RESUMEN
To investigate the role of the Ca2+-binding protein calmodulin on histamine release in the rat peritoneal mast cell, we exposed cells to exogenous calmodulin in the presence of a variety of histamine secretagogues. Histamine release stimulated by compound 48/80, polymyxin B and ionophore A23187 was inhibited while concanavalin A-stimulated release was not affected. Calmodulin in the presence of the secretagogues did not affect cell viability and calmodulin alone had no effect on histamine release. No direct interaction between calmodulin and the secretagogues was observed. Exogenous calmodulin does not appear to be incorporated into the cell. The inhibition of histamine release by calmodulin can be explained as a labile interaction between the protein and the cell that requires externally-bound Ca2+. These experiments demonstrate the use of exogenous calmodulin as a probe in the study of the mechanism of histamine release.
Asunto(s)
Calmodulina/farmacología , Liberación de Histamina/efectos de los fármacos , Mastocitos/inmunología , Animales , Calcimicina/farmacología , Calcio/farmacología , Concanavalina A/farmacología , Ácido Egtácico/farmacología , Cinética , Masculino , Mastocitos/efectos de los fármacos , Polimixina B/farmacología , Ratas , Ratas Endogámicas , p-Metoxi-N-metilfenetilamina/farmacologíaRESUMEN
The ability of oxidative stress to induce apoptosis (programmed cell death), and the effect of Trolox, a water soluble vitamin E analog, on this induction were studied in vitro in mouse thymocytes. Cells were exposed to oxidative stress by treating them with 0.5-10 microM hydrogen peroxide (H2O2) for 10 min, in phosphate-buffered saline supplemented with 0.1 mM ferrous sulfate. Cells were resuspended in RPMI 1640 medium with 10% serum and incubated at 37 degrees C under 5% CO2 in air. Electron microscopic studies revealed morphological changes characteristic of apoptosis in H2O2-treated cells. H2O2 treatment fragmented the DNA in a manner typical of apoptotic cells, producing a ladder pattern of 200 base pair increments upon agarose gel electrophoresis. The percentage of DNA fragmentation (determined fluorometrically) increased with increasing doses of H2O2 and postexposure incubation times. Pre- or posttreatment of cells with Trolox reduced H2O2-induced DNA fragmentation to control levels and below. The results indicate that oxidative stress induces apoptosis in thymocytes, and this induction can be prevented by Trolox, a powerful inhibitor of membrane damage.
Asunto(s)
Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Cromanos/farmacología , Peróxido de Hidrógeno/toxicidad , Timo/citología , Animales , Células Cultivadas , ADN/efectos de los fármacos , ADN/aislamiento & purificación , Daño del ADN , Peróxido de Hidrógeno/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos , Microscopía Electrónica , Timo/efectos de los fármacos , Timo/ultraestructuraRESUMEN
We investigated the capacity of ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one], a glutathione peroxidase mimic, to protect cells from radiation-induced apoptosis. Incubating mouse thymocytes with 25 microM ebselen immediately after 60Co gamma-radiation exposure (5 Gy) inhibited morphological changes associated with apoptosis. Treatment of thymocytes with ebselen before, during, or after irradiation completely blocked internucleosomal DNA fragmentation, a biochemical marker for apoptosis. We measured peroxides formed in cells during and after irradiation, using the oxidation-sensitive fluorescent probe 2',7'-dichlorofluorescin diacetate. By 2 min postirradiation, levels of peroxides in irradiated thymocytes were approximately 10-11 times greater than those in the same cells before irradiation, and levels continued to increase with time. We also measured membrane lipid peroxidation using cis-parinaric acid, a naturally fluorescent polyunsaturated fatty acid that readily incorporates into cell membranes. The oxidation of cis-parinaric acid also began soon after irradiation and increased with time. Peroxide generation and membrane lipid peroxidation preceded both internucleosomal DNA fragmentation and morphological changes characteristic of apoptosis. Treatment of cells with ebselen reduced peroxide levels and appeared to protect thymocytes from radiation-induced apoptosis by scavenging peroxides generated during and after irradiation. The results suggest that peroxide generation and membrane lipid peroxidation may be important signaling events that trigger apoptosis in irradiated cells.
Asunto(s)
Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Azoles/farmacología , Compuestos de Organoselenio/farmacología , Peróxidos/metabolismo , Animales , Apoptosis/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Células Cultivadas , ADN/efectos de los fármacos , ADN/metabolismo , ADN/efectos de la radiación , Isoindoles , Cinética , Masculino , Ratones , Ratones Endogámicos , Oxidación-Reducción , Timo/efectos de los fármacos , Timo/metabolismo , Timo/efectos de la radiaciónRESUMEN
These experiments measured the effect of gamma radiation on the nuclear envelope using doxyl-fatty acid spin-label probes. Nuclei were isolated from cultured MOLT-4 cells, a radiation-sensitive human T-cell lymphocyte. Membrane fluidity was measured from the electron paramagnetic resonance spectra of the probes. MOLT-4 cells were grown under standard conditions, and suspensions were exposed to 60Co gamma radiation at room temperature. The spectra of 5-doxylstearic acid in the nuclei were those of a strongly immobilized label. A difference in the membrane fluidity was detected in a series of experiments comparing labeled irradiated and nonirradiated nuclei. The change in fluidity was measured by comparing the changes in the order parameter, S, of the spin label in irradiated nuclei with those in control nuclei. The change in the S ratio is dependent on radiation dose, increasing with doses up to 15 Gy. The maximum change of the order parameter with time after irradiation occurs 16-20 h after radiation exposure. These observations are correlated with changes in cell viabilities.
Asunto(s)
Fluidez de la Membrana/efectos de la radiación , Membrana Nuclear/efectos de la radiación , Línea Celular , Radioisótopos de Cobalto , Rayos gamma , Humanos , Técnicas In Vitro , Células Tumorales CultivadasRESUMEN
Beta-glucuronidase, a lysosomal marker enzyme, associates with purified nuclei from HeLa and MOLT-4 cell lines in a radiation dose-dependent manner, up to 300 cGy in MOLT-4 cells, and 1000 cGy in HeLa cells. In MOLT-4 cells (200-cGy exposure), there is a significant increase in beta-glucuronidase activity detected in the nuclear fraction 24 h postirradiation with a maximum association occurring at 72 h. In HeLa cells (1000-cGy exposure), a significant association is first detected 24 h postirradiation with a maximum association at 48 h. The association is not the result of nonspecific contamination occurring during nuclei purification since nuclei from irradiated cells show no greater levels of plasma membrane marker and mitochondrial marker than controls. The nature of the association remains unclear, but activity is not removed by detergents used in the nuclei isolation procedure, and incubation of the nuclei with EDTA reverses the association only modestly. Exposure of nuclei from irradiated cells to anisotonic buffers also results in only a small decrease in beta-glucuronidase activity associated with the nuclei. These observations suggest that lysosomal hydrolases become intimately associated with the nuclei of irradiated cells.
Asunto(s)
Núcleo Celular/efectos de la radiación , Glucuronidasa/metabolismo , Lisosomas/enzimología , Línea Celular , Núcleo Celular/enzimología , Supervivencia Celular/efectos de la radiación , Radioisótopos de Cobalto , Células HeLa , Humanos , Células Tumorales Cultivadas/efectos de la radiaciónRESUMEN
The use of antioxidants to prevent intracellular free radical damage is an area currently attracting considerable research interest. The compound 2',7'-dichlorofluorescin diacetate (DCFH-DA) is a probe for intracellular peroxide formation commonly used in such studies. During our studies we unexpectedly found that incubation of Trolox, a water soluble vitamin E analog, with DCFH-DA in cell-free physiological buffers resulted in the deacetylation and oxidation of DCFH-DA to form the fluorescent compound, 2',7'-dichlorofluorescein (DCF). The reaction was time-, temperature-, and pH-dependent. Fluorescence intensity increased with an increase in either Trolox or DCFH-DA concentration. These results indicate that even at physiological pH, DCFH-DA can be deacetylated to form 2',7'-dichlorofluororescin (DCFH), DCFH can then be oxidized to DCF by abstraction of a hydrogen atom by the phenoxyl radical of Trolox. Exposure of the reaction mixture to 10 Gy of 60Co gamma radiation greatly increased production of DCF. Antioxidant compounds reported to "repair" the Trolox phenoxyl radical (e.g., ascorbic acid, salicylate) can also prevent the Trolox-induced DCFH-DA fluorescence. However, radical (e.g., catechin) or can themselves form a radical (e.g., uric acid, TEMPOL) either have no effect or can increase levels of DCF. These results demonstrate that experimental design must be carefully considered when using DCFH-DA to measure peroxide formation in combination with certain antioxidants.
Asunto(s)
Antioxidantes/química , Cromanos/química , Fluoresceínas/química , Vitamina E/análogos & derivados , Tampones (Química) , Rayos gamma , Concentración de Iones de Hidrógeno , Oxidación-Reducción , Espectrometría de Fluorescencia , Temperatura , Factores de TiempoRESUMEN
4-Hydroxynonenal (HNE) is the major aldehydic product resulting from lipid peroxidation and has been implicated as involved in several pathological conditions. In our continuing studies on the role of membranes and lipid peroxidation in the induction of apoptosis, we investigated the effect of HNE on cultured human malignant immune system cells. Two cell lines were utilized; MOLT-4, a human T-cell leukemia cell line, and Reh, a human B-cell lymphoma cell line. A 10 min treatment with 0.01 mM HNE resulted in the apoptotic death, as determined by flow cytometric and morphological analyses, of both cell lines within 24 h. MOLT-4 cells exhibited the manifestations of impending apoptotic death much sooner than did Reh cells, indicating that MOLT-4 cells were more sensitive or not as efficient at detoxifying HNE than were Reh cells. These results suggest that peroxidative damage to cellular membranes resulting in the production of HNE may be a trigger for the induction of apoptosis in immune system cells.
Asunto(s)
Aldehídos/farmacología , Apoptosis/efectos de los fármacos , Leucemia de Células B/patología , Leucemia de Células T/patología , Peroxidación de Lípido , Anexina A5 , Citometría de Flujo , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Microscopía Fluorescente , Propidio , Células Tumorales CultivadasRESUMEN
Studies were carried out to investigate the protective effects of pyruvate, a key glycolytic intermediate and alpha-keto-monocarboxylate, against oxidative stress-induced apoptosis. Oxidative stress was induced by treating mouse thymocytes with 25 microM hydrogen peroxide for 15 min at 37 degrees C under 5% CO2 in air. Pre- and post-treatment of cells with 10 mM pyruvate inhibited morphological changes, internucleosomal DNA fragmentation, and translocation of phosphatidylserine to the plasma membrane surface, which are characteristic features of apoptosis. L-lactate (10 mM) and acetate (10 mM) were ineffective in inhibiting apoptosis and appeared to be toxic to the cells under similar conditions. The results suggest that pyruvate has therapeutic potential for use in the treatment of oxidative stress-induced disorders associated with increased apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Ácido Pirúvico/farmacología , Acetatos/farmacología , Animales , Anexina A5/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Tamaño de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Fragmentación del ADN/efectos de los fármacos , Citometría de Flujo , Ácido Láctico/farmacología , Masculino , Ratones , Ratones Endogámicos , Estrés Oxidativo/efectos de los fármacos , Fosfatidilserinas/metabolismo , Propidio , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , TimoRESUMEN
The role of cellular membranes in thymocyte apoptosis has been examined. Trolox, a water soluble analogue of vitamin E and inhibitor of membrane damage, inhibits DNA fragmentation in thymocytes exposed to gamma-radiation. Trolox is most effective in inhibiting DNA fragmentation when added to cells within 30 min post-irradiation. Exposure to trolox only during irradiation did not prevent DNA fragmentation, suggesting that it does not work by scavenging free radicals generated during radiation exposure. Incubation of the irradiated cell suspension with trolox for 2 h post-irradiation was sufficient to prevent DNA fragmentation measured at 24 h in irradiated cells. This suggests that trolox irreversibly inhibits a cellular lesion required for apoptosis. The induction of DNA fragmentation appears to be related to a concurrent, pronounced flow of Ca2+ into the cell. At 3 h post-irradiation the amount of Ca2+ in irradiated thymocytes was more than twice that of unirradiated thymocytes. Membrane damage has been shown to affect the transport of Ca2+. Trolox treatment completely blocked the radiation-induced influx of Ca2+ into the thymocytes. These results suggest that membrane damage is a critical lesion that is involved in DNA fragmentation in thymocyte apoptosis.
Asunto(s)
Apoptosis/fisiología , Membrana Celular/fisiología , Linfocitos/efectos de la radiación , Timo/citología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Membrana Celular/efectos de los fármacos , Membrana Celular/efectos de la radiación , Cromanos/uso terapéutico , Radioisótopos de Cobalto , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Rayos gamma , Técnicas In Vitro , Linfocitos/efectos de los fármacos , Masculino , Ratones , Vitamina E/análogos & derivadosRESUMEN
BACKGROUND: Thermotolerance affects cell viability, retards translation of heat shock proteins, and protects RNA slicing mechanisms. We reported previously that heat shocking nonthermotolerant cells causes an intracellular acidification and an increase in cytosolic free Ca2+ ([Ca2+]i) in addition to an induction of heat shock protein 72kDa (HSP-72) production. This study characterized heat-induced changes in cytosolic Ca2+, H+, and HSP-72 synthesis in thermotolerant A-431 cells. METHODS: We studied heat-induced changes in pH(i), [Ca2+]i, and HSP-72 using thermotolerant A-431 cell monolayers. pH(i) and [Ca2+]i were determined using fluorescence probes, and HSP-72 was measured by SDS-PAGE. The mRNA encoding HSP-72 was measured by Northern blots probed with a [32P]-labeled 2.3 kb fragment of an HSP-70 cDNA insert. RESULTS: Heat shocking thermotolerant cells induced the same degree of intracellular acidification as that induced in nonthermotolerant cells, but the heat-induced increase in [Ca2+]i was less in thermotolerant cells. This diminished response was characterized by an increase in Km for external Ca2+ and was blocked by pretreatment with cycloheximide, indicating a newly synthesized protein is involved. Similar to what was seen in nonthermotolerant cells, the heat-induced increase in [Ca2+]i in thermotolerant cells depended on external Na+ concentration and was blocked by dichlorobenzamil, though thermotolerant cells were more sensitive to the inhibitor (IC50 = 0.21 mumol/L for nonthermotolerant, 0.025 mumol/Lm for thermotolerant). Thermotolerant cells contained high resting levels of HSP-72. Heat shocking these cells attenuated the HSF translocation from cytosol to nucleus and did not induce a further synthesis of HSP-72 mRNA and protein. CONCLUSIONS: The results suggest that thermotolerance desensitizes the machinery required for Ca2+ entry. Low [Ca2+]i levels probably result in diminished HSP-72 mRNA production and less HSP-72 synthesis.
Asunto(s)
Aclimatación/fisiología , Equilibrio Ácido-Base/fisiología , Calcio/metabolismo , Supervivencia Celular/fisiología , Proteínas de Choque Térmico/biosíntesis , Calor , Carcinoma de Células Escamosas , Citosol/fisiología , Proteínas del Choque Térmico HSP72 , Humanos , Células Tumorales CultivadasRESUMEN
The Persian Gulf War resulted in injuries of US Coalition personnel by fragments of depleted uranium (DU). Fragments not immediately threatening the health of the individuals were allowed to remain in place, based on long-standing treatment protocols designed for other kinds of metal shrapnel injuries. However, questions were soon raised as to whether this approach is appropriate for a metal with the unique radiological and toxicological properties of DU. The Armed Forces Radiobiology Research Institute (AFRRI) is investigating health effects of embedded fragments of DU to determine whether current surgical fragment removal policies remain appropriate for this metal. These studies employ rodents implanted with DU pellets as well as cultured human cells exposed to DU compounds. Results indicate uranium from implanted DU fragments distributed to tissues far-removed from implantation sites, including bone, kidney, muscle, and liver. Despite levels of uranium in the kidney that were nephrotoxic after acute exposure, no histological or functional kidney toxicity was observed. However, results suggest the need for further studies of long-term health impact, since DU was found to be mutagenic, and it transformed human osteoblast cells to a tumorigenic phenotype. It also altered neurophysiological parameters in rat hippocampus, crossed the placental barrier, and entered fetal tissue. This report summarizes AFRRI's depleted uranium research to date.
Asunto(s)
Agencias Gubernamentales , Uranio/farmacocinética , Uranio/toxicidad , Academias e Institutos , Animales , Línea Celular , Células Cultivadas , Humanos , Riñón/efectos de la radiación , Medicina Militar , Monitoreo de Radiación/métodos , Radiobiología , Ratas , Distribución Tisular , Toxicología/métodos , Estados Unidos , Heridas PenetrantesRESUMEN
In our studies of the health effects of internalized depleted uranium, we developed a simple and rapid light microscopic method to stain specifically intracellular uranium deposits. Using J774 cells, a mouse macrophage line, treated with uranyl nitrate and the pyridylazo dye 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol, uranium uptake by the cells was followed. Specificity of the stain for uranium was accomplished by using masking agents to prevent the interaction of the stain with other metals. Prestaining wash consisting of a mixture of sodium citrate and ethylenediaminetetraacetic acid eliminated staining of metals other than uranium. The staining solution consisted of the pyridylazo dye in borate buffer along with a quaternary ammonium salt, ethylhexadecyldimethylammonium bromide, and the aforementioned sodium citrate/ethylenediaminetetraacetic acid mixture. The buffer was essential for maintaining the pH within the optimum range of 8 to 12, and the quaternary ammonium salt prevented precipitation of the dye. Staining was conducted at room temperature and was complete in 30 min. Staining intensity correlated with both uranyl nitrate concentration and incubation time. Our method provides a simple procedure for detecting intracellular uranium deposits in macrophages.
Asunto(s)
Macrófagos/metabolismo , Coloración y Etiquetado/métodos , Uranio/metabolismo , Animales , Compuestos Azo/química , Células Cultivadas , Citratos/química , Ácido Edético/química , Macrófagos/citología , Macrófagos/efectos de los fármacos , Metales/análisis , Metales/metabolismo , Ratones , Sensibilidad y Especificidad , Citrato de Sodio , Soluciones , Uranio/análisis , Uranio/farmacologíaRESUMEN
N(omega)-nitro-L-arginine (LNNA) inhibits the synthesis of heat shock proteins in animals and cultured cells exposed to heat stress. Heat shock protein synthesis is known to be Ca2+-dependent. In this study, we have characterized the effect of LNNA on [Ca2+]i before and after heat stress in human colon carcinoma T84 cells. In untreated cells incubated in the presence of external Ca2+, the resting [Ca2+]i was 201+/-3 nM. If these cells were exposed to 45 degrees C for 10 min, [Ca2+]i increased by 50+/-2%. Preincubation with LNNA (100 microM) without subsequent heating led to a decrease in [Ca2+]i in a LNNA concentration-dependent manner. Preincubation with LNNA followed by heating increased [Ca2+]i to levels 88+/-5% greater than cells heated without LNNA pretreatment. Incubating cells in medium without external Ca2+ (no heating, no LNNA treatment) lowered resting [Ca2+]i to 115+/-2 nM and greatly reduced the increase in [Ca2+]i observed if cells were heated in the presence of Ca2+, indicating that external Ca2+ plays an important role in the maintenance of [Ca2+]i in T84 cells. With external Ca2+ absent, LNNA pretreatment further reduced [Ca2+]i in unheated cells, and heating failed to enhance [Ca2+]i. We determined (with external Ca2+ present) that the heat-stress induced increase in [Ca2+]i in T84 cells was blocked by dichlorobenzamil, a Na+/Ca2+ exchanger inhibitor, suggesting that the exchanger mediates Ca2+ entry. The median inhibitory concentration (IC50) in cells not treated with LNNA was 0.970+/-0.028 microM. With LNNA pretreatment, the IC50 was 5.099+/-0.107 microM. Heat stress of T84 cells did not affect the binding affinity of the Na+/Ca2+ exchanger for external Ca2+, but it increased the maximal velocity of the exchanger. In unheated cells, preincubation with LNNA decreased the binding affinity of the exchanger for Ca2+, but after heat treatment, both the binding affinity and maximal velocity of the exchanger increased. Our data are consistent with the idea that LNNA affects the activity of the Na+/Ca2+ exchanger. We also determined there are intracellular Ca2+ pools in T84 cells sensitive to thapsigargin, monensin, and ionomycin. Treatment with TMB-8, a blocker of Ca2+ sequestration and mobilization, or ionomycin inhibited the LNNA-induced decrease in [Ca2+]i observed in the absence of external Ca2+, suggesting that LNNA promotes Ca2+ sequestration.
Asunto(s)
Calcio/farmacocinética , Inhibidores Enzimáticos/farmacología , Respuesta al Choque Térmico/efectos de los fármacos , Nitroarginina/farmacología , Amilorida/análogos & derivados , Amilorida/farmacología , Calcio/metabolismo , Compartimento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon , Citosol/metabolismo , Humanos , Ionóforos/farmacología , Monensina/farmacología , Óxido Nítrico Sintasa/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Tapsigargina/farmacología , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimologíaRESUMEN
Corticotropin-releasing factor (CRF) has been shown to attenuate vascular leakage in injured skin, mucous membrane, muscle, lung, and brain. We previously reported that CRF increases cytosolic free calcium concentrations ([Ca2+]i), cellular cAMP, and inositol trisphosphates in human epidermoid A-431 cells. This study identified protein kinase C isoforms in A-431 cells and investigated the effect of CRF on PKC activity and isoform translocation. PKC alpha, beta, gamma, delta, and zeta isoforms were present in cytosolic and membrane fractions, but the epsilon isoform was detected only in the membrane fraction. Exposure of cells to CRF at 420 pM for 1 min increased PKC activity and led to the translocation of PKC alpha and beta isoforms from cytosol to membrane. The translocation was dependent on an increase in [Ca2+]i, because cells treated with 100 microM BAPTA-am (an intracellular Ca2+ chelator), then exposed to CRF, showed neither increases in PKC activity nor translocation of PKC isoforms. This suggests that a CRF-induced increase in [Ca2+]i mediates the increase in PKC activity.
Asunto(s)
Calcio/antagonistas & inhibidores , Hormona Liberadora de Corticotropina/farmacología , Proteína Quinasa C/efectos de los fármacos , Translocación Genética , Animales , Calcio/metabolismo , Carcinoma de Células Escamosas/enzimología , Carcinoma de Células Escamosas/metabolismo , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Humanos , Proteína Quinasa C/metabolismo , Ratas , Células Tumorales CultivadasRESUMEN
Depleted uranium is now widely used in the armor of military vehicles as well as in kinetic-energy penetrators designed to defeat enemy armor. As a result, the potential that personnel will be wounded by depleted uranium fragments has increased. Because toxicities associated with depleted uranium fragments may ultimately require different treatment protocols than those used for traditional metal fragment injuries, a method to rapidly detect the presence of depleted uranium in surgically excised shrapnel fragments is required. By treating the shrapnel fragment with an extracting agent, such as nitric acid, for 5 minutes in an ultrasonic cleaner, sufficient metal is solubilized to allow for colorimetric detection using a pyridylazo dye. Although several metals are capable of being detected under these conditions, the reaction can be made specific for depleted uranium through the use of masking agents such as sodium citrate and ethylenediaminetetraacetic acid. This procedure allows for the rapid (< 15 minutes) extraction and detection of depleted uranium in metal shrapnel fragments.
Asunto(s)
Compuestos Azo , Quelantes , Medicina Militar/métodos , Radioisótopos/análisis , Uranio/análisis , Heridas por Arma de Fuego/diagnóstico , Compuestos Azo/química , Quelantes/química , Colorimetría , Humanos , Espectrofotometría Atómica , Ultrasonido , Estados UnidosRESUMEN
During the Persian Gulf War, soldiers may have inhaled, ingested, and/or experienced wound contamination by depleted uranium (DU), which is used in military projectiles and armor. DU is produced by depleting natural uranium of 234U and 235U during the uranium-enrichment process. Although the long-term effects of significant DU exposures require investigation, many veterans express fears about its impact on health. An assay by which DU exposure can be assessed would not only be a useful research tool, but the information could help mitigate the concerns of exposed individuals. In this study, urine samples from individuals enrolled in the Depleted Uranium Follow-Up Program at the Baltimore Veterans Administration Medical Center were examined for uranium content. Isotopic composition of urine uranium was determined by measuring the 235U/238U ratio, using an inductively coupled plasma mass spectrometer. Using this method, natural and depleted uranium could be readily differentiated. By demonstrating the absence of DU in soldiers who suspect exposure by inhalation or ingestion, the assay should reduce psychological stress in these individuals.
Asunto(s)
Exposición a Riesgos Ambientales/análisis , Uranio/orina , Heridas Penetrantes/orina , Biomarcadores/orina , Humanos , Exposición por Inhalación , Medio Oriente , GuerraRESUMEN
The basal levels of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate (InsP3) in A-431 cells incubated in Na(+)-Hanks' solution were, respectively, 1.23 +/- 0.18, 0.17 +/- 0.03, and 0.69 +/- 0.07% of the total radioactivity in the cell. When cells were heated, InsP3 increased in a temperature-dependent manner related to the duration of heating. The active form of InsP3, inositol 1,4,5-trisphosphate, increased 237 +/- 17% after heating (45 degrees C, 20 min) then returned to baseline within 15 min after the return to 37 degrees C. The heat-induced increase in InsP3 was not observed in the absence of extracellular Ca2+ or with amiloride treatment. Treatment with the nonhydrolyzable GTP analogue 5'-guanylylimidodiphosphate stimulated that component of the InsP3 increase due to G proteins. U-73122, an inhibitor of phospholipase C-mediated processes, blocked the increase in InsP3 resulting from heat exposure. Both pertussis toxin (30 ng/ml, 24 h), an inhibitor of G inhibitory protein, and cholera toxin (1 microgram/ml, 1 h), a stimulator of G stimulatory protein, increased InsP3 in unheated cells, and heating failed to induce a further increase, suggesting that heat activates G proteins. Likewise, 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), 3-isobutyl-1-methylxanthine, Ro 20-1724, or forskolin increased InsP3 in unheated cells, and heat did not cause an additional increase. The InsP3 increase induced by 8-BrcAMP was inhibited by removal of extracellular Ca2+ or treatment with verapamil, suggesting that an influx of extracellular Ca2+ stimulates InsP3 production.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Calcio/metabolismo , Carcinoma de Células Escamosas/metabolismo , Calor , Inositol 1,4,5-Trifosfato/metabolismo , Membranas Intracelulares/metabolismo , Choque/metabolismo , Ácidos/metabolismo , Carcinoma de Células Escamosas/patología , AMP Cíclico/farmacología , Proteínas de Unión al GTP/fisiología , Humanos , Concentración Osmolar , Células Tumorales Cultivadas , Fosfolipasas de Tipo C/farmacologíaRESUMEN
Nucleoplasmin is a thermostable karyophilic protein widely used in nuclear transport studies. An expression vector was constructed that contains a string of 10 histidine residues ligated, in frame, to the amino terminal end of the Xenopus nucleoplasmin gene. The vector was then transformed into Escherichia coli strain BL21(DE3). This strain possesses the gene for T7 RNA polymerase under control of the lacUV5 promoter. The induction of the RNA polymerase and subsequent production of nucleoplasmin occurs after exposure to isopropyl-beta-D-thiogalactopyranoside. The nucleoplasmin, produced in milligram quantities per liter of culture, is then isolated by a rapid purification method that includes metal chelation chromatography to purify the oligohistidine-linked nucleoplasmin. Nuclear transport studies indicate that fluorescently labeled nucleoplasmin is translocated to the nuclear interior of permeabilized V79A03 cells, while nucleoplasmin that lacks a nuclear localization signal (core nucleoplasmin) is not imported. The use of this method to produce nuclear transport-competent nucleoplasmin avoids the lengthy purification procedure used to isolate nucleoplasmin from Xenopus laevis oocytes as well as the cost of purchasing and maintaining a toad colony.
Asunto(s)
Proteínas Nucleares/biosíntesis , Proteínas Nucleares/aislamiento & purificación , Fosfoproteínas , Xenopus laevis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico , Núcleo Celular/metabolismo , Escherichia coli/genética , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Nucleoplasminas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Transformación GenéticaRESUMEN
The radiolytic decomposition products of [35S]-methionine have been used to radiolabel proteins in vitro. The process occurs in a time-, temperature-, and pH-dependent manner. Maximum labeling of bovine serum albumin occurs after a 24 h incubation at 37 degrees C and pH 8.5. Once incorporated, the radiolabel cannot be removed by extended incubation at various temperatures, multiple freeze/thaw cycles, or boiling, indicating that the 35S moiety is covalently attached to the protein. A wide variety of proteins have been radiolabeled. The method is simple to perform and yields radiolabeled proteins of high specific activity.
Asunto(s)
Marcaje Isotópico/métodos , Metionina/química , Proteínas , Radioisótopos de Azufre , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Albúmina Sérica Bovina/química , Temperatura , Factores de TiempoRESUMEN
Extracts were prepared from Micrococcus cryophilus, several strains of Branhamella catarrhalis and Neisseria spp. Esterases, NADP-dependent isocitrate dehydrogenase and malate dehydrogenase activities were assayed after electrophoresis of extracts of polyacrylamide gels. Except for Neisseria perflava and N. sicca which resolved activity bands for the acetate-esterase only, the remaining bacteria exhibited species-specific esterase patterns also for the propionate and butyrate substrates. The multiple esterase patterns from B. catarrhalis ATCC25238 were qualitatively and quantitatively different from those of B. catarrhalis ATCC23246. This finding and other evidence supports a taxonomic shift of the latter to a species level of that genus. The atypical neisserias N. caviae and N. ovis appeared to exhibit an intrageneric specificity in their esterase patterns with those from B. catarrhalis but not to the other Neisseria spp. tested. The malate dehydrogenase patterns from the atypical neisserias and B. catarrhalis ATCC23246 were qualitatively similar; however, the patterns of isocitrate dehydrogenase activity were variable for these species. Micrococcus cryophilus was distinct in its esterase and dehydrogenase bands, strongly suggesting its taxon unrelatedness to the genus Branhammella or the atypical neisserias. Of the enzymes assayed, esterase proved to be the most reliable for taxonomic identifications.