Biomedical subjects

F Solano

Publications and source records attributed to F Solano.

At least 91 records · Page 5Linked to original sources

PubMed1991

Opposite effect of cytochalasin B on agonist-induced respiratory burst in neutrophils and monocytes.

The effects of cytochalasin B on the respiratory burst, calcium transients and cell shape change of neutrophils and monocytes has been studied. Cytochalasin B enhanced fMLP-induced respiratory burst in neutrophils whereas the opposite effect, i.e. an inhibition close to 50%, was elicited in fMLP-stimulated monocytes. The differences did not depend on calcium homeostasis. On the basis of cell shape changes, and the well known effect of cytochalasin B on actin polymerization, the opposite effects of cytochalasin B could stem from differences between both cell types concerning the pattern of cytoskeleton-membrane interaction which could affect the recruitment and assembly of membrane-bound or cytosolic components of NADPH-oxidase.

Calcium↗

PubMed1991

Induction of programmed cell death (apoptosis) in mature lymphocytes.

The apoptosis of human peripheral blood lymphocytes was analyzed by the breakdown of DNA into oligonucleosome-sized fragments. The mature lymphocytes were rendered sensitive to apoptosis by either the omission of fetal bovine serum in the culture medium or the addition of polymyxin B. In the first case it was counteracted by phorbol myristate acetate. The possible involvement of protein kinase C in cell survival is pointed out.

Blood↗

PubMed1991

Effect of tumour-promoting phorbol ester on calcium homeostasis in human platelets.

The more interesting features of the effects or PMA on [Ca2+]i and ATP release were the following: 1. preincubation with PMA inhibited thrombin-evoked calcium transients; 2. PMA stimulated slightly the release of calcium and ATP whereas inhibited calcium and ATP pools sensitive to thrombin; 3. A23187 reversed the inhibitory effect of PMA; 4. subsaturating thrombin concentrations gave results similar to PMA on thrombin-induced calcium and ATP release but not on [Ca2+]i.

Adenosine Triphosphate↗

PubMed1991

After dopachrome.

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Indolequinones↗

PubMed1990

Regulation of mammalian melanogenesis. I: Partial purification and characterization of a dopachrome converting factor: dopachrome tautomerase.

A protein that catalyzes the decoloration of dopachrome has been partially purified from B16 mouse melanoma tumors. The enzyme is preferentially associated to the melanosomes, but it is also found in the microsomal and cytosolic fractions of cellular homogenates. The protein is clearly different from tyrosinase, and should be related to the dopachrome oxidoreductase (Barber et al. (1984) J. Invest. Dermatol. 83, 145-149) and the dopachrome conversion factor (Korner and Pawelek (1980) J. Invest. Dermatol. 75, 192-195) since the reaction product of dopachrome conversion is 5,6-dihydroxyindole-2-carboxylic acid. The protein appears to have an oligomeric structure, with a molecular mass slightly higher than 300 kDa estimated by gel filtration, whereas the molecular mass of the monomer might be approx. 46 kDa estimated by SDS-PAGE electrophoresis. Its Km for dopachrome is around 100 microM. The enzyme is competitively inhibited by indoles and is unaffected by metal chelators. It also has the ability to increase the amount of melanin formed from L-tyrosine by melanoma tyrosinase, and therefore, cannot be considered an 'indole blocking factor' as was suggested for the related dopachrome oxidoreductase. Since the reaction catalyzed by the enzyme is a tautomeric shift on dopachrome, we would propose dopachrome tautomerase (EC 5.3.2.3) as the most precise and informative name.

Animals↗

PubMed1990

Regulation of mammalian melanogenesis. II: The role of metal cations.

Melanogenesis can be divided into two phases. The first one involves two tyrosinase-catalyzed oxidations from tyrosine to dopaquinone and a very fast chemical step leading to dopachrome. The second phase, from dopachrome to melanin, can proceed spontaneously through several incompletely known reactions. However, some metal transition ions and protein factors different from tyrosinase might regulate the reaction rate and determine the structure and relative concentrations of the intermediates. The study of the effects of some divalent metal ions (Zn, Cu, Ni and Co) on some steps of the melanogenesis pathway has been approached using different radiolabeled substrates. Zn(II) inhibited tyrosine hydroxylation whereas Ni(II) and Co(II) were activators. Ni(II), Cu(II) and Co(II) accelerated chemical reactions from dopachrome but inhibited its decarboxylation. Dopachrome tautomerase also decreased decarboxylation. When metal ions and this enzyme act together, the inhibition of decarboxylation was greater than that produced by each agent separately, but amount of carboxylated units incorporated to the melanin was not higher than the amount incorporated in the presence of only cations. The amount of total melanin formed from tyrosine was increased by the presence of both agents. The action of Zn(II) was different from other ions also in the second phase of melanogenesis, and its effect on decarboxylation was less pronounced. Since tyrosine hydroxylation is the rate-limiting step in melanogenesis, Zn(II) inhibited the pathway. This ion seems to be the most abundant cation in mammalian melanocytes. Therefore, under physiological conditions, the regulatory role of metal ions and dopachrome tautomerase does not seem to be mutually exclusive, but rather complementary.

Animals↗

PubMed1990

A new spectrophotometric assay for dopachrome tautomerase.

The existence of a new enzyme involved in mammalian melanogenesis has been recently reported. The names dopachrome oxidoreductase and dopachrome tautomerase have been proposed for the enzyme. So far, this enzyme has been assayed at 475 nm on the basis of its ability to catalyze dopachrome decoloration. This method presents two major problems, derived from the instability of the substrate (dopachrome): (1) dopachrome must be prepared immediately before use, and (2) the rate of dopachrome decoloration in the absence of the enzyme is not negligible, and, furthermore, is enhanced by non-enzymatic agents. In order to overcome these problems, we present a new procedure that combines: (1) a quantitative, fast and easy way to prepare dopachrome from L-dopa by sodium periodate oxidation; (2) a spectrophotometric method in the UV region, at 308 nm, based on following the absorbance increase due to the enzyme-specific tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid as opposed to the absorbance decrease due to the spontaneous decarboxylative transformation of dopachrome into 5,6-dihydroxyindole. The advantages of these methods as compared to the previously used procedures are discussed.

Animals↗

PubMed1990

Distribution of extracutaneous melanin pigment in Sparus auratus, Mugil cephalus, and Dicertranchus labrax (Pisces, Teleostei).

The morphological and biochemical characteristics of pigment accumulations found in the kidney, liver, spleen, and mesentery of three different species of teleost fishes have been studied. There are significant differences in number, distribution, and morphology of pigment accumulations in different organs of the three species. Biochemical studies have shown the existence of tyrosinase activity in the mesentery of Mugil cephalus and in the kidney and mesentery of Sparus auratus. No tyrosinase activity was found in any internal organs of Dicertranchus labrax. That activity was assayed using three methods: tyrosine hidroxylation, dopa oxidation, and melanin formation. The morphological and biochemical observations are in agreement. In those organs in which we have demonstrated melanin synthetic activity, the pigment cells are morphologically and like melanophores, while in the organs that show no melanin synthetic activity, the pigment cells resemble macrophages.

Animals↗

PubMed1990

Transport of L-tyrosine by B16/F10 malignant melanocytes: characterization of the process.

The main characteristics of L-tyrosine (L-Tyr) uptake by B16/F10 malignant melanocytes are reported. This amino acid can be taken up by two systems, both of them being saturable. The first one would be system L. This system can be studied in cells preloaded with amino acids that are a good substrate for system L, such as L-methionine or L-tryptophan. The kinetic parameters for L-Tyr uptake by this transport system are Vm = 6.5 pmol L-Tyr/10(3) cells.min and Km around 130 microM. The second system, probably the system ASC, shows lower capacity but higher affinity than the former. This system can be detected only in cells previously depleted of amino acids, showing approximate kinetic values of Vm 0.05 pmol L-Tyr/10(3) cells.min and Km around 5 microM. It is shown that the increase in cell density yields a decrease in the rate of L-Tyr uptake by system L, but this increase does not affect the high affinity system, alpha-MSH does not affect significantly the L-Tyr uptake by both systems. 2-Amino bicyclo-(2,2,1)-heptane-2-carboxylic acid produces a remarkable inhibition of the rate of L-Tyr uptake, but alpha-methylaminoisobutyric acid does not affect the rate of transport of this amino acid. The absence of sodium produces a slight but reliable decrease in the rate of L-Tyr uptake, supporting the involvement of two different transport systems. The ionophores monensin and nigericin enhance the transport by system L, but this effect is suppressed by the presence of ouabain. This finding indicates that the (Na+ -K+)-ATPase is essential for the stimulating action of ionophores.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1990

Transport of L-tyrosine by B16/F10 melanoma cells: the effect of the intracellular content of other amino acids.

The uptake of L-Tyr by B16/F10 malignant melanocytes in culture has been studied. These melanoma cells can either be depleted of amino acids by 1 h preincubation in Hanks' isotonic medium or preloaded with a specific amino acid by 1 h preincubation in the same solution containing 2 mM of the amino acid to be preloaded. By means of these pretreatments, it is shown that the rate of L-Tyr uptake is greatly dependent on the content of other amino acids inside the cells. The L-Tyr uptake is higher in cells preloaded with amino acids transported by the L and ASC systems than in cells depleted of amino acids or preloaded with amino acids transported by the A system. It is concluded that L-Tyr is mainly taken up by an exchange mechanism with other amino acids mediated by the L1 system, although the ASC system can also participate in the process. In agreement with that, the homo-exchange performed by cells preloaded with unlabelled L-Tyr is more efficient than any other hetero-exchange, although L-Dopa, the product of tyrosine hydroxylation in melanin synthesis, is almost as efficient as L-Tyr. Apart from aromatic amino acids, melanoma cells preloaded with L-Met and L-His also yield a high initial rate of L-Tyr uptake. The results herein suggest that melanoma cells do not have transport systems specific for L-Tyr, even if this amino acid is needed to carry out the differential pathway of this type of cells, melanosynthesis.

Amino Acids↗

PubMed1990

Determination of the myeloperoxidase-hydrogen peroxide-halide system activity following the respiratory burst of human neutrophils.

A sensitive assay was developed for the determination of the activity of cell derived myeloperoxidase-hydrogen peroxide-halide system associated with the respiratory burst of human neutrophils. The methodological approach relies on the oxidation of 2-nitro-5-thiobenzoic acid (TNB) which could be continuously monitored by means of a chart recorder attached to a photometer. The oxidation of TNB by neutrophils stimulated with either phorbol myristate acetate or N-formyl-Met-Leu-Phe showed the following features: a) Superoxide dismutase failed to modify the rate of TNB oxidation; b) it was strongly inhibited by catalase but not by boiled catalase; c) azide and cyanide inhibited the reaction; d) it required a halide cofactor, namely chloride, and e) the onset of TNB oxidation was strongly potentiated by the calcium ionophore A23187. These results support the involvement of the myeloperoxidase system in the oxidation of TNB. Therefore, the procedure described here seems to be a suitable method for the above indicated purpose.

Azides↗

PubMed1989

Action of endogenous proteases on the distribution of tyrosinase isozymes in Harding-Passey mouse melanoma.

A high percentage of the total tyrosinase found in Harding-Passey mouse melanoma occurs as a soluble form. This paper shows that melanosomal tyrosinase can be solubilized by several endogenous proteases to yield active tyrosinase. This enzyme, once proteolytically solubilized, can be further degraded, leading to enzyme inactivation. The nature and specificity of the main proteases involved in the solubilization process change depending on the size and necrosis stage of the tumour. Cathepsin B could be the main protease responsible for the solubilization in small tumours (less than 0.5 g). Large tumours are rich in necrotic cells, and cathepsin D and serine-proteases are the main hydrolytic enzymes involved in the proteolytic action on melanosomes. These results support the view that the high activity of tyrosinase found in the soluble fraction of malignant melanoma is mainly an artefact resulting from degradation of melanosomes by a variety of endogenous proteases, rather than the result of the actual occurrence of high levels of an independent cytosolic isozyme.

Animals↗

PubMed1989

A reexamination of the melanin formation assay of tyrosinase and an extension to estimate phaeomelanin formation.

This paper presents some modifications of the melanin formation assay for tyrosinase from the point of view of both eu- and phaeomelanosynthesis. On the one hand, eumelanosynthesis can be estimated using neutral paper filters, such as the 3MM Whatman filters so far employed. The main advantages of this sort of paper are the very low blank values obtained in the absence of tyrosinase and its greater mechanical resistance in the successive washing steps. It is shown that the sensitivity of the assay can be enhanced by the addition of 1 mM Ni(II) to the incubation mixture or of NaOH to stop the enzymatic reaction and allow the incorporation of indolic intermediates into the polymer. Furthermore, the accuracy is also enhanced by the proposed modifications, since all reactions from dopaquinone are standardized, and the assay becomes only dependent on the tyrosinase activity. On the other hand, phaeomelanosynthesis cannot be estimated using neutral paper because of the slow rate of polymerization of the intermediates and the poor absorption of thiol-dopa conjugates to this kind of paper. It is shown that synthesis of this type of melanin can be estimated in the presence of glutathione by means of a cationic filter paper and by washing the excess of the radioactive substrate with distilled water instead of acidic media. Thus, the assay may be adapted to measure eu- or phaeomelanosynthetic activity by introducing slight modifications. This assay must be used with caution if detergent-solubilized tyrosinase is used, because detergents strongly inhibit melanin absorption to paper filters.

Animals↗

PubMed1988

The role of sulfhydryl compounds in mammalian melanogenesis: the effect of cysteine and glutathione upon tyrosinase and the intermediates of the pathway.

The effect of cysteine and glutathione on mammalian melanogenesis has been studied. It has been shown that their action is mediated by two different mechanisms. (a) The reaction of the thiol groups with dopaquinone after the tyrosinase-catalyzed oxidation of tyrosine and dopa. This mechanism leads to the formation of sulfhydryl-dopa conjugates and finally sulfur-containing pigments, phaeomelanins instead of eumelanins. This fact might produce an inhibition of melanogenesis due to the slower rate of chemical reactions involved in the polymerization of such thiol-conjugates when compared to that of indoles. (b) The direct interaction between the sulfhydryl compounds and the tyrosinase active site. This interaction may regulate the activity of the enzyme. It is shown that Harding-Passey mouse melanoma tyrosinase is more sensitive to sulfhydryl compounds than mushroom tyrosinase. Cysteine always produces an inhibition of the tyrosinase hydroxylase and dopa oxidase activities of melanoma tyrosinase, this inhibition becoming greater as the cysteine concentration increases. On the other hand, glutathione produces an activation of the tyrosine hydroxylase activity below 3 mM and an inhibition at higher concentrations. The limit between the enzymatic activation and inhibition appears at glutathione concentrations similar to the physiological levels of this compound found in melanocytes. Although the switch from eumelanogenesis to phaeomelanogenesis occurs at much lower concentrations of glutathione, taking into account these data it is discussed that this sulfhydryl compound may regulate not only the type but also the amount of melanin formed inside melanocytes.

Animals↗

PubMed1988

The effect of hyperthermia on ornithine decarboxylase activity in different rat tissues.

Hyperthermia produced a decrease of ornithine decarboxylase activity in different tissues of adult rats. The fall in ornithine decarboxylase was dependent on time of exposure and temperature. The decay of ornithine decarboxylase activity in liver, brain, kidney, heart, spleen and testes was rather similar. The t1/2 for liver ornithine decarboxylase determined by the hyperthermic treatment (40 degrees ambient temperature) was 20 min. Ornithine decarboxylase activity was recovered in all tissues exposed to the hyperthermic shock after a period of 4 hours, although the degree of recovery was dependent on the type of tissue. The effect that hyperthermia produces on ornithine decarboxylase activity in rats could be related to an inhibition in the synthesis of active enzyme rather than to a specific degradation or inactivation of ornithine decarboxylase molecule.

Amino Acids↗

PubMed1988

Kinetic study of the inhibition of rat liver ornithine decarboxylase by diamines; considerations on the mechanism of interaction between enzyme and inhibitor.

1. Partially purified rat liver ornithine decarboxylase is inhibited by several diamines including putrescine, 1,3-diaminopropane, cadaverine and p-phenylenediamine. 2. The inhibition is dependent on pH, being strong at pH above 8 and negligible below pH 6.5. 3. The kinetic study of the inhibition showed that while the aromatic diamine behaved as a simple competitive inhibitor, the aliphatic diamines presented a more complex pattern of inhibition in which two molecules of inhibitor might bind to the enzyme active site. 4. The Ki values for the different inhibitors were calculated and the degree of affinity for the enzyme was p-phenylenediamine greater than putrescine greater than cadaverine greater than 1,3-diaminopropane. 5. A molecular mechanism explaining how one or two molecules of inhibitor can bind to the enzyme is proposed.

Animals↗

PubMed1988

Half-lives of tyrosinase isozymes from Harding-Passey mouse melanoma.

The half-lives of tyrosinase isozymes, a key enzyme in melanogenesis, have been determined using two different approaches: (a) cycloheximide treatment of mice bearing growing tumors and measurement of the residual enzymatic activity. This approach detected two soluble forms of cytosolic tyrosinase with half-lives of 1/2 and 8 h, respectively. The melanosomal isozyme showed a t1/2 of 3 1/2 h. (b) Metabolic labelling of tyrosinase with [35S]Met and immunoprecipitation analysis using a monoclonal antityrosinase. This method gave values slightly longer, 5 h and 9 1/2 h for the melanosomal and cytosolic tyrosinase, respectively. The origin of soluble tyrosinase and its utility to employ that enzymatic activity in melanoma chemotherapy using catechols as tyrosinase-dependent precursors of cytotoxic quinones is discussed.

Animals↗

PubMed1988

Assays for mammalian tyrosinase: a comparative study.

This work describes a comparative study of the tyrosinase activity determined using three methods which are the most extensively employed; two radiometric assays using L-tyrosine as substrate (tyrosine hydroxylase and melanin formation activities) and one spectrophotometric assay using L-dopa (dopa oxidase activity). The three methods were simultaneously employed to measure the activities of the soluble, melanosomal, and microsomal tyrosinase isozymes from Harding-Passey mouse melanoma through their purification processes. The aim of this study was to find any correlation among the tyrosinase activities measured by the three different assays and to determine whether that correlation varied with the isozyme and its degree of purification. The results show that mammalian tyrosinase has a greater turnover number for L-dopa than for L-tyrosine. Thus, enzyme activity, expressed as mumol of substrate transformed per min, is higher in assays using L-dopa as substrate than those using L-tyrosine. Moreover, the percentage of hydroxylated L-tyrosine that is converted into melanin is low and is affected by several factors, apparently decreasing the tyrosinase activity measured by the melanin formation assay. Bearing these considerations in mind, average interassay factors are proposed. Their values are 10 to transform melanin formation into tyrosine hydroxylase activity, 100 to transform tyrosine hydroxylase into dopa oxidase activity, and 1,000 to transform melanin formation into dopa oxidase activity. Variations in these values due to the presence in the tyrosinase preparations of either inhibitors or regulatory factors in melanogenesis independent of tyrosinase are also discussed.

Animals↗