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1.
Biophys J ; 79(2): 853-62, 2000 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-10920017

RESUMEN

A combined allosteric and competitive model describes the interaction between extracellular Na(+) and Rb(+) during ion transport mediated by the Na, K-ATPase. The model was developed from experiments based on (86)Rb uptake by whole cells transfected with rat isoforms of the enzyme. In the absence of Na(+), only a single transport site for extracellular Rb(+) exists. After the occupation of the Na(+)-specific allosteric site, the Rb(+) transport pocket opens to allow occupation by an additional Rb(+) and the subsequent transport of the two Rb(+) ions into the cells. Na(+) can also directly compete with Rb(+) for binding to at least one of the transport sites. While the model derived here applies to each of the three rat isoforms of the Na, K-ATPase expressed in HeLa cells, subtle differences exist among the isoforms. The alpha(3)* isoform has an increased intrinsic affinity for Rb(+) and a lower affinity for the allosteric Na(+) site than alpha(1) or alpha(2)*. The stimulation of uptake observed according to the best-fit model is due to the displacement by Rb(+) of inhibitory Na(+) bound to the transport site.


Asunto(s)
Potasio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sodio/metabolismo , Regulación Alostérica , Sitio Alostérico , Animales , Unión Competitiva , Células HeLa , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Modelos Químicos , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rubidio/farmacocinética , Transfección
2.
Met Based Drugs ; 6(4-5): 301-9, 1999.
Artículo en Inglés | MEDLINE | ID: mdl-18475905

RESUMEN

Having identified dicyanogold(I) as a common metabolite of gold-based antiarthritis drugs, we are investigating the effects of the compound on the production of lymphokines. Handel, et al. 1 suggested that the transcription factor AP-1, critical to the production of a number of cytokines, might be the target for gold compounds because of a critical cysteine within its DNA binding region. Using Jurkat cells, an established cell line as a model for CD4(+) lymphocytes, we have shown that dicyanogold inhibits the binding of AP-1 to DNA and inhibits the synthesis of IL-2 mRNA and protein. In a macrophage line, THP-1, which synthesizes IL-1beta in response to mitogen, we have shown that dicyanogold inhibits the binding of a second transcription factor, CREB to DNA. Incubation of THP-1 cells with dicyanogold inhibits the production of IL-1beta mRNA. These results suggest that the mechanism of action of gold drugs may be through their interaction with transcription factors necessary for the immune activation seen in Rheumatoid Arthritis.

3.
Met Based Drugs ; 4(2): 97-109, 1997.
Artículo en Inglés | MEDLINE | ID: mdl-18475775

RESUMEN

Cisplatin is an extremely effective cancer chemotherapeutic agent, but its use is often accompanied by toxicity. Second generation drugs such as carboplatin are becoming more widely used because of reduced toxicity. Since biotransformation products have been implicated in the toxic responses, we have begun to investigate the reactions of cisplatin and carboplatin with potential biological ligands. Reaction products were characterized using HPLC with inductively coupled plasma - mass spectrometry (HPLC-ICP-MS), (1)H and (13)C NMR and fast atom bombardment - mass spectrometry (FAB-MS). Three Pt-creatinine complexes, cis-[Pt(NH(3))(2)Cl(Creat)](+), cis-[Pt(NH(3))(2)(H(2)O)(Creat)](2+) and cis-[Pt(NH(3))(2)(Creat)(2)](2+), were synthesized and the platinum was shown to coordinate to the ring nitrogen, N(3). Human urine samples from patients on cisplatin chemotherapy were shown to contain cisplatin, its hydrolysis product and biotransformation products containing Pt-creatinine, Pt-urea and Pt-uric acid complexes. Urine from carboplatin patients shows fewer biotransformation products. Studies with control and diabetic (protected against cisplatin toxicity) rats showed systematic differences in the biotransformation products formed on administration of cisplatin.

4.
Am J Physiol ; 273(6): C2065-79, 1997 12.
Artículo en Inglés | MEDLINE | ID: mdl-9435514

RESUMEN

A competition assay of 86Rb+ uptake in HeLa cells transfected with ouabain-resistant Na(+)-K(+)-ATPase mutants revealed a stimulation of 86Rb+ uptake at low external concentrations (1 mM) of competitor (K+). Of the models that were tested, those that require that two K+ be bound before transport occurs gave the worst fits. Random and ordered binding schemes described the data equally well. General models in which both binding and transport were allowed to be cooperative yielded parameter errors larger than the parameters themselves and could not be utilized. Models that assumed noncooperative transport always showed positive cooperativity in binding. E327Q and E327L mutated forms of rat alpha 2 had lower apparent affinities for the first K+ bound than did wild-type rat alpha 2 modified to be ouabain resistant. The mutations did not affect the apparent affinity of the second K+ bound. Models that assumed noncooperativity in binding always showed positively cooperative transport, i.e., enzymes with two K+ bound had a higher flux than those with one K+ bound. Increases in external Na+ decreased the apparent affinity for K+ for all models and decreased the ratio of the apparent influx rate constants for E327L.


Asunto(s)
Ácido Glutámico , Modelos Químicos , Mutagénesis Sitio-Dirigida , Potasio/farmacología , Radioisótopos de Rubidio/farmacocinética , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sustitución de Aminoácidos , Animales , Unión Competitiva , Células HeLa , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Ouabaína/farmacología , Cloruro de Potasio/farmacología , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transfección
5.
Met Based Drugs ; 1(5-6): 363-74, 1994.
Artículo en Inglés | MEDLINE | ID: mdl-18476256

RESUMEN

We have determined the framework structure of Myochrysine (disodium gold(I)thiomalate) in the solid state and extremely concentrated aqueous solution, previously. It consists of an open chain polymer with linear gold coordination to two thiolates from the thiomalic acid moieties which bridge between pairs of gold atoms providing an Au-S-Au angle of 95 degrees . The question remained: was this structure relevant to the dilute solutions of drugs administered and the still lower concentrations of gold found in the bodies of patients (typically 1 ppm Au in blood and urine or 5 muM in Au). We have provided an answer to that question using extended X-ray absorption spectroscopy (EXAFS) and capillary zone electrophoresis (CZE). EXAFS studies confirm that the polymeric structure with two sulfur atoms per gold atom persists from molar concentrations down to millimolar concentrations. CZE is able to separate and detect Myochrysine at millimolar levels. More importantly, at micromolar levels Myochrysine solutions exhibit identical CZE behavior to that measured at millimolar levels. Thus, aqueous solutions of the drug remain oligomeric at concentrations commensurate with those found in patient blood and urine.The reactivity of Myochrysine with cyanide, a species especially prevalent in smoking patients, was explored using CZE. Cyanide freely replaces thiomalic acid to form [Au(CN)(2)](-) and thiomalic acid via a mixed ligand intermediate. The overall apparent equilibrium constant (K(app)) for the reaction is 6x10(-4)M(-1). Further reaction of [Au(CN)(2)](-) with a large excess of L, where L is cysteine, N-acetylcysteine, or glutathione, shows that these amino acids readily replace cyanide to form [AuL(2)](-). These species are thus potential metabolites and could possibly be active forms of gold in vivo. That all of these species are readily separated and quantified using CZE demonstrates that capillary electrophoresis is an accessible and powerful tool to add to those used for the study of gold-based antiarthritis drugs.

6.
Met Based Drugs ; 1(5-6): 433-43, 1994.
Artículo en Inglés | MEDLINE | ID: mdl-18476261

RESUMEN

We have shown that dicyanogold(I), [Au(CN)(2)](-) is a common metabolite found in blood and urine samples of patients treated with different gold based drugs. Some patients have high levels of gold within their red blood cells (RBCs). Size exclusion and C18 reversed phase chromatography show that the majority of the gold in RBC lysates is bound to protein, but small molecules such as dicyanogold(I) and gold-glutathione complexes are also present. Dicyanogold incubation with red blood cells in vitro leads to a rapid and complete uptake of gold. This uptake is unaffected by DIDS, an inhibitor of the anion channel, but is blocked by the addition of external cyanide. Dicyanogold is also readily taken up by H9 cells, a continuous CD(4+) cell line. This uptake is significantly inhibited by N-ethylmaleimide, suggesting a requirement for sulfhydryl groups. Dicyanogold inhibits the replication of the AIDS virus, HIV, in a cell culture model.

7.
Met Based Drugs ; 1(5-6): 517, 1994.
Artículo en Inglés | MEDLINE | ID: mdl-18476275
8.
Met Based Drugs ; 1(5-6): 521, 1994.
Artículo en Inglés | MEDLINE | ID: mdl-18476279
9.
J Chromatogr ; 615(1): 83-9, 1993 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-8340466

RESUMEN

A sensitive method is described for measuring cisplatin and some possible metabolites. The method combines reversed-phase ion-pairing liquid chromatography (LC) with inductively coupled plasma mass spectrometry (ICP-MS) for platinum-specific detection. Separation conditions for cisplatin hydrolysis products and the reaction products of cisplatin with methionine, cysteine, and glutathione have been investigated with sodium dodecylsulfate or heptanesulfonate as the ion-pairing agent. The detection limit for cisplatin was found to be 0.1 ng, corresponding to a concentration detection limit of 1 ng/ml when using an injection volume of 100 microliters. This study has demonstrated the usefulness of LC-ICP-MS for cisplatin metabolism studies.


Asunto(s)
Cisplatino/análisis , Cromatografía Líquida de Alta Presión , Cisteína/análisis , Glutatión/análisis , Hidrólisis , Espectrometría de Masas , Metionina/análisis , Compuestos Organoplatinos/análisis , Compuestos de Sulfhidrilo/análisis
10.
J Rheumatol ; 20(2): 268-72, 1993 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-8474063

RESUMEN

Gold based drugs and their metabolites have been characterized using reversed phase, ion pairing chromatography with an inductively coupled plasma mass spectrometer as an element specific detector. For a patient receiving gold sodium thiomalate the principal gold species in the urine is [Au(CN)2]-, which is also seen in a low molecular weight infiltrate of the blood. The same compound is also identified in the urine and blood of a patient taking auranofin and in patients taking solganol. This represents the first identification of a specific gold metabolite in biological fluids taken from patients undergoing gold therapy and the first evidence that different gold drugs have common metabolites.


Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Cianatos/farmacocinética , Oro/farmacocinética , Aniones , Artritis Reumatoide/sangre , Artritis Reumatoide/orina , Auranofina/sangre , Auranofina/orina , Aurotioglucosa/sangre , Aurotioglucosa/orina , Cianatos/sangre , Cianatos/orina , Oro/sangre , Oro/orina , Tiomalato Sódico de Oro/sangre , Tiomalato Sódico de Oro/orina , Humanos
11.
J Pharm Biomed Anal ; 10(4): 279-87, 1992 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-1420457

RESUMEN

A sensitive method for the determination of gold-based drugs auranofin, myochrysine, and their metabolites has been developed. These gold-containing compounds were separated by reversed-phase ion-pair chromatography with tetrabutylammonium chloride as the ion-pairing agent. Gold-specific on-line detection utilized inductively coupled plasma mass spectrometry (ICP-MS). The separation conditions of pH, methanol content, concentration of the ion-pairing agent and ionic strength have been investigated. The detection limit for auranofin, the last peak in the chromatogram, was 0.3 ng. These methods were applied to the analysis of gold-containing species in urine from arthritis patients on auranofin, myochrysine or solganol therapy. The recovery of the total gold-containing species from urine was greater than 90%. Dicyanogold(I) anion, [Au(CN)2]-, was detected in the urine of several patients.


Asunto(s)
Auranofina/orina , Cromatografía/métodos , Tiomalato Sódico de Oro/orina , Espectrometría de Masas , Auranofina/metabolismo , Cromatografía Líquida de Alta Presión , Oro/análisis , Tiomalato Sódico de Oro/metabolismo , Humanos
12.
Genomics ; 9(2): 344-54, 1991 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-1840565

RESUMEN

We have characterized a mRNA sequence containing the entire coding region of a mouse carboxylesterase (EC 3.1.1.1). pEs-N, an 1840-bp composite of five overlapping cDNA clones, contains an open reading frame of 554 amino acids that display a high degree of similarity with rat and rabbit carboxylesterases. Genetic mapping studies place this carboxylesterase in cluster 1 of the esterase region on chromosome 8. Results of blot hybridization analysis of genomic DNA probed with a pEs-N cDNA under both low and high stringency conditions suggest membership in a carboxylesterase multigene family, as would be expected for a nonspecific carboxylesterase. A message size of 1850-1900 nucleotides was revealed by RNA blot hybridization analysis. S1 nuclease protection analyses with a probe representing a segment of pEs-N detected message in liver, kidney, and lung, but not in spleen, brain, testes, and submandibular gland, with higher levels in female than in male kidney. Additional S1 nuclease-protected mRNA species were found, suggesting the expression of distinct members of a multigene family. In vitro translation of a full-size transcript of pEs-N resulted in a product of 51.5 kDa. Upon the addition of microsomes, this product was processed into a protein of 60.4 kDa, which is within the size range of monomeric units of mouse carboxylesterases.


Asunto(s)
Hidrolasas de Éster Carboxílico/genética , ADN/aislamiento & purificación , Familia de Multigenes , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Carboxilesterasa , Hidrolasas de Éster Carboxílico/metabolismo , ADN/genética , Esterasas/genética , Esterasas/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Especificidad de Órganos/genética , Biosíntesis de Proteínas , ARN Mensajero/biosíntesis , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Endonucleasas Específicas del ADN y ARN con un Solo Filamento
13.
Proc Soc Exp Biol Med ; 191(2): 179-86, 1989 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-2498886

RESUMEN

Gold sodium thiomalate was incubated with one cadmium-sensitive cell line and two cadmium-resistant variants. The resistant lines have been reported to synthesize metallothionein (MT) in response to both cadmium and zinc, whereas the sensitive line does not. All cell lines showed a dose-dependent inhibition of growth as a result of gold sodium thiomalate treatment. However, daily comparisons of cell numbers indicate that the cadmium-resistant lines actually increase in number at the highest gold concentrations, whereas numbers of cells in the nonresistant line decrease. MT biosynthesis was measured by monitoring the incorporation of [35S )cysteine into low molecular weight protein. None of the cells synthesized MT in response to gold. When incubated with both zinc and gold, MT was synthesized by both of the cadmium resistant lines; however, the amount of MT synthesized was reduced in the presence of gold which appears to inhibit the uptake of [35S]cysteine by all the cell lines. Although MT is synthesized in the presence of zinc and gold sodium thiomalate, the MT does not have a significant effect on the ability of these cells to withstand high concentrations of gold.


Asunto(s)
Cadmio/farmacología , Tiomalato Sódico de Oro/envenenamiento , Ovario/efectos de los fármacos , Compuestos de Zinc , Animales , División Celular/efectos de los fármacos , Línea Celular , Cloruros/farmacología , Cromatografía , Anticonceptivos Orales Combinados , Cricetinae , Cisteína/metabolismo , Resistencia a Medicamentos , Femenino , Mesocricetus , Metalotioneína/biosíntesis , Ovario/citología , Ovario/metabolismo , Zinc/farmacología
14.
Science ; 225(4660): 430-2, 1984 Jul 27.
Artículo en Inglés | MEDLINE | ID: mdl-6429854

RESUMEN

Auranofin, 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranosato-S-(triethy lphosphine)- gold(I), an experimental antiarthritis pharmaceutical, metabolized in contact with hamster or rat gut wall to yield the deacetylated form of the drug. This product, 1-thio-beta-D-glucopyranosato-S-(triethylphosphine)gold(I), passed through hamster or rat intestinal wall in an everted gut experiment. The metabolite was separated by high-performance liquid chromatography and characterized by retention time, chemical reactivity to yield a known product, and comparison to a synthetic sample of the metabolite.


Asunto(s)
Antiinflamatorios/metabolismo , Aurotioglucosa/análogos & derivados , Oro/análogos & derivados , Absorción Intestinal , Animales , Auranofina , Aurotioglucosa/metabolismo , Cromatografía Líquida de Alta Presión , Cricetinae , Mesocricetus , Ratas , Ratas Endogámicas
15.
Dev Biol ; 99(2): 277-86, 1983 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-6413281

RESUMEN

The effects of different glycosaminoglycans (GAGs) on myogenesis were tested by culturing embryonic chick myoblasts on tissue culture dishes to which either hyaluronic acid (HA) or chondroitin sulfate (ChS) was covalently bound. Both in cell number and in apparent cell type distribution, the population of cells bound to GAGs is similar to that on gelatin and significantly different from that observed with uncoated dishes. When plated on ChS, myoblasts proliferate, align, and fuse at a rate similar to cells plated on gelatin. The final fused cells appear as sheets rather than long, thin myotubes. On HA, the cells proliferate but are inhibited from differentiation. The extent of inhibition is dependent on the amount of HA present. The inhibition of myogenesis is maintained through four subcultures on HA, but can be reversed at any time by culturing cells on gelatin. These experiments indicate that different GAGs have different effects on myogenesis and that HA can actively inhibit the process.


Asunto(s)
Glicosaminoglicanos/farmacología , Músculos/fisiología , Animales , Adhesión Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Fusión Celular , Células Cultivadas , Embrión de Pollo , Sulfatos de Condroitina/farmacología , Creatina Quinasa/metabolismo , Ácido Hialurónico/farmacología , Cinética , Músculos/citología , Músculos/efectos de los fármacos
16.
Natl Cancer Inst Monogr ; (48): 277-94, 1978 May.
Artículo en Inglés | MEDLINE | ID: mdl-372817

RESUMEN

The acetylcholine receptor in skeletal muscle is an integral plasma membrane glycoprotein. Its biosynthesis and incorporation into plasma membrane and its degradation are being studied with the use of biochemical, biophysical, and microscopic techniques. In this report, previously published data are combined with new information to yield a consistent and fairly detailed description ofthe mechanisms involved in receptor metabolism. It is proposed that the biosynthesis, transport, and incorporation of the receptor into plasma membranes involve a mechanism similar, or identical, to that used by the cell for production and secretion of secretory proteins. The receptor is degraded by a random-hit process, which involves internalization, transport to secondary lysosomes, and hydrolysis. Sites of regulation of receptor metabolism are discussed in the context of regulation of the number and distribution of receptors in plasma membranes, particularly with respect to the formation and stability of neuromuscular junctions.


Asunto(s)
Acetilcolina/metabolismo , Músculos/metabolismo , Receptores Colinérgicos/metabolismo , Animales , Bungarotoxinas/metabolismo , Embrión de Pollo , Técnicas de Cultivo , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Desarrollo de Músculos , Proteínas Musculares/metabolismo , Unión Neuromuscular/metabolismo , Ratas , Receptores Colinérgicos/biosíntesis
17.
J Cell Physiol ; 86(3 Pt 1): 561-5, 1975 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-1202033

RESUMEN

A cell preparation method by which large numbers of embryonic chick skeletal muscle cells may be obtained is described. The procedure requires fewer manipulations and much less time than standard trypsinization. By the criteria used, both methods are comparable with respect to percent viable cells and survival of plated cells. However, in addition to the ease of preparation, the mechanical dissociation method offers the significant advantage that the cell suspension is greatly enriched for myoblasts without the necessity of an additional preplating step.


Asunto(s)
Separación Celular/métodos , Células Cultivadas , Músculos/citología , Supervivencia Celular , Músculos/análisis , Polirribosomas/análisis , Tripsina
18.
J Cell Physiol ; 86(3 Pt 1): 553-60, 1975 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-811675

RESUMEN

The results reported here have shown that there are significant differences between polysome patterns obtained from cultured cells and from freshly isolated muscle tissue. Polysomes from embryonic homogenates show different patterns with different levels of myosin synthesis, but this does not appear to be the case with cultured cells. Experiments utilizing cell-free protein synthesizing systems indicate that the polysomes isolated from myoblast cultures can synthesize myosin at levels similar to those obtained from myotube cultures, suggesting that the myoblasts contain significant amounts of the messenger RNA for myosin. In contrast, the polysomes isolated from BrdUrd-inhibited cultures synthesize a comparatively low level of myosin. These findings illustrate a significant difference between myoblasts and BrdUrd-inhibited cells.


Asunto(s)
Músculos/citología , Miosinas/biosíntesis , Polirribosomas/metabolismo , Aminoácidos , Animales , Sangre , Bromodesoxiuridina/farmacología , Diferenciación Celular , Fusión Celular , Sistema Libre de Células , Células Cultivadas , Embrión de Pollo , Creatina , Medios de Cultivo , Ácido Edético/farmacología , Músculos/metabolismo , Ribonucleasas/farmacología
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