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1.
Biochim Biophys Acta ; 856(2): 193-201, 1986 Apr 14.
Artículo en Inglés | MEDLINE | ID: mdl-3955038

RESUMEN

The lipid composition of whole sheep platelets and their subcellular fractions was determined. The basic lipids show similar distributions in granules, microsomes, plasma membranes and whole platelets. Phospholipid (about 70% of total lipids) and cholesterol (25% of total lipids) are the principal lipid components. Free cholesterol represents about 98% of the total, whereas cholesteryl ester is a minor component. The phospholipid composition found in intact platelets and their subcellular particles is about: 35% phosphatidylethanolamine (PE), 30% phosphatidylcholine (PC), 20% sphingomyelin and 15% phosphatidylserine (PS). We also investigated aminophospholipid topology in intact platelet plasma membranes and platelet liposomes by using the nonpenetrating chemical probe trinitrobenzenesulfonic acid (TNBS), because they are the major components of total lipids. In intact platelets, PS is not accessible to TNBS during the initial 15 min of incubation, whereas 18% PE is labelled after 15 min. In contrast, in phospholipid extracted from platelets 80% PE and 67% PS react with TNBS within 5 min, while 27 and 25% PE and 15 and 19% PS from liposomes and isolated plasma membranes, respectively, were modified after 15 min of incubation. In view of this chemical modification, it is concluded that 22% of PE and less than 1% of PS are located on the external surface of intact platelet plasma membranes. The asymmetric orientation of aminophospholipids is similar between liposomes and isolated plasma membrane. PS (23 and 28%) and PE (34 and 31%) are scarcely represented outside the bilayer. The data found are consistent with the nonrandom phospholipid distribution of blood cell surface membranes.


Asunto(s)
Plaquetas/análisis , Lípidos/sangre , Liposomas , Lípidos de la Membrana/sangre , Fosfatidiletanolaminas/sangre , Fosfatidilserinas/sangre , Animales , Fraccionamiento Celular , Membrana Celular/análisis , Colesterol/sangre , Gránulos Citoplasmáticos/análisis , Fosfolípidos/sangre , Ovinos , Fracciones Subcelulares/análisis , Ácido Trinitrobencenosulfónico/farmacología
2.
Biochim Biophys Acta ; 1415(1): 163-73, 1998 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-9858719

RESUMEN

Acetylcholinesterase (AChE, EC 3.1.1.7) was extracted from sheep platelets by successive homogenizations, yielding low-salt soluble (LSS), high-salt soluble (HSS) and detergent-soluble (DS) fractions. These accounted, respectively, for about 30%, 7% and 60% of total AChE activity. Applications of hydrophobic chromatography on phenyl-agarose to three solubilized fractions revealed that hydrophilic forms were almost exclusively located in the LSS fraction ( approximately 27% of total AChE), whereas most amphiphilic forms were present in DS extracts ( approximately 59% of total AChE), the remaining forms being distributed among aqueous soluble fractions. Enzyme molecular forms in the solubilized extracts were identified by centrifugation in 5-20% sucrose gradients containing Triton X-100 or Brij 97 to differentiate between hydrophilic or amphiphilic species. A predominance of hydrophilic dimeric forms ( approximately 22%), with small amounts of hydrophilic monomers (5%) and amphiphilic dimers and monomers (3%), was found in soluble AChE (LSS fraction). Amphiphilic AChE forms extracted in the HSS and DS fractions had a single peak in the sedimentation profiles with sedimentation coefficients of about 6S in gradients with Triton X-100; these were slightly shifted in the presence of Brij 97. After treatment with dithiothreitol, this molecular form solubilized in DS was converted to another molecular form with a lower sedimentation coefficient. Our results show that amphiphilic globular dimers are the dominant molecular form in sheep platelet AChE, suggesting a partial conversion of this membrane-bound form into soluble dimers and monomers, mainly with a hydrophilic character, through the action of either endogenous proteases and phospholipases or residual endogenous reducing agents.


Asunto(s)
Acetilcolinesterasa/sangre , Plaquetas/enzimología , Acetilcolinesterasa/química , Acetilcolinesterasa/aislamiento & purificación , Animales , Cromatografía Liquida , Ovinos , Solubilidad
3.
Biochim Biophys Acta ; 1419(2): 195-206, 1999 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-10407071

RESUMEN

To date, although at least 75 different PTPases (protein-tyrosine-phosphate-phosphohydrolase, EC 3.1.3.48) have been identified, those detected in platelets are rather scarce. Based on previous results from our laboratory, we investigated the existence of new PTPases in platelets. Triton X-114 phase partitioning of Triton X-100-solubilized human and sheep platelet membranes allowed PTPase to be recovered in the detergent-rich (40-35%, respectively) and -poor phases (60-65%, respectively). Sedimentation analyses of both phases from the sheep species revealed hydrophilic 6S and 3.7S, and amphiphilic 7.5S and 10.3S PTPase forms. Sedimentation analyses of human platelet membrane-associated or cytosolic PTPase revealed hydrophilic 6.7S and 4.3S, and amphiphilic 5.5S and 10.8S forms, or hydrophilic 4S, 5.9S and 6.9S forms, respectively. Western blot analysis using monoclonal antibodies (MoAb) against human PTP1B, PTP1C, PTP1D and RPTPalpha (mouse anti-human PTPase MoAbs) showed that RPTPalpha was not present in platelets and that the PTP1C type and PTP1D type (but probably not the PTP1B type) were expressed in sheep species. Immunoblots also revealed that all PTPases detected were mainly membrane-associated, with similar percentages of cellular distribution in both species. All PTPases were mainly recovered in the detergent-poor phases from the Triton X-114 phase partitioning, although PTP1D from human species was also significantly present (30%) in the detergent-rich phase. Additionally, all PTPases sedimented within the same PTPase peak in sucrose gradients (sedimentation coefficients around 4S). These findings indicate that amphiphilic and hydrophilic PTPases different from PTP1B, PTP1C, PTP1D or RPTPalpha, with higher sedimentation coefficients and with higher activity when O-phosphotyrosine or a synthetic peptide phosphorylated on tyrosine were used as substrates, are present in platelets.


Asunto(s)
Plaquetas/enzimología , Proteínas Tirosina Fosfatasas/química , Animales , Membrana Celular/enzimología , Centrifugación por Gradiente de Densidad , Humanos , Péptidos y Proteínas de Señalización Intracelular , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/análisis , Ovinos
4.
Biochim Biophys Acta ; 1419(2): 313-24, 1999 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-10407082

RESUMEN

Incubation of sheep platelet crude membranes with xanthine oxidase (XO)/hypoxanthine/Fe(2+)-ADP revealed: (i) a fast peroxidative response - with a maximal linear rate of 14 nmol malondialdehyde (MDA) equivalents/mg protein, as evidenced by the thiobarbituric acid test - and a decrease in the polyunsaturated fatty acid (PUFA) content of the platelet crude membranes; (ii) a decrease in the lipid fluidity in the deep lipid core of the membranes but not at the membrane surface; (iii) a dramatic inhibitory effect on glucose 6-phosphatase (Glc-6-Pase) but not on acetylcholinesterase activity. Platelets were also aged by storage at 4 degrees C in their own plasma or in Seto additive solution. In these media, platelet aggregates were visible and the effects on platelet phospholipids, PUFA, lipid extract fluorescence, crude membrane fluidity and membrane-bound enzyme activities were assessed for comparison with those observed in in vitro lipid peroxidation. The sensitivity of membranes from stored platelets to lipid peroxidation was also assessed. Storage of platelets in plasma for 5 days was associated with different changes in their crude membranes such as decreases in arachidonic acid contents, the decrease not being avoided by the presence of phospholipase A(2) inhibitors, increases in MDA equivalents, conjugated dienes and lipid extract fluorescence, decreases in the amounts of MDA equivalents formed by platelet crude membranes treated with the oxidizing agents, changes in membrane fluidity and inhibition of Glc-6-Pase. All these alterations were less pronounced or even abolished after platelet storage in Seto. These findings suggest that platelet lipid peroxidation due to XO/hypoxanthine/Fe(2+)-ADP and platelet membrane alterations observed after platelet ageing under storage at 4 degrees C share common features. Also, as regards the prevention of peroxidative processes, Seto solution permits better storage of sheep platelets than plasma.


Asunto(s)
Plaquetas/metabolismo , Senescencia Celular , Peroxidación de Lípido , Acetilcolinesterasa/química , Animales , Plaquetas/química , Membrana Celular/química , Criopreservación , Inhibidores Enzimáticos/química , Ácidos Grasos/análisis , Solución Hipertónica de Glucosa , Glucosa-6-Fosfatasa/antagonistas & inhibidores , Técnicas In Vitro , Fluidez de la Membrana , Fosfolípidos/análisis , Fosfolípidos/química , Plasma , Ovinos , Sustancias Reactivas al Ácido Tiobarbitúrico/análisis , Factores de Tiempo
5.
Mol Endocrinol ; 6(12): 2210-8, 1992 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-1491699

RESUMEN

It is well established that the LH/CG receptor expressed in gonadal cells is an 85- to 92-kilodalton (kDa) glycoprotein. Additionally, however, a number of reports have noted the existence of other putative receptor species, but few attempts have been made to characterize these variant receptor species. A cell line [293L(wt1)] had previously been isolated which expresses large numbers of high affinity cell surface LH/CG receptors. Visualization of the LH/CG receptor species expressed in these cells and in rat luteal cells using ligand blots revealed 85- and 90-kDa LH/CG receptors, respectively, while immunoblots revealed another 68-kDa glycoprotein receptor in both cell types. The presence of both the 85- and 68-kDa receptor species was confirmed using immunoprecipitation and affinity purification of metabolically labeled 293L(wt1) cells. Enzymatic deglycosylations established that the 85-kDa receptor is a sialoprotein, while the 68-kDa species contains exposed high mannose residues. Protease digestion before LH/CG receptor immunoprecipitations localized the 85-kDa receptor on the plasma membrane, while the 68-kDa receptor was shown to be located intracellularly. Pulse-chase experiments were then used to positively establish that the 68-kDa receptor protein is actually a precursor of the 85-kDa LH/CG receptor species.


Asunto(s)
Precursores de Proteínas/metabolismo , Receptores de Gonadotropina/genética , Receptores de HL/genética , Proteínas Recombinantes de Fusión/genética , Animales , Carbohidratos/análisis , Línea Celular , Cuerpo Lúteo/citología , Cuerpo Lúteo/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Riñón , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratas , Receptores de Gonadotropina/metabolismo , Receptores de HL/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Transfección
6.
Mol Endocrinol ; 7(7): 823-32, 1993 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-8413307

RESUMEN

Much of the definitive work on G-protein-coupled receptor phosphorylation and its impact on receptor function has been performed with the catecholamine receptors. Evidence for receptor phosphorylation is lacking, however, for G-protein-coupled receptors that bind larger ligands, such as LH/CG. Using immunoprecipitation techniques and a clonal cell line stably transfected with the LH/CG receptor, we show here for the first time that exposure of cells to hCG induces phosphorylation of its cognate receptor. The hCG-induced increase in receptor phosphorylation requires receptor activation because it cannot be elicited with a hCG antagonist and is mediated at least in part by the cAMP second messenger system. This hypothesis is supported by the finding that the hCG-induced receptor phosphorylation is greatly reduced (but not abolished) in a cell line that overexpresses cAMP phosphodiesterase and that receptor phosphorylation can be induced by activation of endogenous cAMP synthesis with prostaglandin E2 or by addition of 8-bromo-cAMP. Last, we show that LH/CG receptor phosphorylation can be induced with a phorbol ester, but not with a calcium ionophore. We also examined a potential correlation between LH/CG receptor phosphorylation and uncoupling of the receptor from its effector. Although the phorbol ester-induced phosphorylation of the LH/CG receptor can be correlated with uncoupling, other experiments indicate that hCG-induced uncoupling of the LH/CG receptor can occur under conditions where the cAMP-mediated receptor phosphorylation is greatly reduced (or abolished).


Asunto(s)
Ésteres del Forbol/farmacología , Receptores de Gonadotropina/metabolismo , Receptores de HL/metabolismo , Calcimicina/farmacología , Células Cultivadas , Gonadotropina Coriónica/farmacología , AMP Cíclico/farmacología , Diglicéridos/fisiología , Glicosilación , Humanos , Fosfatos de Inositol/fisiología , Riñón/citología , Fosforilación/efectos de los fármacos , Pruebas de Precipitina , Receptores de Gonadotropina/análisis , Receptores de Gonadotropina/genética , Receptores de HL/análisis , Receptores de HL/genética , Sistemas de Mensajero Secundario/fisiología , Transfección
7.
Exp Hematol ; 23(3): 258-64, 1995 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-7875242

RESUMEN

The mammalian erythrocyte is an ideal model for studies of membrane aging under conditions of storage. The present study describes the variations in the membrane lipid composition of three density groups (light, 1.110 < d < 1.125; intermediate, 1.125 < d < 1.130, and dense, 1.130 < d < 1.140) of sheep erythrocytes separated by centrifugation in a discontinuous Ficoll density gradient after storage at 4 degrees C in a nutrient medium for up to 6 days. The only changes in phospholipid composition took place in the erythrocyte light fraction where sphingomyelin (SM) and phosphatidic acid increased (p < 0.05), whereas phosphatidylethanolamine (PE) decreased (p < 0.05). Moreover, polyunsaturated fatty acids (20:4 and 22:6) decreased during storage, whereas lipid fluorescence increased (p < 0.01) after 24 hours of storage in all the fractions separated. These observations suggest a lipid peroxidation process in all three erythrocyte groups during storage.


Asunto(s)
Eritrocitos/metabolismo , Ácidos Grasos/análisis , Fosfolípidos/análisis , Animales , Membrana Celular/metabolismo , Senescencia Celular , Centrifugación por Gradiente de Densidad , Eritrocitos/citología , Ovinos
8.
Endocrinology ; 132(3): 1007-16, 1993 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-8440169

RESUMEN

In most experiments done in cell-free systems, the LH/CG-induced desensitization of the ovarian LH/CG-responsive adenylyl cyclase has been reported to be dependent on GTP. Little is known, however, about the molecular basis of this phenomenon or about the FSH-induced desensitization of the FSH-responsive adenylyl cyclase. We report here that, contrary to most previous findings, ATP is required for desensitization of the LH/CG- and FSH-responsive adenylyl cyclase in human kidney cells stably transfected with the complementary DNAs for the rat LH/CG or FSH receptor. This requirement does not seem to be peculiar to transfected cells because under our experimental conditions ATP is also preferred over GTP for the human CG-induced desensitization of the LH/CG-responsive adenylyl cyclase in highly purified plasma membranes from MA-10 Leydig tumor cells. Maximal desensitization of both FSH- and LH/CG-sensitive adenylyl cyclase in membranes from the transfected cells was achieved with millimollar concentrations of Mg2+ and ATP and did not appear to correlate with activation of the enzyme. In both of these systems, GTP, uridine triphosphate, and cytidine triphosphate were not able to substitute for ATP. In MA-10 membranes, however, there was some desensitization even without added nucleotide triphosphates, and ATP was more potent than GTP. Last, desensitization of the gonadotropin-sensitive adenylyl cyclase could not be explained by a decrease in the functional activities of stimulatory guanine nucleotide binding protein or of the catalytic moiety of the enzyme. A change in the functional properties of the gonadotropin receptors appears to be the most likely mechanism for desensitization.


Asunto(s)
Adenilil Ciclasas/metabolismo , Gonadotropina Coriónica/farmacología , Hormona Folículo Estimulante/farmacología , Hormona Luteinizante/farmacología , Receptores de HL/fisiología , Transfección , Adenosina Trifosfato/farmacología , Línea Celular , Membrana Celular/enzimología , Guanosina Trifosfato/farmacología , Humanos , Riñón , Cinética , Tumor de Células de Leydig , Cloruro de Magnesio/farmacología , Masculino , Receptores de HL/genética , Proteínas Recombinantes/metabolismo , Ribonucleótidos/farmacología , Neoplasias Testiculares , Células Tumorales Cultivadas
9.
Free Radic Biol Med ; 26(9-10): 1218-30, 1999 May.
Artículo en Inglés | MEDLINE | ID: mdl-10381193

RESUMEN

Incubation of human or sheep platelet crude membranes with xanthine oxidase/hypoxanthine in the presence of Fe2+/ADP inactivated phosphotyrosine phosphatase (PTPase, protein-tyrosine-phosphate-phosphohydrolase, EC 3.1.3.48) activity in a time-dependent manner, this inhibition being significant within 5 min of treatment. The dynamics of protein thiols differed depending on the platelet species, but in any case decreases in protein thiols were only visible 20-45 min after the start of the treatment. The inhibition of PTPase activity in general showed good a correlation with the production of thiobarbituric acid-reactive substances (TBARS). The results with several antioxidants suggest that the inhibition of PTPase activity is related to the generation of alkoxyl and/or peroxyl radicals. Furthermore, the formation of fluorescent products and changes in amino groups were observed only after long incubation times with the oxidizing agents, these fluorescent products and the residual enzyme activity remaining in the membrane fraction. Treatment of platelet membranes with trans-2-nonenal and n-heptaldehyde, but not with acetaldehyde, also inhibited membrane-associated PTPase activity. However, the amount of protein thiols was reduced only by treatment with trans-2-nonenal. Fluorescence product formation was always higher with trans-2-nonenal, these products being mainly located in the protein fraction. The results with aldehydes suggest that secondary degraded products of lipid hydroperoxides affect PTPase activity. Kinetic studies of PTPase activity indicated that with all treatments enzyme inhibition is mainly due to a decrease in the Vmax value. The results of fluorescence anisotropy measurements of labeled platelet membranes did not support the notion of a contribution of the lipid organization to peroxidation-mediated PTPase inhibition. All the above results indicate that platelet membrane-associated PTPase inhibition due to treatment with xanthine oxidase/ hypoxanthine in the presence of Fe2+/ADP is a very complex, time-dependent process, and that it is probably related, at least after long periods of peroxidation, to changes in protein thiols and amino groups. We predict that the sensitivity of PTPase to lipid peroxidation must be physiologically relevant because of the increasing importance of tyrosine phosphorylation in signal transduction, in general, and in platelet activation and aggregation in particular.


Asunto(s)
Plaquetas/enzimología , Peroxidación de Lípido , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Aldehídos/farmacología , Animales , Antioxidantes/farmacología , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Membrana Celular/metabolismo , Polarización de Fluorescencia , Radicales Libres/metabolismo , Humanos , Hipoxantina/farmacología , Técnicas In Vitro , Cinética , Peroxidación de Lípido/efectos de los fármacos , Malondialdehído/sangre , Glicoproteínas de Membrana Plaquetaria/metabolismo , Proteínas Tirosina Fosfatasas/sangre , Ovinos , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Xantina Oxidasa/farmacología
10.
Mech Ageing Dev ; 71(3): 189-98, 1993 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-8133676

RESUMEN

This study examined the profile in sheep erythrocyte fatty acids from animals of different ages during storage at 4 degrees C in a nutritive medium for up to 6 days. The changes found in the fatty acyl profile were a decrease (P < 0.01) in the percentage of arachidonic acid and an increase (P < 0.01) in the percentage of minor fatty acids (representing < 2% in each case; 20:2, 22:0 and 22:1) with respect to fresh erythrocytes in all age groups. However, the saturated/unsaturated ratios and unsaturated index started almost constant in all cases. The changes observed occurred after 24-48 h of storage, with significant increases (P < 0.01) in the fluorescence detected in the lipid extracts from stored erythrocytes during this period. The above findings suggest peroxidative damage and changes in the erythrocyte lipid membrane during storage.


Asunto(s)
Envejecimiento/fisiología , Eritrocitos/química , Ácidos Grasos/química , Animales , Cromatografía de Gases , Eritrocitos/fisiología , Ácidos Grasos/fisiología , Lípidos/química , Lípidos/fisiología , Ovinos/fisiología
11.
Biochimie ; 72(10): 745-50, 1990 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-2078591

RESUMEN

Development and aging processes in mammals are associated with changes in several physiological parameters. The aim of the present study was to investigate the changes in erythrocyte lipid composition during sheep development. In all the age groups studied, cholesterol/phospholipid ratios remained constant, at close to unity, while phospholipid patterns (sphingomyelin: 45-51%, phosphatidylethanolamine: 26-33%, phosphatidylserine: 13-19% and phosphatidylcholine: less than 2%) changed during development, with a statistically significant decrease (P less than 0.01) in phosphatidylserine and an increase in sphingomyelin content. These data suggest an increase in the rigidity of the erythrocyte lipid bilayer in adult sheep when compared with 1-month-old animals due to a decrease in the phosphatidylserine/sphingomyelin ratio. Fatty acid profiles consistently showed 5 main acids: oleic (52-54%), stearic (17-18%), linoleic (9-15%), palmitic (8.5-11%) and arachidonic acid (2-3%), mainly with significant variations (P less than 0.01) in palmitic and linoleic acid contents, respectively reaching the highest and lowest percentages in the youngest sheep. However, the developmental process seems to have no influence on the aminophospholipid topology of erythrocytes. This study suggests that the animals' developmental process has a marked effect on the lipid composition of erythrocyte membranes, which could affect cell functions.


Asunto(s)
Envejecimiento/fisiología , Envejecimiento Eritrocítico/fisiología , Membrana Eritrocítica/química , Ácidos Grasos/metabolismo , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/química , Fosfolípidos/metabolismo , Animales , Membrana Eritrocítica/metabolismo , Fluidez de la Membrana , Lípidos de la Membrana/análisis , Fosfatidilserinas/análisis , Ovinos/crecimiento & desarrollo , Esfingomielinas/análisis , Ácido Trinitrobencenosulfónico
12.
Thromb Haemost ; 80(4): 668-76, 1998 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-9798989

RESUMEN

To relate the improvement of platelet storage in synthetic media with possible structural changes, we conducted serial studies on the membranes of platelets and microparticles shed during platelet storage for up to 5 days at 4 degrees C either in plasma or in Seto solution. Spontaneous microparticle formation proceeded linearly for up to 2 days in both storage media, although the processes seemed to be different because microparticles from Seto solution had a higher lipid/protein ratio than those released in plasma. Microparticles were heterogeneous structures showing beta-N-acetylhexosaminidase, glucose-6-phosphatase and succinate dehydrogenase activities. After 2-5 days of storage, microparticles contained 60% of total cellular acetylcholinesterase (AChE), were doubly enriched in cholesterol. and showed identical phospholipid profiles but with a decrease in the lipid unsaturation index with respect to fresh platelets. Fluorescence anisotropy studies pointed to a remarkable increase in the deep lipid core fluidity of microparticles during storage of platelets in plasma. With respect to platelets, only those stored in plasma showed significant changes in lipid contents, with a 3-fold decrease in the phospholipid to protein ratio, a decrease in phosphatidylethanolamine (PE) levels and a parallel increase in phosphatidylcholine (PC) percentages in their phospholipid profile, together with a significant reduction in the lipid unsaturation index after 1 day of storage. The fluidity of the negatively charged surface of the platelet membranes decreased in platelets stored for 5 days in both media, whereas the fluidity of the membrane deep core was only increased in platelets stored in plasma. These findings suggest that Seto solution permits better storage of platelets for 5 days than plasma and support the notion that lipid peroxidation could play an important role in the structural changes observed.


Asunto(s)
Plaquetas , Conservación de la Sangre , Membrana Celular , Animales , Solución Hipertónica de Glucosa , Lípidos , Fluidez de la Membrana , Soluciones Preservantes de Órganos , Plasma , Ovinos
13.
Reprod Toxicol ; 6(1): 51-5, 1992.
Artículo en Inglés | MEDLINE | ID: mdl-1562798

RESUMEN

The effects of hourly injections of moderate doses of cocaine hydrochloride (0.5 and 10 mg/kg body weight) over 5 h on testicular structure and testosterone levels were studied in male Wistar rats. Cocaine produced a rapid disruption of spermatogenesis; the number of normal seminiferous tubules declined to 50% (low dose) and 40% (high dose), and regressive tubules (tubules with cellular degeneration, cell sloughing, or abnormal cell structures) increased to 50% (low dose) and 60% (high dose) after treatment with cocaine. The mean tubular diameter, the surface occupied by the tubules, and the volume of seminiferous tubules per pair of testes were significantly reduced (P less than 0.01) after both doses of cocaine. Cocaine produced ultrastructural changes in the cells of the seminiferous epithelium (spermatogonia, spermatids, and Sertoli cells) including vacuoles, abundant lipid droplets, and giant mitochondria. Lower doses of cocaine increased serum testosterone levels (P less than 0.025) while higher doses did not. These findings indicate an acute effect of cocaine on the structure of the rat testis.


Asunto(s)
Cocaína/farmacología , Testículo/efectos de los fármacos , Testículo/patología , Animales , Hormona Luteinizante/sangre , Masculino , Ratas , Ratas Endogámicas , Testículo/ultraestructura , Testosterona/sangre
14.
Artículo en Inglés | MEDLINE | ID: mdl-7553349

RESUMEN

GTP has been shown to inhibit AlF4(-)-stimulated, and to activate forskolin-stimulated adenylyl cyclase activity in the presence of Mg2+ in cell membranes from human embryonic kidney 293 cells. The maximal inhibitory response of AlF4(-)-stimulated adenylyl cyclase activity by GTP was not dependent on the concentration of Mg2+, but was so in the case of forskolin-activated activity at all forskolin concentrations assayed. Mn2+ ions stimulated AlF4(-)- or forskolin-activated adenylyl cyclase activity to a greater extent than Mg2+. The inhibition of AlF4(-)-stimulated cyclase by GTP was still observed with Mn2+, but the activation of forskolin-stimulated cyclase by GTP was not. When assayed together, Mn2+ and Mg2+ showed non-additive behaviours with respect to the amount of cyclic AMP formed after AlF4(-)-stimulation of adenylyl cyclase. The temperature dependence of the activation of adenylyl cyclase by forskolin, AlF4- or under basal conditions was observed to be somehow different in the presence of Mn2+ than in the presence of Mg2+ ions. Cholera toxin treatment produced a markedly increased cyclase activity, specially when assayed with AlF4-. In the case of forskolin-activated adenylyl cyclase, UTP and CTP were unable to reproduce the cyclase activation detected with GTP. However, in the case of AlF4(-)-stimulated adenylyl cyclase, UTP was as good as GTP at inhibiting cyclase activity, and CTP virtually eliminated the activation of the cyclase with AlF4-.


Asunto(s)
Adenilil Ciclasas/efectos de los fármacos , Compuestos de Aluminio/farmacología , Colforsina/farmacología , Fluoruros/farmacología , Guanosina Trifosfato/farmacología , Riñón/efectos de los fármacos , Línea Celular , Activación Enzimática , Humanos , Riñón/citología , Riñón/embriología , Riñón/enzimología , Magnesio/farmacología , Manganeso/farmacología
15.
Comp Biochem Physiol B Biochem Mol Biol ; 110(1): 91-101, 1995 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-7858952

RESUMEN

The biochemical characterization of detergent-solubilized acetylcholinesterase (AChE) from subcellular particles of sheep platelets and the effects of different effectors on AChE activity from solubilized platelet crude membranes have been undertaken and studied. Solubilization of AChE with detergent increased the thermal stability of the enzyme from all particulate fractions. Solubilized AChE from the mitochondria-granule fraction was the most thermostable at 55 degrees C. The Km values against acetylthiocholine chloride and the Arrhenius plot obtained were very similar for the AChE from all the solubilized fractions. There were no differences in the ability of solubilized AChE from different subcellular fractions to bind concanavalin A (Con A). In solubilized platelet crude membranes, benzyl alcohol was a potent AChE inhibitor at a concentration of 10(-2) M, whereas ethanol was not. Mg2+ cations and, to a lesser extent, Ca2+ and Mn2+ cations, activated AChE at concentrations higher than 1 mM. Serine hydrolase inhibitors and cholinesterase-specific inhibitors were very effective in the inactivation of AChE, whereas EDTA and EGTA had no effect. Of all the monosaccharides tested, only N-acetylneuraminic acid exerted an inhibitory effect on AChE activity. Immobilized-lectin binding studies demonstrated the interaction of solubilized crude membrane-bound AChE with Con A, lentil lectin and wheat germ agglutinin. Taken together, these data suggest the presence of a unique form of the membrane-bound AChE which has at least alpha-mannose and N-acetylglucosamine residues in the glycan chain.


Asunto(s)
Acetilcolinesterasa/sangre , Plaquetas/enzimología , Detergentes , Acetilglucosamina/análisis , Acetiltiocolina/metabolismo , Animales , Alcohol Bencilo , Alcoholes Bencílicos/farmacología , Plaquetas/ultraestructura , Carbohidratos/análisis , Membrana Celular/enzimología , Inhibidores de la Colinesterasa , Estabilidad de Enzimas , Calor , Cinética , Manosa/análisis , Ácido N-Acetilneuramínico , Ovinos , Ácidos Siálicos/farmacología , Solubilidad , Fracciones Subcelulares/enzimología , Termodinámica
16.
Comp Biochem Physiol B Biochem Mol Biol ; 117(3): 437-44, 1997 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-9253182

RESUMEN

Using O-phosphotyrosine as a substrate, we characterized the phosphotyrosine phosphatase (PTPase; protein-tyrosine-phosphate-phosphohydrolase, EC 3.1.3.48) activity from sheep platelets. PTPase was found to be located in three particulate subcellular fractions and in the cytosol, with K(m) values in the millimolar range. PTPase was strongly inhibited by vanadate, molybdate and HgCl2 and only weakly inhibited by Zn2+. Other divalent cations and NaF had no significant effect on the activity associated with the membrane fraction but were slightly stimulatory as regards cytosolic activity. Heparin inhibited cytosolic activity 2-fold more than membrane-bound activity and dithiothreitol only inhibited cytosolic PTPase. Polycationic compounds were seen to be weak stimulators of all the PTPase activity. Solubilization of the PTPase from membranes always required a detergent. When subjected to Triton X-114 phase partitioning, PTPase was recovered in the detergent-rich (35%) and in the detergent-poor (65%) phases. Sedimentation analysis of the cytosolic PTPase showed a peak of 3.2S that remained unmodified when Triton X-100 or Brij 97 sucrose gradients were used. Sedimentation analysis of the membrane-associated PTPase showed 6S and 3.7S peaks unchanged in Triton X-100 or Brij 97 gradients together with 7.5S and 10.3S shoulders that shifted to smaller sedimentation coefficients in Brij 97 sucrose gradients. These results support the view that sheep platelets contain amphiphilic and hydrophilic forms of PTPase.


Asunto(s)
Plaquetas/enzimología , Proteínas Tirosina Fosfatasas/sangre , Ovinos/sangre , Animales , Membrana Celular/enzimología , Ácidos Cólicos , Detergentes , Octoxinol , Polietilenglicoles , Solubilidad
17.
Lipids ; 26(11): 878-83, 1991 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-1805091

RESUMEN

The fatty acid composition of individual phospholipids in subcellular fractions of sheep platelets and the asymmetrical distribution of phosphatidylethanolamine (PE) fatty acyl chains across the plasma membrane were examined. The main fatty acids of total lipid extracts were oleic (18:1; 32-41%), linoleic (18:2, 10-17%), stearic (18:0; 13-15%), palmitic (16:0; 11-15%) and arachidonic (20:4; 8-12%) acids, with a saturated/unsaturated ratio of about 0.4. Each phospholipid class had a distinct fatty acid pattern. Sphingomyelin (SM) showed the highest degree of saturation (50%), with large proportions of behenic (22:0), 18:0 and 16:0 acids. The main fatty acid in PE, phosphatidylserine (PS) and phosphatidylcholine (PC) was 18:1n-9. Our findings suggest that fatty acids are asymmetrically distributed between the choline versus the non-choline phospholipids, and also between plasma membranes and intracellular membranes. The transbilayer distribution of PE fatty acids in plasma membranes from non-stimulated sheep platelets was investigated using trinitrobenzene-sulfonic acid (TNBS). A significant degree of asymmetry was found, which is a new observation in a non-polar cell. The PE molecules from the inner monolayer contained higher amounts of 18:2 and significantly less 18:1 and 20:5 than those found in the outer monolayer, although no major differences were detected in the transbilayer distribution of total unsaturated versus saturated PE acyl chains.


Asunto(s)
Plaquetas/química , Ácidos Grasos/análisis , Membranas/química , Fosfolípidos/química , Animales , Membrana Celular/química , Aparato de Golgi/química , Membranas Intracelulares/química , Membrana Dobles de Lípidos/química , Mitocondrias/química , Fosfatidiletanolaminas/química , Fosfatidilserinas/química , Ovinos , Fracciones Subcelulares/química
18.
An Med Interna ; 11(4): 177-80, 1994 Apr.
Artículo en Español | MEDLINE | ID: mdl-8043737

RESUMEN

The present study proved that desmopressin (DDAVP) (1 microgram DDAVP/12 h/5 días) does not affect ovary, testis and adrenal development in immature Wistar rats (17 days old), because the DDAVP does not modify the weight of the aforementioned organs as compared with the control group. Nevertheless, the male adults Wistar rats (80 days old) showed lower serum testosterone concentrations than the control group, after injection of 4 micrograms/day (2 micrograms/12 h) or 8 micrograms/day (4 micrograms/12 h) of DDAVP during a 5 days period time. Moreover, paradoxical significant lower concentrations of serum testosterone were found in 4 micrograms DDAVP/day-treated rats than in 8 micrograms DDAVP/day-treated ones. The former also showed a decreased number of spermatozoa as compared with the latter and with the control group. The percentage of mobile spermatozoa was lower in rats treated with both concentrations of DDAVP as compared with the control group. Therefore, desmopressin does not delay gonadal and adrenal growth in immature rats, but, at low doses, it affects the testicular function and the mobility of the spermatozoa in male adult rats.


Asunto(s)
Glándulas Suprarrenales/crecimiento & desarrollo , Desamino Arginina Vasopresina/farmacología , Motilidad Espermática/efectos de los fármacos , Testículo/efectos de los fármacos , Factores de Edad , Animales , Peso Corporal , Desamino Arginina Vasopresina/administración & dosificación , Femenino , Masculino , Tamaño de los Órganos , Ratas , Ratas Wistar , Testículo/crecimiento & desarrollo , Testículo/fisiología
19.
Cell Death Differ ; 17(12): 1842-54, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20523355

RESUMEN

Transient reactive oxygen species (ROS) production is currently proving to be an important mechanism in the regulation of intracellular signalling, but reports showing the involvement of ROS in important biological processes, such as cell differentiation, are scarce. In this study, we show for the first time that ROS production is required for megakaryocytic differentiation in K562 and HEL cell lines and also in human CD34(+) cells. ROS production is transiently activated during megakaryocytic differentiation, and such production is abolished by the addition of different antioxidants (such as N-acetyl cysteine, trolox, quercetin) or the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium. The inhibition of ROS formation hinders differentiation. RNA interference experiments have shown that a p22(phox)-dependent NADPH oxidase activity is responsible for ROS production. In addition, the activation of ERK, AKT and JAK2 is required for differentiation, but the activation of phosphatidylinositol 3-kinase and c-Jun N-terminal kinase seems to be less important. When ROS production is prevented, the activation of these signalling pathways is partly inhibited. Taken together, these results show that NADPH oxidase ROS production is essential for complete activation of the main signalling pathways involved in megakaryocytopoiesis to occur. We suggest that this might also be important for in vivo megakaryocytopoiesis.


Asunto(s)
Megacariocitos/citología , NADPH Oxidasas/metabolismo , Antígenos CD34/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Cromanos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Janus Quinasa 2/metabolismo , Megacariocitos/metabolismo , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , Compuestos Onio/farmacología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quercetina/farmacología , Interferencia de ARN , ARN Interferente Pequeño , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal
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