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P A Riley

Publications and source records attributed to P A Riley.

At least 37 records · Page 2Linked to original sources
PubMed1997

Evidence of the indirect formation of the catecholic intermediate substrate responsible for the autoactivation kinetics of tyrosinase.

Tyrosinase (EC 1.14.18.1) exhibits unusual kinetic properties in the oxidation of monohydric phenol substrates consisting of a lag period that increases with increasing substrate concentration. The cause of this is an autocatalytic process dependent on the generation of a dihydric phenol substrate, which acts as an activator of the enzyme. Experiments with N-substituted dihydric phenol substrates (N-methyldopamine, N-acetyldopamine) demonstrate that oxygen consumption is retarded in the N-acetyl substituted material due to a diminished rate of cyclization. The oxygen uptake exhibited a similar pattern when N-acetyltyramine was oxidized, and this was reflected by a prolongation of the lag period. N,N-Dipropyldopamine was oxidized with normal kinetics but with an oxygen stoichiometry of 0.5 mol of oxygen/mol of substrate. We show that this is the result of the formation of a stable indoliumolate product with oxidation-reduction properties that prevent the formation of dopaminochrome, thus blocking further stages in the tyrosinase-catalyzed oxidation. Evidence that the indoliumolate product is formed by cyclization of the ortho-quinone is presented by pulse radiolysis studies, which demonstrate the formation of the ortho-quinone (by disproportionation of the corresponding semiquinones), which cyclizes to give the indoliumolate. The rate constant for cyclization was shown to be 48 s-1 (at pH 6.0). Tyrosinase-catalyzed oxidation of the monohydric phenol analogue, N, N-dimethyltyramine, was shown to require the addition of a dihydric phenol. Oxygen utilization then exhibited a stoichiometry of 1.0, indicating that the reactions proceed only as far as the cyclization. The analogous stable cyclic indoliumolate product was shown to be formed, with UV absorption and NMR spectra closely similar to the indoliumolate derived from N,N-dipropyldopamine. This material was methylated by catechol O-methyltransferase but was unreactive to redox reagents. The formation of the cyclic product accounts for the indefinite lag when N,N-dimethyltyramine is used as the substrate for tyrosinase in the absence of a dihydric phenol cofactor.

Catechols↗
PubMed1997

Melanogenesis-targeted anti-melanoma pro-drug development: effect of side-chain variations on the cytotoxicity of tyrosinase-generated ortho-quinones in a model screening system.

A set of 26 substituted phenols, 10 of which were synthesised in our laboratories, were tested for their rate of oxidation by mushroom tyrosinase in vitro as determined by oximetry and spectrophotometry and for their cytotoxic action in a model system. With one exception (4-hydroxybenzoic acid) all the agents tested were oxidised to the corresponding ortho-quinones. The maximum rates of oxidation varied between 15.1 +/- 0.59 nmoles oxygen consumed per minute (4-(2-thioethylthio)phenol) and 372.9 +/- 5.61 nmoles O2/ min. (4-(2-Hydroxyethylthio)phenol) in a reaction system comprising 300 units tyrosinase and 200 microM substrate. The rates of generation of quinone were in close agreement with these oximetric data. Some anomalies in oxygen stoichiometry were observed due to reoxidation of reaction products. Four categories of compounds were tested: those known to undergo side-chain cyclisation (such as tyrosine) (Group A), alkylphenols of increasing chain length with or without terminal hydroxyl groups (Group B), compounds with charged or bulky side-chains (Group C) and agents with oxy-, thio- and selenyl-ether side-chains (Groups D, E and F). In the majority of cases, the cytotoxicity, measured by the reduction of thymidine incorporation in cells exposed for 30 min to the agent in the presence of tyrosinase, reflected the rate of oxidation and is ascribed to the toxic action of the derived ortho-quinone. Tyrosinase-dependent cytotoxicity was absent in cyclising (Group A) and in Group C compounds. Toxicity, expressed by comparison with 4-hydroxyanisole (4HA) (IC50 = 11.7 microM), ranged between 0.36 (4-hydroxybenzyl alcohol) and 1.07 (3-(4-hydroxyphenyl)propanol) for Group B compounds, and be-tween 0.83 (4-ethoxyphenol) and 2.08 (4-(2-hydroxyethylthio)phenol) for groups D, E and F. Addition of glutathione to the toxicity assay system abrogated the cytotoxic action and, on the basis of spectrophotometric data, this is ascribed to the prevention of cellular thiol depletion by the ortho-quinone products of tyrosinase oxidation of the phenolic substrates. The lack of toxicity of the group C compounds may be due to the inability of their derived quinones to gain access to the cells. Addition of catalase or deferoxamine to the incubation medium was without effect on tyrosinase-dependent toxicity.

Animals↗
PubMed1997

Melanin.

Melanin is an irregular light-absorbing polymer containing indoles and other intermediate products derived from the oxidation of tyrosine. Melanin is widely dispersed in the animal and plant kingdoms. It is the major pigment present in the surface structures of vertebrates. The critical step in melanin biogenesis is the oxidation of tyrosine by the enzyme tyrosinase. In vertebrates this enzyme is active only in specialized organelles in retinal pigment epithelium and melanocytes. In mammals melanin is formed as intracellular granules. Melanin granules are transferred from melanocytes to epithelial cells and form the predominant pigment of hair and epidermis. Melanin has many biological functions. Reactive quinone intermediates in the melanin biosynthetic pathway exhibit antibiotic properties and the polymer is an important strengthening element of plant cell walls and insect cuticle. Light absorption by melanin has several biological functions, including photoreceptor shielding, thermoregulation, photoprotection, camouflage and display. Melanin is a powerful cation chelator and may act as a free radical sink. Melanin is used commercially as a component of photoprotective creams, although mainly for its free radical scavenging rather than its light absorption properties. The pigment is also a potential target for anti-melanoma therapy.

Anti-Bacterial Agents↗
PubMed1997

Comparison of delta-aminolaevulinic acid and its methyl ester as an inducer of porphyrin synthesis in cultured cells.

This study was carried out to test the hypothesis that induction of intracellular porphyrin synthesis by delta-aminolaevulinic acid (ALA) used to sensitize cells in photodynamic therapy would be more efficient if the ALA was used in an esterified form. Contrary to expectation, the generation of tetrapyrroles (TP) by cultured epithelial cells (CNCM-I-221) exposed to equimolar concentrations (0.6 mM) of ALA or its methyl ester (ALA-ME) showed that the mean total TP production rate during 6 h incubation in serum-free medium was 0.13 fmol cell(-1) h(-1) for ALA-exposed cells compared with 0.04 fmol cell(-1) h(-1) for cells exposed to ALA-ME. Fluorescein diacetate uptake and conversion to fluorescein indicated intracellular non-specific esterase activity, implying that ALA-ME conversion to ALA can occur. Cells exposed to ALA-ME exhibited loss of a greater proportion of total tetrapyrroles in the form of extracellular protoporphyrin IX (PPIX; 22.8%) compared with 11.6% in ALA-treated cells with a corresponding reduction in cell-associated PPIX (P < 0.05). A variable initial elevation in haem levels in ALA-ME-treated cells was observed, but did not reach statistically significant levels.

Aminolevulinic Acid↗
PubMed1997

Naevogenesis: a hypothesis concerning the control of proliferation of melanocytes with special reference to the growth of intradermal naevi.

The pigmentary function of epidermal melanocytes depends on the donation of melanin granules to the surrounding surface structures. This involves transfer of cytoplasm (cytocrine transfer) from melanocytes to keratocytes, a process which requires competence of the donor cells and the availability of adjacent competent recipient cells. Donor cell competence involves the extension of dendrites and recipient cell competence consists of the ability of these cells to phagocytose peripheral portions of the melanocyte cytoplasm. Since there is a highly regulated mechanism for the control of cellular size which operates by inhibiting proliferation of cells that are below a critical volume, it is proposed that the continual removal of portions of the melanocyte cytoplasm by cytocrine transfer is responsible for inhibiting growth of the epidermal melanocyte population, accounting for their relatively low population density. It is proposed that inhibition of cytocrine transfer permits the proliferation of melanocytes. Cytocrine transfer may be inhibited by loss of competence of donor or recipient cells or by their relative displacement. Displacement of melanocytes into the dermis, out of range of potential recipient keratocytes, would, according to this hypothesis, result in melanocyte proliferation leading to the generation of localized aggregations of melanocytes (melanocytomas). It is proposed that this is the origin of acquired benign pigmented moles.

Cell Communication↗
PubMed1996

Growth hormone-responsive DT-diaphorase-mediated bioreduction of tetrazolium salts.

Microculture tetrazolium assays (MTAs) rely upon the bioreduction of tetrazolium salts to their intensely coloured formazans. Although these assays are being extensively used, the intracellular mechanisms responsible for the formazan production are not known. MTAs currently provide the basis for uniquely precise in vitro bioassays for human growth hormone (hGH) which use the Nb2 cells. We have compared two contrasting tetrazolium salts, namely 3-(4,5-dimethyl-thiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) and 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-++ +sulfophenyl) tetrazolium, inner salt (MTS), in this system. An intermediate electron acceptor (IEA) is obligatory for the MTS- but not the MTT-bioassay. We report that inhibitors of DT-diaphorase abolished MTS- but not MTT-formazan production. We conclude that substitution of MTT with MTS/menadione resulted in formazan production via a different electron transfer pathway which is exclusively mediated by DT-diaphorase.

Animals↗
PubMed1996

A clinical overview of Tilarin.

Tilarin is a nasal spray containing 1% nedocromil sodium, a non-toxic pyranoquinoline dicarboxylate compound with potent antiallergic antiinflammatory properties. As a first-line topical treatment for seasonal allergic rhinitis (SAR) the pharmacokinetics of nedocromil sodium nasal formulation are such that it rivals sodium cromoglycate for safety. Less than 8% of the total dose of nedocromil sodium is systemically absorbed from the nasal mucosa, and this is reversibly bound to plasma proteins and is cleared rapidly from the circulation. Nedocromil sodium is eliminated unmetabolised in the urine and faeces, with an elimination half-life of 5.3 +/- 0.9 minutes. No significant adverse effects have been reported following intranasal administration of 1% nedocromil sodium four times daily, to a total of 964 patients with allergic rhinitis during clinical trials. Laboratory studies have shown that nedocromil sodium has a more wide-ranging pharmacological antiinflammatory profile than sodium cromoglycate and this is manifest in its clinical efficacy in allergic asthma and rhinoconjunctivitis. Analysis of pooled data from a series of double-blind, placebo-controlled group comparative studies in SAR patients demonstrated that, despite a significantly lower use of rescue antihistamines than with placebo treatment (31% reduction; p = 0.005), four times daily dosage with nedocromil sodium 1% nasal spray significantly reduced daily symptoms of rhinitis (p < 0.001) and was considered effective by the majority of patients (p < 0.001). Specific examples of the therapeutic efficacy of nedocromil sodium compared with placebo in patients with grass or ragweed pollen SAR can be found in the literature. One ragweed study (1) included four times daily sodium cromoglycate 4% nasal spray as an active comparator and showed a consistent, if non-significant, trend in favour of nedocromil sodium 1%, which was the more effective drug in comparison to placebo. An Italian paediatric study (2) compared nedocromil sodium 1% nasal spray with placebo in 149 children of whom 72% were under twelve years of age. After one week, the clinicians observed a significant reduction (p = 0.03) in sneezing with nedocromil sodium and after four weeks, patient (p < 0.01) and clinican (p < 0.001) opinions favoured the active treatment. Overall, the clinical profile of topical nedocromil sodium in SAR demonstrates fast relief of existing symptoms, sustained efficacy with four times daily use during peak pollen challenge, and a reduced need for concomitant symptomatic therapies. Nedocromil sodium 1% nasal spray is well tolerated, with minimal side-effects, and is acceptable to a wide age-range of patients.

Administration, Intranasal↗
PubMed1996

Commercialization of health services: implications for the laboratories.

The commercialization of health services has wide ranging implications for all medical specialties as well as for patients. Factors that must be considered include not only the financial implications, but also questions of quality and academic interests such as teaching and training. Laboratories must provide a service that the purchaser wishes to buy and must be successful in overcoming competition from the private sector. Each component part of the overall service must be analyzed in order that the laboratory is efficiently structured to provide an optimum service. A good understanding of management issues and a flexible approach are paramount in the provision of efficient, cost-effective and quality service for the ultimate benefit of the patient.

Cost-Benefit Analysis↗
PubMed1996

Evaluation of the use of Bactec anaerobic blood cultures in the detection of bacteraemia and fungaemia in children.

In an attempt to reduce costs, the role of Bactec anaerobic blood culture in the detection of bacteraemia and fungaemia in children was evaluated. Results from 3167 sets of aerobic and anaerobic blood cultures from children admitted to the University Hospital, Kuala Lumpur during a one year period, were analysed. Four hundred and eight (12.9%) sets of blood cultures were positive, of which 348 sets (11.0%) from 201 patients were clinically significant. Of the 348 significant positive sets, organisms were isolated on 177 (50.9%) occasions from both aerobic and anaerobic bottles, on 136 (39.1%) occasions from the aerobic bottle only and 35 (10.0%) occasions from the anaerobic bottle only. No strict anaerobes were isolated, but clinically significant isolates recovered from the anaerobic bottle only included Klebsiella pneumoniae, Salmonella species, Enterobacter cloacae, Staphylococcus aureus, coagulase negative staphylococci, Streptococcus pneumoniae and Group B streptococcus. Patients with bacteraemia diagnosed solely by anaerobic culture were distributed evenly across the various paediatric subspecialities. When results from the anaerobic bottles were excluded, the overall isolation rate was reduced from 11% to 9.9%. Potential financial savings resulting from omission of anaerobic cultures must be balanced against the small number of bacteraemic episodes that could be missed. Undiagnosed bacteraemia may result in increased morbidity and mortality with its own attendant financial implications.

Anaerobiosis↗
PubMed1995

Synthesis and biological evaluation of substituted phosphate triester alkyl lyso phospholipids (ALPs) as novel potential anti-neoplastic agents.

Phosphate triester derivatives of the anti-neoplastic alkyl lyso phospholipid (ALP) have been prepared as novel potential therapeutic agents. In particular, symmetrical phosphate triesters have been prepared, using phosphorochloridate chemistry. The compounds have been fully characterised by a range of techniques, and assayed for their inhibition of DNA synthesis by mammalian cells in culture. The compounds are generally inhibitory towards DNA synthesis in the microM range. However, the magnitude of the effect varies greatly with the phosphate structure; alkynyl and glycol substituted phosphates being especially potent.

Animals↗
PubMed1995

Tyrosinase-mediated cytotoxicity of 4-substituted phenols: quantitative structure-thiol-reactivity relationships of the derived o-quinones.

Rate constants have been determined for reactions between biologically significant thiols, represented by cysteine and glutathione, and a series of 10 4-substituted o-quinones, and unsubstituted o-quinone itself, generated by rapid disproportionation of the semiquinones formed from the corresponding catechols by pulse radiolysis. The quantitative structure-reactivity relationships were investigated by examining the correlation between the rate constants and various Hammett and other parameters characterizing the electronic nature of the substituents. From these relationships, it can be concluded that the o-quinone reactivity with thiols increases with the electron-withdrawing capacity of the substituent groups and that this effect is principally due to resonance effects. Such relationships allow the prediction of likely reactivities with cellular thiols of further o-quinones whose 4-substituents have known electronic parameters. These reactivities are likely to be one of the critical factors determining overall cytotoxicity, assisting in the choice of improved melanogenesis-targeted anti-melanoma drugs.

Antineoplastic Agents↗
PubMed1994

Oxidation of monohydric phenol substrates by tyrosinase: effect of dithiothreitol on kinetics.

The effect of thiol compounds on the monophenolase activity of tyrosinase was investigated using 4-hydroxyanisole as the substrate and dithiothreitol (DTT) as the model thiol compound. We have demonstrated three actions of DTT on tyrosinase-catalysed reactions: (1) direct reduction of the copper at the active site of the enzyme; (2) generation of secondary, oxidizable species by adduct formation with the o-quinone reaction product, 4-MOB, which leads to an increase in the total oxygen utilization by the reaction system; and (3) reversible inhibition of the enzyme. We confirm our previous observation that, at approx. 10 mol of DTT/mol of enzyme, the lag phase associated with monohydric phenol oxidation by tyrosinase is abolished. We suggest that this is due to reduction of the copper at the active site of the enzyme by DTT, since (a) reduction of active-site copper in situ by DTT was demonstrated by [Cu(I)]2-carbon monoxide complex formation and (b) abolition of the lag at low DTT concentration occurs without effect on the maximum rate of reaction or on the total amount of oxygen utilized. At concentrations of DTT above that required to abolish the lag, we found that the initial velocity of the reaction increased with increasing DTT, with a concomitant increase in the total oxygen utilization. This is due to the formation of DTT-4-methoxy-o-benzoquinone (4-MOB) adducts which provide additional dihydric phenol substrate either directly or by reducing nascent 4-MOB. We present n.m.r. evidence for the formation of mono- and di-aromatic DTT adducts with 4-MOB, consistent with a suggested reoxidation scheme in the presence of tyrosinase. Inhibition of the enzyme at concentrations of DTT above 300 pmol/unit of enzyme was released on exhaustion of DTT by adduct formation with 4-MOB as it was generated.

Anisoles↗
PubMed1994

Catechol-O-methyltransferase as a target for melanoma destruction?

Catechols may interfere in melanogenesis by causing increased levels of toxic quinones. Several catechols and known inhibitors of the enzyme catechol-O-methyltransferase (COMT) were therefore tested for their toxicity towards a pigmented melanoma cell line, UCLA-SO-(M14). The inhibition of thymidine incorporation as a result of exposure to the compounds was measured. All agents were compared to 4-hydroxyanisole (4HA), a depigmenting agent extensively studied as an antimelanoma drug. The compounds were also tested on the epithelial cell line, CNCM-I-(221) in the presence and absence of tyrosinase. All the compounds were more effective than 4HA towards the M14-cells at either 10(-4) M or 10(-5) M. The toxicity of 4HA towards the 221-cells was shown to be completely dependent on the presence of tyrosinase. Effects of the test agents on the 221-cells were also observed in the absence of tyrosinase. Although some of them were shown to be good substrates for tyrosinase only small changes in toxicity were observed as a result of the presence of the enzyme in comparison with 4HA. No direct correlation of the toxicity of the agents and COMT inhibition was observed. The possible mode of action of the compounds through inhibition of COMT and interference in melanogenesis is discussed together with other possibilities and factors involved.

Anisoles↗
PubMed1994

The roles of neuromelanin, binding of metal ions, and oxidative cytotoxicity in the pathogenesis of Parkinson's disease: a hypothesis.

A characteristic feature of both Parkinson's disease (idiopathic paralysis agitans) and normal aging is loss of pigmented neurons in the substantia nigra. This has been found to correlate with the accumulation of neuromelanin and with oxidative stress in this brain region, but a clear association between these factors has not been established. Based on our recent demonstration that neuromelanin is a true melanin, containing bound metal ions in situ, we present a general model for its accumulation in vivo and the hypotheses (1) that it has a cytoprotective function in the sequestration of redox-active metal ions under normal conditions but (2) that it has a cytotoxic role in the pathogenesis of Parkinson's disease. Thus, neuromelanin accumulates normally through the autooxidation of catecholamines and serves tightly to bind redox-active metal ions, processes which would accelerate under conditions of intracellular or extracellular oxidative stress. Based on the known properties of melanin, however, neuromelanin also has the potential for exacerbating oxidative stress, eg by generating H2O2 when it is intact or by releasing redox-active metal ions if it loses its integrity; these reactions also would modulate the reactivity of the neuromelanin. By overwhelming intracellular antioxidative defense mechanisms, such a positive-feedback cycle could turn a condition of chronic or repeated oxidative stress in vulnerable neurons into an acute crisis, leading to cellular death. If the cumulative stress in duration and/or degree is severe enough, neuronal depletion could be sufficient to cause Parkinson's disease during life. One possible trigger for this cascade is suggested by the increased nigral iron contents in postmortem parkinsonian brains and the correlation of this disease with urban living where exposure to heavy metal ions is high: the saturation of neuromelanin with redox-active metal ions. Parkinson's disease therefore may be a form of accelerated aging in the substantia nigra associated with environmental toxins in which neuromelanin has a central, active role.

Aging↗
PubMed1994

Free radicals in biology: oxidative stress and the effects of ionizing radiation.

The most important electron acceptor in the biosphere is molecular oxygen which, by virtue of its bi-radical nature, readily accepts unpaired electrons to give rise to a series of partially reduced species collectively known as reduced (or 'reactive') oxygen species (ROS). These include superoxide (O.2-), hydrogen peroxide (H2O2), hydroxyl radical (HO.) and peroxyl (ROO.) and alkoxyl (RO.) radicals which may be involved in the initiation and propagation of free radical chain reactions and which are potentially highly damaging to cells. Mechanisms have evolved to restrict and control such processes, partly by compartmentation, and partly by antioxidant defences such as chain-breaking antioxidant compounds capable forming stable free radicals (e.g. ascorbate, alpha-tocopherol) and the evolution of enzyme systems (e.g. superoxide dismutase, catalase, peroxidases) that diminish the intracellular concentration of the ROS. Although some ROS perform useful functions, the production of ROS exceeding the ability of the organism to mount an antioxidant defence results in oxidative stress and the ensuing tissue damage may be involved in certain disease processes. Evidence that ROS are involved in primary pathological mechanisms is a feature mainly of extraneous physical or chemical perturbations of which radiation is perhaps the major contributor. One of the important radiation-induced free-radical species is the hydroxyl radical which indiscriminately attacks neighbouring molecules often at near diffusion-controlled rates. Hydroxyl radicals are generated by ionizing radiation either directly by oxidation of water, or indirectly by the formation of secondary partially ROS. These may be subsequently converted to hydroxyl radicals by further reduction ('activation') by metabolic processes in the cell. Secondary radiation injury is therefore influenced by the cellular antioxidant status and the amount and availability of activating mechanisms. The biological response to radiation may be modulated by alterations in factors affecting these secondary mechanisms of cellular injury.

Cell Death↗
PubMed1994

Is Medicare contracting right for your managed care organization?

Americans older than 65 years represent the fastest-growing segment of the population. Today, the elderly comprise 12% of the population but account for one-third of health care spending. By the turn of the century, they will use half of these resources. Ironically, the elderly have been affected little by the managed care revolution; only 2.6 million have enrolled in HMOs that have contracts with the federal Medicare program to provide comprehensive health services to that population. In this article, the author explains the different types of Medicare managed care contracts, the advantages of these contracts, and how to succeed in this growing area of opportunity.

Capitation Fee↗