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R K Hughes

Publications and source records attributed to R K Hughes.

At least 19 recordsLinked to original sources
PubMed2001

Probing a novel potato lipoxygenase with dual positional specificity reveals primary determinants of substrate binding and requirements for a surface hydrophobic loop and has implications for the role of lipoxygenases in tubers.

A new potato tuber lipoxygenase full-length cDNA sequence (lox1:St:2) has been isolated from potato tubers and used to express in Escherichia coli and characterize a novel recombinant lipoxygenase (potato 13/9-lipoxygenase). Like most plant lipoxygenases it produced carbonyl compounds from linoleate (the preferred substrate) and was purified in the Fe(II) (ferrous) state. Typical of other potato tuber lipoxygenases, it produced 5-HPETE [5(S)-hydroperoxy-(6E, 8Z, 11Z, 14Z)-eicosatetraenoic acid] from arachidonate. In contrast to any other potato tuber lipoxygenase, it exhibited dual positional specificity and produced roughly equimolar amounts of 13- and 9-hydroperoxides (or only a slight molar excess of 9-hydroperoxides) from linoleate. We have used a homology model of pea 9/13-lipoxygenase to superimpose and compare the linoleate-binding pockets of different potato lipoxygenases of known positional specificity. We then tested this model by using site-directed mutagenesis to identify some primary determinants of linoleate binding to potato 13/9-lipoxygenase and concluded that the mechanism determining positional specificity described for a cucumber lipoxygenase does not apply to potato 13/9-lipoxygenase. This supports our previous studies on pea seed lipoxygenases for the role of pocket volume rather than inverse orientation as a determinant of dual positional specificity in plant lipoxygenases. We have also used deletion mutagenesis to identify a critical role in catalysis for a surface hydrophobic loop in potato 13/9-lipoxygenase and speculate that this may control substrate access. Although potato 13/9-lipoxygenase represents only a minor isoform in tubers, such evidence for a single lipoxygenase species with dual positional specificity in tubers has implications for the proposed role of potato lipoxygenases in the plant.

Binding Sites↗
PubMed2001

Mutagenesis and modelling of linoleate-binding to pea seed lipoxygenase.

We have produced a model to define the linoleate-binding pocket of pea 9/13-lipoxygenase and have validated it by the construction and characterization of eight point mutants. Three of the mutations reduced, to varying degrees, the catalytic centre activity (kcat) of the enzyme with linoleate. In two of the mutants, reductions in turnover were associated with changes in iron-coordination. Multiple sequence alignments of recombinant plant and mammalian lipoxygenases of known positional specificity, and the results from numerous other mutagenesis and modelling studies, have been combined to discuss the possible role of the mutated residues in pea 9/13-lipoxygenase catalysis. A new nomenclature for recombinant plant lipoxygenases based on positional specificity has subsequently been proposed. The null-effect of mutating pea 9/13-lipoxygenase at the equivalent residue to that which controlled dual positional specificity in cucumber 13/9-lipoxygenase, strongly suggests that the mechanisms controlling dual positional specificity in pea 9/13-lipoxygenase and cucumber 13/9-lipoxygenase are different. This was supported from modelling of another isoform of pea lipoxygenase, pea 13/9-lipoxygenase. Dual positional specificity in pea lipoxygenases is more likely to be determined by the degree of penetration of the methyl terminus of linoleate and the volume of the linoleate-binding pocket rather than substrate orientation. A single model for positional specificity, that has proved to be inappropriate for arachidonate-binding to mammalian 5-, 12- and 15-lipoxygenases, would appear to be true also for linoleate-binding to plant 9- and 13-lipoxygenases.

Amino Acid Sequence↗
PubMed1999

Co-oxidation of beta-carotene catalyzed by soybean and recombinant pea lipoxygenases.

A number of products including apocarotenal, epoxycarotenal, apocarotenone, and epoxycarotenone generated by lipoxygenase (LOX) catalyzed co-oxidation of beta-carotene have been tentatively identified through the use of GC/MS and HPLC combined with photodiode array detection. Because of the large number of high molecular weight products detected and their probable chemical structures, a co-oxidation mechanism is proposed that involves random attack along the alkene chain of the carotenoid by a LOX-generated linoleoylperoxyl radical. It is suggested that a direct release from the enzyme of the radical, which initiates the co-oxidation of beta-carotene, is greater for pea LOX-3 than for pea LOX-2 or soybean LOX-1. It is proposed that further products may be formed by free radical propagated reactions and that the formation of 1,10- and 1,14-dicarbonyl compounds may arise by secondary oxidation of the primary products.

Antioxidants↗
PubMed1998

Characterization of authentic recombinant pea-seed lipoxygenases with distinct properties and reaction mechanisms.

The two major isoforms of lipoxygenase (LOX-2 and LOX-3) from pea (Pisum sativum L. cv. Birte) seeds have been cloned and expressed from full-length cDNAs as soluble, active, non-fusion proteins in Escherichia coli. A comparison of both isoforms purified to apparent homogeneity from E. coli and pea seeds has confirmed the authenticity of the recombinant products and established the properties of the native enzymes. Despite 86% similarity at the amino acid sequence level, the enzymes have distinct properties. They have been characterized in terms of specific activity, Fe content, optimum pH, substrate and product specificity, apparent Km and Vmax for the preferred substrate, linoleic acid, and interfacial behaviour with linoleic acid. We have used this evidence, in addition to EPR spectroscopy of the hydroperoxide-activated enzymes and estimates of kcat/Km, to propose different reaction mechanisms for linoleic acid oxidation for the two isoforms. The differences relate primarily to carbonyl production from linoleic acid for which we propose a mechanism. This implicates the release of a peroxyl radical in an aerobic hydroperoxidase reaction, as the source of the carbonyl compounds formed by dismutation of the liberated peroxyl radical.

Catalysis↗
PubMed1992

Use of rosy mutant strains of Drosophila melanogaster to probe the structure and function of xanthine dehydrogenase.

The usefulness in structure/function studies of molybdenum-containing hydroxylases in work with rosy mutant strains of Drosophila melanogaster has been investigated. At least 23 such strains are available, each corresponding to a single known amino acid change in the xanthine dehydrogenase sequence. Sequence comparisons permit identification, with some certainty, of regions associated with the iron-sulphur centres and the pterin molybdenum cofactor of the enzyme. Procedures have been developed and rigorously tested for the assay in gel-filtered extracts of the flies, of different catalytic activities of xanthine dehydrogenase by the use of various oxidizing and reducing substrates. These methods have been applied to 11 different rosy mutant strains that map to different regions of the sequence. All the mutations studied cause characteristic activity changes in the enzyme. In general these are consistent with the accepted assignment of the cofactors to the different domains and with the known reactivities of the molybdenum, flavin and iron-sulphur centres. Most results are interpretable in terms of the mutation affecting electron transfer to or from one redox centre only. The activity data provide evidence that FAD and the NAD+/NADH binding sites are retained in mutants mapping to the flavin domain. Therefore, despite some indications from sequence comparisons, it is concluded that the structure of this domain of xanthine dehydrogenase cannot be directly related to that of other flavoproteins for which structural data are available. The data also indicate that the artificial electron acceptor phenazine methosulphate acts at the iron-sulphur centres and suggest that these centres may not be essential for electron transfer between molybdenum and flavin. The work emphasizes the importance of combined genetic and biochemical study of rosy mutant xanthine dehydrogenase variants in probing the structure and function of enzymes of this class.

Amino Acid Sequence↗
PubMed1992

Xanthine dehydrogenase from Drosophila melanogaster: purification and properties of the wild-type enzyme and of a variant lacking iron-sulfur centers.

Xanthine dehydrogenase has been purified to homogeneity by conventional procedures from the wild-type strain of the fruit fly Drosophila melanogaster, as well as from a rosy mutant strain (E89----K, ry5231) known to carry a point mutation in the iron-sulfur domain of the enzyme. The wild-type enzyme had all the specific properties that are peculiar to the molybdenum-containing hydroxylases. It had normal contents of molybdenum, the pterin molybdenum cofactor, FAD, and iron-sulfur centers. EPR studies showed its molybdenum center to be quite indistinguishable from that of milk xanthine oxidase. As isolated, only about 10% of the enzyme was present in the functional form, with most or all of the remainder as the inactive desulfo form. It is suggested that this may be present in vivo. Extensive proteolysis accompanied by the development of oxidase activity took place during isolation, but dehydrogenase activity was retained. EPR properties of the reduced iron-sulfur centers, Fe-SI and Fe-SII, in the enzyme are very similar to those of the corresponding centers in milk xanthine oxidase. The E89----K mutant enzyme variant was in all respects closely similar to the wild-type enzyme, with the exception that it lacked both of the iron-sulfur centers. This was established both by its having the absorption spectrum of a simple flavoprotein and by the complete absence of EPR signals characteristic of iron-sulfur centers in the reduced enzyme. Despite the lack of iron-sulfur centers, the mutant enzyme had xanthine:NAD+ oxidoreductase activity indistinguishable from that of the wild-type enzyme. Stopped-flow measurements indicated that, as for the wild-type enzyme, reduction of the mutant enzyme was rate-limiting in turnover. Thus, the iron-sulfur centers appear irrelevant to the normal turnover of the wild-type enzyme with these substrates. However, activity to certain oxidizing substrates, particularly phenazine methosulfate, is abolished in the mutant enzyme variant. This is one of the first examples of deletion by genetic means of iron-sulfur centers from an iron-sulfur protein. The relevance of our findings both to the roles of iron-sulfur centers in other systems and to the nature of the oxidizing substrate for the Drosophila enzyme in vivo are briefly discussed.

Animals↗
PubMed1979

Malignant fibrous histiocytoma of the lung.

Two malignant fibrous histiocytomas arising primarily in the lung are described. The first was a large tumor of the right lower lobe in a 53-year-old man. The other tumor was found incidentally on routine roentgenograms in a 25-year-old woman and involved the left main pulmonary artery. The lesions could be resected but both patients developed early cerebral metastases. The neoplasms were predominantly fibroblastic, had a characteristic storiform pattern, and included large histiocytes with bizarre nuclei and a vacuolated cytoplasm. The ultrastructure of the cells in the fibroblastic areas was characterized by irregular nuclei and a cytoplasm with a well-developed endoplasmic reticulum and dilated cisternae. Some cells lacked the prominent endoplasmic reticulum of fibroblasts and others were characteristic histiocytes with numerous cytoplasmic lysosomes. The cases appear to be the first reported primary malignant fibrous histiocytomas of the lung.

Adult↗
PubMed1977

How to shorten, lengthen, or untwist saphenous vein grafts.

A simple method is described to correct saphenous vein bypass grafts that inadvertently have been made too long or too short or have become twisted. The essential feature of the technique is the use of a Satinsky vascular clamp to hold the divided ends of the vein and maintain their alignment during the repair. The most accessible portion of the vein is used as the site for the repair, leaving the aortic and coronary artery ends of the graft intact. While we have not had need to use the technique frequently, we have found it to be a simple method and believe it to be useful when such instances arise.

Humans↗
PubMed1977

The effectiveness of topical cardiac hypothermia.

The effectiveness of cooling the subendocardial myocardium by five different methods was evaluated in a group of 100 patients. The most effective and consistent method to cool the heart was by total body hypothermia with the heat exchanger in the cardiopulmonary bypass system. Myocardial temperature became equal to vena caval blood temperature after only a one minute lag. The least effective methods of myocardial cooling were those in which a bath of chilled fluid enveloped the outside surface of the heart, with and without aortic cross-clamping. The drop in ventricular septal temperature was so small that topical hypothermia, by itself, may be worthless. Two methods in wich chilled fluid was perfused through the coronary system produced a significant lowering of myocardial temperature. One of these methods employs coronary perfusion with a cold cardioplegic solution in addition to total body hypothermia. It is our current choice for myocardial protection during cross-clamping of the ascending aorta.

Body Temperature↗
PubMed1977

Myocardial revascularization in patients 70 years of age and older.

Myocardial revascularization has been carried out by us in 67 patients 70 years of age or older. Advanced coronary artery disease was found at angiography in more than two thirds of the patients. The postoperative morbidity and mortality compare very favorably with those in younger patients. The early and late mortality in the 67 patients was 4.5 percent and 6.0 percent, respectively. Fifty-seven survivors have been followed an average of 21 months; for most patients there has been a pronounced improvement in clinical classification. Properly selected, patients of advanced age can undergo successful revascularization surgical procedures. The adequacy of function of the left ventricle, proper timing of the surgical operation and an aggressive yet realistic approach seem to be major determinants for a good result.

Aged↗
PubMed1976

Long-term follow-up of patients with coronary artery bypass grafts.

A follow-up study of 1532 patients who had coronary artery bypass grafts from 1969 through 1974 revealed that the 5-year survival rate was substantially better than that recorded in previously published series of patients with similar disease demonstrated angiographically who did not undergo operation. Within this group of operated patients, those who had complete myocardial revascularization (i.e., three grafts for those with three-vessel disease) experienced a significantly better long-term prognosis than those who had fewer grafts than the number of vessels obstructed. The mortality curve in the completely revascularized patients was very similar to that of the general population of the United States, corrected to correspond to this patient sample in age and sex.

Coronary Artery Bypass↗
PubMed1976

Experience with fifty repeat procedures for myocardial revascularization.

Fifty coronary reoperations were performed in 49 patients. The reasons for the operations were occluded or stenotic grafts in 23 patients, an inadequate first operation in 13, progression of coronary atherosclerosis in 3, and combinations of these reasons in 11. Mediastinal adhesions made the operations difficult and produced hazards. Six patients died from the operation. Seven surgical mishaps occurred, including damage to five functioning grafts from the previous operation and laceration of two ventricles. Nine patients had less than complete operations because angiographically demonstrated targets could not be found. Repeat angiography was performed on 9 of the surviving patients. Ten of the 14 new or revised grafts were found to be functioning. Although a repeat operation is more difficult technically and carries additional risks as compared with a first operation, the indications are thought to be the same for both first and secondary revascularization procedures. The increased risks of the repeat operations are compelling arguments to strive for complete revascularization at an initial operation in order to avoid the necessity of the second one.

Adult↗