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Rungusa Pantan Amnart Onsa-ard Jiraporn Tocharus Orawan Wonganan Apichart Suksamrarn Chainarong Tocharus 《Life sciences》2014
Aims
The aim of this study is to investigate the vasorelaxant effect of 16-O-acetyldihydroisosteviol (ADIS) and its underlying mechanisms in isolated rat aorta.Main methods
Rat aortic rings were isolated, suspended in organ baths containing Kreb's solution, maintained at 37 °C, and mounted on tungsten wire and continuously bubbled with a mixture of 95% O2 and 5% CO2 under a resting tension of 1 g. The vasorelaxant effects of ADIS were investigated by means of isometric tension recording experiment.Key findings
ADIS (0.1 μM–3 mM) induced relaxation of aortic rings pre-contracted by phenylephrine (PE, 10 μM) and KCl (80 mM) with intact-endothelium (Emax = 79.26 ± 3.74 and 79.88 ± 3.79, respectively) or denuded-endothelium (Emax = 88.05 ± 3.69 and 78.22 ± 6.86, respectively). In depolarization Ca2+-free solution, ADIS inhibits calcium chloride (CaCl2)-induced contraction in endothelium-denuded rings in a concentration-dependent manner. In addition, ADIS attenuates transient contractions in Ca2+-free medium containing EGTA (1 mM) induced by PE (10 μM) and caffeine (20 mM). By contrast, relaxation was not affected by tetraethylammonium (TEA, 5 mM), 4-aminopyridine (4-AP, 1 mM), glibenclamide (10 μM), barium chloride (BaCl2, 1 mM), and 1H-[1,2,3]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ, 1 μM).Significance
These findings reveal the vasorelaxant effect of ADIS, through endothelium-independent pathway. It acts as a Ca2 + channel blocker through both intracellular and extracellular Ca2 + release. 相似文献3.
M.C. Ribeiro M.S. Costa-Alves M. Wengert J.R. Meyer-Fernandes P. Zancan C. Caruso-Neves A.A.S. Pinheiro 《Biochimica et Biophysica Acta (BBA)/General Subjects》2012
Background
The concentration of extracellular nucleotides is regulated by enzymes that have their catalytic site facing the extracellular space, the so-called ecto-enzymes.Methods
We used LLC-PK1 cells, a well-characterized porcine renal proximal tubule cell line, to biochemically characterize ecto-ATPase activity in the luminal surface. The [γ-32P]Pi released after reaction was measured in aliquots of the supernatant by liquid scintillation.Results
This activity was linear with time up to 20 min of reaction and stimulated by divalent metals. The ecto-ATPase activity measured in the presence of 5 mM MgCl2 was (1) optimum at pH 8, (2) insensitive to different inhibitors of intracellular ATPases, (3) inhibited by 1 mM suramin, an inhibitor of ecto-ATPases, (4) sensitive to high concentrations of sodium azide (NaN3) and (5) also able to hydrolyze ADP in the extracellular medium. The ATP:ADP hydrolysis ratio calculated was 4:1. The ecto-ADPase activity was also inhibited by suramin and NaN3. The dose–response of ATP revealed a hyperbolic profile with maximal velocity of 25.2 ± 1.2 nmol Pi x mg− 1 x min− 1 and K0.5 of 0.07 ± 0.01 mM. When cells were submitted to ischemia, the E-NTPDase activity was reduced with time, achieving 71% inhibition at 60 min of ischemia.Conclusion
Our results suggest that the ecto-ATPase activity of LLC-PK1 cells has the characteristics of a type 3 E-NTPDase which is inhibited by ischemia.General Significance
This could represent an important pathophysiologic mechanism that explains the increase in ATP concentration in the extracellular milieu in the proximal tubule during ischemia. 相似文献4.
Hussein Kaddour Jacques Vergne Guy Herve Marie-Christine Maurel 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
Viroids are the smallest pathogens known to date. They infect plants and cause considerable economic losses. The members of the Avsunviroidae family are known for their capability to form hammerhead ribozymes (HHR) that catalyze self-cleavage during their rolling circle replication.Methods
In vitro inhibition assays, based on the self-cleavage kinetics of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid (CChMVd-HHR) were performed in the presence of various putative inhibitors.Results
Aminated compounds appear to be inhibitors of the self-cleavage activity of the CChMVd HHR. Surprisingly the spermine, a known activator of the autocatalytic activity of another hammerhead ribozyme in the presence or absence of divalent cations, is a potent inhibitor of the CChMVd-HHR with Ki of 17 ± 5 μM. Ruthenium hexamine and TMPyP4 are also efficient inhibitors with Ki of 32 ± 5 μM and IC50 of 177 ± 5 nM, respectively.Conclusions
This study shows that polyamines are inhibitors of the CChMVd-HHR self-cleavage activity, with an efficiency that increases with the number of their amino groups.General significance
This fundamental investigation is of interest in understanding the catalytic activity of HHR as it is now known that HHR are present in the three domains of life including in the human genome. In addition these results emphasize again the remarkable plasticity and adaptability of ribozymes, a property which might have played a role in the early developments of life and must be also of significance nowadays for the multiple functions played by non-coding RNAs. 相似文献5.
Aims
We previously reported that fluvoxamine, a selective serotonin reuptake inhibitor with high affinity for the σ1-receptor (σ1R), ameliorates cardiac hypertrophy and dysfunction via σ1R stimulation. Although σ1R on non-cardiomyocytes interacts with the IP3 receptor (IP3R) to promote mitochondrial Ca2 + transport, little is known about its physiological and pathological relevance in cardiomyocytes.Main methods
Here we performed Ca2 + imaging and measured ATP production to define the role of σ1Rs in regulating sarcoplasmic reticulum (SR)-mitochondrial Ca2 + transport in neonatal rat ventricular cardiomyocytes treated with angiotensin II to promote hypertrophy.Key finding
These cardiomyocytes exhibited imbalances in expression levels of σ1R and IP3R and impairments in both phenylephrine-induced mitochondrial Ca2 + mobilization from the SR and ATP production. Interestingly, σ1R stimulation with fluvoxamine rescued impaired mitochondrial Ca2 + mobilization and ATP production, an effect abolished by treatment of cells with the σ1R antagonist, NE-100. Under physiological conditions, fluvoxamine stimulation of σ1Rs suppressed intracellular Ca2 + mobilization through IP3Rs and ryanodine receptors (RyRs). In vivo, chronic administration of fluvoxamine to TAC mice also rescued impaired ATP production.Significance
These results suggest that σ1R stimulation with fluvoxamine promotes SR-mitochondrial Ca2 + transport and mitochondrial ATP production, whereas σ1R stimulation suppresses intracellular Ca2 + overload through IP3Rs and RyRs. These mechanisms likely underlie in part the anti-hypertrophic and cardioprotective action of the σ1R agonists including fluvoxamine. 相似文献6.
Hideaki Tagashira Chen Zhang Ying-mei Lu Hideyuki Hasegawa Hiroshi Kanai Feng Han Kohji Fukunaga 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
We previously reported that the σ1-receptor (σ1R) is down-regulated following cardiac hypertrophy and dysfunction in transverse aortic constriction (TAC) mice. Here we address how σ1R stimulation with the selective σ1R agonist SA4503 restores hypertrophy-induced cardiac dysfunction through σ1R localized in the sarcoplasmic reticulum (SR).Methods
We first confirmed anti-hypertrophic effects of SA4503 (0.1–1 μM) in cultured cardiomyocytes exposed to angiotensin II (Ang II). Then, to confirm the ameliorative effects of σ1R stimulation in vivo, we administered SA4503 (1.0 mg/kg) and the σ1R antagonist NE-100 (1.0 mg/kg) orally to TAC mice for 4 weeks (once daily).Results
σ1R stimulation with SA4503 significantly inhibited Ang II-induced cardiomyocyte hypertrophy. Ang II exposure for 72 h impaired phenylephrine (PE)-induced Ca2 + mobilization from the SR into both the cytosol and mitochondria. Treatment of cardiomyocytes with SA4503 largely restored PE-induced Ca2 + mobilization into mitochondria. Exposure of cardiomyocytes to Ang II for 72 h decreased basal ATP content and PE-induced ATP production concomitant with reduced mitochondrial size, while SA4503 treatment completely restored ATP production and mitochondrial size. Pretreatment with NE-100 or siRNA abolished these effects. Chronic SA4503 administration also significantly attenuated myocardial hypertrophy and restored ATP production in TAC mice. SA4503 administration also decreased hypertrophy-induced impairments in LV contractile function.Conclusions
σ1R stimulation with the specific agonist SA4503 ameliorates cardiac hypertrophy and dysfunction by restoring both mitochondrial Ca2 + mobilization and ATP production via σ1R stimulation.General significance
Our observations suggest that σ1R stimulation represents a new therapeutic strategy to rescue the heart from hypertrophic dysfunction. 相似文献7.
N. Sindhura Reddy S. Sowmya Joel D. Bumgardner K.P. Chennazhi Raja Biswas R. Jayakumar 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
The objective of this study was to fabricate, characterize and evaluate in vitro, an injectable calcium sulfate bone cement beads loaded with an antibiotic nanoformulation, capable of delivering antibiotic locally for the treatment of periodontal disease.Methods
Tetracycline nanoparticles (Tet NPs) were prepared using an ionic gelation method and characterized using DLS, SEM, and FTIR to determine size, morphology, stability and chemical interaction of the drug with the polymer. Further, calcium sulfate (CaSO4) control and CaSO4-Tet NP composite beads were prepared and characterized using SEM, FTIR and XRD. The drug release pattern, material properties and antibacterial activity were evaluated. In addition, protein adsorption, cytocompatibility and alkaline phosphatase activity of the CaSO4-Tet NP composite beads in comparison to the CaSO4 control were analyzed.Results
Tet NPs showed a size range of 130 ± 20 nm and the entrapment efficiency calculated was 89%. The composite beads showed sustained drug release pattern. Further the drug release data was fitted into various kinetic models wherein the Higuchi model showed higher correlation value (R2 = 0.9279) as compared to other kinetic models. The composite beads showed antibacterial activity against Staphylococcus aureus and Escherichia coli. The presence of Tet NPs in the composite bead didn't alter its cytocompatibility. In addition, the composite beads enhanced the ALP activity of hPDL cells.Conclusions
The antibacterial and cytocompatible CaSO4-Tet NP composite beads could be beneficial in periodontal management to reduce the bacterial load at the infection site.General significance
Tet NPs would deliver antibiotic locally at the infection site and the calcium sulfate cement, would itself facilitate tissue regeneration. 相似文献8.
Harmanpreet Kaur Rebecca Heeney Rupp Carriveau Gabriel Sosne Bulent Mutus 《Biochimica et Biophysica Acta (BBA)/General Subjects》2010
Background
Thymosin beta 4 (Tβ4) is a major actin sequestering peptide present in most mammalian cells. It also acts as an anti-inflammatory agent and promotes corneal wound healing.Methods
In the present study, we constructed a four channel cylindrical flow chambers out of polydimethylsiloxane (PDMS) on microscope coverslips. The platelet-binding proteins–fibrinogen and collagen–were immobilized onto the middle ~ 25% of the inner cylindrical surface. The flow method introduced here was employed to determine the effect of Tβ4, on the deposition of ADP-activated platelets onto fibrinogen cross-linked flow chambers.Results
The binding data from the flow chambers indicated that the both the rate constant of platelet deposition (average: 0.026 ± 0.0015 s− 1, corresponding to a half-life of 26.7 s) and the total number of deposited platelets were independent of the platelet binding protein and the activating agent. Our results show that low concentrations of Tβ4 (0.2 μM to 0.5 μM) increased both the rate constant of platelet deposition by ~ 1.5-fold (i.e. half-life decreased from 26.7 s to 17.6 s) and the total number of deposited platelets by ~ 3-fold. However at higher concentrations (> 1 μM) the Tβ4-potentiating effect was diminished to near control levels. Tβ4 did interact with fibrinogen with an estimated KD of ~ 126 ± 18 nM or 66 ± 20 nM under equilibrium or flow, respectively.Conclusion
These results suggest that Tβ4 could potentially increase the affinity of platelet receptors for their ligands thus promoting platelet deposition. Tβ4 could also bind to fibrinogen and as its concentration increased would prevent platelet–fibrinogen interactions resulting in the attenuation of platelet deposition.General significance
This work suggests that Tβ4 might have a dual role in platelet function. 相似文献9.
Background
Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. The majority of chitinase inhibitors reported are natural products like argifin, argifin linear fragments, argadin, allosamidin and disulfide-cyclized peptides. Here, we report a novel peptidic inhibitor API (Aspartic Protease Inhibitor), isolated from Bacillus licheniformis that inhibits chitinase A (ChiA) from Serratia marcescens.Methods
The binding affinity of API with ChiA and type of inhibition was determined by the inhibition kinetics assays. Fluorescence and CD spectroscopic analysis and chemical modification of API with different affinity reagents elucidated the mechanism of binding of API with ChiA.Results and conclusions
The peptide has an amino acid sequence N-Ile1-Cys2-Glu3-Ala4-Glu5-His6-Lys7-Trp8-Gly9-Asp10-Tyr11-Leu12-Asp13-C. The ChiA–API kinetic interactions reveal noncompetitive, irreversible and tight binding nature of API with I50 = 600 nM and Ki = 510 nM in the presence of chromogenic substrate p-nitrophenyl-N,N′-diacetyl-β-chitobioside[p-NP-(GlcNAc)2]. The inhibition progress curves show a two-step slow tight binding inhibition mechanism with the rate constant k5 = 8.7 ± 1 × 10− 3 s− 1 and k6 = 7.3 ± 0.6 × 10− 5 s− 1. CD-spectra and tryptophanyl fluorescence analysis of ChiA incubated with increasing API concentrations confirms conformational changes in enzyme structure which may be due to irreversible denaturation of enzyme upon binding of API. Chemical modifications by WRK abolished the anti-chitinase activity of API and revealed the involvement of carboxyl groups in the enzyme inactivation. Abolished isoindole fluorescence of OPTA-labeled ChiA demonstrates the irreversible denaturation of ChiA upon incubation with API for prolonged time and distortion of active site of the enzyme.General significance
The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors. 相似文献10.
Carrie A. May John K. Grady Thomas M. Laue Maura Poli Paolo Arosio N. Dennis Chasteen 《Biochimica et Biophysica Acta (BBA)/General Subjects》2010
Background
Ferritin exhibits complex behavior in the ultracentrifuge due to variability in iron core size among molecules. A comprehensive study was undertaken to develop procedures for obtaining more uniform cores and assessing their homogeneity.Methods
Analytical ultracentrifugation was used to measure the mineral core size distributions obtained by adding iron under high- and low-flux conditions to horse spleen (apoHoSF) and human H-chain (apoHuHF) apoferritins.Results
More uniform core sizes are obtained with the homopolymer human H-chain ferritin than with the heteropolymer horse spleen HoSF protein in which subpopulations of HoSF molecules with varying iron content are observed. A binomial probability distribution of H- and L-subunits among protein shells qualitatively accounts for the observed subpopulations. The addition of Fe2+ to apoHuHF produces iron core particle size diameters from 3.8 ± 0.3 to 6.2 ± 0.3 nm. Diameters from 3.4 ± 0.6 to 6.5 ± 0.6 nm are obtained with natural HoSF after sucrose gradient fractionation. The change in the sedimentation coefficient as iron accumulates in ferritin suggests that the protein shell contracts ∼ 10% to a more compact structure, a finding consistent with published electron micrographs. The physicochemical parameters for apoHoSF (15%/85% H/L subunits) are M = 484,120 g/mol, ν? = 0.735 mL/g, s20,w = 17.0 S and D20,w = 3.21 × 10−7 cm2/s; and for apoHuHF M = 506,266 g/mol, ν? = 0.724 mL/g, s20,w = 18.3 S and D20,w = 3.18 × 10−7 cm2/s.Significance
The methods presented here should prove useful in the synthesis of size controlled nanoparticles of other minerals. 相似文献11.
Fredy D.A. Silva Ilka M. Vasconcelos Marina D.P. Lobo Patrícia G. de Castro Vladimir G. Magalhães Cléverson D.T. de Freitas Célia R.R.S. Carlini Paulo M. Pinto Leila M. Beltramini José H.A. Filho Eduardo B. Barros Luciana M.R. Alencar Thalles B. Grangeiro José T.A. Oliveira 《Biochimica et Biophysica Acta (BBA)/General Subjects》2012
Background
Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx).Methods
Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography.Results
Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56–4.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% α-helix, 39% β-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys52 residue and the amino acids Pro45, Thr49 and Arg128 are conserved as in other 2-Cys-Prx.General significance
The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins. 相似文献12.
Salvatore Nesci Vittoria VentrellaFabiana Trombetti Maurizio PiriniAlessandra Pagliarani 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
The macrolide antibiotics oligomycin, venturicidin and bafilomycin, sharing the polyketide ring and differing in the deoxysugar moiety, are known to block the transmembrane ion channel of ion-pumping ATPases; oligomycins are selective inhibitors of mitochondrial ATP synthases.Methods
The inhibition mechanism of macrolides was explored on swine heart mitochondrial F1FO-ATPase by kinetic analyses. The amphiphilic membrane toxicant tributyltin (TBT) and the thiol reducing agent dithioerythritol (DTE) were used to elucidate the nature of the macrolide–enzyme interaction.Results
When individually tested, the macrolide antibiotics acted as uncompetitive inhibitors of the ATPase activity. Binary mixtures of macrolide inhibitors I1 and I2 pointed out a non-exclusive mechanism, indicating that each macrolide binds to its binding site on the enzyme. When co-present, the two macrolides acted synergistically in the formed quaternary complex (ESI1I2), thus mutually strengthening the enzyme inhibition. The enzyme inhibition by macrolides displaying a shared mechanism was dose-dependently reduced by TBT ≥ 1 μM. The TBT-driven enzyme desensitization was reversed by DTE.Conclusions
The macrolides tested share uncompetitive inhibition mechanism by binding to a specific site in a common macrolide-binding region of FO. The oxidation of highly conserved thiols in the ATP synthase c-ring of FO weakens the interaction between the enzyme and the macrolides. The native macrolide-inhibited enzyme conformation can be restored by reducing crucial thiols oxidized by TBT.General significance
The findings, by elucidating the macrolide inhibitory mechanism on FO, indirectly cast light on the F1FO torque generation involving crucial amino acid residues and may address drug design and antimicrobial therapy. 相似文献13.
Hideaki Tagashira Yasuharu Shinoda Norifumi Shioda Kohji Fukunaga 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
Amyotrophic lateral sclerosis (ALS) is a disease caused by motor neuron degeneration. Recently, a novel SIGMAR1 gene variant (p.E102Q) was discovered in some familial ALS patients.Methods
We address mechanisms underlying neurodegeneration caused by the mutation using Neuro2A cells overexpressing σ1RE102Q, a protein of a SIGMAR1 gene variant (p.E102Q) and evaluate potential amelioration by ATP production via methyl pyruvate (MP) treatment.Results
σ1RE102Q overexpression promoted dissociation of the protein from the endoplasmic reticulum (ER) membrane and cytoplasmic aggregation, which in turn impaired mitochondrial ATP production and proteasome activity. Under ER stress conditions, overexpression of wild-type σ1R suppressed ER stress-induced mitochondrial injury, whereas σ1RE102Q overexpression aggravated mitochondrial damage and induced autophagic cell death. Moreover, σ1RE102Q-overexpressing cells showed aberrant extra-nuclear localization of the TAR DNA-binding protein (TDP-43), a condition exacerbated by ER stress. Treatment of cells with the mitochondrial Ca2 + transporter inhibitor Ru360 mimicked the effects of σ1RE102Q overexpression, indicating that aberrant σ1R-mediated mitochondrial Ca2 + transport likely underlies TDP-43 extra-nuclear localization, segregation in inclusion bodies, and ubiquitination. Finally, enhanced ATP production promoted by methyl pyruvate (MP) treatment rescued proteasome impairment and TDP-43 extra-nuclear localization caused by σ1RE102Q overexpression.Conclusions
Our observations suggest that neurodegeneration seen in some forms of ALS are due in part to aberrant mitochondrial ATP production and proteasome activity as well as TDP-43 mislocalization resulting from the SIGMAR1 mutation.General significance
ATP supplementation by MP represents a potential therapeutic strategy to treat ALS caused by SIGMAR1 mutation. 相似文献14.
Stefan Dröse Peter J. Hanley Ulrich Brandt 《Biochimica et Biophysica Acta (BBA)/General Subjects》2009
Background
Reactive oxygen species (ROS) are among the main determinants of cellular damage during ischemia and reperfusion. There is also ample evidence that mitochondrial ROS production is involved in signaling during ischemic and pharmacological preconditioning. In a previous study we analyzed the mitochondrial effects of the efficient preconditioning drug diazoxide and found that it increased the mitochondrial oxidation of the ROS-sensitive fluorescent dye 2′,7′-dichlorodihydrofluorescein (H2DCF) but had no direct impact on the H2O2 production of submitochondrial particles (SMP) or intact rat heart mitochondria (RHM).Methods
H2O2 generation of bovine SMP and tightly coupled RHM was monitored under different conditions using the amplex red/horseradish peroxidase assay in response to diazoxide and a number of inhibitors.Results
We show that diazoxide reduces ROS production by mitochondrial complex I under conditions of reverse electron transfer in tightly coupled RHM, but stimulates mitochondrial ROS production at the Qo site of complex III under conditions of oxidant-induced reduction; this stimulation is greatly enhanced by uncoupling. These opposing effects can both be explained by inhibition of complex II by diazoxide. 5-Hydroxydecanoate had no effect, and the results were essentially identical in the presence of Na+ or K+ excluding a role for putative mitochondrial KATP-channels.General significance
A straightforward rationale is presented to mechanistically explain the ambivalent effects of diazoxide reported in the literature. Depending on the metabolic state and the membrane potential of mitochondria, diazoxide-mediated inhibition of complex II promotes transient generation of signaling ROS at complex III (during preconditioning) or attenuates the production of deleterious ROS at complex I (during ischemia and reperfusion). 相似文献15.
Estela D'Abril Ruíz-Leyja Rafael Villalobos-Molina Juan Javier López-Guerrero Itzell Alejandrina Gallardo-Ortíz Samuel Enoch Estrada-Soto Maximiliano Ibarra-Barajas 《Life sciences》2013
Aims
Hypertension is associated with the impairment of renal cyclooxygenase (COX) activity, which regulates vascular tone, salt and water balance and renin release. We aimed to evaluate the functional role of COX isoforms in kidneys isolated from spontaneously hypertensive rats (SHR) after α1-adrenoceptor (α1-AR) stimulation.Main methods
Male six-month-old SHR and normotensive Wistar-Kyoto rats (WKY) were used. The kidneys were isolated to measure perfusion pressure and COX-1- or COX-2-derived prostanoids in response to α1-AR activation.Key findings
The basal perfusion pressure was higher in SHR kidneys compared with WKY kidneys (95 ± 11 vs. 68 ± 6 mm Hg, P < 0.05). Phenylephrine induced a greater vasopressor response in SHR kidneys (EC50 of 1.89 ± 0.58 nmol) than WKY kidneys (EC50 of 3.30 ± 0.54 nmol, P < 0.05 vs. SHR). COX-1 inhibition decreased the α1-AR-induced vasoconstrictor response in WKY but did not affect SHR response, while COX-2 inhibition diminished the response in SHR. Both basal prostacyclin (PGI2) and thromboxane A2 (TxA2) values were higher in SHR kidney perfusates (P < 0.05) and were reduced by COX-1 and COX-2 inhibitors in both strains. Furthermore, phenylephrine increased PGI2 through COX-2 in WKY and through COX-1 in SHR, but the agonist did not significantly modify TxA2 in both strains.Significance
The data suggest that COX-1contributes to vasoconstrictor effects in WKY kidneys and that COX-2 has the same effect in SHR kidneys. The results also suggest that basal release of COX-2-derived vasoconstrictor prostanoids is involved in renal vascular hypersensitivity in SHR. 相似文献16.
Chantal Eid Miryana HémadiNguyêt-Thanh Ha-Duong Jean-Michel El Hage Chahine 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
Dietary and recycled iron are in the Fe2 + oxidation state. However, the metal is transported in serum by transferrin as Fe3 +. The multi-copper ferroxidase ceruloplasmin is suspected to be the missing link between acquired Fe2 + and transported Fe3 +.Methods
This study uses the techniques of chemical relaxation and spectrophotometric detection.Results
Under anaerobic conditions, ceruloplasmin captures and oxidizes two Fe2 +. The first uptake occurs in domain 6 (< 1 ms) at the divalent iron-binding site. It is accompanied by Fe2 + oxidation by Cu2 +D6. Fe3 + is then transferred from the binding site to the holding site. Cu+D6 is then re-oxidized by a Cu2 + of the trinuclear cluster in about 200 ms. The second Fe2 + uptake and oxidation involve domain 4 and are under the kinetic control of a 200 s change in the protein conformation. With transferrin and in the formed ceruloplasmin–transferrin adduct, two Fe3 + are transferred from their holding sites to two C-lobes of two transferrins. The first transfer (~ 100 s) is followed by conformation changes (500 s) leading to the release of monoferric transferrin. The second transfer occurs in two steps in the 1000–10,000 second range.Conclusion
Fe3 + is transferred after Fe2 + uptake and oxidation by ceruloplasmin to the C-lobe of transferrin in a protein–protein adduct. This adduct is in a permanent state of equilibrium with all the metal-free or bounded ceruloplasmin and transferrin species present in the medium.General significance
Ceruloplasmin is a go-between dietary or recycled Fe2 + and transferrin transported Fe3 +. 相似文献17.
Background
Malaria is a devastating disease and Plasmodium falciparum is the most lethal parasite infecting humans. Understanding the biology of this parasite is vital in identifying potential novel drug targets. During every 48-hour intra-erythrocytic asexual replication cycle, a single parasite can produce up to 32 progeny. This extensive proliferation implies that parasites require substantial amounts of lipid precursors for membrane biogenesis. Glycerol kinase is a highly conserved enzyme that functions at the interface of lipid synthesis and carbohydrate metabolism. P. falciparum glycerol kinase catalyzes the ATP-dependent phosphorylation of glycerol to glycerol-3-phosphate, a major phospholipid precursor.Methods
The P. falciparum glycerol kinase gene was disrupted using double crossover homologous DNA recombination to generate a knockout parasite line. Southern hybridization and mRNA analysis were used to verify gene disruption. Parasite growth rates were monitored by flow cytometry. Radiolabelling studies were used to assess incorporation of glycerol into parasite phospholipids.Results
Disruption of the P. falciparum glycerol kinase gene produced viable parasites, but their growth was significantly reduced to 56.5 ± 1.8% when compared to wild type parasites. 14C-glycerol incorporation into the major phospholipids of the parasite membrane, phosphatidylcholine and phosphatidylethanolamine, was 48.4 ± 10.8% and 53.1 ± 5.7% relative to an equivalent number of wild type parasites.Conclusions
P. falciparum glycerol kinase is required for optimal intra-erythrocytic asexual parasite development. Exogenous glycerol may be used as an alternative carbon source for P. falciparum phospholipid biogenesis, despite the lack of glycerol kinase to generate glycerol-3-phosphate.General significance
These studies provide new insight into glycerolipid metabolism in P. falciparum. 相似文献18.
Francis Jackson de Oliveira Paludo Juliane Bentes Picanço Paulo Roberto Vargas Fallavena Lucas da Rosa Fraga Pietra Graebin Otávio de Toledo Nóbrega Fernando Suparregui Dias Clarice Sampaio Alho 《Gene》2013
Aim
To analyze the effect of the two different versions of the manganese superoxide dismutase gene (SOD2) on sepsis. The SOD2 gene presents the 47C > T single nucleotide polymorphism (SNP; ID: rs4880) which produces MnSOD with different activities. The − 9Val MnSOD (47T allele) is less efficient than the − 9Ala version (47C allele). During sepsis there are abundance of ROS, high SOD2 expression and excess of H2O2 synthesis. High concentrations of H2O2 could affect the sepsis scenario and/or the sepsis outcome.Methods
We determined the 47C > T single nucleotide polymorphism (SNP) frequencies in 529 critically ill patients with or without sepsis, facing outcome. To collect information on population frequencies, we obtained a pilot 47C > T genotypic and allelic frequencies in a random group of 139 healthy subjects.Results
We compared the 47C allele carriers (47CC + 47CT genotypes) with 47TT homozygotes and noticed a significant association between 47C allele carriers and septic shock in septic patients (P = 0.025). With an adjusted binary multivariate logistic regression, incorporating 47C > T SNP and the main clinical predictors, we showed high SOFA scores [P < 0.001, OR = 9.107 (95% CI = 5.319–15.592)] and 47C allele [P = 0.011, OR = 2.125 (95% CI = 1.190–3.794)] were significantly associated with septic shock outcome. With this information we presented a hypothesis suggesting that this negative outcome from sepsis is possibly explained by effects on cellular stress caused by 47C allele.Conclusion
In our population there was a significant higher frequency of septic shock in septic patients with the 47C allele of the SOD2 gene. This higher 47C allele frequency in septic patients with negative outcome could be explained by effects of higher activity MnSOD on cellular stress during the sepsis. 相似文献19.
20.
Christian J. Steib Leonore Gmelin Susanne Pfeiler Julia Schewe Stephan Brand Burkhard Göke Alexander L. Gerbes 《Life sciences》2013