ABSTRACT
Myoendothelial junctions are specialised projections of cell : cell contact through the internal elastic lamina between endothelial cells and vascular smooth muscle cells. These junctions allow for endothelial cells and vascular smooth muscle cells to make direct membrane apposition and are involved in cell : cell communication. In this study, we evaluated for the presence of myoendothelial junctions in murine corporal tissue and used plasminogen activator inhibitor (PAI)-1-deficient mice, which lack myoendothelial junctions, to determine whether myoendothelial junctions affect erectile function. Transmission electron microscopy demonstrated the presence of myoendothelial junctions in the corporal tissue of wild-type mice and confirmed the decreased junction numbers in the tissue of PAI-1(-/-) mice. A potential role for myoendothelial junctions in tumescence was established; in that, PAI-1(-/-) mice demonstrated a significantly longer time to achieve maximal intracavernous pressure. Treatment of PAI-1(-/-) mice with recombinant PAI-1 restored the number of myoendothelial junctions in the corporal tissue and also induced a significant decrease in time to maximal corporal pressures. Myoendothelial junctions were similarly identified in the human corporal tissue. These results suggest a critical role for myoendothelial junctions in erectile pathophysiology and therapies aimed at restoring myoendothelial junction numbers in the corporal tissue may provide a novel therapy for erectile dysfunction.
Subject(s)
Endothelium, Vascular/drug effects , Erectile Dysfunction/drug therapy , Intercellular Junctions/drug effects , Muscle, Smooth, Vascular/drug effects , Penile Erection/drug effects , Serpin E2/deficiency , Animals , Cell Communication , Endothelium, Vascular/physiology , Endothelium, Vascular/ultrastructure , Erectile Dysfunction/etiology , Intercellular Junctions/physiology , Intercellular Junctions/ultrastructure , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron, Transmission , Muscle, Smooth, Vascular/physiology , Muscle, Smooth, Vascular/ultrastructure , Recombinant Proteins , Serpin E2/therapeutic useABSTRACT
The new era of spintronics promises the development of nanodevices, where the electron spin will be used to store information and charge currents will be replaced by spin currents. For this, ferromagnetic semiconductors at room temperature are needed. We report on significant room-temperature spin polarization of EuS in Co/EuS multilayers recorded by x-ray magnetic circular dichroism (XMCD). The films were found to contain a mixture of divalent and trivalent europium, but only Eu(++) is responsible for the ferromagnetic behavior of EuS. The magnetic XMCD signal of Eu at room temperature could unambiguously be assigned to magnetic ordering of EuS and was found to be only one order of magnitude smaller than that at 2.5 K. The room temperature magnetic moment of EuS is as large as the one of bulk ferromagnetic Ni. Our findings pave the path for fabrication of room-temperature spintronic devices using spin polarized EuS layers.
ABSTRACT
BACKGROUND: Extracorporeal circulation (ECC) and hypothermia are routinely used in cardiac surgery to maintain stable circulatory parameters and to increase the ischaemic tolerance of the patient. However, ECC and hypothermia cause platelet activation and dysfunction possibly followed by a devastating coagulopathy. Stimulation of the adenosinediphosphate (ADP) receptor P(2)Y(12) plays a pivotal role in platelet activation. This experimental study tested P(2)Y(12) receptor blockade as an approach to protect platelets during ECC. METHODS: Human blood was treated with the short-acting P(2)Y(12) blocker cangrelor (1 µM, t(1/2)<5 min) or the P(2)Y(12) inhibitor 2-MeSAMP (100 µM) and circulated in an ex vivo ECC model at normothermia (37°C) and hypothermia (28°C). Before and after circulation, markers of platelet activation and of coagulation (thrombin-antithrombin complex generation) were analysed. During hypothermic ECC in pigs, the effect of reversible P(2)Y(12) blockade on platelet function was evaluated by cangrelor infusion (0.075 µg kg(-1) min(-1)). RESULTS: During ex vivo hypothermic ECC, P(2)Y(12) blockade inhibited platelet granule release (P<0.01), platelet-granulocyte binding (P<0.05), and platelet loss (P<0.001), whereas no effects on platelet-ECC binding, platelet CD42bα expression, glycoprotein IIb/IIIa activation, or thrombin-antithrombin complex generation were observed. During hypothermic ECC in pigs, cangrelor inhibited platelet-fibrinogen binding (P<0.05) and ADP-induced platelet aggregation (P<0.001). Platelet function was rapidly restored after termination of cangrelor infusion. CONCLUSIONS: P(2)Y(12) blockade by cangrelor prevents platelet activation during ECC and hypothermia. Owing to its short half-life, platelet inhibition can be well controlled, thus potentially reducing bleeding complications. This novel pharmacological strategy has the potential to reduce complications associated with ECC and hypothermia.
Subject(s)
Adenosine Monophosphate/analogs & derivatives , Blood Coagulation Disorders/prevention & control , Blood Platelets/drug effects , Extracorporeal Circulation , Hypothermia, Induced , Purinergic P2Y Receptor Antagonists/pharmacology , Adenosine Diphosphate/blood , Adenosine Monophosphate/pharmacology , Animals , Antithrombin III/metabolism , Blood Platelets/physiology , Cardiopulmonary Bypass , Cytoplasmic Granules/drug effects , Humans , Peptide Hydrolases/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/drug effects , Platelet Glycoprotein GPIIb-IIIa Complex/physiology , Platelet Glycoprotein GPIb-IX Complex/analysis , SwineABSTRACT
Nitric oxide signaling, through eNOS (or possibly nNOS), and gap junction communication are essential for normal vascular function. While each component controls specific aspects of vascular function, there is substantial evidence for cross-talk between nitric oxide signaling and the gap junction proteins (connexins), and more recently, protein-protein association between eNOS and connexins. This review will examine the evidence for interaction between these pathways in normal and diseased arteries, highlight the questions that remain about the mechanisms of their interaction, and explore the possible interaction between nitric oxide signaling and the newly discovered pannexin channels. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.
Subject(s)
Gap Junctions/physiology , Nitric Oxide/metabolism , Animals , Atherosclerosis/metabolism , Caveolin 1/metabolism , Cells, Cultured , Connexins/metabolism , Cyclic GMP/metabolism , Diabetes Mellitus/metabolism , Gap Junctions/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Hypertension/metabolism , Mice , Models, Biological , Nerve Tissue Proteins/metabolism , Nitric Oxide Synthase Type III/metabolism , Protein Binding , Signal TransductionABSTRACT
Nodular lymphoid hyperplasia is a rare, benign, lymphoproliferative disease usually found in the gastrointestinal tract. It has never been reported in the sinuses. We present an unusual case of pulmonary nodular lymphoid hyperplasia with locally invasive sinus involvement and describe the CT, MRI and positron emission tomography findings.
Subject(s)
Lung Diseases/complications , Lung Diseases/diagnostic imaging , Paranasal Sinus Diseases/complications , Paranasal Sinus Diseases/diagnostic imaging , Pseudolymphoma/complications , Pseudolymphoma/diagnostic imaging , Female , Humans , Middle Aged , Neoplasm Invasiveness , RadiographyABSTRACT
The ADM1 was employed to assess the effect of variations in solids hydrolysis and acetoclastic methanogen process characterizations on municipal digester stability relating to excess acetate utilization capacity. First-order single- and dual-pathway hydrolysis rate functions and single and competitive acetoclastic methanogen rate functions were implemented in the ADM1. The acetate capacity number (ACN), defined as the ratio between the maximum acetate utilization rate and the average acetate production rate, was used to index digester instability. Simulations of a single CSTR at steady state indicate a similar ACN can be obtained with a 12-day SRT digester dominated by Methanosarcina sp and a 24-day SRT digester dominated by Methanosaeta sp. An increase in ACN with a decrease in SRT representing Methanosarcina sp. selection was observed for particulate feed loadings from 40 g COD/L to 90 g COD/L. Feeding frequency and dual-pathway hydrolysis were found to have less effect on the ACN than the competitive acetoclastic model structure.
Subject(s)
Anaerobiosis , Environmental Restoration and Remediation , Methanosarcina/metabolism , Models, Theoretical , Water Pollutants , HydrolysisABSTRACT
OBJECTIVES: Extracorporeal circulation (ECC) induces platelet activation and inflammation with potentially life-threatening organ dysfunction. Short-acting GP IIb/IIIa inhibitors like tirofiban and eptifibatide protect platelets during ECC without increasing bleeding complications and may reduce inflammation. This study investigates anti-thrombotic and anti-inflammatory effects of different platelet inhibitors. METHODS: Control (untreated) and treated (using either 150 ng/mL tirofiban, 2.5 microg/mL eptifibatide, 0.7 microg/mL milrinone, 15 microg/mL dipyridamol, or 300 KIU/mL aprotinin) heparinized blood of healthy volunteers (n = 6) was recirculated in a well-established ECC model (Chandler loop). Percentage of platelet aggregates, P-selectin-expressing (activated) platelets, CD15-positive aggregates (indicating proinflammatory platelet-granulocyte binding), and platelet counts were determined before (baseline) and after 30 minutes recirculation in unstimulated and ADP-stimulated samples using flow cytometry. Statistical analysis was performed using multifactor ANOVA after transforming the data (logarithms for counts and log odds for percentages). Least square means were backtransformed to obtain appropriate means and their 95 % confidence intervals. Multiple post-hoc comparisons were performed by Tukey's HSD test with a global alpha of 5 %. RESULTS: Significant inhibition was observed for: 1) ECC-induced platelet aggregation by tirofiban (unstimulated: 2.2-fold/stimulated: 2.46-fold), eptifibatide (unstimulated: 1.96-fold/stimulated: 2.65-fold), and milrinone (unstimulated: 1.87-fold/stimulated: 1.37-fold); 2) ECC-induced P-selectin expression by tirofiban (unstimulated: 3.95-fold/stimulated: 2.54-fold), and eptifibatide (unstimulated: 5.87-fold/stimulated: 3.28-fold); 3) ECC-induced platelet loss by tirofiban (1.27-fold), and eptifibatide (1.25-fold); 4) ECC-induced platelet-granulocyte binding by tirofiban (unstimulated: 2.25-fold/stimulated: 1.59-fold), but not by eptifibatide. CONCLUSIONS: Amongst the investigated drugs only GP IIb/IIIa inhibitors decreased activation, aggregation, and loss of platelets during ECC but acted differently on platelet-granulocyte interaction. A short-acting GP IIb/IIIa inhibitor with the potential to inhibit platelet activation and platelet-leukocyte interaction should be considered both for platelet protection and inhibition of platelet-mediated inflammation during ECC.
Subject(s)
Binding, Competitive/drug effects , Blood Platelets/drug effects , Extracorporeal Circulation , Granulocytes/drug effects , Platelet Aggregation Inhibitors/pharmacology , Tyrosine/analogs & derivatives , Aprotinin/pharmacology , Biomarkers/blood , Blood Platelets/metabolism , Dipyridamole/pharmacology , Eptifibatide , Flow Cytometry , Granulocytes/metabolism , Humans , Male , Milrinone/pharmacology , P-Selectin/biosynthesis , P-Selectin/drug effects , Peptides/pharmacology , Phosphodiesterase Inhibitors/pharmacology , Platelet Aggregation/drug effects , Platelet Count , Platelet Glycoprotein GPIIb-IIIa Complex/antagonists & inhibitors , Serine Proteinase Inhibitors/pharmacology , Tirofiban , Tyrosine/pharmacologyABSTRACT
BAY 59-7939 is an oral, direct Factor Xa (FXa) inhibitor in development for the prevention and treatment of arterial and venous thrombosis. BAY 59-7939 competitively inhibits human FXa (K(i) 0.4 nm) with > 10 000-fold greater selectivity than for other serine proteases; it also inhibited prothrombinase activity (IC(50) 2.1 nm). BAY 59-7939 inhibited endogenous FXa more potently in human and rabbit plasma (IC(50) 21 nm) than rat plasma (IC(50) 290 nm). It demonstrated anticoagulant effects in human plasma, doubling prothrombin time (PT) and activated partial thromboplastin time at 0.23 and 0.69 microm, respectively. In vivo, BAY 59-7939 reduced venous thrombosis (fibrin-rich, platelet-poor thrombi) dose dependently (ED(50) 0.1 mg kg(-1) i.v.) in a rat venous stasis model. BAY 59-7939 reduced arterial (fibrin- and platelet-rich) thrombus formation in an arteriovenous (AV) shunt in rats (ED(50) 5.0 mg kg(-1) p.o.) and rabbits (ED(50) 0.6 mg kg(-1) p.o.). Slight inhibition of FXa (32% at ED(50)) reduced thrombus formation in the venous model; to affect arterial thrombosis in the rat and rabbit, stronger inhibition of FXa (74%, 92% at ED(50)) was required. Calculated plasma levels in rabbits at the ED(50) were 14-fold lower than in the rat AV shunt model, correlating with the 14-fold lower IC(50) of FXa inhibition in rabbit compared with rat plasma; this may suggest a correlation between FXa inhibition and antithrombotic activity. Bleeding times in rats and rabbits were not significantly affected at antithrombotic doses (3 mg kg(-1) p.o., AV shunt). Based on these results, BAY 59-7939 was selected for clinical development.
Subject(s)
Factor Xa Inhibitors , Fibrinolytic Agents/pharmacology , Morpholines/pharmacology , Venous Thrombosis/drug therapy , Animals , Blood Coagulation Tests , Disease Models, Animal , Dose-Response Relationship, Drug , Fibrinolytic Agents/therapeutic use , Humans , Inhibitory Concentration 50 , Rabbits , Rats , Rivaroxaban , Serine Proteinase Inhibitors/pharmacology , Serine Proteinase Inhibitors/therapeutic use , Species Specificity , Thiophenes , Thromboplastin/metabolism , Venous Thrombosis/prevention & controlABSTRACT
OBJECTIVES: The serine-protease inhibitor aprotinin protects platelet function during cardiopulmonary bypass. However, its safety and efficacy during deep hypothermic circulatory arrest (DHCA) is controversial, and aprotinin is suspected to cause thrombosis especially during hypothermia. The platelet GP IIb/IIIa inhibitor eptifibatide has been assumed to preserve platelet function during cardiopulmonary bypass without increasing bleeding complications. The aim of this study was to compare the effect of aprotinin and eptifibatide on platelet function under conditions of DHCA. METHODS: Heparinized blood from healthy volunteers (n = 10) was incubated in stasis for 30 minutes at 18 degrees C to simulate DHCA and compared to samples incubated at 37 degrees C. The effect of eptifibatide (2.5 microg/ml) and aprotinin (300 KIU/ml) on platelets under these conditions was analyzed by flow cytometry. Platelet aggregates were identified using CD41-antibody binding and size. GPIIb/IIIa function was evaluated with the activation-specific antibody PAC-1 after stimulation with 10 microM ADP. Aggregate numbers and antibody mean-fluorescence are reported as mean +/- standard deviation. RESULTS: Hypothermia induced a 2.5-fold increase of aggregates ( p < 0.001) and a 2.6-fold increase of GPIIb/IIIa activation ( p < 0.001). This effect was not influenced by aprotinin but almost completely inhibited by eptifibatide ( p < 0.001). CONCLUSIONS: Aprotinin has no procoagulatory effect on platelet function during hypothermia but is not protective either. Eptifibatide inhibits hypothermia-induced platelet aggregation in vitro and may prevent aggregate sequestration in the microvasculature and consecutive ischemic organ damage in vivo.
Subject(s)
Aprotinin/pharmacology , Cold Temperature , Peptides/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation/drug effects , Platelet Glycoprotein GPIIb-IIIa Complex/antagonists & inhibitors , Blood Platelets/drug effects , Blood Specimen Collection , Eptifibatide , Flow Cytometry , Humans , Hypothermia/blood , In Vitro Techniques , MaleABSTRACT
After blunt chest trauma, a patient with chronic coronary heart disease sustained an isolated rupture of the right coronary artery. All findings suggested a heart contusion complicated by a non-compromising pericardial effusion and aggravated by anticoagulation with phenprocoumon. After right-ventricular failure occurred, emergency coronary revascularization could not prevent a fatal outcome. This case emphasizes that a coronary artery lesion may be considered in those cases of thoracic trauma with preexisting coronary calcification.
Subject(s)
Arteries/injuries , Coronary Vessels/injuries , Heart Rupture/etiology , Thoracic Injuries/complications , Wounds, Nonpenetrating/complications , Aged , Coronary Disease/complications , Heart Rupture/diagnosis , Humans , Male , Tomography, X-Ray Computed , Ventricular Dysfunction, Right/complicationsABSTRACT
OBJECTIVES: To determine the effectiveness of BAY 41-2272 on penile erections in an in vivo rabbit model. The nitric oxide (NO)-dependent increase of intracellular cyclic guanosine monophosphate (cGMP) by cGMP-phosphodiesterase (PDE5) inhibition has been shown to be an effective mechanism in the treatment of erectile dysfunction. Direct, NO-independent stimulation of soluble guanylyl cyclase should also lead to elevated cGMP levels in tissues and could be an attractive alternative therapeutic option for the treatment of erectile dysfunction. BAY 41-2272 is a novel non-NO-based direct stimulator of soluble guanylyl cyclase that activates purified enzyme in a synergistic fashion with NO. METHODS: BAY 41-2272 was administered to conscious rabbits intravenously (IV) and orally (PO). Erection was assessed in a time-dependent manner by measuring the length of the uncovered penile mucosa. Erections were evaluated in the absence and presence of NO (with intravenous sodium nitroprusside [SNP] as the NO donor). RESULTS: BAY 41-2272 only induced weak penile erections in conscious rabbits after IV (1 mg/kg) and PO (10 mg/kg) administration in the absence of an NO donor. However, the efficacy of BAY 41-2272 was potentiated by the simultaneous administration of SNP. Through simultaneous SNP administration, the effective doses of BAY 41-2272 were reduced significantly (minimal effective dose 0.1 mg/kg IV and 1 mg/kg PO). CONCLUSIONS: The results of this study clearly demonstrated the effect of BAY 41-2272 on penile erection in the conscious rabbit model after PO and IV administration. The time-course and onset of erection was concurrent with the stimulation by exogenous NO (SNP), suggesting that this new pharmacologic mechanism of soluble guanylyl cyclase stimulation could be used in the treatment of erectile dysfunction. Because the effect is increased by SNP, it can be expected that BAY 41-2272 would have enhanced activity during sexual arousal, when NO is produced endogenously.
Subject(s)
Guanylate Cyclase/drug effects , Nitric Oxide/pharmacology , Penile Erection/drug effects , Pyrazoles/pharmacology , Pyridines/pharmacology , Administration, Oral , Animals , Dose-Response Relationship, Drug , Guanylate Cyclase/physiology , Injections, Intravenous , Male , Models, Animal , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/physiology , Nitric Oxide/administration & dosage , Nitric Oxide Donors/pharmacology , Penile Erection/physiology , Penis/blood supply , Penis/drug effects , Penis/physiology , Pyrazoles/administration & dosage , Pyrazoles/therapeutic use , Pyridines/administration & dosage , Pyridines/therapeutic use , RabbitsABSTRACT
A new local delivery device (LDD) capable of releasing silver in periodontal pockets has been developed and tested pre-clinically. Silver has potent antimicrobial effects on Gram-negative periodontal pathogens with a mean in vitro minimum bactericidal concentration (MBC) < or =0.5 microg/ml. This phase 1 study assessed the safety, pharmacokinetics and bioavailability of silver ions delivered intracrevicularly with a resorbable LDD (PocketGuard) in a group of 9 volunteers affected with periodontitis. In each subject, a PLGA/PEG LDD loaded with 12% silver nitrate (w/w) was inserted in each of 4 selected pockets > or =5 mm. Serum, gingival fluid and subgingival plaque samples were evaluated before and at various time points after LDD placement for 21 days. At each time point, the concentration of silver in gingival crevicular fluid (GCF) was quantified with an Inductively Coupled Plasma-Mass Spectrometer. Subgingival plaque samples were processed for evaluation of total anaerobic and aerobic counts (CFU/ml). The maximum mean silver concentration in GCF was 1,493 +/- 709 microg/ml (range 589-2,245). It decayed exponentially with a half-life of 7.1 +/- 6.1 days (2.7-20.4). Average silver concentrations in excess of 10 microg/ml were detected in each patient for 14 days after LDD placement with the average concentration for all patients in excess of 25 microg/mL at day 21. Total anaerobic counts decreased an average of 1.7 +/- 1.9 x 10(6) CFU/ml (p= 0.0078) from baseline to day 7, indicating that the silver was biologically active. A mild increase in cervical root discoloration was observed at day 21:0.25 +/- 0.31 stain index units. Discoloration that did not resolve spontaneously could be removed at the end of the study with polishing. No systemic effects were observed. It is concluded that local silver concentrations above the MBC in serum were maintained for at least 21 days. A specific microbiologic effect was also observed.
Subject(s)
Anti-Infective Agents, Local/administration & dosage , Drug Delivery Systems , Periodontal Pocket/drug therapy , Silver Nitrate/administration & dosage , Absorbable Implants , Anti-Infective Agents, Local/analysis , Anti-Infective Agents, Local/blood , Anti-Infective Agents, Local/pharmacokinetics , Bacteria, Aerobic/drug effects , Bacteria, Aerobic/growth & development , Bacteria, Anaerobic/drug effects , Bacteria, Anaerobic/growth & development , Biocompatible Materials , Biological Availability , Colony Count, Microbial , Dental Plaque/chemistry , Dental Plaque/microbiology , Female , Gingival Crevicular Fluid/chemistry , Humans , Male , Mass Spectrometry , Middle Aged , Polyethylene Glycols , Polyglactin 910 , Safety , Silver Nitrate/analysis , Silver Nitrate/blood , Silver Nitrate/pharmacokinetics , Statistics as Topic , Tooth Discoloration/chemically induced , Tooth Root/drug effectsABSTRACT
Nitric oxide (NO) is a widespread, potent, biological mediator that has many physiological and pathophysiological roles. Research in the field of NO appears to have followed a straightforward path, and the findings have been progressive: NO and cyclic GMP are involved in vasodilatation; glycerol trinitrate relaxes vascular smooth muscles by bioconversion to NO; mammalian cells synthesize NO; and last, NO mediates vasodilatation by stimulating the soluble guanylate cyclase (sGC), a heterodimeric (alpha/beta) haem protein that converts GTP to cGMP2-4. Here we report the discovery of a regulatory site on sGC. Using photoaffinity labelling, we have identified the cysteine 238 and cysteine 243 region in the alpha1-subunit of sGC as the target for a new type of sGC stimulator. Moreover, we present a pyrazolopyridine, BAY 41-2272, that potently stimulates sGC through this site by a mechanism that is independent of NO. This results in antiplatelet activity, a strong decrease in blood pressure and an increase in survival in a low-NO rat model of hypertension, and as such may offer an approach for treating cardiovascular diseases.
Subject(s)
Guanylate Cyclase/chemistry , Nitric Oxide/chemistry , Amino Acid Sequence , Animals , Antihypertensive Agents/therapeutic use , Binding Sites , Blood Pressure/drug effects , Cyclic N-Oxides/pharmacology , Cysteine/chemistry , Disease Models, Animal , Enzyme Activation , Female , Guanylate Cyclase/metabolism , Heme/chemistry , Humans , Imidazoles/pharmacology , In Vitro Techniques , Indazoles/pharmacology , Molecular Sequence Data , Photoaffinity Labels , Platelet Aggregation Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyridines/pharmacology , Rats , SolubilityABSTRACT
OBJECTIVE: The p53 status is increasingly regarded as a marker predictive of response to particular cancer therapies, but for this approach it is self-evident that the p53 status must be determined correctly. METHODS: We have tested ovarian cancers with single-strand conformation polymorphism analysis (SSCP), immunohistochemical staining with DO-1 anti-p53 antibody (IHC), and yeast p53 functional assay (FASAY). RESULTS: These techniques commonly used to detect p53 mutations showed important differences in their sensitivity. Of 53 tumors tested with three indirect techniques, 27 (50%), 33 (62%) and 41 (77%) were positive by SSCP, IHC, and FASAY, respectively. In a subset of 32 tumors strongly suspected of containing mutations, 25 (78%), 26 (81%), 29 (91%) and 30 (94%) were positive by SSCP, immunostaining, DNA sequencing and yeast assay, respectively. CONCLUSIONS: Under comparable routine conditions, the FASAY reached the highest sensitivity. Since no single technique detected all mutations, we recommend the use of at least two different techniques in situations where the p53 status will affect patient management.
Subject(s)
DNA Mutational Analysis/methods , Genes, p53/genetics , Mutation , Ovarian Neoplasms/genetics , Alleles , Female , Humans , Immunohistochemistry , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Sequence Analysis, DNA , Yeasts/geneticsABSTRACT
SARs around a novel type of guanylate cyclase stimulator which act by a mechanism different from classical NO-donors are described. Several pyrazolopyridinylpyrimidines are shown to relax aortic rings and revealed a long-lasting blood pressure lowering effect in rats after oral application.
Subject(s)
Enzyme Activators/pharmacology , Guanylate Cyclase/metabolism , Indazoles/pharmacology , Animals , Aorta/drug effects , Aorta/physiology , Enzyme Activators/chemical synthesis , Enzyme Activators/chemistry , Indazoles/chemical synthesis , Indazoles/chemistry , Pyrazoles/chemistry , Pyrazoles/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Rabbits , Structure-Activity Relationship , Vasodilation/drug effectsABSTRACT
BACKGROUND: The most important receptor for nitric oxide is the soluble guanylate cyclase (sGC), a heme containing heterodimer. Recently, a pyrazolopyridine derivative BAY 41-2272, structurally related to YC-1, was identified stimulating soluble guanylate cyclase in an NO-independent manner, which results in vasodilatation and antiplatelet activity. The study described here addresses the identification of the NO-independent site on soluble guanylate cyclase. RESULTS: We developed a photoaffinity label (3H-meta-PAL) for the direct and NO-independent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 by introducing an azido-group into the tritium labeled compound. The synthesized photoaffinitylabel directly stimulates the purified sGC and shows in combination with NO a synergistic effect on sGC activity. Irradiation with UV light of 3H-meta-PAL together with the highly purified sGC leads to a covalent binding to the alpha1-subunit of the enzyme. This binding is blocked by unlabeled meta-PAL, YC-1 and BAY 41-2272. For further identification of the NO-independent regulatory site the 3H-meta-PAL labeled sGC was fragmented by CNBr digest. The 3H-meta-PAL binds to a CNBr fragment, consisting of the amino acids 236-290 of the alpha1-subunit. Determination of radioactivity of the single PTH-cycles from the sequencing of this CNBr fragment detected the cysteines 238 and 243 as binding residues of the 3H-meta-PAL. CONCLUSIONS: Our data demonstrate that the region surrounding the cysteines 238 and 243 in the alpha1-subunit of the sGC could play an important role in regulation of sGC activity and could be the target of this new type of sGC stimulators.
Subject(s)
Enzyme Activators/pharmacology , Guanylate Cyclase/metabolism , Indazoles/pharmacology , Nitric Oxide/metabolism , Pyrazoles/pharmacology , Pyridines/pharmacology , Animals , Cells, Cultured , Enzyme Activation , Guanylate Cyclase/genetics , Insecta/cytology , Photoaffinity Labels , Recombinant Proteins/drug effects , Recombinant Proteins/metabolismABSTRACT
BACKGROUND: Improvement of thrombolysis may be achieved by concomitant strong platelet inhibition. To monitor platelet function in patients with myocardial infarction (n=46) who were treated with the fibrinolytic agent reteplase, the glycoprotein (GP) IIb/IIIa blocker abciximab, and the ADP receptor antagonist ticlopidine, we developed a flow cytometric assay. METHODS AND RESULTS: Binding of abciximab to platelets was directly monitored as the percentage of platelets stained by a goat anti-mouse antibody. Blood drawn 10 minutes and 2 hours after the start of therapy with reteplase and abciximab and during the 12-hour infusion of abciximab demonstrated a maximal blockade of GP IIb/IIIa (10 minutes, 86.2+/-10.3%; 12 hours, 85.8+/-7.1%). Starting at 24 hours, abciximab binding gradually decreased (24 hours, 74.6+/-16.2%; 48 hours, 66.8+/-14.9%; 72 hours, 60.5+/-16.7%; 96 hours, 49.4+/-17.8%; 120 hours, 35.8+/-16. 4%; and 144 hours, 29.9+/-15.3%). Binding of a chicken anti-fibrinogen antibody to platelets, indicating the level of functional blockade of GP IIb/IIIa, was inversely correlated with the binding of abciximab (r=-0.72, P:<0.0001). In blood drawn at 10 minutes, platelet aggregation was maximally inhibited but recovered within 48 hours even if the majority of GP IIb/IIIa receptors were still blocked by abciximab. Reteplase did not influence abciximab binding and did not activate platelets, as measured by P-selectin expression, fibrinogen binding, and platelet aggregation. Platelet inhibition that was achieved during the first 24 hours by abciximab was directly maintained by additional treatment with ticlopidine. CONCLUSIONS: Flow cytometric monitoring of platelet function allows differentiation of the effects of reteplase, abciximab, and ticlopidine. The combination of abciximab and ticlopidine is an attractive therapeutic strategy that provides a fast and continuous platelet inhibition.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Blood Platelets/drug effects , Fibrinolytic Agents/therapeutic use , Immunoglobulin Fab Fragments/therapeutic use , Myocardial Infarction/blood , Myocardial Infarction/drug therapy , Platelet Aggregation Inhibitors/therapeutic use , Platelet Glycoprotein GPIIb-IIIa Complex/drug effects , Recombinant Proteins/therapeutic use , Ticlopidine/therapeutic use , Tissue Plasminogen Activator/therapeutic use , Abciximab , Aged , Antibodies, Monoclonal/blood , Blood Platelets/physiology , Female , Fibrinolytic Agents/blood , Flow Cytometry , Humans , Immunoglobulin Fab Fragments/blood , Male , Middle Aged , Platelet Aggregation/drug effects , Platelet Aggregation Inhibitors/blood , Recombinant Proteins/blood , Ticlopidine/blood , Time Factors , Tissue Plasminogen Activator/bloodABSTRACT
The blockade of the platelet integrin glycoprotein (GP) IIb/IIIa has proved to be an effective antiplatelet therapy. Profound thrombocytopenia has repeatedly been described as an adverse effect in patients treated with GP IIb/IIIa inhibitors, but its mechanism has not been elucidated yet. With use of flow cytometry, the activation status of platelets was monitored in 26 patients presenting with acute myocardial infarction who were treated with the GP IIb/IIIa inhibitor abciximab alone or in combination with the fibrinolytic agent reteplase. Fibrinogen and PAC-1 (a GP IIb/IIIa activation-specific monoclonal antibody) binding, as well as P-selectin expression on unstimulated platelets were constant in 25 patients throughout a follow-up of 7 days. In 1 patient (D.F.), the percentage of platelet-binding fibrinogen increased from 2.2% to 17.8%, for PAC-1 from 2.8% to 13.2%, and for P-selectin expression from 10.2% to 58.3% 10 minutes after the start of treatment. Furthermore, D.F. had a decrease in single platelet count in ethylenediaminetetraacetic acid-, citrate-, and heparin-anticoagulated and native blood. Blood films revealed platelet aggregates. In vitro testing of D.F.'s blood 2 and 4 weeks after initial admission demonstrated a reinduction of fibrinogen and PAC-1 binding to platelets, an increase of P-selectin expression, and formation of platelet aggregates following exposition of platelets to abciximab in vitro. In summary, this report describes the induction of platelet activation by a GP IIb/IIIa inhibitor in vivo and reinduction in vitro in direct association with thrombocytopenia. Platelet activation by GP IIb/IIIa inhibitors may be one potential mechanism for GP IIb/IIIa inhibitor-induced thrombocytopenia.
Subject(s)
Antibodies, Monoclonal/adverse effects , Immunoglobulin Fab Fragments/adverse effects , Myocardial Infarction/drug therapy , Platelet Activation/drug effects , Platelet Aggregation Inhibitors/adverse effects , Platelet Glycoprotein GPIIb-IIIa Complex/antagonists & inhibitors , Thrombocytopenia/chemically induced , Abciximab , Adult , Aged , Antibodies, Monoclonal/therapeutic use , Drug Therapy, Combination , Dual Specificity Phosphatase 2 , Female , Fibrinogen/metabolism , Fibrinolytic Agents/adverse effects , Fibrinolytic Agents/therapeutic use , Humans , Immunoglobulin Fab Fragments/therapeutic use , Male , Middle Aged , Myocardial Infarction/blood , P-Selectin/blood , Platelet Aggregation/drug effects , Platelet Aggregation Inhibitors/therapeutic use , Platelet Count/drug effects , Protein Phosphatase 2 , Protein Tyrosine Phosphatases/blood , Recombinant Proteins/adverse effects , Recombinant Proteins/therapeutic use , Recurrence , Thrombocytopenia/blood , Thrombolytic Therapy , Tissue Plasminogen Activator/adverse effects , Tissue Plasminogen Activator/therapeutic useABSTRACT
Lumbar isokinetic strength and the influence of age, bodyweight and testing velocity in patients with chronic low back pain in comparison with persons without pain. Lumbar isokinetic strength parameters of 80 patients with chronic low back pain and 70 persons without pain were compared and the influence of age, bodyweight and testing velocity was evaluated. The patients with chronic low back pain showed less strength than the persons without pain. All parameters of extension discriminated between the two groups whereas only some of the flexion parameters did. The isokinetic strength of the lumbal extension muscles was higher than the strength of the flexion muscles. In patients with chronic low back pain, isokinetic strength of lumbar extension muscles was more reduced than the strength of flexion muscles in comparison with persons without pain. At 90 degrees/sec in comparison to 60 degrees/sec, lower extension forces, higher flexion forces and changed ratios of flexion and extension muscles were measured. Age had an influence only on women. There were no changes in ratios of flexion and extension muscles with increasing age. Bodyweight showed weak correlations with isokinetic flexion forces. The influence of different factors on isokinetic force varies between patients with chronic low back patients and healthy subjects.