adhesion molecule-1.

    Science.gov (United States)

    Bayat, Behnaz; Werth, Silke; Sachs, Ulrich J H; Newman, Debra K; Newman, Peter J; Santoso, Sentot

    2010-04-01

    The human neutrophil-specific adhesion molecule CD177 (also known as the NB1 alloantigen) becomes upregulated on the cell surface in a number of inflammatory settings. We recently showed that CD177 functions as a novel heterophilic counterreceptor for the endothelial junctional protein PECAM-1 (CD31), an interaction that is mediated by membrane-proximal PECAM-1 IgD 6, which is known to harbor an S(536)N single nucleotide polymorphism of two major isoforms V(98)N(536)G(643) and L(98)S(536)R(643) and a yet-to-be-determined region on CD177. In vitro transendothelial migration experiments revealed that CD177(+) neutrophils migrated significantly faster through HUVECs expressing the LSR, compared with the VNG, allelic variant of PECAM-1 and that this correlated with the decreased ability of anti-PECAM-1 Ab of ITIM tyrosine phosphorylation in HUVECs expressing the LSR allelic variant relative to the VNG allelic variant. Moreover, engagement of PECAM-1 with rCD177-Fc (to mimic heterophilic CD177 binding) suppressed Ab-induced tyrosine phosphorylation to a greater extent in cells expressing the LSR isoform compared with the VNG isoform, with a corresponding increased higher level of beta-catenin phosphorylation. These data suggest that heterophilic PECAM-1/CD177 interactions affect the phosphorylation state of PECAM-1 and endothelial cell junctional integrity in such a way as to facilitate neutrophil transmigration in a previously unrecognized allele-specific manner.

  3. The Natural Stilbenoid Piceatannol Decreases Activity and Accelerates Apoptosis of Human Neutrophils: Involvement of Protein Kinase C

    Directory of Open Access Journals (Sweden)

    Viera Jancinova

    2013-01-01

    Full Text Available Neutrophils are able to release cytotoxic substances and inflammatory mediators, which, along with their delayed apoptosis, have a potential to maintain permanent inflammation. Therefore, treatment of diseases associated with chronic inflammation should be focused on neutrophils; formation of reactive oxygen species and apoptosis of these cells represent two promising targets for pharmacological intervention. Piceatannol, a naturally occurring stilbenoid, has the ability to reduce the toxic action of neutrophils. This substance decreased the amount of oxidants produced by neutrophils both extra- and intracellularly. Radicals formed within neutrophils (fulfilling a regulatory role were reduced to a lesser extent than extracellular oxidants, potentially dangerous for host tissues. Moreover, piceatannol did not affect the phosphorylation of p40phox—a component of NADPH oxidase, responsible for the assembly of functional oxidase in intracellular (granular membranes. The stilbenoid tested elevated the percentage of early apoptotic neutrophils, inhibited the activity of protein kinase C (PKC—the main regulatory enzyme in neutrophils, and reduced phosphorylation of PKC isoforms α, βII, and δ on their catalytic region. The results indicated that piceatannol may be useful as a complementary medicine in states associated with persisting neutrophil activation and with oxidative damage of tissues.

  4. Human neutrophil peptide-1 promotes alcohol-induced hepatic fibrosis and hepatocyte apoptosis.

    Directory of Open Access Journals (Sweden)

    Rie Ibusuki

    Full Text Available Neutrophil infiltration of the liver is a typical feature of alcoholic liver injury. Human neutrophil peptide (HNP-1 is an antimicrobial peptide secreted by neutrophils. The aim of this study was to determine if HNP-1 affects ethanol-induced liver injury and to examine the mechanism of liver injury induced by HNP-1.Transgenic (TG mice expressing HNP-1 under the control of a β-actin-based promoter were established. Ethanol was orally administered to HNP-1 TG or wild-type C57BL/6N (WT mice. SK-Hep1 hepatocellular carcinoma cells were used to investigate the effect of HNP-1 on hepatocytes in vitro.After 24 weeks of ethanol intake, hepatic fibrosis and hepatocyte apoptosis were significantly more severe in TG mice than in WT mice. Levels of CD14, TLR4, and IL-6 in liver tissues were higher in TG mice than in WT mice. Apoptosis was accompanied by higher protein levels of caspase-3, caspase-8, and cleaved PARP in liver tissue. In addition, phosphorylated ASK1, ASK1, phosphorylated JNK, JNK1, JNK2, Bax, Bak and Bim were all more abundant in TG mice than in WT mice. In contrast, the level of anti-apoptotic Bcl2 in the liver was significantly lower in TG mice than in WT mice. Analysis of microRNAs in liver tissue showed that miR-34a-5p expression was significantly higher in TG mice than in WT mice. Furthermore, in the presence of ethanol, HNP-1 increased the apoptosis with the decreased level of Bcl2 in a concentration-dependent manner in vitro.HNP-1 secreted by neutrophils may exacerbate alcohol-induced hepatic fibrosis and hepatocyte apoptosis with a decrease in Bcl2 expression and an increase in miR-34a-5p expression.

  5. Decreased activity of neutrophils in the presence of diferuloylmethane (curcumin) involves protein kinase C inhibition.

    Science.gov (United States)

    Jancinová, Viera; Perecko, Tomás; Nosál, Radomír; Kostálová, Daniela; Bauerová, Katarína; Drábiková, Katarína

    2009-06-10

    Diferuloylmethane (curcumin) has been shown to act beneficially in arthritis, particularly through downregulated expression of proinflammatory cytokines and collagenase as well as through the modulated activities of T lymphocytes and macrophages. In this study its impact on activated neutrophils was investigated both in vitro and in experimental arthritis. Formation of reactive oxygen species in neutrophils was recorded on the basis of luminol- or isoluminol-enhanced chemiluminescence. Phosphorylation of neutrophil protein kinases C alpha and beta II was assessed by Western blotting, using phosphospecific antibodies. Adjuvant arthritis was induced in Lewis rats by heat-killed Mycobacterium butyricum. Diferuloylmethane or methotrexate was administered over a period of 28 days after arthritis induction. Under in vitro conditions, diferuloylmethane (1-100 microM) reduced dose-dependently oxidant formation both at extra- and intracellular level and it effectively reduced protein kinase C activation. Adjuvant arthritis was accompanied by an increased number of neutrophils in blood and by a more pronounced spontaneous as well as PMA (phorbol myristate acetate) stimulated chemiluminescence. Whereas the arthritis-related alterations in neutrophil count and in spontaneous chemiluminescence were not modified by diferuloylmethane, the increased reactivity of neutrophils to PMA was less evident in diferuloylmethane-treated animals. The effects of diferuloylmethane were comparable with those of methotrexate. Diferuloylmethane was found to be a potent inhibitor of neutrophil functions both in vitro and in experimental arthritis. As neutrophils are considered to be cells with the greatest capacity to inflict damage within diseased joints, the observed effects could represent a further mechanism involved in the antirheumatic activity of diferuloylmethane.

  6. Ly6G-mediated depletion of neutrophils is dependent on macrophages.

    Science.gov (United States)

    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.

  7. Suppression of neutrophil accumulation in mice by cutaneous application of geranium essential oil

    Directory of Open Access Journals (Sweden)

    Oshima Haruyuki

    2005-02-01

    Full Text Available Abstract Background Previous studies suggested that essential oils suppressed the adherence response of human neutrophils in vitro and that intraperitoneal application of geranium oil suppressed the neutrophil accumulation into peritoneal cavity in vivo. Usually, essential oils are applied through skin in aromatherapy in inflammatory symptoms. The purpose of this study is to assess the effects of cutaneous application of essential oils on the accumulation of neutrophils in inflammatory sites in skin of mice. Methods Inflammation with accumulation of inflammatory cells was induced by injection of curdlan, a (1→3-β-D-glucan in skin or peritoneal cavity of mice. Essential oils were applied cutaneously to the mice immediately and 3 hr after intradermal injection of curdlan. The skin with inflammatory lesion was cut off 6 hr after injection of curdlan, and the homogenates were used for myeloperoxidase (MPO: a marker enzyme of neutrophil granule assay. Results The MPO activity of the skin lesion induced by curdlan was suppressed dose-dependently by cutaneous application of geranium oil. Other oils such as lavender, eucalyptus and tea tree oils also suppressed the activity, but their activities seemed weaker than geranium. Juniper oil didn't suppress the activity Conclusion Cutaneous application of essential oils, especially geranium oil, can suppress the inflammatory symptoms with neutrophil accumulation and edema.

  8. Neutrophil depletion improves diet-induced non-alcoholic fatty liver disease in mice.

    Science.gov (United States)

    Ou, Rongying; Liu, Jia; Lv, Mingfen; Wang, Jingying; Wang, Jinmeng; Zhu, Li; Zhao, Liang; Xu, Yunsheng

    2017-07-01

    Non-alcoholic fatty liver disease is highly associated with morbidity and mortality in population. Although studies have already demonstrated that the immune response plays a pivotal role in the development of non-alcoholic fatty liver disease, the comprehensive regulation is unclear. Therefore, present study was carried out to investigate the non-alcoholic fatty liver disease development under neutrophil depletion. To achieve the aim of the study, C57BL/6 J mice were fed with high fat diet for 6 weeks before treated with neutrophil deplete antibody 1A8 or isotype control (200 μg/ mouse every week) for another 4 weeks. Treated with 1A8 antibody, obese mice exhibited better whole body metabolic parameters, including reduction of body weight gain and fasting blood glucose levels. Neutrophil depletion also effectively reduced hepatic structural disorders, dysfunction and lipid accumulation. Lipid β-oxidative markers, phosphorylated-AMP-activated protein kinase α and phosphorylated-acetyl-CoA carboxylase levels were increased in 1A8 antibody-treated obese mouse group. The mitochondrial number and function were also reversed after 1A8 antibody treatment, including increased mitochondrial number, reduced lipid oxidative damage and enhanced mitochondrial activity. Furthermore, the expression of inflammatory cytokines, tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1 were obviously reduced after neutrophil depletion, accompanied with decreased F4/80 mRNA level and macrophage percentage in liver. The decreased NF-κB signaling activity was also involved in the beneficial effect of neutrophil depletion. Taken together, neutrophil depletion could attenuate metabolic syndromes and hepatic dysfunction.

  9. Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases

    DEFF Research Database (Denmark)

    Issinger, O G; Beier, H; Speichermann, N

    1980-01-01

    Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were...... by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins...... from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates...

  10. Neutrophils alter the inflammatory milieu by signal-dependent translation of constitutive messenger RNAs

    Science.gov (United States)

    Lindemann, Stephan W.; Yost, Christian C.; Denis, Melvin M.; McIntyre, Thomas M.; Weyrich, Andrew S.; Zimmerman, Guy A.

    2004-05-01

    The mechanisms by which neutrophils, key effector cells of the innate immune system, express new gene products in inflammation are largely uncharacterized. We found that they rapidly translate constitutive mRNAs when activated, a previously unrecognized response. One of the proteins synthesized without a requirement for transcription is the soluble IL-6 receptor , which translocates to endothelial cells and induces a temporal switch to mononuclear leukocyte recruitment. Its synthesis is regulated by a specialized translational control pathway that is inhibited by rapamycin, a bacterial macrolide with therapeutic efficacy in transplantation, inflammatory syndromes, and neoplasia. Signal-dependent translation in activated neutrophils may be a critical mechanism for alteration of the inflammatory milieu and a therapeutic target.

  11. The atherogenic bacterium Porphyromonas gingivalis evades circulating phagocytes by adhering to erythrocytes

    DEFF Research Database (Denmark)

    Belstrøm, Daniel; Holmstrup, Palle; Damgaard, Christian

    2011-01-01

    A relationship between periodontitis and coronary heart disease has been investigated intensively. A pathogenic role for the oral bacterium Porphyromonas gingivalis has been suggested for both diseases. We examined whether complement activation by P. gingivalis strain ATCC 33277 allows...... the bacterium to adhere to human red blood cells (RBCs) and thereby evade attack by circulating phagocytes. On incubation with normal human serum, the P. gingivalis strain efficiently fixed complement component 3 (C3). Incubation of bacteria with washed whole blood cells suspended in autologous serum resulted...... in a dose- and time-dependent adherence to RBCs. The adherence required functionally intact complement receptor 1 (CR1; also called CD35) on the RBCs and significantly inhibited the uptake of P. gingivalis by neutrophils and B cells within 1 min of incubation (by 64% and 51%, respectively...

  12. 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.

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

    OpenAIRE

    1980-01-01

    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 macrophag...

  14. Hypercholesterolemia Increases the Production of Leukotriene B4 in Neutrophils by Enhancing the Nuclear Localization of 5-Lipoxygenase

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    Xiao-Feng Lai

    2014-11-01

    Full Text Available Aims: Neutrophils can synthesize leukotriene B4 (LTB4 by activating the 5-lipoxygenase (5-LOsignaling pathway. LTB4 is a pro-inflammatory mediator associated with the etiology and progression of atherosclerosis. It can increase function and number of neutrophils in an autocrine manner. Since hypercholesterolemia is associated with an increase in the number and function of neutrophils, we hypothesized that this effect could be mediated through increased production of LTB4 in neutrophils. Methods/Results: Hypercholesterolemia was modeled in Wistar rats by feeding them with a high cholesterol diet. The induction of hypercholesterolemia caused an increase in the plasma levels of LTB4, following lipopolysaccharide stimulation. This effect was recapitulated in vitro, both in the presence and absence of stimulation with the activator of 5-LO, A23187. Neutrophils in hypercholesterolemia rats expressed similar total levels of 5-LO as control rats, but displayed increased nuclear localization of 5-LO, as well as elevated levels of phosphorylated 5-LO and ERK1/2. In vitro, MβCD/cholesterol complexes enriched cholesterol in neutrophils, resulted in similar changes in 5-LO/LTB4. In addition, these alterations could be inhibited with the ERK inhibitor PD98059. Conclusion: Hypercholesterolemia increases LTB4 production in neutrophils by increasing the nuclear localization of 5-LO, which is the result of its phosphorylation by activated ERK1/2.

  15. Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo

    Science.gov (United States)

    von Brühl, Marie-Luise; Stark, Konstantin; Steinhart, Alexander; Chandraratne, Sue; Konrad, Ildiko; Lorenz, Michael; Khandoga, Alexander; Tirniceriu, Anca; Coletti, Raffaele; Köllnberger, Maria; Byrne, Robert A.; Laitinen, Iina; Walch, Axel; Brill, Alexander; Pfeiler, Susanne; Manukyan, Davit; Braun, Siegmund; Lange, Philipp; Riegger, Julia; Ware, Jerry; Eckart, Annekathrin; Haidari, Selgai; Rudelius, Martina; Schulz, Christian; Echtler, Katrin; Brinkmann, Volker; Schwaiger, Markus; Preissner, Klaus T.; Wagner, Denisa D.; Mackman, Nigel; Engelmann, Bernd

    2012-01-01

    Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation through the release of neutrophil extracellular traps (NETs). Correspondingly, neutropenia, genetic ablation of FXII, or disintegration of NETs each confers protection against DVT amplification. Platelets associate with innate immune cells via glycoprotein Ibα and contribute to DVT progression by promoting leukocyte recruitment and stimulating neutrophil-dependent coagulation. Hence, we identified a cross talk between monocytes, neutrophils, and platelets responsible for the initiation and amplification of DVT and for inducing its unique clinical features. PMID:22451716

  16. Neither eosinophils nor neutrophils require ATG5-dependent autophagy for extracellular DNA trap formation.

    Science.gov (United States)

    Germic, Nina; Stojkov, Darko; Oberson, Kevin; Yousefi, Shida; Simon, Hans-Uwe

    2017-11-01

    The importance of extracellular traps (ETs) in innate immunity is well established, but the molecular mechanisms responsible for their formation remain unclear and in scientific dispute. ETs have been defined as extracellular DNA scaffolds associated with the granule proteins of eosinophils or neutrophils. They are capable of killing bacteria extracellularly. Based mainly on results with phosphoinositide 3-kinase (PI3K) inhibitors such as 3-methyladenine (3-MA) and wortmannin, which are commonly used to inhibit autophagy, several groups have reported that autophagy is required for neutrophil extracellular trap (NET) formation. We decided to investigate this apparent dependence on autophagy for ET release and generated genetically modified mice that lack, specifically in eosinophils or neutrophils, autophagy-related 5 (Atg5), a gene encoding a protein essential for autophagosome formation. Interestingly, neither eosinophils nor neutrophils from Atg5-deficient mice exhibited abnormalities in ET formation upon physiological activation or exposure to low concentrations of PMA, although we could confirm that human and mouse eosinophils and neutrophils, after pre-treatment with inhibitors of class III PI3K, show a block both in reactive oxygen species (ROS) production and in ET formation. The so-called late autophagy inhibitors bafilomycin A1 and chloroquine, on the other hand, were without effect. These data indicate that ET formation occurs independently of autophagy and that the inhibition of ROS production and ET formation in the presence of 3-MA and wortmannin is probably owing to their additional ability to block the class I PI3Ks, which are involved in signalling cascades initiated by triggers of ET formation. © 2017 John Wiley & Sons Ltd.

  17. 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

  18. Phosphorylation of plasma membrane aquaporin regulates temperature-dependent opening of tulip petals.

    Science.gov (United States)

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2004-05-01

    The opening and closing of tulip petals was reproduced in the dark by changing the temperature from 5 degrees C to 20 degrees C for opening and 20 degrees C to 5 degrees C for closing. The opening process was accompanied by (3)H(2)O transport through the stem from the incubation medium to the petals. A Ca(2+)-channel blocker and a Ca(2+)-chelator inhibited petal opening and (3)H(2)O transport. Several proteins in the isolated plasma membrane fraction were phosphorylated in the presence of 25 micro M Ca(2+) at 20 degrees C. The 31-kDa protein that was phosphorylated, was suggested immunologically as the putative plasma membrane aquaporin (PM-AQP). This phosphorylated PM-AQP clearly reacted with the anti-phospho-Ser. In-gel assay revealed the presence of a 45-kDa Ca(2+)-dependent protein kinase in the isolated plasma membrane. Phosphorylation of the putative PM-AQP was thought to activate the water channel composed of PM-AQP. Dephosphorylation of the phosphorylated PM-AQP was also observed during petal closing at 5 degrees C, suggesting the inactivation of the water channel.

  19. Activation of Triggering Receptor Expressed on Myeloid Cells-1 on Human Neutrophils by Marburg and Ebola Viruses

    National Research Council Canada - National Science Library

    Mohamadzadeh, Mansour; Coberley, Sadie S; Olinger, Gene G; Kalina, Warren V; Ruthel, Gordon; Fullter, Claudette L; Swenson, Dana L; Pratt, William D; Kuhns, Douglas B; Schmaljohn, Alan L

    2006-01-01

    .... Here, we report that MARV and EBOV activate TREM-1 on human neutrophils, resulting in DAP12 phosphorylation, TREM-1 shedding, mobilization of intracellular calcium, secretion of proinflammatory...

  20. Hydrogen sulfide reduces neutrophil recruitment in hind-limb ischemia-reperfusion injury in an L-selectin and ADAM-17 dependent manner

    Science.gov (United States)

    Ball, Carissa J.; Reiffel, Alyssa J.; Chintalapani, Sathvika; Kim, Minsoo; Spector, Jason A.; King, Michael R.

    2012-01-01

    Background Reperfusion following ischemia leads to neutrophil recruitment injured tissue. Selectins and β2 integrins regulate neutrophil interaction with the endothelium during neutrophil rolling and firm adhesion. Excessive neutrophil infiltration into tissue is thought to contribute to IRI damage. NaHS mitigates the damage caused by ischemia-reperfusion injury (IRI). This study's objective was to determine the effect of hydrogen sulfide (NaHS) on neutrophil adhesion receptor expression. Methods Human neutrophils were either left untreated or incubated in 20 μM NaHS, and/or 50 μg/mL pharmacological ADAM-17 inhibitor TAPI-0; activated by IL-8, fMLP, or TNF-α; and labeled against PSGL-1, LFA-1, Mac-1 α, L-selectin and β2 integrin epitopes CBRM1/5 or KIM127 for flow cytometry. Cohorts of 3 C57BL/6 mice received an intravenous dose of saline vehicle, or 20 μM NaHS with or without 50 μg/mL TAPI-0 before unilateral tourniquet induced hind-limb ischemia for 3 hours followed by 3 hours of reperfusion. Bilateral gastrocnemius muscles were processed for histology before neutrophil infiltration quantification. Results NaHS treatment significantly increased L-selectin shedding from human neutrophils following activation by fMLP and IL-8 in an ADAM-17 dependent manner. Mice treated with NaHS to raise bloodstream concentration by 20 μM prior to ischemia or reperfusion showed a significant reduction in neutrophil recruitment into skeletal muscle tissue following tourniquet-induced hindlimb IRI. Conclusions NaHS administration results in the downregulation of L-selectin expression in activated human neutrophils. This leads to a reduction in neutrophil extravasation and tissue infiltration and may partially account for the protective effects of NaHS seen in the setting of IRI. PMID:23446563

  1. The selective estrogen receptor modulator raloxifene inhibits neutrophil extracellular trap formation.

    Directory of Open Access Journals (Sweden)

    Roxana Flores

    2016-12-01

    Full Text Available Raloxifene is a selective estrogen receptor modulator typically prescribed for the prevention/treatment of osteoporosis in postmenopausal women. Although raloxifene is known to have anti-inflammatory properties, its effect on human neutrophils, the primary phagocytic leukocytes of the immune system, remain poorly understood. Here, through a screen of pharmacologically active small molecules, we find that raloxifene prevents neutrophil cell death in response to the classical activator phorbol 12-myristate 13-acetate (PMA, a compound known to induce formation of DNA-based neutrophil extracellular traps (NETs. Inhibition of PMA-induced NET production by raloxifene was confirmed using quantitative and imaging-based assays. Human neutrophils from both male and female donors express the nuclear estrogen receptors ERα and ERβ, known targets of raloxifene. Like raloxifene, selective antagonists of these receptors inhibit PMA-induced NET production. Furthermore, raloxifene inhibited PMA-induced ERK phosphorylation but not reactive oxygen species (ROS production, pathways known to be key modulators of NET production. Finally, we found that raloxifene inhibited PMA-induced, NET-based killing of the leading human bacterial pathogen, methicillin-resistant Staphylococcus aureus (MRSA. Our results reveal that raloxifene is a potent modulator of neutrophil function and NET production.

  2. 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

  3. Osthol attenuates neutrophilic oxidative stress and hemorrhagic shock-induced lung injury via inhibition of phosphodiesterase 4.

    Science.gov (United States)

    Tsai, Yung-Fong; Yu, Huang-Ping; Chung, Pei-Jen; Leu, Yann-Lii; Kuo, Liang-Mou; Chen, Chun-Yu; Hwang, Tsong-Long

    2015-12-01

    Oxidative stress caused by neutrophils is an important pathogenic factor in trauma/hemorrhagic (T/H)-induced acute lung injury (ALI). Osthol, a natural coumarin found in traditional medicinal plants, has therapeutic potential in various diseases. However, the pharmacological effects of osthol in human neutrophils and its molecular mechanism of action remain elusive. In this study, our data showed that osthol potently inhibited the production of superoxide anion (O2(•-)) and reactive oxidants derived therefrom as well as expression of CD11b in N-formylmethionylleucylphenylalanine (FMLP)-activated human neutrophils. However, osthol inhibited neutrophil degranulation only slightly and it failed to inhibit the activity of subcellular NADPH oxidase. FMLP-induced phosphorylation of extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) was inhibited by osthol. Notably, osthol increased the cAMP concentration and protein kinase A (PKA) activity in activated neutrophils. PKA inhibitors reversed the inhibitory effects of osthol, suggesting that these are mediated through cAMP/PKA-dependent inhibition of ERK and Akt activation. Furthermore, the activity of cAMP-specific phosphodiesterase (PDE) 4, but not PDE3 or PDE7, was significantly reduced by osthol. In addition, osthol reduced myeloperoxidase activity and pulmonary edema in rats subjected to T/H shock. In conclusion, our data suggest that osthol has effective anti-inflammatory activity in human neutrophils through the suppression of PDE4 and protects significantly against T/H shock-induced ALI in rats. Osthol may have potential for future clinical application as a novel adjunct therapy to treat lung inflammation caused by adverse circulatory conditions. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1.

    Science.gov (United States)

    Furihata, Takashi; Maruyama, Kyonoshin; Fujita, Yasunari; Umezawa, Taishi; Yoshida, Riichiro; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2006-02-07

    bZIP-type transcription factors AREBs/ABFs bind an abscisic acid (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression in Arabidopsis. Because AREB1 overexpression could not induce downstream gene expression, activation of AREB1 requires ABA-dependent posttranscriptional modification. We confirmed that ABA activated 42-kDa kinase activity, which, in turn, phosphorylated Ser/Thr residues of R-X-X-S/T sites in the conserved regions of AREB1. Amino acid substitutions of R-X-X-S/T sites to Ala suppressed transactivation activity, and multiple substitution of these sites resulted in almost complete suppression of transactivation activity in transient assays. In contrast, substitution of the Ser/Thr residues to Asp resulted in high transactivation activity without exogenous ABA application. A phosphorylated, transcriptionally active form was achieved by substitution of Ser/Thr in all conserved R-X-X-S/T sites to Asp. Transgenic plants overexpressing the phosphorylated active form of AREB1 expressed many ABA-inducible genes, such as RD29B, without ABA treatment. These results indicate that the ABA-dependent multisite phosphorylation of AREB1 regulates its own activation in plants.

  5. 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

  6. Growth hormone-dependent phosphorylation of tyrosine 333 and/or 338 of the growth hormone receptor

    DEFF Research Database (Denmark)

    VanderKuur, J A; Wang, X; Zhang, L

    1995-01-01

    and a reduction of GH-dependent phosphorylation of the full-length receptor. Consistent with Tyr333 and/or Tyr338 serving as substrates of JAK2, these substitutions resulted in a loss of tyrosyl phosphorylation of truncated receptor in an in vitro kinase assay using substantially purified GH.GHR.JAK2 complexes...

  7. Phosphorylation prevents C/EBPβ from the calpain-dependent degradation

    International Nuclear Information System (INIS)

    Zhang, Yuan-yuan; Li, Shu-fen; Qian, Shu-wen; Zhang, You-you; Liu, Yuan; Tang, Qi-Qun; Li, Xi

    2012-01-01

    Highlights: ► Phosphorylation protected C/EBPβ from μ-calpain-mediated proteolysis in vitro. ► Phosphorylation mimic C/EBPβ was insensitive to calpain accelerator and inhibitor. ► Phosphorylation on Thr 188 contributed more to the stabilization of C/EBPβ. -- Abstract: CCAAT/enhancer-binding protein (C/EBP) β plays an important role in proliferation and differentiation of 3T3-L1 preadipocytes. C/EBPβ is sequentially phosphorylated during the 3T3-L1 adipocyte differentiation program, first by MAPK/Cyclin A/cdk2 on Thr 188 and subsequently by GSK3β on Ser 184 or Thr 179 . Dual phosphorylation is critical for the gain of DNA binding activity of C/EBPβ. In this manuscript, we found that phosphorylation also contributed to the stability of C/EBPβ. Both ex vivo and in vitro experiments showed that phosphorylation by MAPK/Cyclin A/cdk2 and GSK3β protected C/EBPβ from μ-calpain-mediated proteolysis, while phosphorylation on Thr 188 by MAPK/Cyclin A/cdk2 contributed more to the stabilization of C/EBPβ, Further studies indicated that phosphorylation mimic C/EBPβ was insensitive to both calpain accelerator and calpain inhibitor. Thus, phosphorylation might contribute to the stability as well as the gain of DNA binding activity of C/EBPβ.

  8. Angiotensin-(1-7 Promotes Resolution of Neutrophilic Inflammation in a Model of Antigen-Induced Arthritis in Mice

    Directory of Open Access Journals (Sweden)

    Lívia C. Barroso

    2017-11-01

    Full Text Available Defective resolution of inflammation may be crucial for the initiation and development of chronic inflammatory diseases, such as arthritis. Therefore, it has been suggested that therapeutic strategies based on molecules that facilitate inflammation resolution present great potential for the treatment of chronic inflammatory diseases. In this study, we investigated the effects and role of angiotensin-(1-7 [Ang-(1-7] in driving resolution of neutrophilic inflammation in a model of arthritis. For this purpose, male C57BL/6 mice were subjected to antigen-induced arthritis and treated with Ang-(1-7 at the peak of the inflammatory process. Analysis of the number of inflammatory cells, apoptosis, and immunofluorescence for NF-κB was performed in the exudate collected from the knee cavity. Neutrophil accumulation in periarticular tissue was measured by assaying myeloperoxidase activity. Apoptosis of human neutrophil after treatment with Ang-(1-7 was evaluated morphologically and by flow cytometry, and NF-κB phosphorylation by immunofluorescence. Efferocytosis was evaluated in vivo. Therapeutic treatment with Ang-(1-7 at the peak of inflammation promoted resolution, an effect associated with caspase-dependent neutrophils apoptosis and NF-κB inhibition. Importantly, Ang-(1-7 was also able to induce apoptosis of human neutrophils, an effect associated with NF-κB inhibition. The pro-resolving effects of Ang-(1-7 were inhibited by the Mas receptor antagonist A779. Finally, we showed that Ang-(1-7 increased the efferocytic ability of murine macrophages. Our results clearly demonstrate that Ang-(1-7 resolves neutrophilic inflammation in vivo acting in two key step of resolution: apoptosis of neutrophils and their removal by efferocytosis. Ang-(1-7 is a novel mediator of resolution of inflammation.

  9. Quantitative in vivo analyses reveal calcium-dependent phosphorylation sites and identifies a novel component of the Toxoplasma invasion motor complex.

    Directory of Open Access Journals (Sweden)

    Thomas Nebl

    2011-09-01

    Full Text Available Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca²⁺-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of ³²[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT identified ∼546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca²⁺-dependent phosphorylation patterns on three of its components--GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component.

  10. PKN1 Directs Polarized RAB21 Vesicle Trafficking via RPH3A and Is Important for Neutrophil Adhesion and Ischemia-Reperfusion Injury

    Directory of Open Access Journals (Sweden)

    Qianying Yuan

    2017-06-01

    Full Text Available Polarized vesicle transport plays an important role in cell polarization, but the mechanisms underlying this process and its role in innate immune responses are not well understood. Here, we describe a phosphorylation-regulated polarization mechanism that is important for neutrophil adhesion to endothelial cells during inflammatory responses. We show that the protein kinase PKN1 phosphorylates RPH3A, which enhances binding of RPH3A to guanosine triphosphate (GTP-bound RAB21. These interactions are important for polarized localization of RAB21 and RPH3A in neutrophils, which leads to PIP5K1C90 polarization. Consistent with the roles of PIP5K1C90 polarization, the lack of PKN1 or RPH3A impairs neutrophil integrin activation, adhesion to endothelial cells, and infiltration in inflammatory models. Furthermore, myeloid-specific loss of PKN1 decreases tissue injury in a renal ischemia-reperfusion model. Thus, this study characterizes a mechanism for protein polarization in neutrophils and identifies a potential protein kinase target for therapeutic intervention in reperfusion-related tissue injury.

  11. The phosphorylation-dependent regulation of nuclear SREBP1 during mitosis links lipid metabolism and cell growth

    Science.gov (United States)

    Bengoechea-Alonso, Maria Teresa; Ericsson, Johan

    2016-01-01

    ABSTRACT The SREBP transcription factors are major regulators of lipid metabolism. Disturbances in lipid metabolism are at the core of several health issues facing modern society, including cardiovascular disease, obesity and diabetes. In addition, the role of lipid metabolism in cancer cell growth is receiving increased attention. Transcriptionally active SREBP molecules are unstable and rapidly degraded in a phosphorylation-dependent manner by Fbw7, a ubiquitin ligase that targets several cell cycle regulatory proteins for degradation. We have previously demonstrated that active SREBP1 is stabilized during mitosis. We have now delineated the mechanisms involved in the stabilization of SREBP1 in mitotic cells. This process is initiated by the phosphorylation of a specific serine residue in nuclear SREBP1 by the mitotic kinase Cdk1. The phosphorylation of this residue creates a docking site for a separate mitotic kinase, Plk1. Plk1 interacts with nuclear SREBP1 in mitotic cells and phosphorylates a number of residues in the C-terminal domain of the protein, including a threonine residue in close proximity of the Fbw7 docking site in SREBP1. The phosphorylation of these residues by Plk1 blocks the interaction between SREBP1 and Fbw7 and attenuates the Fbw7-dependent degradation of nuclear SREBP1 during cell division. Inactivation of SREBP1 results in a mitotic defect, suggesting that SREBP1 could regulate cell division. We propose that the mitotic phosphorylation and stabilization of nuclear SREBP1 during cell division provides a link between lipid metabolism and cell proliferation. Thus, the current study provides additional support for the emerging hypothesis that SREBP-dependent lipid metabolism may be important for cell growth. PMID:27579997

  12. Depression of efficiency of neutrophils for Candida albicans phagocytosis in personnel working in radiation field

    International Nuclear Information System (INIS)

    Hassan, A.A.

    2000-01-01

    The neutrophil functions, chemotaxis (direct and random migration), phagocytosis using Candida albicans (percent, index), phagocytosis by NBT (percent, score) and adherence were studied on 55 persons working in radiation field (group I) and 40 persons as control (group II). The effect of radiation on blood picture of persons working in this field with special references to leucocytic counts and neutrophil functions was studied. White and red cells counts were 6.275 +- 1.723 and 5.475 +- 1.039 (group I) and 6.440 +- 1.556, 4.704 +- 0.734 for group II, respectively with no significant difference, while in neutrophil function there was a statistically significant difference in all functions between two groups (P < 0.01). This indicates the importance of neutrophil functions in following up persons working in radiation field

  13. A dual phosphorylation switch controls 14-3-3-dependent cell surface expression of TASK-1

    Science.gov (United States)

    Kilisch, Markus; Lytovchenko, Olga; Arakel, Eric C.; Bertinetti, Daniela; Schwappach, Blanche

    2016-01-01

    ABSTRACT The transport of the K+ channels TASK-1 and TASK-3 (also known as KCNK3 and KCNK9, respectively) to the cell surface is controlled by the binding of 14-3-3 proteins to a trafficking control region at the extreme C-terminus of the channels. The current model proposes that phosphorylation-dependent binding of 14-3-3 sterically masks a COPI-binding motif. However, the direct effects of phosphorylation on COPI binding and on the binding parameters of 14-3-3 isoforms are still unknown. We find that phosphorylation of the trafficking control region prevents COPI binding even in the absence of 14-3-3, and we present a quantitative analysis of the binding of all human 14-3-3 isoforms to the trafficking control regions of TASK-1 and TASK-3. Surprisingly, the affinities of 14-3-3 proteins for TASK-1 are two orders of magnitude lower than for TASK-3. Furthermore, we find that phosphorylation of a second serine residue in the C-terminus of TASK-1 inhibits 14-3-3 binding. Thus, phosphorylation of the trafficking control region can stimulate or inhibit transport of TASK-1 to the cell surface depending on the target serine residue. Our findings indicate that control of TASK-1 trafficking by COPI, kinases, phosphatases and 14-3-3 proteins is highly dynamic. PMID:26743085

  14. Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen*

    Science.gov (United States)

    Kozlov, Sergei V.; Waardenberg, Ashley J.; Engholm-Keller, Kasper; Arthur, Jonathan W.; Graham, Mark E.; Lavin, Martin

    2016-01-01

    Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM-dependence

  15. Peptide secreted by human alveolar macrophages releases neutrophil granule contents

    International Nuclear Information System (INIS)

    MacArthur, C.K.; Miller, E.J.; Cohen, A.B.

    1987-01-01

    A monoclonal antibody was developed against an 8000-kDa enzyme-releasing peptide (ERP) released from human alveolar macrophages. ERP was isolated on an immunoaffinity column containing the antibody bound to staphylococcal protein A-Sepharose, and by autoradiography. Release of ERP from the macrophages is not changed by plastic adherence, phagocytosis, calcium ionophore, or phorbol esters. The peptide was not antigenically similar to interferon-γ, tumor necrosis factor, or interleukin lα or 1β. The release of constituents from azurophilic and specific granules was the main identified biologic function of ERP. ERP was a more effective secretagogue in the untreated neutrophils and f-met-leu-phe was more effective in the cytochalasin B-treated neutrophils. Absorption of ERP from macrophage-conditioned medium removed a small amount of the chemotactic activity; however, the immunopurified peptide was not chemotactic or chemokinetic for neutrophils, and at high concentrations, it suppressed base line chemokinesis. Treatment of washed macrophages with trypsin released active ERP of approximately the same m.w. of spontaneously secreted ERP. These studies showed that human alveolar macrophages release a peptide which is a secretagogue for human neutrophils under conditions which may be encountered in the lungs during certain disease states. Proteolytic enzymes which are free in the lungs may release the peptide and lead to the secretion of neutrophil enzymes

  16. 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

  17. Targeting Neutrophilic Inflammation Using Polymersome-Mediated Cellular Delivery.

    Science.gov (United States)

    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.

  18. Phenotypic Diversity and Plasticity in Circulating Neutrophil Subpopulations in Cancer

    Directory of Open Access Journals (Sweden)

    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.

  19. Two Distinct Coagulase-Dependent Barriers Protect Staphylococcus aureus from Neutrophils in a Three Dimensional in vitro Infection Model

    Science.gov (United States)

    Guggenberger, Christoph; Wolz, Christiane; Morrissey, Julie A.; Heesemann, Jürgen

    2012-01-01

    Staphylococcus aureus is a pyogenic abscess-forming facultative pathogenic microorganism expressing a large set of virulence-associated factors. Among these, secreted proteins with binding capacity to plasma proteins (e.g. fibrinogen binding proteins Eap and Emp) and prothrombin activators such as Coagulase (Coa) and vWbp are involved in abscess formation. By using a three-dimensional collagen gel (3D-CoG) supplemented with fibrinogen (Fib) we studied the growth behavior of S. aureus strain Newman and a set of mutants as well as their interaction with mouse neutrophils by real-time confocal microscopy. In 3D-CoG/Fib, S. aureus forms microcolonies which are surrounded by an inner pseudocapsule and an extended outer dense microcolony-associated meshwork (MAM) containing fibrin. Coa is involved in formation of the pseudocapsule whereas MAM formation depends on vWbp. Moreover, agr-dependent dispersal of late stage microcolonies could be observed. Furthermore, we demonstrate that the pseudocapsule and the MAM act as mechanical barriers against neutrophils attracted to the microcolony. The thrombin inhibitor argatroban is able to prevent formation of both pseudocapsule and MAM and supports access of neutrophils to staphylococci. Taken together, this model can simulate specific stages of S. aureus abscess formation by temporal dissection of bacterial growth and recruitment of immune cells. It can complement established animal infection models in the development of new treatment options. PMID:22253592

  20. PKA regulates calcineurin function through the phosphorylation of RCAN1: Identification of a novel phosphorylation site

    International Nuclear Information System (INIS)

    Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon

    2015-01-01

    Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. - Highlights: • We identify novel phosphorylation sites in RCAN1 by LC-MS/MS analysis. • PKA-dependent phosphorylation of RCAN1 at Ser 93 inhibits calcineurin-mediated intracellular signaling. • We show the immunosuppressive function of RCAN1 phosphorylation at Ser 93 in suppressing cytokine expression

  1. ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage

    Science.gov (United States)

    Maya, Ruth; Balass, Moshe; Kim, Seong-Tae; Shkedy, Dganit; Leal, Juan-Fernando Martinez; Shifman, Ohad; Moas, Miri; Buschmann, Thomas; Ronai, Ze'ev; Shiloh, Yosef; Kastan, Michael B.; Katzir, Ephraim; Oren, Moshe

    2001-01-01

    The p53 tumor suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. The rapid activation of p53 by ionizing radiation and radiomimetic agents is largely dependent on the ATM kinase. p53 is phosphorylated by ATM shortly after DNA damage, resulting in enhanced stability and activity of p53. The Mdm2 oncoprotein is a pivotal negative regulator of p53. In response to ionizing radiation and radiomimetic drugs, Mdm2 undergoes rapid ATM-dependent phosphorylation prior to p53 accumulation. This results in a decrease in its reactivity with the 2A10 monoclonal antibody. Phage display analysis identified a consensus 2A10 recognition sequence, possessing the core motif DYS. Unexpectedly, this motif appears twice within the human Mdm2 molecule, at positions corresponding to residues 258–260 and 393–395. Both putative 2A10 epitopes are highly conserved and encompass potential phosphorylation sites. Serine 395, residing within the carboxy-terminal 2A10 epitope, is the major target on Mdm2 for phosphorylation by ATM in vitro. Mutational analysis supports the conclusion that Mdm2 undergoes ATM-dependent phosphorylation on serine 395 in vivo in response to DNA damage. The data further suggests that phosphorylated Mdm2 may be less capable of promoting the nucleo-cytoplasmic shuttling of p53 and its subsequent degradation, thereby enabling p53 accumulation. Our findings imply that activation of p53 by DNA damage is achieved, in part, through attenuation of the p53-inhibitory potential of Mdm2. PMID:11331603

  2. Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay.

    Science.gov (United States)

    Cui, Heying; Loftus, Kyle M; Noell, Crystal R; Solmaz, Sozanne R

    2018-05-03

    Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer. Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin α. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

  3. Cyclin dependent kinase 5 regulates endocytosis in nerve terminals via dynamin I phosphorylation

    International Nuclear Information System (INIS)

    Tan, T.C.; Hansra, G.; Calova, V.; Cousin, M.; Robinson, P.J.

    2002-01-01

    Full text: Synaptic vesicle endocytosis (SVE) in nerve terminals is essential for normal synaptic transmission and for memory retrieval. Dynamin I is a 96kDa nerve terminal phosphoprotein necessary for synaptic vesicle endocytosis in the nerve terminal. Dynamin I is dephosphorylated and rephosphorylated in a cyclical fashion with nerve terminal depolarisation and repolarisation. A number of kinases phosphorylate dynamin I in vitro including PKC, MAP kinase and cdc2. PKC phosphorylates dynamin in the proline rich domain on Ser 795 and is also thought to be the in vivo kinase for dynamin I. Another candidate is the neuron specific kinase cdk5, crucial for CNS development. The aim of this study is to identify the kinase which phosphorylates dynamin I in intact nerve terminals. Here we show that cyclin-dependent kinase 5 (cdk5) phosphorylates dynamin I in the proline-rich tail on Ser-774 or Ser-778. The phosphorylation of these sites but not Ser-795 also occurred in intact nerve terminals suggesting that cdk5 is the physiologically relevant enzyme for dynamin I. Synaptosomes prepared from rat brains (after cervical dislocations) and labelled with 32 Pi, were incubated with 100 M roscovitine (a selective inhibitor of cdks), 10 M Ro 31-8220 (a selective PKC inhibitor) and 100 M PD 98059 (a MEK kinase inhibitor). Dynamin rephosphorylation during repolarisation was reduced in synaptosomes treated with roscovitine and Ro 38-8220 but not in synaptosomes treated with PD 98059. Fluorimetric experiments on intact synaptosomes utilising FM-210 (a fluorescent dye) indicate that endocytosis was reduced in synaptosomes treated with 100 M roscovitine. Our results suggest that dynamin phosphorylation in intact nerve terminals may not be regulated by PKC or MAP kinase and that dynamin phosphorylation by cdk5 may regulate endocytosis. Copyright (2002) Australian Neuroscience Society

  4. Phosphorylation prevents C/EBP{beta} from the calpain-dependent degradation

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yuan-yuan; Li, Shu-fen; Qian, Shu-wen; Zhang, You-you; Liu, Yuan; Tang, Qi-Qun; Li, Xi, E-mail: lixi@shmu.edu.cn

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer Phosphorylation protected C/EBP{beta} from {mu}-calpain-mediated proteolysis in vitro. Black-Right-Pointing-Pointer Phosphorylation mimic C/EBP{beta} was insensitive to calpain accelerator and inhibitor. Black-Right-Pointing-Pointer Phosphorylation on Thr{sub 188} contributed more to the stabilization of C/EBP{beta}. -- Abstract: CCAAT/enhancer-binding protein (C/EBP) {beta} plays an important role in proliferation and differentiation of 3T3-L1 preadipocytes. C/EBP{beta} is sequentially phosphorylated during the 3T3-L1 adipocyte differentiation program, first by MAPK/Cyclin A/cdk2 on Thr{sub 188} and subsequently by GSK3{beta} on Ser{sub 184} or Thr{sub 179}. Dual phosphorylation is critical for the gain of DNA binding activity of C/EBP{beta}. In this manuscript, we found that phosphorylation also contributed to the stability of C/EBP{beta}. Both ex vivo and in vitro experiments showed that phosphorylation by MAPK/Cyclin A/cdk2 and GSK3{beta} protected C/EBP{beta} from {mu}-calpain-mediated proteolysis, while phosphorylation on Thr{sub 188} by MAPK/Cyclin A/cdk2 contributed more to the stabilization of C/EBP{beta}, Further studies indicated that phosphorylation mimic C/EBP{beta} was insensitive to both calpain accelerator and calpain inhibitor. Thus, phosphorylation might contribute to the stability as well as the gain of DNA binding activity of C/EBP{beta}.

  5. Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase

    International Nuclear Information System (INIS)

    Hegazy, M.G.; Reimann, E.M.; Thysseril, T.J.; Schlender, K.K.

    1986-01-01

    Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca 2+ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting 32 P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated 32 P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor

  6. Lipid raft-associated β-adducin is required for PSGL-1-mediated neutrophil rolling on P-selectin.

    Science.gov (United States)

    Xu, Tingshuang; Liu, Wenai; Yang, Chen; Ba, Xueqing; Wang, Xiaoguang; Jiang, Yong; Zeng, Xianlu

    2015-02-01

    Lipid rafts, a liquid-ordered plasma membrane microdomain, are related to cell-surface receptor function. PSGL-1, a major surface receptor protein for leukocyte, also acts as a signaling receptor in leukocyte rolling. To investigate the role of lipid raft in PSGL-1 signaling in human neutrophils, we quantitatively analyzed lipid raft proteome of human promyelocytic leukemia cell line HL-60 cells and identified a lipid raft-associated protein β-adducin. PSGL-1 ligation induced dissociation of the raft-associated protein β-adducin from lipid rafts and actin, as well as phosphorylation of β-adducin, indicating a transient uncoupling of lipid rafts from the actin cytoskeleton. Knockdown of β-adducin greatly attenuated HL-60 cells rolling on P-selectin. We also showed that Src kinase is crucial for PSGL-1 ligation-induced β-adducin phosphorylation and relocation. Taken together, these results show that β-adducin is a pivotal lipid raft-associated protein in PSGL-1-mediated neutrophil rolling on P-selectin. © Society for Leukocyte Biology.

  7. 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.

  8. Protectin DX, a double lipoxygenase product of DHA, inhibits both ROS production in human neutrophils and cyclooxygenase activities

    Science.gov (United States)

    Liu, Miao; Boussetta, Tarek; Makni-Maalej, Karama; Fay, Michèle; Driss, Fathi; El-Benna, Jamel; Lagarde, Michel; Guichardant, Michel

    2014-01-01

    Neutrophils play a major role in inflammation by releasing large amounts of reactive oxygen species (ROS) produced by NADPH oxidase (NOX) and myeloperoxidase (MPO). This ROS overproduction is mediated by phosphorylation of the NOX subunits with an uncontrolled manner. Therefore, targeting neutrophil subunits would represent a promising strategy to moderate NOX activity, lower ROS, and other inflammatory agents, such as cytokines and leukotrienes, produced by neutrophils. For this purpose, we investigated the effects of protectin DX (PDX) - a docosahexaenoic acid (DHA) di-hydroxylated product which inhibits blood platelet aggregation - on neutrophil activation in vitro. We found that PDX decreases ROS production, inhibits NOX activation and MPO release from neutrophils. We also confirm, that PDX is an anti-aggregatory and anti-inflammatory agent by inhibiting both cyclooxygenase-1 and -2 (COX-1 and COX-2, E.C. 1.14.99.1) as well as COX-2 in lipopolysaccharides (LPS)-treated human neutrophils. However, PDX has no effect on the 5-lipoxygenase pathway that produces the chemotactic agent leukotriene B4 (LTB4). Taken together, our results suggest that PDX could be a protective agent against neutrophil invasion in chronic inflammatory diseases. PMID:24254970

  9. Propofol reduced myocardial contraction of vertebrates partly by mediating the cyclic AMP-dependent protein kinase phosphorylation pathway

    International Nuclear Information System (INIS)

    Sun, Xiaotong; Zhang, Xinyu; Bo, Qiyu; Meng, Tao; Lei, Zhen; Li, Jingxin; Hou, Yonghao; Yu, Xiaoqian; Yu, Jingui

    2016-01-01

    Propofol inhibits myocardial contraction in a dose dependent manner. The present study is designed to examine the effect of propofol on PKA mediated myocardial contraction in the absence of adrenoreceptor agonist. The contraction of isolated rat heart was measured in the presence or absence of PKA inhibitor H89 or propofol, using a pressure transducer. The levels of cAMP and PKA kinase activity were detected by ELISA. The mRNA and total protein or phosphorylation level of PKA and downstream proteins were tested in the presence or absence of PKA inhibitor H89 or propofol, using RT-PCR, QPCR and western blotting. The phosphorylation level of PKA was examined thoroughly using immunofluorescence and PKA activity non-radioactive detection kit. Propofol induced a dose-dependent negative contractile response on the rat heart. The inhibitory effect of high concentration propofol (50 μM) with 45% decease of control could be partly reversed by the PKA inhibitor H89 (10 μM) and the depressant effect of propofol decreased from 45% to 10%. PKA kinase activity was inhibited by propofol in a dose-dependent manner. Propofol also induced a decrease in phosphorylation of PKA, which was also inhibited by H89, but did not alter the production of cAMP and the mRNA levels of PKA. The downstream proteins of PKA, PLN and RyR2 were phosphorylated to a lesser extent with propofol or H89 than control. These results demonstrated that propofol induced a negative myocardial contractile response partly by mediating the PKA phosphorylation pathway.

  10. Sonic Hedgehog dependent phosphorylation by CK1α and GRK2 is required for ciliary accumulation and activation of smoothened.

    Directory of Open Access Journals (Sweden)

    Yongbin Chen

    2011-06-01

    Full Text Available Hedgehog (Hh signaling regulates embryonic development and adult tissue homeostasis through the GPCR-like protein Smoothened (Smo, but how vertebrate Smo is activated remains poorly understood. In Drosophila, Hh dependent phosphorylation activates Smo. Whether this is also the case in vertebrates is unclear, owing to the marked sequence divergence between vertebrate and Drosophila Smo (dSmo and the involvement of primary cilia in vertebrate Hh signaling. Here we demonstrate that mammalian Smo (mSmo is activated through multi-site phosphorylation of its carboxyl-terminal tail by CK1α and GRK2. Phosphorylation of mSmo induces its active conformation and simultaneously promotes its ciliary accumulation. We demonstrate that graded Hh signals induce increasing levels of mSmo phosphorylation that fine-tune its ciliary localization, conformation, and activity. We show that mSmo phosphorylation is induced by its agonists and oncogenic mutations but is blocked by its antagonist cyclopamine, and efficient mSmo phosphorylation depends on the kinesin-II ciliary motor. Furthermore, we provide evidence that Hh signaling recruits CK1α to initiate mSmo phosphorylation, and phosphorylation further increases the binding of CK1α and GRK2 to mSmo, forming a positive feedback loop that amplifies and/or sustains mSmo phosphorylation. Hence, despite divergence in their primary sequences and their subcellular trafficking, mSmo and dSmo employ analogous mechanisms for their activation.

  11. Fibroblasts are in a position to provide directional information to migrating neutrophils during pneumonia in rabbit lungs.

    Science.gov (United States)

    Behzad, A R; Chu, F; Walker, D C

    1996-05-01

    Previous findings have shown that pulmonary fibroblasts are associated with preexisting holes in the endothelial and epithelial basal laminae through which neutrophils appear to enter and leave the interstitium as they migrate from capillaries to alveoli. To determine their role in neutrophil migration, fibroblast organization within the interstitium was assessed by transmission electron microscope observations of serial-sectioned rabbit lung tissue. Interstitial fibroblasts were found to physically interconnect the endothelial basal lamina holes to epithelial basal lamina holes. Morphometric assessment of rabbit lung tissue instilled with Streptococcus pneumoniae revealed that approximately 70% of the surface area density of migrating neutrophils is in close contact (15 nm or less) with interstitial fibroblasts and extracellular matrix elements (30 and 40%, respectively). Although migrating neutrophils were close enough to adhere to both fibroblasts and extracellular elements, the interstitial fibroblasts are organized in a manner that would allow them to provide directional information to the neutrophils. A model illustrating this process is proposed.

  12. Regulation of Discrete Functional Responses by Syk and Src Family Tyrosine Kinases in Human Neutrophils

    Directory of Open Access Journals (Sweden)

    Thornin Ear

    2017-01-01

    Full Text Available Neutrophils play a critical role in innate immunity and also influence adaptive immune responses. This occurs in good part through their production of inflammatory and immunomodulatory cytokines, in conjunction with their prolonged survival at inflamed foci. While a picture of the signaling machinery underlying these neutrophil responses is now emerging, much remains to be uncovered. In this study, we report that neutrophils constitutively express various Src family isoforms (STKs, as well as Syk, and that inhibition of these protein tyrosine kinases selectively hinders inflammatory cytokine generation by acting posttranscriptionally. Accordingly, STK or Syk inhibition decreases the phosphorylation of signaling intermediates (e.g., eIF-4E, S6K, and MNK1 involved in translational control. By contrast, delayed apoptosis appears to be independent of either STKs or Syk. Our data therefore significantly extend our understanding of which neutrophil responses are governed by STKs and Syk and pinpoint some signaling intermediates that are likely involved. In view of the foremost role of neutrophils in several chronic inflammatory conditions, our findings identify potential molecular targets that could be exploited for future therapeutic intervention.

  13. cAMP-dependent phosphorylation of bovine lens alpha-crystallin

    International Nuclear Information System (INIS)

    Spector, A.; Chiesa, R.; Sredy, J.; Garner, W.

    1985-01-01

    This communication reports that the A1 and B1 chains of bovine lens alpha-crystallin are phosphorylated. The conclusion is based on the following evidence: (i) When soluble preparations from lens cortex are incubated with [gamma- 32 P]ATP, a cAMP-dependent labeling of a high molecular weight protein is obtained. (ii) After NaDodSO 4 /PAGE, the label is found in two bands with Mr 22,000 and 20,000, corresponding to the B and A chains of alpha-crystallin, respectively. (iii) Isoelectric focusing indicates that the radioactivity is almost exclusively in bands with pI values of 5.58 and 6.70, corresponding to the A1 and B1 chains, respectively. (iv) Similar results are obtained in experiments of [ 32 P]orthophosphate incorporation in lens organ culture. (v) Analyses of the digested protein indicate the label is exclusively in phosphoserine. (vi) 31 P NMR analyses of native, proteolytically digested, and urea-treated alpha-crystallin gives a chemical shift of 4.6 ppm relative to 85% H 3 PO 4 at pH 7.4, suggesting that the phosphate is covalently bound to a serine in the protein. An abundance of approximately one phosphate per four or five monomer units was found. (vii) Similar results were obtained by chemical analyses of independently prepared alpha-crystallin samples. The results are consistent with the view that the A1 and B1 chains arise as result of the phosphorylation of directly synthesized A2 and B2 polypeptides. It is suggested that this metabolically controlled phosphorylation may be associated with the terminal differentiation of the lens epithelial cell and the intracellular organization of the lens fiber cell

  14. Subcellular distribution of cyclin-dependent kinase-like 5 (CDKL5) is regulated through phosphorylation by dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A)

    International Nuclear Information System (INIS)

    Oi, Ami; Katayama, Syouichi; Hatano, Naoya; Sugiyama, Yasunori; Kameshita, Isamu; Sueyoshi, Noriyuki

    2017-01-01

    Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed a typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization. - Highlights: • We investigated the mechanism regulating subcellular localization of CDKL5. • DYRK1A was identified as an enzyme that bound to and phosphorylated CDKL5. • The phosphorylation site of CDKL5 was Ser-308, in the vicinity of the NLS. • When DYRK1A was co-expressed, the cytosolic CDKL5 was significantly increased. • In conclusion, DYRK1A regulates CDKL5 localization via phosphorylation on Ser-308.

  15. 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.

  16. 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

  17. Role of the eosinophil in serum-mediated adherence of equine leukocytes to infective larvae of Strongylus vulgaris.

    Science.gov (United States)

    Klei, T R; Chapman, M R; Dennis, V A

    1992-06-01

    The adherence of equine leukocytes to Strongylus vulgaris infective larvae (L3) in the presence of normal and immune sera was examined in vitro. Immune sera promoted adherence of buffy coat cells from ponies with S. vulgaris-induced eosinophilia (eosinophilic ponies) to S. vulgaris L3. However, eosinophils in the buffy coat cells were the predominant adherent cell type. Studies using leukocyte populations enriched for eosinophils, neutrophils, and mononuclear cells from eosinophilic ponies support the observations using buffy coat cells that eosinophils were the main effector cells. Adherent eosinophils from eosinophilic ponies immobilized L3. Neutrophils were less adherent and did not immobilize L3. Mononuclear cells failed to adhere. Normal eosinophils from strongly-naive ponies did not immobilize S. vulgaris L3 in the presence of immune serum, suggesting the in vivo activation of eosinophils in eosinophilic animals. Immune serum promoted less adherence of buffy coat cells to Strongylus edentatus or mixed species of Cyathostominae L3, suggesting that the serum-mediated cellular adherence phenomenon was species-specific. Normal serum promoted less cellular adherence to S. vulgaris L3 than immune serum. The adherence mediated by normal serum was removed by heat inactivation, suggesting that this nonspecific phenomenon was a complement-mediated reaction. Immune globulins promoted reactions similar to that seen using heat-inactivated immune serum, whereas normal globulins did not promote adherence. Immune globulins absorbed with pieces of S. vulgaris adult worms did not promote the adherence of buffy coat cells to S. vulgaris L3, suggesting that adult and L3 stages share antigens important in this phenomenon that resulted in the removal of specific adherence antibody during absorption.

  18. Subcellular distribution of cyclin-dependent kinase-like 5 (CDKL5) is regulated through phosphorylation by dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A).

    Science.gov (United States)

    Oi, Ami; Katayama, Syouichi; Hatano, Naoya; Sugiyama, Yasunori; Kameshita, Isamu; Sueyoshi, Noriyuki

    2017-01-08

    Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase primarily expressed in the central nervous system and is known to cause X-linked neurodevelopmental disorders such as Rett syndrome. However, the mechanisms regulating CDKL5 have not yet been fully clarified. Therefore, in this study, we investigated the protein kinase that directly phosphorylates CDKL5, identifying it as dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), an enzyme binding to and phosphorylating CDKL5. We showed that subcellular distribution of CDKL5 was regulated by its phosphorylation by DYRK1A. In mouse neuroblastoma Neuro2a cells, CDKL5 was localized in both the cytosol and nucleus, whereas DYRK1A showed a typical nuclear localization. When CDKL5 and DYRK1A were co-expressed, the cytosolic localization of CDKL5 was significantly increased. Results of site-directed mutagenesis revealed that the phosphorylation site was Ser-308, in the vicinity of the nuclear localization signal. A mutation mimicking the phosphorylated serine residue by aspartate substitution (S308D) changed CDKL5 localization to the cytosol, whereas the corresponding alanine-substituted analog, CDKL5(S308A), was primarily localized to the nucleus. Taken together, these results strongly suggested that DYRK1A bound to CDKL5 and phosphorylated it on Ser-308, thus interfering with its nuclear localization. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Lestaurtinib inhibits histone phosphorylation and androgen-dependent gene expression in prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Jens Köhler

    Full Text Available BACKGROUND: Epigenetics is defined as heritable changes in gene expression that are not based on changes in the DNA sequence. Posttranslational modification of histone proteins is a major mechanism of epigenetic regulation. The kinase PRK1 (protein kinase C related kinase 1, also known as PKN1 phosphorylates histone H3 at threonine 11 and is involved in the regulation of androgen receptor signalling. Thus, it has been identified as a novel drug target but little is known about PRK1 inhibitors and consequences of its inhibition. METHODOLOGY/PRINCIPAL FINDING: Using a focused library screening approach, we identified the clinical candidate lestaurtinib (also known as CEP-701 as a new inhibitor of PRK1. Based on a generated 3D model of the PRK1 kinase using the homolog PKC-theta (protein kinase c theta protein as a template, the key interaction of lestaurtinib with PRK1 was analyzed by means of molecular docking studies. Furthermore, the effects on histone H3 threonine phosphorylation and androgen-dependent gene expression was evaluated in prostate cancer cells. CONCLUSIONS/SIGNIFICANCE: Lestaurtinib inhibits PRK1 very potently in vitro and in vivo. Applied to cell culture it inhibits histone H3 threonine phosphorylation and androgen-dependent gene expression, a feature that has not been known yet. Thus our findings have implication both for understanding of the clinical activity of lestaurtinib as well as for future PRK1 inhibitors.

  20. Quantification of deposition of neutrophilic granulocytes on vascular grafts in dogs with 111In-labeled granulocytes

    International Nuclear Information System (INIS)

    Dewanjee, M.K.; Solis, E.; Mackey, S.T.; Socher, S.; Chowdhury, S.; Wu, F.P.; Kaye, M.P.

    1985-01-01

    A new radioisotopic technique has been developed for quantification of deposition of neutrophilic granulocytes on vascular grafts. Nine healthy mongrel dogs underwent bilateral femoral artery resection and reconstruction with grafts of the femoral vein and Gore-Tex. Pure granulocytes that had been separated from whole blood by centrifugal elutriation were labeled with 111 In-tropolone in plasma. The granulocyte harvesting efficiency was 25 +/- 12%, and the labeling efficiency was 87 +/- 7%. Three hours after injection of labeled granulocytes and 2 hours after reperfusion, the grafts were harvested and cut into several segments for study of areas of anastomoses and midsections. On the basis of the radioactivity in the blood and in anastomotic and graft sections, the area of graft sections, and the neutrophilic granulocyte and differential leukocyte counts, the number of neutrophilic granulocytes adherent to a unit area and the total number of neutrophilic granulocytes on graft sections were calculated. These quantifications of the deposition of neutrophilic granulocytes indicated that the midsections of Gore-Tex grafts retained more neutrophilic granulocytes than did the midsections of vein grafts. Although the anastomotic areas retained more neutrophilic granulocytes than did the midsections of vein grafts, the opposite finding prevailed for the Gore-Tex grafts. A major fraction of neutrophilic granulocytes on Gore-Tex grafts was incorporated into the thrombus. Semiquantitative information obtained by scintigraphy of the deposition of neutrophilic granulocytes on vascular grafts also confirmed this observation

  1. Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

    International Nuclear Information System (INIS)

    Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

    1987-01-01

    The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of 3 H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes

  2. Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

    Energy Technology Data Exchange (ETDEWEB)

    Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

    1987-11-01

    The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of /sup 3/H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes.

  3. Phosphorylation of the Transient Receptor Potential Ankyrin 1 by Cyclin-dependent Kinase 5 affects Chemo-nociception

    OpenAIRE

    Hall, Bradford E.; Prochazkova, Michaela; Sapio, Matthew R.; Minetos, Paul; Kurochkina, Natalya; Binukumar, B. K.; Amin, Niranjana D.; Terse, Anita; Joseph, John; Raithel, Stephen J.; Mannes, Andrew J.; Pant, Harish C.; Chung, Man-Kyo; Iadarola, Michael J.; Kulkarni, Ashok B.

    2018-01-01

    Cyclin-dependent kinase 5 (Cdk5) is a key neuronal kinase that is upregulated during inflammation, and can subsequently modulate sensitivity to nociceptive stimuli. We conducted an in silico screen for Cdk5 phosphorylation sites within proteins whose expression was enriched in nociceptors and identified the chemo-responsive ion channel Transient Receptor Potential Ankyrin 1 (TRPA1) as a possible Cdk5 substrate. Immunoprecipitated full length TRPA1 was shown to be phosphorylated by Cdk5 and th...

  4. 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.

  5. 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.

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

    Science.gov (United States)

    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.

  7. Cervico-vaginal immunoglobulin G levels increase post-ovulation independently of neutrophils.

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  10. Bax/Mcl-1 balance affects neutrophil survival in intermittent hypoxia and obstructive sleep apnea: effects of p38MAPK and ERK1/2 signaling.

    Science.gov (United States)

    Dyugovskaya, Larissa; Polyakov, Andrey; Cohen-Kaplan, Victoria; Lavie, Peretz; Lavie, Lena

    2012-10-22

    Prolonged neutrophil survival is evident in various cardiovascular and respiratory morbidities, in hypoxic conditions in-vitro and in patients with obstructive sleep apnea (OSA) characterized by nightly intermittent hypoxia (IH). This may lead to persistent inflammation, tissue injury and dysfunction. We therefore investigated by a translational approach the potential contribution of the intrinsic stress-induced mitochondrial pathway in extending neutrophil survival under IH conditions. Thus, neutrophils of healthy individuals treated with IH in-vitro and neutrophils of OSA patients undergoing nightly IH episodes in-vivo were investigated. Specifically, the balance between pro-apoptotic Bax and anti-apoptotic Mcl-1 protein expression, and the potential involvement of p38MAPK and ERK1/2 signaling pathways in the control of Mcl-1 expression were investigated. Purified neutrophils were exposed to IH and compared to normoxia and to sustained hypoxia (SH) using a BioSpherix-OxyCycler C42 system. Bax and Mcl-1 levels, and p38MAPK and ERK1/2 phosphorylation were determined by western blotting. Also, Bax/Mcl-1 expression and Bax translocation to the mitochondria were assessed by confocal microscopy in pre-apoptotic neutrophils, before the appearance of apoptotic morphology. Co-localization of Bax and mitochondria was quantified by LSM 510 CarlZeiss MicroImaging using Manders Overlap Coefficient. A paired two-tailed t test, with Bonferroni correction for multiple comparisons, was used for statistical analysis. Compared to normoxia, IH and SH up-regulated the anti-apoptotic Mcl-1 by about 2-fold, down-regulated the pro-apoptotic Bax by 41% and 27%, respectively, and inhibited Bax co-localization with mitochondria before visible morphological signs of apoptosis were noted. IH induced ERK1/2 and p38MAPKs phosphorylation, whereas SH induced only p38MAPK phosphorylation. Accordingly, both ERK and p38MAPK inhibitors attenuated the IH-induced Mcl-1 increase. In SH, only p38MAPK

  11. Bax/Mcl-1 balance affects neutrophil survival in intermittent hypoxia and obstructive sleep apnea: effects of p38MAPK and ERK1/2 signaling

    Directory of Open Access Journals (Sweden)

    Dyugovskaya Larissa

    2012-10-01

    Full Text Available Abstract Background Prolonged neutrophil survival is evident in various cardiovascular and respiratory morbidities, in hypoxic conditions in-vitro and in patients with obstructive sleep apnea (OSA characterized by nightly intermittent hypoxia (IH. This may lead to persistent inflammation, tissue injury and dysfunction. We therefore investigated by a translational approach the potential contribution of the intrinsic stress-induced mitochondrial pathway in extending neutrophil survival under IH conditions. Thus, neutrophils of healthy individuals treated with IH in-vitro and neutrophils of OSA patients undergoing nightly IH episodes in-vivo were investigated. Specifically, the balance between pro-apoptotic Bax and anti-apoptotic Mcl-1 protein expression, and the potential involvement of p38MAPK and ERK1/2 signaling pathways in the control of Mcl-1 expression were investigated. Methods Purified neutrophils were exposed to IH and compared to normoxia and to sustained hypoxia (SH using a BioSpherix-OxyCycler C42 system. Bax and Mcl-1 levels, and p38MAPK and ERK1/2 phosphorylation were determined by western blotting. Also, Bax/Mcl-1 expression and Bax translocation to the mitochondria were assessed by confocal microscopy in pre-apoptotic neutrophils, before the appearance of apoptotic morphology. Co-localization of Bax and mitochondria was quantified by LSM 510 CarlZeiss MicroImaging using Manders Overlap Coefficient. A paired two-tailed t test, with Bonferroni correction for multiple comparisons, was used for statistical analysis. Results Compared to normoxia, IH and SH up-regulated the anti-apoptotic Mcl-1 by about 2-fold, down-regulated the pro-apoptotic Bax by 41% and 27%, respectively, and inhibited Bax co-localization with mitochondria before visible morphological signs of apoptosis were noted. IH induced ERK1/2 and p38MAPKs phosphorylation, whereas SH induced only p38MAPK phosphorylation. Accordingly, both ERK and p38MAPK inhibitors attenuated

  12. Phosphorylation-dependent signaling controls degradation of DNA mismatch repair protein PMS2.

    Science.gov (United States)

    Hinrichsen, Inga; Weßbecher, Isabel M; Huhn, Meik; Passmann, Sandra; Zeuzem, Stefan; Plotz, Guido; Biondi, Ricardo M; Brieger, Angela

    2017-12-01

    MutLα, a heterodimer consisting of MLH1 and PMS2, plays an important role in DNA mismatch repair and has been shown to be additionally involved in several other important cellular mechanisms. Previous work indicated that AKT could modulate PMS2 stability by phosphorylation. Still, the mechanisms of regulation of MutLα remain unclear. The stability of MutLα subunits was investigated by transiently overexpression of wild type and mutant forms of MLH1 and PMS2 using immunoblotting for measuring the protein levels after treatment. We found that treatment with the cell-permeable serine/threonine phosphatase inhibitor, Calyculin, leads to degradation of PMS2 when MLH1 or its C-terminal domain is missing or if amino acids of MLH1 essential for PMS2 interaction are mutated. In addition, we discovered that the C-terminal tail of PMS2 is relevant for this Calyculin-dependent degradation. A direct involvement of AKT, which was previously described to be responsible for PMS2 degradation, could not be detected. The multi-kinase inhibitor Sorafenib, in contrast, was able to avoid the degradation of PMS2 which postulates that cellular phosphorylation is involved in this process. Together, we show that pharmacologically induced phosphorylation by Calyculin can induce the selective proteasome-dependent degradation of PMS2 but not of MLH1 and that the PMS2 degradation could be blocked by Sorafenib treatment. Curiously, the C-terminal Lynch Syndrome-variants MLH1 L749P and MLH1 Y750X make PMS2 prone to Calyculin induced degradation. Therefore, we conclude that the specific degradation of PMS2 may represent a new mechanism to regulate MutLα. © 2017 Wiley Periodicals, Inc.

  13. The redox state and the phosphorylation state of the mannitol-specific carrier of the E. coli phosphoenolpyruvate-dependent phosphotransferase system

    NARCIS (Netherlands)

    Robillard, G.T.; Pas, H.H.; Gage, D.; Elferink, M.G.L.

    1988-01-01

    This review summarizes the recent developments in identifying the activity-linked cysteine as one of the phosphorylation sites on the mannitol-specific EII of the E. coli phosphoenolpyruvate-dependent mannitol transport system. Two phosphorylation sites have been identified, one being the HPr/P-HPr

  14. 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.

  15. 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.

  16. Escherichia coli Phosphoenolpyruvate-Dependent Phosphotransferase System : Mechanism of Phosphoryl-Group Transfer from Phosphoenolpyruvate to HPr

    NARCIS (Netherlands)

    Misset, Onno; Robillard, George T.

    1982-01-01

    The mechanism of phosphoryl-group transfer from phosphoenolpyruvate (PEP) to HPr, catalyzed by enzyme I of the Escherichia coli PEP-dependent phosphotransferase system, has been studied in vitro. Steady-state kinetics and isotope exchange measurements revealed that this reaction cannot be described

  17. Cryptococcus neoformans modulates extracellular killing by neutrophils

    Directory of Open Access Journals (Sweden)

    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.

  18. Flow Perturbation Mediates Neutrophil Recruitment and Potentiates Endothelial Injury via TLR2 in Mice: Implications for Superficial Erosion.

    Science.gov (United States)

    Franck, Grégory; Mawson, Thomas; Sausen, Grasiele; Salinas, Manuel; Masson, Gustavo Santos; Cole, Andrew; Beltrami-Moreira, Marina; Chatzizisis, Yiannis; Quillard, Thibault; Tesmenitsky, Yevgenia; Shvartz, Eugenia; Sukhova, Galina K; Swirski, Filip K; Nahrendorf, Matthias; Aikawa, Elena; Croce, Kevin J; Libby, Peter

    2017-06-23

    Superficial erosion currently causes up to a third of acute coronary syndromes; yet, we lack understanding of its mechanisms. Thrombi because of superficial intimal erosion characteristically complicate matrix-rich atheromata in regions of flow perturbation. This study tested in vivo the involvement of disturbed flow and of neutrophils, hyaluronan, and Toll-like receptor 2 ligation in superficial intimal injury, a process implicated in superficial erosion. In mouse carotid arteries with established intimal lesions tailored to resemble the substrate of human eroded plaques, acute flow perturbation promoted downstream endothelial cell activation, neutrophil accumulation, endothelial cell death and desquamation, and mural thrombosis. Neutrophil loss-of-function limited these findings. Toll-like receptor 2 agonism activated luminal endothelial cells, and deficiency of this innate immune receptor decreased intimal neutrophil adherence in regions of local flow disturbance, reducing endothelial cell injury and local thrombosis ( P <0.05). These results implicate flow disturbance, neutrophils, and Toll-like receptor 2 signaling as mechanisms that contribute to superficial erosion, a cause of acute coronary syndrome of likely growing importance in the statin era. © 2017 American Heart Association, Inc.

  19. Multiple lupus-associated ITGAM variants alter Mac-1 functions on neutrophils.

    Science.gov (United States)

    Zhou, Yebin; Wu, Jianming; Kucik, Dennis F; White, Nathan B; Redden, David T; Szalai, Alexander J; Bullard, Daniel C; Edberg, Jeffrey C

    2013-11-01

    Multiple studies have demonstrated that single-nucleotide polymorphisms (SNPs) in the ITGAM locus (including the nonsynonymous SNPs rs1143679, rs1143678, and rs1143683) are associated with systemic lupus erythematosus (SLE). ITGAM encodes the protein CD11b, a subunit of the β2 integrin Mac-1. The purpose of this study was to determine the effects of ITGAM genetic variation on the biologic functions of neutrophil Mac-1. Neutrophils from ITGAM-genotyped and -sequenced healthy donors were isolated for functional studies. The phagocytic capacity of neutrophil ITGAM variants was probed with complement-coated erythrocytes, serum-treated zymosan, heat-treated zymosan, and IgG-coated erythrocytes. The adhesion capacity of ITGAM variants, in adhering to either purified intercellular adhesion molecule 1 or tumor necrosis factor α-stimulated endothelial cells, was assessed in a flow chamber. Expression levels of total CD11b and activation of CD11b were assessed by flow cytometry. Mac-1-mediated neutrophil phagocytosis, determined in cultures with 2 different complement-coated particles, was significantly reduced in individuals with nonsynonymous variant alleles of ITGAM. This reduction in phagocytosis was related to variation at either rs1143679 (in the β-propeller region) or rs1143678/rs1143683 (highly linked SNPs in the cytoplasmic/calf-1 regions). Phagocytosis mediated by Fcγ receptors was also significantly reduced in donors with variant ITGAM alleles. Similarly, firm adhesion of neutrophils was significantly reduced in individuals with variant ITGAM alleles. These functional alterations were not attributable to differences in total receptor expression or activation. The nonsynonymous ITGAM variants rs1143679 and rs1143678/rs113683 contribute to altered Mac-1 function on neutrophils. These results underscore the need to consider multiple nonsynonymous SNPs when assessing the functional consequences of ITGAM variation on immune cell processes and the risk of SLE

  20. Identification and quantitation of signal molecule-dependent protein phosphorylation

    KAUST Repository

    Groen, Arnoud J.

    2013-09-03

    Phosphoproteomics is a fast-growing field that aims at characterizing phosphorylated proteins in a cell or a tissue at a given time. Phosphorylation of proteins is an important regulatory mechanism in many cellular processes. Gel-free phosphoproteome technique involving enrichment of phosphopeptide coupled with mass spectrometry has proven to be invaluable to detect and characterize phosphorylated proteins. In this chapter, a gel-free quantitative approach involving 15N metabolic labelling in combination with phosphopeptide enrichment by titanium dioxide (TiO2) and their identification by MS is described. This workflow can be used to gain insights into the role of signalling molecules such as cyclic nucleotides on regulatory networks through the identification and quantification of responsive phospho(proteins). © Springer Science+Business Media New York 2013.

  1. 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 ...

  2. Production of extracellular traps against Aspergillus fumigatus in vitro and in infected lung tissue is dependent on invading neutrophils and influenced by hydrophobin RodA.

    Directory of Open Access Journals (Sweden)

    Sandra Bruns

    2010-04-01

    Full Text Available Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunocompromised patients. Macrophages and neutrophils are known to kill conidia, whereas hyphae are killed mainly by neutrophils. Since hyphae are too large to be engulfed, neutrophils possess an array of extracellular killing mechanisms including the formation of neutrophil extracellular traps (NETs consisting of nuclear DNA decorated with fungicidal proteins. However, until now NET formation in response to A. fumigatus has only been demonstrated in vitro, the importance of neutrophils for their production in vivo is unclear and the molecular mechanisms of the fungus to defend against NET formation are unknown. Here, we show that human neutrophils produce NETs in vitro when encountering A. fumigatus. In time-lapse movies NET production was a highly dynamic process which, however, was only exhibited by a sub-population of cells. NETosis was maximal against hyphae, but reduced against resting and swollen conidia. In a newly developed mouse model we could then demonstrate the existence and measure the kinetics of NET formation in vivo by 2-photon microscopy of Aspergillus-infected lungs. We also observed the enormous dynamics of neutrophils within the lung and their ability to interact with and phagocytose fungal elements in situ. Furthermore, systemic neutrophil depletion in mice almost completely inhibited NET formation in lungs, thus directly linking the immigration of neutrophils with NET formation in vivo. By using fungal mutants and purified proteins we demonstrate that hydrophobin RodA, a surface protein making conidia immunologically inert, led to reduced NET formation of neutrophils encountering Aspergillus fungal elements. NET-dependent killing of Aspergillus-hyphae could be demonstrated at later time-points, but was only moderate. Thus, these data establish that NET formation occurs in vivo during host defence against A

  3. 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.

  4. 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.

  5. The Response of Macrophages and Neutrophils to Hypoxia in the Context of Cancer and Other Inflammatory Diseases

    Directory of Open Access Journals (Sweden)

    Antje Egners

    2016-01-01

    Full Text Available Lack of oxygen (hypoxia is a hallmark of a multitude of acute and chronic diseases and can be either beneficial or detrimental for organ restitution and recovery. In the context of inflammation, hypoxia is particularly important and can significantly influence the course of inflammatory diseases. Macrophages and neutrophils, the chief cellular components of innate immunity, display distinct properties when exposed to hypoxic conditions. Virtually every aspect of macrophage and neutrophil function is affected by hypoxia, amongst others, morphology, migration, chemotaxis, adherence to endothelial cells, bacterial killing, differentiation/polarization, and protumorigenic activity. Prominent arenas of macrophage and neutrophil function, for example, acute/chronic inflammation and the microenvironment of solid tumors, are characterized by low oxygen levels, demonstrating the paramount importance of the hypoxic response for proper function of these cells. Members of the hypoxia-inducible transcription factor (HIF family emerged as pivotal molecular regulators of macrophages and neutrophils. In this review, we will summarize the molecular responses of macrophages and neutrophils to hypoxia in the context of cancer and other chronic inflammatory diseases and discuss the potential avenues for therapeutic intervention that arise from this knowledge.

  6. Soluble interleukin 6 receptor (sIL-6R) mediates colonic tumor cell adherence to the vascular endothelium: a mechanism for metastatic initiation?

    LENUS (Irish Health Repository)

    Dowdall, J F

    2012-02-03

    The mechanisms by which surgery increases metastatic proliferation remain poorly characterized, although endotoxin and immunocytes play a role. Recent evidence suggests that endothelial adherence of tumor cells may be important in the formation of metastases. Soluble receptors of interleukin-6 (sIL-6R) shed by activated neutrophils exert IL-6 effects on endothelial cells, which are unresponsive under normal circumstances. This study examined the hypothesis that sIL-6R released by surgical stress increases tumor cell adherence to the endothelium. Neutrophils (PMN) were stimulated with lipopolysaccharide, C-reactive protein (CRP), and tumor necrosis factor-alpha. Soluble IL-6R release was measured by enzyme-linked immunosorbent assay. Colonic tumor cells transfected with green fluorescent protein and endothelial cells were exposed to sIL-6R, and tumor cell adherence and transmigration were measured by fluorescence microscopy. Basal release of sIL-6R from PMN was 44.7 +\\/- 8.2 pg\\/ml at 60 min. This was significantly increased by endotoxin and CRP (131 +\\/- 16.8 and 84.1 +\\/- 5.3, respectively; both P < 0.05). However, tumor necrosis factor-alpha did not significantly alter sIL-6R release. Endothelial and tumor cell exposure to sIL-6R increased tumor cell adherence by 71.3% within 2 h but did not significantly increase transmigration, even at 6 h. Mediators of surgical stress induce neutrophil release of a soluble receptor for IL-6 that enhances colon cancer cell endothelial adherence. Since adherence to the endothelium is now considered to be a key event in metastatic genesis, these findings have important implications for colon cancer treatment strategies.

  7. Acetaldehyde dissociates the PTP1B–E-cadherin–β-catenin complex in Caco-2 cell monolayers by a phosphorylation-dependent mechanism

    Science.gov (United States)

    Sheth, Parimal; Seth, Ankur; Atkinson, Katherine J.; Gheyi, Tarun; Kale, Gautam; Giorgianni, Francesco; Desiderio, Dominic M.; Li, Chunying; Naren, Anjaparavanda; Rao, Radhakrishna

    2006-01-01

    Interactions between E-cadherin, β-catenin and PTP1B (protein tyrosine phosphatase 1B) are crucial for the organization of AJs (adherens junctions) and epithelial cell–cell adhesion. In the present study, the effect of acetaldehyde on the AJs and on the interactions between E-cadherin, β-catenin and PTP1B was determined in Caco-2 cell monolayers. Treatment of cell monolayers with acetaldehyde induced redistribution of E-cadherin and β-catenin from the intercellular junctions by a tyrosine phosphorylation-dependent mechanism. The PTPase activity associated with E-cadherin and β-catenin was significantly reduced and the interaction of PTP1B with E-cadherin and β-catenin was attenuated by acetaldehyde. Acetaldehyde treatment resulted in phosphorylation of β-catenin on tyrosine residues, and abolished the interaction of β-catenin with E-cadherin by a tyrosine kinase-dependent mechanism. Protein binding studies showed that the treatment of cells with acetaldehyde reduced the binding of β-catenin to the C-terminal region of E-cadherin. Pairwise binding studies using purified proteins indicated that the direct interaction between E-cadherin and β-catenin was reduced by tyrosine phosphorylation of β-catenin, but was unaffected by tyrosine phosphorylation of E-cadherin-C. Treatment of cells with acetaldehyde also reduced the binding of E-cadherin to GST (glutathione S-transferase)–PTP1B. The pairwise binding study showed that GST–E-cadherin-C binds to recombinant PTP1B, but this binding was significantly reduced by tyrosine phosphorylation of E-cadherin. Acetaldehyde increased the phosphorylation of β-catenin on Tyr-331, Tyr-333, Tyr-654 and Tyr-670. These results show that acetaldehyde induces disruption of interactions between E-cadherin, β-catenin and PTP1B by a phosphorylation-dependent mechanism. PMID:17087658

  8. Flow Perturbation Mediates Neutrophil Recruitment and Potentiates Endothelial Injury via TLR2 in Mice – Implications for Superficial Erosion

    Science.gov (United States)

    Franck, Grégory; Mawson, Thomas; Sausen, Grasiele; Salinas, Manuel; Masson, Gustavo Santos; Cole, Andrew; Beltrami-Moreira, Marina; Chatzizisis, Yiannis; Quillard, Thibault; Tesmenitsky, Yevgenia; Shvartz, Eugenia; Sukhova, Galina K.; Swirski, Filip K.; Nahrendorf, Matthias; Aikawa, Elena; Croce, Kevin J.; Libby, Peter

    2017-01-01

    Rationale Superficial erosion currently causes up to a third of acute coronary syndromes (ACS), yet we lack understanding of its mechanisms. Thrombi due to superficial intimal erosion characteristically complicate matrix-rich atheromata in regions of flow perturbation. Objective This study tested in vivo the involvement of disturbed flow, and of neutrophils, hyaluronan, and TLR2 ligation in superficial intimal injury, a process implicated in superficial erosion. Methods and Results : In mouse carotid arteries with established intimal lesions tailored to resemble the substrate of human eroded plaques, acute flow perturbation promoted downstream endothelial cell (EC) activation, neutrophil accumulation, EC death and desquamation, and mural thrombosis. Neutrophil loss-of-function limited these findings. TLR2 agonism activated luminal ECs, and deficiency of this innate immune receptor decreased intimal neutrophil adherence in regions of local flow disturbance, reducing EC injury and local thrombosis (p<0.05). Conclusions These results implicate flow disturbance, neutrophils, and TLR2 signaling as mechanisms that contribute to superficial erosion, a cause of ACS of likely growing importance in the statin era. PMID:28428204

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

    OpenAIRE

    Pablo Alarcón; Carolina Manosalva; Carolina Manosalva; Ivan Conejeros; María D. Carretta; Tamara Muñoz-Caro; Liliana M. R. Silva; Anja Taubert; Carlos Hermosilla; María A. Hidalgo; Rafael A. Burgos

    2017-01-01

    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 indic...

  10. Postirradiation changes in the amount and contact properties of bone marrow neutrophils in a short-term culture

    International Nuclear Information System (INIS)

    Chukhlovin, A.B.; Nikolaevskaya, L.V.

    1988-01-01

    A study was made of the adherence to plastic Petri dishes and viability ( as non-pyknotic cell counts) of rat bone marrow cells cultured for 5 to 22 h in 199 media containing 15% fresh isologous serum. An overall decrease in the number of viable myelokaryocytes including mature neutrophils was observed in the cultures irradiated with doses of 3 to 12 Gy. In addition gamma irradiation increased substantially the adherence of neutrophyls and to a lesser extent of other myelokaryocytes. A possibility of early radiation-induced disturbances in granulocytic maturation is discussed

  11. Ionizing radiation-dependent and independent phosphorylation of the 32-kDa subunit of replication protein A during mitosis.

    LENUS (Irish Health Repository)

    Stephan, Holger

    2009-10-01

    The human single-stranded DNA-binding protein, replication protein A (RPA), is regulated by the N-terminal phosphorylation of its 32-kDa subunit, RPA2. RPA2 is hyperphosphorylated in response to various DNA-damaging agents and also phosphorylated in a cell-cycle-dependent manner during S- and M-phase, primarily at two CDK consensus sites, S23 and S29. Here we generated two monoclonal phospho-specific antibodies directed against these CDK sites. These phospho-specific RPA2-(P)-S23 and RPA2-(P)-S29 antibodies recognized mitotically phosphorylated RPA2 with high specificity. In addition, the RPA2-(P)-S23 antibody recognized the S-phase-specific phosphorylation of RPA2, suggesting that during S-phase only S23 is phosphorylated, whereas during M-phase both CDK sites, S23 and S29, are phosphorylated. Immunofluorescence microscopy revealed that the mitotic phosphorylation of RPA2 starts at the onset of mitosis, and dephosphorylation occurs during late cytokinesis. In mitotic cells treated with ionizing radiation (IR), we observed a rapid hyperphosphorylation of RPA2 in addition to its mitotic phosphorylation at S23 and S29, associated with a significant change in the subcellular localization of RPA. Our data also indicate that the RPA2 hyperphosphorylation in response to IR is facilitated by the activity of both ATM and DNA-PK, and is associated with activation of the Chk2 pathway.

  12. 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...

  13. Raf kinase inhibitory protein function is regulated via a flexible pocket and novel phosphorylation-dependent mechanism.

    Science.gov (United States)

    Granovsky, Alexey E; Clark, Matthew C; McElheny, Dan; Heil, Gary; Hong, Jia; Liu, Xuedong; Kim, Youngchang; Joachimiak, Grazyna; Joachimiak, Andrzej; Koide, Shohei; Rosner, Marsha Rich

    2009-03-01

    Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics.

  14. Active nuclear transcriptome analysis reveals inflammasome-dependent mechanism for early neutrophil response to Mycobacterium marinum.

    Science.gov (United States)

    Kenyon, Amy; Gavriouchkina, Daria; Zorman, Jernej; Napolitani, Giorgio; Cerundolo, Vincenzo; Sauka-Spengler, Tatjana

    2017-07-26

    The mechanisms governing neutrophil response to Mycobacterium tuberculosis remain poorly understood. In this study we utilise biotagging, a novel genome-wide profiling approach based on cell type-specific in vivo biotinylation in zebrafish to analyse the initial response of neutrophils to Mycobacterium marinum, a close genetic relative of M. tuberculosis used to model tuberculosis. Differential expression analysis following nuclear RNA-seq of neutrophil active transcriptomes reveals a significant upregulation in both damage-sensing and effector components of the inflammasome, including caspase b, NLRC3 ortholog (wu: fb15h11) and il1β. Crispr/Cas9-mediated knockout of caspase b, which acts by proteolytic processing of il1β, results in increased bacterial burden and less infiltration of macrophages to sites of mycobacterial infection, thus impairing granuloma development. We also show that a number of immediate early response genes (IEGs) are responsible for orchestrating the initial neutrophil response to mycobacterial infection. Further perturbation of the IEGs exposes egr3 as a key transcriptional regulator controlling il1β transcription.

  15. Achyrocline satureioides (Lam. D.C. Hydroalcoholic Extract Inhibits Neutrophil Functions Related to Innate Host Defense

    Directory of Open Access Journals (Sweden)

    Eric Diego Barioni

    2013-01-01

    Full Text Available Achyrocline satureioides (Lam. D.C. is a herb native to South America, and its inflorescences are popularly employed to treat inflammatory diseases. Here, the effects of the in vivo actions of the hydroalcoholic extract obtained from inflorescences of A. satureioides on neutrophil trafficking into inflamed tissue were investigated. Male Wistar rats were orally treated with A. satureioides extract, and inflammation was induced one hour later by lipopolysaccharide injection into the subcutaneous tissue. The number of leukocytes and the amount of chemotactic mediators were quantified in the inflammatory exudate, and adhesion molecule and toll-like receptor 4 (TLR-4 expressions and phorbol-myristate-acetate- (PMA- stimulated oxidative burst were quantified in circulating neutrophils. Leukocyte-endothelial interactions were quantified in the mesentery tissue. Enzymes and tissue morphology of the liver and kidney were evaluated. Treatment with A. satureioides extract reduced neutrophil influx and secretion of leukotriene B4 and CINC-1 in the exudates, the number of rolling and adhered leukocytes in the mesentery postcapillary venules, neutrophil L-selectin, β2-integrin and TLR-4 expression, and oxidative burst, but did not cause an alteration in the morphology and activities of liver and kidney. Together, the data show that A. satureioides extract inhibits neutrophil functions related to the innate response and does not cause systemic toxicity.

  16. 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

  17. 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

  18. Elevated glucose concentrations promote receptor-independent activation of adherent human neutrophils: an experimental and computational approach

    DEFF Research Database (Denmark)

    Kummer, Ursula; Zobeley, Jürgen; Brasen, Jens Christian

    2007-01-01

    of NO and superoxide formation were observed. However, these changes were not observed for sorbitol, a nonmetabolizable carbohydrate. Glucose transport appears to be important in this process as phloretin interferes with the glucose-specific receptor-independent activation of neutrophils. However, LY83583...

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

    Directory of Open Access Journals (Sweden)

    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.

  20. Properties of phosphorylated thymidylate synthase

    DEFF Research Database (Denmark)

    Frączyk, Tomasz; Ruman, Tomasz; Wilk, Piotr

    2015-01-01

    by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were...... also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent....

  1. Neuronal phosphorylated RNA-dependent protein kinase in Creutzfeldt-Jakob disease.

    LENUS (Irish Health Repository)

    Paquet, Claire

    2009-02-01

    The mechanisms of neuronal apoptosis in Creutzfeldt-Jakob disease (CJD) and their relationship to accumulated prion protein (PrP) are unclear. A recent cell culture study showed that intracytoplasmic PrP may induce phosphorylated RNA-dependent protein kinase (PKR(p))-mediated cell stress. The double-stranded RNA protein kinase PKR is a proapoptotic and stress kinase that accumulates in degenerating neurons in Alzheimer disease. To determine whether neuronal apoptosis in human CJD is associated with activation of the PKR(p) signaling pathway, we assessed in situ end labeling and immunocytochemistry for PrP, glial fibrillary acidic protein, CD68, activated caspase 3, and phosphorylated PKR (Thr451) in samples of frontal, occipital, and temporal cortex, striatum, and cerebellum from 6 patients with sporadic CJD and 5 controls. Neuronal immunostaining for activated PKR was found in all CJD cases. The most staining was in nuclei and, in contrast to findings in Alzheimer disease, cytoplasmic labeling was not detected. Both the number and distribution of PKR(p)-positive neurons correlated closely with the extent of neuronal apoptosis, spongiosis, astrocytosis, and microglial activation and with the phenotype and disease severity. There was no correlation with the type, topography, or amount of extracellular PrP deposits. These findings suggest that neuronal apoptosis in human CJD may result from PKR(p)-mediated cell stress and are consistent with recent studies supporting a pathogenic role for intracellular or transmembrane PrP.

  2. Phosphorylation-dependent down-regulation of apolipoprotein A5 by insulin

    Energy Technology Data Exchange (ETDEWEB)

    Nowak, Maxine; Helleboid-Chapman, Audrey; Jakel, Heidelinde; Rommens, Corinne; Martin, Genevieve; Duran-Sandoval, Daniel; Staels, Bart; Rubin, Edward M.; Pennacchio, Len A.; Taskinen, Marja-Riitta; Fruchart-Najib, Jamila; Fruchart, Jean-Charles

    2004-02-15

    The apolipoprotein A5 (APOA5) gene has been shown to be important in lowering plasma triglyceride levels. Since several studies have shown that hyperinsulinemia is associated with hypertriglyceridemia, we sought to determine whether APOA5 gene is regulated by insulin. We show here that cell and mouse treatments with insulin down-regulated APOA5 expression in a dose-dependent manner. Furthermore, we determined that insulin decreases APOA5 promoter activity and subsequent deletion analyses revealed an E-box-containing fragment. We showed that Upstream Stimulatory Factors, USF1/USF2, bind to the identified E-box in the APOA5 promoter. Moreover, in cotransfection studies, USF1 stimulates APOA5 promoter activity. The treatment with insulin reduces the binding of USF1/USF2 to APOA5 promoter. The inhibition of PI3K pathway with wortmannin abolished the insulin s effect on APOA5 gene transcription. Using oligoprecipitation method of USF from nuclear extracts, we demonstrated that phosphorylated USF1 failed to bind to APOA5 promoter. This indicates that the APOA5 gene transrepression by insulin involves a phosphorylation of USF through PI3K, that modulate their binding to APOA5 promoter and results in APOA5 down-regulation. The effect of exogenous hyperinsulinemia in healthy men shows a decrease of the plasma ApoAV level. These data suggest a potential mechanism involving APOA5 gene in hypertriglyceridemia associated with hyperinsulinemia.

  3. Raf Kinase Inhibitory Protein Function Is Regulated via a Flexible Pocket and Novel Phosphorylation-Dependent Mechanism▿ †

    Science.gov (United States)

    Granovsky, Alexey E.; Clark, Matthew C.; McElheny, Dan; Heil, Gary; Hong, Jia; Liu, Xuedong; Kim, Youngchang; Joachimiak, Grazyna; Joachimiak, Andrzej; Koide, Shohei; Rosner, Marsha Rich

    2009-01-01

    Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics. PMID:19103740

  4. Multiple isoelectric forms of poliovirus RNA-dependent RNA polymerase: Evidence for phosphorylation

    International Nuclear Information System (INIS)

    Ransone, L.J.; Dasgupta, A.

    1989-01-01

    Poliovirus-specific RNA-dependent RNA polymerase (3Dpol) was purified to apparent homogeneity. A single polypeptide of an apparent molecular weight of 63,000 catalyzes the synthesis of dimeric and monomeric RNA products in response to the poliovirion RNA template. Analysis of purified 3Dpol by two-dimensional electrophoresis showed multiple forms of 3Dpol, suggesting posttranslational modification of the protein in virus-infected cells. The two major forms of 3Dpol appear to have approximate pI values of 7.1 and 7.4. Incubation of purified 3Dpol with calf intestinal phosphatase resulted in almost complete disappearance of the pI 7.1 form and a concomitant increase in the intensity of the pI 7.4 form of 3Dpol. Addition of 32P-labeled Pi during infection of HeLa cells with poliovirus resulted in specific labeling of 3Dpol and 3CD, a viral protein which contains the entire 3Dpol sequence. Both 3Dpol and 3CD appear to be phosphorylated at serine residues. Ribosomal salt washes prepared from both mock- and poliovirus-infected cells contain phosphatases capable of dephosphorylating quantitatively the phosphorylated form (pI 7.1) of 3Dpol

  5. Postprandial triglyceride-rich lipoproteins promote lipid accumulation and apolipoprotein B-48 receptor transcriptional activity in human circulating and murine bone marrow neutrophils in a fatty acid-dependent manner.

    Science.gov (United States)

    Ortega-Gómez, Almudena; Varela, Lourdes M; López, Sergio; Montserrat de la Paz, Sergio; Sánchez, Rosario; Muriana, Francisco J G; Bermúdez, Beatriz; Abia, Rocío

    2017-09-01

    Postprandial triglyceride-rich lipoproteins (TRLs) promote atherosclerosis. Recent research points the bone marrow (BM) as a primary site in atherosclerosis. We elucidated how the acute administration of monounsaturated fatty acids (MUFAs) MUFAs, omega-3 polyunsaturated fatty acids (PUFAs) PUFAs and saturated fatty acids (SFAs) affects human circulating and murine BM neutrophil lipid accumulation and functionality. Postprandial hypertriglyceridemia was induced in healthy subjects and Apoe -/- mice by the acute administration of dietary fats enriched in MUFAs, PUFAs, or SFAs. Postprandial hypertriglyceridemia increased apolipoprotein-B48 receptor (ApoB48R) transcriptional activity that was linearly correlated with intracellular triglycerides (TGs) TGs accumulation in human circulating and murine BM neutrophils. MUFA and omega-3 PUFAs attenuated ApoB48R gene expression and intracellular TG accumulation compared to SFAs. TRLs induced apoB48R-dependent TG accumulation in human neutrophils ex vivo. Murine BM neutrophils showed a decrease in surface L-selectin and an increase in TNF-α and IL-1β mRNA expressions only after SFAs administration. TRLs enriched in SFAs induced BM neutrophil degranulation ex vivo suggesting cell priming/activation. Postprandial TRLs disrupts the normal biology and function of circulating and BM neutrophils. MUFA- and omega-3 PUFA-rich dietary fats such as virgin olive oil or fish oil has the potential to prevent excessive neutrophil lipid accumulation and activation by targeting the fatty acid composition of TRLs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. The emerging role of neutrophils in thrombosis – The journey of TF through NETs

    Directory of Open Access Journals (Sweden)

    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.

  7. (±)-2-Chloropropionic acid elevates reactive oxygen species formation in human neutrophil granulocytes

    International Nuclear Information System (INIS)

    Aam, B.B.; Fonnum, F.

    2006-01-01

    (±)-2-Chloropropionic acid (2-CPA) is a neurotoxic compound which kills cerebellar granule cells in vivo, and makes cerebellar granule cells in vitro produce reactive oxygen species (ROS). We have studied the effect of 2-CPA on ROS formation in human neutrophil granulocytes in vitro. We found an increased formation of ROS after 2-CPA exposure using three different methods; the fluorescent probe DCFH-DA and the chemiluminescent probes lucigenin and luminol. Four different inhibitors of ROS formation were tested on the cells in combination with 2-CPA to characterize the signalling pathways. The spin-trap s-PBN, the ERK1/2 inhibitor U0126 and the antioxidant Vitamin E inhibited the 2-CPA-induced ROS formation completely, while the mitochondrial transition permeability pore blocker cyclosporine A inhibited the ROS formation partly. We also found that 2-CPA induced an increased nitric oxide production in the cells by using the Griess reagent. The level of reduced glutathione, measured with the DTNB assay, was decreased after exposure to high concentrations of 2-CPA. Western blotting analysis showed that 2-CPA exposure led to an elevated phosphorylation of ERK MAP kinase. This phosphorylation was inhibited by U0126. Based on these experiments it seems like the mechanisms for 2-CPA induced toxicity involves ROS formation and is similar in neutrophil granulocytes as earlier shown in cerebellar granule cells. This also implies that 2-CPA may be immunotoxic

  8. Physiology of polymorphonuclear neutrophils Fisiología de los polimorfonucleares neutrófilos

    Directory of Open Access Journals (Sweden)

    Diana García de Olarte

    1991-02-01

    Full Text Available

    Phagocytic cells. particularly neutrophils. are a fundamental part of the host response against aggression by infectious as well as non-infectious agents. and they are Involved In the generation of tissue damage during Inflammatory response. Cell responses of neutrophils depend on a series of closely related events like adherence to and diapedesis through endothelial cells. migration toward the sites of inflammation. phagocytósis and destruction of opsonized particles. All these actions are performed through the perfect integration between the systems of cellular activation and microbicidal mechanisms. Both oxygen-dependent and independent. A large portion of the biochemical. molecular and genetic mechanisms that lead to the physiologic response of neutrophils has been elucidated which permits the identification and understanding of the pathogenesis of disorders affecting these cells.

    Las células fagocíticas, en particular los neutrófilos son una pieza fundamental en la respuesta del huésped contra la agresión por diversos agentes, Infecciosos O no y están Involucradas en la generación de daño tisular durante la inflamación. Las respuestas celulares de los PMN dependen de una serie de hechos íntimamente relacionados, como la adherencia al endotelio vascular, la diapedesis a través de las células endoteliales, la migración hacia los sitios de Inflamación y la fagocitosis y ulterior destrucción de las partículas opsonizadas. Todo esto se logra mediante la integración perfecta entre los sistemas de activación celular y los mecanismos microbicidas, dependientes O no del oxígeno. Se ha esclarecido una gran parte de los mecanismos bioquímicos, moleculares y genéticos que llevan a la respuesta fisiológica de los neutrófilos lo cual ha permitido Identificar y entender la patogénesis de

  9. Employment-based reinforcement of adherence to depot naltrexone in unemployed opioid-dependent adults: a randomized controlled trial.

    Science.gov (United States)

    Everly, Jeffrey J; DeFulio, Anthony; Koffarnus, Mikhail N; Leoutsakos, Jeannie-Marie S; Donlin, Wendy D; Aklin, Will M; Umbricht, Annie; Fingerhood, Michael; Bigelow, George E; Silverman, Kenneth

    2011-07-01

    Naltrexone can be used to treat opioid dependence, but patients refuse to take it. Extended-release depot formulations may improve adherence, but long-term adherence rates to depot naltrexone are not known. This study determined long-term rates of adherence to depot naltrexone and whether employment-based reinforcement can improve adherence. Participants who were inducted onto oral naltrexone were assigned randomly to contingency (n = 18) or prescription (n = 17) groups. Participants were offered six depot naltrexone injections and invited to work at the therapeutic workplace on week days for 26 weeks, where they earned stipends for participating in job skills training. Contingency participants were required to accept naltrexone injections to maintain workplace access and to maintain maximum pay. Prescription participants could work independently of whether they accepted injections. The therapeutic workplace, a model employment-based intervention for drug addiction and unemployment. Opioid-dependent unemployed adults. Depot naltrexone injections accepted and opiate-negative urine samples. Contingency participants accepted significantly more naltrexone injections than prescription participants (81% versus 42%), and were more likely to accept all injections (66% versus 35%). At monthly assessments (with missing urine samples imputed as positive), the groups provided similar percentages of samples negative for opiates (74% versus 62%) and for cocaine (56% versus 54%). Opiate-positive samples were more likely when samples were also positive for cocaine. Employment-based reinforcement can maintain adherence to depot naltrexone. Future research should determine whether persistent cocaine use compromises naltrexone's effect on opiate use. Workplaces may be useful for promoting sustained adherence to depot naltrexone. © 2011 The Authors, Addiction © 2011 Society for the Study of Addiction.

  10. 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.

  11. 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.

  12. The Vi capsular polysaccharide enables Salmonella enterica serovar typhi to evade microbe-guided neutrophil chemotaxis.

    Directory of Open Access Journals (Sweden)

    Tamding Wangdi

    2014-08-01

    Full Text Available Salmonella enterica serovar Typhi (S. Typhi causes typhoid fever, a disseminated infection, while the closely related pathogen S. enterica serovar Typhimurium (S. Typhimurium is associated with a localized gastroenteritis in humans. Here we investigated whether both pathogens differ in the chemotactic response they induce in neutrophils using a single-cell experimental approach. Surprisingly, neutrophils extended chemotactic pseudopodia toward Escherichia coli and S. Typhimurium, but not toward S. Typhi. Bacterial-guided chemotaxis was dependent on the presence of complement component 5a (C5a and C5a receptor (C5aR. Deletion of S. Typhi capsule biosynthesis genes markedly enhanced the chemotactic response of neutrophils in vitro. Furthermore, deletion of capsule biosynthesis genes heightened the association of S. Typhi with neutrophils in vivo through a C5aR-dependent mechanism. Collectively, these data suggest that expression of the virulence-associated (Vi capsular polysaccharide of S. Typhi obstructs bacterial-guided neutrophil chemotaxis.

  13. The Application of Dextran Sedimentation as an Initial Step in Neutrophil Purification Promotes Their Stimulation, due to the Presence of Monocytes

    Science.gov (United States)

    Ferrante, Antonio

    2017-01-01

    The purification of human neutrophils for in vitro studies is challenging as they are easily activated through ex vivo manipulations. The technique of erythrocyte sedimentation combined with density gradient centrifugation remains widely practiced and was the subject of this study. Since in the sedimentation step the leukocytes are incubated with dextran, we have raised the likelihood that cellular activation would occur with mediator release leading to neutrophil activation. By comparing the activity of neutrophils purified from whole blood by the classical 2-step method of dextran sedimentation followed by low-density Ficoll-Hypaque (1.077 g/mL) medium, and the 1-step high-density Ficoll-Hypaque (1.114 g/mL) gradient centrifugation, we found that neutrophils from the 2-step method had a significant increase in cell surface CD11b expression and CD62L shedding and a marked increase in adhesion. Decreased random migration and chemotaxis and raised baseline oxidative burst activity were also observed. The effect was not specific to dextran, as using Ficoll for erythrocyte sedimentation replicated the elevated neutrophil adherence. Through the depletion of monocytes, lymphocytes, and platelets prior to sedimentation, we deduced that monocytes were responsible for the neutrophil activation. Our findings suggest that care needs to be exercised in choosing the method of neutrophil purification for functional studies. PMID:29164154

  14. 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.

  15. 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.

  16. Myosin light chain kinase phosphorylation in tracheal smooth muscle

    International Nuclear Information System (INIS)

    Stull, J.T.; Hsu, L.C.; Tansey, M.G.; Kamm, K.E.

    1990-01-01

    Purified myosin light chain kinase from smooth muscle is phosphorylated by cyclic AMP-dependent protein kinase, protein kinase C, and the multifunctional calmodulin-dependent protein kinase II. Because phosphorylation in a specific site (site A) by any one of these kinases desensitizes myosin light chain kinase to activation by Ca2+/calmodulin, kinase phosphorylation could play an important role in regulating smooth muscle contractility. This possibility was investigated in 32 P-labeled bovine tracheal smooth muscle. Treatment of tissues with carbachol, KCl, isoproterenol, or phorbol 12,13-dibutyrate increased the extent of kinase phosphorylation. Six primary phosphopeptides (A-F) of myosin light chain kinase were identified. Site A was phosphorylated to an appreciable extent only with carbachol or KCl, agents which contract tracheal smooth muscle. The extent of site A phosphorylation correlated to increases in the concentration of Ca2+/calmodulin required for activation. These results show that cyclic AMP-dependent protein kinase and protein kinase C do not affect smooth muscle contractility by phosphorylating site A in myosin light chain kinase. It is proposed that phosphorylation of myosin light chain kinase in site A in contracting tracheal smooth muscle may play a role in the reported desensitization of contractile elements to activation by Ca2+

  17. Abscisic acid-dependent multisite phosphorylation regulates the activity of a transcription activator AREB1

    OpenAIRE

    Furihata, Takashi; Maruyama, Kyonoshin; Fujita, Yasunari; Umezawa, Taishi; Yoshida, Riichiro; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2006-01-01

    bZIP-type transcription factors AREBs/ABFs bind an abscisic acid (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression in Arabidopsis. Because AREB1 overexpression could not induce downstream gene expression, activation of AREB1 requires ABA-dependent posttranscriptional modification. We confirmed that ABA activated 42-kDa kinase activity, which, in turn, phosphorylated Ser/Thr residues of R-X-X-S/T sites in the conserved regions of AREB1. Amino acid subs...

  18. Potent homocysteine-induced ERK phosphorylation in cultured neurons depends on self-sensitization via system Xc-

    International Nuclear Information System (INIS)

    Gu Li; Hu Xiaoling; Xue Zhanxia; Yang Jun; Wan Lishu; Ren Yan; Hertz, Leif; Peng Liang

    2010-01-01

    Homocysteine is increased during pathological conditions, endangering vascular and cognitive functions, and elevated homocysteine during pregnancy may be correlated with an increased incidence of schizophrenia in the offspring. This study showed that millimolar homocysteine concentrations in saline medium cause phosphorylation of extracellular-signal regulated kinases 1 and 2 (ERK 1/2 ) in cerebellar granule neurons, inhibitable by metabotropic but not ionotropic glutamate receptor antagonists. These findings are analogous to observations by , that similar concentrations cause neuronal death. However, these concentrations are much higher than those occurring clinically during hyperhomocysteinemia. It is therefore important that a ∼ 10-fold increase in potency occurred in the presence of the glutamate precursor glutamine, when ERK 1/2 phosphorylation became inhibitable by NMDA or non-NMDA antagonists and dependent upon epidermal growth factor (EGF) receptor transactivation. However, glutamate release to the medium was reduced, suggesting that reversal of the cystine/glutamate antiporter, system X c - could be involved in potentiation of the response by causing a localized release of initially accumulated homocysteine. In agreement with this hypothesis further enhancement of ERK 1/2 phosphorylation occurred in the additional presence of cystine. Pharmacological inhibition of system X c - prevented the effect of micromolar homocysteine concentrations, and U0126-mediated inhibition of ERK 1/2 phosphorylation enhanced homocysteine-induced death. In conclusion, homocysteine interacts with system X c - like quisqualate (Venkatraman et al. 1994), by 'self-sensitization' with initial accumulation and subsequent release in exchange with cystine and/or glutamate, establishing high local homocysteine concentrations, which activate adjacent ionotropic glutamate receptors and cause neurotoxicity.

  19. Syndecan-4 Phosphorylation Is a Control Point for Integrin Recycling

    Science.gov (United States)

    Morgan, Mark R.; Hamidi, Hellyeh; Bass, Mark D.; Warwood, Stacey; Ballestrem, Christoph; Humphries, Martin J.

    2013-01-01

    Summary Precise spatiotemporal coordination of integrin adhesion complex dynamics is essential for efficient cell migration. For cells adherent to fibronectin, differential engagement of α5β1 and αVβ3 integrins is used to elicit changes in adhesion complex stability, mechanosensation, matrix assembly, and migration, but the mechanisms responsible for receptor regulation have remained largely obscure. We identify phosphorylation of the membrane-intercalated proteoglycan syndecan-4 as an essential switch controlling integrin recycling. Src phosphorylates syndecan-4 and, by driving syntenin binding, leads to suppression of Arf6 activity and recycling of αVβ3 to the plasma membrane at the expense of α5β1. The resultant elevation in αVβ3 engagement promotes stabilization of focal adhesions. Conversely, abrogation of syndecan-4 phosphorylation drives surface expression of α5β1, destabilizes adhesion complexes, and disrupts cell migration. These data identify the dynamic spatiotemporal regulation of Src-mediated syndecan-4 phosphorylation as an essential switch controlling integrin trafficking and adhesion dynamics to promote efficient cell migration. PMID:23453597

  20. Insulin treatment promotes tyrosine phosphorylation of PKR and inhibits polyIC induced PKR threonine phosphorylation.

    Science.gov (United States)

    Swetha, Medchalmi; Ramaiah, Kolluru V A

    2015-11-01

    Tyrosine phosphorylation of insulin receptor beta (IRβ) in insulin treated HepG2 cells is inversely correlated to ser(51) phosphorylation in the alpha-subunit of eukaryotic initiation factor 2 (eIF2α) that regulates protein synthesis. Insulin stimulates interaction between IRβ and PKR, double stranded RNA-dependent protein kinase, also known as EIF2AK2, and phosphorylation of tyrosine residues in PKR, as analyzed by immunoprecipitation and pull down assays using anti-IRβ and anti-phosphotyrosine antibodies, recombinant IRβ and immunopurified PKR. Further polyIC or synthetic double stranded RNA-induced threonine phosphorylation or activation of immunopurified and cellular PKR is suppressed in the presence of insulin treated purified IRβ and cell extracts. Acute, but not chronic, insulin treatment enhances tyrosine phosphorylation of IRβ, its interaction with PKR and tyrosine phosphorylation of PKR. In contrast, lipopolysaccharide that stimulates threonine phosphorylation of PKR and eIF2α phosphorylation and AG 1024, an inhibitor of the tyrosine kinase activity of IRβ, reduces PKR association with the receptor, IRβ in HepG2 cells. These findings therefore may suggest that tyrosine phosphorylated PKR plays a role in the regulation of insulin induced protein synthesis and in maintaining insulin sensitivity, whereas, suppression of polyIC-mediated threonine phosphorylation of PKR by insulin compromises its ability to fight against virus infection in host cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. 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...

  2. 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.

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  5. Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis.

    Science.gov (United States)

    Douglas, Pauline; Ye, Ruiqiong; Trinkle-Mulcahy, Laura; Neal, Jessica A; De Wever, Veerle; Morrice, Nick A; Meek, Katheryn; Lees-Miller, Susan P

    2014-06-25

    The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser2056, Thr2647 and Thr2609 in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser3205 and Thr3950 in mitosis. Phosphorylation of Thr3950 is DNA-PK-dependent, whereas phosphorylation of Ser3205 requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser3205 in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser3205 in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr68 in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs' role in mitosis may be mechanistically distinct from its well-established role in NHEJ.

  6. Perspectives Regarding Adherence to Prescribed Treatment in Highly Adherent HIV-Infected Gay Men.

    Science.gov (United States)

    Brion, John M; Menke, Edna M

    2008-01-01

    Focus group methodology was used to describe the medication adherence experience of 24 HIV-infected gay men who reported being adherent to their medication regimens. A conceptualization of medication adherence as an evolving process consisted of challenges to adherence (learning the diagnosis, starting the medications, struggling with the medications, dealing with side effects, coping with stigma) as well as those factors supportive of adherence (believing in medications, finding motivating factors, using reminders, depending on others, owning the disease). Themes associated with challenges to adherence focused on diagnosis and the physical and emotional adjustments individuals made to incorporate antiretroviral medications into their daily lives and move toward medication adherence. The factors supportive of adherence were related to the ongoing behaviors identified with establishing and maintaining adherence behaviors. What can be taken from the study is that adherence is a complex and dynamic process rather than a static behavior.

  7. 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

  8. AMPA receptor phosphorylation and recognition memory: learning-related, time-dependent changes in the chick brain following filial imprinting.

    Science.gov (United States)

    Solomonia, Revaz O; Meparishvili, Maia; Mikautadze, Ekaterine; Kunelauri, Nana; Apkhazava, David; McCabe, Brian J

    2013-04-01

    There is strong evidence that a restricted part of the chick forebrain, the intermediate medial mesopallium (IMM), stores information acquired through the learning process of visual imprinting. We have previously demonstrated that at 1 h but not 24 h after imprinting training, a learning-specific increase in the amount of membrane Thr286-autophosphorylated α-calcium/calmodulin-dependent protein kinase II (αCaMKII), and in the proportion of total αCaMKII that is phosphorylated, occurs in the IMM but not in a control brain region, the posterior pole of the nidopallium (PPN). αCaMKII directly phosphorylates Ser831 in the GluA1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. In the present study we have inquired whether the learning-related increase in αCaMKII autophosphorylation is followed by changes in the Ser831 phosphorylation of GluA1 (P-GluA1) and in the total amount of this subunit (T-GluA1). Trained chicks together with untrained control chicks were killed either 1 or 24 h after training. Tissue was removed from the IMM together with tissue from the PPN as a control. Amounts of P-GluA1 and T-GluA1 were measured. In the left IMM of the 1 h group the P-GluA1/T-GluA1 ratio increased in a learning-specific way. No learning-related changes were observed in other brain regions at 1 h or in any region 24 h after training. The results indicate that a time- and regionally-dependent, learning-specific increase in GluA1 phosphorylation occurs early in recognition memory formation.

  9. Phosphorylation of chicken growth hormone

    International Nuclear Information System (INIS)

    Aramburo, C.; Montiel, J.L.; Donoghue, D.; Scanes, C.G.; Berghman, L.R.

    1990-01-01

    The possibility that chicken growth hormone (cGH) can be phosphorylated has been examined. Both native and biosynthetic cGH were phosphorylated by cAMP-dependent protein kinase (and γ- 32 P-ATP). The extent of phosphorylation was however less than that observed with ovine prolactin. Under the conditions employed, glycosylated cGH was not phosphorylated. Chicken anterior pituitary cells in primary culture were incubated in the presence of 32 P-phosphate. Radioactive phosphate was incorporated in vitro into the fraction immunoprecipitable with antisera against cGH. Incorporation was increased with cell number and time of incubation. The presence of GH releasing factor (GRF) increased the release of 32 P-phosphate labeled immunoprecipitable GH into the incubation media but not content of immunoprecipitable GH in the cells. The molecular weight of the phosphorylated immunoreactive cGH in the cells corresponded to cGH dimer

  10. Protein phosphorylation systems in postmortem human brain

    International Nuclear Information System (INIS)

    Walaas, S.I.; Perdahl-Wallace, E.; Winblad, B.; Greengard, P.

    1989-01-01

    Protein phosphorylation systems regulated by cyclic adenosine 3',5'-monophosphate (cyclic AMP), or calcium in conjunction with calmodulin or phospholipid/diacylglycerol, have been studied by phosphorylation in vitro of particulate and soluble fractions from human postmortem brain samples. One-dimensional or two-dimensional gel electrophoretic protein separations were used for analysis. Protein phosphorylation catalyzed by cyclic AMP-dependent protein kinase was found to be highly active in both particulate and soluble preparations throughout the human CNS, with groups of both widely distributed and region-specific substrates being observed in different brain nuclei. Dopamine-innervated parts of the basal ganglia and cerebral cortex contained the phosphoproteins previously observed in rodent basal ganglia. In contrast, calcium/phospholipid-dependent and calcium/calmodulin-dependent protein phosphorylation systems were less prominent in human postmortem brain than in rodent brain, and only a few widely distributed substrates for these protein kinases were found. Protein staining indicated that postmortem proteolysis, particularly of high-molecular-mass proteins, was prominent in deeply located, subcortical regions in the human brain. Our results indicate that it is feasible to use human postmortem brain samples, when obtained under carefully controlled conditions, for qualitative studies on brain protein phosphorylation. Such studies should be of value in studies on human neurological and/or psychiatric disorders

  11. 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.

  12. Age- and brain region-dependent α-synuclein oligomerization is attributed to alterations in intrinsic enzymes regulating α-synuclein phosphorylation in aging monkey brains.

    Science.gov (United States)

    Chen, Min; Yang, Weiwei; Li, Xin; Li, Xuran; Wang, Peng; Yue, Feng; Yang, Hui; Chan, Piu; Yu, Shun

    2016-02-23

    We previously reported that the levels of α-syn oligomers, which play pivotal pathogenic roles in age-related Parkinson's disease (PD) and dementia with Lewy bodies, increase heterogeneously in the aging brain. Here, we show that exogenous α-syn incubated with brain extracts from older cynomolgus monkeys and in Lewy body pathology (LBP)-susceptible brain regions (striatum and hippocampus) forms higher amounts of phosphorylated and oligomeric α-syn than that in extracts from younger monkeys and LBP-insusceptible brain regions (cerebellum and occipital cortex). The increased α-syn phosphorylation and oligomerization in the brain extracts from older monkeys and in LBP-susceptible brain regions were associated with higher levels of polo-like kinase 2 (PLK2), an enzyme promoting α-syn phosphorylation, and lower activity of protein phosphatase 2A (PP2A), an enzyme inhibiting α-syn phosphorylation, in these brain extracts. Further, the extent of the age- and brain-dependent increase in α-syn phosphorylation and oligomerization was reduced by inhibition of PLK2 and activation of PP2A. Inversely, phosphorylated α-syn oligomers reduced the activity of PP2A and showed potent cytotoxicity. In addition, the activity of GCase and the levels of ceramide, a product of GCase shown to activate PP2A, were lower in brain extracts from older monkeys and in LBP-susceptible brain regions. Our results suggest a role for altered intrinsic metabolic enzymes in age- and brain region-dependent α-syn oligomerization in aging brains.

  13. Potent inhibition of human neutrophil activations by bractelactone, a novel chalcone from Fissistigma bracteolatum

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Yang-Chang [Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical University, Taichung 404, Taiwan (China); Sureshbabu, Munisamy; Fang, Yao-Ching; Wu, Yi-Hsiu [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Lan, Yu-Hsuan [School of Pharmacy, China Medical University, Taichung 404, Taiwan (China); Chang, Fang-Rong [Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Chang, Ya-Wen [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Hwang, Tsong-Long, E-mail: htl@mail.cgu.edu.tw [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan, Taoyuan 333, Taiwan (China)

    2013-02-01

    Fissistigma bracteolatum is widely used in traditional medicine to treat inflammatory diseases. However, its active components and mechanisms of action remain unclear. In this study, (3Z)-6,7-dihydroxy-4-methoxy-3-(phenylmethylidene)-5-(3-phenylpropanoyl) -1-benzofuran-2(3H) (bractelactone), a novel chalcone from F. bracteolatum, showed potent inhibitory effects against superoxide anion (O{sub 2}{sup ·−}) production, elastase release, and CD11b expression in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-induced human neutrophils. However, bractelactone showed only weak inhibition of phorbol myristate acetate-caused O{sub 2}{sup ·−} production. The peak cytosolic calcium concentration ([Ca{sup 2+}]{sub i}) was unaltered by bractelactone in FMLP-induced neutrophils, but the decay time of [Ca{sup 2+}]{sub i} was significantly shortened. In a calcium-free solution, changes in [Ca{sup 2+}]{sub i} caused by the addition of extracellular Ca{sup 2+} were inhibited by bractelactone in FMLP-activated cells. In addition, bractelactone did not alter the phosphorylation of p38 MAPK, ERK, JNK, or AKT or the concentration of cAMP. These results suggest that bractelactone selectively inhibits store-operated calcium entry (SOCE). In agreement with this concept, bractelactone suppressed sustained [Ca{sup 2+}]{sub i} changes in thapsigargin-activated neutrophils. Furthermore, bractelactone did not alter FMLP-induced formation of inositol 1,4,5-triphosphate. Taken together, our results demonstrate that the anti-inflammatory effects of bractelactone, an active ingredient of F. bracteolatum, in human neutrophils are through the selective inhibition of SOCE. Highlights: ► Bractelactone isolated from Fissistigma bracteolatum. ► Bractelactone inhibited FMLP-induced human neutrophil activations. ► Bractelactone had no effect on IP3 formation. ► Bractelactone did not alter MAPKs, AKT, and cAMP pathways. ► Bractelactone inhibited store-operated calcium entry.

  14. ABA-Induced Stomatal Closure Involves ALMT4, a Phosphorylation-Dependent Vacuolar Anion Channel of Arabidopsis[OPEN

    Science.gov (United States)

    Baetz, Ulrike; Huck, Nicola V.; Zhang, Jingbo

    2017-01-01

    Stomatal pores are formed between a pair of guard cells and allow plant uptake of CO2 and water evaporation. Their aperture depends on changes in osmolyte concentration of guard cell vacuoles, specifically of K+ and Mal2−. Efflux of Mal2− from the vacuole is required for stomatal closure; however, it is not clear how the anion is released. Here, we report the identification of ALMT4 (ALUMINUM ACTIVATED MALATE TRANSPORTER4) as an Arabidopsis thaliana ion channel that can mediate Mal2− release from the vacuole and is required for stomatal closure in response to abscisic acid (ABA). Knockout mutants showed impaired stomatal closure in response to the drought stress hormone ABA and increased whole-plant wilting in response to drought and ABA. Electrophysiological data show that ALMT4 can mediate Mal2− efflux and that the channel activity is dependent on a phosphorylatable C-terminal serine. Dephosphomimetic mutants of ALMT4 S382 showed increased channel activity and Mal2− efflux. Reconstituting the active channel in almt4 mutants impaired growth and stomatal opening. Phosphomimetic mutants were electrically inactive and phenocopied the almt4 mutants. Surprisingly, S382 can be phosphorylated by mitogen-activated protein kinases in vitro. In brief, ALMT4 likely mediates Mal2− efflux during ABA-induced stomatal closure and its activity depends on phosphorylation. PMID:28874508

  15. Inflammation kinase PKR phosphorylates α-synuclein and causes α-synuclein-dependent cell death

    DEFF Research Database (Denmark)

    Reimer, Lasse; Lund, Louise Buur; Betzer, Cristine

    2018-01-01

    , and acute brain slices), while overexpression of constitutively active PKR increases Ser129 α-syn phosphorylation. Treatment with pre-formed α-synuclein fibrils, proteostatic stress-promoting MG-132 and known PKR activators, herpes simplex virus-1-∆ICP34.5 and LPS, as well as PKR inducer, IFN-β-1b, lead...... on Ser129. Although the inflammation-associated serine-threonine kinase, PKR (EIF2AK2), promotes cellular protection against infection, we demonstrate a pro-degenerative role of activated PKR in an α-synuclein-dependent cell model of multiple system atrophy, where inhibition and silencing of PKR decrease...

  16. Calcium-dependent isolation of the 36-kilodalton substrate of pp60/sup src/-kinase. Fractionation of the phosphorylated and unphosphorylated species

    International Nuclear Information System (INIS)

    Soric, J.; Gordon, J.A.

    1986-01-01

    In this paper, the authors present a new and simple purification of the 36-kDa protein, a major substrate of both viral and growth factor-receptor associated tyrosine protein kinases, and its complex from normal and Schmidt-Ruppin strain Rous sarcoma virus-transformed chicken embryo fibroblasts that employs a DEAE-Sephacel column and introduces the calcium-dependent adsorption of 36-kDa protein. The use of EGTA step gradients differentially elutes the 36-kDa molecules from the DEAE-Sephacel column. An average total yield of the 36-kDa protein in all fractions approached 80% and represented 0.78% of the [ 35 S]methionine-labeled cellular protein. A purity of 95-99% was obtained with a yield of 60% in the central elution fractions from normal or Rous sarcoma virus-transformed chicken embryo fibroblasts. Furthermore, 2 mM EGTA elutes poorly phosphorylated molecules while heavily phosphorylated 36-kDa protein requires 4 or 6 M EGTA; a small residual fraction is released at 8-10 mM EGTA. If the EGTA step gradients were neutralized with Ca 2+ ion, elution of the 36-kDa protein is inhibited. Tyrosine phosphorylation of the 36-kDa protein is increased 2-3-fold following a short term incubation of whole cells with micromolar vanadate. The elution pattern (but not intensity) of the 36-kDa protein obtained from lysates of vanadate-treated cells was identical to untreated cell lysates. The additional phosphorylation appears to result from a recruitment of unphosphorylated 36-kDa protein as the position (but not intensity) of the phosphorylated tryptic peptides is unchanged. They conclude that the function of the 36-kDa protein may be calcium ion-dependent and may be influenced by the phosphorylation state of the protein

  17. Snf1 Phosphorylates Adenylate Cyclase and Negatively Regulates Protein Kinase A-dependent Transcription in Saccharomyces cerevisiae.

    Science.gov (United States)

    Nicastro, Raffaele; Tripodi, Farida; Gaggini, Marco; Castoldi, Andrea; Reghellin, Veronica; Nonnis, Simona; Tedeschi, Gabriella; Coccetti, Paola

    2015-10-09

    In eukaryotes, nutrient availability and metabolism are coordinated by sensing mechanisms and signaling pathways, which influence a broad set of cellular functions such as transcription and metabolic pathways to match environmental conditions. In yeast, PKA is activated in the presence of high glucose concentrations, favoring fast nutrient utilization, shutting down stress responses, and boosting growth. On the contrary, Snf1/AMPK is activated in the presence of low glucose or alternative carbon sources, thus promoting an energy saving program through transcriptional activation and phosphorylation of metabolic enzymes. The PKA and Snf1/AMPK pathways share common downstream targets. Moreover, PKA has been reported to negatively influence the activation of Snf1/AMPK. We report a new cross-talk mechanism with a Snf1-dependent regulation of the PKA pathway. We show that Snf1 and adenylate cyclase (Cyr1) interact in a nutrient-independent manner. Moreover, we identify Cyr1 as a Snf1 substrate and show that Snf1 activation state influences Cyr1 phosphorylation pattern, cAMP intracellular levels, and PKA-dependent transcription. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. The inward rectifier potassium channel Kir2.1 is expressed in mouse neutrophils from bone marrow and liver.

    Science.gov (United States)

    Masia, Ricard; Krause, Daniela S; Yellen, Gary

    2015-02-01

    Neutrophils are phagocytic cells that play a critical role in innate immunity by destroying bacterial pathogens. Channels belonging to the inward rectifier potassium channel subfamily 2 (Kir2 channels) have been described in other phagocytes (monocytes/macrophages and eosinophils) and in hematopoietic precursors of phagocytes. Their physiological function in these cells remains unclear, but some evidence suggests a role in growth factor-dependent proliferation and development. Expression of functional Kir2 channels has not been definitively demonstrated in mammalian neutrophils. Here, we show by RT-PCR that neutrophils from mouse bone marrow and liver express mRNA for the Kir2 subunit Kir2.1 but not for other subunits (Kir2.2, Kir2.3, and Kir2.4). In electrophysiological experiments, resting (unstimulated) neutrophils from mouse bone marrow and liver exhibit a constitutively active, external K(+)-dependent, strong inwardly rectifying current that constitutes the dominant current. The reversal potential is dependent on the external K(+) concentration in a Nernstian fashion, as expected for a K(+)-selective current. The current is not altered by changes in external or internal pH, and it is blocked by Ba(2+), Cs(+), and the Kir2-selective inhibitor ML133. The single-channel conductance is in agreement with previously reported values for Kir2.1 channels. These properties are characteristic of homomeric Kir2.1 channels. Current density in short-term cultures of bone marrow neutrophils is decreased in the absence of growth factors that are important for neutrophil proliferation [granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF)]. These results demonstrate that mouse neutrophils express functional Kir2.1 channels and suggest that these channels may be important for neutrophil function, possibly in a growth factor-dependent manner. Copyright © 2015 the American Physiological Society.

  19. p21-Activated kinase (PAK regulates cytoskeletal reorganization and directional migration in human neutrophils.

    Directory of Open Access Journals (Sweden)

    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.

  20. 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

  1. Twitchin can regulate the ATPase cycle of actomyosin in a phosphorylation-dependent manner in skinned mammalian skeletal muscle fibres.

    Science.gov (United States)

    Avrova, Stanislava V; Rysev, Nikita A; Matusovsky, Oleg S; Shelud'ko, Nikolay S; Borovikov, Yurii S

    2012-05-01

    The effect of twitchin, a thick filament protein of molluscan muscles, on the actin-myosin interaction at several mimicked sequential steps of the ATPase cycle was investigated using the polarized fluorescence of 1.5-IAEDANS bound to myosin heads, FITC-phalloidin attached to actin and acrylodan bound to twitchin in the glycerol-skinned skeletal muscle fibres of mammalian. The phosphorylation-dependent multi-step changes in mobility and spatial arrangement of myosin SH1 helix, actin subunit and twitchin during the ATPase cycle have been revealed. It was shown that nonphosphorylated twitchin inhibited the movements of SH1 helix of the myosin heads and actin subunits and decreased the affinity of myosin to actin by freezing the position and mobility of twitchin in the muscle fibres. The phosphorylation of twitchin reverses this effect by changing the spatial arrangement and mobility of the actin-binding portions of twitchin. In this case, enhanced movements of SH1 helix of the myosin heads and actin subunits are observed. The data imply a novel property of twitchin incorporated into organized contractile system: its ability to regulate the ATPase cycle in a phosphorylation-dependent fashion by changing the affinity and spatial arrangement of the actin-binding portions of twitchin. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Measures and predictors of varenicline adherence in the treatment of nicotine dependence.

    Science.gov (United States)

    Peng, Annie R; Morales, Mark; Wileyto, E Paul; Hawk, Larry W; Cinciripini, Paul; George, Tony P; Benowitz, Neal L; Nollen, Nicole L; Lerman, Caryn; Tyndale, Rachel F; Schnoll, Robert

    2017-12-01

    While adherence to medication in smoking cessation clinical trials is strongly associated with clinical outcome, very few studies have evaluated the validity of pill count as a measure of adherence relative to a biological assay, and evaluated a broad range of correlates of adherence. In a smoking cessation clinical trial of varenicline, we compared pill counts collected over 4 different time periods to varenicline salivary levels taken after 2weeks of treatment, as well as evaluated predictors of adherence to varenicline. Using a binary measure of adherence based on salivary varenicline levels, adherence was higher among older, white, and more educated participants. Relative to 3, 7, and 14-day pill count, 12-week pill count was the only significant measure able to discriminate adherence as defined by salivary varenicline levels (assessed by area under the receiver operating characteristic curve; AUC=0.59, p=0.004). Seventy-two percent of participants who indicated adherence on 12-week pill count were classified as adherent based on varenicline saliva levels (sensitivity=0.80; specificity=0.40). There was modest variability in the relationship between 12-week pill count and varenicline levels across race and rate of nicotine metabolism. Lastly, General Estimating Equation models demonstrated that longitudinal changes in withdrawal, craving, negative and positive affect, and side effect count and severity were not related to adherence based on salivary varenicline levels. These results indicate that 12-week pill count was the best, albeit a relatively weak, measure of varenicline adherence; additional factors associated with treatment adherence need to be identified. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Syk-dependent tyrosine phosphorylation of 3BP2 is required for optimal FcRγ-mediated phagocytosis and chemokine expression in U937 cells.

    Science.gov (United States)

    Chihara, Kazuyasu; Kato, Yuji; Yoshiki, Hatsumi; Takeuchi, Kenji; Fujieda, Shigeharu; Sada, Kiyonao

    2017-09-13

    The adaptor protein c-Abl SH3 domain binding protein-2 (3BP2) is tyrosine phosphorylated by Syk in response to cross-linking of antigen receptors, which in turn activates various immune responses. Recently, a study using the mouse model of cherubism, a dominant inherited disorder caused by mutations in the gene encoding 3BP2, showed that 3BP2 is involved in the regulation of phagocytosis mediated by Fc receptor for IgG (FcγR) in macrophages. However, the molecular mechanisms underlying 3BP2-mediated regulation of phagocytosis and the physiological relevance of 3BP2 tyrosine phosphorylation remains elusive. In this study, we established various gene knockout U937 cell lines using the CRISPR/Cas9 system and found that 3BP2 is rapidly tyrosine phosphorylated by Syk in response to cross-linking of FcγRI. Depletion of 3BP2 caused significant reduction in the Fc receptor γ chain (FcRγ)-mediated phagocytosis in addition to the FcγRI-mediated induction of chemokine mRNA for IL-8, CCL3L3 and CCL4L2. Syk-dependent tyrosine phosphorylation of 3BP2 was required for overcoming these defects. Finally, we found that the PH and SH2 domains play important roles on FcγRI-mediated tyrosine phosphorylation of 3BP2 in HL-60 cells. Taken together, these results indicate that Syk-dependent tyrosine phosphorylation of 3BP2 is required for optimal FcRγ-mediated phagocytosis and chemokine expression.

  6. Grain dust induces IL-8 production from bronchial epithelial cells: effect on neutrophil recruitment.

    Science.gov (United States)

    Park, H S; Suh, J H; Kim, S S; Kwon, O J

    2000-06-01

    There have been several investigations suggesting an involvement of activated neutrophils in the development of grain dust (GD)-induced occupational asthma. Interleukin-8 in the sputa from GD-induced asthmatic patients increased significantly after the exposure to GD. To confirm IL-8 production from bronchial epithelial cells when exposed to GD, and to evaluate the role of IL-8 on neutrophil recruitment. We cultured Beas-2B, a bronchial epithelial cell line. To observe GD-induced responses, four different concentrations ranging from 1 to 200 microg/mL of GD were incubated for 24 hours and compared with those without incubation of GD. To evaluate the effect of pro-inflammatory cytokines on IL-8 production and neutrophil chemotaxis, epithelial cells were incubated with peripheral blood mononuclear cell (PBMC) culture supernatant derived from subjects with GD-induced asthma exposed to 10 microg/mL of GD, and then compared with those without addition of PBMC supernatant. The level of released IL-8 in the supernatant was measured by enzyme-linked immunosorbent assay. Neutrophil chemotactic activity of the culture supernatant was determined by modified Boyden chamber method. Interleukin-8 production and neutrophil chemotactic activity from bronchial epithelial cells significantly increased with additions of GD in a dose-dependent manner (P < .05, respectively), and were significantly augmented with additions of PBMC supernatant (P < .05, respectively) at each concentration. Close correlation was noted between neutrophil chemotactic activity and IL-8 level (r = 0.87, P < .05). Compared with the untreated sample, pre-treatment of anti-IL-8 antibody induced a significant suppression (up to 67.2%) of neutrophil chemotactic activity in a dose-dependent manner. These results suggest that IL-8 produced from bronchial epithelial cells may be a major cytokine, which induces neutrophil migration into the airways when exposed to GD.

  7. Neutrophils degrade subendothelial matrices in the presence of alpha-1-proteinase inhibitor. Cooperative use of lysosomal proteinases and oxygen metabolites.

    OpenAIRE

    Weiss, S J; Regiani, S

    1984-01-01

    Triggered neutrophils rapidly degraded labeled matrices secreted by cultured, venous endothelial cells via a process dependent on elastase but not oxygen metabolites. In the presence of high concentrations of alpha-1-proteinase inhibitor, the ability of the stimulated neutrophil to solubilize the matrix was impaired. However, at lower concentrations of alpha-1-proteinase inhibitor the neutrophil could enhance the degradative potential of its released elastase by a H2O2-dependent process. Coin...

  8. Cholinergic regulation of protein phosphorylation in bovine adrenal chromaffin cells

    International Nuclear Information System (INIS)

    Haycock, J.W.; Browning, M.D.; Greengard, P.

    1988-01-01

    Chromaffin cells were isolated from bovine adrenal medullae and maintained in primary culture. After prelabeling with 32 PO 4 , exposure of the chromaffin cells to acetylcholine increased the phosphorylation of a M/sub r/ ≅ 100,000 protein and a M/sub r/ ≅ 60,000 protein (tyrosine hydroxylase), visualized after separation of total cellular proteins in NaDodSO 4 /polyacrylamide gels. Immunoprecipitation with antibodies to three known phosphoproteins (100-kDa, 87-kDa, and protein III) revealed an acetylcholine-dependent phosphorylation of these proteins. These three proteins were also shown to be present in bovine adrenal chromaffin cells by immunolabeling techniques. 100-kDa is a M/sub r/ ≅ 100,000 protein selectively phosphorylated by calcium/calmodulin-dependent protein kinase III, 87-kDa is a M/sub r/ ≅ 87,000 protein selectively phosphorylated by protein kinase C, and protein III is a phosphoprotein doublet of M/sub r/ ≅ 74,000 (IIIa) and M/sub r/ ≅ 55,000 (IIIb) phosphorylated by cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase I. The data demonstrate that cholinergic activation of chromaffin cells increases the phosphorylation of several proteins and that several protein kinase systems may be involved in these effects

  9. Oxygen dependency of neutrophilic Fe(II) oxidation by Leptothrix differs from abiotic reaction

    NARCIS (Netherlands)

    Vollrath, S.; Behrends, T.; Van Cappellen, P.

    2012-01-01

    Neutrophilic Fe(II) oxidizing microorganisms are found in many natural environments. It has been hypothesized that, at low oxygen concentrations, microbial iron oxidation is favored over abiotic oxidation. Here, we compare the kinetics of abiotic Fe(II) oxidation to oxidation in the presence of

  10. 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

  11. 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.

  12. The Golgi localization of phosphatidylinositol transfer protein beta requires the protein kinase C-dependent phosphorylation of serine 262 and is essential for maintaining plasma membrane sphingomyelin levels.

    Science.gov (United States)

    van Tiel, Claudia M; Westerman, Jan; Paasman, Marten A; Hoebens, Martha M; Wirtz, Karel W A; Snoek, Gerry T

    2002-06-21

    Recombinant mouse phosphatidylinositol transfer protein (PI-TP)beta is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser(262) was identified as the major site of phosphorylation and Ser(165) as a minor phosphorylation site. The phospholipid transfer activities of wild-type PI-TP beta and PI-TP beta(S262A) were identical, whereas PI-TP beta(S165A) was completely inactive. PKC-dependent phosphorylation of Ser(262) also had no effect on the transfer activity of PI-TP beta. To investigate the role of Ser(262) in the functioning of PI-TP beta, wtPI-TP beta and PI-TP beta(S262A) were overexpressed in NIH3T3 fibroblast cells. Two-dimensional PAGE analysis of cell lysates was used to separate PI-TP beta from its phosphorylated form. After Western blotting, wtPI-TP beta was found to be 85% phosphorylated, whereas PI-TP beta(S262A) was not phosphorylated. In the presence of the PKC inhibitor GF 109203X, the phosphorylated form of wtPI-TP beta was strongly reduced. Immunolocalization showed that wtPI-TP beta was predominantly associated with the Golgi membranes. In the presence of the PKC inhibitor, wtPI-TP beta was distributed throughout the cell similar to what was observed for PI-TP beta(S262A). In contrast to wtPI-TP beta overexpressors, cells overexpressing PI-TP beta(S262A) were unable to rapidly replenish sphingomyelin in the plasma membrane upon degradation by sphingomyelinase. This implies that PKC-dependent association with the Golgi complex is a prerequisite for PI-TP beta to express its effect on sphingomyelin metabolism.

  13. The calcium-dependent protein kinase RcCDPK2 phosphorylates sucrose synthase at Ser11 in developing castor oil seeds.

    Science.gov (United States)

    Fedosejevs, Eric T; Gerdis, Suzanne A; Ying, Sheng; Pyc, Michal; Anderson, Erin M; Snedden, Wayne A; Mullen, Robert T; She, Yi-Min; Plaxton, William C

    2016-10-15

    Imported sucrose is cleaved by sucrose synthase (SUS) as a critical initial reaction in the biosynthesis of storage end-products by developing seeds. Although SUS is phosphorylated at a conserved seryl residue by an apparent CDPK (Ca 2+ -dependent protein kinase) in diverse plant tissues, the functions and mechanistic details of this process remain obscure. Thus, the native CDPK that phosphorylates RcSUS1 (Ricinus communis SUS1) at Ser 11 in developing COS (castor oil seeds) was highly purified and identified as RcCDPK2 by MS/MS. Purified RcSUS1-K (-kinase) and heterologously expressed RcCDPK2 catalyzed Ca 2+ -dependent Ser 11 phosphorylation of RcSUS1 and its corresponding dephosphopeptide, while exhibiting a high affinity for free Ca 2+ ions [K 0.5 (Ca 2+ ) < 0.4 µM]. RcSUS1-K activity, RcCDPK2 expression, and RcSUS1 Ser 11 phosphorylation peaked during early COS development and then declined in parallel. The elimination of sucrose import via fruit excision triggered RcSUS1 dephosphorylation but did not alter RcSUS1-K activity, suggesting a link between sucrose signaling and posttranslational RcCDPK2 control. Both RcCDPK2-mCherry and RcSUS1-EYFP co-localized throughout the cytosol when transiently co-expressed in tobacco suspension cells, although RcCDPK2-mCherry was also partially localized to the nucleus. Subcellular fractionation revealed that ∼20% of RcSUS1-K activity associates with microsomal membranes in developing COS, as does RcSUS1. In contrast with RcCDPK1, which catalyzes inhibitory phosphorylation of COS bacterial-type phosphoenolpyruvate carboxylase at Ser 451 , RcCDPK2 exhibited broad substrate specificity, a wide pH-activity profile centered at pH 8.5, and insensitivity to metabolite effectors or thiol redox status. Our combined results indicate a possible link between cytosolic Ca 2+ -signaling and the control of photosynthate partitioning during COS development. © 2016 The Author(s); published by Portland Press Limited on behalf of the

  14. Plk4-dependent phosphorylation of STIL is required for centriole duplication

    Directory of Open Access Journals (Sweden)

    Anne-Sophie Kratz

    2015-02-01

    Full Text Available Duplication of centrioles, namely the formation of a procentriole next to the parental centriole, is regulated by the polo-like kinase Plk4. Only a few other proteins, including STIL (SCL/TAL1 interrupting locus, SIL and Sas-6, are required for the early step of centriole biogenesis. Following Plk4 activation, STIL and Sas-6 accumulate at the cartwheel structure at the initial stage of the centriole assembly process. Here, we show that STIL interacts with Plk4 in vivo. A STIL fragment harboring both the coiled-coil domain and the STAN motif shows the strongest binding affinity to Plk4. Furthermore, we find that STIL is phosphorylated by Plk4. We identified Plk4-specific phosphorylation sites within the C-terminal domain of STIL and show that phosphorylation of STIL by Plk4 is required to trigger centriole duplication.

  15. 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...

  16. Calcium-regulated in vivo protein phosphorylation in Zea mays L. root tips

    Science.gov (United States)

    Raghothama, K. G.; Reddy, A. S.; Friedmann, M.; Poovaiah, B. W.

    1987-01-01

    Calcium dependent protein phosphorylation was studied in corn (Zea mays L.) root tips. Prior to in vivo protein phosphorylation experiments, the effect of calcium, ethyleneglycol-bis-(beta-aminoethyl ether)-N-N' -tetraacetic acid (EGTA) and calcium ionophore (A-23187) on phosphorus uptake was studied. Calcium increased phosphorus uptake, whereas EGTA and A-23187 decreased it. Consequently, phosphorus concentration in the media was adjusted so as to attain similar uptake in different treatments. Phosphoproteins were analyzed by two-dimensional gel electrophoresis. Distinct changes in phosphorylation were observed following altered calcium levels. Calcium depletion in root tips with EGTA and A-23187 decreased protein phosphorylation. However, replenishment of calcium following EGTA and ionophore pretreatment enhanced phosphorylation of proteins. Preloading of the root tips with 32P in the presence of EGTA and A-23187 followed by a ten minute calcium treatment, resulted in increased phosphorylation indicating the involvement of calcium, calcium and calmodulin-dependent kinases. Calmodulin antagonist W-7 was effective in inhibiting calcium-promoted phosphorylation. These studies suggest a physiological role for calcium-dependent phosphorylation in calcium-mediated processes in plants.

  17. 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.

  18. Earthworm coelomocyte extracellular traps: structural and functional similarities with neutrophil NETs.

    Science.gov (United States)

    Homa, Joanna

    2018-03-01

    Invertebrate immunity is associated with natural mechanisms that include cellular and humoral elements, similar to those that play a role in vertebrate innate immune responses. Formation of extracellular traps (ETs) is a newly discovered mechanism to combat pathogens, operating not only in vertebrate leucocytes but also in invertebrate immune cells. The ET components include extracellular DNA (exDNA), antimicrobial proteins and histones. Formation of mammalian ETs depends on enzymes such as neutrophil elastase, myeloperoxidase, the citrullination of histones and protease activity. It was confirmed that coelomocytes-immunocompetent cells of the earthworm Eisenia andrei-are also able to release ETs in a protease-dependent manner, dependent or independent of the formation of reactive oxygen species and rearrangement of the cell cytoskeleton. Similar to vertebrate leukocytes (e.g., neutrophil), coelomocytes are responsible for many immune functions like phagocytosis, cytotoxicity and secretion of humoral factors. ETs formed by coelomocyte analogues to neutrophil ETs consist of exDNA, histone H3 and attached to these structures proteins, e.g., heat shock proteins HSP27. The latter fact confirms that mechanisms of ET release are conserved in evolution. The study on Annelida adds this animal group to the list of invertebrates capable of ET release, but most importantly provides insides into innate mechanisms of ET formation in lower animal taxa.

  19. 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.

  20. 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

  1. 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.

  2. Interleukin-8: an expanding universe beyond neutrophil chemotaxis and activation.

    Science.gov (United States)

    Mukaida, N

    2000-12-01

    Since the discovery 13 years ago of interleukin (IL)-8 as a potent neutrophil chemotactic factor, accumulating evidence has established it as a crucial mediator in neutrophil-dependent acute inflammation. Numerous observations have demonstrated that various types of cells can produce a large amount of IL-8, either in response to various stimuli or constitutively, after malignant transformation. Recent studies of IL-8-mediated signaling have revealed that IL-8 activates a wide range of signaling molecules in a coordinate manner. IL-8 has been proven to have diverse actions on various types of leukocytic and nonleukocytic cells besides neutrophils. The author reviews recent progress in IL-8 signal transduction and biological actions on nonneutrophilic leukocytes, including T lymphocytes, monocytes, and hematopoietic progenitor cells. Potential involvement of IL-8 in viral infections and tumor progression is also discussed.

  3. Development of a phosphorylated Momordica charantia protein system for inhibiting susceptible dose-dependent C. albicans to available antimycotics: An allosteric regulation of protein.

    Science.gov (United States)

    Qiao, Yuanbiao; Song, Li; Zhu, Chenchen; Wang, Qian; Guo, Tianyan; Yan, Yanhua; Li, Qingshan

    2017-11-15

    A regulatory Momordica charantia protein system was constructed allosterically by in vitro protein phosphorylation, in an attempt to evaluate antimycological pluripotency against dose-dependent susceptibilities in C. albicans. Fungal strain lineages susceptible to ketoconazole, econazole, miconazole, 5-flucytosine, nystatin and amphotericin B were prepared in laboratory, followed by identification via antifungal susceptibility testing. Protein phosphorylation was carried out in reactions with 5'-adenylic, guanidylic, cytidylic and uridylic acids and cyclic adenosine triphosphate, through catalysis of cyclin-dependent kinase 1, protein kinase A and protein kinase C respectively. Biochemical analysis of enzymatic reactions indicated the apparent Michaelis-Menten constants and maximal velocity values of 16.57-91.97mM and 55.56-208.33μM·min -1 , together with an approximate 1:1 reactant stoichiometric ratio. Three major protein phosphorylation sites were theoretically predicted at Thr255, Thr102 and Thr24 by a KinasePhos tool. Additionally, circular dichroism spectroscopy demonstrated that upon phosphorylation, protein folding structures were decreased in random coil, β6-sheet and α1-helix partial regions. McFarland equivalence standard testing yielded the concentration-dependent inhibition patterns, while fungus was grown in Sabouraud's dextrose agar. The minimal inhibitory concentrations of 0.16-0.51μM (at 50% response) were obtained for free protein and phosphorylated counterparts. With respect to the 3-cycling susceptibility testing regimen, individuals of total protein forms were administrated in-turn at 0.14μM/cycle. Relative inhibition ratios were retained to 66.13-81.04% of initial ones regarding the ketoconazole-susceptible C. albicans growth. An inhibitory protein system, with an advantage of decreasing antifungal susceptibilities to diverse antimycotics, was proposed because of regulatory pluripotency whereas little contribution to susceptibility in

  4. Sulforaphane restores cellular glutathione levels and reduces chronic periodontitis neutrophil hyperactivity in vitro.

    Directory of Open Access Journals (Sweden)

    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.

  5. 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.

  6. Rapid changes in protein phosphorylation associated with gravity perception in corn roots

    International Nuclear Information System (INIS)

    McFadden, J.J.; Poovaiah, B.W.

    1987-01-01

    A previous paper from this laboratory showed calcium- and calmodulin-dependent in vivo protein phosphorylation in corn root tips. The authors show that rapid changes in calcium-dependent protein phosphorylation are involved in light-dependent graviperception in corn root tips. Corn seedlings (Zea mays L, cv Merit) were grown in the dark for 3 d, then apical root segments were harvested in dim green light to measure in vivo protein phosphorylation. Segments were incubated with 0.5 mCi 32 P for 1 h, then immediately frozen in liquid N 2 or first treated with either 7 min light, or 7 min light plus 1 mM EGTA and 10 μM A23187. Labeled proteins were separated by 2D gel electrophoresis and detected by autoradiography. Light caused rapid and specific promotion of phosphorylation of 5 polypeptides. The increases in protein phosphorylation were reversed by treating with EGTA and A23187. The authors postulate that these changes in protein phosphorylation are an essential part of the light-dependent gravity response in Merit roots

  7. A role for protein phosphatase-2A in p38 mitogen-activated protein kinase-mediated regulation of the c-Jun NH(2)-terminal kinase pathway in human neutrophils.

    Science.gov (United States)

    Avdi, Natalie J; Malcolm, Kenneth C; Nick, Jerry A; Worthen, G Scott

    2002-10-25

    Human neutrophil accumulation in inflammatory foci is essential for the effective control of microbial infections. Although exposure of neutrophils to cytokines such as tumor necrosis factor-alpha (TNFalpha), generated at sites of inflammation, leads to activation of MAPK pathways, mechanisms responsible for the fine regulation of specific MAPK modules remain unknown. We have previously demonstrated activation of a TNFalpha-mediated JNK pathway module, leading to apoptosis in adherent human neutrophils (Avdi, N. J., Nick, J. A., Whitlock, B. B., Billstrom, M. A., Henson, P. M., Johnson, G. L., and Worthen, G. S. (2001) J. Biol. Chem. 276, 2189-2199). Herein, evidence is presented linking regulation of the JNK pathway to p38 MAPK and the Ser/Thr protein phosphatase-2A (PP2A). Inhibition of p38 MAPK by SB 203580 and M 39 resulted in significant augmentation of TNFalpha-induced JNK and MKK4 (but not MKK7 or MEKK1) activation, whereas prior exposure to a p38-activating agent (platelet-activating factor) diminished the TNFalpha-induced JNK response. TNFalpha-induced apoptosis was also greatly enhanced upon p38 inhibition. Studies with a reconstituted cell-free system indicated the absence of a direct inhibitory effect of p38 MAPK on the JNK module. Neutrophil exposure to the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A induced JNK activation. Increased phosphatase activity following TNFalpha stimulation was shown to be PP2A-associated and p38-dependent. Furthermore, PP2A-induced dephosphorylation of MKK4 resulted in its inactivation. Thus, in neutrophils, p38 MAPK, through a PP2A-mediated mechanism, regulates the JNK pathway, thus determining the extent and nature of subsequent responses such as apoptosis.

  8. 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.

  9. Oxidative stress augments toll-like receptor 8 mediated neutrophilic responses in healthy subjects

    Directory of Open Access Journals (Sweden)

    Matsunaga Kazuto

    2009-06-01

    Full Text Available Abstract Background Excessive oxidative stress has been reported to be generated in inflamed tissues and contribute to the pathogenesis of inflammatory lung diseases, exacerbations of which induced by viral infections are associated with toll-like receptor (TLR activation. Among these receptors, TLR8 has been reported as a key receptor that recognizes single-strand RNA virus. However, it remains unknown whether TLR8 signaling is potentiated by oxidative stress. The aim of this study is to examine whether oxidative stress modulates TLR8 signaling in vitro. Methods Human peripheral blood neutrophils were obtained from healthy non-smokers and stimulated with TLR 7/8 agonist imidazoquinoline resiquimod (R848 in the presence or absence of hydrogen peroxide (H2O2. Neutrophilic responses including cytokine release, superoxide production and chemotaxis were examined, and the signal transduction was also analyzed. Results Activation of TLR8, but not TLR7, augmented IL-8 release. The R848-augmented IL-8 release was significantly potentiated by pretreatment with H2O2 (p L-cysteine reversed this potentiation. The combination of H2O2 and R848 significantly potentiated NF-kB phosphorylation and IkBα degradation. The H2O2-potentiated IL-8 release was suppressed by MG-132, a proteosome inhibitor, and by dexamethasone. The expressions of TLR8, myeloid differentiation primary response gene 88 (MyD88, and tumor necrosis factor receptor-associated factor 6 (TRAF6 were not affected by H2O2. Conclusion TLR8-mediated neutrophilic responses were markedly potentiated by oxidative stress, and the potentiation was mediated by enhanced NF-kB activation. These results suggest that oxidative stress might potentiate the neutrophilic inflammation during viral infection.

  10. Differential effector responses by circulating/blood and tissue/peritoneal neutrophils following burn combined with Enterococcus faecalis infection.

    Science.gov (United States)

    Fazal, Nadeem; Shelip, Alla; Siddiqui, Erum; Ali, Ashraf; Azim, Anser C; Al-Ghoul, Walid M

    2012-03-01

    Recently we found that superimposition of Enterococcus faecalis infection on burn injury caused an eruption of host mortality not seen with either individual challenge. We hypothesized that the Enterococcus bacteria, and/or factors related to these organisms, aggravate burn-induced modulations in host defense by neutrophils. Our study focuses on alterations in neutrophils' oxidative, proteolytic, and adhesive functions and transendothelial migration of neutrophils in burn rats inoculated with E. faecalis. Rats were subjected to burn (30% total body surface area) and then intra-abdominally inoculated with E. faecalis (10(4)CFU kg(-1) b.w). Polymorphonuclear neutrophils (PMNs) were harvested from circulating/blood and tissue/peritoneal cavity at day-2 post injury. Extracellular release of O(-)(2) anion production was determined by luminometry, and intracellular production of reactive oxygen species was measured by digital imaging technique. Fluoroscan analysis and confocal microscopy determined intracellular elastase production. The expression of adhesion molecule CD11b/CD18 was performed by flow cytometry. Calcein AM-labeled PMNs were co-cultured with TNF-α-stimulated rat lung microvascular endothelial cells, and their ability to adhere was assessed by fluorometry and digital imaging and finally, chemotaxis was measured by neutrophil transmigration assays. The results showed differential effector responses by circulatory and/or tissue PMNs. Tissue/peritoneal PMNs produced more O(-)(2), less intracellular elastase, and increased expression of CD11b/CD18 accompanied with increased adhesivity of MIP-2-stimulated PMNs to endothelial cells as compared to circulatory/blood PMNs. This differential effect was more pronounced following burn plus E. faecalis infection, indicating that the combined injury changed neutrophil functions. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  11. Neutrophils reduce the parasite burden in Leishmania (Leishmania amazonensis-infected macrophages.

    Directory of Open Access Journals (Sweden)

    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.

  12. 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.

  13. 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.

  14. Effects of acrolein on leukotriene biosynthesis in human neutrophils.

    Science.gov (United States)

    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

  15. Selective inhibition reveals cyclin-dependent kinase 2 as another kinase that phosphorylates the androgen receptor at serine 81

    Czech Academy of Sciences Publication Activity Database

    Jorda, Radek; Bučková, Zuzana; Řezníčková, Eva; Bouchal, J.; Kryštof, Vladimír

    2018-01-01

    Roč. 1865, č. 2 (2018), s. 354-363 ISSN 0167-4889 R&D Projects: GA MŠk(CZ) LO1204; GA MŠk(CZ) LO1304 Institutional support: RVO:61389030 Keywords : Androgen receptor * Cyclin-dependent kinase * Inhibitor * Phosphorylation * Serine 81 Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 4.521, year: 2016

  16. 2',3-dihydroxy-5-methoxybiphenyl suppresses fMLP-induced superoxide anion production and cathepsin G release by targeting the β-subunit of G-protein in human neutrophils.

    Science.gov (United States)

    Liao, Hsiang-Ruei; Chen, Ih-Sheng; Liu, Fu-Chao; Lin, Shinn-Zhi; Tseng, Ching-Ping

    2018-06-15

    This study investigates the effect and the underlying mechanism of 2',3-dihydroxy-5-methoxybiphenyl (RIR-2), a lignan extracted from the roots of Rhaphiolepis indica (L.) Lindl. ex Ker var. tashiroi Hayata ex Matsum. & Hayata (Rosaceae), on N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced respiratory burst and cathepsin G in human neutrophils. Signaling pathways regulated by RIR-2 which modulated fMLP-induced respiratory burst were evaluated by an interaction between β subunit of G-protein (Gβ) with downstream signaling induced by fMLP and by immunoblotting analysis of the downstream targets of Gβ-protein. RIR-2 inhibited fMLP-induced superoxide anion production (IC 50 :2.57 ± 0.22 μM), cathepsin G release (IC 50 :18.72 ± 3.76 μM) and migration in a concentration dependent manner. RIR-2 specifically suppresses fMLP-induced Src family kinases phosphorylation by inhibiting the interaction between Gβ-protein with Src kinases without inhibiting Src kinases activities, therefore, RIR-2 attenuated the downstream targets of Src kinase, such as phosphorylation of Raf/ERK, AKT, P38, PLCγ2, PKC and translocation Tec, p47 ph ° x and P40 ph ° x from the cytosol to the inner leaflet of the plasma membrane. Furthermore, RIR-2 attenuated fMLP-induced intracellular calcium mobilization by inhibiting the interaction between Gβ-protein with PLCβ2. RIR-2 was not a competitive or allosteric antagonist of fMLP. On the contrary, phorbol 12-myristate 13-acetate (PMA)-induced phosphorylation of Src, AKT, P38, PKC and membrane localization of p47 ph ° x and P40 ph ° x remained unaffected. RIR-2 specifically modulates fMLP-mediated neutrophil superoxide anion production and cathepsin G release by inhibiting the interaction between Gβ-protein with downstream signaling which subsequently interferes with the activation of intracellular calcium, PLCγ2, AKT, p38, PKC, ERK, p47 ph ° x and p40 phox . Copyright © 2018 Elsevier B.V. All rights reserved.

  17. Thymosin-β4 (Tβ4) Blunts PDGF-Dependent Phosphorylation and Binding of AKT to Actin in Hepatic Stellate Cells

    Science.gov (United States)

    Reyes-Gordillo, Karina; Shah, Ruchi; Popratiloff, Anastas; Fu, Sidney; Hindle, Anna; Brody, Frederick; Rojkind, Marcos

    2011-01-01

    Hepatic stellate cell transdifferentiation is a key event in the fibrogenic cascade. Therefore, attempts to prevent and/or revert the myofibroblastic phenotype could result in novel therapeutic approaches to treat liver cirrhosis. The expression of platelet-derived growth factor (PDGF)-β receptor and the proliferative response to platelet-derived growth factor-ββ (PDGF-ββ) are hallmarks of the transdifferentiation of hepatic stellate cells (HSC). In this communication, we investigated whether thymosin-β4 (Tβ4), a chemokine expressed by HSC could prevent PDGF-BB-mediated proliferation and migration of cultured HSC. Using early passages of human HSC, we showed that Tβ4 inhibited cell proliferation and migration and prevented the expression of PDGF-β receptor (PDGF-βr), α-smooth muscle actin and α1(I) collagen mRNAs. Tβ4 also inhibited the reappearance of PDGF-βr after its PDGF-BB-dependent degradation. These PDGF-dependent events were associated with the inhibition of AKT phosphorylation at both T308 and S473 amino acid residues. The lack of AKT phosphorylation was not due to the inhibition of PDGF-βr phosphorylation, the activation of phosphoinositide 3-kinase (PI3K), pyruvate dehydrogenase kinase isozyme 1 (PDK1), and mammalian target of rapamycin (mTOR). We found that PDGF-BB induced AKT binding to actin, and that Tβ4 prevented this effect. Tβ4 also prevented the activation of freshly isolated HSC cultured in the presence of Dulbecco's modified Eagle's medium or Dulbecco's minimal essential medium containing 10% fetal bovine serum. In conclusion, overall, our findings suggest that Tβ4 by sequestering actin prevents binding of AKT, thus inhibiting its phosphorylation. Therefore, Tβ4 has the potential to be an antifibrogenic agent. PMID:21514425

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

    Directory of Open Access Journals (Sweden)

    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.

  19. 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

  20. In Vitro Evaluation of the Link Between Cell Activation State and Its Rheological Impact on the Microscale Flow of Neutrophil Suspensions

    Science.gov (United States)

    Akenhead, Michael L.; Horrall, Nolan M.; Rowe, Dylan; Sethu, Palaniappan; Shin, Hainsworth Y.

    2015-01-01

    Activated neutrophils have been reported to affect peripheral resistance, for example, by plugging capillaries or adhering to the microvasculature. In vivo and ex vivo data indicate that activated neutrophils circulating in the blood also influence peripheral resistance. We used viscometry and microvascular mimics for in vitro corroboration. The rheological impact of differentiated neutrophil-like HL-60 promyelocytes (dHL60s) or human neutrophil suspensions stimulated with 10 nM fMet-Leu-Phe (fMLP) was quantified using a cone-plate rheometer (450 s−1 shear rate). To evaluate their impact on microscale flow resistance, we used 10-μm Isopore® membranes to model capillaries as well as single 200 × 50 μm microchannels and networks of twenty 20 × 50 μm microfluidic channels to mimic noncapillary microvasculature. Stimulation of dHL60 and neutrophil populations significantly altered their flow behavior as evidenced by their impact on suspension viscosity. Notably, hematocrit abrogated the impact of leukocyte activation on blood cell suspension viscosity. In micropore filters, activated cell suspensions enhanced flow resistance. This effect was further enhanced by the presence of erythrocytes. The resistance of our noncapillary microvascular mimics to flow of activated neutrophil suspensions was significantly increased only with hematocrit. Notably, it was elevated to a higher extent within the micronetwork chambers compared to the single-channel chambers. Collectively, our findings provide supportive evidence that activated neutrophils passing through the microcirculation may alter hemodynamic resistance due to their altered rheology in the noncapillary microvasculature. This effect is another way neutrophil activation due to chronic inflammation may, at least in part, contribute to the elevated hemodynamic resistance associated with cardiovascular diseases (e.g., hypertension and hypercholesterolemia). PMID:26065495

  1. In Vitro Evaluation of the Link Between Cell Activation State and Its Rheological Impact on the Microscale Flow of Neutrophil Suspensions.

    Science.gov (United States)

    Akenhead, Michael L; Horrall, Nolan M; Rowe, Dylan; Sethu, Palaniappan; Shin, Hainsworth Y

    2015-09-01

    Activated neutrophils have been reported to affect peripheral resistance, for example, by plugging capillaries or adhering to the microvasculature. In vivo and ex vivo data indicate that activated neutrophils circulating in the blood also influence peripheral resistance. We used viscometry and microvascular mimics for in vitro corroboration. The rheological impact of differentiated neutrophil-like HL-60 promyelocytes (dHL60s) or human neutrophil suspensions stimulated with 10 nM fMet-Leu-Phe (fMLP) was quantified using a cone-plate rheometer (450 s(-1) shear rate). To evaluate their impact on microscale flow resistance, we used 10-μm Isopore® membranes to model capillaries as well as single 200 × 50 μm microchannels and networks of twenty 20 × 50 μm microfluidic channels to mimic noncapillary microvasculature. Stimulation of dHL60 and neutrophil populations significantly altered their flow behavior as evidenced by their impact on suspension viscosity. Notably, hematocrit abrogated the impact of leukocyte activation on blood cell suspension viscosity. In micropore filters, activated cell suspensions enhanced flow resistance. This effect was further enhanced by the presence of erythrocytes. The resistance of our noncapillary microvascular mimics to flow of activated neutrophil suspensions was significantly increased only with hematocrit. Notably, it was elevated to a higher extent within the micronetwork chambers compared to the single-channel chambers. Collectively, our findings provide supportive evidence that activated neutrophils passing through the microcirculation may alter hemodynamic resistance due to their altered rheology in the noncapillary microvasculature. This effect is another way neutrophil activation due to chronic inflammation may, at least in part, contribute to the elevated hemodynamic resistance associated with cardiovascular diseases (e.g., hypertension and hypercholesterolemia).

  2. Neutrophil activation during acetaminophen hepatotoxicity and repair in mice and humans

    Energy Technology Data Exchange (ETDEWEB)

    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

  3. The Golgi localization of phosphatidylinositol transfer protein beta requires the protein kinase C-dependent phosphorylation of serine 262 and is essential for maintaining plasma membrane sphingomyelin levels

    NARCIS (Netherlands)

    van Tiel, Claudia M.; Westerman, Jan; Paasman, Marten A.; Hoebens, Martha M.; Wirtz, Karel W. A.; Snoek, Gerry T.

    2002-01-01

    Recombinant mouse phosphatidylinositol transfer protein (PI-TP)beta is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser(262) was identified as the major site of phosphorylation and Ser(165)

  4. 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

    2015-06-16

    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.

  5. Tamoxifen induces apoptotic neutrophil efferocytosis in horses.

    Science.gov (United States)

    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.

  6. With Friends Like These: The Complex Role of Neutrophils in the Progression of Severe Pneumonia

    Directory of Open Access Journals (Sweden)

    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.

  7. 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

  8. Neutrophil Responses to Sterile Implant Materials.

    Directory of Open Access Journals (Sweden)

    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.

  9. Neutrophil migration under normal and sepsis conditions.

    Science.gov (United States)

    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.

  10. PKB/SGK-dependent GSK3-phosphorylation in the regulation of LPS-induced Ca2+ increase in mouse dendritic cells.

    Science.gov (United States)

    Russo, Antonella; Schmid, Evi; Nurbaeva, Meerim K; Yang, Wenting; Yan, Jing; Bhandaru, Madhuri; Faggio, Caterina; Shumilina, Ekaterina; Lang, Florian

    2013-08-02

    The function of dendritic cells (DCs) is modified by glycogen synthase kinase GSK3 and GSK3 inhibitors have been shown to protect against inflammatory disease. Regulators of GSK3 include the phosphoinositide 3 kinase (PI3K) pathway leading to activation of protein kinase B (PKB/Akt) and serum and glucocorticoid inducible kinase (SGK) isoforms, which in turn phosphorylate and thus inhibit GSK3. The present study explored, whether PKB/SGK-dependent inhibition of GSK3 contributes to the regulation of cytosolic Ca(2+) concentration following stimulation with bacterial lipopolysaccharides (LPS). To this end DCs from mutant mice, in which PKB/SGK-dependent GSK3α,β regulation was disrupted by replacement of the serine residues in the respective SGK/PKB-phosphorylation consensus sequence by alanine (gsk3(KI)), were compared to DCs from respective wild type mice (gsk3(WT)). According to Western blotting, GSK3 phosphorylation was indeed absent in gsk3(KI) DCs. According to flow cytometry, expression of antigen-presenting molecule major histocompatibility complex II (MHCII) and costimulatory molecule CD86, was similar in unstimulated and LPS (1μg/ml, 24h)-stimulated gsk3(WT) and gsk3(KI) DCs. Moreover, production of cytokines IL-6, IL-10, IL-12 and TNFα was not significantly different in gsk3(KI) and gsk3(WT) DCs. In gsk3(WT) DCs, stimulation with LPS (1μg/ml) within 10min led to transient phosphorylation of GSK3. According to Fura2 fluorescence, LPS (1μg/ml) increased cytosolic Ca(2+) concentration, an effect significantly more pronounced in gsk3(KI) DCs than in gsk3(WT) DCs. Conversely, GSK3 inhibitor SB216763 (3-[2,4-Dichlorophenyl]-4-[1-methyl-1H-indol-3-yl]-1H-pyrrole-2,5-dione, 10μM, 30min) significantly blunted the increase of cytosolic Ca(2+) concentration following LPS exposure. In conclusion, PKB/SGK-dependent GSK3α,β activity participates in the regulation of Ca(2+) signaling in dendritic cells. Copyright © 2013 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    1993-02-01

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

  12. Mitochondrial membrane potential in human neutrophils is maintained by complex III activity in the absence of supercomplex organisation

    NARCIS (Netherlands)

    van Raam, Bram J.; Sluiter, Wim; de Wit, Elly; Roos, Dirk; Verhoeven, Arthur J.; Kuijpers, Taco W.

    2008-01-01

    BACKGROUND: Neutrophils depend mainly on glycolysis for their energy provision. Their mitochondria maintain a membrane potential (Deltapsi(m)), which is usually generated by the respiratory chain complexes. We investigated the source of Deltapsi(m) in neutrophils, as compared to peripheral blood

  13. Role of the Mycoplasma pneumoniae/Interleukin-8/Neutrophil Axis in the Pathogenesis of Pneumonia.

    Directory of Open Access Journals (Sweden)

    Zhengrong Chen

    Full Text Available Neutrophil infiltration is the characteristic pathological feature of M. pneumoniae pneumonia (MPP. This study aimed to explore the associations among neutrophil activity, clinical presentation, and role of the M. pneumoniae/interleukin-8 (IL-8/neutrophil axis in the pathogenesis of MPP. A total of 42 patients with MPP were prospectively enrolled in the study. Neutrophil activity, including matrix metalloproteinase-9 (MMP-9, myeloperoxidase (MPO, and neutrophil elastase (NE, were measured. Clinical information was collected for all patients and control group. In vitro, IL-8 production was measured at different time points after M. pneumoniae infection of bronchial epithelial cells, and neutrophil activity was analyzed after IL-8 stimulation. The percentage of neutrophil in the bronchoalveolar lavage fluid was higher in the group of patients with high levels of M. pneumoniae DNA than in those with low levels of M. pneumoniae DNA (P < 0.05. IL-8, MMP-9, and NE in patients with MPP significantly increased compared with controls and decreased after treatment (P < 0.05. MPO and MMP-9 were associated with duration of fever (r = 0.332, P < 0.05 and length of stay (r = 0.342, P < 0.05, respectively. In vitro, M. pneumoniae induced IL-8 production by bronchial epithelial cells in a time dependent manner. MPO, MMP-9 and NE production by neutrophils significantly increased compared with medium controls after IL-8 stimulation. In summary, the M. pneumoniae/IL-8/neutrophil axis likely plays a vital role in the pathogenesis of MPP.

  14. Cisplatinum and Taxol Induce Different Patterns of p53 Phosphorylation

    Directory of Open Access Journals (Sweden)

    Giovanna Damia

    2001-01-01

    Full Text Available Posttranslational modifications of p53 induced by two widely used anticancer agents, cisplatinum (DDP and taxol were investigated in two human cancer cell lines. Although both drugs were able to induce phosphorylation at serine 20 (Ser20, only DDP treatment induced p53 phosphorylation at serine 15 (Ser15. Moreover, both drug treatments were able to increase p53 levels and consequently the transcription of waf1 and mdm-2 genes, although DDP treatment resulted in a stronger inducer of both genes. Using two ataxia telangiectasia mutated (ATM cell lines, the role of ATM in druginduced p53 phosphorylations was investigated. No differences in drug-induced p53 phosphorylation could be observed, indicating that ATM is not the kinase involved in these phosphorylation events. In addition, inhibition of DNA-dependent protein kinase activity by wortmannin did not abolish p53 phosphorylation at Ser15 and Ser20, again indicating that DNA-PK is unlikely to be the kinase involved. After both taxol and DDP treatments, an activation of hCHK2 was found and this is likely to be responsible for phosphorylation at Ser20. In contrast, only DDP was able to activate ATR, which is the candidate kinase for phosphorylation of Ser15 by this drug. This data clearly suggests that differential mechanisms are involved in phosphorylation and activation of p53 depending on the drug type.

  15. An oral Hemokine™, α-methylhydrocinnamate, enhances myeloid and neutrophil recovery following irradiation in vivo

    Science.gov (United States)

    Faller, Douglas V.; Castaneda, Serguei A.; Zhou, Daohong; Vedamony, Merriline; Newburger, Peter E.; White, Gary L.; Kosanke, Stanley; Plett, P. Artur; Orschell, Christie M.; Boosalis, Michael S.; Perrine, Susan P.

    2017-01-01

    An oral therapeutic which reduces duration of cytopenias and is active following accidental radiation exposures is an unmet need in radiation countermeasures. Alpha methylhydrocinnamate (ST7) prolongs STAT-5 phosphorylation, reduces growth-factor dependency of multi-lineage cell lines, and stimulates erythropoiesis. Here, ST7 and its isomers were studied for their effects on myeloid progenitors and hematopoietic stem cells (HSCs) following radiation, in nonhuman primates, and murine irradiation models. Addition of ST7 or ST7-S increased CFU-GM production by 1.7-fold (p200/mm3 and neutropenia recovered in 6 days with ST7 treatment and 18 days in controls (p<0.05). In lethally-irradiated mice, marrow pathology at 15 days was hypocellular (10% cellularity) in controls, but normal (55–75% cellularity) with complete neutrophil maturation with ST7-S treatment. Following lethal irradiation, ST7, given orally for 4 days, reduced mortality, with 30% survival in ST7-animals vs 8% in controls, (p<0.05). Collectively, the studies indicate that ST7 and ST7-S enhance myeloid recovery post-radiation and merit further evaluation to accelerate hematologic recovery in conditions of radiation-related and other marrow hypoplasias. PMID:27888688

  16. Frontline Science: HMGB1 induces neutrophil dysfunction in experimental sepsis and in patients who survive septic shock.

    Science.gov (United States)

    Grégoire, Murielle; Tadié, Jean-Marc; Uhel, Fabrice; Gacouin, Arnaud; Piau, Caroline; Bone, Nathaniel; Le Tulzo, Yves; Abraham, Edward; Tarte, Karin; Zmijewski, Jaroslaw W

    2017-06-01

    Sepsis is accompanied by the initial activation of proinflammatory pathways and long-lasting immunosuppression that appears to contribute to late-occurring mortality. Although high-mobility group box 1 (HMGB1) is involved in many aspects of inflammation, its role in sepsis-induced immune suppression remains unclear. In this study, we examined HMGB1's contribution to neutrophil NADPH oxidase activity dysfunction and associated neutrophil-dependent bacterial clearance in mice subjected to sepsis and in patients who survive septic shock. Using a murine model of polymicrobial septic peritonitis, we demonstrated that treatment with anti-HMGB1 Ab significantly diminished sepsis-induced dysfunction of neutrophil NADPH oxidase activity. In a subsequent set of experiments, we found that blocking HMGB1 preserved the ability of neutrophils from patients recovering from septic shock to activate NADPH oxidase. Taken together, our data suggest that HMGB1 accumulation in the late phase of sepsis plays a specific role in the development of postsepsis immunosuppression and specifically affects neutrophil-dependent antibacterial defense mechanisms. Thus, blocking HMGB1 may be a promising therapeutic intervention to diminish the adverse effects of sepsis-induced immunosuppression. © Society for Leukocyte Biology.

  17. Possible role of calcium dependent protein phosphorylation in the modulation of wound induced HRGP gene activation in potatoes after gamma irradiation

    International Nuclear Information System (INIS)

    Ussuf, K.K.; Laxmi, N.H.; Nair, P.M.

    1996-01-01

    Hydroxyproline rich glycoprotein (HRGP) gene is induced in both control and gamma irradiated potato tubers after wounding. The enhanced RNA synthesis in response to wounding correlated well with the accumulation of both HRGP gene transcripts and protein. Initially, the level of HRGP gene expression in gamma irradiated potatoes in response to wounding was 30% more than the corresponding controls. After post irradiation storage of 3-5 weeks, HRGP gene expression in response to wounding was significantly lower than the unirradiated samples. This low level of HRGP gene expression in irradiated potatoes was partially retrieved by 5 mM Ca 2+ treatment. Prior treatment with trifluoperazine, a calcium channel blocker resulted in 35% reduction in wound induced HRGP gene expression in control potatoes, further providing evidence for the involvement of Ca 2+ dependency for HRGP gene activation. A comparative study on in vivo protein phosphorylation induced by wounding in control and irradiated potatoes exhibited significant differences. A good correlation was observed in the modulation of phosphorylation and HRGP gene expression by Ca 2+ in irradiated potatoes. Wound induced signal transduction system and subsequent Ca 2+ dependent protein phosphorylation for the activation of HRGP gene is affected in potatoes after gamma irradiation, thus impairing the wound healing process adversely. (author). 25 refs., 5 figs

  18. Neutrophil Reverse Migration Becomes Transparent with Zebrafish

    Directory of Open Access Journals (Sweden)

    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.

  19. Ascorbic acid transport and accumulation in human neutrophils

    International Nuclear Information System (INIS)

    Washko, P.; Rotrosen, D.; Levine, M.

    1989-01-01

    The transport, accumulation, and distribution of ascorbic acid were investigated in isolated human neutrophils utilizing a new ascorbic acid assay, which combined the techniques of high performance liquid chromatography and coulometric electrochemical detection. Freshly isolated human neutrophils contained 1.0-1.4 mM ascorbic acid, which was localized greater than or equal to 94% to the cytosol, was not protein bound, and was present only as ascorbic acid and not as dehydroascorbic acid. Upon addition of ascorbic acid to the extracellular medium in physiologic amounts, ascorbic acid was accumulated in neutrophils in millimolar concentrations. Accumulation was mediated by a high affinity and a low affinity transporter; both transporters were responsible for maintenance of concentration gradients as large as 50-fold. The high affinity transporter had an apparent Km of 2-5 microns by Lineweaver-Burk and Eadie-Hofstee analyses, and the low affinity transporter had an apparent Km of 6-7 mM by similar analyses. Each transporter was saturable and temperature dependent. In normal human blood the high affinity transporter should be saturated, whereas the low affinity transporter should be in its linear phase of uptake

  20. Flavonoids Inhibit the Respiratory Burst of Neutrophils in Mammals

    Directory of Open Access Journals (Sweden)

    Milan Ciz

    2012-01-01

    Full Text Available Neutrophils represent the front-line defence cells in protecting organisms against infection and play an irreplaceable role in the proper performance of the immune system. As early as within the first minutes of stimulation, neutrophilic NADPH oxidase is activated, and cells release large quantities of highly toxic reactive oxygen species (ROS. These oxidants can be highly toxic not only for infectious agents but also for neighboring host tissues. Since flavonoids exhibit antioxidant and anti-inflammatory effects, they are subjects of interest for pharmacological modulation of ROS production. The present paper summarizes contemporary knowledge on the effects of various flavonoids on the respiratory burst of mammalian neutrophils. It can be summarized that the inhibitory effects of flavonoids on the respiratory burst of phagocytes are mediated via inhibition of enzymes involved in cell signaling as well as via modulation of redox status. However, the effects of flavonoids are even more complex, and several sites of action, depending upon the flavonoid structure and way of application, are included.

  1. Cxcl8b and Cxcr2 Regulate Neutrophil Migration through Bloodstream in Zebrafish

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    Constanza Zuñiga-Traslaviña

    2017-01-01

    Full Text Available Neutrophils play an essential role during an inflammatory response, which is dependent on their rapid recruitment from the bone marrow to the vasculature. However, there is no information about the molecular signals that regulate neutrophil entry to circulation during an inflammatory process in humans. This is mainly due to the lack of a suitable model of study that contains similar set of molecules and that allows in vivo analyses. In this study, we used the zebrafish to assess the role of Cxcl8a, Cxcl8b, and Cxcr2 in neutrophil migration to blood circulation after injury. Using Tg(BACmpx:GFPi114 transgenic embryos and two damage models (severe and mild, we developed in vivo lack of function assays. We found that the transcription levels of cxcl8a, cxcl8b, and cxcr2 were upregulated in the severe damage model. In contrast, only cxcr2 and cxcl8a mRNA levels were increased during mild damage. After knocking down Cxcl8a, neutrophil quantity decreased at the injury site, while Cxcl8b decreased neutrophils in circulation. When inhibiting Cxcr2, we observed a decrease in neutrophil entry to the bloodstream. In conclusion, we identified different functions for both Cxcl8 paralogues, being the Cxcl8b/Cxcr2 axis that regulates neutrophil entry to the bloodstream, while Cxcl8a/Cxcr2 regulates the migration to the affected area.

  2. Employment-based reinforcement of adherence to an FDA approved extended release formulation of naltrexone in opioid-dependent adults: a randomized controlled trial.

    Science.gov (United States)

    DeFulio, Anthony; Everly, Jeffrey J; Leoutsakos, Jeannie-Marie S; Umbricht, Annie; Fingerhood, Michael; Bigelow, George E; Silverman, Kenneth

    2012-01-01

    Naltrexone provides excellent opioid blockade, but its clinical utility is limited because opioid-dependent patients typically refuse it. An injectable suspension of naltrexone for extended release (XR-NTX) was recently approved by the FDA for treatment of opioid dependence. XR-NTX treatment may require concurrent behavioral intervention to maximize adherence and effectiveness, thus we sought to evaluate employment-based reinforcement as a method of improving adherence to XR-NTX in opiate dependent adults. Opioid-dependent adults (n=38) were detoxified and inducted onto oral naltrexone, then randomly assigned to contingency or prescription conditions. Participants received up to six doses of XR-NTX at four-week intervals. All participants could earn vouchers for attendance and performance at a therapeutic workplace. Contingency participants were required to accept XR-NTX injections to access the workplace and earn vouchers. Prescription participants could earn vouchers independent of their acceptance of XR-NTX injections. Contingency participants accepted significantly more naltrexone injections than prescription participants (87% versus 52%, p=.002), and were more likely to accept all injections (74% versus 26%, p=.004). Participants in the two conditions provided similar percentages of samples negative for opiates (72% versus 65%) and for cocaine (58% versus 54%). Opiate positivity was significantly more likely when samples were also cocaine positive, independent of naltrexone blockade (p=.002). Long-term adherence to XR-NTX in unemployed opiate dependent adults is low under usual care conditions. Employment-based reinforcement can maintain adherence to XR-NTX. Ongoing cocaine use appears to interfere with the clinical effectiveness of XR-NTX on opiate use. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  3. Just showing up is not enough: Homework adherence and outcome in cognitive-behavioral therapy for cocaine dependence.

    Science.gov (United States)

    Decker, Suzanne E; Kiluk, Brian D; Frankforter, Tami; Babuscio, Theresa; Nich, Charla; Carroll, Kathleen M

    2016-10-01

    Homework in cognitive-behavioral therapy (CBT) provides opportunities to practice skills. In prior studies, homework adherence was associated with improved outcome across a variety of disorders. Few studies have examined whether the relationship between homework adherence and outcome is maintained after treatment end or is independent of treatment attendance. This study combined data from 4 randomized clinical trials of CBT for cocaine dependence to examine relationships among homework adherence, participant variables, and cocaine use outcomes during treatment and at follow-up. The data set included only participants who attended at least 2 CBT sessions to allow for assignment and return of homework (N = 158). Participants returned slightly less than half (41.1%) of assigned homework. Longitudinal random effects regression suggested a greater reduction in cocaine use during treatment and through 12-month follow-up for participants who completed half or more of assigned homework (3-way interaction), F(2, 910.69) = 4.28, p = .01. In multiple linear regression, the percentage of homework adherence was associated with greater number of cocaine-negative urine toxicology screens during treatment, even when accounting for baseline cocaine use frequency and treatment attendance; at 3 months follow-up, multiple logistic regression indicated homework adherence was associated with cocaine-negative urine toxicology screen, controlling for baseline cocaine use and treatment attendance. These results extend findings from prior studies regarding the importance of homework adherence by demonstrating associations among homework and cocaine use outcomes during treatment and up to 12 months after, independent of treatment attendance and baseline cocaine use severity. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

  4. 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 ...

  5. 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

  6. Activation of macrophages by a laccase-polymerized polyphenol is dependent on phosphorylation of Rac1.

    Science.gov (United States)

    Tajima, Katsuya; Akanuma, Satoshi; Matsumoto-Akanuma, Akiko; Yamanaka, Daisuke; Ishibashi, Ken-Ichi; Adachi, Yoshiyuki; Ohno, Naohito

    2018-01-15

    Various physiologically active effects of polymerized polyphenols have been reported. In this study, we synthesized a polymerized polyphenol (mL2a-pCA) by polymerizing caffeic acid using mutant Agaricus brasiliensis laccase and analyzed its physiological activity and mechanism of action. We found that mL2a-pCA induced morphological changes and the production of cytokines and chemokines in C3H/HeN mouse-derived resident peritoneal macrophages in vitro. The mechanisms of action of polymerized polyphenols on in vitro mouse resident peritoneal cells have not been characterized in detail previously. Herein, we report that the mL2a-pCA-induced production of interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) in C3H/HeN mouse-derived resident peritoneal cells was inhibited by treatment with the Rac1 inhibitor NSC23766 trihydrochloride. In addition, we found that mL2a-pCA activated the phosphorylation Rac1. Taken together, the results show that mL2a-pCA induced macrophage activation via Rac1 phosphorylation-dependent pathways. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. 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

  8. Mitochondrial membrane potential in human neutrophils is maintained by complex III activity in the absence of supercomplex organisation

    NARCIS (Netherlands)

    B.J. van Raam (Bram); W.J. Sluiter (Wim); F.R.C. de Wit (Frank); D. Roos (Dirk); A.J. Verhoeven (Arthur); T.W. Kuijpers (Taco W.)

    2008-01-01

    textabstractBackground: Neutrophils depend mainly on glycolysis for their enegry provision. Their mitochondria maintain a membrace potential (ΔΨm), which is usually generated by the repiratory chain complexes. We investigated the source of ΔΨm in neutrophils, as compared to peripheral blood

  9. The effect of the anaesthetic agent isoflurane on the rate of neutrophil apoptosis in vitro.

    LENUS (Irish Health Repository)

    Tyther, R

    2012-02-03

    BACKGROUND: Volatile anaesthetic agents influence neutrophil function, and potentially, the inflammatory response to surgery. AIM: The objective of this study was to determine the effect of isoflurane (1-4%) on human polymorphonuclear neutrophil apoptosis in vitro. METHODS: Venous blood from 12 healthy volunteers was exposed to 0, 1, and 4% isoflurane delivered via a 14G Wallace flexihub internal jugular cannula, at a fresh gas flow of 0.51\\/min for 5 minutes. Isolated neutrophils were assessed for apoptosis at 1, 12, and 24 hours in culture using dual staining with annexin V-FITC and propidium iodide (Annexin-V FITC assay). Data were analysed using paired, one-tailed Student\\'s t-tests. p<0.05 was considered significant. RESULTS: At 1 hour apoptosis was inhibited in the 1% (5.1 [6.8]%; p=0.017) and 4% (4.8 [4.5]%; p=0.008) isoflurane groups compared to control (11.3 [6.9]%). At 12 and 24 hours, a dose-dependent inhibition of apoptosis was demonstrated, i.e. 4% > 1% > 0%. CONCLUSION: Human neutrophil apoptosis is inhibited in a concentration-dependent manner in vitro by isoflurane in clinical concentrations.

  10. 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.

  11. Glycogen phosphorylation and Lafora disease.

    Science.gov (United States)

    Roach, Peter J

    2015-12-01

    Covalent phosphorylation of glycogen, first described 35 years ago, was put on firm ground through the work of the Whelan laboratory in the 1990s. But glycogen phosphorylation lay fallow until interest was rekindled in the mid 2000s by the finding that it could be removed by a glycogen-binding phosphatase, laforin, and that mutations in laforin cause a fatal teenage-onset epilepsy, called Lafora disease. Glycogen phosphorylation is due to phosphomonoesters at C2, C3 and C6 of glucose residues. Phosphate is rare, ranging from 1:500 to 1:5000 phosphates/glucose depending on the glycogen source. The mechanisms of glycogen phosphorylation remain under investigation but one hypothesis to explain C2 and perhaps C3 phosphate is that it results from a rare side reaction of the normal synthetic enzyme glycogen synthase. Lafora disease is likely caused by over-accumulation of abnormal glycogen in insoluble deposits termed Lafora bodies in neurons. The abnormality in the glycogen correlates with elevated phosphorylation (at C2, C3 and C6), reduced branching, insolubility and an enhanced tendency to aggregate and become insoluble. Hyperphosphorylation of glycogen is emerging as an important feature of this deadly childhood disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation*

    OpenAIRE

    Nojiri, Mari; Loyet, Kelly M.; Klenchin, Vadim A.; Kabachinski, Gregory; Martin, Thomas F. J.

    2009-01-01

    CAPS (Ca2+-dependent activator protein for secretion) functions in priming Ca2+-dependent vesicle exocytosis, but the regulation of CAPS activity has not been characterized. Here we show that phosphorylation by protein kinase CK2 is required for CAPS activity. Dephosphorylation eliminated CAPS activity in reconstituting Ca2+-dependent vesicle exocytosis in permeable and intact PC12 cells. Ser-5, -6, and -7 and Ser-1281 were identified by mass spectrometry as the major phosphorylation sites in...

  13. Vinculin but not alpha-actinin is a target of PKC phosphorylation during junctional assembly induced by calcium

    DEFF Research Database (Denmark)

    Perez-Moreno, M; Avila, A; Islas, S

    1998-01-01

    and alpha-actinin, two actin binding proteins of the adherent junctions. We found that during the junctional sealing induced by Ca2+, both proteins move towards the cell periphery and, while there is a significant increase in the phosphorylation of vinculin, alpha-actinin remains unchanged. The increased...

  14. Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds.

    Science.gov (United States)

    Ying, Sheng; Hill, Allyson T; Pyc, Michal; Anderson, Erin M; Snedden, Wayne A; Mullen, Robert T; She, Yi-Min; Plaxton, William C

    2017-06-01

    Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a crucial branch point of central plant metabolism. In developing castor oil seeds ( Ricinus communis ) a novel, allosterically desensitized 910-kD Class-2 PEPC hetero-octameric complex, arises from a tight interaction between 107-kD plant-type PEPC and 118-kD bacterial-type (BTPC) subunits. The native Ca 2+ -dependent protein kinase (CDPK) responsible for in vivo inhibitory phosphorylation of Class-2 PEPC's BTPC subunit's at Ser-451 was highly purified from COS and identified as RcCDPK1 (XP_002526815) by mass spectrometry. Heterologously expressed RcCDPK1 catalyzed Ca 2+ -dependent, inhibitory phosphorylation of BTPC at Ser-451 while exhibiting: ( i ) a pair of Ca 2+ binding sites with identical dissociation constants of 5.03 μM, ( ii ) a Ca 2+ -dependent electrophoretic mobility shift, and ( iii ) a marked Ca 2+ -independent hydrophobicity. Pull-down experiments established the Ca 2+ -dependent interaction of N-terminal GST-tagged RcCDPK1 with BTPC. RcCDPK1-Cherry localized to the cytosol and nucleus of tobacco bright yellow-2 cells, but colocalized with mitochondrial-surface associated BTPC-enhanced yellow fluorescent protein when both fusion proteins were coexpressed. Deletion analyses demonstrated that although its N-terminal variable domain plays an essential role in optimizing Ca 2+ -dependent RcCDPK1 autophosphorylation and BTPC transphosphorylation activity, it is not critical for in vitro or in vivo target recognition. Arabidopsis ( Arabidopsis thaliana ) CPK4 and soybean ( Glycine max ) CDPKβ are RcCDPK1 orthologs that effectively phosphorylated castor BTPC at Ser-451. Overall, the results highlight a potential link between cytosolic Ca 2+ signaling and the posttranslational control of respiratory CO 2 refixation and anaplerotic photosynthate partitioning in support of storage oil and protein biosynthesis in developing COS. © 2017 American Society of Plant

  15. Protein phosphatases active on acetyl-CoA carboxylase phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase

    International Nuclear Information System (INIS)

    Witters, L.A.; Bacon, G.W.

    1985-01-01

    The protein phosphatases in rat liver cytosol, active on rat liver acetyl-CoA carboxylase (ACC) phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase, have been partially purified by anion-exchange and gel filtration chromatography. The major phosphatase activities against all three substrates copurify through fractionation and appear to be identical to protein phosphatases 2A1 and 2A2. No unique protein phosphatase active on 32 P-ACC phosphorylated by the casein kinases was identified

  16. 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.

  17. Inhibition by sodium nitroprusside of the expression of inducible nitric oxide synthase in rat neutrophils.

    OpenAIRE

    Mariotto, S; Cuzzolin, L; Adami, A; Del Soldato, P; Suzuki, H; Benoni, G

    1995-01-01

    A well-known nitric oxide (NO)-releasing compound, sodium nitroprusside (SNP), decreases in a dose-dependent manner NO synthase (NOS) activity induced in rat neutrophils by treatment with lipopolysaccharide (LPS). This inhibitory action of SNP seems not to be due to its direct effect on the enzyme activity. The strong nitrosonium ion (NO+) character of SNP could be responsible for its inhibition of NOS induction in neutrophils.

  18. 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.

  19. 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.

  20. Protein kinase Cα phosphorylates a novel argininosuccinate synthase site at serine 328 during calcium-dependent stimulation of endothelial nitric-oxide synthase in vascular endothelial cells.

    Science.gov (United States)

    Haines, Ricci J; Corbin, Karen D; Pendleton, Laura C; Eichler, Duane C

    2012-07-27

    Endothelial nitric-oxide synthase (eNOS) utilizes l-arginine as its principal substrate, converting it to l-citrulline and nitric oxide (NO). l-Citrulline is recycled to l-arginine by two enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase, providing the substrate arginine for eNOS and NO production in endothelial cells. Together, these three enzymes, eNOS, AS, and argininosuccinate lyase, make up the citrulline-NO cycle. Although AS catalyzes the rate-limiting step in NO production, little is known about the regulation of AS in endothelial cells beyond the level of transcription. In this study, we showed that AS Ser-328 phosphorylation was coordinately regulated with eNOS Ser-1179 phosphorylation when bovine aortic endothelial cells were stimulated by either a calcium ionophore or thapsigargin to produce NO. Furthermore, using in vitro kinase assay, kinase inhibition studies, as well as protein kinase Cα (PKCα) knockdown experiments, we demonstrate that the calcium-dependent phosphorylation of AS Ser-328 is mediated by PKCα. Collectively, these findings suggest that phosphorylation of AS at Ser-328 is regulated in accordance with the calcium-dependent regulation of eNOS under conditions that promote NO production and are in keeping with the rate-limiting role of AS in the citrulline-NO cycle of vascular endothelial cells.

  1. Modulation of IgE-dependent COX-2 gene expression by reactive oxygen species in human neutrophils.

    Science.gov (United States)

    Vega, Antonio; Chacón, Pedro; Alba, Gonzalo; El Bekay, Rajaa; Martín-Nieto, José; Sobrino, Francisco

    2006-07-01

    Cyclooxygenase (COX) is a key enzyme in prostaglandin (PG) synthesis. Up-regulation of its COX-2 isoform is responsible for the increased PG release, taking place under inflammatory conditions, and also, is thought to be involved in allergic and inflammatory diseases. In the present work, we demonstrate that COX-2 expression becomes highly induced by anti-immunoglobulin E (IgE) antibodies and by antigens in human neutrophils from allergic patients. This induction was detected at mRNA and protein levels and was accompanied by a concomitant PGE(2) and thromboxane A(2) release. We also show evidence that inhibitors of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, such as 4-(2-aminoethyl)benzenesulphonyl fluoride and 4-hydroxy-3-methoxyaceto-phenone, completely cancelled anti-IgE-induced COX-2 protein up-regulation, suggesting that this process is mediated by reactive oxygen species (ROS) derived from NADPH oxidase activity. Moreover, the mitogen-activated protein kinases (MAPKs), p38 and extracellular signal-regulated kinase, and also, the transcription factor, nuclear factor (NF)-kappaB, are involved in the up-regulation of COX-2 expression, as specific chemical inhibitors of these two kinases, such as SB203580 and PD098059, and of the NF-kappaB pathway, such as N(alpha)-benzyloxycarbonyl-l-leucyl-l-leucyl-l-leucinal, abolished IgE-dependent COX-2 induction. Evidence is also presented, using Fe(2)(+)/Cu(2)(+) ions, that hydroxyl radicals generated from hydrogen peroxide through Fenton reactions could constitute candidate modulators able to directly trigger anti-IgE-elicited COX-2 expression through MAPK and NF-kappaB pathways. Present results underscore a new role for ROS as second messengers in the modulation of COX-2 expression by human neutrophils in allergic conditions.

  2. Phosphorylation of rat brain purified mitochondrial Voltage-Dependent Anion Channel by c-Jun N-terminal kinase-3 modifies open-channel noise.

    Science.gov (United States)

    Gupta, Rajeev

    2017-09-02

    The drift kinetic energy of ionic flow through single ion channels cause vibrations of the pore walls which are observed as open-state current fluctuations (open-channel noise) during single-channel recordings. Vibration of the pore wall leads to transitions among different conformational sub-states of the channel protein in the open-state. Open-channel noise analysis can provide important information about the different conformational sub-state transitions and how biochemical modifications of ion channels would affect their transport properties. It has been shown that c-Jun N-terminal kinase-3 (JNK3) becomes activated by phosphorylation in various neurodegenerative diseases and phosphorylates outer mitochondrion associated proteins leading to neuronal apoptosis. In our earlier work, JNK3 has been reported to phosphorylate purified rat brain mitochondrial voltage-dependent anion channel (VDAC) in vitro and modify its conductance and opening probability. In this article we have compared the open-state noise profile of the native and the JNK3 phosphorylated VDAC using Power Spectral Density vs frequency plots. Power spectral density analysis of open-state noise indicated power law with average slope value α ≈1 for native VDAC at both positive and negative voltage whereas average α value open-state noise arises due to coupling of ionic transport and conformational sub-states transitions in open-state and this coupling is perturbed as a result of channel phosphorylation. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Rapid changes in plasma membrane protein phosphorylation during initiation of cell wall digestion

    International Nuclear Information System (INIS)

    Blowers, D.P.; Boss, W.F.; Trewavas, A.J.

    1988-01-01

    Plasma membrane vesicles from wild carrot cells grown in suspension culture were isolated by aqueous two-phase partitioning, and ATP-dependent phosphorylation was measured with [γ- 32 P]ATP in the presence and absence of calcium. Treatment of the carrot cells with the cell wall digestion enzymes, driselase, in a sorbitol osmoticum for 1.5 min altered the protein phosphorylation pattern compared to that of cells treated with sorbitol alone. Driselase treatment resulted in decreased phosphorylation of a band of M r 80,000 which showed almost complete calcium dependence in the osmoticum treated cells; decreased phosphorylation of a band of M r 15,000 which showed little calcium activation, and appearance of a new band of calcium-dependent phosphorylation at M r 22,000. However, protein phosphorylation was decreased. Adding driselase to the in vitro reaction mixture caused a general decrease in the membrane protein phosphorylation either in the presence or absence of calcium which did not mimic the in vivo response. Cells labeled in vivo with inorganic 32 P also showed a response to the Driselase treatment. An enzymically active driselas preparation was required for the observed responses

  4. 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

  5. Damage to Candida albicans Hyphae and Pseudohyphae by the Myeloperoxidase System and Oxidative Products of Neutrophil Metabolism In Vitro

    OpenAIRE

    Diamond, Richard D.; Clark, Robert A.; Haudenschild, Christian C.

    1980-01-01

    In previous studies, we noted that Candida hyphae and pseudohyphae could be damaged and probably killed by neutrophils, primarily by oxygen-dependent nonphagocytic mechanisms. In extending these studies, amount of damage to hyphae again was measured by inhibition of [14C]cytosine uptake. Neutrophils from only one of four patients with chronic granulomatous disease damaged hyphae at all, and neutrophils from this single patient damaged hyphae far less efficiently than simultaneously tested neu...

  6. Porphyromonas gingivalis-induced production of reactive oxygen species, tumor necrosis factor-α, interleukin-6, CXCL8 and CCL2 by neutrophils from localized aggressive periodontitis and healthy donors

    DEFF Research Database (Denmark)

    Damgaard, C; Kantarci, A; Holmstrup, P

    2017-01-01

    BACKGROUND AND OBJECTIVES: Porphyromonas gingivalis is regarded as a significant contributor in the pathogenesis of periodontitis and certain systemic diseases, including atherosclerosis. P. gingivalis occasionally translocates from periodontal pockets into the circulation, where it adheres to red...... (ROS) in response to challenge with P. gingivalis. In addition, the impact of RBC interaction with P. gingivalis was investigated. The actions of resolvin E1 (RvE1), a known regulator of P. gingivalis induced neutrophil responses, on the cytokine and ROS responses elicited by P. gingivalis in cultures...... of neutrophils were investigated. RESULTS: Upon stimulation with P. gingivalis, neutrophils from subjects with LAgP and healthy controls released similar quantities of IL-6, TNF-α, CXCL8, CCL2 and intracellular ROS. The presence of RBCs amplified the release of IL-6, TNF-α and CCL2 statistically significant...

  7. Identification of a phosphorylation-dependent nuclear localization motif in interferon regulatory factor 2 binding protein 2.

    Directory of Open Access Journals (Sweden)

    Allen C T Teng

    Full Text Available Interferon regulatory factor 2 binding protein 2 (IRF2BP2 is a muscle-enriched transcription factor required to activate vascular endothelial growth factor-A (VEGFA expression in muscle. IRF2BP2 is found in the nucleus of cardiac and skeletal muscle cells. During the process of skeletal muscle differentiation, some IRF2BP2 becomes relocated to the cytoplasm, although the functional significance of this relocation and the mechanisms that control nucleocytoplasmic localization of IRF2BP2 are not yet known.Here, by fusing IRF2BP2 to green fluorescent protein and testing a series of deletion and site-directed mutagenesis constructs, we mapped the nuclear localization signal (NLS to an evolutionarily conserved sequence (354ARKRKPSP(361 in IRF2BP2. This sequence corresponds to a classical nuclear localization motif bearing positively charged arginine and lysine residues. Substitution of arginine and lysine with negatively charged aspartic acid residues blocked nuclear localization. However, these residues were not sufficient because nuclear targeting of IRF2BP2 also required phosphorylation of serine 360 (S360. Many large-scale phosphopeptide proteomic studies had reported previously that serine 360 of IRF2BP2 is phosphorylated in numerous human cell types. Alanine substitution at this site abolished IRF2BP2 nuclear localization in C(2C(12 myoblasts and CV1 cells. In contrast, substituting serine 360 with aspartic acid forced nuclear retention and prevented cytoplasmic redistribution in differentiated C(2C(12 muscle cells. As for the effects of these mutations on VEGFA promoter activity, the S360A mutation interfered with VEGFA activation, as expected. Surprisingly, the S360D mutation also interfered with VEGFA activation, suggesting that this mutation, while enforcing nuclear entry, may disrupt an essential activation function of IRF2BP2.Nuclear localization of IRF2BP2 depends on phosphorylation near a conserved NLS. Changes in phosphorylation status

  8. Modulation of opiate-related signaling molecules in morphine-dependent conditioned behavior: conditioned place preference to morphine induces CREB phosphorylation.

    Science.gov (United States)

    Morón, José A; Gullapalli, Srinivas; Taylor, Chirisse; Gupta, Achla; Gomes, Ivone; Devi, Lakshmi A

    2010-03-01

    Opiate addiction is a chronic, relapsing behavioral disorder where learned associations that develop between the abused opiate and the environment in which it is consumed are brought about through Pavlovian (classical) conditioning processes. However, the signaling mechanisms/pathways regulating the mechanisms that underlie the responses to opiate-associated cues or the development of sensitization as a consequence of repeated context-independent administration of opiates are unknown. In this study we examined the phosphorylation levels of various classic signaling molecules in brain regions implicated in addictive behaviors after acute and repeated morphine administration. An unbiased place conditioning protocol was used to examine changes in phosphorylation that are associated with (1) the expression of the rewarding effects of morphine and (2) the sensitization that develops to this effect. We also examined the effects of a delta-receptor antagonist on morphine-induced conditioned behavior and on the phosphorylation of classic signaling molecules in view of data showing that blockade of delta-opioid receptor (deltaOR) prevents the development of sensitization to the rewarding effects of morphine. We find that CREB phosphorylation is specifically induced upon the expression of a sensitized response to morphine-induced conditioned behavior in brain areas related to memory consolidation, such as the hippocampus and cortex. A similar effect is also observed, albeit to a lesser extent, in the case of the GluR1 subunit of AMPA glutamate receptor. These increases in the phosphorylation levels of CREB and pGluR1 are significantly blocked by pretreatment with a deltaOR antagonist. These results indicate a critical role for phospho-CREB, AMPA, and deltaOR activities in mediating the expression of a sensitized response to morphine-dependent conditioned behavior.

  9. Modulation of P1798 lymphosarcoma proliferation by protein phosphorylation

    International Nuclear Information System (INIS)

    Michnoff, C.A.H.

    1983-01-01

    The role of protein kinases in modulating cell proliferation was examined. Studies characterized the regulation of cell proliferation by adenosine 3':5'-monophosphate-dependent protein kinase (cA-Pk). Calcium/calmodulin-dependent myosin light chain kinase (MLCK) was isolated and examined as a potential substrate regulated by cA-PK in the rapidly proliferating P1798 lymphosarcoma. Modulation of cell proliferation by cA-PK was characterized by quantitating cell division by [methyl- 3 H] thymidine ([ 3 H]-dT) incorporation into DNA, cAMP accumulations, and activation of cA-PK using P1798 lymphosarcoma cells. Epinephrine and prostaglandin E 1 (PGE 1 ) were demonstrated to suppress [ 3 H]-dT incorporation into DNA, to stimulate cAMP accumulation, and to activate cA-PK with dose-dependency. Calcium/calmodulin-dependent MLCK was partially purified from P1798 lymphosarcoma. P1798 MLCK phosphorylated myosin regulatory light chains (P-LC) from thymus, cardiac and skeletal muscles. One mol [ 32 Pi] was transferred into one mol cardiac or skeletal P-LC by P1798 MLCK. Apparent Km values of 65 μM and 51 μM were determined for ATP and cardiac P-LC, respectively. The apparent molecular weight of P1798 MLCK was 135,000. P1798 MLCK was phosphorylated by cA-PK. Phosphorylated MLCK showed a 41% decrease in calcium-dependent activity. Two additional protein kinases from P1798 lymphosarcoma phosphorylated cardiac and skeletal light chains

  10. Activation and regulation of arachidonic acid release in rabbit peritoneal neutrophils

    International Nuclear Information System (INIS)

    Tao, W.

    1988-01-01

    Arachidonic acid release in rabbit neutrophils can be enhanced by the addition of chemotactic fMet-Leu-Phe, platelet-activating factor, PAF, or the calcium ionophore A23187. Over 80% of the release [ 3 H]arachidonic acid comes from phosphatidylcholine and phosphatidylinositol. The release is dose-dependent and increases with increasing concentration of the stimulus. The A23187-induced release increases with increasing time of the stimulation. [ 3 H]arachidonic acid release, but not the rise in the concentration of intracellular calcium, is inhibited in pertussis toxin-treated neutrophils stimulated with PAF. The [ 3 H]arachidonic acid released by A23187 is potentiated while that release by fMET-Leu-Phe or PAF is inhibited in phorbol 12-myristate 13-acetate, PMA, treated rabbit neutrophils. The protein kinase C inhibitor 1-(5-isoquinoline sulfonyl)-2-methylpiperazine, H-7, has no effect on the potentiation by PMA of the A23187-induced release, it prevents the inhibition by PMA of the release produced by PAF or fMet-Leu-Phe. In addition, PMA increases arachidonic acid release in H-7-treated cells stimulated with fMet-Leu-Phe. The diacylglycerol kinase inhibitor R59022 increases the level of diacylglycerol in neutrophils stimulated with fMet-Leu-Phe. Furthermore, R59022 potentiates [ 3 H] arachidonic acid release produced by fMet-Leu-Phe. This potentiation is not inhibited by H-7, in fact, it is increased in H-7-treated neutrophils

  11. Beliefs about medications predict adherence to antidepressants in older adults.

    Science.gov (United States)

    Fawzi, Waleed; Abdel Mohsen, Mohamed Yousry; Hashem, Abdel Hamid; Moussa, Suaad; Coker, Elizabeth; Wilson, Kenneth C M

    2012-01-01

    Adherence to treatment is a complex and poorly understood phenomenon. This study investigates the relationship between older depressed patients' adherence to antidepressants and their beliefs about and knowledge of the medication. Assessment was undertaken of 108 outpatients over the age of 55 years diagnosed with depressive disorder and treated for at least four weeks with antidepressants. Adherence was assessed using two self-report measures: the Medication Adherence Rating Scale (MARS) and a Global Adherence Measure (GAM). Potential predictors of adherence investigated included sociodemographic, medication and illness variables. In addition, 33 carers were interviewed regarding general medication beliefs. 56% of patients reported 80% or higher adherence on the GAM. Sociodemographic variables were not associated with adherence on the MARS. Specific beliefs about medicines, such as "my health depends on antidepressants" (necessity) and being less worried about becoming dependant on antidepressants (concern) were highly correlated with adherence. General beliefs about medicines causing harm or being overprescribed, experiencing medication side-effects and severity of depression also correlated with poor adherence. Linear regression with the MARS as the dependent variable explained 44.3% of the variance and showed adherence to be higher in subjects with healthy specific beliefs who received more information about antidepressants and worse with depression severity and autonomic side-effects. Our findings strongly support a role for specific beliefs about medicines in adherence. Challenging patients' beliefs, providing information about treatment and discussing side-effects could improve adherence. Poor response to treatment and medication side-effects can indicate poor adherence and should be considered before switching medications.

  12. Inhibition by sodium nitroprusside of the expression of inducible nitric oxide synthase in rat neutrophils.

    Science.gov (United States)

    Mariotto, S; Cuzzolin, L; Adami, A; Del Soldato, P; Suzuki, H; Benoni, G

    1995-01-01

    A well-known nitric oxide (NO)-releasing compound, sodium nitroprusside (SNP), decreases in a dose-dependent manner NO synthase (NOS) activity induced in rat neutrophils by treatment with lipopolysaccharide (LPS). This inhibitory action of SNP seems not to be due to its direct effect on the enzyme activity. The strong nitrosonium ion (NO+) character of SNP could be responsible for its inhibition of NOS induction in neutrophils. PMID:7542530

  13. Limb Remote Ischemic Postconditioning Reduces Ischemia-Reperfusion Injury by Inhibiting NADPH Oxidase Activation and MyD88-TRAF6-P38MAP-Kinase Pathway of Neutrophils

    Directory of Open Access Journals (Sweden)

    Gangling Chen

    2016-11-01

    Full Text Available Limb remote ischemic postconditioning (LRIP has been confirmed to reduce the ischemia-reperfusion injury but its mechanisms are still not clear. This study clarified the mechanism of LRIP based on the nicotinamide-adenine dinucleotide phosphate (NADPH oxidase and Myeloid differentiation factor 88 (MyD88-Tumor necrosis factor (TNF receptor-associated factor 6 (TRAF6-P38 pathway of neutrophils. Rat middle cerebral artery occlusion (MCAO model was used in this study. Ischemia-reperfusion injury was carried out by MCAO 1.5 h followed by 24 h reperfusion. LRIP operation was performed to the left femoral artery at 0, 1 or 3 h after reperfusion. Behavioral testing, including postural reflex test, vibrissae-elicited forelimb placing test and tail hang test, showed that LRIP operated at 0 h of reperfusion could significantly ameliorate these behavioral scores. Pathological examinations, infarct size, Myeloperoxidase (MPO activity showed that LRIP operated at 0 h of reperfusion could significantly ameliorate the pathological scores, reduce the infarct size and MPO activity in the brain and increase the MPO activity in the left leg. By using Neutrophil counting, immunofluorescence and real-time PCR techniques, we found that LRIP operated at 0 h of reperfusion could reduce neutrophil counts in the peripheral blood and downregulate the activation of neutrophil in the peripheral blood and rat brain. Western blots revealed that MyD88, TRAF6, p38 mitogen-activated protein kinase (p38-MAPK in neutrophils and the phosphorylation of p47phox (Ser 304 and Ser 345 in neutrophil could be downregulated by LRIP. Our study suggests that LRIP inhibits the number and activation of neutrophils in the rat brain and peripheral blood linked to down-regulating the activation of NADPH oxidase in neutrophils by MyD88/TRAF6/p38-MAPK pathway.

  14. Sphingosine 1-phosphate induces neutrophil chemoattractant IL-8: repression by steroids.

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    Md Mostafizur Rahman

    Full Text Available The bioactive sphingolipid sphingosine 1-phosphate (S1P is found in increased amounts in the airways of asthmatics. S1P can regulate airway smooth muscle functions associated with asthmatic inflammation and remodeling, including cytokine secretion. To date however, whether S1P induces secretion of an important chemokine responsible for neutrophilia in airway inflammation--IL-8--was unexplored. The aim of this study was to investigate whether S1P induces IL-8 gene expression and secretion to enhance neutrophil chemotaxis in vitro, as well as examine the molecular mechanisms responsible for repression by the corticosteroid dexamethasone. We show that S1P upregulates IL-8 secretion from ASM cells and enhance neutrophil chemotaxis in vitro. The corticosteroid dexamethasone significantly represses IL-8 mRNA expression and protein secretion in a concentration- and time-dependent manner. Additionally, we reveal that S1P-induced IL-8 secretion is p38 MAPK and ERK-dependent and that these key phosphoproteins act on the downstream effector mitogen- and stress-activated kinase 1 (MSK1 to control secretion of the neutrophil chemoattractant cytokine IL-8. The functional relevance of this in vitro data was demonstrated by neutrophil chemotaxis assays where S1P-induced effects can be significantly attenuated by pretreatment with dexamethasone, pharmacological inhibition of p38 MAPK- or ERK-mediated pathways, or by knocking down MSK-1 with siRNA. Taken together, our study reveals the molecular pathways responsible for IL-8 secretion from ASM cells in response to S1P and indicates ways in which the impact on IL-8-driven neutrophilia may be lessened.

  15. Medication adherence in inflammatory bowel disease

    Directory of Open Access Journals (Sweden)

    Webber Chan

    2017-10-01

    Full Text Available Inflammatory bowel disease (IBD is a chronic idiopathic inflammatory condition with intestinal and extraintestinal manifestations. Medications are the cornerstone of treatment of IBD. However, patients often adhere to medication poorly. Adherence to medications is defined as the process by which patients take their medications as prescribed. Treatment non-adherence is a common problem among chronic diseases, averaging 50% in developed countries and is even poorer in developing countries. In this review, we will examine the adherence data in IBD which vary greatly depending on the study population, route of administration, and methods of adherence measurement used. We will also discuss the adverse clinical outcomes related to non-adherence to medical treatment including increased disease activity, flares, loss of response to anti-tumor necrosis factor therapy, and so forth. There are many methods to measure medication adherence namely direct and indirect methods, each with their advantages and drawbacks. Finally, we will explore different intervention strategies to improve adherence to medications.

  16. Live-cell calcium imaging of adherent and non-adherent GL261 cells reveals phenotype-dependent differences in drug responses.

    Science.gov (United States)

    Strong, Averey D; Daniels, Richard L

    2017-08-02

    The tumor-derived GL261 cell line is used as a model for studying glioblastoma and other high-grade gliomas, and can be cultured adherently or as free-floating aggregates known as neurospheres. These different culture conditions give rise to distinct phenotypes, with increased tumorigenicity displayed by neurosphere-cultured cells. An important technique for understanding GL261 pathobiology is live cell fluorescent imaging of intracellular calcium. However, live cell imaging of GL261 neurospheres presents a technical challenge, as experimental manipulations where drugs are added to the extracellular media cause the cells to move during analysis. Here we present a method to immobilize GL261 neurospheres with low melting point agarose for calcium imaging using the fluorescent calcium sensor fura-2. GL261 cells were obtained from the NCI-Frederick Cancer Research Tumor Repository and cultured as adherent cells or induced to form neurospheres by placing freshly trypsinized cells into serum-free media containing fibroblast growth factor 2, epidermal growth factor, and B-27 supplement. Prior to experiments, adherent cells were loaded with fura-2 and cultured on 8-well chamber slides. Non-adherent neurospheres were first loaded with fura-2, placed in droplets onto an 8-well chamber slide, and finally covered with a thin layer of low melting point agarose to immobilize the cells. Ratiometric pseudocolored images were obtained during treatment with ATP, capsaicin, or vehicle control. Cells were marked as responsive if fluorescence levels increased more than 30% above baseline. Differences between treatment groups were tested using Student's t-tests and one-way ANOVA. We found that cellular responses to pharmacological treatments differ based on cellular phenotype. Adherent cells and neurospheres both responded to ATP with a rise in intracellular calcium. Notably, capsaicin treatment led to robust responses in GL261 neurospheres but not adherent cells. We demonstrate the use

  17. Vimentin expression influences flow dependent VASP phosphorylation and regulates cell migration and proliferation

    International Nuclear Information System (INIS)

    Lund, Natalie; Henrion, Daniel; Tiede, Petra; Ziche, Marina; Schunkert, Heribert; Ito, Wulf D.

    2010-01-01

    The cytoskeleton plays a central role for the integration of biochemical and biomechanical signals across the cell required for complex cellular functions. Recent studies indicate that the intermediate filament vimentin is necessary for endothelial cell morphogenesis e.g. in the context of leukocyte transmigration. Here, we present evidence, that the scaffold provided by vimentin is essential for VASP localization and PKG mediated VASP phosphorylation and thus controls endothelial cell migration and proliferation. Vimentin suppression using siRNA technique significantly decreased migration velocity by 50% (videomicroscopy), diminished transmigration activity by 42.5% (Boyden chamber) and reduced proliferation by 43% (BrdU-incorporation). In confocal microscopy Vimentin colocalized with VASP and PKG in endothelial cells. Vimentin suppression was accompanied with a translocation of VASP from focal contacts to the perinuclear region. VASP/Vimentin and PKG/Vimentin colocalization appeared to be essential for proper PKG mediated VASP phosphorylation because we detected a diminished expression of PKG and p Ser239 -VASP in vimentin-suppressed cells, Furthermore, the induction of VASP phosphorylation in perfused arteries was markedly decreased in vimentin knockout mice compared to wildtypes. A link is proposed between vimentin, VASP phosphorylation and actin dynamics that delivers an explanation for the important role of vimentin in controlling endothelial cell morphogenesis.

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

    Directory of Open Access Journals (Sweden)

    Grant S. Nichols

    2015-01-01

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

  19. 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

  20. Inhibitory Effects of Cytosolic Ca2+ Concentration by Ginsenoside Ro Are Dependent on Phosphorylation of IP3RI and Dephosphorylation of ERK in Human Platelets

    Directory of Open Access Journals (Sweden)

    Hyuk-Woo Kwon

    2015-01-01

    Full Text Available Intracellular Ca2+ ([Ca2+]i is platelet aggregation-inducing molecule and is involved in activation of aggregation associated molecules. This study was carried out to understand the Ca2+-antagonistic effect of ginsenoside Ro (G-Ro, an oleanane-type saponin in Panax ginseng. G-Ro, without affecting leakage of lactate dehydrogenase, dose-dependently inhibited thrombin-induced platelet aggregation, and the half maximal inhibitory concentration was approximately 155 μM. G-Ro inhibited strongly thrombin-elevated [Ca2+]i, which was strongly increased by A-kinase inhibitor Rp-8-Br-cAMPS compared to G-kinase inhibitor Rp-8-Br-cGMPS. G-Ro increased the level of cAMP and subsequently elevated the phosphorylation of inositol 1, 4, 5-triphosphate receptor I (IP3RI (Ser1756 to inhibit [Ca2+]i mobilization in thrombin-induced platelet aggregation. Phosphorylation of IP3RI (Ser1756 by G-Ro was decreased by PKA inhibitor Rp-8-Br-cAMPS. In addition, G-Ro inhibited thrombin-induced phosphorylation of ERK 2 (42 kDa, indicating inhibition of Ca2+ influx across plasma membrane. We demonstrate that G-Ro upregulates cAMP-dependent IP3RI (Ser1756 phosphorylation and downregulates phosphorylation of ERK 2 (42 kDa to decrease thrombin-elevated [Ca2+]i, which contributes to inhibition of ATP and serotonin release, and p-selectin expression. These results indicate that G-Ro in Panax ginseng is a beneficial novel Ca2+-antagonistic compound and may prevent platelet aggregation-mediated thrombotic disease.

  1. Leishmania major surface protease Gp63 interferes with the function of human monocytes and neutrophils in vitro

    DEFF Research Database (Denmark)

    Sørensen, A L; Hey, A S; Kharazmi, A

    1994-01-01

    In the present study the effect of Leishmania major surface protease Gp63 on the chemotaxis and oxidative burst response of human peripheral blood monocytes and neutrophils was investigated. It was shown that prior incubation of cells with Gp63 inhibited chemotaxis of neutrophils but not monocytes...... towards the chemotactic peptide f-met-leu-phe. On the other hand, chemotaxis of both neutrophils and monocytes towards zymosan-activated serum containing C5a was inhibited by Gp63. Monocyte and neutrophil chemiluminescence response to opsonized zymosan was reduced by preincubation of the cells with Gp63...... in a concentration-dependent manner. Notably, monocytes were inhibited to a much greater degree than neutrophils by a given concentration of Gp63, and they were also inhibited at much lower concentrations of the protease. The inhibitory effect of Gp63 on chemotaxis and chemiluminescence was completely abolished...

  2. IFN-γ protects from lethal IL-17 mediated viral encephalomyelitis independent of neutrophils

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    Savarin Carine

    2012-05-01

    Full Text Available Abstract Background The interplay between IFN-γ, IL-17 and neutrophils during CNS inflammatory disease is complex due to cross-regulatory factors affecting both positive and negative feedback loops. These interactions have hindered the ability to distinguish the relative contributions of neutrophils, Th1 and Th17 cell-derived effector molecules from secondary mediators to tissue damage and morbidity. Methods Encephalitis induced by a gliatropic murine coronavirus was used as a model to assess the direct contributions of neutrophils, IFN-γ and IL-17 to virus-induced mortality. CNS inflammatory conditions were selectively manipulated by adoptive transfer of virus-primed wild-type (WT or IFN-γ deficient (GKO memory CD4+ T cells into infected SCID mice, coupled with antibody-mediated neutrophil depletion and cytokine blockade. Results Transfer of GKO memory CD4+ T cells into infected SCID mice induced rapid mortality compared to recipients of WT memory CD4+ T cells, despite similar virus control and demyelination. In contrast to recipients of WT CD4+ T cells, extensive neutrophil infiltration and IL-17 expression within the CNS in recipients of GKO CD4+ T cells provided a model to directly assess their contribution(s to disease. Recipients of WT CD4+ T cells depleted of IFN-γ did not express IL-17 and were spared from mortality despite abundant CNS neutrophil infiltration, indicating that mortality was not mediated by excessive CNS neutrophil accumulation. By contrast, IL-17 depletion rescued recipients of GKO CD4+ T cells from rapid mortality without diminishing neutrophils or reducing GM-CSF, associated with pathogenic Th17 cells in CNS autoimmune models. Furthermore, co-transfer of WT and GKO CD4+ T cells prolonged survival in an IFN-γ dependent manner, although IL-17 transcription was not reduced. Conclusions These data demonstrate that IL-17 mediates detrimental clinical consequences in an IFN-γ-deprived environment, independent of

  3. Effective reaction rates in diffusion-limited phosphorylation-dephosphorylation cycles

    Science.gov (United States)

    Szymańska, Paulina; Kochańczyk, Marek; Miekisz, Jacek; Lipniacki, Tomasz

    2015-02-01

    We investigate the kinetics of the ubiquitous phosphorylation-dephosphorylation cycle on biological membranes by means of kinetic Monte Carlo simulations on the triangular lattice. We establish the dependence of effective macroscopic reaction rate coefficients as well as the steady-state phosphorylated substrate fraction on the diffusion coefficient and concentrations of opposing enzymes: kinases and phosphatases. In the limits of zero and infinite diffusion, the numerical results agree with analytical predictions; these two limits give the lower and the upper bound for the macroscopic rate coefficients, respectively. In the zero-diffusion limit, which is important in the analysis of dense systems, phosphorylation and dephosphorylation reactions can convert only these substrates which remain in contact with opposing enzymes. In the most studied regime of nonzero but small diffusion, a contribution linearly proportional to the diffusion coefficient appears in the reaction rate. In this regime, the presence of opposing enzymes creates inhomogeneities in the (de)phosphorylated substrate distributions: The spatial correlation function shows that enzymes are surrounded by clouds of converted substrates. This effect becomes important at low enzyme concentrations, substantially lowering effective reaction rates. Effective reaction rates decrease with decreasing diffusion and this dependence is more pronounced for the less-abundant enzyme. Consequently, the steady-state fraction of phosphorylated substrates can increase or decrease with diffusion, depending on relative concentrations of both enzymes. Additionally, steady states are controlled by molecular crowders which, mostly by lowering the effective diffusion of reactants, favor the more abundant enzyme.

  4. Phosphorylation regulates SIRT1 function.

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    Tsutomu Sasaki

    Full Text Available BACKGROUND: SIR2 is an NAD(+-dependent deacetylase [1]-[3] implicated in the regulation of lifespan in species as diverse as yeast [4], worms [5], and flies [6]. We previously reported that the level of SIRT1, the mammalian homologue of SIR2 [7], [8], is coupled to the level of mitotic activity in cells both in vitro and in vivo[9]. Cells from long-lived mice maintained SIRT1 levels of young mice in tissues that undergo continuous cell replacement by proliferating stem cells. Changes in SIRT1 protein level were not associated with changes in mRNA level, suggesting that SIRT1 could be regulated post-transcriptionally. However, other than a recent report on sumoylation [10] and identification of SIRT1 as a nuclear phospho-protein by mass spectrometry [11], post-translational modifications of this important protein have not been reported. METHODOLOGY/PRINCIPAL FINDINGS: We identified 13 residues in SIRT1 that are phosphorylated in vivo using mass spectrometry. Dephosphorylation by phosphatases in vitro resulted in decreased NAD(+-dependent deacetylase activity. We identified cyclinB/Cdk1 as a cell cycle-dependent kinase that forms a complex with and phosphorylates SIRT1. Mutation of two residues phosphorylated by Cyclin B/Cdk1 (threonine 530 and serine 540 disturbs normal cell cycle progression and fails to rescue proliferation defects in SIRT1-deficient cells [12], [13]. CONCLUSIONS/SIGNIFICANCE: Pharmacological manipulation of SIRT1 activity is currently being tested as a means of extending lifespan in mammals. Treatment of obese mice with resveratrol, a pharmacological activator of SIRT1, modestly but significantly improved longevity and, perhaps more importantly, offered some protection against the development of type 2 diabetes mellitus and metabolic syndrome [14]-[16]. Understanding the endogenous mechanisms that regulate the level and activity of SIRT1, therefore, has obvious relevance to human health and disease. Our results identify

  5. PARTICIPATION OF TLR4 IN ENGULFMENT OF ESCHERICHIA COLI BY HUMAN BLOOD NEUTROPHILS IN PRESENCE OF LIPOPOLYSACCHARIDES

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    S. V. Zubova

    2012-01-01

    Full Text Available Abstract. TLR4 is a key player in signaling system of host cells. Possible role of TLR4 is actively discussed, e.g. its significance for phagocytosis. A capacity of neutrophils to engulf FITC-labeled E. coli bacteria upon activation with LPS of different origin was studied in presence of anti-TLR4 Mab’s (HTA125 clone. It was shown that, in whole blood, TLR4 does not play any essential role in engulfment of bacteria by the neutrophils. Phagocytic activity of neutrophils in blood increases increased after their priming with E. coli endotoxins. LPS from Rb. сapsulatus did not affect phagocytosis. In presence of endotoxins, the degree of TLR4 involvement in neutrophil phagocytosis depends on LPS structure.

  6. Swimming Motility Mediates the Formation of Neutrophil Extracellular Traps Induced by Flagellated Pseudomonas aeruginosa.

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    Madison Floyd

    2016-11-01

    Full Text Available Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections often characterized by robust neutrophilic infiltration. Neutrophils provide the first line of defense against P. aeruginosa. Aside from their defense conferred by phagocytic activity, neutrophils also release neutrophil extracellular traps (NETs to immobilize bacteria. Although NET formation is an important antimicrobial process, the details of its mechanism are largely unknown. The identity of the main components of P. aeruginosa responsible for triggering NET formation is unclear. In this study, our focus was to identify the main bacterial factors mediating NET formation and to gain insight into the underlying mechanism. We found that P. aeruginosa in its exponential growth phase promoted strong NET formation in human neutrophils while its NET-inducing ability dramatically decreased at later stages of bacterial growth. We identified the flagellum as the primary component of P. aeruginosa responsible for inducing NET extrusion as flagellum-deficient bacteria remained seriously impaired in triggering NET formation. Purified P. aeruginosa flagellin, the monomeric component of the flagellum, does not stimulate NET formation in human neutrophils. P. aeruginosa-induced NET formation is independent of the flagellum-sensing receptors TLR5 and NLRC4 in both human and mouse neutrophils. Interestingly, we found that flagellar motility, not flagellum binding to neutrophils per se, mediates NET release induced by flagellated bacteria. Immotile, flagellar motor-deficient bacterial strains producing paralyzed flagella did not induce NET formation. Forced contact between immotile P. aeruginosa and neutrophils restored their NET-inducing ability. Both the motAB and motCD genetic loci encoding flagellar motor genes contribute to maximal NET release; however the motCD genes play a more important role. Phagocytosis of P. aeruginosa and superoxide production by neutrophils were also

  7. Regulation of AKT phosphorylation at Ser473 and Thr308 by endoplasmic reticulum stress modulates substrate specificity in a severity dependent manner.

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    Hong Wa Yung

    2011-03-01

    Full Text Available Endoplasmic reticulum (ER stress is a common factor in the pathophysiology of diverse human diseases that are characterised by contrasting cellular behaviours, from proliferation in cancer to apoptosis in neurodegenerative disorders. Coincidently, dysregulation of AKT/PKB activity, which is the central regulator of cell growth, proliferation and survival, is often associated with the same diseases. Here, we demonstrate that ER stress modulates AKT substrate specificity in a severity-dependent manner, as shown by phospho-specific antibodies against known AKT targets. ER stress also reduces both total and phosphorylated AKT in a severity-dependent manner, without affecting activity of the upstream kinase PDK1. Normalisation to total AKT revealed that under ER stress phosphorylation of Thr308 is suppressed while that of Ser473 is increased. ER stress induces GRP78, and siRNA-mediated knock-down of GRP78 enhances phosphorylation at Ser473 by 3.6 fold, but not at Thr308. Substrate specificity is again altered. An in-situ proximity ligation assay revealed a physical interaction between GRP78 and AKT at the plasma membrane of cells following induction of ER stress. Staining was weak in cells with normal nuclear morphology but stronger in those displaying rounded, condensed nuclei. Co-immunoprecipitation of GRP78 and P-AKT(Ser473 confirmed the immuno-complex consists of non-phosphorylated AKT (Ser473 and Thr308. The interaction is likely specific as AKT did not bind to all molecular chaperones, and GRP78 did not bind to p70 S6 kinase. These findings provide one mechanistic explanation for how ER stress contributes to human pathologies demonstrating contrasting cell fates via modulation of AKT signalling.

  8. Regulation of AKT Phosphorylation at Ser473 and Thr308 by Endoplasmic Reticulum Stress Modulates Substrate Specificity in a Severity Dependent Manner

    Science.gov (United States)

    Yung, Hong Wa

    2011-01-01

    Endoplasmic reticulum (ER) stress is a common factor in the pathophysiology of diverse human diseases that are characterised by contrasting cellular behaviours, from proliferation in cancer to apoptosis in neurodegenerative disorders. Coincidently, dysregulation of AKT/PKB activity, which is the central regulator of cell growth, proliferation and survival, is often associated with the same diseases. Here, we demonstrate that ER stress modulates AKT substrate specificity in a severity-dependent manner, as shown by phospho-specific antibodies against known AKT targets. ER stress also reduces both total and phosphorylated AKT in a severity-dependent manner, without affecting activity of the upstream kinase PDK1. Normalisation to total AKT revealed that under ER stress phosphorylation of Thr308 is suppressed while that of Ser473 is increased. ER stress induces GRP78, and siRNA-mediated knock-down of GRP78 enhances phosphorylation at Ser473 by 3.6 fold, but not at Thr308. Substrate specificity is again altered. An in-situ proximity ligation assay revealed a physical interaction between GRP78 and AKT at the plasma membrane of cells following induction of ER stress. Staining was weak in cells with normal nuclear morphology but stronger in those displaying rounded, condensed nuclei. Co-immunoprecipitation of GRP78 and P-AKT(Ser473) confirmed the immuno-complex consists of non-phosphorylated AKT (Ser473 and Thr308). The interaction is likely specific as AKT did not bind to all molecular chaperones, and GRP78 did not bind to p70 S6 kinase. These findings provide one mechanistic explanation for how ER stress contributes to human pathologies demonstrating contrasting cell fates via modulation of AKT signalling. PMID:21445305

  9. Neutrophil Extracellular DNA Traps Induce Autoantigen Production by Airway Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Youngwoo Choi

    2017-01-01

    Full Text Available The hypothesis of autoimmune involvement in asthma has received much recent interest. Autoantibodies, such as anti-cytokeratin (CK 18, anti-CK19, and anti-α-enolase antibodies, react with self-antigens and are found at high levels in the sera of patients with severe asthma (SA. However, the mechanisms underlying autoantibody production in SA have not been fully determined. The present study was conducted to demonstrate that neutrophil extracellular DNA traps (NETs, cytotoxic molecules released from neutrophils, are a key player in the stimulation of airway epithelial cells (AECs to produce autoantigens. This study showed that NETs significantly increased the intracellular expression of tissue transglutaminase (tTG but did not affect that of CK18 in AECs. NETs induced the extracellular release of both tTG and CK18 in a concentration-dependent manner. Moreover, NETs directly degraded intracellular α-enolase into small fragments. However, antibodies against neutrophil elastase (NE or myeloperoxidase (MPO attenuated the effects of NETs on AECs. Furthermore, each NET isolated from healthy controls (HC, nonsevere asthma (NSA, and SA had different characteristics. Taken together, these findings suggest that AECs exposed to NETs may exhibit higher autoantigen production, especially in SA. Therefore, targeting of NETs may represent a new therapy for neutrophilic asthma with a high level of autoantigens.

  10. IL-1 Coordinates the Neutrophil Response to C. albicans in the Oral Mucosa.

    Directory of Open Access Journals (Sweden)

    Simon Altmeier

    2016-09-01

    Full Text Available Mucosal infections with Candida albicans belong to the most frequent forms of fungal diseases. Host protection is conferred by cellular immunity; however, the induction of antifungal immunity is not well understood. Using a mouse model of oropharyngeal candidiasis (OPC we show that interleukin-1 receptor (IL-1R signaling is critical for fungal control at the onset of infection through its impact on neutrophils at two levels. We demonstrate that both the recruitment of circulating neutrophils to the site of infection and the mobilization of newly generated neutrophils from the bone marrow depended on IL-1R. Consistently, IL-1R-deficient mice displayed impaired chemokine production at the site of infection and defective secretion of granulocyte colony-stimulating factor (G-CSF in the circulation in response to C. albicans. Strikingly, endothelial cells were identified as the primary cellular source of G-CSF during OPC, which responded to IL-1α that was released from keratinocytes in the infected tissue. The IL-1-dependent crosstalk between two different cellular subsets of the nonhematopoietic compartment was confirmed in vitro using a novel murine tongue-derived keratinocyte cell line and an established endothelial cell line. These data establish a new link between IL-1 and granulopoiesis in the context of fungal infection. Together, we identified two complementary mechanisms coordinating the neutrophil response in the oral mucosa, which is critical for preventing fungal growth and dissemination, and thus protects the host from disease.

  11. IL-1 Coordinates the Neutrophil Response to C. albicans in the Oral Mucosa.

    Science.gov (United States)

    Altmeier, Simon; Toska, Albulena; Sparber, Florian; Teijeira, Alvaro; Halin, Cornelia; LeibundGut-Landmann, Salomé

    2016-09-01

    Mucosal infections with Candida albicans belong to the most frequent forms of fungal diseases. Host protection is conferred by cellular immunity; however, the induction of antifungal immunity is not well understood. Using a mouse model of oropharyngeal candidiasis (OPC) we show that interleukin-1 receptor (IL-1R) signaling is critical for fungal control at the onset of infection through its impact on neutrophils at two levels. We demonstrate that both the recruitment of circulating neutrophils to the site of infection and the mobilization of newly generated neutrophils from the bone marrow depended on IL-1R. Consistently, IL-1R-deficient mice displayed impaired chemokine production at the site of infection and defective secretion of granulocyte colony-stimulating factor (G-CSF) in the circulation in response to C. albicans. Strikingly, endothelial cells were identified as the primary cellular source of G-CSF during OPC, which responded to IL-1α that was released from keratinocytes in the infected tissue. The IL-1-dependent crosstalk between two different cellular subsets of the nonhematopoietic compartment was confirmed in vitro using a novel murine tongue-derived keratinocyte cell line and an established endothelial cell line. These data establish a new link between IL-1 and granulopoiesis in the context of fungal infection. Together, we identified two complementary mechanisms coordinating the neutrophil response in the oral mucosa, which is critical for preventing fungal growth and dissemination, and thus protects the host from disease.

  12. 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.

  13. 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.

  14. Ck2-Dependent Phosphorylation Is Required to Maintain Pax7 Protein Levels in Proliferating Muscle Progenitors.

    Directory of Open Access Journals (Sweden)

    Natalia González

    Full Text Available Skeletal muscle regeneration and long term maintenance is directly link to the balance between self-renewal and differentiation of resident adult stem cells known as satellite cells. In turn, satellite cell fate is influenced by a functional interaction between the transcription factor Pax7 and members of the MyoD family of muscle regulatory factors. Thus, changes in the Pax7-to-MyoD protein ratio may act as a molecular rheostat fine-tuning acquisition of lineage identity while preventing precocious terminal differentiation. Pax7 is expressed in quiescent and proliferating satellite cells, while its levels decrease sharply in differentiating progenitors Pax7 is maintained in cells (reacquiring quiescence. While the mechanisms regulating Pax7 levels based on differentiation status are not well understood, we have recently described that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4, thus promoting proteasome-dependent Pax7 degradation in differentiating satellite cells. Here we show that Pax7 levels are maintained in proliferating muscle progenitors by a mechanism involving casein kinase 2-dependent Pax7 phosphorylation at S201. Point mutations preventing S201 phosphorylation or casein kinase 2 inhibition result in decreased Pax7 protein in proliferating muscle progenitors. Accordingly, this correlates directly with increased Pax7 ubiquitination. Finally, Pax7 down regulation induced by casein kinase 2 inhibition results in precocious myogenic induction, indicating early commitment to terminal differentiation. These observations highlight the critical role of post translational regulation of Pax7 as a molecular switch controlling muscle progenitor fate.

  15. Coccidioides Endospores and Spherules Draw Strong Chemotactic, Adhesive, and Phagocytic Responses by Individual Human Neutrophils.

    Directory of Open Access Journals (Sweden)

    Cheng-Yuk Lee

    Full Text Available Coccidioides spp. are dimorphic pathogenic fungi whose parasitic forms cause coccidioidomycosis (Valley fever in mammalian hosts. We use an innovative interdisciplinary approach to analyze one-on-one encounters between human neutrophils and two forms of Coccidioides posadasii. To examine the mechanisms by which the innate immune system coordinates different stages of the host response to fungal pathogens, we dissect the immune-cell response into chemotaxis, adhesion, and phagocytosis. Our single-cell technique reveals a surprisingly strong response by initially quiescent neutrophils to close encounters with C. posadasii, both from a distance (by complement-mediated chemotaxis as well as upon contact (by serum-dependent adhesion and phagocytosis. This response closely resembles neutrophil interactions with Candida albicans and zymosan particles, and is significantly stronger than the neutrophil responses to Cryptococcus neoformans, Aspergillus fumigatus, and Rhizopus oryzae under identical conditions. The vigorous in vitro neutrophil response suggests that C. posadasii evades in vivo recognition by neutrophils through suppression of long-range mobilization and recruitment of the immune cells. This observation elucidates an important paradigm of the recognition of microbes, i.e., that intact immunotaxis comprises an intricate spatiotemporal hierarchy of distinct chemotactic processes. Moreover, in contrast to earlier reports, human neutrophils exhibit vigorous chemotaxis toward, and frustrated phagocytosis of, the large spherules of C. posadasii under physiological-like conditions. Finally, neutrophils from healthy donors and patients with chronic coccidioidomycosis display subtle differences in their responses to antibody-coated beads, even though the patient cells appear to interact normally with C. posadasii endospores.

  16. 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...

  17. ROS-activated ATM-dependent phosphorylation of cytoplasmic substrates identified by large scale phosphoproteomics screen

    DEFF Research Database (Denmark)

    Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper

    2016-01-01

    ATM (ataxia-telangiectasia, mutated) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signalling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoi......ATM (ataxia-telangiectasia, mutated) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signalling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle...... checkpoints, initiating DNA repair and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach...... to identify cytoplasmic proteins altered in their phosphorylation state in control and A-T (ataxia-telangiectasia) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites...

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

    Directory of Open Access Journals (Sweden)

    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

  19. Tyrosine Phosphorylation in Toll-Like Receptor Signaling

    Science.gov (United States)

    Chattopadhyay, Saurabh; Sen, Ganes C.

    2014-01-01

    There is a wealth of knowledge about how different Ser/Thr protein kinases participate in Toll-like receptor (TLR) signaling. In many cases, we know the identities of the Ser/Thr residues of various components of the TLR-signaling pathways that are phosphorylated, the functional consequences of the phosphorylation and the responsible protein kinases. In contrast, the analysis of Tyr-phosphorylation of TLRs and their signaling proteins is currently incomplete, because several existing analyses are not systematic or they do not rely on robust experimental data. Nevertheless, it is clear that many TLRs require, for signaling, ligand-dependent phosphorylation of specific Tyr residues in their cytoplasmic domains; the list includes TLR2, TLR3, TLR4, TLR5, TLR8 and TLR9. In this article, we discuss the current status of knowledge on the effect of Tyr-phosphorylation of TLRs and their signaling proteins on their biochemical and biological functions, the possible identities of the relevant protein tyrosine kinases (PTKs) and the nature of regulations of PTK-mediated activation of TLR signaling pathways. PMID:25022196

  20. Phosphorylated intermediate of (Ca2+ + K+)-stimulated Mg2+-dependent transport ATPase in endoplasmic reticulum from rat pancreatic acinar cells

    International Nuclear Information System (INIS)

    Imamura, K.; Schulz, I.

    1985-01-01

    Formation and decomposition of the phosphorylated intermediate of endoplasmic reticulum (Ca 2+ + Mg 2+ )-ATPase from pancreatic acinar cells have been studied using lithium dodecyl sulfate- and tetradecyltrimethylammonium bromide-polyacrylamide gel electrophoresis. Incorporation of 32 P from [gamma- 32 P]ATP is Ca 2+ -dependent (approximate Km for free [Ca 2+ ] = 2-3 x 10(-8) mol/liter). Formation of the 100-kDa phosphoprotein is rapid, reaching maximal 32 P incorporation within 1 s at room temperature. At 4 degrees C, phosphorylation is slower and dephosphorylation is drastically decreased. For dephosphorylation, Mg 2+ and monovalent cations such as K + or Na + are necessary. Vanadate inhibits both 32 P incorporation and 32 P liberation dose dependently (Km = 3 x 10(-6) mol/liter), whereas mitochondrial inhibitors and ouabain have no effect. The phosphoprotein is stable at pH 2 and destabilizes with increasing pH being completely decomposed at pH 9. Reduction of 32 P incorporation in the presence of high concentrations of cold ATP and hydroxylamine suggests formation of acylphosphate present in the ATPase intermediate. The characteristics of Ca 2+ , cation, and pH dependencies of the ATPase activity are similar to those previously described for MgATP-dependent Ca 2+ transport into rough endoplasmic reticulum from pancreatic acinar cells. The data suggest that the 100-kDa phosphoprotein as described in this study is the intermediate of this Ca2+ transport ATPase

  1. Carp macrophages and neutrophilic granulocytes secrete an interleukin-1-like factor.

    NARCIS (Netherlands)

    Verburg-van Kemenade, B.M.L.; Weyts, F.A.A.; Debets, R.; Flik, G.

    1995-01-01

    Carp, Cyprinus carpio L, macrophages and neutrophilic granulocytes obtained from pronephros were cultured. Supernatant was harvested after 48 h and tested for interleukin-1 (IL-1) bioactivity. A concentration-dependent stimulation of proliferation was found of carp Ig− lymphocytes as well as of the

  2. An unusual protein kinase phosphorylates the chemotactic receptor of Dictystelium discoideum

    International Nuclear Information System (INIS)

    Meier, K.; Klein, C.

    1988-01-01

    The authors report the cAMP-dependent phosphorylation of the chemotactic receptor of Dictyostelium discoideum in partially purified plasma membranes. The protein kinase responsible for receptor phosphorylation is associated with this fraction and preferentially phosphorylates the ligand-occupied form of the receptor. 8-Azido[ 32 P]cAMP labeling of the cell surface has shown that the cAMP receptor exists in two forms. A 45-kDa protein is predominant on unstimulated cells. cAMP stimulation results in an increased receptor phosphorylation such that the receptor migrates on NaDodSO 4 /PAGE as a 47-kDa protein. Phosphorylation of the chemotactic receptor is not detected in membrane preparations unless cAMP is added to the incubation mixture. Only under those conditions is the phosphorylated 47-kDa form observed. The requirement for cAMP reflects the fact that the kinase involved preferentially uses the ligand-occupied receptor as a substrate. In vitro phosphorylation of the receptor does not involve tyrosine residues. The enzyme does not appear to be a cAMP- or cGMP-dependent protein kinase nor is it sensitive to guanine nucleotides, Ca 2+ /calmodulin, Ca 2+ /phospholipid, or EGTA. Similarities with the β-adrenergic receptor protein kinase are discussed

  3. ATM regulates NF-κB-dependent immediate-early genes via RelA Ser 276 phosphorylation coupled to CDK9 promoter recruitment

    Science.gov (United States)

    Fang, Ling; Choudhary, Sanjeev; Zhao, Yingxin; Edeh, Chukwudi B; Yang, Chunying; Boldogh, Istvan; Brasier, Allan R.

    2014-01-01

    Ataxia-telangiectasia mutated (ATM), a member of the phosphatidylinositol 3 kinase-like kinase family, is a master regulator of the double strand DNA break-repair pathway after genotoxic stress. Here, we found ATM serves as an essential regulator of TNF-induced NF-kB pathway. We observed that TNF exposure of cells rapidly induced DNA double strand breaks and activates ATM. TNF-induced ROS promote nuclear IKKγ association with ubiquitin and its complex formation with ATM for nuclear export. Activated cytoplasmic ATM is involved in the selective recruitment of the E3-ubiquitin ligase β-TrCP to phospho-IκBα proteosomal degradation. Importantly, ATM binds and activates the catalytic subunit of protein kinase A (PKAc), ribosmal S6 kinase that controls RelA Ser 276 phosphorylation. In ATM knockdown cells, TNF-induced RelA Ser 276 phosphorylation is significantly decreased. We further observed decreased binding and recruitment of the transcriptional elongation complex containing cyclin dependent kinase-9 (CDK9; a kinase necessary for triggering transcriptional elongation) to promoters of NF-κB-dependent immediate-early cytokine genes, in ATM knockdown cells. We conclude that ATM is a nuclear damage-response signal modulator of TNF-induced NF-κB activation that plays a key scaffolding role in IκBα degradation and RelA Ser 276 phosphorylation. Our study provides a mechanistic explanation of decreased innate immune response associated with A-T mutation. PMID:24957606

  4. Pseudomonas aeruginosa ExoU augments neutrophil transepithelial migration.

    Science.gov (United States)

    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.

  5. Pseudomonas aeruginosa ExoU augments neutrophil transepithelial migration.

    Directory of Open Access Journals (Sweden)

    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.

  6. Flow Cytometric Evaluation of Human Neutrophil Apoptosis During Nitric Oxide Generation In Vitro: The Role of Exogenous Antioxidants

    Directory of Open Access Journals (Sweden)

    Zofia Sulowska

    2005-01-01

    in vitro. The effect of exogenous supply of NO donors such as SNP, SIN-1, and GEA-3162 on the course of human neutrophil apoptosis and the role of extracellular antioxidants in this process was investigated. Isolated from peripheral blood, neutrophils were cultured in the presence or absence of NO donor compounds and antioxidants for 8, 12, and 20 hours. Apoptosis of neutrophils was determined in vitro by flow cytometric analysis of cellular DNA content and Annexin V protein binding to the cell surface. Exposure of human neutrophils to GEA-3162 and SIN-1 significantly accelerates and enhances their apoptosis in vitro in a time-dependent fashion. In the presence of SNP, intensification of apoptosis has not been revealed until 12 hours after the culture. The inhibition of GEA-3162- and SIN-1-mediated neutrophil apoptosis by superoxide dismutase (SOD but not by catalase (CAT was observed. Our results show that SOD and CAT can protect neutrophils against NO-donors-induced apoptosis and suggest that the interaction of NO and oxygen metabolites signals may determine the destructive or protective role of NO donor compounds during apoptotic neutrophil death.

  7. Social and structural barriers for adherence to methadone maintenance treatment among Vietnamese opioid dependence patients.

    Science.gov (United States)

    Tran, Bach Xuan; Nguyen, Long Hoang; Tran, Tung Thanh; Latkin, Carl A

    2018-01-01

    Methadone maintenance treatment (MMT) services may reduce the risk of HIV transmission if patients completely adhere to the treatment. Identifying adherence patterns and potential related factors is vital for the sustainability of MMT program in Vietnam. This study examined social and structural factors associated with adherence to MMT among patients in different service delivery models. A total of 510 patients at three MMT clinics in Hanoi were interviewed. Measures of self-reported adherence included the number of missed doses in the past 7 days and the level of adherence in the past 30 days using a visual analog scale (VAS) scoring from 0 (non-adherence) to 100 (perfect adherence). Multivariate regressions were employed to identify factors associated with non-adherence to MMT. A total of 17.7% of participants reported incomplete MMT adherence in the last 30 days and 8.3% reported missing a dose in the last seven days, respectively. Living with HIV/AIDS, poor self-care and usual activities, and disclosure of health issues to spouses or intimate partners were associated with non-adherence. Those patients with pain or depression were more likely to report better adherence. Disclosing health status to spouse/partner increased the risk of incomplete adherence, while disclosing to friends reduced the number of missed dose in the last seven days. Patients attending clinics with comprehensive services had a lower VAS score of adherence compared to those enrolling in clinics with only MMT and general health care. Sustaining the compliance of patients to MMT is principal in the rapid expansion of this service in Vietnam. It is necessary to address the complexity of health care demands of drug users, their difficulties to be rehabilitated into workforce and society, and the stigmatization to maximize the outcomes of MMT program.

  8. Effect of the dimetilsulfoxido in the response chemiluminescent and the consumption of oxygen of neutrophils activated human

    International Nuclear Information System (INIS)

    Garcia, J.

    2001-01-01

    Dimethylsulfoxide (DMSO), a hydroxyl radical scavenger, exerted a dose dependent inhibition on the luminol and lucigenin-enhanced chemiluminescent responses of human neutrophils activated with soluble and particulate stimulants. DMSO inhibition of the luminol chemiluminescense induced by calcium ionophore A23187 was probably due to OH scavenging, whereas inhibition of the lucigenin chemiluminescence suggested DMSO negatively affects the NADPH-dependent membrane oxidase of neutrophils. In agreement with this, DMSO moderately inhibited O2 consumption in PMN suspensions stimulated with chemotactic peptide and opsonized zymosan-induced luminol chemiluminescense was observed only when added before or in conjunction with stimulants, whereas A23187-induced chemiluminescense was inhibited by DMSO regardless of time of addition. Washing of DMSO-treated PMN resulted in increased luminol enhanced chemiluminescense in response to chemotactic peptide and opsonized zymosan. This is consistent with the idea that DMSO may be interfering with activation of the membrane subunits of the oxidase by translocation and docking of the cytoplasmic, regulatory subunits. These data imply that DMSO inhibits neutrophil chemiluminescense both by OH scavenging and interfering with oxidase activation. Key words:Dimethylsulfoxide, chemiluminescent, luminol, lucigenin,neutrophils [es

  9. Protective Effect of Phillyrin on Lethal LPS-Induced Neutrophil Inflammation in Zebrafish

    Directory of Open Access Journals (Sweden)

    Liling Yang

    2017-10-01

    Full Text Available Background/Aims: Forsythia suspensa Vahl. (Oleaceae fruits are widely used in traditional Chinese medicine to treat pneumonia, typhoid, dysentery, ulcers and oedema. Antibacterial and anti-inflammatory activities have been reported for phillyrin (PHN, the main ingredient in Forsythia suspensa Vahl fruits, in vitro. However, the underlying mechanisms in vivo remain poorly defined. In this study, we discovered that PHN exerted potent anti-inflammatory effects in lethal LPS-induced neutrophil inflammation by suppressing the MyD88-dependent signalling pathway in zebrafish. Methods: LPS-yolk microinjection was used to induce a lethal LPS-infected zebrafish model. The effect of PHN on the survival of zebrafish challenged with lethal LPS was evaluated using survival analysis. The effect of PHN on neutrophil inflammation grading in vivo was assessed by tracking neutrophils with a transgenic line. The effects of PHN on neutrophil production and migration were analysed by SB+ cell counts during consecutive hours after modelling. Additionally, key cytokines and members of the MyD88 signalling pathway that are involved in inflammatory response were detected using quantitative RT-PCR. To assess gene expression changes during consecutive hours after modelling, the IL-1β, IL-6, TNF-α, MyD88, TRIF, ERK1/2, JNK, IκBa and NF-κB expression levels were measured. Results: PHN could protect zebrafish against a lethal LPS challenge in a dose-dependent manner, as indicated by decreased neutrophil infltration, reduced tissue necrosis and increased survival rates. Up-regulated IL-1β, IL-6 and TNF-α expression also showed the same tendencies of depression by PHN. Critically, PHN significantly inhibited the LPS-induced activation of MyD88, IκBa, and NF-κB but did not affect the expression of ERK1/2 MAPKs or JNK MAPKs in LPS-stimulated zebrafish. Additionally, PHN regulated the MyD88/IκBα/NF-κB signalling pathway by controlling IκBα, IL-1β, IL-6, and TNF

  10. Site-specific mapping of the human SUMO proteome reveals co-modification with phosphorylation

    DEFF Research Database (Denmark)

    Hendriks, Ivo A; Lyon, David; Young, Clifford

    2017-01-01

    that were co-modified by ubiquitylation, acetylation and methylation. Notably, 9% of the identified SUMOylome occurred proximal to phosphorylation, and numerous SUMOylation sites were found to be fully dependent on prior phosphorylation events. SUMO-proximal phosphorylation occurred primarily in a proline......-directed manner, and inhibition of cyclin-dependent kinases dynamically affected co-modification. Collectively, we present a comprehensive analysis of the SUMOylated proteome, uncovering the structural preferences for SUMO and providing system-wide evidence for a remarkable degree of cross-talk between...

  11. 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.

  12. Blockade by fenspiride of endotoxin-induced neutrophil migration in the rat.

    Science.gov (United States)

    Cunha, F Q; Boukili, M A; da Motta, J I; Vargaftig, B B; Ferreira, S H

    1993-07-06

    Fenspiride, an antiinflammatory drug with low anti-cyclooxygenase activity, administered orally at 60-200 mg/kg inhibited neutrophil migration into peritoneal and air pouches cavities as well as exudation into peritoneal cavities induced by endotoxin but not induced by carrageenin. Up to 100 microM, fenspiride failed to inhibit the in vitro release of a neutrophil chemotactic activity by endotoxin-stimulated macrophages and the in vivo migration into the peritoneal cavities induced by the supernatant of those macrophages. The release of tumour necrosis factor by stimulated macrophages was inhibited by fenspiride in a dose-dependent manner. These results suggest that the antiinflammatory effects of fenspiride are associated with the inhibition of the tumour necrosis factor release by resident macrophages.

  13. Identification of Mitosis-Specific Phosphorylation in Mitotic Chromosome-Associated Proteins.

    Science.gov (United States)

    Ohta, Shinya; Kimura, Michiko; Takagi, Shunsuke; Toramoto, Iyo; Ishihama, Yasushi

    2016-09-02

    During mitosis, phosphorylation of chromosome-associated proteins is a key regulatory mechanism. Mass spectrometry has been successfully applied to determine the complete protein composition of mitotic chromosomes, but not to identify post-translational modifications. Here, we quantitatively compared the phosphoproteome of isolated mitotic chromosomes with that of chromosomes in nonsynchronized cells. We identified 4274 total phosphorylation sites and 350 mitosis-specific phosphorylation sites in mitotic chromosome-associated proteins. Significant mitosis-specific phosphorylation in centromere/kinetochore proteins was detected, although the chromosomal association of these proteins did not change throughout the cell cycle. This mitosis-specific phosphorylation might play a key role in regulation of mitosis. Further analysis revealed strong dependency of phosphorylation dynamics on kinase consensus patterns, thus linking the identified phosphorylation sites to known key mitotic kinases. Remarkably, chromosomal axial proteins such as non-SMC subunits of condensin, TopoIIα, and Kif4A, together with the chromosomal periphery protein Ki67 involved in the establishment of the mitotic chromosomal structure, demonstrated high phosphorylation during mitosis. These findings suggest a novel mechanism for regulation of chromosome restructuring in mitosis via protein phosphorylation. Our study generated a large quantitative database on protein phosphorylation in mitotic and nonmitotic chromosomes, thus providing insights into the dynamics of chromatin protein phosphorylation at mitosis onset.

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds1[OPEN

    Science.gov (United States)

    Hill, Allyson T.; Anderson, Erin M.; She, Yi-Min

    2017-01-01

    Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a crucial branch point of central plant metabolism. In developing castor oil seeds (Ricinus communis) a novel, allosterically desensitized 910-kD Class-2 PEPC hetero-octameric complex, arises from a tight interaction between 107-kD plant-type PEPC and 118-kD bacterial-type (BTPC) subunits. The native Ca2+-dependent protein kinase (CDPK) responsible for in vivo inhibitory phosphorylation of Class-2 PEPC’s BTPC subunit’s at Ser-451 was highly purified from COS and identified as RcCDPK1 (XP_002526815) by mass spectrometry. Heterologously expressed RcCDPK1 catalyzed Ca2+-dependent, inhibitory phosphorylation of BTPC at Ser-451 while exhibiting: (i) a pair of Ca2+ binding sites with identical dissociation constants of 5.03 μM, (ii) a Ca2+-dependent electrophoretic mobility shift, and (iii) a marked Ca2+-independent hydrophobicity. Pull-down experiments established the Ca2+-dependent interaction of N-terminal GST-tagged RcCDPK1 with BTPC. RcCDPK1-Cherry localized to the cytosol and nucleus of tobacco bright yellow-2 cells, but colocalized with mitochondrial-surface associated BTPC-enhanced yellow fluorescent protein when both fusion proteins were coexpressed. Deletion analyses demonstrated that although its N-terminal variable domain plays an essential role in optimizing Ca2+-dependent RcCDPK1 autophosphorylation and BTPC transphosphorylation activity, it is not critical for in vitro or in vivo target recognition. Arabidopsis (Arabidopsis thaliana) CPK4 and soybean (Glycine max) CDPKβ are RcCDPK1 orthologs that effectively phosphorylated castor BTPC at Ser-451. Overall, the results highlight a potential link between cytosolic Ca2+ signaling and the posttranslational control of respiratory CO2 refixation and anaplerotic photosynthate partitioning in support of storage oil and protein biosynthesis in developing COS. PMID:28363991

  16. Biomaterial-induced alterations of neutrophil superoxide production.

    Science.gov (United States)

    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.

  17. Superoxide anion production by human neutrophils activated by Trichomonas vaginalis.

    Science.gov (United States)

    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.

  18. Advanced Role of Neutrophils in Common Respiratory Diseases

    Directory of Open Access Journals (Sweden)

    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.

  19. 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

  20. Neutrophil extracellular trap formation in supragingival biofilms.

    Science.gov (United States)

    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

  1. Neutrophil elastase-induced elastin degradation mediates macrophage influx and lung injury in 60% O2-exposed neonatal rats.

    Science.gov (United States)

    Masood, Azhar; Yi, Man; Belcastro, Rosetta; Li, Jun; Lopez, Lianet; Kantores, Crystal; Jankov, Robert P; Tanswell, A Keith

    2015-07-01

    Neutrophil (PMNL) influx precedes lung macrophage (LM) influx into the lung following exposure of newborn pups to 60% O2. We hypothesized that PMNL were responsible for the signals leading to LM influx. This was confirmed when inhibition of PMNL influx with a CXC chemokine receptor-2 antagonist, SB-265610, also prevented the 60% O2-dependent LM influx, LM-derived nitrotyrosine formation, and pruning of small arterioles. Exposure to 60% O2 was associated with increased lung contents of neutrophil elastase and α-elastin, a marker of denatured elastin, and a decrease in elastin fiber density. This led us to speculate that neutrophil elastase-induced elastin fragments were the chemokines that led to a LM influx into the 60% O2-exposed lung. Inhibition of neutrophil elastase with sivelestat or elafin attenuated the LM influx. Sivelestat also attenuated the 60% O2-induced decrease in elastin fiber density. Daily injections of pups with an antibody to α-elastin prevented the 60% O2-dependent LM influx, impaired alveologenesis, and impaired small vessel formation. This suggests that neutrophil elastase inhibitors may protect against neonatal lung injury not only by preventing structural elastin degradation, but also by blocking elastin fragment-induced LM influx, thus preventing tissue injury from LM-derived peroxynitrite formation. Copyright © 2015 the American Physiological Society.

  2. Gβ1 is required for neutrophil migration in zebrafish.

    Science.gov (United States)

    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.

  3. Nucleobindin co-localizes and associates with cyclooxygenase (COX-2 in human neutrophils.

    Directory of Open Access Journals (Sweden)

    Patrick Leclerc

    2008-05-01

    Full Text Available The inducible cyclooxygenase isoform (COX-2 is associated with inflammation, tumorigenesis, as well as with physiological events. Despite efforts deployed in order to understand the biology of this multi-faceted enzyme, much remains to be understood. Nucleobindin (Nuc, a ubiquitous Ca(2+-binding protein, possesses a putative COX-binding domain. In this study, we investigated its expression and subcellular localization in human neutrophils, its affinity for COX-2 as well as its possible impact on PGE(2 biosynthesis. Complementary subcellular localization approaches including nitrogen cavitation coupled to Percoll fractionation, immunofluorescence, confocal and electron microscopy collectively placed Nuc, COX-2, and all of the main enzymes involved in prostanoid synthesis, in the Golgi apparatus and endoplasmic reticulum of human neutrophils. Immunoprecipitation experiments indicated a high affinity between Nuc and COX-2. Addition of human recombinant (hr Nuc to purified hrCOX-2 dose-dependently caused an increase in PGE(2 biosynthesis in response to arachidonic acid. Co-incubation of Nuc with COX-2-expressing neutrophil lysates also increased their capacity to produce PGE(2. Moreover, neutrophil transfection with hrNuc specifically enhanced PGE(2 biosynthesis. Together, these results identify a COX-2-associated protein which may have an impact in prostanoid biosynthesis.

  4. PPARγ1 phosphorylation enhances proliferation and drug resistance in human fibrosarcoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Pang, Xiaojuan; Shu, Yuxin; Niu, Zhiyuan; Zheng, Wei; Wu, Haochen [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Lu, Yan, E-mail: luyan@nju.edu.cn [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Shen, Pingping, E-mail: ppshen@nju.edu.cn [State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing (China); Model Animal Research Center (MARC), Nanjing University, Nanjing (China)

    2014-03-10

    Post-translational regulation plays a critical role in the control of cell growth and proliferation. The phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ) is the most important post-translational modification. The function of PPARγ phosphorylation has been studied extensively in the past. However, the relationship between phosphorylated PPARγ1 and tumors remains unclear. Here we investigated the role of PPARγ1 phosphorylation in human fibrosarcoma HT1080 cell line. Using the nonphosphorylation (Ser84 to alanine, S84A) and phosphorylation (Ser84 to aspartic acid, S84D) mutant of PPARγ1, the results suggested that phosphorylation attenuated PPARγ1 transcriptional activity. Meanwhile, we demonstrated that phosphorylated PPARγ1 promoted HT1080 cell proliferation and this effect was dependent on the regulation of cell cycle arrest. The mRNA levels of cyclin-dependent kinase inhibitor (CKI) p21{sup Waf1/Cip1} and p27{sup Kip1} descended in PPARγ1{sup S84D} stable HT1080 cell, whereas the expression of p18{sup INK4C} was not changed. Moreover, compared to the PPARγ1{sup S84A}, PPARγ1{sup S84D} up-regulated the expression levels of cyclin D1 and cyclin A. Finally, PPARγ1 phosphorylation reduced sensitivity to agonist rosiglitazone and increased resistance to anticancer drug 5-fluorouracil (5-FU) in HT1080 cell. Our findings establish PPARγ1 phosphorylation as a critical event in human fibrosarcoma growth. These findings raise the possibility that chemical compounds that prevent the phosphorylation of PPARγ1 could act as anticancer drugs. - Highlights: • Phosphorylation attenuates PPARγ1 transcriptional activity. • Phosphorylated PPARγ1 promotes HT1080 cells proliferation. • PPARγ1 phosphorylation regulates cell cycle by mediating expression of cell cycle regulators. • PPARγ1 phosphorylation reduces sensitivity to agonist and anticancer drug. • Our findings establish PPARγ1 phosphorylation as a critical event in HT1080

  5. Phosphorylation of αB-crystallin: Role in stress, aging and patho-physiological conditions.

    Science.gov (United States)

    Bakthisaran, Raman; Akula, Kranthi Kiran; Tangirala, Ramakrishna; Rao, Ch Mohan

    2016-01-01

    αB-crystallin, once thought to be a lenticular protein, is ubiquitous and has critical roles in several cellular processes that are modulated by phosphorylation. Serine residues 19, 45 and 59 of αB-crystallin undergo phosphorylation. Phosphorylation of S45 is mediated by p44/42 MAP kinase, whereas S59 phosphorylation is mediated by MAPKAP kinase-2. Pathway involved in S19 phosphorylation is not known. The review highlights the role of phosphorylation in (i) oligomeric structure, stability and chaperone activity, (ii) cellular processes such as apoptosis, myogenic differentiation, cell cycle regulation and angiogenesis, and (iii) aging, stress, cardiomyopathy-causing αB-crystallin mutants, and in other diseases. Depending on the context and extent of phosphorylation, αB-crystallin seems to confer beneficial or deleterious effects. Phosphorylation alters structure, stability, size distribution and dynamics of the oligomeric assembly, thus modulating chaperone activity and various cellular processes. Phosphorylated αB-crystallin has a tendency to partition to the cytoskeleton and hence to the insoluble fraction. Low levels of phosphorylation appear to be protective, while hyperphosphorylation has negative implications. Mutations in αB-crystallin, such as R120G, Q151X and 464delCT, associated with inherited myofibrillar myopathy lead to hyperphosphorylation and intracellular inclusions. An ongoing study in our laboratory with phosphorylation-mimicking mutants indicates that phosphorylation of R120GαB-crystallin increases its propensity to aggregate. Phosphorylation of αB-crystallin has dual role that manifests either beneficial or deleterious consequences depending on the extent of phosphorylation and interaction with cytoskeleton. Considering that disease-causing mutants of αB-crystallin are hyperphosphorylated, moderation of phosphorylation may be a useful strategy in disease management. This article is part of a Special Issue entitled Crystallin

  6. Polo-like kinase 2-dependent phosphorylation of NPM/B23 on serine 4 triggers centriole duplication.

    Directory of Open Access Journals (Sweden)

    Annekatrin Krause

    Full Text Available Duplication of the centrosome is well controlled during faithful cell division while deregulation of this process leads to supernumary centrosomes, chromosome missegregation and aneuploidy, a hallmark of many cancer cells. We previously reported that Polo-like kinase 2 (Plk2 is activated near the G1/S phase transition, and regulates the reproduction of centrosomes. In search for Plk2 interacting proteins we have identified NPM/B23 (Nucleophosmin as a novel Plk2 binding partner. We find that Plk2 and NPM/B23 interact in vitro in a Polo-box dependent manner. An association between both proteins was also observed in vivo. Moreover, we show that Plk2 phosphorylates NPM/B23 on serine 4 in vivo in S-phase. Notably, expression of a non-phosphorylatable NPM/B23 S4A mutant interferes with centriole reduplication in S-phase arrested cells and leads to a dilution of centriole numbers in unperturbed U2OS cells. The corresponding phospho-mimicking mutants have the opposite effect and their expression leads to the accumulation of centrioles. These findings suggest that NPM/B23 is a direct target of Plk2 in the regulation of centriole duplication and that phosphorylation on serine 4 can trigger this process.

  7. Rapid Diagnostic Device for Subclinical Mastitis Based on Electrochemical Detection of Superoxide Produced from Neutrophils in Fresh Milk

    Science.gov (United States)

    Okada, Kohei; Fukuda, Junji; Suzuki, Hiroaki

    Electrochemical microdevices were fabricated to identify mastitic cows based on the increased number of neutrophils in raw milk. Because neutrophils produce superoxide (O2·-), the amount of O2·- can be used as an early indicator for subclinical mastitis. In the microdevices, O2·- was detected on a gold electrode using superoxide dismutase immobilized via a self-assembled monolayer of cysteine. In a preliminary test using xanthine oxidase to produce O2·-, one of the devices detected the production and rapid extinction of O2·-. When neutrophils obtained from a mastitic cow were concentrated by centrifugation and introduced into the device, a current increase distinctly different from the background was observed. Furthermore, a micropillar structure was fabricated on the gold electrode to trap and collect neutrophils, thereby facilitating the concentration of these cells around the electrode. The measured current clearly depended on the number of neutrophils in raw milk samples, demonstrating the applicability of the device for rapid diagnosis of subclinical mastitis.

  8. Haloperidol Regulates the State of Phosphorylation of Ribosomal Protein S6 via Activation of PKA and Phosphorylation of DARPP-32

    Science.gov (United States)

    Valjent, Emmanuel; Bertran-Gonzalez, Jesus; Bowling, Heather; Lopez, Sébastien; Santini, Emanuela; Matamales, Miriam; Bonito-Oliva, Alessandra; Hervé, Denis; Hoeffer, Charles; Klann, Eric; Girault, Jean-Antoine; Fisone, Gilberto

    2011-01-01

    Administration of typical antipsychotic drugs, such as haloperidol, promotes cAMP-dependent signaling in the medium spiny neurons (MSNs) of the striatum. In this study, we have examined the effect of haloperidol on the state of phosphorylation of the ribosomal protein S6 (rpS6), a component of the small 40S ribosomal subunit. We found that haloperidol increases the phosphorylation of rpS6 at the dual site Ser235/236, which is involved in the regulation of mRNA translation. This effect was exerted in the MSNs of the indirect pathway, which express specifically dopamine D2 receptors (D2Rs) and adenosine A2 receptors (A2ARs). The effect of haloperidol was decreased by blockade of A2ARs or by genetic attenuation of the Gαolf protein, which couples A2ARs to activation of adenylyl cyclase. Moreover, stimulation of cAMP-dependent protein kinase A (PKA) increased Ser235/236 phosphorylation in cultured striatal neurons. The ability of haloperidol to promote rpS6 phosphorylation was abolished in knock-in mice deficient for PKA activation of the protein phosphatase-1 inhibitor, dopamine- and cAMP-regulated phosphoprotein of 32 kDa. In contrast, pharmacological or genetic inactivation of p70 rpS6 kinase 1, or extracellular signal-regulated kinases did not affect haloperidol-induced rpS6 phosphorylation. These results identify PKA as a major rpS6 kinase in neuronal cells and suggest that regulation of protein synthesis through rpS6 may be a potential target of antipsychotic drugs. PMID:21814187

  9. 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...

  10. Anaplasma phagocytophilum inhibits human neutrophil apoptosis via upregulation of bfl-1, maintenance of mitochondrial membrane potential and prevention of caspase 3 activation.

    Science.gov (United States)

    Ge, Yan; Yoshiie, Kiyotaka; Kuribayashi, Futoshi; Lin, Mingqun; Rikihisa, Yasuko

    2005-01-01

    The inhibition of neutrophil apoptosis plays a central role in human granulocytic anaplasmosis. Intracellular signalling pathways through which the obligatory intracellular bacterium Anaplasma phagocytophilum inhibits the spontaneous apoptosis of human peripheral blood neutrophils were investigated. bfl-1 mRNA levels in uninfected neutrophils after 12 h in culture were reduced to approximately 5-25% of 0 h levels, but remained high in infected neutrophils. The eukaryotic RNA synthesis inhibitor, actinomycin D, prevented the maintenance of bfl-1 mRNA levels by A. phagocytophilum. Differences in mcl-1, bax, bcl-w, bad or bak mRNA levels in infected versus uninfected neutrophils were not remarkable. By using mitochondrial fluorescent dyes, Mitotracker Red and JC-1, it was found that most uninfected neutrophils lost mitochondrial membrane potential after 10-12 h incubation, whereas A. phagocytophilum-infected neutrophils maintained high membrane potential. Caspase 3 activity and the degree of apoptosis were lower in dose-dependent manner in A. phagocytophilum-infected neutrophils at 16 h post infection, as compared to uninfected neutrophils. Anti-active caspase 3 antibody labelling showed less positively stained population in infected neutrophils compared to those in uninfected neutrophils after 12 h incubation. These results suggest that A. phagocytophilum inhibits human neutrophil apoptosis via transcriptional upregulation of bfl-1 and inhibition of mitochondria-mediated activation of caspase 3.

  11. GMP-140 binds to a glycoprotein receptor on human neutrophils: Evidence for a lectin-like interaction

    International Nuclear Information System (INIS)

    Moore, K.L.; Varki, A.; McEver, R.P.

    1991-01-01

    GMP-140 is a rapidly inducible receptor for neutrophils and monocytes expressed on activated platelets and endothelial cells. It is a member of the selectin family of lectin-like cell surface molecules that mediate leukocyte adhesion. We used a radioligand binding assay to characterize the interaction of purified GMP-140 with human neutrophils. Unstimulated neutrophils rapidly bound [125I]GMP-140 at 4 degrees C, reaching equilibrium in 10-15 min. Binding was Ca2+ dependent, reversible, and saturable at 3-6 nM free GMP-140 with half-maximal binding at approximately 1.5 nM. Receptor density and apparent affinity were not altered when neutrophils were stimulated with 4 beta-phorbol 12-myristate 13-acetate. Treatment of neutrophils with proteases abolished specific binding of [125I]GMP-140. Binding was also diminished when neutrophils were treated with neuraminidase from Vibrio cholerae, which cleaves alpha 2-3-, alpha 2-6-, and alpha 2-8-linked sialic acids, or from Newcastle disease virus, which cleaves only alpha 2-3- and alpha 2-8-linked sialic acids. Binding was not inhibited by an mAb to the abundant myeloid oligosaccharide, Lex (CD15), or by the neoglycoproteins Lex-BSA and sialyl-Lex-BSA. We conclude that neutrophils constitutively express a glycoprotein receptor for GMP-140, which contains sialic acid residues that are essential for function. These findings support the concept that GMP-140 interacts with leukocytes by a lectin-like mechanism

  12. Ketamine produces antidepressant-like effects through phosphorylation-dependent nuclear export of histone deacetylase 5 (HDAC5) in rats

    Science.gov (United States)

    Choi, Miyeon; Lee, Seung Hoon; Wang, Sung Eun; Ko, Seung Yeon; Song, Mihee; Choi, June-Seek; Duman, Ronald S.; Son, Hyeon

    2015-01-01

    Ketamine produces rapid antidepressant-like effects in animal assays for depression, although the molecular mechanisms underlying these behavioral actions remain incomplete. Here, we demonstrate that ketamine rapidly stimulates histone deacetylase 5 (HDAC5) phosphorylation and nuclear export in rat hippocampal neurons through calcium/calmodulin kinase II- and protein kinase D-dependent pathways. Consequently, ketamine enhanced the transcriptional activity of myocyte enhancer factor 2 (MEF2), which leads to regulation of MEF2 target genes. Transfection of a HDAC5 phosphorylation-defective mutant (Ser259/Ser498 replaced by Ala259/Ala498, HDAC5-S/A), resulted in resistance to ketamine-induced nuclear export, suppression of ketamine-mediated MEF2 transcriptional activity, and decreased expression of MEF2 target genes. Behaviorally, viral-mediated hippocampal knockdown of HDAC5 blocked or occluded the antidepressant effects of ketamine both in unstressed and stressed animals. Taken together, our results reveal a novel role of HDAC5 in the actions of ketamine and suggest that HDAC5 could be a potential mechanism contributing to the therapeutic actions of ketamine. PMID:26647181

  13. Pulmonary endothelial activation caused by extracellular histones contributes to neutrophil activation in acute respiratory distress syndrome.

    Science.gov (United States)

    Zhang, Yanlin; Guan, Li; Yu, Jie; Zhao, Zanmei; Mao, Lijun; Li, Shuqiang; Zhao, Jinyuan

    2016-11-21

    During the acute respiratory distress syndrome (ARDS), neutrophils play a central role in the pathogenesis, and their activation requires interaction with the endothelium. Extracellular histones have been recognized as pivotal inflammatory mediators. This study was to investigate the role of pulmonary endothelial activation during the extracellular histone-induced inflammatory response in ARDS. ARDS was induced in male C57BL/6 mice by intravenous injection with lipopolysaccharide (LPS) or exogenous histones. Concurrent with LPS administration, anti-histone H4 antibody (anti-H4) or non-specific IgG was administered to study the role of extracellular histones. The circulating von Willebrand factor (vWF) and soluble thrombomodulin (sTM) were measured with ELISA kits at the preset time points. Myeloperoxidase (MPO) activity in lung tissue was measured with a MPO detection kit. The translocation of P-selectin and neutrophil infiltration were measured by immunohistochemical detection. For in vitro studies, histone H4 in the supernatant of mouse lung vascular endothelial cells (MLVECs) was measured by Western blot. The binding of extracellular histones with endothelial membrane was examined by confocal laser microscopy. Endothelial P-selectin translocation was measured by cell surface ELISA. Adhesion of neutrophils to MLVECs was assessed with a color video digital camera. The results showed that during LPS-induced ARDS extracellular histones caused endothelial and neutrophil activation, as seen by P-selectin translocation, release of vWF, an increase of circulating sTM, lung neutrophil infiltration and increased MPO activity. Extracellular histones directly bound and activated MLVECs in a dose-dependent manner. On the contrary, the direct stimulatory effect of exogenous histones on neutrophils was very limited, as measured by neutrophil adhesion and MPO activity. With the contribution of activated endothelium, extracellular histones could effectively activating

  14. 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.

  15. Proteasome phosphorylation regulates cocaine-induced sensitization.

    Science.gov (United States)

    Gonzales, Frankie R; Howell, Kristin K; Dozier, Lara E; Anagnostaras, Stephan G; Patrick, Gentry N

    2018-04-01

    Repeated exposure to cocaine produces structural and functional modifications at synapses from neurons in several brain regions including the nucleus accumbens. These changes are thought to underlie cocaine-induced sensitization. The ubiquitin proteasome system plays a crucial role in the remodeling of synapses and has recently been implicated in addiction-related behavior. The ATPase Rpt6 subunit of the 26S proteasome is phosphorylated by Ca 2+ /calmodulin-dependent protein kinases II alpha at ser120 which is thought to regulate proteasome activity and distribution in neurons. Here, we demonstrate that Rpt6 phosphorylation is involved in cocaine-induced locomotor sensitization. Cocaine concomitantly increases proteasome activity and Rpt6 S120 phosphorylation in cultured neurons and in various brain regions of wild type mice including the nucleus accumbens and prefrontal cortex. In contrast, cocaine does not increase proteasome activity in Rpt6 phospho-mimetic (ser120Asp) mice. Strikingly, we found a complete absence of cocaine-induced locomotor sensitization in the Rpt6 ser120Asp mice. Together, these findings suggest a critical role for Rpt6 phosphorylation and proteasome function in the regulation cocaine-induced behavioral plasticity. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. 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

  17. Enhanced {sup 18}F-FDG uptake in activated neutrophils is unaffected by respiratory burst inhibition with RGD

    Energy Technology Data Exchange (ETDEWEB)

    Paik, J. Y.; Lee, K. H.; Go, B. H.; Jeong, K. H.; Kim, H. K.; Choi, J. S.; Choi, Y.; Kim, P. T [Samsung Medical Center, Seoul (Korea, Republic of)

    2004-07-01

    Respiratory burst generation is an important response of activated neutrophils and is associated with enhanced glucose metabolism. Since such activation in dependent on adhesion through integrins, we investigated how integrin occupation with RGD influences respiratory burst response and {sup 18}F-FDG uptake in neutrophils. Human neutrophils separated from healthy volunteers were incubated in RPMI media. For RGD peptide inhibitory experiments, neutrophils were preincubated with 200 {mu} g/ml of cRGD peptides ad 37.deg. for 2 hr prior. Respiratory burst generation and uptake of {sup 18}F-FDG was then measured with or without PMA stimulation. Cellular total hexokinase levels were assayed with a colorimetric method. Treatment with RGD in the basal state resulted in a significant but relatively small increase in neutrophil superoxide release to 1.5{+-}0.25 fold o control levels (p<0.005). Whereas PMA stimulation caused a marked increase in superoxide generation, pretreatment with RGD caused a significant attenuation of this response to 35.6{+-}0.2% (p<0.005). PMA stimulation resulted in a significant increase in {sup 18}F-FDG uptake. However, unlike the attenution of superoxide generation, neutrophils pretreated with RGD before PMA stimulation showed an identical magnitude of enhanced {sup 18}F-FDG uptake (201.8{+-}20.5 of controls, p=0.0001). In addition, hexokinase levels were increased to comparable levels of approximately 1.5 fold for PMA stimulated neutrophils irrespective of RGD pretreatment. In conclusion, soluble RGD blocks stimulation of respiratory burst activation in neutrophils but does not inhibit stimulation of cellular glucose metabolism. This dissociation may contribute to maximally enhanced neutrophil FDG uptake in inflammatory lesions regardless of the occupancy of their integrin receptors.

  18. 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.

  19. Tyrosine phosphorylation switching of a G protein.

    Science.gov (United States)

    Li, Bo; Tunc-Ozdemir, Meral; Urano, Daisuke; Jia, Haiyan; Werth, Emily G; Mowrey, David D; Hicks, Leslie M; Dokholyan, Nikolay V; Torres, Matthew P; Jones, Alan M

    2018-03-30

    Heterotrimeric G protein complexes are molecular switches relaying extracellular signals sensed by G protein-coupled receptors (GPCRs) to downstream targets in the cytoplasm, which effect cellular responses. In the plant heterotrimeric GTPase cycle, GTP hydrolysis, rather than nucleotide exchange, is the rate-limiting reaction and is accelerated by a receptor-like regulator of G signaling (RGS) protein. We hypothesized that posttranslational modification of the Gα subunit in the G protein complex regulates the RGS-dependent GTPase cycle. Our structural analyses identified an invariant phosphorylated tyrosine residue (Tyr 166 in the Arabidopsis Gα subunit AtGPA1) located in the intramolecular domain interface where nucleotide binding and hydrolysis occur. We also identified a receptor-like kinase that phosphorylates AtGPA1 in a Tyr 166 -dependent manner. Discrete molecular dynamics simulations predicted that phosphorylated Tyr 166 forms a salt bridge in this interface and potentially affects the RGS protein-accelerated GTPase cycle. Using a Tyr 166 phosphomimetic substitution, we found that the cognate RGS protein binds more tightly to the GDP-bound Gα substrate, consequently reducing its ability to accelerate GTPase activity. In conclusion, we propose that phosphorylation of Tyr 166 in AtGPA1 changes the binding pattern with AtRGS1 and thereby attenuates the steady-state rate of the GTPase cycle. We coin this newly identified mechanism "substrate phosphoswitching." © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Tousled-like kinase-dependent phosphorylation of Rad9 plays a role in cell cycle progression and G2/M checkpoint exit.

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    Ryan Kelly

    Full Text Available Genomic integrity is preserved by checkpoints, which act to delay cell cycle progression in the presence of DNA damage or replication stress. The heterotrimeric Rad9-Rad1-Hus1 (9-1-1 complex is a PCNA-like clamp that is loaded onto DNA at structures resulting from damage and is important for initiating and maintaining the checkpoint response. Rad9 possesses a C-terminal tail that is phosphorylated constitutively and in response to cell cycle position and DNA damage. Previous studies have identified tousled-like kinase 1 (TLK1 as a kinase that may modify Rad9. Here we show that Rad9 is phosphorylated in a TLK-dependent manner in vitro and in vivo, and that T355 within the C-terminal tail is the primary targeted residue. Phosphorylation of Rad9 at T355 is quickly reduced upon exposure to ionizing radiation before returning to baseline later in the damage response. We also show that TLK1 and Rad9 interact constitutively, and that this interaction is enhanced in chromatin-bound Rad9 at later stages of the damage response. Furthermore, we demonstrate via siRNA-mediated depletion that TLK1 is required for progression through S-phase in normally cycling cells, and that cells lacking TLK1 display a prolonged G2/M arrest upon exposure to ionizing radiation, a phenotype that is mimicked by over-expression of a Rad9-T355A mutant. Given that TLK1 has previously been shown to be transiently inactivated upon phosphorylation by Chk1 in response to DNA damage, we propose that TLK1 and Chk1 act in concert to modulate the phosphorylation status of Rad9, which in turn serves to regulate the DNA damage response.

  1. L-Arginine Enhances Protein Synthesis by Phosphorylating mTOR (Thr 2446 in a Nitric Oxide-Dependent Manner in C2C12 Cells

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    Ruxia Wang

    2018-01-01

    Full Text Available Muscle atrophy may arise from many factors such as inactivity, malnutrition, and inflammation. In the present study, we investigated the stimulatory effect of nitric oxide (NO on muscle protein synthesis. Primarily, C2C12 cells were supplied with extra L-arginine (L-Arg in the culture media. L-Arg supplementation increased the activity of inducible nitric oxide synthase (iNOS, the rate of protein synthesis, and the phosphorylation of mTOR (Thr 2446 and p70S6K (Thr 389. L-NAME, an NOS inhibitor, decreased NO concentrations within cells and abolished the stimulatory effect of L-Arg on protein synthesis and the phosphorylation of mTOR and p70S6K. In contrast, SNP (sodium nitroprusside, an NO donor, increased NO concentrations, enhanced protein synthesis, and upregulated mTOR and p70S6K phosphorylation, regardless of L-NAME treatment. Blocking mTOR with rapamycin abolished the stimulatory effect of both L-Arg and SNP on protein synthesis and p70S6K phosphorylation. These results indicate that L-Arg stimulates protein synthesis via the activation of the mTOR (Thr 2446/p70S6K signaling pathway in an NO-dependent manner.

  2. Synaptotagmin-7 phosphorylation mediates GLP-1-dependent potentiation of insulin secretion from β-cells

    DEFF Research Database (Denmark)

    Wu, Bingbing; Wei, Shunhui; Petersen, Natalia

    2015-01-01

    Glucose stimulates insulin secretion from β-cells by increasing intracellular Ca(2+). Ca(2+) then binds to synaptotagmin-7 as a major Ca(2+) sensor for exocytosis, triggering secretory granule fusion and insulin secretion. In type-2 diabetes, insulin secretion is impaired; this impairment...... is ameliorated by glucagon-like peptide-1 (GLP-1) or by GLP-1 receptor agonists, which improve glucose homeostasis. However, the mechanism by which GLP-1 receptor agonists boost insulin secretion remains unclear. Here, we report that GLP-1 stimulates protein kinase A (PKA)-dependent phosphorylation...... of synaptotagmin-7 at serine-103, which enhances glucose- and Ca(2+)-stimulated insulin secretion and accounts for the improvement of glucose homeostasis by GLP-1. A phospho-mimetic synaptotagmin-7 mutant enhances Ca(2+)-triggered exocytosis, whereas a phospho-inactive synaptotagmin-7 mutant disrupts GLP-1...

  3. Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity

    DEFF Research Database (Denmark)

    de Jong, Arthur P H; Meijer, Marieke; Saarloos, Ingrid

    2016-01-01

    Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAG-induced potentiation of vesicle priming, but the role of most presynaptic PKC substrates is not unde......Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAG-induced potentiation of vesicle priming, but the role of most presynaptic PKC substrates...... is not understood. Here, we show that a mutation in synaptotagmin-1 (Syt1(T112A)), which prevents its PKC-dependent phosphorylation, abolishes DAG-induced potentiation of synaptic transmission in hippocampal neurons. This mutant also reduces potentiation of spontaneous release, but only if alternative Ca(2+)sensors...

  4. Effect of binding in cyclic phosphorylation-dephosphorylation process and in energy transformation.

    Science.gov (United States)

    Sarkar, A; Beard, D A; Franza, B R

    2006-07-01

    The effects of binding on the phosphorylation-dephosphorylation cycle (PDPC) - one of the key components of the signal transduction processes - is analyzed based on a mathematical model. The model shows that binding of proteins, forming a complex, diminishes the ultrasensitivity of the PDPC to the differences in activity between kinase and phosphatase in the cycle. It is also found that signal amplification depends upon the strength of the binding affinity of the protein (phosphorylated or dephosphorylated) to other proteins . It is also observed that the amplification of signal is not only dependent on phosphorylation potential but also on binding properties and resulting adjustments in binding energies.

  5. 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.

  6. 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.

  7. 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

  8. Human Neutrophils Use Different Mechanisms To Kill Aspergillus fumigatus Conidia and Hyphae: Evidence from Phagocyte Defects.

    Science.gov (United States)

    Gazendam, Roel P; van Hamme, John L; Tool, Anton T J; Hoogenboezem, Mark; van den Berg, J Merlijn; Prins, Jan M; Vitkov, Ljubomir; van de Veerdonk, Frank L; van den Berg, Timo K; Roos, Dirk; Kuijpers, Taco W

    2016-02-01

    Neutrophils are known to play a pivotal role in the host defense against Aspergillus infections. This is illustrated by the prevalence of Aspergillus infections in patients with neutropenia or phagocyte functional defects, such as chronic granulomatous disease. However, the mechanisms by which human neutrophils recognize and kill Aspergillus are poorly understood. In this work, we have studied in detail which neutrophil functions, including neutrophil extracellular trap (NET) formation, are involved in the killing of Aspergillus fumigatus conidia and hyphae, using neutrophils from patients with well-defined genetic immunodeficiencies. Recognition of conidia involves integrin CD11b/CD18 (and not dectin-1), which triggers a PI3K-dependent nonoxidative intracellular mechanism of killing. When the conidia escape from early killing and germinate, the extracellular destruction of the Aspergillus hyphae needs opsonization by Abs and involves predominantly recognition via Fcγ receptors, signaling via Syk, PI3K, and protein kinase C to trigger the production of toxic reactive oxygen metabolites by the NADPH oxidase and myeloperoxidase. A. fumigatus induces NET formation; however, NETs did not contribute to A. fumigatus killing. Thus, our findings reveal distinct killing mechanisms of Aspergillus conidia and hyphae by human neutrophils, leading to a comprehensive insight in the innate antifungal response. Copyright © 2016 by The American Association of Immunologists, Inc.

  9. Na,K-ATPase regulates intercellular communication in the vascular wall via cSrc kinase dependent connexin43 phosphorylation

    DEFF Research Database (Denmark)

    Hangaard, Lise; Bouzinova, Elena; Stæhr, Christian Albeck

    2017-01-01

    Communication between vascular smooth muscle cells (VSMCs) is dependent on gap junctions and is regulated by the Na-K-ATPase. The Na-K-ATPase is therefore important for synchronized VSMC oscillatory activity, i.e., vasomotion. The signaling between the Na-K-ATPase and gap junctions is unknown. We...... coupling in rat mesenteric small arteries in vitro. Phosphorylation of cSrc kinase and connexin43 (Cx43) were semiquantified by Western blotting. Micromole concentration of ouabain reduced the amplitude of norepinephrine-induced vasomotion and desynchronized Ca2+ transients in VSMC in the arterial wall...

  10. Ferroxitosis: A cell death from modulation of oxidative phosphorylation and PKM2-dependent glycolysis in melanoma

    Science.gov (United States)

    Lakhter, Alexander J.; Hamilton, James; Dagher, Pierre C.; Mukkamala, Suresh; Hato, Takashi; Dong, X. Charlie; Mayo, Lindsey D.; Harris, Robert A.; Shekhar, Anantha; Ivan, Mircea; Brustovetsky, Nickolay; Naidu, Samisubbu R.

    2014-01-01

    Reliance on glycolysis is a characteristic of malignancy, yet the development of resistance to BRAF inhibitors in melanoma is associated with gain of mitochondrial function. Concurrent attenuation of oxidative phosphorylation and HIF-1α/PKM2-dependent glycolysis promotes a non-apoptotic, iron- and oxygen-dependent cell death that we term ferroxitosis. The redox cycling agent menadione causes a robust increase in oxygen consumption, accompanied by significant loss of intracellular ATP and rapid cell death. Conversely, either hypoxic adaptation or iron chelation prevents menadione-induced ferroxitosis. Ectopic expression of K213Q HIF-1α mutant blunts the effects of menadione. However, knockdown of HIF-1α or PKM2 restores menadione-induced cytotoxicity in hypoxia. Similarly, exposure of melanoma cells to shikonin, a menadione analog and a potential PKM2 inhibitor, is sufficient to induce ferroxitosis under hypoxic conditions. Collectively, our findings reveal that ferroxitosis curtails metabolic plasticity in melanoma. PMID:25587028

  11. 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

  12. CYP4F18-Deficient Neutrophils Exhibit Increased Chemotaxis to Complement Component C5a

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    Rachel Vaivoda

    2015-01-01

    Full Text Available CYP4Fs were first identified as enzymes that catalyze hydroxylation of leukotriene B4 (LTB4. CYP4F18 has an unusual expression in neutrophils and was predicted to play a role in regulating LTB4-dependent inflammation. We compared chemotaxis of wild-type and Cyp4f18 knockout neutrophils using an in vitro assay. There was no significant difference in the chemotactic response to LTB4, but the response to complement component C5a increased 1.9–2.25-fold in knockout cells compared to wild-type (P < 0.01. This increase was still observed when neutrophils were treated with inhibitors of eicosanoid synthesis. There were no changes in expression of other CYP4 enzymes in knockout neutrophils that might compensate for loss of CYP4F18 or lead to differences in activity. A mouse model of dextran sodium sulfate colitis was used to investigate the consequences of increased C5a-dependent chemotaxis in vivo, but there was no significant difference in weight loss, disease activity, or colonic tissue myeloperoxidase between wild-type and Cyp4f18 knockout mice. This study demonstrates the limitations of inferring CYP4F function based on an ability to use LTB4 as a substrate, points to expanding roles for CYP4F enzymes in immune regulation, and underscores the in vivo challenges of CYP knockout studies.

  13. Phosphorylation of a splice variant of collapsin response mediator protein 2 in the nucleus of tumour cells links cyclin dependent kinase-5 to oncogenesis.

    Science.gov (United States)

    Grant, Nicola J; Coates, Philip J; Woods, Yvonne L; Bray, Susan E; Morrice, Nicholas A; Hastie, C James; Lamont, Douglas J; Carey, Francis A; Sutherland, Calum

    2015-11-10

    Cyclin-dependent protein kinase-5 (CDK5) is an unusual member of the CDK family as it is not cell cycle regulated. However many of its substrates have roles in cell growth and oncogenesis, raising the possibility that CDK5 modulation could have therapeutic benefit. In order to establish whether changes in CDK5 activity are associated with oncogenesis one could quantify phosphorylation of CDK5 targets in disease tissue in comparison to appropriate controls. However the identity of physiological and pathophysiological CDK5 substrates remains the subject of debate, making the choice of CDK5 activity biomarkers difficult. Here we use in vitro and in cell phosphorylation assays to identify novel features of CDK5 target sequence determinants that confer enhanced CDK5 selectivity, providing means to select substrate biomarkers of CDK5 activity with more confidence. We then characterize tools for the best CDK5 substrate we identified to monitor its phosphorylation in human tissue and use these to interrogate human tumour arrays. The close proximity of Arg/Lys amino acids and a proline two residues N-terminal to the phosphorylated residue both improve recognition of the substrate by CDK5. In contrast the presence of a proline two residues C-terminal to the target residue dramatically reduces phosphorylation rate. Serine-522 of Collapsin Response Mediator-2 (CRMP2) is a validated CDK5 substrate with many of these structural criteria. We generate and characterise phosphospecific antibodies to Ser522 and show that phosphorylation appears in human tumours (lung, breast, and lymphoma) in stark contrast to surrounding non-neoplastic tissue. In lung cancer the anti-phospho-Ser522 signal is positive in squamous cell carcinoma more frequently than adenocarcinoma. Finally we demonstrate that it is a specific and unusual splice variant of CRMP2 (CRMP2A) that is phosphorylated in tumour cells. For the first time this data associates altered CDK5 substrate phosphorylation with

  14. Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

    Science.gov (United States)

    Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

    2005-01-01

    The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

  15. Tumor-Associated Macrophages and Neutrophils in Tumor Microenvironment

    Directory of Open Access Journals (Sweden)

    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.

  16. Granule protein processing and regulated secretion in neutrophils

    Directory of Open Access Journals (Sweden)

    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.

  17. The Resolution of Inflammation: A Mathematical Model of Neutrophil and Macrophage Interactions

    KAUST Repository

    Dunster, J. L.

    2014-07-23

    © 2014, Society for Mathematical Biology. There is growing interest in inflammation due to its involvement in many diverse medical conditions, including Alzheimer’s disease, cancer, arthritis and asthma. The traditional view that resolution of inflammation is a passive process is now being superceded by an alternative hypothesis whereby its resolution is an active, anti-inflammatory process that can be manipulated therapeutically. This shift in mindset has stimulated a resurgence of interest in the biological mechanisms by which inflammation resolves. The anti-inflammatory processes central to the resolution of inflammation revolve around macrophages and are closely related to pro-inflammatory processes mediated by neutrophils and their ability to damage healthy tissue. We develop a spatially averaged model of inflammation centring on its resolution, accounting for populations of neutrophils and macrophages and incorporating both pro- and anti-inflammatory processes. Our ordinary differential equation model exhibits two outcomes that we relate to healthy and unhealthy states. We use bifurcation analysis to investigate how variation in the system parameters affects its outcome. We find that therapeutic manipulation of the rate of macrophage phagocytosis can aid in resolving inflammation but success is critically dependent on the rate of neutrophil apoptosis. Indeed our model predicts that an effective treatment protocol would take a dual approach, targeting macrophage phagocytosis alongside neutrophil apoptosis.

  18. Yersinia pestis subverts the dermal neutrophil response in a mouse model of bubonic plague.

    Science.gov (United States)

    Shannon, Jeffrey G; Hasenkrug, Aaron M; Dorward, David W; Nair, Vinod; Carmody, Aaron B; Hinnebusch, B Joseph

    2013-08-27

    The majority of human Yersinia pestis infections result from introduction of bacteria into the skin by the bite of an infected flea. Once in the dermis, Y. pestis can evade the host's innate immune response and subsequently disseminate to the draining lymph node (dLN). There, the pathogen replicates to large numbers, causing the pathognomonic bubo of bubonic plague. In this study, several cytometric and microscopic techniques were used to characterize the early host response to intradermal (i.d.) Y. pestis infection. Mice were infected i.d. with fully virulent or attenuated strains of dsRed-expressing Y. pestis, and tissues were analyzed by flow cytometry. By 4 h postinfection, there were large numbers of neutrophils in the infected dermis and the majority of cell-associated bacteria were associated with neutrophils. We observed a significant effect of the virulence plasmid (pCD1) on bacterial survival and neutrophil activation in the dermis. Intravital microscopy of i.d. Y. pestis infection revealed dynamic interactions between recruited neutrophils and bacteria. In contrast, very few bacteria interacted with dendritic cells (DCs), indicating that this cell type may not play a major role early in Y. pestis infection. Experiments using neutrophil depletion and a CCR7 knockout mouse suggest that dissemination of Y. pestis from the dermis to the dLN is not dependent on neutrophils or DCs. Taken together, the results of this study show a very rapid, robust neutrophil response to Y. pestis in the dermis and that the virulence plasmid pCD1 is important for the evasion of this response. Yersinia pestis remains a public health concern today because of sporadic plague outbreaks that occur throughout the world and the potential for its illegitimate use as a bioterrorism weapon. Since bubonic plague pathogenesis is initiated by the introduction of Y. pestis into the skin, we sought to characterize the response of the host's innate immune cells to bacteria early after

  19. 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.

  20. Ser46 phosphorylation and prolyl-isomerase Pin1-mediated isomerization of p53 are key events in p53-dependent apoptosis induced by mutant huntingtin.

    Science.gov (United States)

    Grison, Alice; Mantovani, Fiamma; Comel, Anna; Agostoni, Elena; Gustincich, Stefano; Persichetti, Francesca; Del Sal, Giannino

    2011-11-01

    Huntington disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the gene coding for huntingtin protein. Several mechanisms have been proposed by which mutant huntingtin (mHtt) may trigger striatal neurodegeneration, including mitochondrial dysfunction, oxidative stress, and apoptosis. Furthermore, mHtt induces DNA damage and activates a stress response. In this context, p53 plays a crucial role in mediating mHtt toxic effects. Here we have dissected the pathway of p53 activation by mHtt in human neuronal cells and in HD mice, with the aim of highlighting critical nodes that may be pharmacologically manipulated for therapeutic intervention. We demonstrate that expression of mHtt causes increased phosphorylation of p53 on Ser46, leading to its interaction with phosphorylation-dependent prolyl isomerase Pin1 and consequent dissociation from the apoptosis inhibitor iASPP, thereby inducing the expression of apoptotic target genes. Inhibition of Ser46 phosphorylation by targeting homeodomain-interacting protein kinase 2 (HIPK2), PKCδ, or ataxia telangiectasia mutated kinase, as well as inhibition of the prolyl isomerase Pin1, prevents mHtt-dependent apoptosis of neuronal cells. These results provide a rationale for the use of small-molecule inhibitors of stress-responsive protein kinases and Pin1 as a potential therapeutic strategy for HD treatment.

  1. 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.

  2. Phosphatidylinositol-3-kinase-dependent phosphorylation of SLP-76 by the lymphoma-associated ITK-SYK fusion-protein

    International Nuclear Information System (INIS)

    Hussain, Alamdar; Faryal, Rani; Nore, Beston F.; Mohamed, Abdalla J.; Smith, C.I. Edvard

    2009-01-01

    Recurrent chromosomal translocations have long been implicated in various types of lymphomas and other malignancies. Novel recurrent t(5;9)(q33;q22) has been recently discovered in un-specified peripheral T-cell lymphoma. To elucidate the role of this translocation, the corresponding fusion construct encoding the N-terminal portion of the ITK kinase and the C-terminal catalytic region of the SYK kinase was generated. We herein show that the ITK-SYK fusion-protein is constitutively active. Moreover, we demonstrate that ITK-SYK is phosphorylated on key tyrosine residues and is capable of potently phosphorylating the related adapter proteins BLNK and SLP-76. In transiently transfected cells, SYK was phosphorylated at Y352 but not detectably at the activation-loop tyrosines Y525/Y526. In contrast, ITK-SYK was phosphorylated both at Y212 and the activation-loop tyrosines Y385/Y386, corresponding to Y352 and Y525/Y526 in SYK, respectively. In resting primary lymphocytes, ITK-SYK predominantly localizes to the cell surface. In addition, we demonstrate that following stimulation, the ITK-SYK fusion-protein in cell lines translocates to the cell membrane and, moreover, that this phenomenon as well as SLP-76 phosphorylation are blocked upon phosphatidylinositol-3-kinase (PI3-kinase) inhibition.

  3. Phosphorylation-dependent trafficking of plasma membrane proteins in animal and plant cells.

    Science.gov (United States)

    Offringa, Remko; Huang, Fang

    2013-09-01

    In both unicellular and multicellular organisms, transmembrane (TM) proteins are sorted to and retained at specific membrane domains by endomembrane trafficking mechanisms that recognize sorting signals in the these proteins. The trafficking and distribution of plasma membrane (PM)-localized TM proteins (PM proteins), especially of those PM proteins that show an asymmetric distribution over the PM, has received much attention, as their proper PM localization is crucial for elementary signaling and transport processes, and defects in their localization often lead to severe disease symptoms or developmental defects. The subcellular localization of PM proteins is dynamically regulated by post-translational modifications, such as phosphorylation and ubiquitination. These modificaitons mostly occur on sorting signals that are located in the larger cytosolic domains of the cargo proteins. Here we review the effects of phosphorylation of PM proteins on their trafficking, and present the key examples from the animal field that have been subject to studies for already several decades, such as that of aquaporin 2 and the epidermal growth factor receptor. Our knowledge on cargo trafficking in plants is largely based on studies of the family of PIN FORMED (PIN) carriers that mediate the efflux of the plant hormone auxin. We will review what is known on the subcellular distribution and trafficking of PIN proteins, with a focus on how this is modulated by phosphorylation, and identify and discuss analogies and differences in trafficking with the well-studied animal examples. © 2013 Institute of Botany, Chinese Academy of Sciences.

  4. Tau Phosphorylation by GSK3 in Different Conditions

    Directory of Open Access Journals (Sweden)

    Jesús Avila

    2012-01-01

    Full Text Available Almost a 20% of the residues of tau protein are phosphorylatable amino acids: serine, threonine, and tyrosine. In this paper we comment on the consequences for tau of being a phosphoprotein. We will focus on serine/threonine phosphorylation. It will be discussed that, depending on the modified residue in tau molecule, phosphorylation could be protective, in processes like hibernation, or toxic like in development of those diseases known as tauopathies, which are characterized by an hyperphosphorylation and aggregation of tau.

  5. 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.

  6. Replication Protein A (RPA) Phosphorylation Prevents RPA Association with Replication Centers

    OpenAIRE

    Vassin, Vitaly M.; Wold, Marc S.; Borowiec, James A.

    2004-01-01

    Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (RPA2D) or alanine (RPA2A). Although RPA2D was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2D mutant was selectively unable to associate with re...

  7. Type 1 Diabetes Prone NOD Mice Have Diminished Cxcr1 mRNA Expression in Polymorphonuclear Neutrophils and CD4+ T Lymphocytes.

    Directory of Open Access Journals (Sweden)

    Karine Haurogné

    Full Text Available In humans, CXCR1 and CXCR2 are two homologous proteins that bind ELR+ chemokines. Both receptors play fundamental roles in neutrophil functions such as migration and reactive oxygen species production. Mouse Cxcr1 and Cxcr2 genes are located in an insulin-dependent diabetes genetic susceptibility locus. The non obese diabetic (NOD mouse is a spontaneous well-described animal model for insulin-dependent type 1 diabetes. In this disease, insulin deficiency results from the destruction of insulin-producing beta cells by autoreactive T lymphocytes. This slow-progressing disease is dependent on both environmental and genetic factors. Here, we report descriptive data about the Cxcr1 gene in NOD mice. We demonstrate decreased expression of mRNA for Cxcr1 in neutrophils and CD4+ lymphocytes isolated from NOD mice compared to other strains, related to reduced NOD Cxcr1 gene promoter activity. Looking for Cxcr1 protein, we next analyze the membrane proteome of murine neutrophils by mass spectrometry. Although Cxcr2 protein is clearly found in murine neutrophils, we did not find evidence of Cxcr1 peptides using this method. Nevertheless, in view of recently-published experimental data obtained in NOD mice, we argue for possible Cxcr1 involvement in type 1 diabetes pathogenesis.

  8. Improved recovery of functionally active eosinophils and neutrophils using novel immunomagnetic technology.

    Science.gov (United States)

    Son, Kiho; Mukherjee, Manali; McIntyre, Brendan A S; Eguez, Jose C; Radford, Katherine; LaVigne, Nicola; Ethier, Caroline; Davoine, Francis; Janssen, Luke; Lacy, Paige; Nair, Parameswaran

    2017-10-01

    Clinically relevant and reliable reports derived from in vitro research are dependent on the choice of cell isolation protocols adopted between different laboratories. Peripheral blood eosinophils are conventionally isolated using density-gradient centrifugation followed by immunomagnetic selection (positive/negative) while neutrophils follow a more simplified dextran-sedimentation methodology. With the increasing sophistication of molecular techniques, methods are now available that promise protocols with reduced user-manipulations, improved efficiency, and better yield without compromising the purity of enriched cell populations. These recent techniques utilize immunomagnetic particles with multiple specificities against differential cell surface markers to negatively select non-target cells from whole blood, greatly reducing the cost/time taken to isolate granulocytes. Herein, we compare the yield efficiencies, purity and baseline activation states of eosinophils/neutrophils isolated using one of these newer protocols that use immunomagnetic beads (MACSxpress isolation) vs. the standard isolation procedures. The study shows that the MACSxpress method consistently allowed higher yields per mL of peripheral blood compared to conventional methods (Peosinophils (95.0±1.7%) and neutrophils (94.2±10.1%) assessed by two methods: Wright's staining and flow cytometry. In addition, enumeration of CD63 + (marker for eosinophil activation) and CD66b + (marker for neutrophil activation) cells within freshly isolated granulocytes, respectively, confirmed that conventional protocols using density-gradient centrifugation caused cellular activation of the granulocytes at baseline compared to the MACSxpress method. In conclusion, MACSxpress isolation kits were found to be superior to conventional techniques for consistent purifications of eosinophils and neutrophils that were suitable for activation assays involving degranulation markers. Copyright © 2017 Elsevier B.V. All

  9. Protein phosphorylation and its role in archaeal signal transduction

    Science.gov (United States)

    Esser, Dominik; Hoffmann, Lena; Pham, Trong Khoa; Bräsen, Christopher; Qiu, Wen; Wright, Phillip C.; Albers, Sonja-Verena; Siebers, Bettina

    2016-01-01

    Reversible protein phosphorylation is the main mechanism of signal transduction that enables cells to rapidly respond to environmental changes by controlling the functional properties of proteins in response to external stimuli. However, whereas signal transduction is well studied in Eukaryotes and Bacteria, the knowledge in Archaea is still rather scarce. Archaea are special with regard to protein phosphorylation, due to the fact that the two best studied phyla, the Euryarchaeota and Crenarchaeaota, seem to exhibit fundamental differences in regulatory systems. Euryarchaeota (e.g. halophiles, methanogens, thermophiles), like Bacteria and Eukaryotes, rely on bacterial-type two-component signal transduction systems (phosphorylation on His and Asp), as well as on the protein phosphorylation on Ser, Thr and Tyr by Hanks-type protein kinases. Instead, Crenarchaeota (e.g. acidophiles and (hyper)thermophiles) only depend on Hanks-type protein phosphorylation. In this review, the current knowledge of reversible protein phosphorylation in Archaea is presented. It combines results from identified phosphoproteins, biochemical characterization of protein kinases and protein phosphatases as well as target enzymes and first insights into archaeal signal transduction by biochemical, genetic and polyomic studies. PMID:27476079

  10. The impact of cellular debris on Pseudomonas aeruginosa adherence to silicone hydrogel contact lenses and contact lens storage cases.

    Science.gov (United States)

    Burnham, Geoffrey W; Cavanagh, H Dwight; Robertson, Danielle M

    2012-01-01

    To evaluate neutrophil-enhanced Pseudomonas aeruginosa (PA) biofilm formation on silicone hydrogel contact lenses and to determine the effect of epithelial biodebris on PA adherence in contact lens storage cases. A fully invasive PA corneal isolate stably conjugated to green fluorescent protein was used. Unworn lotrafilcon A contact lenses were incubated at various ratios of PA to polymorphonuclear neutrophil (PMN) for 24 hours at 37°C. Lens-associated PA was evaluated using laser scanning confocal microscopy and nonviable PA were visualized using propidium iodide. Viable bacteria were enumerated by colony-forming unit (CFU) analysis. For acute epithelial cell studies, PA viability was determined after coincubation with freeze-thaw epithelial cell lysates in 96-well polystyrene plates. Levels of residual cellular debris and bacterial viability were further assessed in used contact lens storage cases. Laser scanning confocal microscopy demonstrated that cotreatment with PMA-stimulated neutrophils increased PA adherence over 24 hours to lens surfaces with a striking alteration of PA architecture. Propidium iodide staining showed that the adherent bacteria consisted of a mixture of viable and nonviable PA; a PMN-associated increase in viable PA was confirmed by CFU (PA:PMN 0.1:1, P = 0.025; PA:PMN 1:1, P = 0.005). Acute epithelial cell debris studies revealed a significant increase in viable PA in 96-well plates in the presence of epithelial freeze-thaw lysates (PA:debris 1:1, P = 0.002; PA:debris 100:1, P = 0.002). Crystal violet staining of used lens storage cases revealed residual cellular debris at all time points, which was independent of microbial contamination; all lens cases used for periods of 9 months or more were uniformly associated with high levels of viable microorganisms. These results demonstrate that prolonged corneal inflammation with the presence of PMNs when confronted with simultaneous PA challenge in extended contact lens wear has the potential

  11. The physiological link between metabolic rate depression and tau phosphorylation in mammalian hibernation.

    Directory of Open Access Journals (Sweden)

    Jens T Stieler

    Full Text Available Abnormal phosphorylation and aggregation of tau protein are hallmarks of a variety of neurological disorders, including Alzheimer's disease (AD. Increased tau phosphorylation is assumed to represent an early event in pathogenesis and a pivotal aspect for aggregation and formation of neurofibrillary tangles. However, the regulation of tau phosphorylation in vivo and the causes for its increased stage of phosphorylation in AD are still not well understood, a fact that is primarily based on the lack of adequate animal models. Recently we described the reversible formation of highly phosphorylated tau protein in hibernating European ground squirrels. Hence, mammalian hibernation represents a model system very well suited to study molecular mechanisms of both tau phosphorylation and dephosphorylation under in vivo physiological conditions. Here, we analysed the extent and kinetics of hibernation-state dependent tau phosphorylation in various brain regions of three species of hibernating mammals: arctic ground squirrels, Syrian hamsters and black bears. Overall, tau protein was highly phosphorylated in torpor states and phosphorylation levels decreased after arousal in all species. Differences between brain regions, hibernation-states and phosphosites were observed with respect to degree and kinetics of tau phosphorylation. Furthermore, we tested the phosphate net turnover of tau protein to analyse potential alterations in kinase and/or phosphatase activities during hibernation. Our results demonstrate that the hibernation-state dependent phosphorylation of tau protein is specifically regulated but involves, in addition, passive, temperature driven regulatory mechanisms. By determining the activity-state profile for key enzymes of tau phosphorylation we could identify kinases potentially involved in the differentially regulated, reversible tau phosphorylation that occurs during hibernation. We show that in black bears hibernation is associated with

  12. 3-phosphorylated and -thiophosphorylated 2-thiazolidine- and 2-oxazolidine-thiones

    International Nuclear Information System (INIS)

    Vorob'eva, N.N.; Razvodovskaya, L.V.; Negrebetskii, V.V.; Grapov, A.F.; Mel'nikov, N.N.

    1987-01-01

    We investigated the phosphorylation and thiophosphorylation of 2-thiazolidine- and 2-oxazolidine-thiones. The presence in the heterocycle of the ambident triad HN-C=S can also lead to two series of phosphorylation products formed at the nitrogen and at the sulfur atom. It was therefore of interest to determine the dependence of the site of the phosphorylation on the structures of the heterocycle and off the phosphorylating agent. The formation of the N-phosphorylation products is confirmed by the 1 H NMR spectra, in which the signals of protons of the methylene group of the heteroring (C 4 H 2 ) are split on account of interaction with the phosphorus atom ( 3 JPH 0.5-2.3 Hz). We observed analogous values of 3 JPH constants for 2-aminothiazolines phosphorylated on the endocyclic nitrogen atom. In the 13 C NMR spectra of these compounds there are also coupling constants for the interaction of the carbon atoms C 4 and C 5 of the heterocycle with the phosphorus atom. The existence of the compounds as N-phosphorylated heterocycles is evidenced also by the 31 P chemical shifts

  13. 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

  14. Loss of neutrophil polarization in colon carcinoma liver metastases of mice with an inducible, liver-specific IGF-I deficiency.

    Science.gov (United States)

    Rayes, Roni F; Milette, Simon; Fernandez, Maria Celia; Ham, Boram; Wang, Ni; Bourdeau, France; Perrino, Stephanie; Yakar, Shoshana; Brodt, Pnina

    2018-03-20

    The growth of cancer metastases in the liver depends on a permissive interaction with the hepatic microenvironment and neutrophils can contribute to this interaction, either positively or negatively, depending on their phenotype. Here we investigated the role of IGF-I in the control of the tumor microenvironment in the liver, using mice with a conditional, liver-specific, IGF-I deficiency (iLID) induced by a single tamoxifen injection. In mice that had a sustained (3 weeks) IGF-I deficiency prior to the intrasplenic/portal inoculation of colon carcinoma MC-38 cells, we observed an increase in neutrophil accumulation in the liver relative to controls. However, unlike controls, these neutrophils did not acquire the (anti-inflammatory) tumor-promoting phenotype, as evidenced by retention of high ICAM-1 expression and nitric oxide production and low CXCR4, CCL5, and VEGF expression and arginase production, all characteristic of the (pro-inflammatory) phenotype. This coincided with an increase in apoptotic tumor cells and reduced metastasis. Neutrophils isolated from these mice also had reduced IGF-IR expression levels. These changes were not observed in iLID mice with a short-term (2 days) IGF-I depletion, despite a 70% reduction in their circulating IGF-I levels, indicating that a sustained IGF-I deficiency was necessary to alter the neutrophil phenotype. Similar results were obtained with the highly metastatic Lewis lung carcinoma subline H-59 cells and in mice injected with an IGF-Trap that blocks IGF-IR signaling by reducing ligand bioavailability. Our results implicate the IGF axis in neutrophil polarization and the induction of a pro-metastatic microenvironment in the liver.

  15. 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.

  16. Expression of IL-17A concentration and effector functions of peripheral blood neutrophils in food allergy hypersensitivity patients.

    Science.gov (United States)

    Żbikowska-Gotz, Magdalena; Pałgan, Krzysztof; Gawrońska-Ukleja, Ewa; Kuźmiński, Andrzej; Przybyszewski, Michał; Socha, Ewa; Bartuzi, Zbigniew

    2016-03-01

    Lymphocytes Th17 and other types of immune system cells produce IL17. By induction of cytokines and chemokines, the IL17 cytokine is involved in mechanisms of allergic reaction with participation of neutrophil granulocytes. It affects activation, recruitment, and migration of neutrophils to the tissues, regulating inflammatory reaction intensity. Excited neutrophils secrete inter alia elastase and reactive oxygen species (ROS)--significant mediators of inflammation process responsible for tissues damage.The aim of the study was to evaluate the concentrations of serum interleukin 17A, serum neutrophil elastase, and ROS production by neutrophils in patients with food allergy.The study included 30 patients with food allergy diagnosed based on interview, clinical symptoms, positive SPT, placebo controlled double-blind oral provocation trial, and the presence of asIgE in blood serum against selected food allergens using fluoro-immuno-enzymatic method FEIA UNICap 100. The control group consisted of 10 healthy volunteers. The concentrations of IL17A were determined in all patients using ELISA method with eBioscience kits, and elastase using BenderMed Systems kits. Chemiluminescence of non-stimulated neutrophils was evaluated using luminol-dependent kinetic method for 40 min on Luminoskan (Labsystems luminometer).The results of serum IL-17A concentrations and the values of chemiluminescence obtained by non-activated neutrophils, as well as elastase concentrations, were higher in patients with food allergic hypersensitivity compared to healthy volunteers.This study demonstrates a significance of IL-17A and activated neutrophil granulocytes in the course of diseases with food allergic hypersensitivity. © The Author(s) 2015.

  17. ATM-Dependent Phosphorylation of MEF2D Promotes Neuronal Survival after DNA Damage

    Science.gov (United States)

    Chan, Shing Fai; Sances, Sam; Brill, Laurence M.; Okamoto, Shu-ichi; Zaidi, Rameez; McKercher, Scott R.; Akhtar, Mohd W.; Nakanishi, Nobuki

    2014-01-01

    Mutations in the ataxia telangiectasia mutated (ATM) gene, which encodes a kinase critical for the normal DNA damage response, cause the neurodegenerative disorder ataxia-telangiectasia (AT). The substrates of ATM in the brain are poorly understood. Here we demonstrate that ATM phosphorylates and activates the transcription factor myocyte enhancer factor 2D (MEF2D), which plays a critical role in promoting survival of cerebellar granule cells. ATM associates with MEF2D after DNA damage and phosphorylates the transcription factor at four ATM consensus sites. Knockdown of endogenous MEF2D with a short-hairpin RNA (shRNA) increases sensitivity to etoposide-induced DNA damage and neuronal cell death. Interestingly, substitution of endogenous MEF2D with an shRNA-resistant phosphomimetic MEF2D mutant protects cerebellar granule cells from cell death after DNA damage, whereas an shRNA-resistant nonphosphorylatable MEF2D mutant does not. In vivo, cerebella in Mef2d knock-out mice manifest increased susceptibility to DNA damage. Together, our results show that MEF2D is a substrate for phosphorylation by ATM, thus promoting survival in response to DNA damage. Moreover, dysregulation of the ATM–MEF2D pathway may contribute to neurodegeneration in AT. PMID:24672010

  18. 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

  19. Intramuscular administration of a synthetic CpG-oligodeoxynucleotide modulates functional responses of neutrophils of neonatal foals.

    Directory of Open Access Journals (Sweden)

    Noah D Cohen

    Full Text Available Neutrophils play an important role in protecting against infection. Foals have age-dependent deficiencies in neutrophil function that may contribute to their predisposition to infection. Thus, we investigated the ability of a CpG-ODN formulated with Emulsigen to modulate functional responses of neutrophils in neonatal foals. Eighteen foals were randomly assigned to receive either a CpG-ODN with Emulsigen (N = 9 or saline intramuscularly at ages 1 and 7 days. At ages 1, 3, 9, 14, and 28, blood was collected and neutrophils were isolated from each foal. Neutrophils were assessed for basal and Rhodococcus equi-stimulated mRNA expression of the cytokines interferon-γ (IFN-γ, interleukin (IL-4, IL-6, and IL-8 using real-time PCR, degranulation by quantifying the amount of β-D glucuronidase activity, and reactive oxygen species (ROS generation using flow cytometry. In vivo administration of the CpG-ODN formulation on days 1 and 7 resulted in significantly (P<0.05 increased IFN-γ mRNA expression by foal neutrophils on days 3, 9, and 14. Degranulation was significantly (P<0.05 lower for foals in the CpG-ODN-treated group than the control group at days 3 and 14, but not at other days. No effect of treatment on ROS generation was detected. These results indicate that CpG-ODN administration to foals might improve innate and adaptive immune responses that could protect foals against infectious diseases and possibly improve responses to vaccination.

  20. The LPS-induced neutrophil recruitment into rat air pouches is mediated by TNFα: likely macrophage origin

    Directory of Open Access Journals (Sweden)

    C-D. Arreto

    1997-01-01

    Full Text Available The role of resident cells during the lipopolysaccharide (LPS-induced neutrophil recruitment into rat air pouches was investigated. In this model, LPS (Escherichia coli, O55: B5 strain; 2–2000 ng induced a dose– and time-dependent neutrophil recruitment accompanied by the generation of a tumour necrosis factor-α (TNFα-like activity. Dexamethasone (0.05–5 mug and cycloheximide (6 ng, injected 2 h before LPS into the pouches, inhibited the neutrophil recruitment and the generation of the TNFα-like activity, while the H1-receptor antagonist mepyramine (1 and 4 mg/kg, i.p., 0.5 h before LPS and the PAF-receptor antagonist WEB 2170 (0.05 and 1 mg/kg, i.p., 0.5 h before LPS had no effect. Purified alveolar macrophages (AM were used to replenish the pouches of cycloheximide-treated recipient rats. AM provided by PBS-treated animals led to the recovery of the LPS-induced neutrophil recruitment and of the TNFα-like formation contrasting with those from cycloheximide-treated animals (1 mg/kg, i.p.. When delivered in situ, liposome-encapsulated clodronate, a macrophage depletor, significantly impaired both the LPSinduced neutrophil recruitment and the TNFα-like activity. An anti-murine TNFα polyclonal antibody (0.5 h before LPS was also effective. These results emphasize the pivotal role of macrophages for LPS-induced neutrophil recruitment via the formation of TNFα.

  1. 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.

  2. 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.

  3. 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...

  4. Inflammatory Ly6Chigh Monocytes Protect against Candidiasis through IL-15-Driven NK Cell/Neutrophil Activation.

    Science.gov (United States)

    Domínguez-Andrés, Jorge; Feo-Lucas, Lidia; Minguito de la Escalera, María; González, Leticia; López-Bravo, María; Ardavín, Carlos

    2017-06-20

    Neutrophils play a crucial role in defense against systemic candidiasis, a disease associated with a high mortality rate in patients receiving immunosuppressive therapy, although the early immune mechanisms that boost the candidacidal activity of neutrophils remain to be defined in depth. Here, we used a murine model of systemic candidiasis to explore the role of inflammatory Ly6C high monocytes in NK cell-mediated neutrophil activation during the innate immune response against C. albicans. We found that efficient anti-Candida immunity required a collaborative response between the spleen and kidney, which relied on type I interferon-dependent IL-15 production by spleen inflammatory Ly6C high monocytes to drive efficient activation and GM-CSF release by spleen NK cells; this in turn was necessary to boost the Candida killing potential of kidney neutrophils. Our findings unveil a role for IL-15 as a critical mediator in defense against systemic candidiasis and hold promise for the design of IL-15-based antifungal immunotherapies. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. In cellulo phosphorylation of XRCC4 Ser320 by DNA-PK induced by DNA damage

    International Nuclear Information System (INIS)

    Sharma, Mukesh Kumar; Imamichi, Shoji; Fukuchi, Mikoto; Samarth, Ravindra Mahadeo; Tomita, Masanori; Matsumoto, Yoshihisa

    2016-01-01

    XRCC4 is a protein associated with DNA Ligase IV, which is thought to join two DNA ends at the final step of DNA double-strand break repair through non-homologous end joining. In response to treatment with ionizing radiation or DNA damaging agents, XRCC4 undergoes DNA-PK-dependent phosphorylation. Furthermore, Ser260 and Ser320 (or Ser318 in alternatively spliced form) of XRCC4 were identified as the major phosphorylation sites by purified DNA-PK in vitro through mass spectrometry. However, it has not been clear whether these sites are phosphorylated in vivo in response to DNA damage. In the present study, we generated an antibody that reacts with XRCC4 phosphorylated at Ser320 and examined in cellulo phosphorylation status of XRCC4 Ser320. The phosphorylation of XRCC4 Ser320 was induced by γ-ray irradiation and treatment with Zeocin. The phosphorylation of XRCC4 Ser320 was detected even after 1 Gy irradiation and increased in a manner dependent on radiation dose. The phosphorylation was observed immediately after irradiation and remained mostly unchanged for up to 4 h. The phosphorylation was inhibited by DNA-PK inhibitor NU7441 and was undetectable in DNA-PKcs-deficient cells, indicating that the phosphorylation was mainly mediated by DNA-PK. These results suggested potential usefulness of the phosphorylation status of XRCC4 Ser320 as an indicator of DNA-PK functionality in living cells

  6. 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.

  7. PAK6 Phosphorylates 14-3-3γ to Regulate Steady State Phosphorylation of LRRK2

    Directory of Open Access Journals (Sweden)

    Laura Civiero

    2017-12-01

    Full Text Available Mutations in Leucine-rich repeat kinase 2 (LRRK2 are associated with Parkinson's disease (PD and, as such, LRRK2 is considered a promising therapeutic target for age-related neurodegeneration. Although the cellular functions of LRRK2 in health and disease are incompletely understood, robust evidence indicates that PD-associated mutations alter LRRK2 kinase and GTPase activities with consequent deregulation of the downstream signaling pathways. We have previously demonstrated that one LRRK2 binding partner is P21 (RAC1 Activated Kinase 6 (PAK6. Here, we interrogate the PAK6 interactome and find that PAK6 binds a subset of 14-3-3 proteins in a kinase dependent manner. Furthermore, PAK6 efficiently phosphorylates 14-3-3γ at Ser59 and this phosphorylation serves as a switch to dissociate the chaperone from client proteins including LRRK2, a well-established 14-3-3 binding partner. We found that 14-3-3γ phosphorylated by PAK6 is no longer competent to bind LRRK2 at phospho-Ser935, causing LRRK2 dephosphorylation. To address whether these interactions are relevant in a neuronal context, we demonstrate that a constitutively active form of PAK6 rescues the G2019S LRRK2-associated neurite shortening through phosphorylation of 14-3-3γ. Our results identify PAK6 as the kinase for 14-3-3γ and reveal a novel regulatory mechanism of 14-3-3/LRRK2 complex in the brain.

  8. Solid-phase assay for the phosphorylation of proteins blotted on nitrocellulose membrane filters

    International Nuclear Information System (INIS)

    Valtorta, F.; Schiebler, W.; Jahn, R.; Ceccarelli, B.; Greengard, P.

    1986-01-01

    A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters, and the blotted polypeptides are phyosphorylated with the catalytic subunit of cyclic AMP (adenosine 3':5'-monophosphate)-dependent protein kinase. The method was developed for the assay of dephosphosynapsin I, but it has also proven suitable for the phosphorylation of other proteins. The patterns of phosphorylation of tissue samples phosphorylated using the new method are similar to those obtained using the conventional test tube assay. Once phosphorylated, the adsorbed proteins can be digested with proteases and subjected to phosphopeptide mapping. The phosphorylated blotted proteins can also be analyzed by overlay techniques for the immunological detection of polypeptides

  9. Histones and their phosphorylation during germination of rice seeds

    International Nuclear Information System (INIS)

    Iqbal Ahmed, C.M.; Padayatti, J.D.

    1980-01-01

    Histones from nuclei of rice embryos were identified by their mobilities on 15% acid-urea polyacrylamide gel electrophoreogram, incorporation of ( 3 H)lysine and ( 14 C) arginine and lack of incorporation of ( 3 H)tryptophan. The ratio of histone to DNA in ungerminated embryos was 2.7 which decreased during germination reaching unity by 48 hr. There was preferential phosphorylation of lysine-rich histones, which paralleled the synthesis of DNA. In the presence of cytosine arabinoside and mitomycin-C, which inhibited the synthesis of DNA to the extend of 75-80% the phosphorylation of lysine-rich histone was reduced by 50-60% suggesting the dependence of phosphorylation on the ongoing synthesis of DNA. (auth.)

  10. Akt2-Dependent Phosphorylation of Radixin in Regulation of Mrp-2 Trafficking in WIF-B Cells.

    Science.gov (United States)

    Suda, Jo; Rockey, Don C; Karvar, Serhan

    2016-02-01

    The dominant ezrin/radixin/moesin protein in hepatocytes is radixin, which plays an important role in mediating the binding of F-actin to the plasma membrane after a conformational activation by phosphorylation at Thr564. Here we have investigated the importance of Akt-mediated radixin Thr564 phosphorylation on Mrp-2 distribution and function in WIF-B cells. Mrp-2 is an adenosine triphosphate (ATP)-binding cassette transporter that plays an important role in detoxification and chemoprotection by transporting a wide range of compounds, especially conjugates of lipophilic substances with glutathione, organic anions, and drug metabolites such as glucuronides. Akt1 and Akt2 expression were manipulated using dominant active and negative constructs as well as Akt1 and Akt2 siRNA. Cellular distribution of radixin and Mrp-2 was visualized by fluorescence microscopy. A 5-chloromethylfluorescein diacetate, which is a substrate of the Mrp-2 and is actively transported in canalicular lumina, was used to measure Mrp-2 function. Radixin phosphorylation was significantly increased in wild-type and dominant active Akt2 transfected cells. Furthermore, radixin and Mrp-2 were localized at the canalicular membrane, similar to control cells. In contrast, overexpression of dominant negative Akt2, siRNA knockdown of Akt2 and a specific Akt inhibitor prevented radixin phosphorylation and led to alteration of normal radixin and Mrp-2 localization; inhibition of Akt2, but not Akt1 function led to radixin localization to the cytoplasmic space. In addition, dominant negative and Akt2 knockdown led to a dramatically impaired hepatocyte secretory response, while wild-type and dominant active Akt2 transfected cells exhibited increased 5-chloromethylfluorescein diacetate excretion. In contrast to Akt2, Akt1 was not associated with radixin phosphorylation. These studies, therefore, identify Akt2 as a critical kinase that regulates radixin phosphorylation and leads to Mrp-2 translocation and

  11. Phosphorylation of a splice variant of collapsin response mediator protein 2 in the nucleus of tumour cells links cyclin dependent kinase-5 to oncogenesis

    International Nuclear Information System (INIS)

    Grant, Nicola J.; Coates, Philip J.; Woods, Yvonne L.; Bray, Susan E.; Morrice, Nicholas A.; Hastie, C. James; Lamont, Douglas J.; Carey, Francis A.; Sutherland, Calum

    2015-01-01

    Cyclin-dependent protein kinase-5 (CDK5) is an unusual member of the CDK family as it is not cell cycle regulated. However many of its substrates have roles in cell growth and oncogenesis, raising the possibility that CDK5 modulation could have therapeutic benefit. In order to establish whether changes in CDK5 activity are associated with oncogenesis one could quantify phosphorylation of CDK5 targets in disease tissue in comparison to appropriate controls. However the identity of physiological and pathophysiological CDK5 substrates remains the subject of debate, making the choice of CDK5 activity biomarkers difficult. Here we use in vitro and in cell phosphorylation assays to identify novel features of CDK5 target sequence determinants that confer enhanced CDK5 selectivity, providing means to select substrate biomarkers of CDK5 activity with more confidence. We then characterize tools for the best CDK5 substrate we identified to monitor its phosphorylation in human tissue and use these to interrogate human tumour arrays. The close proximity of Arg/Lys amino acids and a proline two residues N-terminal to the phosphorylated residue both improve recognition of the substrate by CDK5. In contrast the presence of a proline two residues C-terminal to the target residue dramatically reduces phosphorylation rate. Serine-522 of Collapsin Response Mediator-2 (CRMP2) is a validated CDK5 substrate with many of these structural criteria. We generate and characterise phosphospecific antibodies to Ser522 and show that phosphorylation appears in human tumours (lung, breast, and lymphoma) in stark contrast to surrounding non-neoplastic tissue. In lung cancer the anti-phospho-Ser522 signal is positive in squamous cell carcinoma more frequently than adenocarcinoma. Finally we demonstrate that it is a specific and unusual splice variant of CRMP2 (CRMP2A) that is phosphorylated in tumour cells. For the first time this data associates altered CDK5 substrate phosphorylation with

  12. 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...

  13. [Immunologic indexes, enzyme status of lymphocytes and functional activity of blood neutrophils in children with infectious mononucleosis caused by Epstein-Barr virus].

    Science.gov (United States)

    Kurtasova, L M; Tolstikova, A E; Savchenko, A A

    2013-01-01

    Explore the immunological parameters, levels of activity of NAD(P)-dependent dehydrogenases lymphocytes, interferon status parameters, phagocytic activity and chemiluminescence response of neutrophils in the blood of children in the acute phase of infectious mononucleosis caused by the Epstein-Barr virus. 65 children at the age of 4-6 years old with infectious mononucleosis caused by EBV in acute phase were observed. Such indexes as cell-mediated, humoral and interferon immunity, NAD(P)-depended dehydrogenases activity in blood lymphocyte, phagocytes activity, levels of spontaneous and induced chemiluminescence ofperipheral blood neutrophils were studied. Children with EVB-infection have immunophenotype spectrum changes and changes of enzymes status of blood lymphocytes against the increasing in leucocytes and the useful increasing in lymphocytes. The useful increasing in IgA, IgM, IgG contenting in serum blood were found. The decreasing of spontaneous production of IFN alpha and the decreasing of induced production of IFNalpha, IFNgamma were determined. The breach of phagocytes activity and chemiluminescent response of blood neutrophils were found. The children in the acute phase of infectious mononucleosis caused by the Epstein-Barr virus, there are changes in the immune status, changes the activity of NAD(P)-dependent dehydrogenases in blood lymphocytes, marked changes in functional and metabolic state of peripheral blood neutrophils.

  14. Bad phosphorylation as a target of inhibition in oncology.

    Science.gov (United States)

    Bui, Ngoc-Linh-Chi; Pandey, Vijay; Zhu, Tao; Ma, Lan; Basappa; Lobie, Peter E

    2018-02-28

    Bcl-2 agonist of cell death (BAD) is a BH3-only member of the Bcl-2 family which possesses important regulatory function in apoptosis. BAD has also been shown to possess many non-apoptotic functions closely linked to cancer including regulation of glycolysis, autophagy, cell cycle progression and immune system development. Interestingly, BAD can be either pro-apoptotic or pro-survival depending on the phosphorylation state of three specific serine residues (human S75, S99 and S118). Expression of BAD and BAD phosphorylation patterns have been shown to influence tumor initiation and progression and play a predictive role in disease prognosis, drug response and chemosensitivity in various cancers. This review aims to summarize the current evidence on the functional role of BAD phosphorylation in human cancer and evaluate the potential utility of modulating BAD phosphorylation in cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. 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.

  16. An intense and short-lasting burst of neutrophil activation differentiates early acute myocardial infarction from systemic inflammatory syndromes.

    Directory of Open Access Journals (Sweden)

    Norma Maugeri

    Full Text Available BACKGROUND: Neutrophils are involved in thrombus formation. We investigated whether specific features of neutrophil activation characterize patients with acute coronary syndromes (ACS compared to stable angina and to systemic inflammatory diseases. METHODS AND FINDINGS: The myeloperoxidase (MPO content of circulating neutrophils was determined by flow cytometry in 330 subjects: 69 consecutive patients with acute coronary syndromes (ACS, 69 with chronic stable angina (CSA, 50 with inflammation due to either non-infectious (acute bone fracture, infectious (sepsis or autoimmune diseases (small and large vessel systemic vasculitis, rheumatoid arthritis. Four patients have also been studied before and after sterile acute injury of the myocardium (septal alcoholization. One hundred thirty-eight healthy donors were studied in parallel. Neutrophils with normal MPO content were 96% in controls, >92% in patients undergoing septal alcoholization, 91% in CSA patients, but only 35 and 30% in unstable angina and AMI (STEMI and NSTEMI patients, compared to 80%, 75% and 2% of patients with giant cell arteritis, acute bone fracture and severe sepsis. In addition, in 32/33 STEMI and 9/21 NSTEMI patients respectively, 20% and 12% of neutrophils had complete MPO depletion during the first 4 hours after the onset of symptoms, a feature not observed in any other group of patients. MPO depletion was associated with platelet activation, indicated by P-selectin expression, activation and transactivation of leukocyte β2-integrins and formation of platelet neutrophil and -monocyte aggregates. The injection of activated platelets in mice produced transient, P-selectin dependent, complete MPO depletion in about 50% of neutrophils. CONCLUSIONS: ACS are characterized by intense neutrophil activation, like other systemic inflammatory syndromes. In the very early phase of acute myocardial infarction only a subpopulation of neutrophils is massively activated, possibly via

  17. [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

  18. 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.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Novel Phosphorylation and Ubiquitination Sites Regulate Reactive Oxygen Species-dependent Degradation of Anti-apoptotic c-FLIP Protein*

    Science.gov (United States)

    Wilkie-Grantham, Rachel P.; Matsuzawa, Shu-Ichi; Reed, John C.

    2013-01-01

    The cytosolic protein c-FLIP (cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein) is an inhibitor of death receptor-mediated apoptosis that is up-regulated in a variety of cancers, contributing to apoptosis resistance. Several compounds found to restore sensitivity of cancer cells to TRAIL, a TNF family death ligand with promising therapeutic potential, act by targeting c-FLIP ubiquitination and degradation by the proteasome. The generation of reactive oxygen species (ROS) has been implicated in c-FLIP protein degradation. However, the mechanism by which ROS post-transcriptionally regulate c-FLIP protein levels is not well understood. We show here that treatment of prostate cancer PPC-1 cells with the superoxide generators menadione, paraquat, or buthionine sulfoximine down-regulates c-FLIP long (c-FLIPL) protein levels, which is prevented by the proteasome inhibitor MG132. Furthermore, pretreatment of PPC-1 cells with a ROS scavenger prevented ubiquitination and loss of c-FLIPL protein induced by menadione or paraquat. We identified lysine 167 as a novel ubiquitination site of c-FLIPL important for ROS-dependent degradation. We also identified threonine 166 as a novel phosphorylation site and demonstrate that Thr-166 phosphorylation is required for ROS-induced Lys-167 ubiquitination. The mutation of either Thr-166 or Lys-167 was sufficient to stabilize c-FLIP protein levels in PPC-1, HEK293T, and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants protected cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL. PMID:23519470

  2. Reversal of DDK-Mediated MCM Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability Independently of ATR/Chk1.

    Science.gov (United States)

    Alver, Robert C; Chadha, Gaganmeet Singh; Gillespie, Peter J; Blow, J Julian

    2017-03-07

    Dbf4-dependent kinases (DDKs) are required for the initiation of DNA replication, their essential targets being the MCM2-7 proteins. We show that, in Xenopus laevis egg extracts and human cells, hyper-phosphorylation of DNA-bound Mcm4, but not phosphorylation of Mcm2, correlates with DNA replication. These phosphorylations are differentially affected by the DDK inhibitors PHA-767491 and XL413. We show that DDK-dependent MCM phosphorylation is reversed by protein phosphatase 1 (PP1) targeted to chromatin by Rif1. Loss of Rif1 increased MCM phosphorylation and the rate of replication initiation and also compromised the ability of cells to block initiation when challenged with replication inhibitors. We also provide evidence that Rif1 can mediate MCM dephosphorylation at replication forks and that the stability of dephosphorylated replisomes strongly depends on Chk1 activity. We propose that both replication initiation and replisome stability depend on MCM phosphorylation, which is maintained by a balance of DDK-dependent phosphorylation and Rif1-mediated dephosphorylation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  3. NR4A orphan nuclear receptor family members, NR4A2 and NR4A3, regulate neutrophil number and survival.

    Science.gov (United States)

    Prince, Lynne R; Prosseda, Svenja D; Higgins, Kathryn; Carlring, Jennifer; Prestwich, Elizabeth C; Ogryzko, Nikolay V; Rahman, Atiqur; Basran, Alexander; Falciani, Francesco; Taylor, Philip; Renshaw, Stephen A; Whyte, Moira K B; Sabroe, Ian

    2017-08-24

    The lifespan of neutrophils is plastic and highly responsive to factors that regulate cellular survival. Defects in neutrophil number and survival are common to both hematologic disorders and chronic inflammatory diseases. At sites of inflammation, neutrophils respond to multiple signals that activate protein kinase A (PKA) signaling, which positively regulates neutrophil survival. The aim of this study was to define transcriptional responses to PKA activation and to delineate the roles of these factors in neutrophil function and survival. In human neutrophil gene array studies, we show that PKA activation upregulates a significant number of apoptosis-related genes, the most highly regulated of these being NR4A2 and NR4A3 Direct PKA activation by the site-selective PKA agonist pair N6/8-AHA (8-AHA-cAMP and N6-MB-cAMP) and treatment with endogenous activators of PKA, including adenosine and prostaglandin E2, results in a profound delay of neutrophil apoptosis and concomitant upregulation of NR4A2/3 in a PKA-dependent manner. NR4A3 expression is also increased at sites of neutrophilic inflammation in a human model of intradermal inflammation. PKA activation also promotes survival of murine neutrophil progenitor cells, and small interfering RNA to NR4A2 decreases neutrophil production in this model. Antisense knockdown of NR4A2 and NR4A3 homologs in zebrafish larvae significantly reduces the absolute neutrophil number without affecting cellular migration. In summary, we show that NR4A2 and NR4A3 are components of a downstream transcriptional response to PKA activation in the neutrophil, and that they positively regulate neutrophil survival and homeostasis. © 2017 by The American Society of Hematology.

  4. Human neutrophil clearance of bacterial pathogens triggers anti-microbial γδ T cell responses in early infection.

    Directory of Open Access Journals (Sweden)

    Martin S Davey

    2011-05-01

    Full Text Available Human blood Vγ9/Vδ2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vγ9/Vδ2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8. In turn, Vγ9/Vδ2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN-γ and tumor necrosis factor (TNF-α. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP, requires cell-cell contact of Vγ9/Vδ2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1, and results in a TNF-α dependent proliferation of Vγ9/Vδ2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting γδ T cell responses in vitro. Patients with acute peritoneal-dialysis (PD-associated bacterial peritonitis--characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity--show a selective activation of local Vγ9/Vδ2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The γδ T cell-driven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of γδ T cells and TNF-α and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive γδ T cells in

  5. Human Neutrophil Clearance of Bacterial Pathogens Triggers Anti-Microbial γδ T Cell Responses in Early Infection

    Science.gov (United States)

    Roberts, Gareth W.; Heuston, Sinéad; Brown, Amanda C.; Chess, James A.; Toleman, Mark A.; Gahan, Cormac G. M.; Hill, Colin; Parish, Tanya; Williams, John D.; Davies, Simon J.; Johnson, David W.; Topley, Nicholas; Moser, Bernhard; Eberl, Matthias

    2011-01-01

    Human blood Vγ9/Vδ2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vγ9/Vδ2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8). In turn, Vγ9/Vδ2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), requires cell-cell contact of Vγ9/Vδ2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1), and results in a TNF-α dependent proliferation of Vγ9/Vδ2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting γδ T cell responses in vitro. Patients with acute peritoneal-dialysis (PD)-associated bacterial peritonitis – characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity – show a selective activation of local Vγ9/Vδ2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The γδ T cell-driven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of γδ T cells and TNF-α and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive γδ T cells in early

  6. Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase

    Science.gov (United States)

    Bachmann, M.; Shiraishi, N.; Campbell, W. H.; Yoo, B. C.; Harmon, A. C.; Huber, S. C.; Davies, E. (Principal Investigator)

    1996-01-01

    Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signals and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major regulatory phosphorylation site as Ser-543, which is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of NR, using recombinant NR fragments containing or lacking the phosphorylation site sequence. Studies with NR partial reactions indicated that the block in electron flow caused by phosphorylation also could be localized to the hinge 1 region. A synthetic peptide (NR6) based on the phosphorylation site sequence was phosphorylated readily by NR kinase (NRk) in vitro. NR6 kinase activity tracked the ATP-dependent inactivation of NR during several chromatographic steps and completely inhibited inactivation/phosphorylation of native NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a basic residue at P-3 (i.e., three amino acids N-terminal to the phosphorylated serine) and a hydrophobic residue at P-5. Both forms are strictly calcium dependent but belong to distinct families of protein kinases because they are distinct immunochemically.

  7. Damage to Aspergillus fumigatus and Rhizopus oryzae Hyphae by Oxidative and Nonoxidative Microbicidal Products of Human Neutrophils In Vitro

    OpenAIRE

    Diamond, Richard D.; Clark, Robert A.

    1982-01-01

    Our previous studies established that human neutrophils could damage and probably kill hyphae of Aspergillus fumigatus and Rhizopus oryzae in vitro, primarily by oxygen-dependent mechanisms active at the cell surface. These studies were extended, again quantitating hyphal damage by reduction in uptake of 14C-labeled uracil or glutamine. Neither A. fumigatus nor R. oryzae hyphae were damaged by neutrophils from patients with chronic granulomatous disease, confirming the importance of oxidative...

  8. Effects of gadolinium oxide nanoparticles on the oxidative burst from human neutrophil granulocytes

    International Nuclear Information System (INIS)

    Abrikossova, Natalia; Skoglund, Caroline; Ahrén, Maria; Uvdal, Kajsa; Bengtsson, Torbjörn

    2012-01-01

    We have previously shown that gadolinium oxide (Gd 2 O 3 ) nanoparticles are promising candidates to be used as contrast agents in magnetic resonance (MR) imaging applications. In this study, these nanoparticles were investigated in a cellular system, as possible probes for visualization and targeting intended for bioimaging applications. We evaluated the impact of the presence of Gd 2 O 3 nanoparticles on the production of reactive oxygen species (ROS) from human neutrophils, by means of luminol-dependent chemiluminescence. Three sets of Gd 2 O 3 nanoparticles were studied, i.e. as synthesized, dialyzed and both PEG-functionalized and dialyzed Gd 2 O 3 nanoparticles. In addition, neutrophil morphology was evaluated by fluorescent staining of the actin cytoskeleton and fluorescence microscopy. We show that surface modification of these nanoparticles with polyethylene glycol (PEG) is essential in order to increase their biocompatibility. We observed that the as synthesized nanoparticles markedly decreased the ROS production from neutrophils challenged with prey (opsonized yeast particles) compared to controls without nanoparticles. After functionalization and dialysis, more moderate inhibitory effects were observed at a corresponding concentration of gadolinium. At lower gadolinium concentration the response was similar to that of the control cells. We suggest that the diethylene glycol (DEG) present in the as synthesized nanoparticle preparation is responsible for the inhibitory effects on the neutrophil oxidative burst. Indeed, in the present study we also show that even a low concentration of DEG, 0.3%, severely inhibits neutrophil function. In summary, the low cellular response upon PEG-functionalized Gd 2 O 3 nanoparticle exposure indicates that these nanoparticles are promising candidates for MR-imaging purposes. (paper)

  9. Effect of fluticasone propionate on neutrophil chemotaxis, superoxide generation, and extracellular proteolytic activity in vitro.

    Science.gov (United States)

    Llewellyn-Jones, C G; Hill, S L; Stockley, R A

    1994-03-01

    Corticosteroids are widely used in the treatment of many inflammatory conditions but the exact mode of action on neutrophil function is uncertain. Fluticasone propionate is a new topically active synthetic steroid which can be measured in body fluids and which undergoes first pass metabolism. The effects of fluticasone propionate on the function of neutrophils isolated from normal, healthy control subjects and on the chemotactic activity of sputum sol phase were assessed. Preincubation of neutrophils with fluticasone propionate reduced the chemotactic response to 10(-8) mol/l F-Met-Leu-Phe (FMLP) and to a 1:5 dilution of sputum sol phase in a dose dependent manner. Furthermore, when fluticasone propionate was added to sputum from eight patients with stable chronic obstructive bronchitis the chemotactic activity of a 1:5 dilution of the sol phase fell from a mean (SE) value of 22.2 (1.21) cells/field to 19.6 (0.89), 17.1 (0.74), and 11.9 (0.6) cells field at 1 mumol/l, 10 mumol/l, and 100 mumol/l, respectively. In further experiments fluticasone propionate preincubated with neutrophils inhibited fibronectin degradation by resting cells and by cells stimulated by FMLP (15.2% inhibition of resting cells, 5.1% inhibition of stimulated cells with 1 mumol/l fluticasone propionate, 24% and 18.7% inhibition respectively at 100 mumol/l fluticasone propionate. Fluticasone propionate had no effect on generation of superoxide anion by resting or stimulated cells. These results indicate that fluticasone propionate has a direct suppressive effect on several aspects of neutrophil function and may suggest a role for this agent in the modulation of neutrophil mediated damage to connective tissue.

  10. PEGylated single-walled carbon nanotubes activate neutrophils to increase production of hypochlorous acid, the oxidant capable of degrading nanotubes

    Energy Technology Data Exchange (ETDEWEB)

    Vlasova, Irina I., E-mail: irina.vlasova@yahoo.com [Research Institute for Physico-Chemical Medicine, Federal Medico-Biological Agency, Moscow (Russian Federation); Vakhrusheva, Tatyana V. [Research Institute for Physico-Chemical Medicine, Federal Medico-Biological Agency, Moscow (Russian Federation); Sokolov, Alexey V.; Kostevich, Valeria A. [Research Institute for Physico-Chemical Medicine, Federal Medico-Biological Agency, Moscow (Russian Federation); Research Institute for Experimental Medicine, Russian Academy of Medical Science, Saint Petersburg (Russian Federation); Gusev, Alexandr A.; Gusev, Sergey A. [Research Institute for Physico-Chemical Medicine, Federal Medico-Biological Agency, Moscow (Russian Federation); Melnikova, Viktoriya I. [Institute of Developmental Biology, Russian Academy of Science, Moscow (Russian Federation); Lobach, Anatolii S. [Institute of Problems of Chemical Physics, Russian Academy of Science, Chernogolovka (Russian Federation)

    2012-10-01

    Perspectives for the use of carbon nanotubes in biomedical applications depend largely on their ability to degrade in the body into products that can be easily cleared out. Carboxylated single-walled carbon nanotubes (c-SWCNTs) were shown to be degraded by oxidants generated by peroxidases in the presence of hydrogen peroxide. In the present study we demonstrated that conjugation of poly(ethylene glycol) (PEG) to c-SWCNTs does not interfere with their degradation by peroxidase/H{sub 2}O{sub 2} system or by hypochlorite. Comparison of different heme-containing proteins for their ability to degrade PEG-SWCNTs has led us to conclude that the myeloperoxidase (MPO) product hypochlorous acid (HOCl) is the major oxidant that may be responsible for biodegradation of PEG-SWCNTs in vivo. MPO is secreted mainly by neutrophils upon activation. We hypothesize that SWCNTs may enhance neutrophil activation and therefore stimulate their own biodegradation due to MPO-generated HOCl. PEG-SWCNTs at concentrations similar to those commonly used in in vivo studies were found to activate isolated human neutrophils to produce HOCl. Both PEG-SWCNTs and c-SWCNTs enhanced HOCl generation from isolated neutrophils upon serum-opsonized zymosan stimulation. Both types of nanotubes were also found to activate neutrophils in whole blood samples. Intraperitoneal injection of a low dose of PEG-SWCNTs into mice induced an increase in percentage of circulating neutrophils and activation of neutrophils and macrophages in the peritoneal cavity, suggesting the evolution of an inflammatory response. Activated neutrophils can produce high local concentrations of HOCl, thereby creating the conditions favorable for degradation of the nanotubes. -- Highlights: ► Myeloperoxidase (MPO) product hypochlorous acid is able to degrade CNTs. ► PEGylated SWCNTs stimulate isolated neutrophils to produce hypochlorous acid. ► SWCNTs are capable of activating neutrophils in blood samples. ► Activation of

  11. 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.

  12. Skeletal muscle eEF2 and 4EBP1 phosphorylation during endurance exercise is dependent on intensity and muscle fiber type

    DEFF Research Database (Denmark)

    Rose, Adam John; Bisiani, Bruno; Vistisen, Bodil

    2009-01-01

    that the increase in skeletal muscle eEF2 Thr(56) phosphorylation was restricted to type I myofibers. Taken together, these data suggest that the depression of skeletal muscle protein synthesis with endurance-type exercise may be regulated at both initiation (i.e. 4EBP1) and elongation (i.e. eEF2) steps, with eEF2......Protein synthesis in skeletal muscle is known to decrease during exercise and it has been suggested that this may depend on the magnitude of the relative metabolic stress within the contracting muscle. To examine the mechanisms behind this, the effect of exercise intensity on skeletal muscle......) increased during exercise but was not influenced by exercise intensity, and was lower than rest 30min after exercise. On the other hand, 4EBP1 phosphorylation at Thr(37/46) decreased during exercise and this decrease was greater at higher exercise intensities, and was similar to rest 30min after exercise...

  13. Cyclin A regulates a cell-cycle-dependent expression of CKAP2 through phosphorylation of Sp1

    International Nuclear Information System (INIS)

    Kang, Du-Seock; Hong, Kyeong-Man; Park, Joobae; Bae, Chang-Dae

    2012-01-01

    Highlights: ► We identified a GC box and a CHR element in human CKAP2 minimal promoter. ► The CHR element repressed the CKAP2 minimal promoter activity at the G1/S phase. ► The GC box was essential for the basic promoter activity of human CKAP2. ► The GC box was also essential for the cyclic expression of human CKAP2. ► The phosphorylation of Sp1, mediated by Cyclin A, underlies the cyclic expression. -- Abstract: CKAP2 plays crucial roles in proper chromosome segregation and maintaining genomic stability. CKAP2 protein showed cell-cycle-dependent expression, which reached a maximum level at the G2/M phase and disappeared at the onset of G1 phase. To elucidate the mechanisms underlying cell cycle-dependent expression of CKAP2, we cloned and analyzed the human CKAP2 promoter. The upstream 115-bp region from the transcription start site was sufficient for minimal CKAP2 promoter activity. We identified 2 regulatory sequences; a CHR (−110 to −104 bp) and a GC box (−41 to −32 bp). We confirmed Sp1 bound to the GC box using a supershift assay and a ChIP assay. Mutation in the GC box resulted in a near complete loss of CKAP2 promoter activity while mutation in the CHR decreased the promoter activity by 50%. The CHR mutation showed enhanced activity at the G1/S phase, but still retained cyclic activity. The Chromatin IP revealed that the amount of Sp1 bound to the GC box gradually increased and reached a maximum level at the G2/M phase. The amount of Sp1 bound to the GC box was greatly reduced when Cyclin A was depleted, which was restored by adding Cyclin A/Cdk2 complex back into the nuclear extracts. Together, we concluded that the GC box was responsible for the cyclic activity of human CKAP2 promoter through the phosphorylation of Sp1, possibly by Cyclin A/Cdk complex.

  14. Changes in Neutrophil Functions in Astronauts

    Science.gov (United States)

    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.

  15. Inhibition of peptide aggregation by means of enzymatic phosphorylation

    Directory of Open Access Journals (Sweden)

    Kristin Folmert

    2016-11-01

    Full Text Available As is the case in numerous natural processes, enzymatic phosphorylation can be used in the laboratory to influence the conformational populations of proteins. In nature, this information is used for signal transduction or energy transfer, but has also been shown to play an important role in many diseases like tauopathies or diabetes. With the goal of determining the effect of phosphorylation on amyloid fibril formation, we designed a model peptide which combines structural characteristics of α-helical coiled-coils and β-sheets in one sequence. This peptide undergoes a conformational transition from soluble structures into insoluble amyloid fibrils over time and under physiological conditions and contains a recognition motif for PKA (cAMP-dependent protein kinase that enables enzymatic phosphorylation. We have analyzed the pathway of amyloid formation and the influence of enzymatic phosphorylation on the different states along the conformational transition from random-coil to β-sheet-rich oligomers to protofilaments and on to insoluble amyloid fibrils, and we found a remarkable directing effect from β-sheet-rich structures to unfolded structures in the initial growth phase, in which small oligomers and protofilaments prevail if the peptide is phosphorylated.

  16. Keratin 8 phosphorylation in vitro by cAMP-dependent protein kinase occurs within the amino- and carboxyl-terminal end domains.

    Science.gov (United States)

    Ando, S; Tokui, T; Yano, T; Inagaki, M

    1996-04-05

    We reported earlier that phosphorylation in vitro of keratin filaments reconstituted from rat type I keratin 18 and type II keratin 8 by cAPM-dependent protein kinase induces disassembly of the keratin filament structure. Keratin 8 rather than keratin 18 was the major target of the kinase. We have now identified the sites on rat keratin 8 for cAMP-dependent protein kinase. Sequential analysis of the purified phosphoropeptides, together with the known primary sequence, revealed that four major sites, Ser-12, Ser-23, Ser-36, and Ser-50, and three minor sites, Ser-8, Ser-33, Ser-42, are located in the amino-terminal head domain, while three minor sites, Ser-416, Ser-423 and Ser-425 locate in the carboxyl-terminal tail domain.

  17. Microbicidal activity of neutrophils is inhibited by isolates from recurrent vaginal candidiasis (RVVC) caused by Candida albicans through fungal thioredoxin reductase.

    Science.gov (United States)

    Ratti, Bianca Altrão; Godoy, Janine Silva Ribeiro; de Souza Bonfim Mendonça, Patrícia; Bidóia, Danielle Lazarin; Nakamura, Tânia Ueda; Nakamura, Celso Vataru; Lopes Consolaro, Marcia Edilaine; Estivalet Svidzinski, Terezinha Inez; de Oliveira Silva, Sueli

    2015-01-01

    Vulvovaginal candidiasis (VVC) is characterized by an infection of the vulva and vagina, mainly caused by Candida albicans, a commensal microorganism that inhabits the vaginal, digestive, and respiratory mucosae. Vulvovaginal candidiasis affects approximately 75% of women, and 5% develop the recurrent form (RVVC). The aim of the present study was to evaluate whether neutrophils microbicidal response is triggered when activated with RVVC isolates caused by C. albicans. Our results showed that RVVC isolates induced neutrophil migration but significantly decrease the microbicidal activity of neutrophils, compared with VVC and ASS isolates. The microbicidal activity of neutrophils is highly dependent on the production of reactive oxygen species/reactive nitrogen species (ROS/RNS). However, this isolate induced detoxification of ROS/RNS produced by neutrophils, reflected by the high level of thiol groups and by the oxygen consumption. Therefore, RVVC isolates induced biochemical changes in the inflammatory response triggered by neutrophils, and these effects were mainly related to the detoxification of ROS/RNS through the thioredoxin reductase (TR), a key antioxidant enzyme in fungi. This might be one of the resistance mechanisms triggered by RVVC caused by C. albicans. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Extracellular lipase of Pseudomonas aeruginosa: biochemical characterization and effect on human neutrophil and monocyte function in vitro

    DEFF Research Database (Denmark)

    Jaeger, K E; Kharazmi, A; Høiby, N

    1991-01-01

    concentrations of this lipase preparation were preincubated with human peripheral blood neutrophils and monocytes. The chemotaxis and chemiluminescence of these cells were then determined. It was shown that lipase inhibited the monocyte chemotaxis and chemiluminescence, whereas it had no or very little effect...... on neutrophils. The inhibitory effect was concentration dependent and was abolished by heat treatment of the enzyme at 100 degrees C. Since monocytes are one of the important cells of the host defence system the inhibition of the function of these cells may contribute to the pathogenesis of infections caused...

  19. Butyrate-Loaded Chitosan/Hyaluronan Nanoparticles: A Suitable Tool for Sustained Inhibition of ROS Release by Activated Neutrophils

    DEFF Research Database (Denmark)

    Sacco, Pasquale; Decleva, Eva; Tentor, Fabio

    2017-01-01

    that butyrate inhibits neutrophil ROS release in a dose and time-dependent fashion. Given the short half-life of butyrate, chitosan/hyaluronan nanoparticles are next designed and developed as controlled release carriers able to provide cells with a long-lasting supply of this SCFA. Notably, while the inhibition...... of neutrophil ROS production by free butyrate declines over time, that of butyrate-loaded chitosan/hyaluronan nanoparticles (B-NPs) is sustained. Additional valuable features of these nanoparticles are inherent ROS scavenger activity, resistance to cell internalization, and mucoadhesiveness. B-NPs appear...

  20. Inflammation triggers emergency granulopoiesis through a density-dependent feedback mechanism.

    Directory of Open Access Journals (Sweden)

    Derek W Cain

    Full Text Available Normally, neutrophil pools are maintained by homeostatic mechanisms that require the transcription factor C/EBPα. Inflammation, however, induces neutrophilia through a distinct pathway of "emergency" granulopoiesis that is dependent on C/EBPβ. Here, we show in mice that alum triggers emergency granulopoiesis through the IL-1RI-dependent induction of G-CSF. G-CSF/G-CSF-R neutralization impairs proliferative responses of hematopoietic stem and progenitor cells (HSPC to alum, but also abrogates the acute mobilization of BM neutrophils, raising the possibility that HSPC responses to inflammation are an indirect result of the exhaustion of BM neutrophil stores. The induction of neutropenia, via depletion with Gr-1 mAb or myeloid-specific ablation of Mcl-1, elicits G-CSF via an IL-1RI-independent pathway, stimulating granulopoietic responses indistinguishable from those induced by adjuvant. Notably, C/EBPβ, thought to be necessary for enhanced generative capacity of BM, is dispensable for increased proliferation of HSPC to alum or neutropenia, but plays a role in terminal neutrophil differentiation during granulopoietic recovery. We conclude that alum elicits a transient increase in G-CSF production via IL-1RI for the mobilization of BM neutrophils, but density-dependent feedback sustains G-CSF for accelerated granulopoiesis.

  1. Protein phosphorylation in pancreatic islets induced by 3-phosphoglycerate and 2-phosphoglycerate

    International Nuclear Information System (INIS)

    Pek, S.B.; Usami, Masaru; Bilir, N.; Fischer-Bovenkerk, C.; Ueda, Tetsufumi

    1990-01-01

    The authors have shown previously that 3-phosphoglycerate, which is a glycolytic metabolite of glucose, induces protein phosphorylation in bovine and rat brain and in rat heart, kidney, liver, lung, and whole pancreas. Since glycolytic metabolism of glucose is of paramount importance in insulin release, they considered the possibility that 3-phosphoglycerate may act as a coupling factor, and they searched for evidence for the existence of 3-phosphoglycerate-dependent protein phosphorylation systems in freshly isolated normal rat pancreatic islets. Membrane and cytosol fractions were incubated with [γ- 32 P]ATP and appropriate test substances and were subjected to NaDodSO 4 /PAGE and autoradiography. As little as 0.005 mM 3-phosphoglycerate or 2-phosphoglycerate stimulated the phosphorylation of 65-kDa cytosol protein by as early as 0.25 min. The phosphate bond of the 65-kDa phosphoprotein was sufficiently stable to withstand dialysis; the radioactivity could not be chased out by subsequent exposure to ATP, ADP, 3-phosphoglycerate, or 2,3-bisphosphoglycerate. Moreover, cAMP, cGMP, phorbol 12-myristate 13-acetate, or calcium failed to stimulate the phosphorylation of the 65-kDa protein. Phosphoglycerate-dependent protein phosphorylation in islets may have relevance to stimulation of insulin secretion

  2. Survival and differentiation defects contribute to neutropenia in glucose-6-phosphatase-β (G6PC3) deficiency in a model of mouse neutrophil granulocyte differentiation.

    Science.gov (United States)

    Gautam, S; Kirschnek, S; Gentle, I E; Kopiniok, C; Henneke, P; Häcker, H; Malleret, L; Belaaouaj, A; Häcker, G

    2013-08-01

    Differentiation of neutrophil granulocytes (neutrophils) occurs through several steps in the bone marrow and requires a coordinate regulation of factors determining survival and lineage-specific development. A number of genes are known whose deficiency disrupts neutrophil generation in humans and in mice. One of the proteins encoded by these genes, glucose-6-phosphatase-β (G6PC3), is involved in glucose metabolism. G6PC3 deficiency causes neutropenia in humans and in mice, linked to enhanced apoptosis and ER stress. We used a model of conditional Hoxb8 expression to test molecular and functional differentiation as well as survival defects in neutrophils from G6PC3(-/-) mice. Progenitor lines were established and differentiated into neutrophils when Hoxb8 was turned off. G6PC3(-/-) progenitor cells underwent substantial apoptosis when differentiation was started. Transgenic expression of Bcl-XL rescued survival; however, Bcl-XL-protected differentiated cells showed reduced proliferation, immaturity and functional deficiency such as altered MAP kinase signaling and reduced cytokine secretion. Impaired glucose utilization was found and was associated with ER stress and apoptosis, associated with the upregulation of Bim and Bax; downregulation of Bim protected against apoptosis during differentiation. ER-stress further caused a profound loss of expression and secretion of the main neutrophil product neutrophil elastase during differentiation. Transplantation of wild-type Hoxb8-progenitor cells into irradiated mice allowed differentiation into neutrophils in the bone marrow in vivo. Transplantation of G6PC3(-/-) cells yielded few mature neutrophils in bone marrow and peripheral blood. Transgenic Bcl-XL permitted differentiation of G6PC3(-/-) cells in vivo. However, functional deficiencies and differentiation abnormalities remained. Differentiation of macrophages from Hoxb8-dependent progenitors was only slightly disturbed. A combination of defects in differentiation

  3. A CK2 site is reversibly phosphorylated in the photosystem II subunit CP29

    NARCIS (Netherlands)

    Testi, Maria Grazia; Croce, Roberta; Polverino-De Laureto, Patrizia; Bassi, Roberto

    1996-01-01

    Protein phosphorylation is a major mechanism in the regulation of protein function. In chloroplast thylakoids several photosystem II subunits, including the major antenna light-harvesting complex II and several core complex components, are reversibly phosphorylated depending on the redox state of

  4. PKC-Dependent GlyT1 Ubiquitination Occurs Independent of Phosphorylation: Inespecificity in Lysine Selection for Ubiquitination.

    Directory of Open Access Journals (Sweden)

    Susana P Barrera

    Full Text Available Neurotransmitter transporter ubiquitination is emerging as the main mechanism for endocytosis and sorting of cargo into lysosomes. In this study, we demonstrate PKC-dependent ubiquitination of three different isoforms of the glycine transporter 1 (GlyT1. Incubation of cells expressing transporter with the PKC activator phorbol ester induced a dramatic, time-dependent increase in GlyT1 ubiquitination, followed by accumulation of GlyT1 in EEA1 positive early endosomes. This occurred via a mechanism that was abolished by inhibition of PKC. GlyT1 endocytosis was confirmed in both retinal sections and primary cultures of mouse amacrine neurons. Replacement of only all lysines in the N-and C-termini to arginines prevented ubiquitination and endocytosis, displaying redundancy in the mechanism of ubiquitination. Interestingly, a 40-50% reduction in glycine uptake was detected in phorbol-ester stimulated cells expressing the WT-GlyT1, whereas no significant change was for the mutant protein, demonstrating that endocytosis participates in the reduction of uptake. Consistent with previous findings for the dopamine transporter DAT, ubiquitination of GlyT1 tails functions as sorting signal to deliver transporter into the lysosome and removal of ubiquitination sites dramatically attenuated the rate of GlyT1 degradation. Finally, we showed for the first time that PKC-dependent GlyT1 phosphorylation was not affected by removal of ubiquitination sites, suggesting separate PKC-dependent signaling events for these posttranslational modifications.

  5. Dynamic phosphorylation of Ebola virus VP30 in NP-induced inclusion bodies.

    Science.gov (United States)

    Lier, Clemens; Becker, Stephan; Biedenkopf, Nadine

    2017-12-01

    Zaire Ebolavirus (EBOV) causes a severe feverish disease with high case fatality rates. Transcription of EBOV is dependent on the activity of the nucleocapsid protein VP30 which represents an essential viral transcription factor. Activity of VP30 is regulated via phosphorylation at six N-terminal serine residues. Recent data demonstrated that dynamic phosphorylation and dephosphorylation of serine residue 29 is essential for transcriptional support activity of VP30. To analyze the spatio/temporal dynamics of VP30 phosphorylation, we generated a peptide antibody recognizing specifically VP30 phosphorylated at serine 29. Using this antibody we could demonstrate that (i) the majority of VP30 molecules in EBOV-infected cells is dephosphorylated at the crucial position serine 29, (ii) both, VP30 phosphorylation and dephosphorylation take place in viral inclusion bodies that are induced by the nucleoprotein NP and (iii) NP influences the phosphorylation state of VP30. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Class IA phosphoinositide 3-kinase β and δ regulate neutrophil oxidase activation in response to Aspergillus fumigatus hyphae.

    Science.gov (United States)

    Boyle, Keith B; Gyori, David; Sindrilaru, Anca; Scharffetter-Kochanek, Karin; Taylor, Philip R; Mócsai, Attila; Stephens, Len R; Hawkins, Phillip T

    2011-03-01

    An effective immune response to the ubiquitous fungus Aspergillus fumigatus is dependent upon production of reactive oxygen species (ROS) by the NADPH oxidase. This is evidenced by the acute sensitivity of oxidase-deficient humans and mice to invasive aspergillosis. Neutrophils are recruited to the lungs shortly postinfection and respond by phagocytosing conidia and mediating extracellular killing of germinated hyphae in a ROS-dependent manner. However, the signaling mechanisms regulating the generation of ROS in response to hyphae are poorly understood. PI3Ks are important regulators of numerous cellular processes, with much recent work describing unique roles for the different class I PI3K isoforms. We showed by live-cell imaging that the lipid products of class I PI3Ks accumulated at the hyphal-bound neutrophil plasma membrane. Further, we used pharmacological and genetic approaches to demonstrate essential, but overlapping, roles for PI3Kβ and PI3Kδ in the ROS and spreading responses of murine neutrophils to Aspergillus hyphae. Hyphal-induced ROS responses were substantially inhibited by deletion of the common β2-integrin subunit CD18, with only a minor, redundant role for Dectin-1. However, addition of soluble algal glucans plus the genetic deletion of CD18 were required to significantly inhibit activation of the PI3K-effector protein kinase B. Hyphal ROS responses were also totally dependent on the presence of Syk, but not its ITAM-containing adaptor proteins FcRγ or DAP12, and the Vav family of Rac-guanine nucleotide exchange factors. These results start to define the signaling network controlling neutrophil ROS responses to A. fumigatus hyphae.

  7. The effect of N-nitrosodimethylamine (NDMA) on Bax and Mcl-1 expression in human neutrophils.

    Science.gov (United States)

    Jablonski, Jakub; Jablonska, Ewa; Leonik, Agnieszka

    2011-12-01

    In the present study we examined a role of pro-apoptotic Bax and anti-apoptotic Mcl-1 proteins, participating in the regulation of intrinsic apoptosis pathway in human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA), the environmental xenobiotic. For the purpose comparison, the same studies were conducted in autologous peripheral blood mononuclear cells (PBMCs). The production of cytochrome c by PMNs was also determined. A deficit of anti-apoptotic Mcl-1 and overexpression of the pro-apoptotic protein Bax suggest that the apoptosis process in human neutrophils exposed to NDMA is dependent on changes in the expression of these proteins. PMNs were more sensitive to NDMA than PBMCs.

  8. Neutrophils in oral paracoccidioidomycosis and the involvement of Nrf2.

    Directory of Open Access Journals (Sweden)

    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.

  9. 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

  10. Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C.

    Science.gov (United States)

    Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar

    2003-08-01

    DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.

  11. 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 ...

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

    Lifescience Database Archive (English)

    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 ...

  13. 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 ...

  14. Phosphorylated nano-diamond/ Polyimide Nanocomposites

    International Nuclear Information System (INIS)

    Beyler-Çiǧil, Asli; Çakmakçi, Emrah; Kahraman, Memet Vezir

    2014-01-01

    In this study, a novel route to synthesize polyimide (PI)/phosphorylated nanodiamond films with improved thermal and mechanical properties was developed. Surface phosphorylation of nano-diamond was performed in dichloromethane. Phosphorylation dramatically enhanced the thermal stability of nano-diamond. Poly(amic acid) (PAA), which is the precursor of PI, was successfully synthesized with 3,3',4,4'-Benzophenonetetracarboxylic dianhydride (BTDA) and 4,4'-oxydianiline (4,4'-ODA) in the solution of N,N- dimethylformamide (DMF). Pure BTDA-ODA polyimide films and phosphorylated nanodiamond containing BTDA-ODA PI films were prepared. The PAA displayed good compatibility with phosphorylated nano-diamond. The morphology of the polyimide (PI)/phosphorylated nano-diamond was characterized by scanning electron microscopy (SEM). Chemical structure of polyimide and polyimide (PI)/phosphorylated nano-diamond was characterized by FTIR. SEM and FTIR results showed that the phosphorylated nano-diamond was successfully prepared. Thermal properties of the polyimide (PI)/phosphorylated nanodiamond was characterized by thermogravimetric analysis (TGA). TGA results showed that the thermal stability of (PI)/phosphorylated nano-diamond film was increased

  15. Phosphorylation of ribosomal proteins induced by auxins in maize embryonic tissues

    International Nuclear Information System (INIS)

    Perez, L.; Aguilar, R.; Mendez, A.P.; de Jimenez, E.S.

    1990-01-01

    The effect of auxin on ribosomal protein phosphorylation of germinating maize (Zea mays) tissues was investigated. Two-dimensional gel electrophoresis and autoradiography of [ 32 P] ribosomal protein patterns for natural and synthetic auxin-treated tissues were performed. Both the rate of 32 P incorporation and the electrophoretic patterns were dependent on 32 P pulse length, suggesting that active protein phosphorylation-dephosphorylation occurred in small and large subunit proteins, in control as well as in auxin-treated tissues. The effect of ribosomal protein phosphorylation on in vitro translation was tested. Measurements of poly(U) translation rates as a function of ribosome concentration provided apparent K m values significantly different for auxin-treated and nontreated tissues. These findings suggest that auxin might exert some kind of translational control by regulating the phosphorylated status of ribosomal proteins

  16. Spontaneous neutrophil activation in HTLV-1 infected patients

    Directory of Open Access Journals (Sweden)

    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.

  17. Prometaphase arrest-dependent phosphorylation of Bcl-2 family proteins and activation of mitochondrial apoptotic pathway are associated with 17α-estradiol-induced apoptosis in human Jurkat T cells.

    Science.gov (United States)

    Han, Cho Rong; Jun, Do Youn; Kim, Yoon Hee; Lee, Ji Young; Kim, Young Ho

    2013-10-01

    In Jurkat T cell clone (JT/Neo), G2/M arrest, apoptotic sub-G1 peak, mitochondrial membrane potential (Δψm) loss, and TUNEL-positive DNA fragmentation were induced following exposure to 17α-estradiol (17α-E2), whereas none of these events (except for G2/M arrest) were induced in Jurkat cells overexpressing Bcl-2 (JT/Bcl-2). Under these conditions, phosphorylation at Thr161 and dephosphorylation at Tyr15 of Cdk1, upregulation of cyclin B1 level, histone H1 phosphorylation, Cdc25C phosphorylation at Thr-48, Bcl-2 phosphorylation at Thr-56 and Ser-70, Mcl-1 phosphorylation, and Bim phosphorylation were detected in the presence of Bcl-2 overexpression. However, the 17α-E2-induced upregulation of Bak levels, activation of Bak, activation of caspase-3, and PARP degradation were abrogated by Bcl-2 overexpression. In the presence of the G1/S blocking agent hydroxyurea, 17α-E2 failed to induce G2/M arrest and all apoptotic events including Cdk1 activation and phosphorylation of Bcl-2, Mcl-1 and Bim. The 17α-E2-induced phosphorylation of Bcl-2 family proteins and mitochondrial apoptotic events were suppressed by a Cdk1 inhibitor but not by aurora A and aurora B kinase inhibitors. Immunofluorescence microscopic analysis showed that an aberrant bipolar microtubule array, incomplete chromosome congression at the metaphase plate, and prometaphase arrest, which was reversible, were the underlying factors for 17α-E2-induced mitotic arrest. The in vitro microtubule polymerization assay showed that 17α-E2 could directly inhibit microtubule formation. These results show that the apoptogenic activity of 17α-E2 was due to the impaired mitotic spindle assembly causing prometaphase arrest and prolonged Cdk1 activation, the phosphorylation of Bcl-2, Mcl-1 and Bim, and the activation of Bak and mitochondria-dependent caspase cascade. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. 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

  19. Formation of neutrophil extracellular traps under low oxygen level

    Directory of Open Access Journals (Sweden)

    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

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

    Lifescience Database Archive (English)

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

    Lifescience Database Archive (English)

    Full Text Available His.Bld.20.AllAg.Neutrophils mm9 Histone Blood Neutrophils SRX760584,SRX760581,SRX7...60583,SRX760580,SRX760582 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Bld.20.AllAg.Neutrophils.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available His.Bld.10.AllAg.Neutrophils mm9 Histone Blood Neutrophils SRX760584,SRX760583,SRX7...60581,SRX760582,SRX760580 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Bld.10.AllAg.Neutrophils.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available His.Bld.05.AllAg.Neutrophils mm9 Histone Blood Neutrophils SRX760584,SRX760581,SRX7...60583,SRX760582,SRX760580 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Bld.05.AllAg.Neutrophils.bed ...

  4. Novel insight into neutrophil immune responses by dry mass determination of Candida albicans morphotypes.

    Directory of Open Access Journals (Sweden)

    Ava Hosseinzadeh

    Full Text Available The common fungal pathogen Candida albicans has the ability to grow as a yeast or as a hypha and can alternate between these morphotypes. The overall biomass of both morphotypes increases with growth. However, only yeasts, but not hyphae, exist as discrete cellular entities. Multiplicity of infection (MOI is a useful parameter to determine the initial inoculum of yeasts for in vitro infection assays. Since the amount of hyphae is difficult to quantify, comparable starting conditions in such assays cannot be determined accurately for yeasts and hyphae using MOI. To circumvent this problem, we have established a set of correlation coefficients to convert fungal metabolic activity and optical density to dry mass. Using these correlations, we were able to accurately compare ROS production and IL-8 release by polymorphonuclear neutrophils upon infection with equal dry mass amounts of yeast and hyphal morphotypes. Neutrophil responses depended on the initial form of infection, irrespective of C. albicans wild-type yeasts transforming to hyphal growth during the assay. Infection with a high mass of live C. albicans yeasts resulted in lower neutrophil ROS and this decrease stems from efficient ROS detoxification by C. albicans without directly affecting the phagocyte ROS machinery. Moreover, we show that dead C. albicans induces significantly less ROS and IL-8 release than live fungi, but thimerosal-killed C. albicans were still able to detoxify neutrophil ROS. Thus, the dry mass approach presented in this study reveals neutrophil responses to different amounts and morphotypes of C. albicans and serves as a template for studies that aim to identify morphotype-specific responses in a variety of immune cells.

  5. 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....

  6. Clinical Efficacy of Andrographolide Sulfonate in the Treatment of Severe Hand, Foot, and Mouth Disease (HFMD) is Dependent upon Inhibition of Neutrophil Activation.

    Science.gov (United States)

    Wen, Tao; Xu, Wenjun; Liang, Lianchun; Li, Junhong; Ding, Xiaorong; Chen, Xiao; Hu, Jianhua; Lv, Aiping; Li, Xiuhui

    2015-08-01

    Andrographolide sulfonate treatment has been shown to improve clinical severe hand, foot, and mouth disease (HFMD) efficacies when combined with conventional therapy. However, the mechanisms for its therapeutic effects remain elusive. In this study, we aimed to investigate whether andrographolide sulfonate exerts its efficacy by acting on neutrophil activation. We obtained serial plasma samples at two time points (before and after 5 days of therapy) from 28 HFMD patients who received conventional therapy and 18 patients who received combination therapy (andrographolide sulfonate plus conventional therapy). Then, we measured plasma myeloperoxidase (MPO), S100A8/A9, histone, and inflammatory cytokine levels. Furthermore, we examined if andrographolide sulfonate had direct effects on neutrophil activation in vitro. We observed that MPO and S100A8/A9 levels were markedly elevated in the HFMD patients before clinical treatment. At 5 days post-medication, the MPO, S100A8/A9, histone, and interleukin-6 levels were markedly lower in the combination therapy group compared with the conventional therapy group. In vitro studies showed that andrographolide sulfonate inhibited lipopolysaccharide-stimulated neutrophil activation, demonstrated by the decreased production of reactive oxygen species and cytokines. These data indicate that neutrophil activation modulation by andrographolide sulfonate may be a critical determinant for its clinical HFMD treatment efficacy. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  7. 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 neutrophilsneutrophil 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

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

    Lifescience Database Archive (English)

    Full Text Available ALL.Bld.20.AllAg.Neutrophils mm9 All antigens Blood Neutrophils SRX760584,SRX760581...,SRX760583,SRX760580,SRX801903,SRX760582 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Bld.20.AllAg.Neutrophils.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available His.Bld.05.AllAg.Neutrophils hg19 Histone Blood Neutrophils SRX956551,SRX956548,SRX...956545,SRX956544,SRX956550,SRX956547 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Bld.05.AllAg.Neutrophils.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available ALL.Bld.50.AllAg.Neutrophils mm9 All antigens Blood Neutrophils SRX760581,SRX760584...,SRX760583,SRX760580,SRX801903,SRX760582 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Bld.50.AllAg.Neutrophils.bed ...

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

    Lifescience Database Archive (English)

    Full Text Available His.Bld.10.AllAg.Neutrophils hg19 Histone Blood Neutrophils SRX956551,SRX956548,SRX...956545,SRX956544,SRX956550,SRX956547 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/His.Bld.10.AllAg.Neutrophils.bed ...

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

    Lifescience Database Archive (English)

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  13. 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

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

    Lifescience Database Archive (English)

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  15. Saponarin activates AMPK in a calcium-dependent manner and suppresses gluconeogenesis and increases glucose uptake via phosphorylation of CRTC2 and HDAC5.

    Science.gov (United States)

    Seo, Woo-Duck; Lee, Ji Hae; Jia, Yaoyao; Wu, Chunyan; Lee, Sung-Joon

    2015-11-15

    This study investigated the molecular mechanism of saponarin, a flavone glucoside, in the regulation of insulin sensitivity. Saponarin suppressed the rate of gluconeogenesis and increased cellular glucose uptake in HepG2 and TE671 cells by regulating AMPK. Using an in vitro kinase assay, we showed that saponarin did not directly interact with the AMPK protein. Instead, saponarin increased intracellular calcium levels and induced AMPK phosphorylation, which was diminished by co-stimulation with STO-609, an inhibitor of CAMKKβ. Transcription of hepatic gluconeogenesis genes was upregulated by nuclear translocation of CRTC2 and HDAC5, coactivators of CREB and FoxO1 transcription factors, respectively. This nuclear translocation was inhibited by increased phosphorylation of CRTC2 and HDAC5 by saponarin-induced AMPK in HepG2 cells and suppression of CREB and FoxO1 transactivation activities in cells stimulated by saponarin. The results from a chromatin immunoprecipitation assay confirmed the reduced binding of CRTC2 on the PEPCK and G6Pase promoters. In TE671 cells, AMPK phosphorylated HDAC5, which suppressed nuclear penetration and upregulated GLUT4 transcription, leading to enhanced glucose uptake. Collectively, these results suggest that saponarin activates AMPK in a calcium-dependent manner, thus regulating gluconeogenesis and glucose uptake. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. 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

  17. Emerging roles for protein histidine phosphorylation in cellular signal transduction: lessons from the islet ?-cell

    OpenAIRE

    Kowluru, Anjaneyulu

    2008-01-01

    Protein phosphorylation represents one of the key regulatory events in physiological insulin secretion from the islet ?-cell. In this context, several classes of protein kinases (e.g. calcium-, cyclic nucleotide- and phospholipid-dependent protein kinases and tyrosine kinases) have been characterized in the ?-cell. The majority of phosphorylated amino acids identified include phosphoserine, phosphothreonine and phosphotyrosine. Protein histidine phosphorylation has been implicated in the prok...

  18. Tyrosine phosphorylation of Jak2 in the JH2 domain inhibits cytokine signaling.

    Science.gov (United States)

    Feener, Edward P; Rosario, Felicia; Dunn, Sarah L; Stancheva, Zlatina; Myers, Martin G

    2004-06-01

    Jak family tyrosine kinases mediate signaling by cytokine receptors to regulate diverse biological processes. Although Jak2 and other Jak kinase family members are phosphorylated on numerous sites during cytokine signaling, the identity and function of most of these sites remains unknown. Using tandem mass spectroscopic analysis of activated Jak2 protein from intact cells, we identified Tyr(221) and Tyr(570) as novel sites of Jak2 phosphorylation. Phosphorylation of both sites was stimulated by cytokine treatment of cultured cells, and this stimulation required Jak2 kinase activity. While we observed no gross alteration of signaling upon mutation of Tyr(221), Tyr(570) lies within the inhibitory JH2 domain of Jak2, and mutation of this site (Jak2(Y570F)) results in constitutive Jak2-dependent signaling in the absence of cytokine stimulation and enhances and prolongs Jak2 activation during cytokine stimulation. Mutation of Tyr(570) does not alter the ability of SOCS3 to bind or inhibit Jak2, however. Thus, the phosphorylation of Tyr(570) in vivo inhibits Jak2-dependent signaling independently of SOCS3-mediated inhibition. This Tyr(570)-dependent mechanism of Jak2 inhibition likely represents an important mechanism by which cytokine function is regulated.

  19. Phosphoenolpyruvate-dependent fructose phosphotransferase system in Rhodopseudomonas sphaeroides : The coupling between transport and phosphorylation in inside-out vesicles

    NARCIS (Netherlands)

    Lolkema, Juke S.; Robillard, George T.

    The bacterial phosphotransferase systems are believed to catalyze the concomitant transport and phosphorylation of hexoses and hexitols. The transport is from the outside to the inside of the cell. An absolute coupling between transport and phosphorylation has however been questioned in the

  20. Stimulation of JNK Phosphorylation by the PTTH in Prothoracic Glands of the Silkworm, Bombyx mori

    Directory of Open Access Journals (Sweden)

    Shi-Hong Gu

    2018-02-01

    Full Text Available In this study, phosphorylation of c-Jun N-terminal kinase (JNK by the prothoracicotropic hormone (PTTH was investigated in prothoracic glands (PGs of the silkworm, Bombyx mori. Results showed that JNK phosphorylation was stimulated by the PTTH in time- and dose-dependent manners. In vitro activation of JNK phosphorylation in PGs by the PTTH was also confirmed in an in vivo experiment, in which a PTTH injection greatly increased JNK phosphorylation in PGs of day-6 last instar larvae. JNK phosphorylation caused by PTTH stimulation was greatly inhibited by U73122, a potent and specific inhibitor of phospholipase C (PLC and an increase in JNK phosphorylation was also detected when PGs were treated with agents (either A23187 or thapsigargin that directly elevated the intracellular Ca2+ concentration, thereby indicating involvement of PLC and Ca2+. Pretreatment with an inhibitor (U0126 of mitogen-activated protein kinase (MAPK/extracellular signal-regulated kinase (ERK kinase (MEK and an inhibitor (LY294002 of phosphoinositide 3-kinase (PI3K failed to significantly inhibit PTTH-stimulated JNK phosphorylation, indicating that ERK and PI3K were not related to JNK. We further investigated the effect of modulation of the redox state on JNK phosphorylation. In the presence of either an antioxidant (N-acetylcysteine, NAC or diphenylene iodonium (DPI, PTTH-stimulated JNK phosphorylation was blocked. The JNK kinase inhibitor, SP600125, markedly inhibited PTTH-stimulated JNK phosphorylation and ecdysteroid synthesis. The kinase assay of JNK in PGs confirmed its stimulation by PTTH and inhibition by SP600125. Moreover, PTTH treatment did not affect JNK or Jun mRNA expressions. Based on these findings, we concluded that PTTH stimulates JNK phosphorylation in Ca2+- and PLC-dependent manners and that the redox-regulated JNK signaling pathway is involved in PTTH-stimulated ecdysteroid synthesis in B. mori PGs.

  1. Activation of purified calcium channels by stoichiometric protein phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Nunoki, K.; Florio, V.; Catterall, W.A. (Univ. of Washington, Seattle (USA))

    1989-09-01

    Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of {sup 45}Ca{sup 2+} uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of {sup 45}Ca{sup 2+} uptake was linearly proportional to the extent of phosphorylation of the alpha 1 and beta subunits of the calcium channel up to a stoichiometry of approximately 1 mol of phosphate incorporated into each subunit. The calcium channels activated by phosphorylation were determined to be incorporated into the reconstituted vesicles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd{sup 2+}, Ni{sup 2+}, and Mg{sup 2+}. The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation of the alpha 1 and beta subunits of the purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels.

  2. Activation of purified calcium channels by stoichiometric protein phosphorylation

    International Nuclear Information System (INIS)

    Nunoki, K.; Florio, V.; Catterall, W.A.

    1989-01-01

    Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of 45 Ca 2+ uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of 45 Ca 2+ uptake was linearly proportional to the extent of phosphorylation of the alpha 1 and beta subunits of the calcium channel up to a stoichiometry of approximately 1 mol of phosphate incorporated into each subunit. The calcium channels activated by phosphorylation were determined to be incorporated into the reconstituted vesicles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd 2+ , Ni 2+ , and Mg 2+ . The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation of the alpha 1 and beta subunits of the purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels

  3. A Neutrophil Phenotype Model for Extracorporeal Treatment of Sepsis.

    Directory of Open Access Journals (Sweden)

    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

  4. Regulation of cardiac C-protein phosphorylation

    International Nuclear Information System (INIS)

    Titus, F.L.

    1985-01-01

    Molecular mechanisms of cardiac sympathetic and parasympathetic responses were addressed by studying subcellular changes in protein phosphorylation, cAMP-dependent protein kinase activity and protein phosphatase activity in frog hearts. B-adrenergic agonists increased and muscarinic cholinergic agonists decreased [ 32 P]phosphate incorporation into C-protein, a thick filament component. Regulation of protein phosphatase activity by Iso and methacholine (MCh) was assayed using extracts of drug treated frog hearts and [ 32 P]phospho-C-protein as substrate. Total phosphatase activity decreased 21% in extracts from hearts perfused with 0.1 μM Iso and 17% in hearts exposed to Iso plus 1 μM methacholine. This decrease reflected decreased phosphatase-2A activity. No changes in total phosphatase activity were measurable in broken cells treated with Iso or MCh. The results suggest adrenergic stimulation changes contractile activity in frog hearts by activating cAMP-dependent protein kinase associated with particulate cellular elements and inactivating soluble protein phosphatase-2A. This is the first demonstration of coordinated regulation of these enzymes by B-adrenergic agonists favoring phosphorylation of effector proteins. Coordinated regulation by methacholine in the presence of Iso was not observed

  5. Tampering with springs: phosphorylation of titin affecting the mechanical function of cardiomyocytes.

    Science.gov (United States)

    Hamdani, Nazha; Herwig, Melissa; Linke, Wolfgang A

    2017-06-01

    Reversible post-translational modifications of various cardiac proteins regulate the mechanical properties of the cardiomyocytes and thus modulate the contractile performance of the heart. The giant protein titin forms a continuous filament network in the sarcomeres of striated muscle cells, where it determines passive tension development and modulates active contraction. These mechanical properties of titin are altered through post-translational modifications, particularly phosphorylation. Titin contains hundreds of potential phosphorylation sites, the functional relevance of which is only beginning to emerge. Here, we provide a state-of-the-art summary of the phosphorylation sites in titin, with a particular focus on the elastic titin spring segment. We discuss how phosphorylation at specific amino acids can reduce or increase the stretch-induced spring force of titin, depending on where the spring region is phosphorylated. We also review which protein kinases phosphorylate titin and how this phosphorylation affects titin-based passive tension in cardiomyocytes. A comprehensive overview is provided of studies that have measured altered titin phosphorylation and titin-based passive tension in myocardial samples from human heart failure patients and animal models of heart disease. As our understanding of the broader implications of phosphorylation in titin progresses, this knowledge could be used to design targeted interventions aimed at reducing pathologically increased titin stiffness in patients with stiff hearts.

  6. Study of ATM Phosphorylation by Cdk5 in Neuronal Cells.

    Science.gov (United States)

    She, Hua; Mao, Zixu

    2017-01-01

    The phosphatidylinositol-3-kinase-like kinase ATM (ataxia-telangiectasia mutated) plays a central role in coordinating the DNA damage responses including cell cycle checkpoint control, DNA repair, and apoptosis. Mutations of ATM cause a spectrum of defects ranging from neurodegeneration to cancer predisposition. We previously showed that Cdk5 (cyclin-dependent kinase 5) is activated by DNA damage and directly phosphorylates ATM at serine 794 in postmitotic neurons. Phosphorylation at serine 794 precedes and is required for ATM autophosphorylation at serine 1981, and activates ATM kinase activity. Cdk5-ATM pathway plays a crucial role in DNA damage-induced neuronal injury. This chapter describes protocols used in analyzing ATM phosphorylation by Cdk5 in CGNs (cerebellar granule neurons) and its effects on neuronal survival.

  7. 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 ...

  8. ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis

    Energy Technology Data Exchange (ETDEWEB)

    Inesta-Vaquera, Francisco A. [Departamento de Inmunologia y Oncologia, Centro Nacional de Biotecnologia-CSIC, Campus de Cantoblanco-UAM, 28049 Madrid (Spain); Campbell, David G.; Arthur, J. Simon C. [MRC Protein Phosphorylation Unit, Sir James Black Building, School of Life Sciences, University of Dundee, Dundee DD1 5EH (United Kingdom); Cuenda, Ana, E-mail: acuenda@cnb.csic.es [Departamento de Inmunologia y Oncologia, Centro Nacional de Biotecnologia-CSIC, Campus de Cantoblanco-UAM, 28049 Madrid (Spain)

    2010-08-13

    Research highlights: {yields} hDlg is phosphorylated during mitosis in multiple residues. {yields} Prospho-hDlg is excluded from the midbody during mitosis. {yields} hDlg is not phosphorylated by p38{gamma} or JNK1/2 during mitosis. {yields} ERK5 pathway mediates hDlg phosphorylation in mitosis. -- Abstract: Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.

  9. ERK5 pathway regulates the phosphorylation of tumour suppressor hDlg during mitosis

    International Nuclear Information System (INIS)

    Inesta-Vaquera, Francisco A.; Campbell, David G.; Arthur, J. Simon C.; Cuenda, Ana

    2010-01-01

    Research highlights: → hDlg is phosphorylated during mitosis in multiple residues. → Prospho-hDlg is excluded from the midbody during mitosis. → hDlg is not phosphorylated by p38γ or JNK1/2 during mitosis. → ERK5 pathway mediates hDlg phosphorylation in mitosis. -- Abstract: Human disc-large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is thought to be a tumour suppressor that regulates the cell cycle and proliferation. However, the mechanism and pathways involved in hDlg regulation during these processes is still unclear. Here we report that hDlg is phosphorylated during mitosis, and we establish the identity of at least three residues phosphorylated in hDlg; some are previously unreported. Phosphorylation affects hDlg localisation excluding it from the contact point between the two daughter cells. Our results reveal a previously unreported pathway for hDlg phosphorylation in mitosis and show that ERK5 pathway mediates hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression.

  10. Dopamine signaling negatively regulates striatal phosphorylation of Cdk5 at tyrosine 15 in mice.

    Directory of Open Access Journals (Sweden)

    Yukio eYamamura

    2013-02-01

    Full Text Available Striatal functions depend on the activity balance between the dopamine and glutamate neurotransmissions. Glutamate inputs activate cyclin-dependent kinase 5 (Cdk5, which inhibits postsynaptic dopamine signaling by phosphorylating DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, 32 kDa at Thr75 in the striatum. c-Abelson tyrosine kinase (c-Abl is known to phosphorylate Cdk5 at Tyr15 (Tyr15-Cdk5 and thereby facilitates the Cdk5 activity. We here report that Cdk5 with Tyr15 phosphorylation (Cdk5-pTyr15 is enriched in the mouse striatum, where dopaminergic stimulation inhibited phosphorylation of Tyr15-Cdk5 by acting through the D2 class dopamine receptors. Moreover, in the 1-methyl-4-phenyl-1,2,4,6-tetrahydropyridine mouse model, dopamine deficiency caused increased phosphorylation of both Tyr15-Cdk5 and Thr75-DARPP-32 in the striatum, which could be attenuated by administration of L-3,4-dihydroxyphenylalanine and imatinib (STI-571, a selective c-Abl inhibitor. Our results suggest a functional link of Cdk5-pTyr15 with postsynaptic dopamine and glutamate signals through the c-Abl kinase activity in the striatum.

  11. 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.

  12. Hepatitis C Virus Particle Assembly Involves Phosphorylation of NS5A by the c-Abl Tyrosine Kinase.

    Science.gov (United States)

    Yamauchi, Shota; Takeuchi, Kenji; Chihara, Kazuyasu; Sun, Xuedong; Honjoh, Chisato; Yoshiki, Hatsumi; Hotta, Hak; Sada, Kiyonao

    2015-09-04

    Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is thought to regulate the replication of viral RNA and the assembly of virus particles in a serine/threonine phosphorylation-dependent manner. However, the host kinases that phosphorylate NS5A have not been fully identified. Here, we show that HCV particle assembly involves the phosphorylation of NS5A by the c-Abl tyrosine kinase. Pharmacological inhibition or knockdown of c-Abl reduces the production of infectious HCV (J6/JFH1) particles in Huh-7.5 cells without markedly affecting viral RNA translation and replication. NS5A is tyrosine-phosphorylated in HCV-infected cells, and this phosphorylation is also reduced by the knockdown of c-Abl. Mutational analysis reveals that NS5A tyrosine phosphorylation is dependent, at least in part, on Tyr(330) (Tyr(2306) in polyprotein numbering). Mutation of this residue to phenylalanine reduces the production of infectious HCV particles but does not affect the replication of the JFH1 subgenomic replicon. These findings suggest that c-Abl promotes HCV particle assembly by phosphorylating NS5A at Tyr(330). © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. 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 ...

  14. Measuring medication adherence in patients with incident hypertension: a retrospective cohort study.

    Science.gov (United States)

    Tang, Karen L; Quan, Hude; Rabi, Doreen M

    2017-02-13

    Though pharmacy claims data are commonly used to study medication adherence, there remains no standard operational definition for adherence especially for patients on multiple medications. Even when studies use the same terminology, the actual methods of calculating adherence can differ drastically. It is unclear whether the use of different definitions results in different conclusions regarding adherence and associated outcomes. The objective of our study was to compare adherence rates and associations with mortality using different operational definitions of adherence, and using various methods of handling concurrent medication use. We conducted a cohort study of patients aged ≥65 years from Manitoba, Canada, with incident hypertension diagnosed in 2004 and followed to 2009. We calculated adherence rates to anti-hypertensive medications using different operational definitions of medication adherence (including interval and prescription based medication possession ratios [MPR] and proportion of days covered [PDC]). For those on concurrent medications, we calculated adherence rates using the different methods of handling concurrent medication use, for each definition. We used logistic regression to determine the association between adherence and mortality for each operational definition. Among 2199 patients, 24.1% to 90.5% and 71.2% to 92.7% were considered adherent when using fixed interval and prescription-based interval medication possession ratios [MPRi and MPRp] respectively, depending on how concurrent medications were handled. Adherence was inversely associated with death, with the strongest association for MPRp measures. This association was significant only when considering adherence to any anti-hypertensive [aOR 0.70, 95% CI 0.51, 0.97], or when the mean of the class-specific MPRp's [adjusted OR 0.71, 95% CI 0.53, 0.95] was used. No significant association existed when the highest or lowest class-specific MPRp was used as the adherence estimate. The

  15. Oxidative phosphorylation revisited

    DEFF Research Database (Denmark)

    Nath, Sunil; Villadsen, John

    2015-01-01

    The fundamentals of oxidative phosphorylation and photophosphorylation are revisited. New experimental data on the involvement of succinate and malate anions respectively in oxidative phosphorylation and photophosphorylation are presented. These new data offer a novel molecular mechanistic...

  16. Phosphorylation of the adaptor protein SH2B1β regulates its ability to enhance growth hormone-dependent macrophage motility

    OpenAIRE

    Su, Hsiao-Wen; Lanning, Nathan J.; Morris, David L.; Argetsinger, Lawrence S.; Lumeng, Carey N.; Carter-Su, Christin

    2013-01-01

    Previous studies have shown that growth hormone (GH) recruits the adapter protein SH2B1β to the GH-activated, GH receptor-associated tyrosine kinase JAK2, implicating SH2B1β in GH-dependent actin cytoskeleton remodeling, and suggesting that phosphorylation at serines 161 and 165 in SH2B1β releases SH2B1β from the plasma membrane. Here, we examined the role of SH2B1β in GH regulation of macrophage migration. We show that GH stimulates migration of cultured RAW264.7 macrophages, and primary cul...

  17. Phosphorylation of p300 by ATM controls the stability of NBS1

    International Nuclear Information System (INIS)

    Jang, Eun Ryoung; Choi, Jae Duk; Jeong, Gajin; Lee, Jong-Soo

    2010-01-01

    Acetyltransferase, p300 is a transcriptional cofactor of signal-responsive transcriptional regulation. The surveillance kinase ataxia-telangiectasia mutated (ATM) plays a central role in regulation of a wide range of cellular DNA damage responses. Here, we investigated whether and how ATM mediates phosphorylation of p300 in response to DNA damage and how p300 phosphorylation is functionally linked to DNA damage. ATM-phosphorylated p300 in vitro and in vivo, in response to DNA damage. Phosphorylation of p300 proteins was observed upon γ-irradiation in ATM + cells but not ATM - cells. Importantly, expression of nonphosphorylatable serine to alanine form of p300 (S106A) destabilized both p300 and NBS1 proteins, after DNA damage. These data demonstrate that ATM transduces a DNA damage signal to p300, and that ATM-dependent phosphorylation of p300 is required for stabilization of NBS1 proteins in response to DNA damage.

  18. Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood

    Directory of Open Access Journals (Sweden)

    N.E. Gomes

    2010-09-01

    Full Text Available Lipopolysaccharide (LPS activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R and smooth (S forms signal through Toll-like receptor 4 (TLR4, but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS and nitric oxide (NO generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30% increase followed by a 40% decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50% reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.

  19. Protein phosphorylation in isolated hepatocytes of septic and endotoxemic rats

    International Nuclear Information System (INIS)

    Deaciuc, I.V.; Spitzer, J.A.

    1989-01-01

    The purpose of this study was to investigate possible alterations induced by sepsis and endotoxicosis in the late phase of Ca2+-dependent signaling in rat liver. Hepatocytes isolated from septic or chronically endotoxin (ET)-treated rats were labeled with [32P]H3PO4 and stimulated with various agents. Proteins were resolved by one-dimensional polyacrylamide gel electrophoresis and autoradiographed. Vasopressin (VP)- and phenylephrine (PE)-induced responses were attenuated in both septic and ET-treated rats for cytosolic and membrane proteins compared with their respective controls. Glucagon and 12-O-myristate phorbol-13-acetate (TPA) affected only the phosphorylation of membrane proteins. Glucagon-induced changes in the phosphorylation of membrane proteins were affected by both sepsis and endotoxicosis, whereas TPA-stimulated phosphorylation was lowered only in endotoxicosis. Response to the Ca2+ ionophore A23187 was depressed in septic rats for cytosolic proteins. The phosphorylation of two cytosolic proteins, i.e., 93 and 61 kDa (previously identified as glycogen phosphorylase and pyruvate kinase, respectively), in response to VP, PE, and A23187 was severely impaired by endotoxicosis and sepsis. TPA did not affect the phosphorylation state of these two proteins. The results show that sepsis and endotoxicosis produce perturbations of the phosphorylation step in Ca2+ transmembrane signaling. Such changes can explain alterations of glycogenolysis and gluconeogenesis associated with sepsis and endotoxicosis

  20. D-lactic acid interferes with the effects of platelet activating factor on bovine neutrophils.

    Science.gov (United States)

    Alarcón, P; Conejeros, I; Carretta, M D; Concha, C; Jara, E; Tadich, N; Hidalgo, M A; Burgos, R A

    2011-11-15

    D-lactic acidosis occurs in ruminants, such as cattle, with acute ruminal acidosis caused by ingestion of excessive amounts of highly fermentable carbohydrates. Affected animals show clinical signs similar to those of septic shock, as well as acute laminitis and liver abscesses. It has been proposed that the inflammatory response and susceptibility to infection could both be caused by the inhibition of phagocytic mechanisms. To determine the effects of d-lactic acid on bovine neutrophil functions, we pretreated cells with different concentrations of D-lactic acid and measured intracellular pH using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and calcium flux using FLUO-3 AM-loaded neutrophils. Reactive oxygen species (ROS) production was measured using a luminol chemiluminescence assay, and MMP-9/gelatinase-B granule release was measured by zymography. CD11b and CD62L/l-selectin expression, changes in cell shape, superoxide anion production, phagocytosis of Escherichia coli-Texas red bioparticles, and apoptosis were all measured using flow cytometry. Our results demonstrated that D-lactic acid reduced ROS production, CD11b upregulation and MMP-9 release in bovine neutrophils treated with 100 nM platelet-activating factor (PAF). D-lactic acid induced MMP-9 release and, at higher concentrations, upregulated CD11b expression, decrease L-selectin expression, and induces late apoptosis. We concluded that D-lactic acid can interfere with neutrophil functions induced by PAF, leading to reduced innate immune responses during bacterial infections. Moreover, the increase of MMP-9 release and CD11b expression induced by 10mM D-lactic acid could promote an nonspecific neutrophil-dependent inflammatory reaction in cattle with acute ruminal acidosis. Copyright © 2011 Elsevier B.V. All rights reserved.

  1. Opioid Addiction in Pregnancy: Does Depression Negatively Impact Adherence With Prenatal Care?

    Science.gov (United States)

    Hensley, Lauren; Sulo, Suela; Kozmic, Sarah; Parilla, Barbara V

    We aimed to evaluate whether depression in pregnancy in women with opioid dependency negatively impacts adherence with prenatal care. This was a retrospective chart analysis of opioid-dependent pregnant women over a 6-year period at 2 large referral and tertiary care centers. The primary outcome was adherence with prenatal care based on the concurrent diagnosis of depression. Adherence was assessed by looking at the number of observed versus expected prenatal visits. Secondary outcomes included neonatal intensive care unit (NICU) stay, and incidence and severity of neonatal abstinence syndrome (NAS). A total of 74 patient charts were reviewed. 45/74 (60.8%) of the opioid-dependent pregnant patients were either diagnosed with depression (n = 41), anxiety (n = 2), or scored >10 on the Edinburgh Prenatal Depression Scale (n = 1). Patients with a diagnosis of depression were significantly less adherent with prenatal care; 80% adherent (73% vs 93%; P = 0.03), 90% adherent (62% vs 93%; P = 0.003). A higher number of patients in the depression group had an infant treated for withdrawal (62% vs 38%; P = 0.041), and had longer NICU stays (27% vs 21%; P = 0.018). Analysis of the whole cohort of opioid dependent gravidas revealed Buprenorphine maintenance therapy had the lowest mean NAS score 6.5 ± 4.4, compared with methadone maintenance 10.6 ± 3.6, and no maintenance therapy 9.4 ± 4.0 (P = 0.008). Depression negatively impacts adherence with prenatal care and was significantly associated with a higher incidence of neonatal withdrawal and longer NICU stays. Buprenorphine therapy had the lowest incidence and severity of NAS when compared with methadone and no maintenance therapy.

  2. 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

  3. HSP20 phosphorylation and airway smooth muscle relaxation

    Directory of Open Access Journals (Sweden)

    Mariam Ba

    2009-06-01

    Full Text Available Mariam Ba1, Cherie A Singer1, Manoj Tyagi2, Colleen Brophy3, Josh E Baker4, Christine Cremo4, Andrew Halayko5, William T Gerthoffer21Department of Pharmacology, University of Nevada School of Medicine, Reno, NV, USA; 2Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, AL, USA; 3Harrington Department of Biochemistry, Arizona State University, Tempe, AZ, USA; 4Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV, USA; 5Departments of Physiology and Internal Medicine, University of Manitoba, Winnipeg, MB, CanadaAbstract: HSP20 (HSPB6 is a small heat shock protein expressed in smooth muscles that is hypothesized to inhibit contraction when phosphorylated by cAMP-dependent protein kinase. To investigate this hypothesis in airway smooth muscle (ASM we showed that HSP20 was constitutively expressed as well as being inducible in cultured hASM cells by treatment with 1 µM isoproterenol or 10 µM salmeterol. In contrast, a mixture of proinflammatory mediators (interleukin-1β, tumor necrosis factor α, and interferon γ inhibited expression of HSP20 by about 50% in 48 hours. To determine whether phosphorylation of HSP20 is sufficient to induce relaxation, canine tracheal smooth muscle was treated with a cell permeant phosphopeptide that mimics the phosphorylation of HSP20. The HSP20 phosphopeptide antagonized carbacholinduced contraction by 60% with no change in myosin light chain phosphorylation. Recombinant full length HSP20 inhibited skeletal actin binding to smooth muscle myosin subfragment 1 (S1, and recombinant cell permeant TAT-HSP20 S16D mutant reduced F-actin filaments in cultured hASM cells. Carbachol stimulation of canine tracheal smooth muscle tissue caused redistribution of HSP20 from large macromolecular complexes (200–500 kDa to smaller complexes (<60 kDa. The results are consistent with HSP20 expression and macromolecular structure being dynamically regulated in airway

  4. 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 ...

  5. Inhibition of Ribosome Recruitment Induces Stress Granule Formation Independently of Eukaryotic Initiation Factor 2α Phosphorylation

    Science.gov (United States)

    Mazroui, Rachid; Sukarieh, Rami; Bordeleau, Marie-Eve; Kaufman, Randal J.; Northcote, Peter; Tanaka, Junichi; Gallouzi, Imed

    2006-01-01

    Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2α phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2α phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2α to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2α phosphorylation-dependent and -independent pathways that target translation initiation. PMID:16870703

  6. Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation.

    Science.gov (United States)

    Mazroui, Rachid; Sukarieh, Rami; Bordeleau, Marie-Eve; Kaufman, Randal J; Northcote, Peter; Tanaka, Junichi; Gallouzi, Imed; Pelletier, Jerry

    2006-10-01

    Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2alpha phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2alpha phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2alpha to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2alpha phosphorylation-dependent and -independent pathways that target translation initiation.

  7. Parenteral medium-chain triglyceride-induced neutrophil activation is not mediated by a Pertussis Toxin sensitive receptor.

    Science.gov (United States)

    Versleijen, Michelle W J; van Esterik, Joantine C J; Roelofs, Hennie M J; van Emst-de Vries, Sjenet E; Willems, Peter H G M; Wanten, Geert J A

    2009-02-01

    Lipid-induced immune modulation might contribute to the increased infection rate that is observed in patients using parenteral nutrition. We previously showed that emulsions containing medium-chain triglycerides (LCT/MCTs or pure MCTs), but not pure long-chain triglycerides (LCTs), impair neutrophil functions, modulate cell-signaling and induce neutrophil activation in vitro. It has recently been shown that medium-chain fatty acids are ligands for GPR84, a pertussis toxin (PT)-sensitive G-protein-coupled receptor (GPCR). This finding urged us to investigate whether MCT-induced neutrophil activation is mediated by PT-sensitive GPCRs. Neutrophils isolated from blood of healthy volunteers were pre-incubated with PT (0.5-1 microg/mL, 1.5 h) and analyzed for the effect of this pre-incubation on LCT/MCT (2.5 mmol/L)-dependent modulation of serum-treated zymosan (STZ)-induced intracellular Ca(2+) mobilization and on LCT/MCT (5 mmol/L)-induced expression of cell surface adhesion (CD11b) and degranulation (CD66b) markers and oxygen radical (ROS) production. PT did not inhibit the effects of LCT/MCT on the STZ-induced increase in cytosolic free Ca(2+) concentration. LCT/MCT increased ROS production to 146% of unstimulated cells. However, pre-incubation with PT did not inhibit the LCT/MCT-induced ROS production. Furthermore, the LCT/MCT-induced increase in CD11b and CD66b expression (196% and 235% of unstimulated cells, respectively) was not inhibited by pre-incubation with PT. LCT/MCT-induced neutrophil activation does not involve the action of a PT-sensitive G-protein-coupled receptor.

  8. 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.

  9. 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.

  10. Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

    Directory of Open Access Journals (Sweden)

    Neil Arvin Bretaña

    Full Text Available Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase

  11. Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

    Science.gov (United States)

    Bretaña, Neil Arvin; Lu, Cheng-Tsung; Chiang, Chiu-Yun; Su, Min-Gang; Huang, Kai-Yao; Lee, Tzong-Yi; Weng, Shun-Long

    2012-01-01

    Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD) is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase-specific phosphorylation site

  12. Nanostructured Surfaces to Target and Kill Circulating Tumor Cells While Repelling Leukocytes

    Directory of Open Access Journals (Sweden)

    Michael J. Mitchell

    2012-01-01

    Full Text Available Hematogenous metastasis, the process of cancer cell migration from a primary to distal location via the bloodstream, typically leads to a poor patient prognosis. Selectin proteins hold promise in delivering drug-containing nanocarriers to circulating tumor cells (CTCs in the bloodstream, due to their rapid, force-dependent binding kinetics. However, it is challenging to deliver such nanocarriers while avoiding toxic effects on healthy blood cells, as many possess ligands that adhesively interact with selectins. Herein, we describe a nanostructured surface to capture flowing cancer cells, while preventing human neutrophil adhesion. Microtube surfaces with immobilized halloysite nanotubes (HNTs and E-selectin functionalized liposomal doxorubicin (ES-PEG L-DXR significantly increased the number of breast adenocarcinoma MCF7 cells captured from flow, yet also significantly reduced the number of captured neutrophils. Neutrophils firmly adhered and projected pseudopods on surfaces coated only with liposomes, while neutrophils adherent to HNT-liposome surfaces maintained a round morphology. Perfusion of both MCF7 cells and neutrophils resulted in primarily cancer cell adhesion to the HNT-liposome surface, and induced significant cancer cell death. This work demonstrates that nanostructured surfaces consisting of HNTs and ES-PEG L-DXR can increase CTC recruitment for chemotherapeutic delivery, while also preventing healthy cell adhesion and uptake of therapeutic intended for CTCs.

  13. Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Berbís, M. Álvaro [Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid (Spain); André, Sabine [Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians University, 80539 Munich (Germany); Cañada, F. Javier [Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid (Spain); Pipkorn, Rüdiger [Central Peptide Synthesis Unit, German Cancer Research Center, 69120 Heidelberg (Germany); Ippel, Hans [Department of Biochemistry, CARIM, University of Maastricht, Maastricht (Netherlands); Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455 (United States); Mayo, Kevin H. [Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455 (United States); Kübler, Dieter [Biomolecular Interactions, German Cancer Research Center, 69120 Heidelberg (Germany); Gabius, Hans-Joachim [Institute of Physiological Chemistry, Faculty of Veterinary Medicine, Ludwig-Maximilians University, 80539 Munich (Germany); Jiménez-Barbero, Jesús, E-mail: jjbarbero@cib.csic.es [Chemical and Physical Biology Department, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid (Spain)

    2014-01-03

    Highlights: •Galectin-3 is composed of a carbohydrate recognition domain and an N-terminal tail. •Synthetic peptides derived from the tail are shown to interact with the CRD. •This interaction is modulated by Ser- and Tyr-phosphorylation of the peptides. -- Abstract: Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence unique in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with {sup 15}N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein.

  14. Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner

    International Nuclear Information System (INIS)

    Berbís, M. Álvaro; André, Sabine; Cañada, F. Javier; Pipkorn, Rüdiger; Ippel, Hans; Mayo, Kevin H.; Kübler, Dieter; Gabius, Hans-Joachim; Jiménez-Barbero, Jesús

    2014-01-01

    Highlights: •Galectin-3 is composed of a carbohydrate recognition domain and an N-terminal tail. •Synthetic peptides derived from the tail are shown to interact with the CRD. •This interaction is modulated by Ser- and Tyr-phosphorylation of the peptides. -- Abstract: Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence unique in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with 15 N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein

  15. 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...

  16. Molecular mechanism of APC/C activation by mitotic phosphorylation.

    Science.gov (United States)

    Zhang, Suyang; Chang, Leifu; Alfieri, Claudio; Zhang, Ziguo; Yang, Jing; Maslen, Sarah; Skehel, Mark; Barford, David

    2016-05-12

    In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our

  17. Regulation of gap junction conductance by calcineurin through Cx43 phosphorylation: implications for action potential conduction.

    Science.gov (United States)

    Jabr, Rita I; Hatch, Fiona S; Salvage, Samantha C; Orlowski, Alejandro; Lampe, Paul D; Fry, Christopher H

    2016-11-01

    Cardiac arrhythmias are associated with raised intracellular [Ca 2+ ] and slowed action potential conduction caused by reduced gap junction (GJ) electrical conductance (Gj). Ventricular GJs are composed of connexin proteins (Cx43), with Gj determined by Cx43 phosphorylation status. Connexin phosphorylation is an interplay between protein kinases and phosphatases but the precise pathways are unknown. We aimed to identify key Ca 2+ -dependent phosphorylation sites on Cx43 that regulate cardiac gap junction conductance and action potential conduction velocity. We investigated the role of the Ca 2+ -dependent phosphatase, calcineurin. Intracellular [Ca 2+ ] was raised in guinea-pig myocardium by a low-Na solution or increased stimulation. Conduction velocity and Gj were measured in multicellular strips. Phosphorylation of Cx43 serine residues (S365 and S368) and of the intermediary regulator I1 at threonine35 was measured by Western blot. Measurements were made in the presence and absence of inhibitors to calcineurin, I1 or protein phosphatase-1 and phosphatase-2.Raised [Ca 2 + ] i decreased Gj, reduced Cx43 phosphorylation at S365 and increased it at S368; these changes were reversed by calcineurin inhibitors. Cx43-S368 phosphorylation was reversed by the protein kinase C inhibitor chelerythrine. Raised [Ca 2+ ] i also decreased I1 phosphorylation, also prevented by calcineurin inhibitors, to increase activity of the Ca 2+ -independent phosphatase, PPI. The PP1 inhibitor, tautomycin, prevented Cx43-365 dephosphorylation, Cx43-S368 phosphorylation and Gj reduction in raised [Ca 2+ ] i . PP2A had no role. Conduction velocity was reduced by raised [Ca 2+ ] i and reversed by calcineurin inhibitors. Reduced action potential conduction and Gj in raised [Ca 2+ ] are regulated by calcineurin-dependent Cx43-S365 phosphorylation, leading to Cx43-S368 dephosphorylation. The calcineurin action is indirect, via I1 dephosphorylation and subsequent activation of PP1.

  18. Phosphorylation of Mycobacterium tuberculosis Ser/Thr phosphatase by PknA and PknB.

    Directory of Open Access Journals (Sweden)

    Andaleeb Sajid

    2011-03-01

    Full Text Available The integrated functions of 11 Ser/Thr protein kinases (STPKs and one phosphatase manipulate the phosphorylation levels of critical proteins in Mycobacterium tuberculosis. In this study, we show that the lone Ser/Thr phosphatase (PstP is regulated through phosphorylation by STPKs.PstP is phosphorylated by PknA and PknB and phosphorylation is influenced by the presence of Zn(2+-ions and inorganic phosphate (Pi. PstP is differentially phosphorylated on the cytosolic domain with Thr(137, Thr(141, Thr(174 and Thr(290 being the target residues of PknB while Thr(137 and Thr(174 are phosphorylated by PknA. The Mn(2+-ion binding residues Asp(38 and Asp(229 are critical for the optimal activity of PstP and substitution of these residues affects its phosphorylation status. Native PstP and its phosphatase deficient mutant PstP(c (D38G are phosphorylated by PknA and PknB in E. coli and addition of Zn(2+/Pi in the culture conditions affect the phosphorylation level of PstP. Interestingly, the phosphorylated phosphatase is more active than its unphosphorylated equivalent.This study establishes the novel mechanisms for regulation of mycobacterial Ser/Thr phosphatase. The results indicate that STPKs and PstP may regulate the signaling through mutually dependent mechanisms. Consequently, PstP phosphorylation may play a critical role in regulating its own activity. Since, the equilibrium between phosphorylated and non-phosphorylated states of mycobacterial proteins is still unexplained, understanding the regulation of PstP may help in deciphering the signal transduction pathways mediated by STPKs and the reversibility of the phenomena.

  19. Aggregation of Ribosomal Protein S6 at Nucleolus Is Cell Cycle-Controlled and Its Function in Pre-rRNA Processing Is Phosphorylation Dependent.

    Science.gov (United States)

    Zhang, Duo; Chen, Hui-Peng; Duan, Hai-Feng; Gao, Li-Hua; Shao, Yong; Chen, Ke-Yan; Wang, You-Liang; Lan, Feng-Hua; Hu, Xian-Wen

    2016-07-01

    Ribosomal protein S6 (rpS6) has long been regarded as one of the primary r-proteins that functions in the early stage of 40S subunit assembly, but its actual role is still obscure. The correct forming of 18S rRNA is a key step in the nuclear synthesis of 40S subunit. In this study, we demonstrate that rpS6 participates in the processing of 30S pre-rRNA to 18S rRNA only when its C-terminal five serines are phosphorylated, however, the process of entering the nucleus and then targeting the nucleolus does not dependent its phosphorylation. Remarkably, we also find that the aggregation of rpS6 at the nucleolus correlates to the phasing of cell cycle, beginning to concentrate in the nucleolus at later S phase and disaggregate at M phase. J. Cell. Biochem. 117: 1649-1657, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  20. Far-infrared radiation acutely increases nitric oxide production by increasing Ca2+ mobilization and Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179

    International Nuclear Information System (INIS)

    Park, Jung-Hyun; Lee, Sangmi; Cho, Du-Hyong; Park, Young Mi; Kang, Duk-Hee; Jo, Inho

    2013-01-01

    Highlights: •Far-infrared (FIR) radiation increases eNOS-Ser 1179 phosphorylation and NO production in BAEC. •CaMKII and PKA mediate FIR-stimulated increases in eNOS-Ser 1179 phosphorylation. •FIR increases intracellular Ca 2+ levels. •Thermo-sensitive TRPV Ca 2+ channels are unlikely to be involved in the FIR-mediated eNOS-Ser 1179 phosphorylation pathway. -- Abstract: Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser 1179 ) in a time-dependent manner (up to 40 min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca 2+ levels. Treatment with KN-93, a selective inhibitor of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser 1179 phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser 1179 phosphorylation. This study suggests that FIR radiation increases NO