Pulmonary Vascular remodelling in hypoxic rats: effects of amlodipine, alone and with perindopril

This study investigated whether pulmonary Vascular remodelling in hypoxic pulmonary hypertensive rats (10% oxygen; 4 weeks) could be prevented by treatment, during hypoxia, with amlodipine (IO mg/kg/day, p.o.), either alone or in combination with the angiotensin converting enzyme inhibitor, perindopril (30 mg/kg/day, p.o.). Medial thickening of pulmonary arteries (30-500 mum o.d.) was attenuated by amlodipine whereas it was totally prevented by the combination treatment (amlodipine plus perindopril); neomuscularisation of small alveolar arteries (assessed from critical closing pressure in isolated perfused lungs) was not affected. Pulmonary vascular resistance (isolated perfused lungs) was reduced by both treatment regimes but only combination treatment reduced right ventricular hypertrophy. Taus, amlodipine has anti-remodelling properties in pulmonary hypertensive rats. The finding that combining amlodipine with another anti-remodelling drug produced effects on vascular structure that were additive raises the question of whether combination therapy with two different anti-remodelling drugs may be of value in the treatment of patients with hypoxic (and possibly other forms of) pulmonary hypertension. (C) 2001 Elsevier Science B.V. All rights reserved.

Reversal of cardiovascular remodelling with candesart

Cardiovascular remodelling, defined as ventricular and vascular hypertrophy together with fibrosis, characterises hypertension following inhibition of the production of the endogenous vasodilator, nitric oxide (NO). This study has determined whether the cardiovascular remodelling following chronic NO synthase inhibition can e reversed by administration of the selective angiotensin II AT(1)-receptor antagonist, candesartan. Male Wistar rats were treated with L-nitroarginine methyl ester (L-NAME, 400 mg/l in drinking water) for eight weeks and with candesartan cilexetil (2 mg/kg/day by oral gavage) for the last four weeks. L-NAME-treated rats became hypertensive with systolic blood pressure increasing from 110 +/- 4 mmHg (control) to 170 +/- 10 mmHg. Rats developed left ventricular hypertrophy (control 1.70 +/- 0.06; L-NAME 2.10 +/- 0.04 mg/kg body wt) with markedly increased deposition of perivascular and interstitial collagen. Candesartan returned blood pressure, left ventricular weights and collagen deposition to control values. Echo cardiographic assessment showed concentric hypertrophy with an increased fractional shortening; this was reversed by candesartan treatment. Heart failure was not evident. In the isolated Langendorff heart, diastolic stiffness increased in L-NAME-treated rats while the rate of increase in pressure (+dP/dt) increased after eight weeks only; candesartan reduced collagen deposition and normalised +dP/dt. In isolated left ventricular papillary muscles, the potency (negative log EC50) of noradrenaline as a positive inotropic compound was unchanged, (control 6.56 +/- 0.14); maximal increase in force before ectopic beats was reduced from 5.0 +/- 0.4 mN to 2.0 +/- 0.2 mN. Noradrenaline potency as a vasoconstrictor in thoracic aortic rings was unchanged, but maximal contraction was markedly reduced from 25.2 +/- 2.0 mN to 3.0 +/- 0.3 mN; this was partially reversed by candesartan treatment. Thus, chronic inhibition of NO production with L-NAME induces hypertension, hypertrophy and fibrosis with increased toxicity and significant decreases in vascular responses to noradrenaline. These changes were at least partially reversible by treatment with candesartan, implying a significant role of AT(1)-receptors in L-NAME-induced cardiovascular changes.

Growth and hard tissue remodelling in the dentition of the Australian lungfish, Neoceratodus forsteri (Osteichthyes: Dipnoi)

The extant lungfish, including three genera, the Australian, South American and African lungfishes, retain a dentition that appeared first in the Devonian, in some of the oldest members of this group. The dentition consists of permanent tooth plates with persistent cusps that appear early in development of the fish. The cusps, separate early in development, form ridges that are arranged in a radiating pattern, and fusion of the cusps to each other and to the underlying jaw bone produces a tooth plate. The lungfish dentition is based on a template of mantle dentine that surrounds bone trabeculae enclosed in the tooth plate. The mantle layer is covered by enamel. In most derived dipnoans, this framework encloses two further forms of dentine, known as interdenteonal and circumdenteonal dentines. The tooth plates grow in area and in depth without evidence of macroscopic resorption of dentines or of enamel. Increase in size and changes in shape of lungfish tooth plates is actually achieved by a process involving microscopic remodelling of the bone contained within the margin of each tooth plate, and the later addition of new dentines and enamel within and around the bone. This is accomplished without creating weakness in the structural integrity of the tooth plate and bone complex, and proceeds in line with growth and remodelling of the jaw bones attached to the tooth plates.

Characterization of redox centers in dimethyl sulfide dehydrogenase from Rhodovulum sulfidophilum

Dimethyl sulfide dehydrogenase from the purple phototrophic bacterium Rhodovulum sulfidophilum catalyzes the oxidation of dimethyl sulfide to dimethyl sulfoxide. Recent DNA sequence analysis of the ddh operon, encoding dimethyl sulfide dehydrogenase (ddhABC), and biochemical analysis (1) have revealed that it is a member of the DMSO reductase family of molybdenum enzymes and is closely related to respiratory nitrate reductase (NarGHI). Variable temperature X-band EPR spectra (120122 K) of purified heterotrimeric dimethyl sulfide dehydrogenase showed resonances arising from multiple redox centers, Mo(V), [3Fe-4S](+), [4Fe-4S](+), and a b-type heme. A pH-dependent EPR study of the Mo(V) center in (H2O)-H-1 and (H2O)-H-2 revealed the presence of three Mo(V) species in equilibrium, Mo(V)-OH2, Mo(v)-anion, and Mo(V)-OH. Above pH 8.2 the dominant species was Mo(V)-OH. The maximum specific activity occurred at pH 9.27. Comparison of the rhombicity and anisotropy parameters for the Mo(V) species in DMS dehydrogenase with other molybdenum enzymes of the DMSO reductase family showed that it was most similar to the low-pH nitrite spectrum of Escherichia coli nitrate reductase (NarGHI), consistent with previous sequence analysis of DdhA and NarG. A sequence comparison of DdhB and NarH has predicted the presence of four [Fe-S] clusters in DdhB. A [3Fe-4S](+) cluster was identified in dimethyl sulfide dehydrogenase whose properties resembled those of center 2 of NarH. A [4Fe-4S](+) cluster was also identified with unusual spin Hamiltonian parameters, suggesting that one of the iron atoms may have a fifth non-sulfur ligand. The g matrix for this cluster is very similar to that found for the minor conformation of center 1 in NarH [Guigliarelli, B., Asso, M., More, C., Augher, V., Blasco, F., Pommier, J., Giodano, G., and Bertrand, P. (1992) Eur. J. Biochem. 307,63-68]. Analysis of a ddhC mutant showed that this gene encodes the b-type cytochrome in dimethyl sulfide dehydrogenase. Magnetic circular dichroism studies revealed that the axial ligands to the iron in this cytochrome are a histidine and methionine, consistent with predictions from protein sequence analysis. Redox potentiometry showed that the b-type cytochrome has a high midpoint redox potential (E-o = +315 mV, pH 8).

A system for the heterologous expression of complex redox proteins in Rhodobacter capsulatus: characterisation of recombinant sulphite : cytochrome c oxidoreductase from Starkeya novella

The phototrophic purple non-sulfur bacterium Rhodobacter capsulatus expresses a wide variety of complex redox proteins in response to changing environmental conditions. Here we report the construction and evaluation of an expression system for recombinant proteins in that organism which makes use of the dor promoter from the same organism. A generic expression vector, pDorEX, was constructed and used to express sulphite:cytochrome c oxidoreductase from Starkeya novella, a heterodimeric protein containing both molybdenum and haem c. The recombinant protein was secreted to the periplasm and its biochemical properties were very similar to those of the native enzyme. The pDorEX system therefore seems to be potentially useful for heterologous expression of multi-subunit proteins containing complex redox cofactors. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Redox active macrocyclic receptors for neutral guests

A novel series of triazine-appended macrocyclic complexes has been investigated as potential hydrogen bonding receptors for complementarily disposed heterocycles. Cocrystallization of a melamine-appended azacyclam complex of Cull has been achieved with barbitone, the barbiturate anion and thymine. In each case, a complementary DAD/ADA hydrogen bonding motif between the melamine group and the heterocycle has been identified by X-ray crystallography. Electrochemical studies of the copper macrocycles in both nonaqueous and aqueous solution show anodic shifts of the CuII/I redox couple of more than 60 mV upon addition of guest molecules with matching H-bonding motifs. The Zn-II analogues have been synthesized via transmetalation of the Cull complex, and their guest binding properties investigated by NMR spectroscopy. H-1 NMR shifts of up to 0.8 ppm were observed upon addition of guest, and stability constants are similar to those obtained electrochemically.

Cytotoxic iron chelators: Characterization of the structure, solution chemistry and redox activity of ligands and iron complexes of the di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) analogues

Di-2-pyridyl ketone isonicotinoyl hydrazone (HPKIH) and a range of its analogues comprise a series of monobasic acids that are capable of binding iron (Fe) as tridentate (N,N,O) ligands. Recently, we have shown that these chelators are highly cytotoxic, but show selective activity against cancer cells. Particularly interesting was the fact that cytotoxicity of the HPKIH analogues is maintained even after complexation with Fe. To understand the potent anti-tumor activity of these compounds, we have fully characterized their chemical properties. This included examination of the solution chemistry and X-ray crystal structures of both the ligands and Fe complexes from this class and the ability of these complexes to mediate redox reactions. Potentiometric titrations demonstrated that all chelators are present predominantly in their charge-neutral form at physiological pH (7.4), allowing access across biological membranes. Keto-enol tautomerism of the ligands was identified, with the tautomers exhibiting distinctly different protonation constants. Interestingly, the chelators form low-spin (diamagnetic) divalent Fe complexes in solution. The chelators form distorted octahedral complexes with Fe-II, with two tridentate ligands arranged in a meridional fashion. Electrochemistry of the Fe complexes in both aqueous and non-aqueous solutions revealed that the complexes are oxidized to their ferric form at relatively high potentials, but this oxidation is coupled to a rapid reaction with water to form a hydrated (carbinolamine) derivative, leading to irreversible electrochemistry. The Fe complexes of the HPKIH analogues caused marked DNA degradation in the presence of hydrogen peroxide. This observation confirms that Fe complexes from the HPKIH series mediate Fenton chemistry and do not repel DNA. Collectively, studies on the solution chemistry and structure of these HPKIH analogues indicate that they can bind cellular Fe and enhance its redox activity, resulting in oxidative damage to vital biomolecules.

Discrete cyanide-bridged mixed-valence Co/Fe complexes: Outer-sphere redox behaviour

The outer-sphere redox behaviour of a series of [LnCoIII-NCFeII(CN)(5)](-) (L-n = n-membered pentadentate aza-macrocycle) complexes have been studied as a function of pH and oxidising agent. All the dinuclear complexes show a double protonation process at pH approximate to 2 that produces a shift in their UV/Vis spectra. Oxidation of the different non-protonated and diprotonated complexes has been carried out with peroxodisulfate, and of the non-protonated complexes also with trisoxalatocobaltate(III). The results are in agreement with predictions from the Marcus theory. The oxidation of [Fe(phen)(3)](3+) and [IrCl6](2-) is too fast to be measured, although for the latter the transient observation of the process has been achieved at pH = 0. The study of the kinetics of the outer-sphere redox process, with the S2O82- and [Co(ox)(3)](3-) oxidants, has been carried out as a function of pH, temperature, and pressure. As a whole, the values found for the activation volumes, entropies, and enthalpies are in the following margins, for the diprotonated and non-protonated dinuclear complexes, respectively: DeltaV(not equal) from 11 to 13 and 15 to 20 cm(3) mol(-1); DeltaS(not equal) from 110 to 30 and -60 to -90 J K-1 mol(-1); DeltaH(not equal) from 115 to 80 and 50 to 65 kJ.mol(-1). The thermal activation parameters are clearly dominated by the electrostriction occurring on outer-sphere precursor formation, while the trends found for the values of the volume of activation indicate an important degree of tuning due to the charge distribution during the electron transfer process. The special arrangement on the amine ligands in the isomer trans[(L14CoNCFeII)-N-III(CN)(5)](-) accounts for important differences in solvent-assisted hydrogen bonding occurring within the outer-sphere redox process, as has been established in redox reactions of similar compounds. ((C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003).

Cambial meristem dormancy in trees involves extensive remodelling of the transcriptome

The establishment of the dormant state in meristems involves considerable physiological and metabolic alterations necessary for surviving unfavourable growth conditions. However, a global molecular analysis of dormancy in meristems has been hampered by the difficulty in isolating meristem cells. We used cryosectioning to isolate purified cambial meristem cells from the woody plant Populus tremula during active growth and dormancy. These samples were used to generate meristem-specific cDNA libraries and for cDNA microarray experiments to define the global transcriptional changes underlying cambial dormancy. The results indicate a significant reduction in the complexity of the cambial transcriptome in the dormant state. Although cell division is terminated in the dormant cambium, the cell cycle machinery appears to be maintained in a skeletal state as suggested by the continued presence of transcripts for several cell cycle regulators. The downregulation of PttPIN1 and PttPIN2 transcripts explains the reduced basipetal polar auxin transport during dormancy. The induction of a member of the SINA family of ubiquitin ligases implicated in auxin signalling indicates a potential mechanism for modulation of auxin sensitivity during cambial dormancy. The metabolic alterations during dormancy are mirrored in the induction of genes involved in starch breakdown and the glyoxysomal cycle. Interestingly, the induction of RGA1 like gene suggests modification of gibberellin signalling in cambial dormancy. The induction of genes such as poplar orthologues of FIE and HAP2 indicates a potential role for these global regulators of transcription in orchestrating extensive changes in gene expression during dormancy.

Effects of revascularisation and contractile reserve on left ventricular remodelling in patients with impaired left ventricular function

Objective: We sought to define the influence of revascularisation and contractile reserve on left ventricular (LV) remodelling in patients with LV dysfunction after myocardial infarction. Revascularisation of viable myocardium is associated with improved regional function, but the effect on remodelling is undefined. Methods: We studied 70 patients with coronary artery disease and LV dysfunction, 31 of whom underwent revascularisation. A standard dobutamine stress echocardiogram (DbE) was carried out. All patients underwent standard medical treatment; the decision to revascularise was made clinically, independent of this study. LV volumes and ejection fraction were measured by 3D echocardiography at baseline and after an average of 40 weeks. Results: There was no significant difference in baseline ejection fraction or volumes between patients who underwent revascularisation and the remainder. Compared to medically treated patients, revascularised patients had significant improvements in ejection fraction and end-systolic volume in follow-up. The impact of baseline variables on remodelling was assessed by dividing patients into tertiles of LV ejection fraction and volumes. Revascularised patients in the lowest tertile of ejection fraction at baseline (<38%) had a significant improvement in end-systolic volume and ejection fraction, larger than obtained in medically treated patients with low ejection fraction. Revascularised patients with an ejection fraction >38% did not show significant improvement in volumes compared to baseline. Revascularised patients in the largest tertiles of end-systolic (>88 ml) or end-diastolic volume (>149 ml) at baseline had a significant improvement in end-systolic volume. Conclusion: Remodeling appears to occur independent of the presence of regional contractile reserve but does correlate with the volume response to low-dose dobutamine. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

A numerical approach to modeling the catalytic voltammetry of surface-confined redox enzymes

A finite difference method for simulating voltammograms of electrochemically driven enzyme catalysis is presented. The method enables any enzyme mechanism to be simulated. The finite difference equations can be represented as a matrix equation containing a nonlinear sparse matrix. This equation has been solved using the software package Mathematica. Our focus is on the use of cyclic voltammetry since this is the most commonly employed electrochemical method used to elucidate mechanisms. The use of cyclic voltammetry to obtain data from systems obeying Michaelis-Menten kinetics is discussed, and we then verify our observations on the Michaelis-Menten system using the finite difference simulation. Finally, we demonstrate how the method can be used to obtain mechanistic information on a real redox enzyme system, the complex bacterial molybdoenzyme xanthine dehydrogenase.

The acidic nature of the CcmG redox-active center is important for cytochrome c maturation in Escherichia coli

Cytochrome c biogenesis in Escherichia coli is a complex process requiring at least eight genes (ccmABC DEFGH). One of these genes, ccmG, encodes a thioredoxin-like protein with unusually specific redox activity. Here, we investigate the basis for CcmG function and demonstrate the importance of acidic residues surrounding the redox-active center.

Cyclosporine A-induced changes to erythrocyte redox balance is time course-dependent

Cyclosporine A-treated transplant recipients develop pronounced cardiovascular disease and have increased oxidative stress and altered antioxidant capacity in erythrocytes and plasma. These experiments investigated the time-course of cyclosporine A-induced changes to redox balance in plasma and erythrocytes. Rats were randomly assigned to either a control or cyclosporine A-treated group. Treatment animals received 25 mg/kg of cyclosporine A via intraperitoneal injection for either 7 days or a single dose. Control rats were injected with the same volume of the vehicle. Three hours after the final injections, plasma was analysed for total antioxidant status, a-tocopherol, malondialdehyde, and creatinine. Erythrocytes were analysed for reduced glutathione (GSH), alpha-tocopherol, methaemoglobin, malondialdehyde, and the activities of superoxide dismutase, catalase, GSH peroxidase, and glucose-6-phosphate dehydrogenase (G6PD). Cyclosporine A administration for 7 days resulted in a significant increase (P < 0.05) in plasma malondialdehyde, methaemoglobin, and superoxide dismutase and catalase activities. There was a significant decrease (P < 0.05) in erythrocyte GSH concentration and G6PD activity in cyclosporine A animals. There were no significant differences (P > 0.05) between groups following a single dose of cyclosporine A in any of the measures. In summary, cyclosporine A alters erythrocyte redox balance after 7 days administration, but not after a single dose.

NmlR of Neisseria gonorrhoeae: a novel redox responsive transcription factor from the MerR family

A MerR-like regulator (NmlR -Neisseria merR-like Regulator) identified in the Neisseria gonorrhoeae genome lacks the conserved cysteines known to bind metal ions in characterized proteins of this family. Phylogenetic analysis indicates that NmlR defines a subfamily of MerR-like transcription factors with a distinctive pattern of conserved cysteines within their primary structure. NmlR regulates itself and three other genes in N. gonorrhoeae encoding a glutathione-dependent dehydrogenase (AdhC), a CPx-type ATPase (CopA) and a thioredoxin reductase (TrxB). An nmlR mutant lacked the ability to survive oxidative stress induced by diamide and cumene hydroperoxide. It also had > 50-fold lower NADH-S-nitrosoglutathione oxidoreductase activity consistent with a role for AdhC in protection against nitric oxide stress. The upstream sequences of the NmlR regulated genes contained typical MerR-like operator/promoter arrangements consisting of a dyad symmetry located between the -35 and -10 elements of the target genes. The NmlR target operator/promoters were cloned into a beta-galactosidase reporter system and promoter activity was repressed by the introduction of NmlR in trans. Promoter activity was activated by NmlR in the presence of diamide. Under metal depleted conditions NmlR did not repress P-AdhC (or P-CopA) promoter activity, but this was reversed in the presence of Zn(II), indicating repression was Zn(II)-dependent. Analysis of mutated promoters lacking the dyad symmetry revealed constitutive promoter activity which was independent of NmlR. Gel shift assays further confirmed that NmlR bound to the target promoters possessing the dyad symmetry. Site-directed mutagenesis of the four NmlR cysteine residues revealed that they were essential for activation of gene expression by NmlR.

Tuning the photophysical behavior of luminescent cyclam derivatives by cation binding and excited state redox potential

The emission from two photoactive 14-membered macrocyclic ligands, 6-((naphthalen-1-ylmethyl)-amino)trans-6,13-dimethyl- 13-amino- 1,4,8,11 -tetraaza-cyclotetradecane (L-1) and 6-((anthracen-9-ylmethyl)-amino)trans-6,13 -dimethyl - 13 -amino- 1,4,8, 1 1-tetraaza-cyclotetradecane (L-2) is strongly quenched by a photoinduced electron transfer (PET) mechanism involving amine lone pairs as electron donors. Time-correlated single photon counting (TCSPC), multiplex transient grating (TG), and fluorescence upconversion (FU) measurements were performed to characterize this quenching mechanism. Upon complexation with the redox inactive metal ion, Zn(II), the emission of the ligands is dramatically altered, with a significant increase in the fluorescence quantum yields due to coordination-induced deactivation of the macrocyclic amine lone pair electron donors. For [ZnL2](2+), the substituted exocyclic amine nitrogen, which is not coordinated to the metal ion, does not quench the fluorescence due to an inductive effect of the proximal divalent metal ion that raises the ionization potential. However, for [ZnL1](2+), the naphthalene chromophore is a sufficiently strong excited-state oxidant for PET quenching to occur.