Biomedical subjects

P A Riley

Publications and source records attributed to P A Riley.

At least 91 records · Page 5Linked to original sources

PubMed1988

Major primary cytotoxic product of 4-hydroxyanisole oxidation by mushroom tyrosinase is 4-methoxy ortho benzoquinone.

It has been shown previously that the initial product of mushroom tyrosinase-catalysed oxidation of the monophenol 4-hydroxyanisole (4HA) is 4-methoxy ortho benzoquinone (4-MOB). This study presents evidence that 4-MOB is primarily responsible for the cytotoxicity of 4HA oxidation products in vitro. Equivalent toxicity in a model system was produced by products of tyrosinase catalysed oxidation of 4HA and by synthetic 4-MOB. Cytotoxicity was estimated both by a blebbing assay and by plating efficiency of exposed cells. HPLC analysis of the reaction mixture revealed a positive correlation between cytotoxicity and 4-MOB concentration.

Animals↗

PubMed1988

Radicals in melanin biochemistry.

Melanins are light-absorbant polymeric pigments found widely dispersed in nature. They possess many interesting physicochemical properties. One of these is the expression in the polymer of stable free radicals which appear to have a protective action in cells, probably by acting as a sink for diffusible free-radical species. Polymer formation is thought to occur by a free-radical process in which semiquinones are added to the chain. Semiquinones are formed by redox equilibration interactions between metabolic intermediates formed during the tyrosinase-catalyzed oxidation process. In the continued presence of substrate, steady-state concentrations of reactive species are predicted in the reaction system, and the melanogenic pathway may be considered as potentially hazardous for pigment-generating cells. This feature has been exploited by the use of analogue substrates to generate cytotoxic species as a possible rational approach to the treatment of malignant melanoma. One such substance is 4-hydroxyanisole, the oxidation of which gives rise to semiquinone radical species. The possibility that the anisyl semiquinone initiates a mechanism leading to cell damage has not been excluded. However, the current view is that the major cytotoxicity due to the oxidation products of this compound is the result of the action of the corresponding orthoquinone. A number of mechanisms exist for detoxifying quinones if they reach the cytosol such as O-methylation and the formation of thiol adducts with cysteine or glutathione, and these can be used as markers of melanogenesis. In general, however, only small amounts of reactive intermediates of melanogenesis escape from the confines of the melanosome, probably because of their limited lipid solubility. The selective toxic action of anisyl quinone in the treatment of melanoma may, in part, be due to membrane defects in the melanosomes of malignant melanocytes.

Benzoquinones↗

PubMed1988

The stoichiometry of tyrosinase-catalyzed oxidation of 4-hydroxyanisole.

Oxygen utilisation during tyrosinase-catalysed oxidation of 4-hydroxyanisole was investigated using an electron spin resonance technique which employs quantitative changes in the characteristics of the electron spin resonance spectrum of the spin label 3-carbamoyl-2,5-dihydro-2,2,5-5-tetramethyl-1-H-pyridoyl-1-ylox y (CTPO) to follow changes in the oxygen concentration. Reaction mixtures containing mushroom tyrosinase (15 micrograms ml-1) and differing initial concentrations of 4-hydroxyanisole in aerated phosphate buffer at pH 6.8 were incubated at room temperature. The ratio of utilisation of oxygen was found to be in approximately 1:1 molar ratio with the initial 4-hydroxyanisole concentration in the reaction mixture between 50 and 200 mumol/l 4-hydroxyanisole. The results are consistent with the stoichiometry of oxygen utilisation being accounted for by the oxidation of 4-hydroxyanisole to anisyl quinone.

Anisoles↗

PubMed1988

Quantitative studies of increased cell-to-substratum adhesion at low temperature.

The cell-to-substratum adhesion of an established epithelial cell line cultured for 24 h on glass coverslips was determined at 4 degrees C, 8 degrees C and 37 degrees C using a miniaturised parallel-plate shearing apparatus. The measurements of the minimum shear necessary to dislodge the cells (minimum distraction force, MDF) demonstrated a three- to fourfold increase in the adhesion of the cells at 4 degrees C (6.17 Pa) compared to that at 37 degrees C (1.36 Pa). At 8 degrees C the MDF was 2.31 pascals. Part of the adhesion was resistant to mild trypsinisation. Trypsin-resistant adhesion (TRA) was stabilised by low temperature, and by treatment with concanavalin A (50 micrograms ml-1) or colchicine (200-400 microM). The effects of con A (140 micrograms ml-1) and low temperature (4 degrees C) were additive, giving a combined MDF of greater than 9.27 pascals. On the basis of their different temperature and protease susceptibility it is suggested that trypsin-sensitive adhesion (TSA) and TRA represent separate functional classes of cell-to-substratum attachment corresponding to 'frictional' and 'tractional' adhesion, respectively.

Animals↗

PubMed1987

Synergistic activity of sodium cromoglycate and beta 2-stimulants in rat lung anaphylaxis.

Using a model of passive anaphylactic bronchoconstriction in the rat, the combined inhibitory effects of sodium cromoglycate and the beta 2-adrenoceptor agonists fenoterol and salbutamol were shown to be synergistic. Synergism was most evident in animals strongly sensitised with relatively high levels of IgE antibodies, in which the individual drugs showed reduced potency.

Adrenergic beta-Agonists↗

PubMed1987

Activity of nedocromil sodium in mast-cell-dependent reactions in the rat.

Nedocromil sodium, a pyranoquinoline dicarboxylic acid derivative which differs structurally and physiochemically from sodium cromoglycate, was nonetheless shown to be effective in classical models of anti-allergic activity in the rat. These models, based on immediate hypersensitivity reactions in the passively sensitized rat, involve release of mediators from mast cells following cross-linking of membrane-bound IgE antibodies by specific antigen. In this system, nedocromil sodium exhibited a similar profile of activity to that of sodium cromoglycate. Whilst not predictive for therapeutic activity, rat models of immediate hypersensitivity are believed to reflect one component of obstructive airways disease. The results suggest that nedocromil sodium is worthy of clinical evaluation in this indication.

Animals↗

PubMed1987

The relative significance of growth rate and interdivision time in the size control of cultured mammalian epithelial cells.

Genealogies of a line of mammalian epithelial cells (GPK) have been constructed from time-lapse film of monolayer cultures and measurements made of initial (post-divisional) cell size, final (pre-divisional) cell size and interdivision time (IDT). The mean initial cell volume was 2696 +/- 404 (S.D.) micron3, the mean final volume was 5247 +/- 696 micron3 and the mean IDT was 985 +/- 84 min. Cell size regulation must be by modulation of either the growth rate or the length of the growth period. Increase in size was strongly correlated with the average rate of growth (increase in volume per unit time) (R = 0.94, P much less than 0.001), whilst no correlation was found between increase in size and IDT. Although a negative correlation was found between initial volume and IDT (P less than 0.02), this appeared to be due to differences in IDT between sister cells being correlated with differences in their initial volumes (P less than 0.02), as indicated by the lack of correlation between mean sister IDT and mean sister initial volume. The regulatory effect of growth rate was demonstrated by a negative correlation between growth rate and the initial volume of the cell (P less than 0.005), which is independent of differences between siblings. The mean growth rate of sibling cells was found to be negatively correlated with both the maternal growth rate (P less than 0.01) and the maternal volume increase (P less than 0.005). This implies that the growth rate of division products (which manifest similar growth rates) is influenced by the growth of the progenitor cell.

Cell Division↗

PubMed1987

On the interaction of anisyl-3,4-semiquinone with oxygen.

Pulse radiolysis studies of anisyl-3,4-semiquinone, formed in the metabolic activation of 4-hydroxyanisole, a possible melanocytotoxic drug under current assessment as a treatment for malignant melanoma, have shown this semiquinone to be unreactive towards oxygen (k less than or equal to 10(5) M-1 s-1), although the reverse reaction of O2- with anisyl-3,4-quinone is very rapid (k = 8.7 x 10(8) M-1 s-1). Since 1,4 benzoquinone is also unreactive towards anisyl-3,4-semiquinone (k less than or equal to 10(5) M-1 s-1), the one-electron reduction potential, E17 (anisyl-3,4-quinone/anisyl-3,4-semiquinone), is likely to be considerably more positive than 0.1 V. This suggests that the cytotoxicity mechanism does not involve the generation of O2- and possible subsequent production of H2O2 and/or OH., leading to lipid peroxidation, as previously proposed, but rather involves as yet unknown reactions of anisyl-3,4-quinone. This quinone is unstable in water and its absorption spectrum was measured immediately (less than 0.1 s) following disproportionation of anisyl-3,4-semiquinone, before significant decay of the quinone had occurred.

Oxidation-Reduction↗

PubMed1987

A miniaturised parallel-plate shearing apparatus for the measurement of cell adhesion.

The design and construction of a miniaturised shearing apparatus is described. Cultures (24 h) of an established epithelial cell line (BEB) were exposed to flow conditions in the shearing chamber at 37 degrees C, and subsequently glass coverslip cultures were prepared for photography. The critical shear radius (CSR) was determined by densitometry from a negative film and the minimum distraction force (MDF) at the CSR calculated using predetermined viscosity values of the flow medium. The mean calculated MDF of BEB cells ranged from 1.04-1.36 Nm-2, and was independent of the culture inoculation density (9 to 37 X 10(4) cells cm-2) and the time (5-20 min) of exposure to shearing conditions. The MDF was increased to 3.3 Nm-2 by a 30 min exposure of cultures to concanavalin A (50 micrograms ml-1), and this effect was abolished by treating with mannose (0.05 M). The results demonstrated that the radial flow chamber principle is applicable to the measurement of cell to substratum adhesion of cultured mammalian cells.

Animals↗

PubMed1986

The suppression of endogenous protein degradation by fractions of foetal calf serum: dialysed serum is less able to suppress degradation in aged cells.

We have studied the effect of various fractions of foetal bovine serum upon the endogenous degradation of long labelled proteins in cultured MRC5 cells, and upon other cellular functions. Only heat-inactivated serum was capable of suppressing protein degradation to a similar extent to complete serum. Acid-treated and delipidized sera were moderately effective. Albumin on its own was able to replace 40 per cent of the effect of serum, indicating the exogenous protein might compete with endogenous protein for degradation in lysosomes. Albumin was not capable of supporting DNA synthesis. Dialysed serum showed an age-related effect suppressing protein degradation to a lesser extent and being less effective in supporting DNA synthesis or cellular proliferation in aged cells. All the effects noted were related to lysosomal protein degradation. Serum diffusate did not suppress protein degradation.

Blood↗

PubMed1985

Radicals and melanomas.

The synthesis of melanin involves the oxidation of phenolic substrates by the enzyme tyrosinase. In vertebrates tyrosinase is present only in specialized cells (melanocytes), where it catalyses the oxidation of tyrosine and certain diphenolic intermediate products to quinones which polymerize to give rise to melanin. This specialized metabolic pathway provides a possible approach to the specific chemotherapy of malignant tumours of pigment cells (malignant melanoma). Certain analogues of tyrosine are oxidized by tyrosinase generating reactive orthoquinones with cytotoxic potential. One such analogue, 4-hydroxyanisole, has been investigated as a possible specific melanocytotoxic precursor. The parent compound inhibits DNA synthesis but exhibits little general toxicity, while the tyrosinase oxidation products are highly toxic to cells. The mechanism of this toxicity may involve semiquinone radicals. Encouraging initial results have been obtained from clinical pilot studies using intra-arterial infusion of hydroxyanisole in patients with localized recurrences of malignant melanoma.

Anisoles↗

PubMed1985

Metabolism of 1-naphthol by tyrosinase.

1-Naphthol was metabolized by the polyphenol oxidase, tyrosinase, primarily to 1,2-naphthoquinone and to small amounts of 1,4-naphthoquinone as well as to covalently bound products. The inhibition of covalent binding by ethylenediamine, which reacts specifically with 1,2-naphthoquinone but not 1,4-naphthoquinone, suggested that most of the covalent binding was due to 1,2-naphthoquinone or a metabolite of similar structure. The activation by tyrosinase of 1-naphthol to covalently bound products suggested that it may alter the reaction kinetics of the enzyme. This was investigated by studying the effects of 1-naphthol on the tyrosinase-catalysed oxidation of 4-hydroxyanisole. Preincubation of tyrosinase with 1-naphthol increased the lag period of the oxidation of 4-hydroxyanisole, which may be due to a decrease in the amount of active enzyme, as well as to a reaction of 1-naphthol with 3,4-anisylquinone, an oxidation product of 4-hydroxyanisole. The metabolic activation of 1-naphthol by tyrosinase to covalently bound species suggests that 1-naphthol or a structurally related derivative may be of potential therapeutic application in the treatment of cells high in tyrosinase activity, such as certain melanomas.

Anisoles↗

PubMed1985

In vitro studies on the fibrinolytic, thrombolytic and fibrinogenolytic properties of a tissue plasminogen activator from guinea pig keratocytes.

The fibrinolytic and thrombolytic properties of a tissue plasminogen activator (tPA) purified from the conditioned medium of an established guinea pig keratocyte (GPK) cell line were investigated in in vitro systems and compared with urokinase. Using the fibrin clot lysis assay, GPK activator appears to be similar to human melanoma tPA and not to human urokinase. GPK activator also caused negligible fibrinogen breakdown, when incubated with human plasma at 37 degrees C over 23 hr. Urokinase on the other hand caused significant fibrinogenolysis, under similar conditions. Comparison of the lysis of plasma clots by GPK activator and human urokinase have shown that GPK activator was a much more effective fibrinolytic agent than urokinase, especially at lower concentrations (less than 50 IU/ml). Studies on the thrombolytic effect of GPK activator on the lysis of aged and cross-linked whole human blood clots and plasma clots hanging in artificially circulating human plasma suggest that GPK activator can lyse both these types of clots equally well. The lysis is dose dependent, attaining complete lysis within 3-6 hr with the concentration of GPK activator in the range of 1-5 micrograms/ml plasma. It is concluded that GPK activator has a higher fibrinolytic and thrombolytic activity and lower fibrinogenolytic activity than urokinase.

Animals↗

PubMed1985

Comparison of the cytotoxic activities of chemotherapeutic drugs using a human bladder cancer cell line.

Many chemotherapeutic drugs have been used to treat patients with advanced bladder cancer, but few of these have been evaluated adequately in phase II clinical trials. Continuous cell lines provide one means for comparing the in vitro cytotoxicities of anticancer agents. In this study, a continuous cell line derived from a transitional cell cancer of the human bladder, which still produces tumours histologically similar to the tumour of origin on xenotransplantation, was used to measure the in vitro cytotoxicities of twelve chemotherapeutic drugs by clonogenic assay. The most cytotoxic agents tested were methotrexate, mitoxantrone, adriamycin, mitomycin C and cisplatin. These in vitro findings are compatible with the activity of these drugs given systemically as single agents in phase II clinical trials in patients with advanced bladder cancer.

Animals↗

PubMed1985

The anti-allergic effects of FPL 52694.

The substituted chromone carboxylic acid FPL 52694 inhibited models of IgE-mediated immediate hypersensitivity reactions in the rat by a mechanism similar to that of sodium cromoglycate. The compound was more potent than sodium cromoglycate but unlike cromoglycate was active following oral administration.

Administration, Oral↗

PubMed1985

Quantitative changes in mast cells and microvascular pattern associated with dietary gastric ulceration in rats.

The regional distribution of mast cells and the microvascular pattern in the stomach of Wistar rats fed on an ulcerogenic diet were compared with those of control animals. Mounts of the distended stomach were made and stained for blood vessels and mast cells in the subserosal layer. Measurements made under dark ground illumination in 12 operationally defined regions of the serosa demonstrated that the stomach wall in the region around the oesophagus is more richly vascularised and possesses a greater number of mast cells (60 cells per mm2) than other regions. Changes in the mast cells and microvasculature were observed in rats fed an ulcerogenic diet, notably in the zone around the oesophagus, where more than 30% mast cells were degranulated and the mean vascular diameter increased by 26%. The severity of the vascular changes correlated with the location of the ulcers of which 50% were in the zone surrounding the oesophagus.

Animals↗

PubMed1985

The production and properties of a tissue plasminogen activator from normal epithelial cells grown in microcarrier culture.

A plasminogen activator with different biochemical and physical properties to the one obtained from Bowes melanoma cells has been isolated from a normal epithelial cell line derived from guinea-pig keratocytes (GPK). Cell growth and enzyme production is carried out in microcarrier cultures using up to 15 g Cytodex 3 per litre. Cells are maintained in optimum conditions by the use of a closed perfusion loop and the system has been scaled up to 20 litres. Initially enzyme production was in serum-free medium following a growth phase in serum supplemented medium. However, the discovery that the cells produce the enzyme mainly during the replicative phase has led to investigating the use of serum-substitutes and growth factors on cell growth and enzyme production. The aim is to harvest the enzyme after the growth phase which precludes the use of high (greater than 1%) serum concentrations. The biochemical and biophysical properties of the enzyme are described.

Animals↗

PubMed1985

The effect of zinc on mouse melanoma growth in vitro and in vivo.

The effect of Zn2+ on mouse melanoma growth in vitro and in vivo was studied. Under in vitro conditions the proliferation of a Cloudman mouse melanoma cell line was inhibited by zinc ions at 10(-4) M, as measured by 3H-thymidine incorporation and optical density of NaOH cell digests. However, in vivo it was not possible to suppress both B16 and Cloudman S91 melanoma growth in mice by the administration of zinc ions. There were no significant differences in tumor growth after subcutaneous inoculation between mice constantly receiving 0.1% zinc acetate or 0.05% zinc sulphate in their drinking water and control groups, nor was it possible to decrease the number of lung metastases by zinc treatment after intravenous inoculation of tumor cells. The increased dietary supply of Zn failed to influence the survival time of mice in both melanoma types studied. Preincubation in vitro of cell suspensions in 10(-3) M zinc acetate prior to injection inhibited melanoma development in vivo. This implies that the in vivo zinc levels did not reach the necessary cytotoxic concentration.

Animals↗