
Jose Antonio LozanoUniversity of Murcia | UM
Jose Antonio Lozano
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Publications (144)
Fungi have traditionally been considered opportunistic pathogens in primary infections caused by virulent bacteria, protozoan, or viruses. Consequently, antimycotic chemotherapy is clearly less developed in comparison to its bacterial counterpart. Currently, the three main families of antifungals (polyenes, echinocandins, and azoles) are not suffic...
Current antifungal chemotherapy against the prevalent basidiomycete Cryptococcus neoformans displays some drawbacks. This pathogenic fungus is refractory to echinocandins, whereas conventional treatment with amphotericin B plus 5-fluorocytosine has a limited efficacy. In this study, we explored the potential cryptococcal activity of some natural ag...
The potential fungicidal action of the natural extracts, carnosic acid (obtained from rosemary) and propolis (from honeybees’ panels) against the highly prevalent yeast Candida albicans, used herein as an archetype of pathogenic fungi, was tested. The separate addition of carnosic acid and propolis on exponential cultures of the standard SC5314 C....
Lección inaugural del Curso Académico 2008/2009 en la Universidad de Murcia, a cargo del catedrático José Antonio Lozano Teruel.
The melanocortin 1 receptor, a Gs protein-coupled receptor expressed in epidermal melanocytes, is a major determinant of skin pigmentation and phototype and an important contributor to melanoma risk. MC1R activation stimulates synthesis of black, strongly photoprotective eumelanin pigments. Several MC1R alleles are associated with red hair, fair sk...
Solar UV and other ionizing radiations cause a generation of reactive oxygen species, induce cellular DNA damage and alter skin homeostasis. The use of exogenous antioxidants is increasingly frequents, we attempt to demonstrate that a rosmarinic acid extract acts as photo-protector; both free radical scavenger as an inducer of the body's own endoge...
Available data have led to a controversy on the relationship between human leukocyte antigen (HLA) and cutaneous malignant melanoma susceptibility or prognosis. Moreover, the influence of HLA-C on melanoma has not yet been well established. Therefore, the aim of the current study was to analyze the possible influence of the HLA system on melanoma s...
Polyphenolic compounds are widely distributed in the vegetable kingdom and are therefore consumed regularly in the human diet. Epidemiological studies suggest that foods rich in polyphenolic compounds contribute to reducing the risk of cancer. The purpose of our work is to: 1) study the possible cytotoxicity and antiproliferative effects of 13 poly...
The response of B16F10 melanoma and Melan-a melanocyte cell lines to treatment with five polyhydroxylated flavonoids and gallic acid, after 24 and 72 h of exposure, was determined, and the relationship between any antiproliferative effects observed and the chemical structure is discussed. After 24 h, none of the studied compounds showed significant...
The alpha-melanocyte-stimulating hormone (alphaMSH) receptor (MC1R) is a major determinant of mammalian skin and hair pigmentation. Binding of alphaMSH to MC1R in human melanocytes stimulates cell proliferation and synthesis of photoprotective eumelanin pigments. Certain MC1R alleles have been associated with increased risk of melanoma. This can be...
Although flavonoids seem to be capable of acting at all stages of the carcinogenic process, little information is available on their action in melanoma cell lines. The aim of this study was to assess the response of B16F10 and SK-MEL-1 melanoma cell lines to treatment with six different flavonoids after 24 and 72 h of exposure and to relate the res...
Melanin synthesis in mammals is catalysed by at least three enzymic proteins, tyrosinase (monophenol dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) and tyrosinase-related proteins (tyrps) 1 and 2, whose genes map to the albino, brown and slaty loci in mice, respectively. Tyrosinase catalyses the rate-limiting generation of L-dopaquinon...
The melanosomal proteins encoded by the silver locus play important roles in melanogenesis. The human locus yields two proteins, PMEL17 and GP100, by alternative mRNA splicing. The mouse si locus was reported to encode a Pmel17 protein, and later gp87, a GP100 homologue. When we re‐examined the products of wild‐type and silver ‐mutant mouse si loci...
The melanosomal proteins encoded by the silver (si, SILV, or PMEL17) locus play important roles in melanogenesis and are actively investigated as targets for melanoma immunotherapy. The human silver locus yields two proteins, gp100 and PMEL17, by alternative splicing of a common mRNA precursor. Mouse melanocytes exclusively express the gp100 orthol...
The melanosomal proteins encoded by the silver locus play important roles in melanogenesis. The human locus yields two proteins, PMEL17 and GP100, by alternative mRNA splicing. The mouse si locus was reported to encode a Pmel17 protein, and later gp87, a GP100 homologue. When we re-examined the products of wild-type and silver-mutant mouse si loci,...
Alpha melanocyte-stimulating hormone (MSH) and related proopiomelanocortin-derived (POMC) peptides bind to the melanocortin 1 receptor (MC1-R) of mammalian melanocytes and stimulate proliferation and melanogenesis. POMC transcripts and alpha-MSH-like immunoreactivity have been found in melanoma cells and a possible autocrine loop involving MC1-R an...
alpha melanocyte-stimulating hormone (MSH) and related proopiomelanocortin-derived (POMC) peptides bind to the melanocortin 1 receptor (MC1-R) of mammalian melanocytes and stimulate proliferation and melanogenesis. POMC transcripts and a-MSH-like immunoreactivity have been found in melanoma cells and a possible autocrine loop involving MC1-R and PO...
Melanin synthesis in mammals is catalyzed by three structurally related, membrane-bound proteins, tyrosinase, and the tyrosinase-related proteins 1 and 2 (TRP1 and TRP2). Current evidence suggests that in vivo these proteins may form a multienzyme complex. However, neither the precise composition of the complex, nor the specific interactions betwee...
The inflammatory cytokine tumor necrosis factor-α (TNF-α) is present in the dermal and epidermal layers of normal skin [Kilgus, O., Payer, E., Schreiber, S., Elbe, A., Strohal, R. & Stingl, G. (1993) J. Invest. Dermatol. 100, 674−680]. Its local concentrations are modified by several stimuli, including wound healing and ultraviolet irradiation. Mor...
Colección de preguntas de respuesta múltiple para autoevaluación. Clasificadas por temas y por grados de dificultad
Most of our knowledge of the mammalian tyrosinase related protein (TRP) activities is derived from studies using murine melanoma models, such as B16 or Cloudman S-91 melanocytes. Owing to the high degree of homology between the murine and human enzymes, it has been assumed that their kinetic behaviour could be similar. However, the protein sequence...
Detergent solubilized extracts of the cochleae of adult gerbils (Meriones unguiculatus) contain a tyrosine hydroxylase activity measurable by the radiometric method of Pomerantz. This activity is not related to Fenton-type reactions, since it is not inhibited by free radical scavengers and is heat and protease sensitive. It does not appear to be re...
Current evidence suggests that melanogenesis is controlled by epidermal paracrine modulators. We have analyzed the effects of transforming growth factor-beta1 (TGF-beta1) on the basal melanogenic activities of B16/F10 mouse melanoma cells. TGF-beta1 treatment (48 h) elicited a concentration-dependent decrease in basal tyrosine hydroxylase and 3,4-d...
Current evidence suggests that melanogenesis is controlled by epidermal paracrine modulators. We have analyzed the effects of transforming growth factor-beta 1 (TGF-beta 1) on the basal melanogenic activities of B16/F10 mouse melanoma cells. TGF-beta 1 treatment (48 h) elicited a concentration-dependent decrease in basal tyrosine hydroxylase and 3,...
Dopachrome tautomerase (DCT; EC 5.3.3.12) catalyses the conversion of L-dopachrome into 5,6-dihydroxyindole-2-carboxylic acid in the mammalian eumelanogenic biosynthetic pathway. This enzyme, also named TRP2, belongs to a family of three metalloenzymes termed the tyrosinase-related proteins (TRPs). It is well known that tyrosinase has copper in its...
The melanogenic activities in the eye of the adult gerbil (Meriones unguiculatus) have been investigated and compared to those found in the B16 mouse melanoma model. Eye extracts contain tyrosine hydroxylase, DOPA oxidase, DOPAchrome tautomerase and DHICA oxidase activities. The subcellular distribution of these activities was investigated by diffe...
Melatonin was found to have a small inhibitory effect on tyrosinase activity and a slight stimulatory action on dopachrome tautomerase activity in B16 mouse melanoma cells. These effects were time and dose dependent, with the maximal response being observed after 24–48h treatment and at concentrations of melatonin higher than the physiologic levels...
Within mammalian melanocytes, melanin biosynthesis is controlled by three enzymes structurally related: tyrosinase and two tyrosinase related proteins, TRP1 and TRP2. These melanosomal enzymes are integral membrane proteins with a carboxyl tail oriented to the cytoplasm, a single membrane-spanning helix and the bulk of the protein located inside th...
Penicillin and streptomycin, the most widely used antibiotics in mammalian cell cultures, caused a moderate stimulation in dopa oxidase and tyrosine hydroxylase activities, but a slight inactivation in the dopachrome tautomerase activity of B16/F10 melanoma cells at the routine concentration (100 units/ml penicillin and 100 micrograms/ml streptomyc...
Dopachrome tautomerase (DCT) catalyzes the conversion of L-dopachrome into 5,6-dihydroxyindole-2-carboxylic acid through the melanogenic biosynthetic pathway. This enzyme, also named TRP2, belongs to the family of the tyrosinase related proteins. The three members of the family contain two highly conserved metal-binding sites with three histidines...
Two forms of tyrosinase from B16 mouse melanoma were identified by nonreducing SDS-PAGE after solubilization of crude melanosomal preparations with the nonionic detergent Brij 35. These forms, named LEMT and HEMT (low and high electrophoretic mobility tyrosinase, respectively), were purified by a combination of differential detergent extraction and...
Although melanins can be formed in vitro by the unique action of tyrosinase on L-tyrosine, it is now well accepted that other enzymes termed tyrosinase-related proteins are involved in mammalian melanogenesis. However, some aspects of their roles in the regulation of the pathway are still unknown. The action of dopachrome tautomerase on L-dopachrom...
Since the characterization of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) as a major melanogenic intermediate, the fate of this compound and the mechanisms of its incorporation into the melanin polymer have become major issues in the study of melanogenesis. DHICA is a stable dihydroxyindole with a low rate of spontaneous oxidation, suggesting tha...
Dopachrome tautomerase (DCT) is a recently characterized enzyme contributing to the control of melanogenesis in mammals. The enzyme catalyzes the rearrangement of L-Dopachrome (L-DC) to 5,6-dihydroxyindole 2-carboxylic acid (DHICA), while the spontaneous rearrangement of L-DC leads to 5,6-dihydroxyindole (DHI). Due to the lower reactivity of DHICA...
Mammalian tyrosinase exists in a variety of subcellular locations and maturation states that result from a complex post-translational processing with possible regulatory implications. So far, SDS-PAGE has proven to be the method of choice for the resolution of tyrosinase isoforms. However, the relatively poor sensitivity of the currently available...
B-16 mouse melanoma melanosomes contain two forms of tyrosinase that can be resolved by SDS/PAGE. These forms interact to different extents with the ion-exchanger DEAE-Sephadex and with hydroxyapatite, and have different affinity for the melanosomal membrane and/or the intraorganular matrix. After partial purification and complete separation of the...
In mouse melanoma melanocytes, alpha-melanocyte-stimulating hormone (MSH) stimulates differentiation, melanin synthesis and tyrosinase activity. However, the molecular mechanisms underlying these events have not yet been characterized. We have studied the activation of tyrosinase by MSH. Treatment of B16 melanoma cells with either theophylline, MSH...
Although tyrosinase has been considered for a long time the only enzyme involved in mammalian melanosynthesis, it has been shown that mouse melanoma melanosomes contain high levels of dopachrome tautomerase (DCT2), an enzyme catalyzing DC tautomerization to DHICA. At least in B16 mouse melanoma, DCT is present in higher catalytic amounts than tyros...
1. Intermediates in the process of melanin synthesis formed through oxidation of catechols by tyrosinase produced the inactivation of ornithine decarboxylase (ODC), a key enzyme in the polyamine biosynthesis pathway. 2. The inactivation was dependent on the substrate used (dihydroxybenzylamine > L-3,4-dihydroxyphenylalanine > L-tyrosine) and on the...
The antifungal reagent Fungizone (amphotericin B and deoxycholate) caused an activation in dopachrome tautomerase and dopa oxidase activities of B16/F10 melanoma cells at the routine concentration (2.5 micrograms/ml) used for preventing molds and yeast growth in cultures of animal cells. However, higher amphotericin B concentrations caused a signif...
alpha-MSH was found to decrease the recently characterized dopachrome tautomerase activity in cultures of B16/F10 mouse melanoma cells. Other stimulating agents of melanogenesis, like dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthine, theophylline, retinol, and retinoic acid, caused the same effect. The grade of inhibition depended on the nature of...
In spite of the central role of tyrosinase in mammalian pigmentation, few data are available on its structure and structure-function relationships based on direct analysis of the protein. A number of reasons have been invoked to account for this situation, including the problems for its purification and its resistance to proteases. However, no stud...
The regulation of the final steps of the melanogenesis pathway, after L-2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome) formation, is studied. It is shown that both tyrosinase and dopachrome tautomerase are involved in the process. In vivo, it seems that tyrosinase is involved in the regulation of the amount of melanin formed, whereas dopachro...
Our previous work indicated that IR-alpha-MSH (immunoreactive alpha-melanocyte-stimulating hormone) plasma levels are three times as high in melanoma patients with progressing disease than in disease-free patients, and that the melanoma tumor itself may be the source of IR-alpha-MSH. Further identification of the material in tumor extracts has been...
Tyrosinase induction in murine malignant melanocytes by alpha MSH is well known, but its molecular basis has not been characterized. Treatment of B16 melanoma cells with theophylline or alpha MSH mediates a larger induction of tyrosine hydroxylase than of dopa oxidase activity in total cell extracts, and in the melanosomal and microsomal fractions....
It is shown that dopachrome (2-carboxy-2,3-dihydroindole-5,6-quinone) tautomerase (DCT) is a glycoprotein containing N-linked oligosaccharides. The enzymic activity can be stimulated by partial deglycosylation with a number of glycosylases such as neuraminidase, beta-mannosidase and beta-galactosidase. However, the stability of the enzyme after the...
Dopachrome, an intermediate in melanin biosynthesis, exhibits some unusual properties. At physiologic pH (e.g., pH 6-8) it is unstable and spontaneously loses its carboxyl group to form 5,6-dihydroxyindole (DHI(and CO2. However, over this same pH range, if various metals or a melanocyte-specific enzyme are present, it rapidly rearranges to its isom...
The sensitivity of B16/F10 mouse melanoma cells to oxidative lysis after GSH depletion was studied. Treatment with 25 μM buthionine sulfoximine for 4h caused the almost complete disappearance of the tripeptide, this time being much shorter than that needed for other, non-melanogenic, cells. More than 80% depletion was also achieved by exposure to 2...
A vis-a-vis comparison between the effects of dopachrome tautomerase (DCT) and metal ions, e.g., cupric ions, on the kinetics and mode of rearrangement of dopachrome has been carried out under appropriate analytical conditions. The enzyme-promoted reaction is highly stereospecific for L-dopachrome, is unaffected by metal chelators and has an optima...
Dopachrome tautomerase (EC 5.3.2.3) catalyses the tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) within the melanin-formation pathway. We have analysed a series of substrate analogues and related compounds as possible substrates and inhibitors of tautomerization. The enzyme appears to be highly specific since D-dopac...
Previous studies have shown that melanoma is very sensitive to inhibitors of polyamine biosynthesis. In this work, we have studied changes in polyamine concentrations and the activities of two key enzymes of their biosynthetic pathway at different stages of growth of Harding-Passey melanoma transplanted to mice. We found that the peak activity of o...
The oral administration of alpha-difluoromethylornithine (DFMO), an enzyme-activated inhibitor of ornithine decarboxylase (ODC), produced a marked decrease in the rate of growth of amelanotic Harding-Passey melanoma transplanted in mice. The half-life of this compound in Harding-Pasey melanoma was 30 min. A combined treatment of DFMO and hypertherm...
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 dopachr...
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 prote...
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, deriv...
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...
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...
This paper deals with the kinetic study of a multisubstrate mechanism with enzyme inactivation induced by a suicide substrate. A transient phase approach has been developed that enables the deduction of explicit equations of product concentration vs. time. From these equations kinetic constants which characterize the suicide substrate can be obtain...
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...
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 m...
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 simultan...
The effect of several polyamines on the activity of tyrosinase from different sources has been studied. Diaminoethane, 1,3-diaminopropane and putrescine activated tyrosinase from Harding-Passey mouse melanoma but did not activate frog epidermis or mushroom tyrosinases. 1,3-diaminopropane was the strongest activator (Ka = 0.23 mM). The activation wa...
It was found that L-mimosine is a slow-binding inhibitor of L-DOPA oxidation by mushroom tyrosinase. This inhibition is characterized by a prolonged transient phase. A mechanism is postulated according to the kinetic data.
The present work deals with epinine oxidation by mushroom tyrosinase and sodium metaperiodate. Intermediates produced within short reaction times were characterized by repetitive scanning spectrophotometry and the stoichiometry of the appearance of the respective aminochrome was established. The oxidation pathway from epinine to aminochrome had the...
This paper deals with the kinetic study of reaction mechanisms with enzyme inactivation induced by a suicide substrate in the presence or absence of an auxiliary substrate and in conditions of excess of substrates in relation to the enzyme concentration and vice versa. A transient-phase approach has been developed that enables explicit equations wi...
This paper reports the effect of Cu(II) supplementation on the tyrosinase isozymes from Harding-Passey mouse melanoma. The dopa-oxidase activity of the microsomal and soluble isozymes is increased by incubation with Cu(II), whereas the activity of the unique 'in vivo' melanin-forming isozyme, bound to melanosomes, is not. Other divalent cations are...
Tyrosinase has a suicide inactivation reaction when it acts on o-diphenols. In the present paper, this reaction has been studied using a transient phase approach. Explicit equations of product vs. time have been developed for the multisubstrate mechanism of tyrosinase, and the kinetic parameters which characterize the enzyme acting on the suicide s...
In the pathway of melanin biosynthesis originating from L-tyrosine, the dopachrome accumulation at physiological pH is produced with a pronounced lag period, during which the level of L-dopa increases, following a sigmoidal kinetics to reach a steady-state. A kinetic model has been proposed for the overall pathway of melanization from L-tyrosine to...
A kinetic study of the inactivation of frog epidermis tyrosinase by a suicide substrate dopamine hydrochloride is described. The kinetic parameters and constants which characterize this reaction have been determined and the effects of pH and the stoichiometric inhibition by chloride have been considered.
1.1. Spectra of products obtained during dopa oxidation by mushroom tyrosinase in presence of cysteine or glutathione were recorded for the first minutes of the enzymatic reaction.2.2. Two isosbestic points were defined, indicating the existence of a constant ratio between the disappearance of dopa and the formation of cysteinyl- or glutathione-dop...
A method for the determination of kinetic constants of inhibited acetylcholinesterase, in the presence of substrate, is proposed. Theoretical, experimental and data treatment aspects are described for a suitable analysis of inhibitors acting with one or two steps. Limitations of other methods recently reported are pointed out.