Nano‐analysis reveals high fraction of serotonin release during exocytosis from a gut epithelium model cell

The primary force of muscle contraction is thought to involve a change in the myosin head whilst attached to actin, the energy coming from ATP hydrolysis. This change in attached state could either be a conformational change in the head or an alteration in the binding angle made with actin. A considerable amount is known about one bound state, the so-called strongly attached state, which occurs in the presence of ADP or in the absence of nucleotide. In this state, which probably corresponds to the last attached state of the force-producing cycle, the angle between the long axis myosin head and the actin filament is roughly 45°. Details of other attached states before and during power production have been difficult to obtain because, even at very high protein concentration, the complex is almost completely dissociated by ATP. Electron micrographs of the complex in the presence of ATP have therefore been obtained only after chemically cross-linking myosin subfragment-1 (S1) to actin filaments to prevent dissociation. But it is unclear then whether the variability in attachment angle observed is due merely to the cross-link acting as a hinge.We have recently found low ionic-strength conditions under which, without resorting to cross-linking, a high fraction of S1 is bound to actin during steady state ATP hydrolysis. The structure of this complex is being studied by cryo-electron microscopy of hydrated specimens. Most advantages of frozen specimens over ambient temperature methods such as negative staining have already been documented. These include improved preservation and fixation rates and the ability to observe protein directly rather than a surrounding stain envelope. In the present experiments, hydrated specimens have the additional benefit that it is feasible to use protein concentrations roughly two orders of magnitude higher than in conventional specimens, thereby reducing dissociation of weakly bound complexes.

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Membrane–cytoskeleton interactions in cholesterol-dependent domain formation

Essays in Biochemistry ◽

10.1042/bse0570177 ◽

2015 ◽

Vol 57 ◽

pp. 177-187 ◽

Cited By ~ 9

Author(s):

Jennifer N. Byrum ◽

William Rodgers

Keyword(s):

Biological Properties ◽

Membrane Rafts ◽

Membrane Domains ◽

Biological Functions ◽

Membrane Cytoskeleton ◽

Heterogeneous Structures ◽

Integral Role ◽

Model Cell ◽

Actomyosin Cytoskeleton ◽

Dependent Domain

Since the inception of the fluid mosaic model, cell membranes have come to be recognized as heterogeneous structures composed of discrete protein and lipid domains of various dimensions and biological functions. The structural and biological properties of membrane domains are represented by CDM (cholesterol-dependent membrane) domains, frequently referred to as membrane ‘rafts’. Biological functions attributed to CDMs include signal transduction. In T-cells, CDMs function in the regulation of the Src family kinase Lck (p56lck) by sequestering Lck from its activator CD45. Despite evidence of discrete CDM domains with specific functions, the mechanism by which they form and are maintained within a fluid and dynamic lipid bilayer is not completely understood. In the present chapter, we discuss recent advances showing that the actomyosin cytoskeleton has an integral role in the formation of CDM domains. Using Lck as a model, we also discuss recent findings regarding cytoskeleton-dependent CDM domain functions in protein regulation.

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Differing Susceptibilities of Platelets from Normal Individuals and Patients with von Willebrand’s Disease to Attack by Quinine- and Quinidine-Dependent Antiplatelet Antibodies

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646537 ◽

1989 ◽

Vol 61 (01) ◽

pp. 111-116

Author(s):

Sharron L Pfueller ◽

Robyn A Bilston ◽

Dana Logan ◽

Rosemary David ◽

Ian G Sloan ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Rich Plasma ◽

Serotonin Release ◽

Von Willebrand's Disease ◽

Von Willebrand’S Disease ◽

Antiplatelet Antibodies ◽

Normal Plasma ◽

Igg Binding ◽

Normal Individuals ◽

Drug Dependent

SummaryReactivity of quinine- and quinidine-dependent antiplatelet antibodies has been compared in platelet-rich-plasma (PRP) from normal donors and from patients with von Willebrand’s disease (vWd). One quinine-dependent antibody (Q. Ab) caused platelet aggregation and [14C] serotonin release with only 7 of 12 normal donors, while another Q. Ab and a quinidine-dependent antibody (Qd. Ab) caused aggregation and release with all 12. Drug- dependent IgG binding and PF 3 availability induced by the antibodies were, however, comparable in all donors. Differences in responsiveness were associated with platelets and not plasma. vWd platelets showed normal drug-dependent IgG binding, but decreased aggregation and serotonin release to most drug- dependent antibodies. Responsiveness was not restored by purified vWf:Ag, but, in one case, was corrected by normal plasma or cryoprecipitate. Drug-dependent binding of the Q. Ab which caused variable responsiveness in normals was to the same platelet antigens (GPIb and GPIIIa) in both normal and vWd platelets and did not require plasma components. Reduced PF 3 availability was seen with some antibodies in some vWd patients. Plasma from two of these patients inhibited aggregation of normal platelets to Q. Ab and one of these inhibited aggregation to ADP. Antiplatelet antibodies were detected in these two plasmas by ELISA. Thus some Q. Ab produce different responses with platelets from different donors. In vWd, reduced responsiveness to Q.Ab and Qd. Ab may result from production of inhibitory antiplatelet antibodies.

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Some Effects of Fibrinogen Degradation Products (FDP) on Blood Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649442 ◽

1966 ◽

Vol 15 (03/04) ◽

pp. 413-419 ◽

Cited By ~ 55

Author(s):

Z Jerushalmy ◽

M. B Zucker

Keyword(s):

Platelet Aggregation ◽

Connective Tissue ◽

Degradation Products ◽

Blood Platelets ◽

Adenosine Diphosphate ◽

Serotonin Release ◽

Fibrinogen Degradation Products ◽

Inhibition Of Platelet Aggregation

Summary“Early” fibrinogen degradation products are more potent inhibitors of thrombin-induced clotting than “late” products and also interfere with the ability of thrombin to release serotonin from platelets. “Early” and “intermediate” FDP cause moderate inhibition of platelet aggregation induced by adenosine diphosphate or connective tissue particles. Serotonin release by connective tissue particles is probably not inhibited by FDP.

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The Clinical Usefulness of the Platelet Aggregation Test for the Diagnosis of Heparin-Induced Thrombocytopenia

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651610 ◽

1993 ◽

Vol 69 (04) ◽

pp. 344-350 ◽

Cited By ~ 175

Author(s):

B H Chong ◽

J Burgess ◽

F Ismail

Keyword(s):

Platelet Aggregation ◽

Heparin Therapy ◽

Serotonin Release ◽

Point System ◽

Control Groups ◽

Heparin Induced Thrombocytopenia ◽

Heparin Concentration ◽

Release Test ◽

The Mean ◽

Normal Controls

SummaryThe platelet aggregation test is widely used for the diagnosis of heparin-induced thrombocytopenia (HIT), a potentially serious complication of heparin therapy. We have evaluated its sensitivity and specificity in comparison with those of the 14C-serotonin release test. The sensitivity of the platelet aggregation test was found to vary with the heparin concentration and the donor of the platelets used in the test. The optimal heparin concentrations were between 0.1 and 1.0 U/ml. Using these heparin concentrations, the mean sensitivity varied from 39% (with the least reactive platelets) to 81% (with the most reactive platelets). In comparison, the sensitivity of the release test ranged from 65% to 94%. The specificities of the platelet aggregation test were 82%, 90% and 100% for the following control groups: (1) non-thrombocytopenic patients given heparin, (2) patients with thrombocytopenia due to other causes, and (3) normal controls not given heparin, respectively. The corresponding specificities for the release test was 94%, 90% and 100%. The specificities can be further increased to 100% for all controls with the adoption of a two-point system which defines a positive result as one in which platelet aggregation occurs with a low heparin concentration (0.5 U/ml) but not with 100 U heparin/ml. For optimal results, a two-point platelet aggregation test should be performed with heparin concentrations of 0.5 and 100 U/ml and using platelets of more reactive donors.

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Effects of Prostaglandins, Derivatives of Cyclic 3':5'-AMP, Theophylline, Cholinergic Agents and Colchicine on Clot Retraction in Dilute Platelet-Rich Plasma and Gel-Separated Platelet Test Systems

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651477 ◽

1977 ◽

Vol 38 (02) ◽

pp. 0420-0428 ◽

Cited By ~ 1

Author(s):

J. L Moake ◽

P. L Cimo ◽

K Widmer ◽

D. M Peterson ◽

J. R Gum

Keyword(s):

Platelet Rich Plasma ◽

Inhibitory Effect ◽

Fibrin Clot ◽

Serotonin Release ◽

Dibutyryl Camp ◽

Clot Retraction ◽

Dilute Suspensions ◽

Cholinergic Agents ◽

Camp Levels ◽

Derivatives Of

SummaryIn dilute suspensions of platelet-rich plasma (PRP) or gel-separated platelets (GSP), dibutyryl-cAMP (DBcAMP) and monobutyryl-cAMP inhibited platelet-mediated fibrin clot retraction in concentrations of 2–3 × 10–6M, with complete inhibition at 1–3 × 10–4M. Prostaglandin E1 (PGE1), which inhibited fibrin clot retraction in concentrations greater than 1.5–3 × 10–8M, was a more effective inhibitor than either PGE2 or PGF2α. In the presence of theophylline (10–4M), concentrations of DBcAMP, PGE1 PGE2 and PGF2α necessary to inhibit fibrin clot retraction were reduced 50-fold for DBcAMP and 2.5 to 20-fold for the prostaglandins. In dilute PRP or GSP, inhibition of fibrin clot retraction does not result from inhibition of thrombin-induced platelet aggregation. Thus, compounds which increase platelet cAMP levels result in the inhibition of platelet-mediated fibrin clot retraction, and this inhibitory effect may be mediated, at least in part, through suppression of platelet contractility. Cyclic GMP, dibutyryl-cGMP and carbamylcholine-Cl (which stimulates guanylate cyclase) did not influence fibrin clot retraction, and did not prevent inhibition of fibrin clot retraction by DBcAMP and PGE?. Colchicine, in concentrations known to disrupt platelet microtubules (2.5 × 10–6M to 2.5 x 10–3M), had little inhibitory effect on either fibrin clot retraction or platelet (3H)-serotonin release.

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Platelet Coagulant Activities and Retinal Vein Thrombosis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651475 ◽

1977 ◽

Vol 38 (02) ◽

pp. 0399-0406 ◽

Cited By ~ 10

Author(s):

Peter N. Walsh ◽

Richard E. Goldberg ◽

Richard L. Tax ◽

Larry E. Magargal

Keyword(s):

Platelet Aggregation ◽

Venous Thrombosis ◽

Retinal Vein Occlusion ◽

Serotonin Release ◽

Platelet Factor ◽

Trigger Mechanism ◽

Local Conditions ◽

Plasma Coagulation ◽

Vein Thrombosis ◽

Vein Occlusion

SummaryTo determine whether platelets play a role in the pathogenesis of retinal vein occlusion (RVO), platelets and coagulation were evaluated in 28 patients with RVO. Platelet coagulant activities concerned with the initiation and early stages of intrinsic coagulation were 2–4 fold increased in 9 patients with acute primary RVO but not in patients with acute secondary (10 patients) or chronic (9 patients) RVO. Platelet factor 3 activity, platelet aggregation, serotonin release by platelets and plasma coagulation were normal in all patients. Platelets may provide a trigger mechanism for venous thrombosis in the eye when local conditions permit.

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Physiological Effects of Nonimmune Platelet Associated Immunoglobulin G

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650123 ◽

1981 ◽

Vol 45 (01) ◽

pp. 027-033 ◽

Cited By ~ 10

Author(s):

K Sugiura ◽

M Steiner ◽

M Baldini

Keyword(s):

Shape Change ◽

Fluorescein Isothiocyanate ◽

Recovery Phase ◽

Human Platelets ◽

Serotonin Release ◽

Platelet Volume ◽

Igg Antibody ◽

Heterologous Antiserum ◽

Igg Binding ◽

Series Of Experiments

SummaryThe function of nonimmune IgG associated with platelets is unknown. In a series of experiments we have investigated this problem, relating amount of platelet-associated IgG (PAIgG) to platelet volume, serotonin release, adherence of platelets to monocytes and platelet senescence. Most of these studies were performed with human platelets. Platelets freed of preexisting PAIgG by incubation at 22° C were incubated with IgG in a series of concentrations ranging from 0.4 — 27.0 X10-6 M. The IgG preparations used were demonstrably free of aggregated forms of the protein. The amount of PAIgG bound to platelets was determined by the use of fluorescein isothiocyanate-conjugated anti-IgG antibody (F-anti-IgG antibody) which was quantified in a fluorospectrophotometer. Newly bound IgG was assayed similarly by the use of F-IgG. A dose-dependent increase in platelet volume was associated with the binding of nonimmune IgG by platelets. The process which leveled off at an IgG concentration of 1.2 —1.5 X10-5 M was almost fully reversible and was not due to platelet shape change or aggregation. Release of serotonin from IgG-treated platelets was relatively small but to the extent that it occurred was positively related to the IgG concentration to which platelets were exposed. Adherence to autologous monocytes studied quantitatively by the use of formaldehyde-fixed cells was also positively related to the amount of IgG on the platelets. Normal or IgG-defident serum had a potent inhibitory (noncompetitive) action on the binding of F-IgG and F-anti-human IgG antibody to human platelets. Cohorts of platelets prepared in rabbits during the recovery phase of immunological thrombocytopenia induced by injection of heterologous antiserum, showed an age-dependent increase of PAIgG and of IgG binding. These results suggest that PAIgG plays a role in the clearance of senescent platelets.

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Therapeutic Application of Diacylglycerol Oil for Obesity: Serotonin Hypothesis

Functional Foods in Health and Disease ◽

10.31989/ffhd.v2i1.103 ◽

2012 ◽

Vol 2 (1) ◽

pp. 1 ◽

Cited By ~ 4

Author(s):

Hidekatsu Yanai ◽

Hiroshi Yoshida ◽

Yuji Hirowatari ◽

Norio Tada

Keyword(s):

Energy Expenditure ◽

Molecular Mechanisms ◽

Uncoupling Protein ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Serotonin Release ◽

Intestinal Cell ◽

Therapeutic Application ◽

Postprandial Triglyceride

Characteristics for the serum lipid abnormalities in the obesity/metabolic syndrome are elevated fasting, postprandial triglyceride (TG), and decreased high-density lipoprotein-cholesterol (HDL-C). Diacylglycerol (DAG) oil ingestion has been reported to ameliorate postprandial hyperlipidemia and prevent obesity by increasing energy expenditure, due to the intestinal physiochemical dynamics that differ from triacylglycerol (TAG). Our study demonstrated that DAG suppresses postprandial increase in TG-rich lipoprotein, very low-density lipoprotein (VLDL), and insulin, as compared with TAG in young, healthy individuals. Interestingly, our study also presented that DAG significantly increases plasma serotonin, which is mostly present in the intestine, and mediates thermogenesis, proposing a possible mechanism for a postprandial increase in energy expenditure by DAG. Our other study demonstrated that DAG suppresses postprandial increase in TG, VLDL-C, and remnant-like particle-cholesterol, in comparison with TAG in an apolipoprotein C-II deficient subject, suggesting that DAG suppresses postprandial TG-rich lipoprotein independently of lipoprotein lipase. Further, to understand the molecular mechanisms for DAG-mediated increase in serotonin and energy expenditure, we studied the effects of 1-monoacylglycerol and 2-monoacylglycerol, distinct digestive products of DAG and TAG, respectively, on serotonin release from the Caco-2 cells, the human intestinal cell line. We also studied effects of 1- and 2-monoacylglycerol, and serotonin on the expression of mRNA associated with β-oxidation, fatty acids metabolism, and thermogenesis, in the Caco-2 cells. 1-monoacylglycerol significantly increased serotonin release from the Caco-2 cells, compared with 2-monoacylglycerol by approximately 40%. The expression of mRNA of acyl-CoA oxidase (ACO), fatty acid translocase (FAT), and uncoupling protein-2 (UCP-2), was significantly higher in 1-MOG-treated Caco-2 cells, than 2-MOG-treated cells. The expression of mRNA of ACO, medium-chain acyl-CoA dehydrogenase, FAT, and UCP-2, was significantly elevated in serotonin-treated Caco-2 cells, compared to cells incubated without serotonin. In conclusion, our clinical and in vitro studies suggested a possible therapeutic application of DAG for obesity, and obesity-related metabolic disorders.Key words: Diacylglycerol, intestine, obesity, serotonin, thermogenesis

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