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1.
Influenza Other Respir Viruses ; 4(2): 73-9, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20167047

RESUMEN

BACKGROUND: One of the challenges of the recent pandemic (H1N1) 2009 influenza outbreak was to differentiate the virus from seasonal influenza when confronting clinical cases. The determination of the virus has implications on treatment choice, and obvious epidemiologic significance. OBJECTIVES: We set out to apply a novel electrochemical device to samples derived from clinical cases of pandemic (H1N1) 2009 influenza to examine the ability of the device to differentiate these samples from cases of seasonal influenza. PATIENTS/METHODS: An IRB approved protocol allowed for the use of original nasal wash samples from 24 confirmed human cases pandemic (H1N1) 2009 influenza. Clinical samples from cases of seasonal influenza (Influenza A/H1N1, A/H3N2, and B) were included as controls. Nucleic acids were extracted and samples examined by the ElectraSense Influenza A assay (CombiMatrix, Inc). Samples were also examined by RT-PCR or Luminex assays as a comparator. RESULTS AND CONCLUSIONS: The ElectraSense Influenza A assay correctly identified 23 of 24 samples of laboratory-confirmed pandemic (H1N1) 2009 Influenza. The assay correctly identified all samples of influenza A/H1N1 and A/H3N2, and differentiated these from pandemic (H1N1) 2009 Influenza in all cases. The ElectraSense Influenza A assay proved to be a useful assay to quickly and accurately differentiate pandemic (H1N1) 2009 influenza from seasonal influenza.


Asunto(s)
Técnicas de Laboratorio Clínico/métodos , Técnicas Electroquímicas/métodos , Subtipo H1N1 del Virus de la Influenza A/clasificación , Subtipo H3N2 del Virus de la Influenza A/clasificación , Gripe Humana/diagnóstico , Gripe Humana/virología , Humanos , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Subtipo H3N2 del Virus de la Influenza A/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sensibilidad y Especificidad
2.
Virology ; 388(2): 231-5, 2009 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-19409596

RESUMEN

Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involved in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.


Asunto(s)
Baculoviridae/enzimología , Flavina-Adenina Dinucleótido/metabolismo , Oxidorreductasas/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Baculoviridae/genética , Baculoviridae/metabolismo , Secuencia Conservada , Flavina-Adenina Dinucleótido/genética , Sistemas de Lectura Abierta/genética , Alineación de Secuencia , Análisis Espectral , Proteínas Virales/genética , Proteínas Virales/aislamiento & purificación
3.
Hum Vaccin ; 4(5): 389-92, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18437056

RESUMEN

The presence of circulating tumor cells (CTC) from various cancers has provided a wealth of information and possibilities. As the role of CTC detection in the treatment assessment of metastatic breast cancer becomes standard, there is interest in applying this tool in cancer vaccine development and clinical trial monitoring. Since we lack a proven immunologic assay that correlates with clinical response, CTC detection, quantification and phenotypic characterization may be a useful surrogate for clinical outcome. The Cancer Vaccine Development Program is involved in the development of HER2/neu peptide based vaccine development for the prevention of recurrence in HER2/neu expressing cancers like breast cancer. The CellSearch System (Veridex, LLC Warren, NJ) has been used by our lab in conjunction with in vivo and/or in vitro immunologic measurements to define a monitoring tool that could predict clinical response. Once validated, this assay could significantly shorten clinical trials and lead to more efficient assessment of potentially promising cancer vaccines.


Asunto(s)
Células Sanguíneas , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/inmunología , Vacunas contra el Cáncer/inmunología , Biomarcadores , Recuento de Células , Humanos , Resultado del Tratamiento
4.
Exp Biol Med (Maywood) ; 228(6): 674-82, 2003 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-12773698

RESUMEN

Male and female Hartley strain guinea pigs weighing 280 +/- 10 g were given acetaminophen-treated water ad libitum for 10 days. Sham-treated control animals were given similar quantities of untreated tap water (vehicle-treated control group). On Day 10, hearts were extracted, instrumented, and exposed to an ischemia (low-flow, 20 min)/reperfusion protocol. Our objective was to compare and contrast ventricular function, coronary circulation, and selected biochemical and histological indices in the two treatment groups. Left ventricular developed pressure in the early minutes of reperfusion was significantly greater in the presence of acetaminophen, e.g., at 1 min, 40 +/- 4 vs 21 +/- 3 mmHg (P < 0.05). Coronary perfusion pressure was significantly less from 3 to 40 min of reperfusion in the presence of acetaminophen. Creatine kinase release in vehicle-treated hearts rose from 42 +/- 14 (baseline) to 78 +/- 25 units/liter by the end of ischemia. Corresponding values in acetaminophen-treated hearts were 36 +/- 8 and 44 +/- 14 units/liter. Acetaminophen significantly (P < 0.05) attenuated release of creatine kinase. Chemiluminescence, an indicator of the in vitro production of peroxynitrite via the in vivo release of superoxide and nitric oxide, was also significantly attenuated by acetaminophen. Electron microscopy indicated a well-preserved myofibrillar ultrastructure in the postischemic myocardium of acetaminophen-treated hearts relative to vehicle-treated hearts (e.g., few signs of contraction bands, little or no evidence of swollen mitochondria, and well-defined light and dark bands in sarcomeres with acetaminophen; opposite with vehicle). We conclude that chronic administration of acetaminophen provides cardioprotection to the postischemic, reperfused rodent myocardium.


Asunto(s)
Acetaminofén/farmacología , Cardiotónicos/farmacología , Corazón/efectos de los fármacos , Isquemia Miocárdica/fisiopatología , Daño por Reperfusión Miocárdica/prevención & control , Animales , Circulación Coronaria/efectos de los fármacos , Creatina Quinasa/metabolismo , Femenino , Cobayas , Mediciones Luminiscentes , Masculino , Microscopía Electrónica , Isquemia Miocárdica/enzimología , Isquemia Miocárdica/metabolismo , Ácido Peroxinitroso/metabolismo , Factores de Tiempo , Función Ventricular Izquierda/efectos de los fármacos , Función Ventricular Izquierda/fisiología
5.
Carcinogenesis ; 23(10): 1609-15, 2002 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-12376468

RESUMEN

Reporter gene transactivation by human p53 is compromised in S. cerevisiae lacking the TRR1 gene encoding thioredoxin reductase. The basis for p53 inhibition was investigated by measuring the redox state of thioredoxin and glutathione in wild-type and Deltatrr1 yeast. The Deltatrr1 mutation affected the redox state of both molecules. About 34% of thioredoxin was in the disulfide form in wild-type yeast and increased to 70% in Deltatrr1 yeast. About 18% of glutathione was in the GSSG form in wild-type yeast and increased to 32% in Deltatrr1 yeast. The Deltatrr1 mutation also resulted in a 2.9-fold increase in total glutathione per mg extract protein. Highcopy expression of the GLR1 gene encoding glutathione reductase in Deltatrr1 yeast restored the GSSG:GSH ratio to wild-type levels, but did not restore p53 activity. Also, p53 activity was shown to be unaffected by a Deltaglr1 mutation, even though the mutation was known to result in glutathione oxidation. In summary, the results show that, although glutathione becomes more oxidized in Deltatrr1 cells, glutathione oxidation is neither sufficient nor necessary for p53 inhibition. The results indicate that p53 activity has a specific requirement for an intact thioredoxin system, rather than a general dependence on the intracellular reducing environment.


Asunto(s)
Genes p53 , Glutatión/metabolismo , Saccharomyces cerevisiae/genética , Reductasa de Tiorredoxina-Disulfuro/genética , Tiorredoxinas/metabolismo , Activación Transcripcional/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Disulfuros/metabolismo , Eliminación de Gen , Disulfuro de Glutatión/metabolismo , Humanos , Oxidación-Reducción , Compuestos de Sulfhidrilo/metabolismo
6.
Gut ; 50(2): 201-5, 2002 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11788560

RESUMEN

BACKGROUND: Hydrogen sulphide (H(2)S) is a potent toxin normally present in the colonic lumen which may play a role in ulcerative colitis (UC). Two enzymes, thiol methyltransferase (TMT) and rhodanese (RHOD), are thought to be responsible for sulphide removal but supportive evidence is lacking. AIMS: To determine the distribution of TMT and RHOD in different sites throughout the gastrointestinal tract and their efficacy as detoxifiers of H(2)S. METHODS: Enzyme activities were measured in normal tissue resected from patients with cancer. TMT and RHOD activities were determined using their conventional substrates, 2-mercaptoethanol and sodium thiosulphate, respectively. For measurement of H(2)S metabolism, sodium sulphide was used in the absence of dithiothreitol. Thiopurine methyltransferase (TPMT), which in common with TMT methylates sulphydryl groups but is not thought to act on H(2)S, was also examined. RESULTS: TMT, RHOD, and TPMT activities using their conventional substrates were found throughout the gastrointestinal tract with highest activity in the colonic mucosa. When H(2)S was given as substrate, no reaction product was found with TMT or TPMT but RHOD was extremely active (Km 8.8 mM, Vmax 14.6 nmol/mg/min). Incubation of colonic homogenates with a specific RHOD antibody prevented the metabolism of H(2)S, indicating that RHOD is responsible for detoxifying H(2)S. A purified preparation of RHOD also detoxified H(2)S. CONCLUSIONS: RHOD, located in the submucosa and crypts of the colon, is the principal enzyme involved in H(2)S detoxication. TMT does not participate in the detoxication of H(2)S.


Asunto(s)
Sulfuro de Hidrógeno/metabolismo , Intestinos/enzimología , Metiltransferasas/metabolismo , Tiosulfato Azufretransferasa/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Colitis Ulcerosa/enzimología , Colitis Ulcerosa/etiología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Mucosa Intestinal/enzimología , Masculino , Persona de Mediana Edad , Tiocianatos/sangre
7.
Physiol Genomics ; 7(1): 3-13, 2001 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-11595787

RESUMEN

The genetic response of human cells to sublethal thermal injury was assessed by gene expression profiling, using macroarrays containing 588 complementary known genes. At 1, 4, 8, and 24 h following thermal injury, RNA was isolated, and a cDNA copy was generated incorporating (33)P and hybridized to Atlas arrays. About one-fifth of the genes on the membrane exhibited a significant elevation or depression in expression (>/=2-fold) by 4 h posttreatment. Genes for heat shock proteins (HSPs) were upregulated as well as genes for transcription factors, growth regulation, and DNA repair. Cluster analysis was performed to assess temporal relationships between expression of genes. Translation of mRNA for some expressed genes, including HSP70 and HSP40, was corroborated by Western blotting. Gene expression profiling can be used to determine information about gene responses to thermal injury by retinal pigment epithelium cells following sublethal injury. The induction of gene expression following thermal injury involves a number of genes not previously identified as related to the stress response.


Asunto(s)
Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Respuesta al Choque Térmico/genética , Calor , Western Blotting , División Celular/genética , Línea Celular , Supervivencia Celular , Células/metabolismo , Células/patología , Análisis por Conglomerados , Reparación del ADN/genética , Regulación hacia Abajo , Proteínas de Choque Térmico/análisis , Proteínas de Choque Térmico/genética , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Biosíntesis de Proteínas , ARN Mensajero/análisis , ARN Mensajero/genética , Retina/citología , Retina/metabolismo , Factores de Tiempo , Factores de Transcripción/genética , Transcripción Genética , Regulación hacia Arriba
8.
Basic Res Cardiol ; 96(5): 423-30, 2001 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-11605988

RESUMEN

The acute administration of acetaminophen to isolated, perfused guinea pig hearts appears to have cardioprotective effects against the injury/mechanical dysfunction caused by global, low-flow, myocardial ischemia and reperfusion. In the current study we selected ischemia/reperfusion and administration of sodium pentobarbital as perturbations of the electrical stability of the myocardium. We investigated their ability to induce ventricular arrhythmias and changes in the characteristics of monophasic action potentials in the absence and presence of acetaminophen (0.35 mmol/l). The numbers of ventricular premature beats and ventricular salvos encountered in the presence of pentobarbital were significantly (P < 0.05) reduced by acetaminophen. The combined frequency of these arrhythmias was 0.14+/-0.06/min vs 0.03+/-0.01/min (P < 0.05) in the absence and presence of acetaminophen, respectively. The incidence of ventricular salvos increased steadily in vehicle-treated hearts after administration of pentobarbital. No such trend was seen with acetaminophen. After 10 min of global, low-flow myocardial ischemia, MAP50 and MAP90 (monophasic action potentials at 50 and 90% repolarization, respectively) decreased without acetaminophen (e.g. MAP50, 31+/-4 ms) but did not change during the same time interval with acetaminophen (e.g. MAP50, 57+/-6 ms)(P < 0.05). During ischemia and reperfusion, acetaminophen attenuated the release of hydroxyl radicals and peroxynitrite. Collectively these data reveal cardioprotective, antioxidant behavior of acetaminophen. Under selected conditions (e.g. those causing release of free radicals and other oxidants) such behavior might also prevent ventricular arrhythmias.


Asunto(s)
Acetaminofén/farmacología , Analgésicos no Narcóticos/farmacología , Antioxidantes/farmacología , Cardiotónicos/farmacología , Isquemia Miocárdica/tratamiento farmacológico , Potenciales de Acción/efectos de los fármacos , Animales , Femenino , Moduladores del GABA , Cobayas , Sistema de Conducción Cardíaco/efectos de los fármacos , Radical Hidroxilo/análisis , Técnicas In Vitro , Masculino , Miocardio/química , Pentobarbital , Ácido Peroxinitroso/análisis , Complejos Prematuros Ventriculares/inducido químicamente , Complejos Prematuros Ventriculares/tratamiento farmacológico
9.
Naturwissenschaften ; 88(8): 333-8, 2001 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-11572014

RESUMEN

Ethyl (2E, 4Z)-2,4-decadienoate, a pear-derived volatile, is a species-specific, durable, and highly potent attractant to the codling moth (CM), Cydia pomonella (L.), a serious pest of walnuts, apples, and pears worldwide. This kairomone attracts both CM males and virgin and mated females. It is highly attractive to CM in both walnut and apple orchard contexts, but has shown limited effectiveness in a pear orchard context. Rubber septa lures loaded with ethyl (2E, 4Z)-2,4-decadienoate remained attractive for several months under field conditions. At the same low microgram load rates on septa, the combined gender capture of CM in kairomone-baited traps was similar to the capture rate of males in traps baited with codlemone, the major sex pheromone component. The particular attribute of attracting CM females renders this kairomone a novel tool for monitoring population flight and mating-ovipositional status, and potentially a major new weapon for directly controlling CM populations.


Asunto(s)
Decanoatos/farmacología , Frutas/fisiología , Frutas/parasitología , Mariposas Nocturnas/fisiología , Control Biológico de Vectores , Feromonas/fisiología , Animales , Femenino , Vuelo Animal , Masculino , Oviposición , Feromonas/farmacología
10.
Chest ; 120(2): 362-8, 2001 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-11502630

RESUMEN

STUDY OBJECTIVES: To determine if soluble leukocyte selectin (sL-selectin) levels in serum and pleural fluid (PF) are an inflammatory marker that differentiates pleural effusion transudates from exudates. DESIGN: sL-selectin PF and serum levels were measured in consecutive patients and compared to established criteria. SETTING: A tertiary-care military medical center. PATIENTS: One hundred twenty patients undergoing diagnostic or therapeutic thoracentesis. INTERVENTIONS: PF and serum samples were collected during thoracentesis and analyzed separately for sL-selectin levels. Results were compared with clinical diagnosis and established PF criteria including the criteria of Light et al, cholesterol ratio, total bilirubin ratio, and albumin gradient. MEASUREMENTS AND RESULTS: sL-selectin levels in PF and serum were determined in 109 patients. By clinical diagnosis, mean +/- SD PF sL-selectin levels were 200.2 +/- 124.3 ng/mL in transudates and 496.8 +/- 379.2 ng/mL in exudates (p < 0.001). By the criteria of Light et al, mean PF sL-selectin levels were 195.7 +/- 105.2 ng/mL in transudates and 448.2 +/- 367.6 ng/mL in exudates (p < 0.001). Mean sL-selectin PF to serum ratios were 0.31 +/- 0.17 in transudates and 0.72 +/- 0.31 in exudates (p < 0.001) by clinical criteria, and 0.31 +/- 0.18 in transudates and 0.64 +/- 0.33 in exudates (p < 0.001) by the criteria of Light et al. No significant difference was noted with serum sL-selectin levels between groups. CONCLUSIONS: sL-selectin is an inflammatory marker that differentiates transudates from exudates in pleural effusions and is a sensitive indicator for PF analysis.


Asunto(s)
Biomarcadores/análisis , Selectina L/análisis , Derrame Pleural/química , Exudados y Transudados/química , Humanos , Selectina L/sangre , Pleuresia/diagnóstico , Sensibilidad y Especificidad
11.
Am J Physiol Heart Circ Physiol ; 280(6): H2631-8, 2001 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-11356619

RESUMEN

Acetaminophen is a phenol with antioxidant properties, but little is known about its actions on the mammalian myocardium and coronary circulation. We studied isolated, perfused guinea pig hearts, and tested the hypothesis that acetaminophen-treated hearts would be protected during ischemia-reperfusion. Acetaminophen concentrations in the range of 0.3-0.6 mmol/l caused modest but significant (P < 0.05) coronary vasoconstriction and positive inotropy. The effects were more brisk during constant pressure perfusion than during constant flow. During 20 min of low-flow, global myocardial ischemia and 40 min of reperfusion, hearts treated with acetaminophen retained or recovered a greater percentage of left ventricular function than hearts treated with vehicle. Myofibrillar ultrastructure appeared to be preserved in the reperfused myocardium with acetaminophen. By using chemiluminescence and spin-trap methodologies, we investigated acetaminophen-mediated antioxidant mechanisms to help explain the cardioprotection. The burst of hydroxyl radicals seen between 0 and 10 min of reperfusion was significantly attenuated (P < 0.05) by acetaminophen but not by vehicle. The 3-morpholinosydnominine (SIN-1) generation of peroxynitrite and its oxidative interaction with luminol to produce blue light during ischemia-reperfusion was also blocked by acetaminophen. Our results show that acetaminophen provides significant functional and structural protection to the ischemic-reperfused myocardium, and the mechanism of cardioprotection seems to involve attenuation of the production of both hydroxyl radicals and peroxynitrite.


Asunto(s)
Acetaminofén/farmacología , Citoprotección/efectos de los fármacos , Gentisatos , Isquemia Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Miocardio/metabolismo , Animales , Circulación Coronaria/efectos de los fármacos , Vasos Coronarios/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Cobayas , Corazón/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Hidroxibenzoatos/metabolismo , Radical Hidroxilo/metabolismo , Técnicas In Vitro , Mediciones Luminiscentes , Masculino , Reperfusión Miocárdica , Miocardio/ultraestructura , Miofibrillas/efectos de los fármacos , Miofibrillas/ultraestructura , Nitratos/metabolismo , Perfusión/métodos , Vasoconstricción/efectos de los fármacos , Función Ventricular Izquierda/efectos de los fármacos
12.
Comp Med ; 51(1): 16-21, 2001 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11926296

RESUMEN

Chicken heterophil polymorphonuclear leukocytes (CPMNLs) have NADPH oxidase activity, but lack myeloperoxidase (MPO). Stimulation of CPMNLs by phorbol 12-myristate 13-acetate or chicken opsonified zymosan results in luminol-dependent chemiluminescence (CL) activity, which is small relative to that of human peroxidase-positive neutrophils (HPMNLs), as well as lucigenin-dependent CL, comparable to HPMNL responses. Inhibitors were used to investigate and characterize the CL activity of CPMNLs. Inhibition constants were calculated, using Dixon inhibition analysis, or were reported as the concentration producing 50% inhibition of the magnitude of CL responses. Azide and cyanide are effective inhibitors of luminol CL in HPMNLs, although these peroxidase inhibitors do not inhibit either luminol or lucigenin CL of CPMNLs. Since these agents also inhibit eosinophil peroxidase, lack of inhibition of CPMNL CL indicates that the small percentages of peroxidase-positive eosinophils in CPMNL preparations are not responsible for the luminol CL observed. Iodoacetate and fluoride, pre-oxidase and pre-peroxidase inhibitors of glycolytic metabolism, effectively inhibit lucigenin and luminol CL activities in CPMNLs. Superoxide dismutase competitively inhibits lucigenin and luminol CL in CPMNLs, but catalase is an ineffective inhibitor. Although luminol is efficiently dioxygenated by a MPO-dependent mechanism in HPMNL, use of peroxidase-deficient CPMNLs indicates that this substrate does not exclusively measure peroxidase activity.


Asunto(s)
Pollos/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Acridinas , Animales , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Mediciones Luminiscentes , Luminol , Masculino , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Peroxidasa/metabolismo , Especificidad de la Especie , Acetato de Tetradecanoilforbol/farmacología , Zimosan/farmacología
13.
J Homosex ; 39(2): 1-30, 2000.
Artículo en Inglés | MEDLINE | ID: mdl-10933279

RESUMEN

In comparison to a large body of literature about battered heterosexual women and a growing body about battered lesbians, this is one of the first published studies that investigates the experiences of battered gay and bisexual men. Results indicated that these men suffered patterns, forms, and frequencies of physical, emotional, and sexual abuse similar to what has been documented by research on battered heterosexual and lesbian women. Likewise, the most commonly reported reasons for staying--namely, hope for change and love for partner--appear to be universal to the experience of being battered. Unlike battered heterosexual women, respondents in this study were not likely to report that being financially trapped was a major reason why they had remained. HIV-status, however, appears to significantly influence their decision to remain. Moreover, lack of knowledge about domestic violence and the lack of availability of appropriate resources play a significant role in same-gender domestic violence victims' decisions to remain. Like battered lesbians, battered gay men infrequently sought assistance from battered women's services and perceived these services as not helpful. By contrast, individual counselors and agencies who provided individual counselors were rated as quite helpful.


Asunto(s)
Homosexualidad Masculina , Maltrato Conyugal , Femenino , Homosexualidad Femenina , Humanos , Relaciones Interpersonales , Masculino , Aceptación de la Atención de Salud , Maltrato Conyugal/estadística & datos numéricos
14.
Anal Biochem ; 284(1): 1-5, 2000 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-10933848

RESUMEN

Pathophysiological levels of oxygen radical metabolites have been studied as indicators of trauma caused by burn insult. The 2, 3-diaminonaphthalene assay is routinely used in the determination of nitrite/nitrate levels in biological fluids and cellular extracts as one indicator of nitric oxide activity. Several laboratories, including ours, have noted matrix-based interferences resulting in decreased assay sensitivity during nitrite/nitrate analysis. We evaluated filtration using Millipore Ultrafree-MC 10,000 NMWL filters for the ability to eliminate matrix-based interferences from human serum and tissue culture medium, thereby restoring assay sensitivity.


Asunto(s)
Filtración/métodos , Nitratos/análisis , Nitritos/análisis , Ultrafiltración/métodos , 2-Naftilamina/análogos & derivados , 2-Naftilamina/análisis , Animales , Bovinos , Relación Dosis-Respuesta a Droga , Humanos , Filtros Microporos , Nitratos/sangre , Nitritos/sangre , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Albúmina Sérica/farmacología , Espectrometría de Fluorescencia/métodos
15.
J Agric Food Chem ; 48(7): 2858-61, 2000 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-10898636

RESUMEN

Volatiles were isolated from whole green mature walnuts (Hartley variety) with husks still intact using dynamic headspace sweeping with trapping on Tenax. A total of 45 volatile compounds were identified by GC-MS. Major volatiles identified included (E)-4, 8-dimethyl-1,3,7-nonatriene, pinocarvone, pinocarveol, myrtenal, myrtenol, (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene, caryophyllene epoxide, verbenol, verbenone, and terpinolene. Green walnuts that had been infested with codling moth showed appreciably higher amounts emitted for (E)-4,8-dimethyl-1,3,7-nonatriene, (E, E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene, alpha- and beta-pinenes, sabinene, (E)-beta-ocimene, (E,E)-alpha-farnesene, and linalool. The infested nuts also emitted benzyl methyl ether, isobutyl cyanide, and 1-nitro-3-methylbutane, compounds not found with the healthy nuts. Volatiles from uninfested green walnuts at the maturity stage where the husk was just beginning to split were also analyzed and compared.


Asunto(s)
Nueces/química , Cromatografía de Gases y Espectrometría de Masas , Naftoquinonas/análisis , Naftoquinonas/aislamiento & purificación , Extractos Vegetales/química , Terpenos/análisis , Volatilización
16.
Proc Natl Acad Sci U S A ; 97(12): 6850-5, 2000 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-10823899

RESUMEN

The dopamine transporter (DAT) is a target of amphetamine (AMPH) and cocaine. These psychostimulants attenuate DAT clearance efficiency, thereby increasing synaptic dopamine (DA) levels. Re-uptake rate is determined by the number of functional transporters at the cell surface as well as by their turnover rate. Here, we present evidence that DAT substrates, including AMPH and DA, cause internalization of human DAT, thereby reducing transport capacity. Acute treatment with AMPH reduced the maximal rate of [(3)H]DA uptake, decreased AMPH-induced currents, and significantly redistributed the immunofluorescence of an epitope-tagged DAT from the plasma membrane to the cytosol in human embryonic kidney 293 cells. Conversely, DAT inhibitors, such as cocaine, mazindol, and nomifensine, when administered with AMPH, blocked the reduction in [(3)H]DA uptake and the redistribution of DAT immunofluorescence to the cytosol. The reductions of [(3)H]DA uptake and AMPH-induced DAT internalization also were inhibited by coexpression of a dominant negative mutant of dynamin I (K44A), indicating that endocytosis modulates transport capacity, likely through a clathrin-mediated pathway. With this mechanism of regulation, acute application of AMPH would reduce DA uptake not only by direct competition for uptake, but also by reducing the available cell-surface DAT. Moreover, AMPH-induced internalization might diminish the amount of DAT available for DA efflux, thereby modulating the cytotoxic effects of elevated extracellular DA.


Asunto(s)
Anfetamina/farmacología , Proteínas Portadoras/efectos de los fármacos , Cocaína/farmacología , Glicoproteínas de Membrana , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Línea Celular , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Humanos , Mazindol/farmacología , Proteínas Recombinantes de Fusión/efectos de los fármacos
17.
Toxicon ; 38(3): 381-9, 2000 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-10669027

RESUMEN

Sensitive and specific enzyme-linked immunosorbent assays were developed to detect Clostridium botulinum neurotoxin serotypes A (BoNT A) and B (BoNT B) in assay buffer and human serum. The assay is based upon affinity-purified horse polyclonal antibodies directed against the approximately 50 kDa C-fragments of each toxin. Standard curves were linear over the range of 0.1-10 ng mL. Detection was possible at 0.2 ng mL (20 pg/well) and accurate quantitation at 0.5 ng/mL (50 pg well) in assay buffer and 10% human serum. Variations between triplicates was typically 5-10%. Less than 1% cross reactivity occurred between other serotypes when each assay was performed against serotypes A, B and E. When tested against toxins complexed to their associated nontoxic proteins, interference was absent (BoNT B) or < 25% (BoNT A). These assays demonstrate sensitivity close to that of the mouse bioassay without the use of animals and in a much simpler format than other reported assays of similar sensitivity.


Asunto(s)
Toxinas Botulínicas Tipo A/análisis , Toxinas Botulínicas/análisis , Neurotoxinas/análisis , Animales , Bioensayo , Biotina/química , Cromatografía de Afinidad , Colorimetría , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Humanos , Ratones
18.
Proc Natl Acad Sci U S A ; 96(21): 12004-9, 1999 Oct 12.
Artículo en Inglés | MEDLINE | ID: mdl-10518566

RESUMEN

Genetic variation in CC chemokine receptor 5 (CCR5), the major HIV-1 coreceptor, has been shown to influence HIV-1 transmission and disease progression. However, it is generally assumed that the same CCR5 genotype (or haplotype) has similar phenotypic effects in different populations. To test this assumption, we used an evolutionary-based classification of CCR5 haplotypes to determine their associated HIV-1 disease-modifying effects in a large well-characterized racially mixed cohort of HIV-1-seropositive individuals. We demonstrate that the spectrum of CCR5 haplotypes associated with disease acceleration or retardation differs between African Americans and Caucasians. Also, we show that there is a strong interactive effect between CCR5 haplotypes with different evolutionary histories. The striking population-specific phenotypic effects associated with CCR5 haplotypes emphasize the importance of understanding the evolutionary context in which disease susceptibility genes are expressed.


Asunto(s)
Seropositividad para VIH/genética , VIH-1 , Grupos Raciales/genética , Receptores CCR5/genética , Síndrome de Inmunodeficiencia Adquirida/genética , Síndrome de Inmunodeficiencia Adquirida/metabolismo , Adolescente , Adulto , África , Anciano , Alelos , Asia , Evolución Biológica , Población Negra/genética , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Variación Genética , Genotipo , Seropositividad para VIH/epidemiología , Haplotipos , Humanos , Masculino , Persona de Mediana Edad , Filogenia , Polimorfismo de Longitud del Fragmento de Restricción , Factores de Tiempo , Estados Unidos , Población Blanca/genética
19.
Cancer Res ; 59(13): 3175-9, 1999 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-10397262

RESUMEN

Stimulation of target gene transcription by human p53 is inhibited in budding yeast lacking the TRR1 gene encoding thioredoxin reductase. LexA/p53 fusion proteins were used to study the basis for thioredoxin reductase dependence. A fusion protein containing all 393 of the residues of p53 efficiently and specifically stimulated transcription of a LexOP-LacZ reporter gene in wild-type yeast but was several-fold less effective in delta trr1 yeast lacking the thioredoxin reductase gene. Thus, even when p53 was tethered to a reporter gene by a heterologous DNA-binding domain, reporter gene transactivation remained dependent on thioredoxin reductase. A fusion protein containing only the activation domain of p53 stimulated reporter gene transcription equally in wild-type and delta trr1 cells, suggesting that p53 residues downstream from the activation domain created the requirement for thioredoxin reductase. Experiments using additional LexA/p53 truncation mutations indicated that the p53 negative regulatory domain, rather than the DNA-binding or oligomerization domains, created the requirement for thioredoxin reductase. The fusion protein results suggested that, under oxidative conditions, the negative regulatory domain inhibited the ability of DNA-bound p53 to stimulate transcription. However, deletion of the negative regulatory domain did not alleviate the requirement of non-LexA-containing p53 for thioredoxin reductase. The results, thus, suggest that oxidative conditions inhibit both DNA binding and transactivation by p53, and that inhibition of the latter requires the negative regulatory domain.


Asunto(s)
Genes p53 , Secuencias Reguladoras de Ácidos Nucleicos , Saccharomyces cerevisiae/genética , Activación Transcripcional , Genes Reporteros , Humanos , Plásmidos , Proteínas Recombinantes de Fusión/biosíntesis , Reductasa de Tiorredoxina-Disulfuro/genética , Transcripción Genética , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genética
20.
J Appl Physiol (1985) ; 85(5): 1909-14, 1998 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-9804598

RESUMEN

5-Aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR) is taken up by perfused skeletal muscle and phosphorylated to form 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuraosyl-5'-monopho sph ate (analog of 5'-AMP) with consequent activation of AMP-activated protein kinase, phosphorylation of acetyl-CoA carboxylase, decrease in malonyl-CoA, and increase in fatty acid oxidation. This study was designed to determine the effect of increasing levels of palmitate on the rate of fatty acid oxidation. Malonyl-CoA concentration was manipulated with AICAR at different palmitate concentrations. Rat hindlimbs were perfused with Krebs-Henseleit bicarbonate containing 4% bovine serum albumin, washed bovine red cells, 200 microU/ml insulin, 10 mM glucose, and different concentrations of palmitate (0. 1-1.0 mM) without or with AICAR (2.0 mM). Perfusion with medium containing AICAR was found to activate AMP-activated protein kinase in skeletal muscle, inactivate acetyl-CoA carboxylase, and decrease malonyl-CoA at all concentrations of palmitate. The rate of palmitate oxidation increased as a function of palmitate concentration in both the presence and absence of AICAR but was always higher in the presence of AICAR. These results provide additional evidence that malonyl-CoA is an important regulator of the rate of fatty acid oxidation at palmitate concentrations in the physiological range.


Asunto(s)
Malonil Coenzima A/metabolismo , Músculo Esquelético/metabolismo , Palmitatos/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Quinasas Activadas por AMP , Acetil-CoA Carboxilasa/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Ácidos Grasos/metabolismo , Miembro Posterior/irrigación sanguínea , Miembro Posterior/fisiología , Hipoglucemiantes/farmacología , Masculino , Complejos Multienzimáticos/metabolismo , Oxidación-Reducción , Consumo de Oxígeno/fisiología , Proteínas Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Flujo Sanguíneo Regional/fisiología , Ribonucleótidos/farmacología
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