scholarly journals Effects of cycloheximide or 6-dimethyl aminopurine on the parthenogenetic activation of pig oocytes using pulsatile treatment with nitric oxide donor

2009 ◽  
Vol 54 (No. 7) ◽  
pp. 293-306 ◽  
Author(s):  
T. Krejčová ◽  
J. Petr ◽  
M. Krejčová ◽  
K. Kheilová
Keyword(s):  
Nitric Oxide ◽  
Kinase Inhibitor ◽  
Calcium Ionophore ◽  
Continuous Treatment ◽  
Nitric Oxide Donor ◽  
Activation Rate ◽  
Morula Stage ◽  
Inhibitor Of Protein Synthesis ◽  
Parthenogenetic Embryos

Pig oocytes matured <I>in vitro</I> were parthenogenetically activated using nitric oxide donor SNAP (2mM). Continuous treatment successfully activated the oocytes only after more than 12 hours of exposure. Pulsatile treatments during which oocytes were repeatedly exposed to 2mM SNAP for a short time (10, 20 or 30 minutes) were more efficient with regard to the activation rate, even when the total exposure time did not exceed 4 hours. Parthenogenetic development was very limited after continuous treatment with 2mM SNAP. A significantly higher proportion of developing parthenogenetic embryos was observed after the pulsatile treatment (development to the morula stage 0 vs. 18%; development to the blastocyst 0 vs. 7%; <I>P</I> < 0.05). However, this developmental rate was significantly lower (<I>P</I> < 0.05) than the development induced by conventional activation treatment with calcium ionophore (development to the morula stage, 23%; development to the blastocyst stage, 18%). When we combined pulsatile SNAP-treatment with the effect of protein kinase inhibitor 6-dimethyl aminopurine (6-DMAP) (2mM 6-DMAP for 2 hours) or with the inhibitor of protein synthesis cycloheximide (CHX) (10 µM CHX for 2 hours), we observed a significant increase (<I>P</I> < 0.05) in the activation rate when compared to the respective pulsatile SNAP-treatment without 6-DMAP or CHX (63 vs. 78% of activated oocytes for 6-DMAP; 63 vs. 83% of activated oocytes for CHX). However, the development of parthenogenetic embryos was not enhanced when the pulsatile SNAP-treatment was combined with 6-DMAP or with CHX.

scholarly journals Parthenogenetic Activation of Porcine Oocytes by Calcium Ionophore A23187

2005 ◽  
Vol 48 (1) ◽  
pp. 60-67 ◽  
Author(s):  
S. Yin ◽  
M. Hishinuma ◽  
K. Hamana ◽  
J. Sekine
Keyword(s):  
Calcium Ionophore ◽  
Developmental Rate ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Cleavage Rate ◽  
North Carolina State University ◽  
Activation Rate ◽  
Morula Stage ◽  
Almost All

Abstract. This study was designated to clarify the influence of activation of porcine matured oocytes by calcium ionophore on in vitro development of the parthenotes. The follicular oocytes were matured, activated and cultured in North Carolina State University-23 (NCSU-23) medium supplemented with 10% porcine follicular fluid (pFF). The in vitro-matured oocytes were exposed to calcium ionophore at concentrations of 12.5, 25 or 50 μM for 3, 5, 7 or 9 min. The activation rate of the oocytes increased as concentration of ionophore decreased, being at 27–33 and 68–77 % for the oocytes treated with 50 and 12.5 μM ionophore, respectively. Almost all activated oocytes were haploid. The highest cleavage rate (76%) and developmental rate to morula (41%) were observed in the oocytes treated with 12.5 μM ionophore for 5 min. However, development to blastocyst was observed only in the oocytes treated with 25 μM ionophore for 3 and 5 min (3 and 4% of treated oocytes, respectively). We concluded that the activation treatment of the porcine oocytes with 12.5 μM ionophore for 5 min provided the highest develop-mental rate to morula, but this treatment is not sufficient to overcome a developmental block at the morula stage.

scholarly journals Effects of protein kinase C on parthenogenetic activation of pig oocytes using calcium ionophore or nitric oxide-donor

2008 ◽  
Vol 52 (No. 12) ◽  
pp. 415-422 ◽  
Author(s):  
J. Petr ◽  
E. Chmelíková ◽  
A. Dörflerová ◽  
M. Ješeta ◽  
Z. Kuthanová
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Calcium Ionophore ◽  
Nitric Oxide Donor ◽  
Oocyte Activation ◽  
Parthenogenetic Activation ◽  
Kinase C ◽  
Activation Rate ◽  
Pkc Inhibitors

Porcine oocytes matured <I>in vitr</I>o were activated for parthenogenetic development using either calcium ionophore (50&mu;M for 10 min) or nitric oxide donor SNAP (2mM for 23.5 hours). Protein kinase C (PKC) inhibitors, bisindolylmaleimide I or rottlerin, are able to inhibit parthenogenetic activation induced by calcium ionophore. The rate of activated oocytes decreased from 69% to 2% (<I>P</I> < 0.05) under the effect of bisindolylmaleimide I at a concentration of 0 or 20nM, respectively. The activation rate decreased from 68% to 0% (<I>P</I> < 0.05) under the influence of 0 or 20&mu;M rottlerin, respectively. PKC inhibitors Go6976 or hispidin had no effect on the oocyte activation using calcium ionophore or on oocytes activated by a nitric oxide donor. The activation of oocytes by a nitric oxide donor is not significantly influenced even under the effects of bisindolylmaleimide I or rottlerin. Based on these data we can conclude that the oocyte activation induced by calcium ionophore depends on PKC, especially on PKC-δ. On the other hand, the oocyte activation induced by nitric oxide is independent of the tested isotypes of PKC.

scholarly journals Cephalosporin nitric oxide-donor prodrug DEA-C3D disperses biofilms formed by clinical cystic fibrosis isolates of Pseudomonas aeruginosa

2019 ◽  
Vol 75 (1) ◽  
pp. 117-125 ◽  
Author(s):  
Odel Soren ◽  
Ardeshir Rineh ◽  
Diogo G Silva ◽  
Yuming Cai ◽  
Robert P Howlin ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Clinical Isolates ◽  
Nitric Oxide Donor ◽  
Confocal Laser ◽  
Broth Microdilution Method

Abstract Objectives The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D (‘DiEthylAmin-Cephalosporin-3′-Diazeniumdiolate’) has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro. Methods β-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. Results DEA-C3D was confirmed to selectively release NO in response to contact with bacterial β-lactamase. Despite lacking direct, cephalosporin/β-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. Conclusions DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.

Inhibition of tetrahydrobiopterin biosynthesis impairs endothelium-dependent relaxations in canine basilar artery

1997 ◽  
Vol 273 (2) ◽  
pp. H718-H724 ◽  
Author(s):  
H. Kinoshita ◽  
S. Milstien ◽  
C. Wambi ◽  
Z. S. Katusic
Keyword(s):  
Nitric Oxide ◽  
Superoxide Dismutase ◽  
Nitric Oxide Synthase ◽  
Endothelial Function ◽  
Isometric Force ◽  
Cerebral Arteries ◽  
Calcium Ionophore ◽  
Nitric Oxide Donor ◽  
Ionophore A23187 ◽  
Oxide Synthase

Tetrahydrobiopterin is an essential cofactor in biosynthesis of nitric oxide. The present study was designed to determine the effect of decreased intracellular tetrahydrobiopterin levels on endothelial function of isolated cerebral arteries. Blood vessels were incubated for 6 h in minimum essential medium (MEM) in the presence or absence of a GTP cyclohydrolase I inhibitor, 2,4-diamino-6-hydroxypyrimidine (DAHP, 10(-2) M). Rings with and without endothelium were suspended for isometric force recording in the presence of a cyclooxygenase inhibitor, indomethacin (10(-5) M). In arteries with endothelium, DAHP significantly reduced intracellular levels of tetrahydrobiopterin. DAHP in combination with a precursor of the salvage pathway of tetrahydrobiopterin biosynthesis, sepiapterin (10(-4) M), not only restored but increased levels of tetrahydrobiopterin above control values. In DAHP-treated arteries, endothelium-dependent relaxations to bradykinin (10(-10)-10(-6) M) or calcium ionophore A23187 (10(-9)-10(-6) M) were significantly reduced, whereas endothelium-independent relaxations to a nitric oxide donor, 3-morpholinosydnonimine (10(-9)-10(-4) M), were not affected. When DAHP-treated arteries with endothelium were incubated with sepiapterin (10(-4) M) or superoxide dismutase (150 U/ml), relaxations to bradykinin and A23187 were restored to control levels. In contrast, superoxide dismutase did not affect endothelium-dependent relaxations in arteries incubated in MEM. A nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (10(-4) M), abolished relaxations to bradykinin or A23187 in control arteries and in DAHP-treated arteries. These studies demonstrate that in cerebral arteries, decreased intracellular levels of tetrahydrobiopterin can reduce endothelium-dependent relaxations. Production of superoxide anions during activation of dysfunctional endothelial nitric oxide synthase appears to be responsible for the impairment of endothelial function.

Nitric oxide and cGMP protein kinase activity in aged ventricular myocytes

2001 ◽  
Vol 281 (6) ◽  
pp. H2304-H2309 ◽  
Author(s):  
Qihang Zhang ◽  
Bruno Molino ◽  
Lin Yan ◽  
Todd Haim ◽  
Yakir Vaks ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Ventricular Myocytes ◽  
Nitric Oxide Donor ◽  
Protein Kinase Activity ◽  
Edge Detector ◽  
Negative Effects ◽  
Dependent Protein

We tested the hypothesis that nitric oxide-induced negative functional effects through cGMP would be reduced in aged cardiac myocytes. Maximum rate of shortening ( R max) and percent shortening of ventricular myocytes from young (6 mo) and old (3 y) rabbits were studied using a video edge detector. cGMP-dependent phosphorylation was examined by electrophoresis and autoradiography. Myocytes received a nitric oxide donor S-nitroso- N-acetyl-penicillamine (SNAP, 10−7, 10−6, and 10−5 M) followed by KT-5823 (10−6 M), a cGMP protein kinase inhibitor. Baseline function was similar in young and old myocytes (89.1 ± 4.5 young vs. 86.4 ± 8.3 μm/s old R max, 5.6 ± 0.3 vs. 5.2 ± 0.7%shortening). SNAP (10−5 M) decreased R max in both young (25%, n = 6) and old myocytes (24%, n = 7). SNAP also reduced percent shortening by 28% in young and 23% in old myocytes. The negative effects of SNAP were partially reversed by KT-5823 only in young myocytes. Multiple proteins were phosphorylated by cGMP, and KT-5823 could reduce this effect. The degree of phosphorylation was significantly less in old myocytes. These results suggest that the functional response of ventricular myocytes to nitric oxide was preserved during aging. However, the importance of cGMP-dependent protein phosphorylation was decreased, indicating a shift to other pathways.

Caspase Activation Is Required for Nitric Oxide–Mediated, CD95(APO-1/Fas)–Dependent and Independent Apoptosis in Human Neoplastic Lymphoid Cells

Blood ◽  
1998 ◽  
Vol 91 (11) ◽  
pp. 4311-4320 ◽  
Author(s):  
Katerina Chlichlia ◽  
Marcus E. Peter ◽  
Marian Rocha ◽  
Carsten Scaffidi ◽  
Mariana Bucur ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Present Report ◽  
Lymphoid Cells ◽  
Jurkat Cells ◽  
Target Cells ◽  
No Production ◽  
No Donor ◽  
Synthase Inhibitor ◽  
Inhibitor Of Protein Synthesis

Abstract Nitric oxide (NO), an important effector molecule involved in immune regulation and host defense, was shown to induce apoptosis in lymphoma cells. In the present report the NO donor glycerol trinitrate was found to induce apoptosis in Jurkat cells that are sensitive to CD95-mediated kill. In contrast, a CD95-resistant Jurkat subclone showed substantial protection from apoptosis after exposure to NO. NO induced mRNA expression of CD95 (APO-1/Fas) and TRAIL/APO-2 ligands. Moreover, NO triggered apoptosis in freshly isolated human leukemic lymphocytes which were also sensitive to anti-CD95 treatment. The ability of NO to induce apoptosis was completely blocked by a broad-spectrum ICE (interleukin-1β converting enzyme)-protease/caspase inhibitor and correlated with FLICE/caspase-8 activation. This activation was abrogated in some neoplastic lymphoid cells but not in others by the inhibitor of protein synthesis cycloheximide. Our results were confirmed using an in vitro experimental model of coculture of human lymphoid target cells with activated bovine endothelial cells generating NO as effectors. Furthermore, the inhibition of endogenous NO production with the inducible NO synthase inhibitor NG-monomethyl-L-arginine caused a complete abrogation of the apoptotic effect. Our data provide evidence that NO-induced apoptosis in human neoplastic lymphoid cells strictly requires activation of caspases, in particular FLICE, the most CD95 receptor-proximal caspase. Depending on the cell line tested this activation required or was independent of the CD95 receptor/ligand system.

Protein disulphide isomerase-mediated LA419– NO release provides additional antithrombotic effects to the blockade of the ADP receptor

2007 ◽  
Vol 97 (04) ◽  
pp. 650-657 ◽  
Author(s):  
Gemma Vilahur ◽  
Esther Pena ◽  
Teresa Padró ◽  
Lina Badimon
Keyword(s):  
Nitric Oxide ◽  
Platelet Aggregation ◽  
Vessel Wall ◽  
Oral Treatment ◽  
Nitric Oxide Donor ◽  
Hemodynamic Parameters ◽  
Vascular Events ◽  
Protein Disulphide Isomerase ◽  
Antithrombotic Effects

SummaryDespite the proven efficacy of current antithrombotic therapy in preventing ischemic heart disease (IHD), vascular events still occur. Our aims were i) to evaluate if combined oral treatment of clopidogrel and LA419, a novel nitric oxide donor with antiischemic and antiplatelet properties, provides additional antiplatelet effects to those of the blockade of P2Y12 receptor; and ii) to gain insight into the mechanism behind LA419 antiplatelet effects. Pigs (n=16) were randomized into four groups: 1) placebocontrol; 2) LA419; 3) clopidogrel; and 4) LA419+clopidogrel. Both compounds were administered orally: LA419 0.9 mg kg-1 twice daily for 10 days; clopidogrel 10 mg kg-1 day the three last days. Antithrombotic effects were assessed by measuring platelet deposition (PD) triggered by denuded and disrupted vessel wall placed on the Badimon chamber. LA419 effects on platelet aggregation, hemodynamic parameters, and platelet protein expression upon in vitro thrombin stimulation were also evaluated. Total PD on denuded vessels was similarly reduced by all treatments with respect to placebo (p<0.05). However, combination of LA419+clopidogrel largely reduced PD triggered by disrupted vessel wall by 80% versus placebo (p<0.005), 15% versus clopidogrel alone (p<0.01), and 30% versus LA419 alone (p<0.005). All treatments inhibited collagen- and ADP-induced platelet aggregation, and no variations were detected in hemodynamic parameters. Proteomic analysis revealed that LA419 was associated with an increase in membrane protein disulphide isomerase (protein implicated in nitric oxide release).Treatment with LA419 may result in additional antiplatelet effect to that of clopidogrel in addition to restoring impaired endothelial dependent vasodilation without hemodynamic side effects. Further studies in IHD patients seem warranted.

scholarly journals Thioredoxin 1 Promotes Intracellular Replication and Virulence of Salmonella enterica Serovar Typhimurium

2006 ◽  
Vol 74 (9) ◽  
pp. 5140-5151 ◽  
Author(s):  
Eva Bjur ◽  
Sofia Eriksson-Ygberg ◽  
Fredrik Åslund ◽  
Mikael Rhen
Keyword(s):  
Oxidative Stress ◽  
Nitric Oxide ◽  
Salmonella Enterica ◽  
Nitric Oxide Donor ◽  
Intracellular Replication ◽  
Thioredoxin System ◽  
Serovar Typhimurium ◽  
Thioredoxin 1 ◽  
Disulfide Bond Isomerase

ABSTRACT The effect of the cytoplasmic reductase and protein chaperone thioredoxin 1 on the virulence of Salmonella enterica serovar Typhimurium was evaluated by deleting the trxA, trxB, or trxC gene of the cellular thioredoxin system, the grxA or gshA gene of the glutathione/glutaredoxin system, or the dsbC gene coding for a thioredoxin-dependent periplasmic disulfide bond isomerase. Mutants were tested for tolerance to oxidative and nitric oxide donor substances in vitro, for invasion and intracellular replication in cultured epithelial and macrophage-like cells, and for virulence in BALB/c mice. In these experiments only the gshA mutant, which was defective in glutathione synthesis, exhibited sensitization to oxidative stress in vitro and a small decrease in virulence. In contrast, the trxA mutant did not exhibit any growth defects or decreased tolerance to oxidative or nitric oxide stress in vitro, yet there were pronounced decreases in intracellular replication and mouse virulence. Complementation analyses using defined catalytic variants of thioredoxin 1 showed that there is a direct correlation between the redox potential of thioredoxin 1 and restoration of intracellular replication of the trxA mutant. Attenuation of mouse virulence that was caused by a deficiency in thioredoxin 1 was restored by expression of wild-type thioredoxin 1 in trans but not by expression of a catalytically inactive variant. These results clearly imply that in S. enterica serovar Typhimurium, the redox-active protein thioredoxin 1 promotes virulence, whereas in vitro tolerance to oxidative stress depends on production of glutathione.

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