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P A Riley

Publications and source records attributed to P A Riley.

At least 19 recordsLinked to original sources
PubMed2001

Melanocyte-Directed enzyme prodrug therapy (MDEPT): development of second generation prodrugs for targeted treatment of malignant melanoma.

Evaluation of second generation prodrugs for MDEPT, by oximetry, has highlighted structural properties that are advantageous and disadvantageous for efficient oxidation using mushroom tyrosinase. In particular, a sterically undemanding prodrug bis-(2-chloroethyl)amino-4-hydroxyphenylaminomethanone 28 was synthesised and found to be oxidised by mushroom tyrosinase at a superior rate to tyrosine methyl ester, the carboxylic acid of which is the natural substrate for tyrosinase. The more sterically demanding phenyl mustard prodrugs 9 and 10 were oxidised by mushroom tyrosinase at a similar rate to tyrosine methyl ester. In contrast, tyramine chain elongation via heteroatom insertion was detrimental and the rate of mushroom tyrosinase oxidation of phenyl mustard prodrugs 21 and 22 decreased by 10 nanomol/min.

Agaricales↗
PubMed2000

Tyrosinase kinetics: a semi-quantitative model of the mechanism of oxidation of monohydric and dihydric phenolic substrates.

A mathematical model of phase I melanogenesis is described based on the differential reactivity of tyrosinase according to the redox status of the active site copper atoms shown by Lerch and co-workers (see Lerch, 1981, Metal Ions in Biological Systems (Sigel, H., ed.) Vol. 13, pp. 143-186. New York: Marcel Dekker) in combination with the indirect formation of the catecholic intermediate substrate. In this model the unusual autoactivation kinetics of tyrosinase are explained by recruitment of enzyme from the met -form, in which the active-site copper atoms are in the oxidized (Cu(II)) state, by 2-electron donation from catechol oxidation. Using estimates of the values for the rate constants of the six reactions involved, the general characteristics of the model are shown to be consistent with the kinetic behaviour of tyrosinase in vitro. These include a lag period which is sensitive to catechol addition.

Animals↗
PubMed2000

Spontaneous redox reactions of dopaquinone and the balance between the eumelanic and phaeomelanic pathways.

Eumelanogenesis and phaeomelanogenesis diverge at an early stage in pigment formation, namely at the point where dopaquinone, the initial product of tyrosine oxidation by tyrosinase, undergoes one of two types of reaction: either (1) a reductive endocyclisation in which a Michael addition of the side-chain amino group takes place; or (2) a reductive addition of cysteine to give cysteinyldopa. In the former case, the product cyclodopa, is known rapidly to undergo a redox exchange reaction with dopaquinone to yield dopachrome, the precursor of the eumelanogenic pathway. In the second instance, cysteinyldopa is regarded as leading to the formation of benzothiazoles, which are characteristic of phaeomelanin. The precursor molecule of the phaeomelanic pathway is cysteinyldopaquinone. We have examined quantitatively the role of dopaquinone in the non-enzymatic oxidation of 5-S-cysteinyldopa using pulse radiolysis and have demonstrated that the redox exchange reaction between dopaquinone and 5-S-cysteinyldopa occurs spontaneously with a rate constant of 8.8 x 10(5) M(-1) sec(-1). This study has also enabled an improved estimate of < or = 4 x 10(7) M(-1) sec(-1) to be obtained for the rate constant of the reaction of dopaquinone with cyclodopa. Calculations utilising these figures and estimates of the rate constants for the other reactions in early melanogenesis, demonstrate that, whilst similar pathways are invoked, the phaeomelanic pathway predominates in the presence of cysteine, irrespective of the availability of dopaquinone and thus independently of the rate of tyrosinase-catalysed oxidation. This suggests that the balance between the formation of eumelanin and phaeomelanin is regulated principally by the availability of cysteine at the site of melanogenesis.

Benzoquinones↗
PubMed1999

Melanocyte-directed enzyme prodrug therapy (MDEPT): development of a targeted treatment for malignant melanoma.

A novel prodrug rationally designed to function as a tyrosinase substrate has been synthesised to allow targeted treatment of malignant melanoma. This agent has been evaluated for tyrosinase-mediated drug release, and has been shown to act in the desired manner. Furthermore, differential cytotoxicity has been demonstrated in cell lines which express tyrosinase and those which do not.

Animals↗
PubMed1999

Comparative effect of ALA derivatives on protoporphyrin IX production in human and rat skin organ cultures.

Samples of human and rat skin in short-term organ culture exposed to ALA or a range of hydrophobic derivatives were examined for their effect on the accumulation of protoporphyrin IX (PpIX) measured using fluorescence spectroscopy. With the exception of carbobenzoyloxy-D-phenylalanyl-5-ALA-ethyl ester the data presented indicate that, in normal tissues, ALA derivatives generate protoporphyrin IX more slowly than ALA, suggesting that they are less rapidly taken up and/or converted to free ALA. However, the resultant depot effect may lead to the enhanced accumulation of porphyrin over long exposure periods, particularly in the case of ALA-methyl ester or ALA-hexyl ester, depending on the applied concentration and the exposed tissue. Addition of the iron chelator, CP94, greatly increased PpIX accumulation in human skin exposed to ALA, ALA-methyl ester and ALA-hexyl ester. The effect in rat skin was less marked.

Aminolevulinic Acid↗
PubMed1999

The great DOPA mystery: the source and significance of DOPA in phase I melanogenesis.

One of the important characteristics of tyrosinase is the autocatalytic nature of the oxidation of natural monohydric phenol substrates, such as tyrosine. In vitro tyrosinase exhibits a lag phase in which the maximum velocity of oxidation is attained after a period of induction. This acceleration contrasts with the kinetics of dihydric phenol oxidation which exhibit conventional Michaelis-Menten kinetics. It has been known for half a century that DOPA is a co-factor in the oxidation of tyrosine and addition of a small amount of catechol reduces the length of the lag period. The significance of DOPA is in this action, and DOPA is known to be formed in phase I melanogenesis. Until recently there has been controversy regarding the source of the DOPA in the in vitro reaction system. Most investigators have favoured a mechanism based on the generation of DOPA by a direct hydroxylation of tyrosine. However, recent evidence has suggested that DOPA is indirectly derived by reduction of dopaquinone. In this communication the evidence for the indirect mechanism derived from the use of analogue substrates is reviewed.

Animals↗
PubMed1999

Study of DT-diaphorase in pigment-producing cells.

DT-diaphorase is an FAD-containing enzyme capable of a two-electron reduction of ortho- and paraquinones. Nicotinamide coenzymes (NADH + H+ and NADPH + H+) serve as hydrogen sources in these reactions. The role of DT-diaphorase has been thoroughly investigated in situations when the enzyme is able to reduce exogenous and endogenous quinones, hence protecting the cells against these reactive intermediates. The enzyme has also been studied in connection with its ability to activate some quinoid cytostatics. It is surprising that DT-diaphorase has never been investigated in pigment-producing cells that are known to generate considerable amounts of ortho-quinones. Using a spectrophotometric method we could readily measure the activity of DT-diaphorase in epidermis and various cultured pigment cells. The melanocytes isolated from dark skin showed generally higher DT-diaphorase activity than those from fair skin samples. Also, darkly pigmented congenital naevus cells exhibited higher activity of this enzyme. The most striking was the high DT-diaphorase activity in melanoma cell cultures. In these cells DT-diaphorase activity could be induced by incubation of the cells with 4-hydroxyanisole. A similar effect was seen when a catechol-O-methyltransferase (COMT) inhibitor (3-(3,4-dihydroxy-5-nitrobenzylidene)-2,4-pentanedione (OR-462) was utilised. The induction was inhibited by cyclohexidine.

Anisoles↗
PubMed1998

Tyrosinase kinetics: failure of the auto-activation mechanism of monohydric phenol oxidation by rapid formation of a quinomethane intermediate.

When 3,4-dihydroxybenzylcyanide (DBC) is oxidized by mushroom tyrosinase, the first visible product, identified as the corresponding quinomethane, exhibits an absorption maximum at 480 nm. Pulse-radiolysis experiments, in which the o-quinone is formed by disproportionation of semiquinone radicals generated by single-electron oxidation of DBC, showed that the quinomethane (A480 6440 M-1.cm-1) is formed through the intermediacy of the o-quinone with a rate constant at neutral pH of 7.5 s-1. The oxygen stoichiometry of the formation of the quinomethane by tyrosinase-catalysed oxidation of DBC was 0.5:1. On the basis of oxygen utilization rates the calculated Vmax was 4900 nmol.min-1 and the apparent Km was 374 microM. The corresponding monohydric phenol, 4-hydroxybenzylcyanide (HBC), was not oxidized by tyrosinase unless the enzyme was pre-exposed to DBC, the maximum acceleration of HBC oxidation being obtained by approximately equimolar addition of DBC. These results are consistent with tyrosinase auto-activation on the basis of the indirect formation of the dihydric phenol-activating cofactor. The rapid conversion of the o-quinone to the quinomethane prevents the formation of the catechol by reduction of the o-quinone product of monohydric phenol oxidation from occurring in the case of the compounds studied. In the absence of auto-activation, the kinetic parameters for HBC oxidation by tyrosinase were estimated as Vmax 70 nmol.min-1 and Km 309 microM. The quinomethane was found to decay with a rate constant of 2k 38 M-1.s-1, as determined both by pulse-radiolysis and tyrosinase experiments. The second-order kinetics indicate that a dimer is formed. In the presence of tyrosinase, but not in the pulse-radiolysis experiments, the quinomethane decay was accompanied by a steady-state oxygen uptake concurrently with the generation of a melanoid product measured by its A650, which is ascribed to the formation of an oligomer incorporating the oxidized dimer.

Acetonitriles↗
PubMed1998

Tyrosinase kinetics: failure of acceleration in oxidation of ring-blocked monohydric phenol substrate.

When 2,5,6-trimethyl-4-hydroxyanisole is used as substrate for mushroom tyrosinase the oxidation rate is slow and the kinetics do not exhibit an initial acceleration (lag period), in contrast to the kinetics of oxidation of the parent compound, 4-hydroxyanisole. This finding is interpreted as evidence that the acceleration of oxidation of 4-hydroxyanisole is indirectly contingent on a reductive nucleophile addition to the orthoquinone product of the monohydric phenol, which is prevented by ring methylation. Such a view is consistent with the proposal that the lag-phase characteristic of the kinetics of monohydric phenol oxidation by tyrosinase is due to the activation of previously inactive enzyme by electron donation from an orthodiphenol substrate formed from the orthoquinone oxidation product.

Anisoles↗
PubMed1998

Tyrosinase autoactivation and the problem of the lag period.

Evidence is presented for the binding of the quinone oxidation product of the monohydric phenol substrate, 4-hydroxyanisole, to mushroom tyrosinase. Column chromatography and SDS-PAGE separation showed labelling of the enzyme when incubated with 14C ring-labelled 4-hydroxyanisole. It is proposed that covalent binding to the enzyme and other proteins is through reaction of accessible nucleophilic groups, including thiols and amino groups, with the anisylquinone. This reductive addition enables the indirect generation of the catecholic substrate, which acts as an electron donor for the bicupric active site of met-tyrosinase and explains the lag kinetics of tyrosinase oxidation of non-cyclizing substrates. The effects of diluting the enzyme or the addition of amino acids on the lag period was consistent with a mechanism involving indirect generation of the dihydric phenol, which acts as the met-enzyme-recruiting substrate.

Anisoles↗
PubMed1998

Onychomycosis: diagnosis and systemic treatment.

Onychomycosis, a persistent fungal infection affecting the toenails and fingernails, can interfere with standing, walking, and exercising. Associated physical impairments can result in paresthesia, pain, discomfort, and loss of manual dexterity. Patients may also suffer from loss of self-esteem and social interaction. A definitive diagnosis is crucial for effective treatment, because many other skin and nail disorders mimic onychomycosis. Diagnosis involves microscopic potassium hydroxide preparation, cultures, nail biopsy, and histologic analysis. Treatment can include topical and systemic antifungal therapies as well as nonpharmaceutical methods. This paper discusses pathophysiology, diagnosis, and treatment options for this common nail dystrophy, including the newer antifungal medications now available.

Antifungal Agents↗
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↗