Biomedical subjects
Publications and source records attributed to R Cammack.
The international hydrogenase community stages a meeting every 3-4 years, and this time it was held in the University of Reading. Each congress has revealed new aspects of the complex nature of hydrogenases: their structure, molecular mechanisms and biosynthesis are all yielding up their secrets. This following introduction provides background information, and the highlights of the meeting are presented in the papers that follow.
Sub-Tenon's block for cataract surgery is an increasingly common technique. While this technique has been successfully applied, the optimal local anaesthetic solution is not known. This study was performed to assess any differences in anaesthesia and oculomotor block between 1% ropivacaine and a 2% lignocaine with 0.5% bupivacaine mixture. The results indicate that there was no difference noted in the clinical effect between the solutions.
Susceptibility to oxygen and properties relative to oxygen metabolism were compared in metronidazole-resistant and susceptible strains of Trichomonas vaginalis. The study involved clinical isolates displaying the aerobic type of resistance, as well as resistant strains developed in vitro, both with aerobic (MR-3) and anaerobic (MR-5, MR-100) resistance. Elevated sensitivity to oxygen of the resistant clinical isolates was observed. Progressive increase of susceptibility to oxygen also accompanied in vitro development of resistance. No correlation was found between the activity of NADH oxidase and aerobic resistance, while the in vitro derivative with fully developed anaerobic resistance (MR-100) showed about 50% decrease of NADH oxidase activity. The superoxide dismutase (SOD) activity was elevated in both resistant clinical isolates and in in vitro-developed resistant strains. The changes in levels of ferredoxin were insufficient to support ferredoxin deficiency as a cause of aerobic metronidazole resistance. Western blot analysis and electron paramagnetic resonance spectroscopy of purified hydrogenosomes showed that ferredoxin is expressed in aerobically resistant strains and has intact iron-sulfur clusters. Down-regulation of ferredoxin was demonstrated only in the late phase of development of the anaerobic resistance (MR-100). The results support a link between aerobic resistance and defective oxygen scavenging. The increased levels of intracellular oxygen, beneficial to resistant parasites when they interact with the drug, may have adverse effects on their fitness as shown by their increased sensitivity to oxidative stress.
The 1H electron nuclear double resonance (ENDOR) spectra in frozen solutions of the reduced [2Fe-2S] cluster in ferredoxin from Arthrospira (Spirulina) platensis have been measured at low temperatures (5-20 K) and simulated using orientational selection methods. The analysis confirmed the existence of a single paramagnetic species with iron valence states II and III connected uniquely to the cluster irons. The experimental ENDOR spectra were fitted to a model including the spin distribution on the centre, the orientation of the g-matrix, and the isotropic and anisotropic hyperfine couplings of the nearest protons in the crystallographically determined structure. In order to partially simulate ENDOR line shapes, a statistical distribution of the corresponding torsion angles between the Fe(III) centre and one of the beta-CH2 protons was introduced. From the analysis, four of the larger hyperfine couplings found were assigned to the cysteine beta-protons near the Fe(III) ion of the cluster, with isotropic hyperfine couplings ranging from 1.6 to 4.1 MHz. The spin distribution on the two iron ions was estimated to be +1.85 for the Fe(III) ion and -0.9 for the Fe(II) ion. The Fe(III) ion was identified as being coordinated to the cysteine ligands Cys49 and Cys79, confirming previous NMR results. The direction of the g-tensor with respect to the cluster was deduced. The g1-g2 plane is parallel to the planes through each iron and its adjacent cysteine sulfurs; the g2-g3 plane is nearly perpendicular to the latter planes and deviates by 25 degrees from the FeSSFe plane. The g1 direction is dominated by the bonding geometry of Fe(II) and does not align with the Fe(II)-Fe(III) vector.
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Nitrite is consumed in the diet, through vegetables and drinking water. It is also added to meat products as a preservative. The potential risks of this practice are balanced against the unique protective effect against toxin-forming bacteria such as Clostridium botulinum. The chemistry of nitrite, and compounds derived from it, in food systems and bacterial cells are complex. It is known that the bactericidal species is not nitrite itself, but a compound or compounds derived from it during food preparation. Of a range of nitrosyl compounds tested, the anion of Roussin's black salt [Fe4S3(NO)7]- was the most inhibitory to C. sporogenes. This compound is active against both anaerobic and aerobic food-spoilage bacteria, while some other compounds are selective, indicating multiple sites of action. There are numerous possible targets for inhibition in the bacterial cells, including respiratory chains, iron-sulfur proteins and other metalloproteins, membranes and the genetic apparatus.
Nocardia corallina B-276 possesses a multi-component enzyme, alkene mono-oxygenase (AMO), that catalyses the stereoselective epoxygenation of alkenes. The reductase component of this system has been shown by EPR and fluorescence spectroscopy to contain two prosthetic groups, an FAD centre and a [2Fe-2S] cluster. The role of these centres in the epoxygenation reaction was determined by midpoint potential measurements and electron transfer kinetics. The order of potentials of the prosthetic groups of the reductase were FAD/FAD.=-216 mV, [2Fe-2S]/[2Fe-2S].=-160 mV and FAD./FAD.=-134 mV. Combined, these data implied that the reductase component supplied the energy required for the epoxygenation reaction and allowed a prediction of the mechanism of electron transfer within the AMO complex. The FAD moiety was reduced by bound NADH in a two-electron reaction. The electrons were then transported to the [2Fe-2S] centre one at a time, which in turn reduced the di-iron centre of the epoxygenase. Reduction of the di-iron centre is required for oxygen binding and substrate oxidation.
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The influence of the amino acid residues surrounding the flavin ring in the flavodoxin of the cyanobacterium Anabaena PCC 7119 on the electron spin density distribution of the flavin semiquinone was examined in mutants of the key residues Trp(57) and Tyr(94) at the FMN binding site. Neutral semiquinone radicals of the proteins were obtained by photoreduction and examined by electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies. Significant differences in electron density distribution were observed in the flavodoxin mutants Trp(57) --> Ala and Tyr(94) --> Ala. The results indicate that the presence of a bulky residue (either aromatic or aliphatic) at position 57, as compared with an alanine, decreases the electron spin density in the nuclei of the benzene flavin ring, whereas an aromatic residue at position 94 increases the electron spin density at positions N(5) and C(6) of the flavin ring. The influence of the FMN ribityl and phosphate on the flavin semiquinone was determined by reconstituting apoflavodoxin samples with riboflavin and with lumiflavin. The coupling parameters of the different nuclei of the isoalloxazine group, as detected by ENDOR and HYSCORE, were very similar to those of the native flavodoxin. This indicates that the protein conformation around the flavin ring and the electron density distribution in the semiquinone form are not influenced by the phosphate and the ribityl of FMN.
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The purpose of the study was to compare 1% ropivacaine and hyaluronidase 75 units/ml with a 1:1 mixture of 2% lignocaine and 0.5% bupivacaine and hyaluronidase 75 units/ml for peribulbar anaesthesia in cataract surgery. We conducted a double-blind randomized trial involving 100 patients. Group 1 received a peribulbar injection of 8 ml of 1% ropivacaine and hyaluronidase 75 units/ml. Group 2 received a peribulbar injection of 8 ml of a 1:1 mixture of 2% lignocaine and 0.5% bupivacaine and hyaluronidase 75 units/ml. Parameters measured were ocular and eyelid movement scores, time suitable for surgery, need for supplementary injections, verbal pain score and complications. No statistical differences were found between the two groups regarding any of the study parameters. Both groups had excellent surgical analgesia and akinesia. We conclude that 1% ropivacaine is a suitable agent for single injection peribulbar anaesthesia for cataract surgery.
Nitric oxide and species derived from it have a wide range of biological functions. Some applications of electron paramagnetic resonance (EPR) spectroscopy are reviewed, for observing nitrosyl species in biological systems. Nitrite has long been used as a food preservative owing to its bacteriostatic effect on spoilage bacteria. Nitrosyl complexes such as sodium nitroprusside, which are added experimentally as NO-generators, themselves produce paramagnetic nitrosyl species, which may be seen by EPR. We have used this to observe the effects of nitroprusside on clostridial cells. After growth in the presence of sublethal concentrations of nitroprusside, the cells show they have been converted into other, presumably less toxic, nitrosyl complexes such as (RS)2Fe(NO)2. Nitric oxide is cytotoxic, partly due to its effects on mitochondria. This is exploited in the destruction of cancer cells by the immune system. The targets include iron-sulfur proteins. It appears that species derived from nitric oxide such as peroxynitrite may be responsible. Addition of peroxynitrite to mitochondria led to depletion of the EPR-detectable iron-sulfur clusters. Paramagnetic complexes are formed in vivo from hemoglobin, in conditions such as experimental endotoxic shock. This has been used to follow the course of production of NO by macrophages. We have examined the effects of suppression of NO synthase using biopterin antagonists. Another method is to use an injected NO-trapping agent, Fe-diethyldithiocarbamate (Fe-DETC) to detect accumulated NO by EPR. In this way we have observed the effects of depletion of serum arginine by arginase. In brains from victims of Parkinson's disease, a nitrosyl species, identified as nitrosyl hemoglobin, has been observed in substantia nigra. This is an indication for the involvement of nitric oxide or a derived species in the damage to this organ.
The potent bactericidal activity of sodium nitroprusside (SNP; Na2[Fe(CN)5(NO)]) towards Clostridium sporogenes has been investigated. SNP inhibited cell growth in the concentration range of 10 to 40 microM. Concentrations above 80 microM caused irreversible loss of cell viability and cell lysis. Inhibition of cell growth was similar in complex and in defined media. SNP was found to be unreactive towards individual components of the defined medium, with the exception of cysteine. The chemical characteristics responsible for the potency of SNP were investigated by synthesizing analogs of SNP in which the Fe was replaced by different metals. The inhibitory potency of the pentacyanonitrosyl complexes decreased in the order Fe > Cr > V, which correlates with N-O stretching frequency (vNO). In contrast, the Ru complex which had a vNO comparable to that of Fe was a poor inhibitor. Electron paramagnetic resonance spectroscopy showed that SNP was rapidly reduced to the paramagnetic Fe(I) compound [Fe(CN)4(NO)](2-) on contact with cells. Analysis of fractions from SNP-treated cells showed 90% oxidation of thiols in the cell walls compared with those in control cells. The toxicity of SNP involves S-nitrosation and reduction, the lack of toxicity of the Ru analog being consistent with the fact that it has poor reactivity towards thiols. When C. sporogenes cells were exposed to sublethal concentrations of SNP and viewed under the electron microscope, they showed blisters on the surface. These results point to the cell wall surface as a primary point of attack of the nitrosyl complex.
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Extracellular laccases from submerged cultures of Coriolus versicolor BKM F-116, Panus tigrinus 8/18, Phlebia radiata 79 (ATCC 64658), Phlebia tremellosa 77-51 and from cultures of Pa. tigrinus 8/18, Ph. radiata 79 and Agaricus bisporus D-649 grown on wheat straw (solid-state fermentation) were purified. All enzymes from submerged cultures had a blue colour and characteristic absorption and EPR spectra. Laccases from the solid-state cultures were yellow-brown and had no typical blue oxidase spectra and also showed atypical EPR spectra. Comparison of N-terminal amino acid sequences of purified laccases showed high homology between blue and yellow-brown laccase forms. Formation of yellow laccases as a result of binding of lignin-derived molecules by enzyme protein is proposed.
Nocardia corallina B-276 possesses a constitutive multi-component alkene monooxygenase which catalyses the epoxidation of terminal and sub-terminal alkenes. The epoxygenase component of this system has been purified with an overall yield of 35%. The electron paramagnetic resonance spectrum of the oxidised protein has a weak signal at g = 4.3, which we ascribe to rhombic iron and a free radical signal at g(ave) = 2.01. Upon partial reduction with dithionite using methyl viologen as a mediator, a signal at g(ave) = 1.9 appeared. Upon further reduction with excess dithionite a signal at g = 15 appeared with the concomitant disappearance of the g(ave) = 1.9 signal. These results indicate that the epoxygenase contains a bridged dinuclear iron centre similar to that found in a variety of proteins involved in oxygen transport and activation as well as desaturation of fatty acids. Analysis of the products of the reaction indicates that AMO is capable of stereospecific epoxidation of alkenes producing the R-enantiomer in high yield, a reaction catalysed by very few oxygenase enzymes. Whole cells gave lower enantiomeric excess values for the epoxide and a stereospecific epoxidase enzyme has been proposed to account for this difference. Although alkene monooxygenase was not inhibited by ethyne, a potent inhibitor of soluble methane monooxygenase with which alkene monooxygenase shares many common features, it was weakly inhibited by propyne with an apparent Km value of 340 microM. The mechanistic implications of these physicochemical features of the enzyme are discussed.
The electron spin echo envelope modulation (ESEEM) technique of pulsed EPR spectroscopy was applied to the anionic semiquinone of the cholesterol oxidase flavin cofactor, formed when the enzyme was photoreduced in the presence of 5-deazariboflavin and EDTA. Fourier transforms of the three-pulse ESEEM spectra showed the presence of 14N nuclei magnetically coupled to the paramagnet. In 2H2O buffer the surroundings of the flavin ring were shown to be accessible to solvent exchange, with a deuterium population in close proximity to the paramagnetic centre. Upon binding of the pseudosubstrate, dehydroisoandrosterone, subtle changes were observed in the coupling to nitrogen nuclei, which are interpreted as changes in the electron density distribution of the flavin ring system. The results are discussed in terms of the three-dimensional structure reported for the protein and the flavin ring architecture.
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