ABSTRACT
INTRODUCTION AND OBJECTIVES: Rett Syndrome (RTT) is a neurodevelopmental disorder caused by Methyl CpG Binding Protein 2 (MECP2) gene mutations. Previous studies of MeCP2 in the human brain showed variable and inconsistent mosaic-pattern immunolabelling, which has been interpreted as a reflection of activation-state variability. We aimed to study post mortem MeCP2 and BDNF (MeCP2 target) degradation and brain region-specific detection in relation to RTT pathophysiology. METHODS: We investigated MeCP2 and BDNF stabilities in non-RTT human brains by immunohistochemical labelling and compared them in three brain regions of RTT and controls. RESULTS: In surgically excised samples of human hippocampus and cerebellum, MeCP2 was universally detected. There was no significantly obvious difference between males and females. However, post mortem delay in autopsy samples had substantial influence on MeCP2 detection. Immunohistochemistry studies in RTT patients showed lower MeCP2 detection in glial cells of the white matter. Glial fibrillary acidic protein (GFAP) expression was also reduced in RTT brain samples without obvious change in myelin basic protein (MBP). Neurons did not show any noticeable decrease in MeCP2 detection. BDNF immunohistochemical detection showed an astroglial/endothelial pattern without noticeable difference between RTT and controls. CONCLUSIONS: Our findings indicate that MeCP2 protein is widely expressed in mature human brain cells at all ages. However, our data points towards a possible white matter abnormality in RTT and highlights the importance of studying human RTT brain tissues in parallel with research on animal and cell models of RTT.
Subject(s)
Brain-Derived Neurotrophic Factor/metabolism , Gray Matter/metabolism , Methyl-CpG-Binding Protein 2/metabolism , Rett Syndrome/metabolism , White Matter/metabolism , Adolescent , Adult , Astrocytes/metabolism , Brain-Derived Neurotrophic Factor/genetics , Female , Glial Fibrillary Acidic Protein/metabolism , Gray Matter/pathology , Hippocampus/metabolism , Hippocampus/pathology , Humans , Male , Methyl-CpG-Binding Protein 2/genetics , Neurons/metabolism , Rett Syndrome/genetics , Rett Syndrome/physiopathology , White Matter/pathology , Young AdultABSTRACT
BACKGROUND: Resistance to contemporary broad-spectrum ß-lactam antibiotics mediated by extended-spectrum ß-lactamases (ESBLs) is increasing worldwide. Klebsiella pneumoniae, an important cause of nosocomial and community acquired urinary tract infections has rapidly become the most common ESBL producing organism. We examined ESBL production in urinary isolates of K. pneumoniae in relation to the presence of bla(SHV), bla(TEM) and bla(CTX-M) genes. METHODS: Antibiotic resistance of 51 clinical isolates of K. pneumoniae was determined to amoxicillin, amikacin, ceftazidime, cefotaxime, cefteriaxon, ceftizoxime, gentamicin, ciprofloxacin and nitrofurantoin by disc diffusion. Minimum inhibitory concentrations were also measured for ceftazidime, cefotaxime, cefteriaxon, ceftizoxime and ciprofloxacin. ESBL production was detected by the double disc synergy test and finally, presence of the bla(SHV), bla(TEM) and bla(CTX-M) genes were shown using specific primers and PCR. RESULTS: Disc diffusion results showed that 96.08 % of the isolates were resistant to amoxicillin followed by 78.43 % resistance to nitrofurantoin, 49.02 % to amikacin and ceftazidime, 41.17 % to ceftriaxone, 37.25% resistance to cefotaxime and ceftizoxime, and 29.42 % to gentamicin and ciprofloxacin. Both resistant and intermediately resistant organisms were resistant in MIC determinations. Twenty two isolates (43.14%) carried bla(SHV), 18 (35.29%) had bla(TEM) and 16 (31.37%) harbored bla(CTX-M) genes. ESBL production was present in 14 isolates (27.45 %) of which, 3 did not harbor any of the 3 genes. Among the non-ESBL producers, 9 lacked all 3 genes and 2 carried them all. CONCLUSION: No relation was found between gene presence and ESBL expression.
ABSTRACT
BACKGROUND: Considering the limited available resources, high cost of the helicopter emergency medical service (HEMS), and high load of trauma patients especially in our centers, a careful assessment of HEMS in our center seemed to be necessary for trauma patients. METHODS: From April 2001 to September 2007, the data of all patients transferred by HEMS were extracted including: Annual number of services, clinical category, number of proper or improper services, and rescue time for HEMS and ground ambulance. The criteria for the properly transferred group included: Death or being operated in the first 24 hours of admission, admission in ICU care units, and transfer of more than three patients in one mission. Others were considered as improper group. RESULTS: In this period through 185 flights, 225 victims were transferred. The most common reason of HEMS dispatching was trauma. The most difference of rescue time between ground ambulance and HEMS was recorded in Lamerd that was transferring patients with HEMS needed 3 hours less than ground ambulance. However, in Sarvestan, Dashte-Arjan, and Marvdasht, transferred patients with ground ambulance needed less time than air transfer. Most of transferred patients were from Kazeroon, Nourabad and Lamerd respectively while 46.3% of patients were in the proper group, and the rest were considered as improper group. CONCLUSION: Our study revealed that helicopter dispatch to the cities like Lamerd, Lar, Khonj, Abadeh can be more effective, whereas, for the towns like Marvdasht, Dashte-Arjan, Sarvestan, Sepidan, Saadatshar, Tang Abolhayat use of HEMS should be limited to specific conditions. Our study showed inclusion of physicians in the decision making team increased the number of transferred cases.
ABSTRACT
Optimization of photocatalytic degradation of C.I. Reactive Green 19 (RG 19) under UV light irradiation using ceramic-coated TiO2 nanoparticles in a continuous circulation rectangular photoreactor was studied. The used catalyst was TiO2 Millennium PC-500 (crystallite mean size 8 nm) immobilized on ceramic plates. A central composite design was used for optimization of the UV/TiO2 process. Predicted values of decolorization efficiency were found to be in good agreement with experimental values (R2 = 0.97 and Adj-R2 = 0.91). Optimization results showed that maximum decolorization efficiency was achieved at the optimum conditions of: initial dye concentration 10 mg/L, UV light intensity 47.2 W/m2, flow rate 150 mL/min and reaction time 240 min. Photocatalytic mineralization of RG 19 was monitored by chemical oxygen demand (COD) decrease and changes in the UV-Vis spectrum.
Subject(s)
Diazonium Compounds/chemistry , Photolysis , Titanium/chemistry , Analysis of Variance , Biological Oxygen Demand Analysis , Ceramics , Coloring Agents/chemistry , Nanoparticles/chemistry , Ultraviolet RaysABSTRACT
Investigation was conducted during 2006 and 2007 to detect and determine typing of Cucumber mosaic virus (CMV) in field-collected samples from Razavi Khorasan Province. Leaves showing chlorosis, mosaic, distortion and shoestring symptoms were collected from the Mashhad Regions (Astan-e-ghods and Khaje-rabie), Neishabour, Torbat-e-heidarye, Ghochan, Chenaran, Fariman, Shirvan, Kalat and Kashmar. Samples were transferred to laboratory in order to detect the virus in the collected samples, Samples were tested by DAS-ELISA. Typing was done for those serologically positive-reacted samples by RT-PCR-RFLP. Specific primers have amplified 650 bp fragments of RNA2, in RT-PCR assay. Digestion was performed by restriction enzyme MluI. Present research showed that all of collected samples were in subgroup IA.
Subject(s)
Cucumovirus , DNA, Viral/analysis , Plant Diseases/virology , Serologic Tests/methods , Cucumovirus/genetics , Cucumovirus/isolation & purification , Cucurbitaceae/anatomy & histology , Cucurbitaceae/virology , Iran , Plant Leaves/virologyABSTRACT
The complete nucleotide sequence of the genome of wheat Eqlid mosaic virus (WEqMV) (excluding the poly A tail) comprised 9636 nucleotides including 5' and 3' noncoding regions of 137 and 172 nt, respectively. It contained a single ORF coding for a polyprotein of 3,109 amino acid residues and had a deduced genome organization typical of members of the family Potyviridae and with proteinase cleavage sites very similar to those of the members of the genus Tritimovirus. Pairwise and multiple alignments and phylogenetic analysis showed that WEqMV is a distinct species in the genus Tritimovirus. WEqMV and Wheat streak mosaic virus (WSMV) shared the greatest nucleotide sequence identity in the NIb and HC-Pro cistrons (63.2% and 60.8%, respectively) and the lowest sequence identity in the P1 and CP cistrons (51.2% and 51.1%, respectively). Sequence identity for the complete genome of WEqMV and WSMV was 56.8% at the nucleotide level and 50.7% at the amino acid level. WEqMV had 57.2% nucleotide identity and 50.6% amino acid identity with Oat necrotic mottle virus and 52.5% nucleotide identity and 45.5% amino acid identity with Brome streak mosaic virus. The relationship of WEqMV with other members of the family Potyviridae was more distant. Structural analysis of WEqMV protein showed presence of potential transmembrane helices in 6k1, 6k2, and P3 proteins.
Subject(s)
Genome, Viral , Potyviridae/classification , Potyviridae/genetics , Amino Acid Sequence , Base Sequence , Molecular Sequence Data , Phylogeny , Plant Diseases/virology , Potyviridae/isolation & purification , Sequence Alignment , Triticum/virologyABSTRACT
In this study, we examined whether exogenous beta(2)-microglobulin (beta(2)m) can induce apoptosis in the drug sensitive HL-60 leukemia cell line and its drug resistant variants and investigated the molecular mechanism of beta(2)m-induced apoptosis. Our data revealed that beta(2)m is very significantly down-regulated in two multidrug resistant variants of the HL-60 cells: (a) the MRP1-bearing, Bax-deficient HL-60/ADR cell line, and (b) the P-glycoprotein (P-gp) overexpressing HL-60/VCR cell line. However, exogenous beta(2)m induced similar levels of apoptosis in HL-60 cells and these drug resistant variants. beta(2)m-induced apoptosis in HL-60 and HL-60/VCR cells was associated with decreased mitochondrial membrane potential (Deltapsim) but did not affect Deltapsim in HL-60/ADR cells. Surprisingly, cyclosporin A (CsA), a known inhibitor of the mitochondrial permeability transition (MPT) pore, inhibited beta(2)m-induced apoptosis in HL-60/ADR cells but not in HL-60 and HL-60/VCR cells, suggesting that the pro-apoptotic effect of beta(2)m in these cells is not through MPT pore formation. Furthermore, beta(2)m induced the release of cytochrome c and the apoptosis-inducing factor (AIF) from mitochondria in HL-60 and HL-60/VCR cells, but not in HL-60/ADR cells. Additionally, Z-VAD-fmk, a general inhibitor of caspases which inhibited cytochrome c release in HL-60 and HL-60/VCR cells, had no effect on AIF release in any of these cell lines, but inhibited beta(2)m-induced apoptosis in all three cell lines. However, Western blot analysis revealed that caspases-1, -3, -6, -8, and -9 are not activated during beta(2)m-induced apoptosis in these cells. Therefore, beta(2)m-induces apoptosis through an unknown caspase-dependent mitochondrial pathway in HL-60 and HL-60/VCR cells and by a Bax-independent, non-mitochondrial, caspase-dependent pathway in HL-60/ADR cells.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Apoptosis/physiology , DNA-Binding Proteins/metabolism , Drug Resistance, Multiple/physiology , Drug Resistance, Neoplasm/physiology , Ion Channels , Multidrug Resistance-Associated Proteins , Neoplasm Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2 , Proto-Oncogene Proteins/deficiency , beta 2-Microglobulin/physiology , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Amino Acid Chloromethyl Ketones/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis Inducing Factor , Cyclosporine/pharmacology , Cysteine Endopeptidases/physiology , Cysteine Proteinase Inhibitors/pharmacology , Cytochrome c Group/metabolism , DNA-Binding Proteins/genetics , Doxorubicin/pharmacology , Drug Resistance, Multiple/genetics , Drug Resistance, Neoplasm/genetics , Flavoproteins/metabolism , Gene Expression Regulation, Leukemic , HL-60 Cells/cytology , HL-60 Cells/drug effects , HL-60 Cells/metabolism , Humans , Membrane Potentials , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/metabolism , Mitochondria/physiology , Mitochondrial Membrane Transport Proteins , Mitochondrial Permeability Transition Pore , MutS Homolog 3 Protein , Neoplasm Proteins/deficiency , Neoplasm Proteins/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , RNA, Messenger/biosynthesis , RNA, Neoplasm/biosynthesis , Vinblastine/pharmacology , Vincristine/pharmacology , bcl-2-Associated X Protein , beta 2-Microglobulin/biosynthesis , beta 2-Microglobulin/genetics , beta 2-Microglobulin/pharmacologyABSTRACT
The 13C NMR solution spectra of 30-crown-10 ether and its tetrahydrate show only one resonance at all accessible temperatures. In contrast, the solid state 13C NMR spectrum of the 30-crown-10.4H2O shows two resonances in the ratio of 4:1, separated by 1.2 ppm. In the case of 30-crown-10 itself, six resolvable 13C resonances in the ratio of 4:1:1:2:1:1 are observed in the solid with an overall chemical shift dispersion of 5 ppm. The remarkably different spectral behavior of these two systems in the solid state is discussed in terms of the torsional environments of the crystallographically unique carbons and the results of GIAO calculations of isotropic 13C shieldings for simpler model compounds. Results of dipolar dephased 13C CPMAS spectra indicate that 30-crown-10 does not undergo a large amplitude molecular motion, in contrast to earlier results for 18-crown-6. Only a small amount of residual intensity is found in the dipolar dephased spectrum of 30-crown-10.4H2O, indicating that it also is relatively rigid in the solid.
Subject(s)
Ether/chemistry , Magnetic Resonance Spectroscopy , Carbon Isotopes , Crystallography , Magnetic Resonance Spectroscopy/methodsABSTRACT
The aim of the present study was to examine the effects of atrial natriuretic peptide (ANP) on the responses to coronary artery occlusion. In chloralose-urethane anaesthetised mongrel dogs either saline (controls) or human synthetic ANP was infused intravenously (10 microg kg(-1) + 0.1 microg kg(-1) min(-1)), starting 30 min before and continuing 10 min during a 25 min occlusion of the left anterior descending coronary artery (LAD). ANP infusion resulted in a fall in mean arterial blood pressure (by 17 +/- 2 mmHg, p < 0.05), a transient (max. at 5 min) increase in coronary blood flow (by 24 +/- 5 ml min(-1), p < 0.05), and a reduction in coronary vascular resistance (by 0.27 +/- 0.05 mmHg ml(-1), p < 0.05). When the LAD coronary artery was occluded, there was a less marked elevation in left ventricular end-diastolic pressure (LVEDP) in the ANP-treated dogs than in the controls (9.0 +/- 0.9 versus 12.2 +/- 0.8 mmHg, p < 0.05). Compared to the controls, ANP reduced the number of ventricular premature beats (VPBs, 26 +/- 12 versus 416 +/- 87, p < 0.05), the number of episodes of ventricular tachycardia per dogs (VT, 0.7 +/- 0.3 versus 12.4 +/- 4.2, p < 0.05), and the incidences of VT (45% versus 100%, p < 0.05) and ventricular fibrillation (VF 18% versus 57%, p < 0.05) during occlusion. Reperfusion of the ischaemic myocardium at the end of the occlusion period led to VF in all the control dogs (survival from the combined ischaemia-reperfusion insult was therefore 0%), but VF following reperfusion was much less in the dogs given ANP (survival 64%; p < 0.05). The severity of myocardial ischaemia, as assessed from changes in the epicardial ST-segment and the degree of inhomogeneity, was significantly less marked in dogs given ANP. We conclude that ANP protects the myocardium from the consequences of myocardial ischaemia resulting from acute coronary artery occlusion and reperfusion in anaesthetized dogs.
Subject(s)
Atrial Natriuretic Factor/therapeutic use , Coronary Disease/drug therapy , Hemodynamics/drug effects , Myocardial Ischemia/prevention & control , Analysis of Variance , Animals , Atrial Natriuretic Factor/administration & dosage , Coronary Disease/complications , Dogs , Female , Infusions, Intravenous , Male , Myocardial Ischemia/etiology , Myocardial ReperfusionABSTRACT
The effects of the intracoronary administration of isosorbide-2-mononitrate (ISMN; 3 microg kg(-1) min(-1)), a major metabolite of isosorbide dinitrate, were examined in chloralose-urethane anaesthetized dogs before and during a 25 min, acute occlusion of the left anterior descending coronary artery. The only significant haemodynamic effects of ISMN administration were a slight (-11 +/- 2 mmHg) decrease in arterial blood pressure and a decrease (< 12%) in diastolic coronary vascular resistance. Coronary occlusion in the presence of ISMN led to a markedly reduced incidence and severity of ventricular arrhythmias compared to those in control, saline-infused dogs. There were fewer ectopic beats (62 +/- 35 versus 202 +/- 72; p < 0.05), a lower incidence (25% versus 75%; p < 0.05) and number of episodes (0.7 +/- 0.4 versus 4.3 +/- 2.1; p < 0.05) of ventricular tachycardia and fewer dogs fibrillated during the ischaemic period (17% versus 82%; p < 0.05). More dogs given ISMN survived the combined ischaemia-reperfusion insult (50% versus 0%; p < 0.05). Changes in ST-segment elevation (recorded by epicardial electrodes) and in the degree of inhomogeneity of electrical activation within the ischaemic area were much less pronounced throughout the occlusion period in dogs given ISMN. These results add weight to the hypothesis that the previously reported antiarrhythmic effects of ischaemic preconditioning, and of the intracoronary administration of nicorandil, involve nitric oxide.
Subject(s)
Isosorbide Dinitrate/analogs & derivatives , Myocardial Ischemia/drug therapy , Animals , Coronary Disease/complications , Dogs , Female , Hemodynamics/drug effects , Isosorbide Dinitrate/pharmacology , Male , Myocardial Ischemia/etiology , Myocardial Reperfusion Injury/complications , Severity of Illness Index , Ventricular Fibrillation/drug therapy , Ventricular Fibrillation/etiologyABSTRACT
Growth hormone (GH) controls gene expression in liver. Recent work suggests that this can result in part from the stimulation by GH of the synthesis of liver-specific transcription factors, one of which is HNF-6. The liver-specific factors HNF-4 and C/EBP alpha respectively stimulate and inhibit transcription of the hnf 6 gene. Upon GH stimulation, the affinity of HNF-4 for the hnf 6 promoter is increased and the binding of C/EBP alpha is decreased. GH therefore controls hnf 6 by a combination of stimulatory and derepressive mechanisms. On the other hand, HNF-6 stimulates transcription of the hnf 3beta and hnf 4 genes, the stimulation of hnf 4 resulting most likely from the GH-induced increase in HNF-6 concentration. We conclude that in liver GH is likely to control the synthesis of a whole set of proteins whose genes are regulated by a GH-sensitive network of transcription factors, which regulate each other by feed-back and autoregulatory loops.
Subject(s)
Gene Expression Regulation/physiology , Growth Hormone/physiology , Liver/physiology , Animals , HumansABSTRACT
GH regulates gene expression by modulating the concentration or activity of transcription factors. To identify transcription factors that mediate the effects of GH in liver we analyzed the promoter of the gene coding for hepatocyte nuclear factor-6 (HNF-6), whose expression in liver is stimulated by GH. In protein-DNA interaction studies and in transfection experiments, we found that the liver-enriched transcription factor CCAAT/enhancer-binding protein-alpha (C/EBPalpha) binds to the hnf6 gene and inhibits its expression. This inhibitory effect involved an N-terminal subdomain of C/EBPalpha and two sites in the hnf6 gene promoter. Using liver nuclear extracts from GH-treated hypophysectomized rats, we found that GH induces a rapid, transient decrease in the amount of C/EBPalpha protein. This GH-induced change is concomitant with the transient stimulatory effect of GH on the hnf6 gene. Stimulation of the hnf6 gene by GH therefore involves lifting of the repression exerted by C/EBPalpha in addition to the known GH-induced stimulatory effects of STAT5 (signal transducer and activator of transcription-5) and HNF-4 on that gene. Our data provide further evidence that GH controls a network of liver transcription factors and show that C/EBPalpha participates in this process.
Subject(s)
DNA-Binding Proteins/physiology , Enhancer Elements, Genetic/physiology , Growth Hormone/physiology , Liver/metabolism , Nuclear Proteins/physiology , Transcription Factors/physiology , Animals , Base Sequence , CCAAT-Enhancer-Binding Proteins , DNA Footprinting , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation , Hepatocyte Nuclear Factor 6 , Homeodomain Proteins/genetics , Male , Molecular Sequence Data , Promoter Regions, Genetic , RNA, Messenger/metabolism , Rats , Rats, Wistar , Trans-Activators/geneticsABSTRACT
HNF-6 is a tissue-restricted transcription factor that participates in the regulation of several genes in liver. We reported earlier that in adult rats, HNF-6 mRNA concentration in liver drops to almost undetectable levels after hypophysectomy and returns to normal after 1 week of GH treatment. We now show that this results from a rapid effect of GH, and we characterize its molecular mechanism. In hypophysectomized rats, HNF-6 mRNAs increased within 1 h after a single injection of GH. The same GH-dependent induction was reproduced on isolated hepatocytes. To determine whether GH regulates hnf6 expression at the gene level, we studied its promoter. DNA binding experiments showed that 1) the transcription factors STAT5 (signal transducer and activator of transcription 5) and HNF-4 (hepatocyte nuclear factor 4) bind to sites located around -110 and -650, respectively; and 2) STAT5 binding is induced and HNF-4 binding affinity is increased in liver within 1 h after GH injection to hypophysectomized rats. Using transfection experiments and site-directed mutagenesis, we found that STAT5 and HNF-4 stimulated transcription of an hnf6 gene promoter-reporter construct. Furthermore, GH stimulated transcription of this construct in cells that express GH receptors. Consistent with our earlier finding that HNF-6 stimulates the hnf4 and hnf3beta gene promoters, GH treatment of hypophysectomized rats increased the liver concentration of HNF-4 and HNF-3beta mRNAs. Together, these data demonstrate that GH stimulates transcription of the hnf6 gene by a mechanism involving STAT5 and HNF-4. They show that HNF-6 participates not only as an effector, but also as a target, to the regulatory network of liver transcription factors, and that several members of this network are GH regulated.
Subject(s)
DNA-Binding Proteins/metabolism , Growth Hormone/metabolism , Homeodomain Proteins/genetics , Milk Proteins , Phosphoproteins/metabolism , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/metabolism , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Cells, Cultured , Female , Gene Expression Regulation , Growth Hormone/pharmacology , Hepatocyte Nuclear Factor 4 , Hepatocyte Nuclear Factor 6 , Homeodomain Proteins/drug effects , Homeodomain Proteins/metabolism , Liver/drug effects , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Promoter Regions, Genetic , RNA, Messenger , Rats , Rats, Wistar , STAT5 Transcription Factor , Trans-Activators/drug effects , Transcription, GeneticABSTRACT
The effects on the responses to coronary artery occlusion of a combined ACE/NEP inhibitor (Z13752A) were examined in anaesthetized dogs. A 1 h infusion of Z13752A (128 microgram kg(-1) min(-1) intravenously) decreased arterial blood pressure (by 11+/-3%; P<0. 05) and increased coronary blood flow (by 12+/-4%, P<0.05). There were no other significant haemodynamic changes. Z13752A inhibited both NEP and ACE enzymes both in dog plasma and in tissue (lung ACE; kidney NEP). Pressor responses to angiotensin I in vivo were inhibited and systemic vasodilator responses to bradykinin were potentiated. When the left anterior descending coronary artery was occluded for 25 min, Z13752A markedly reduced the severity of the resultant ventricular arrhythmias. No ventricular fibrillation (VF) occurred (compared to 7/16 in the controls; P<0.05), and ventricular tachycardia (VT) was reduced (VT in 2/9 dogs treated with Z13752A cp. 16/16 of controls; episodes of VT 0.2+/-0.1 c.p. 10.7+/-3.3; P<0. 05). Reperfusion of the ischaemic myocardium led to VF in all control dogs but occurred less frequently in dogs given Z13752A (survival from the combined ischaemia-reperfusion insult 67% c.p. 0% in controls; P<0.05). Z13752A reduced two other indices of ischaemia severity; epicardial ST-segment elevation and inhomogeneity of electrical activation. These protective effects of Z13752A during ischaemia and reperfusion were abolished by the administration of icatibant (0.3 mg kg(-1), i.v.) a selective antagonist of bradykinin at B(2) receptors; the ischaemic changes in dogs given both icatibant and Z13752A were similar to those in the controls. We conclude that this ACE/NEP inhibitor is effective at reducing the consequences of coronary artery occlusion in this canine model and that this protection is primarily due to potentiation of released bradykinin. British Journal of Pharmacology (2000) 129, 671 - 680
Subject(s)
Angiotensin-Converting Enzyme Inhibitors/pharmacology , Arterial Occlusive Diseases/physiopathology , Bradykinin/pharmacology , Coronary Disease/physiopathology , Neprilysin/antagonists & inhibitors , Phenylalanine/analogs & derivatives , Adrenergic beta-Antagonists/pharmacology , Angiotensin I/pharmacology , Angiotensin II/pharmacology , Animals , Arrhythmias, Cardiac/drug therapy , Arrhythmias, Cardiac/enzymology , Arrhythmias, Cardiac/etiology , Arterial Occlusive Diseases/drug therapy , Arterial Occlusive Diseases/enzymology , Blood Pressure/drug effects , Bradykinin/analogs & derivatives , Coronary Circulation/drug effects , Coronary Disease/drug therapy , Coronary Disease/enzymology , Dogs , Dose-Response Relationship, Drug , Female , Kidney/drug effects , Kidney/enzymology , Lung/drug effects , Lung/enzymology , Male , Myocardial Ischemia/drug therapy , Myocardial Ischemia/enzymology , Myocardial Ischemia/etiology , Myocardial Reperfusion Injury/drug therapy , Myocardial Reperfusion Injury/enzymology , Myocardial Reperfusion Injury/etiology , Peptidyl-Dipeptidase A/blood , Peptidyl-Dipeptidase A/metabolism , Phenylalanine/pharmacologyABSTRACT
Bacterial endotoxin reduces the severity of ventricular arrhythmias which occur when a coronary artery is occluded several hours later. We have now examined in anaesthetised dogs the effects on ischaemia and reperfusion-induced arrhythmias, of a non-toxic derivative component of the endotoxin molecule of the lipid-A (monophosphoryl lipid-A). This was given intravenously, in doses of 10 and 100 microg kg(-1), 24 h prior to coronary artery occlusion. Arrhythmia severity was markedly reduced by monophosphoryl lipid-A. During ischaemia, ventricular premature beats were reduced from 315+/-84 in the vehicle controls to 89+/-60 (with the lower dose of monophosphoryl lipid-A) and 53+/-23 (P<0.05) with the higher dose. The incidence of ventricular tachycardia was reduced from 75% to 25% (P<0.05) and 31% (P<0.05), and the number of episodes of ventricular tachycardia from 13.4+/-4.9 per dog to 1.1+/-1.1 (P<0.05) and 1. 2+/-0.9 (P<0.05) after doses of 10 and 100 microg kg(-1), respectively. The incidence of ventricular fibrillation during occlusion and reperfusion in the control group was 96% (15/16), i.e., only 6% (1/16) dogs survived the combined ischaemia-reperfusion insult. Monophosphoryl lipid-A (100 microg kg(-1)) significantly reduced the incidence of occlusion-induced ventricular fibrillation (from 50% to 7%; P<0.05), and increased survival following reperfusion to 54% (P<0.05). Monophosphoryl lipid-A also significantly reduced ischaemia severity as assessed from ST-segment elevation recorded from epicardial electrodes as well as the degree of inhomogeneity of electrical activation within the ischaemia area. There were no haemodynamic differences prior to coronary occlusion between vehicle controls and monophosphoryl lipid-A-treated dogs. These results demonstrate that monophosphoryl lipid-A reduces arrhythmia severity 24 h after administration. Although the precise mechanisms are still unclear, there is some evidence that nitric oxide and prostanoids (most likely prostacyclin) may be involved because the dual inhibition of nitric oxide synthase and cyclooxygenase enzymes by administration of aminoguanidine and meclofenamate abolished the marked antiarrhythmic protection resulted from monophosphoryl lipid-A treatment 24 h previously.
Subject(s)
Anti-Arrhythmia Agents/pharmacology , Arrhythmias, Cardiac/prevention & control , Heart Ventricles/drug effects , Lipid A/analogs & derivatives , Myocardial Ischemia/complications , Myocardial Reperfusion Injury/prevention & control , Animals , Anti-Arrhythmia Agents/therapeutic use , Arrhythmias, Cardiac/etiology , Arterial Occlusive Diseases/physiopathology , Arterial Occlusive Diseases/prevention & control , Coronary Vessels/drug effects , Coronary Vessels/physiopathology , Disease Models, Animal , Dogs , Enzyme Inhibitors/pharmacology , Female , Guanidines/pharmacology , Heart Ventricles/pathology , Heart Ventricles/physiopathology , Hemodynamics/drug effects , Lipid A/pharmacology , Lipid A/therapeutic use , Male , Meclofenamic Acid/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase Type II , Prostaglandin-Endoperoxide Synthases/drug effects , Severity of Illness Index , Tachycardia, Ventricular/prevention & control , Time Factors , Ventricular Fibrillation/prevention & controlABSTRACT
Hepatocyte nuclear factor 6 (HNF-6) is the prototype of a family of tissue-specific transcription factors characterized by a bipartite DNA-binding domain consisting of a single cut domain and a novel type of homeodomain. We have previously cloned rat cDNA species coding for two isoforms, HNF-6alpha (465 residues) and beta (491 residues), which differ only by the length of the spacer between the two DNA-binding domains. We have now localized the rat Hnf6 gene to chromosome 8q24-q31 by Southern blotting of DNA from somatic cell hybrids and by fluorescence in situ hybridization. Cloning and sequencing of the rat gene showed that the two HNF-6 isoforms are generated by alternative splicing of three exons that are more than 10 kb apart from each other. Exon 1 codes for the N-terminal part and the cut domain, exon 2 codes for the 26 HNF-6beta-specific amino acids, and exon 3 codes for the homeodomain and the C-terminal amino acids. The transcription initiation site was mapped by ribonuclease protection and 5' rapid amplification of cDNA ends. Transfection experiments showed that promoter activity was contained within 0.75 kb upstream of the transcription initiation site. This activity was detected by the transfection of liver-derived HepG2 cells, but not of Rat-1 fibroblasts, suggesting that the promoter is sufficient to confer liver-specific expression.