RESUMEN
The in vitro effect of seminal vesicle protein IV (SV-IV) on the cytotoxic activity of human natural or acquired cellular immunity has been investigated by standard immunological procedures, a (51)Cr-release cytotoxicity assay, and labeled-ligand binding experiments. The data obtained demonstrate that: (1) fluoresceinated or [(125)I]-labeled SV-IV binds specifically to the surface of human purified non-adherent mononuclear cells (NA-MNC); (2) SV-IV suppresses the cytotoxicity of natural killer (NK) cells against K562 target cells, that of IL-2-stimulated NK (LAK) cells against DAUDI target cells, and that of VEL antigen-sensitized cytotoxic T lymphocytes (CTLs) against VEL target cells; (3) treatment of K562 target cells alone with SV-IV decreases their susceptibility to NK-induced lysis. These findings indicate that the protein SV-IV has a marked in vitro inhibitory effect on NK, LAK and CTL cytotoxicity, providing a better understanding of its immune regulatory functions.
Asunto(s)
Células Asesinas Activadas por Linfocinas/inmunología , Células Asesinas Naturales/inmunología , Leucocitos Mononucleares/inmunología , Proteínas de Secreción de la Vesícula Seminal/metabolismo , Linfocitos T Citotóxicos/inmunología , Línea Celular Tumoral , Citotoxicidad Inmunológica , Humanos , Inmunidad Celular , Células K562 , Células Asesinas Activadas por Linfocinas/metabolismo , Células Asesinas Naturales/metabolismo , Leucocitos Mononucleares/metabolismo , Proteínas de Secreción de la Vesícula Seminal/inmunología , Proteínas de Secreción de la Vesícula Seminal/aislamiento & purificación , Linfocitos T Citotóxicos/metabolismoRESUMEN
A number of studies performed in our laboratory and elsewhere, showed that resveratrol is able to prevent carcinogenesis and to impair tumor growth and progression. In order to provide additional information on the pleiotropic effects of resveratrol on malignant cells, the present study was performed to test the in vitro influence of the compound on the growth and TLMA of HT-29 and WiDr human colon cancer cell lines. The results confirmed that resveratrol has a direct, dose dependent, inhibitory effect on cell proliferation in both lines. In addition, for the first time, relatively high concentrations of this compound were found to be able to substantially down-regulate telomerase activity. These preliminary results further support the potential role of resveratrol in chemoprevention/chemotherapy of human colon tumor cells and provide the rational basis for novel strategies in cancer control.
Asunto(s)
Anticarcinógenos/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/enzimología , Neoplasias del Colon/patología , Proteínas de Unión al ADN/metabolismo , Estilbenos/farmacología , Telomerasa/metabolismo , Inhibidores de la Angiogénesis/farmacología , Línea Celular Tumoral , Humanos , ResveratrolRESUMEN
Nerve growth factor (NGF) belongs by sequence homology to the neurotrophins, a family of proteins binding the same p75 receptor and closely related members of the Trk family of receptor tyrosine kinases. Fundamental in the vertebrate nervous system, neurotrophin signals have also been suggested as essential for relatively complex nervous systems occurring in invertebrate species that live longer than Caenorhabditis elegans and Drosophila melanogaster. Mammalian neurotrophins have been found to influence invertebrate neuronal growth. However, there are only a few data on the presence of molecules related to neurotrophin signalling components in invertebrates. Our studies provide evidence that analogues of neurotrophins and neurotrophin receptors are expressed in Eisenia foetida earthworms. In particular, NGF-like and Trk-like immunoreactive proteins are both expressed in the nervous system, whereas p75-like positivity identifies tubular structures associated with dorsal pores that are involved in the earthworm response to mechanical irritation or stress.
Asunto(s)
Factor de Crecimiento Nervioso/metabolismo , Oligoquetos/metabolismo , Péptidos/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Animales , Western Blotting , Sistema Nervioso Central/anatomía & histología , Sistema Nervioso Central/química , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/ultraestructura , Inmunohistoquímica , Microscopía Confocal , Microscopía Electrónica , Factor de Crecimiento Nervioso/análisis , Factor de Crecimiento Nervioso/inmunología , Oligoquetos/anatomía & histología , Oligoquetos/química , Oligoquetos/inmunología , Especificidad de Órganos , Péptidos/análisis , Péptidos/inmunología , Receptores de Factor de Crecimiento Nervioso/inmunologíaRESUMEN
Dopaminergic neurons and their projection-systems are important in some fundamental human activities like locomotion, feeding and sex, essential for survival and procreation, and are relevant to pathologies like Parkinson's disease and drug abuse. Three main dopaminergic projection-systems, namely the nigrostriatal, mesocortical and mesolimbic pathways are the major targets of the neuropharmacological actions of psychomotor stimulants such as cocaine and amphetamine. Studies on knockout mice for dopamine or its receptors provide substantial information but fail to reveal the role of individual dopaminergic projection-systems. Mutant animals with defects specific to one or more projection-systems might be useful for studying the role of individual dopaminergic projection-systems. We propose the weaver mutant mouse, with a defective nigrostriatal dopaminergic projection-system and dopamine depletion in the dorsal striatum but with intact mesocorticolimbic projection-systems, as a suitable model to study the role of individual dopaminergic systems in diverse biological processes including Parkinson's disease and drug abuse.
Asunto(s)
Dopamina/fisiología , Ratones Mutantes Neurológicos/fisiología , Sistema Nervioso/crecimiento & desarrollo , Trastornos Relacionados con Sustancias/fisiopatología , Transmisión Sináptica/fisiología , Animales , RatonesRESUMEN
Resveratrol (3,5,4'-trihydroxystilbene), a polyphenol found in grapes and grape products such as red wine, has been reported to exhibit a wide range of biological and pharmacological activities both in vitro and in vivo. Because many of the biological activities of resveratrol, like the inhibition of cyclooxygenase, induction of CD95 signaling-dependent apoptosis, effects on cell division cycle and modulation of NF-kB activation, suggest a possible effect on the immune system, we evaluated the in vitro effects of resveratrol in three immune response models: i) development of cytokine-producing CD4+ and CD8+ T cells induced by stimulation of peripheral blood mononuclear cells (PBMC) with anti-CD3/anti-CD28; ii) specific antigen-induced generation of cytotoxic T lymphocytes; iii) natural killer (NK) activity of PBMC. The results showed that in vitro exposure to resveratrol produces a biphasic effect on the anti-CD3/anti-CD28-induced development of both IFN-gamma- IL2- and IL4-producing CD8+ and CD4+ T cells, with stimulation at low resveratrol concentrations and suppression at high concentrations. Similarly, the compound was found to induce a significant enhancement at low concentrations and suppression at high concentrations of both CTL and NK cell cytotoxic activity. On the whole, the results of the study indicate that resveratrol modulates several human immune cell functions and suggest that this activity may be related to its effects on cytokine production by both CD4+ and CD8+ T cells.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Inmunidad Celular/efectos de los fármacos , Monocitos/efectos de los fármacos , Estilbenos/farmacología , Anticuerpos Monoclonales/farmacología , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/metabolismo , Citocinas/análisis , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Humanos , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Activación de Linfocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Resveratrol , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismoRESUMEN
One of the proposed mechanisms for multidrug resistance relies on the ability of resistant tumor cells to efficiently promote glutathione S-transferase (GST)-catalyzed GSH conjugation of the antitumor drug. This type of conjugation, observed in several families of drugs, has never been documented satisfactorily for anthracyclines. Adriamycin-resistant human breast cancer MCF-7/DOX cells, presenting a comparable GSH concentration, but a 14-fold increase of the GST P1-1 activity relative to the sensitive MCF-7 cells, have been treated with adriamycin in the presence of verapamil, an inhibitor of the 170 P-glycoprotein (P-gp) drug transport protein, and scrutinized for any production of GSH-adriamycin conjugates. HPLC analysis of cell content and culture broths have shown unequivocally that no GSH conjugates are present either inside the cell or in the culture broth. The only anthracycline present inside the cells after 24 hr of incubation was > 98% pure adriamycin. Confocal laser scanning microscopic observation showed that in MCF-7/DOX cells adriamycin was localized mostly in the Golgi apparatus rather than in the nucleus, the preferred site of accumulation for sensitive MCF-7 cells. These findings rule out GSH conjugation or any other significant biochemical transformation as the basis for resistance to adriamycin and as a ground for the anomalous localization of the drug in the cell. Adriamycin, daunomycin, and menogaril did not undergo meaningful conjugation to GSH in the presence of GST P1-1 at pH 7.2. Indeed, their synthetic C(7)-aglycon-GSH conjugates exerted a strong inhibitory effect on GST P1-1, with K(i) at 25 degrees in the 1-2 microM range, scarcely dependent on their stereochemistry at C(7).
Asunto(s)
Neoplasias de la Mama/metabolismo , Doxorrubicina/metabolismo , Glutatión Transferasa/metabolismo , Glutatión/metabolismo , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/farmacología , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Neoplasias de la Mama/enzimología , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Femenino , Glutatión Transferasa/antagonistas & inhibidores , Humanos , Células Tumorales CultivadasRESUMEN
Adriamycin (ADR), one of the major antitumor agents used for the clinical treatment of a wide variety of human cancers and its glutathione(GSH)-conjugated adduct, ADRIGLU, induced apoptosis in K562 erythroleukemia and TVM-A12 clone 2 melanoma human cell lines. We have previously reported that ADR has nuclear localization and that ADRIGLU localizes exclusively in the cytoplasm. During ADR or ADRIGLU treatment, significant depletion of the cell energy state, demonstrated by a reduction in high-energy phosphates (ATP and GTP) and a decrease in energy charge potential (ECP), were recorded between 2 hours and 24 hours, by HPLC analysis. Transmission electron microscopy also revealed that between 2 hours and 24 hours of ADR or ADRIGLU treatment, mitochondria underwent evident morphological changes, from an initial "high amplitude swelling state" to a "shrinkage state" and finally, in early apoptotic cells, to an "abnormal shrinkage state", in which a marked accumulation of pycnotic mitochondria was also observed. Confocal microscopic analysis, using the potential-sensitive dye JC-1, showed that inhibition of cell energy metabolism was preceded by a rapid decrease in mitochondrial transmembrane potential (delta psi m). With the progression of exposure time, the early depolarization of the mitochondrial membrane was followed by a transient reversion to normal delta psi m until, in apoptotic cells, almost all mitochondrial subpopulations appeared to be hyperpolarized. Our results indicated that mitochondria are actively involved in anthracycline-induced programmed cell death, suggesting a novel mechanism that may be common to all forms of apoptosis.
Asunto(s)
Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Doxorrubicina/farmacología , Glutatión/farmacología , Mitocondrias/efectos de los fármacos , Apoptosis/fisiología , Bencimidazoles , Carbocianinas , Metabolismo Energético/efectos de los fármacos , Colorantes Fluorescentes , Humanos , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/fisiología , Células K562/efectos de los fármacos , Células K562/metabolismo , Células K562/patología , Células K562/ultraestructura , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Melanoma/patología , Melanoma/ultraestructura , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Microscopía Confocal , Microscopía Electrónica , Mitocondrias/fisiología , Mitocondrias/ultraestructuraRESUMEN
The influence of chronic maternal morphine on the parvalbumin immunoreactive patterns in developing mouse brain was studied. Female Swiss mice were administered daily saline or morphine (30 or 60 mg/kg) for a period of 7 days before mating, gestation, and 21 days postpartum. Their pups were sacrificed on postnatal day 18 and the brains were examined histologically and immunohistochemically for parvalbumin-positive neurons. Histological observations revealed no significant changes in the cell number of the morphine-exposed neonatal forebrain, whereas the number of parvalbumin-positive neurons increased in layers II-IV of the parietal cortex I. Moreover, the number of parvalbumin-positive dendrites increased remarkably in the cingulate and parietal I cortices of the morphine-exposed neonates, indicating the region-specific increase in the PV immunoreactive profiles. These results are consistent with the key roles played by the above brain regions in the altered behavioral patterns of the maternally addicted neonates, such as impaired somatosensory and cognitive performances. The mechanism of morphine action on parvalbumin expression in neonatal mouse brain is not evident, but alterations in the expression patterns of parvalbumin in specific regions of the developing brain might be one of the cellular mechanisms by which addictive drugs modify the functional aspects of the developing CNS.
Asunto(s)
Encéfalo/fisiología , Morfina/farmacología , Neuronas/fisiología , Parvalbúminas/metabolismo , Efectos Tardíos de la Exposición Prenatal , Envejecimiento , Animales , Animales Recién Nacidos , Encéfalo/efectos de los fármacos , Encéfalo/crecimiento & desarrollo , Dendritas/efectos de los fármacos , Dendritas/fisiología , Femenino , Giro del Cíngulo/fisiología , Inmunohistoquímica , Lactancia , Ratones , Neuronas/citología , Neuronas/efectos de los fármacos , Lóbulo Parietal/fisiología , Embarazo , Corteza Somatosensorial/fisiologíaRESUMEN
Human Raji B lymphoid cells after exposure for 64 h to a 1 mT (rms) 50 Hz sinusoidal magnetic field showed a reorganization of membrane and cytoskeletal components. Atomic force microscopy in air revealed several modifications in 80% of the exposed cells, such as loss of microvilli-like structures followed by progressive appearance of membrane introflections. This change in plasma membrane morphology was also accompanied by a different actin distribution, as detected by phalloidin fluorescence. These observations support our previous hypothesis that electric and magnetic fields may modify the plasma membrane structure.
Asunto(s)
Linfocitos B/efectos de la radiación , Membrana Celular/efectos de la radiación , Citoesqueleto/efectos de la radiación , Campos Electromagnéticos , Linfocitos B/patología , Linfocitos B/ultraestructura , Linfoma de Burkitt , Membrana Celular/ultraestructura , Citoesqueleto/ultraestructura , Humanos , Microscopía de Fuerza Atómica/métodos , Microscopía Confocal/métodos , Células Tumorales CultivadasRESUMEN
We have analysed the effects of cocaine, administered to mice during the in vivo differentiation of effector T cells stimulated by antigen (influenza virus) recognition, on the frequency of IL-2-, IL-4- and interferon-gamma (IFN-gamma)-expressing CD4+ and CD8+ T cells. Each animal was injected intraperitoneally with 10 mg/kg of cocaine 6, 24, 48 and 72 h after immunization with A/PR8 influenza virus (PR8). This enabled the determination of the pharmacological effects of cocaine on T cells during the initial step of the immune response, which is characterized by the production of large amounts of immunoregulatory cytokines. The distribution of IL-2-, IL-4- and IFN-gamma-producing CD4+ and CD8+ T cells was assayed on unseparated PR8-immune spleen cells, obtained from mice treated with cocaine or vehicle, and restimulated in vitro with UV-inactivated PR8 virus. The frequency of T cells singly or co-expressing the above three cytokines was determined at single-cell level by simultaneous flow cytometric analysis of intracellular cytokines and surface antigen expression. In parallel, the levels of IL-2, IL-4 and IFN-gamma in the culture supernatants were quantified by ELISA. The results showed that cocaine, administered during the in vivo virus-induced differentiation of T cells, caused an increase of both the frequencies of CD8+ T cells singly and co-expressing IL-2 and IFN-gamma and the levels of these cytokines in virus-restimulated spleen cell culture supernatants, compared with those of untreated controls. In contrast, no effect was found on IL-4-positive CD8+ T cells and on IL-2-, IFN-gamma- and IL-4-positive CD4+ T cells. Our findings suggest that the immunomodulatory effects of cocaine may be due to the up-regulation of the production of IL-2 and IFN-gamma by CD8+ T cells with a type 0 cytokine profile.
Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Cocaína/farmacología , Citocinas/biosíntesis , Orthomyxoviridae/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Medios de Cultivo Condicionados/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Inmunofenotipificación , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Interleucina-4/biosíntesis , Masculino , Ratones , Ratones Endogámicos BALB C , Bazo/citología , Bazo/efectos de los fármacos , Bazo/metabolismo , Vacunas Virales/inmunologíaRESUMEN
PURPOSE: To investigate the production and repair of lipid oxidative damage in two human cell lines exposed to acute and fractionated dose of ionizing radiation. Radiation dose was in the range from 0.1 to 44 Gy. MATERIALS AND METHODS: K562 and HL60 human cell lines have been used, 24 and 96 h after seeding. Membrane lipid oxidative damage has been detected by the measurement of the fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH), its polarization value and the conjugated dienes concentration. The modification of DPH decay has been previously reported to be directly related to the lipid hydroperoxide concentration. RESULTS: A modification of the DPH decay has been observed as a linear function of the logarithm of the radiation dose and only when the irradiation was performed in the presence of oxygen. The amount of the damage is related to the time after the cell medium change. By exposing the cells to fractionated radiation doses for several days (10 cGy day(-1)), the oxidative damage has been found to be cumulative. After a single acute dose, evidence of repair of the lipid oxidative damage was not obtained. CONCLUSIONS: Following a previously developed method, the membrane damage was attributed to the production of hydroperoxide residues in the lipid acyl chains with the consequence of water penetration into the external portion of the bilayer, from the aqueous environment to the position of hydroperoxides. This damage is not repaired. The results obtained by measuring the DPH fluorescence decay have been compared with those obtained using other current optical and biochemical methods. None of these techniques could detect membrane oxidative damage at doses < 10 Gy. Finally, the different sensitivity of 'young' and 'old' cells to the oxidative damage can be related to different cholesterol concentrations.
Asunto(s)
Peroxidación de Lípido , Lípidos de la Membrana/efectos de la radiación , Membrana Celular/efectos de la radiación , Difenilhexatrieno/metabolismo , Relación Dosis-Respuesta en la Radiación , Colorantes Fluorescentes/metabolismo , Células HL-60/efectos de la radiación , Humanos , Peróxido de Hidrógeno/metabolismo , Células K562/efectos de la radiación , Lípidos de la Membrana/metabolismo , Oxidación-ReducciónRESUMEN
We have studied the patterns of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) co-expression displayed by individual splenic CD4+ and CD8+ T cells in response to influenza virus immunization. Unseparated spleen cells obtained from mice intraperitoneally (i.p.) injected with A/PR8 (H1N1) influenza virus (PR8) were cultured for 24 hr in the presence of ultraviolet-inactivated PR8. As controls, cultures of both naive spleen cells stimulated with PR8 or of immune cells lacking the inactivated virus were used. The frequencies of CD4+ and CD8+ T cells expressing IL-2, IL-4 and IFN-gamma were determined by three-colour flow cytometric analysis of fixed and saponin-permeabilized cells fluorescent-stained for either CD4 or CD8 surface molecules and for one of the following combinations of two intracellular cytokines: IL-2/IL-4, IL-2/IFN-gamma and IL-4/IFN-gamma. The results showed that immunization with influenza virus induces in both CD4+ and CD8+ T cells a heterogeneity of cytokine response patterns that do not follow the type 1/type 2 polarized response model, but with substantial differences between the two populations. In fact, the analysis of the phenotypes of virus-immune CD8+ T cells revealed similar significant proportions of cells either expressing any one of the three cytokines or co-expressing combinations of them (i.e. IL-4/IL-2, IL-4/IFN-gamma and IL-2/IFN-gamma), whereas immune CD4+ T cells were seen to express almost exclusively a single cytokine per cell. The observed patterns of cytokine production suggest that influenza virus immunization induces the expression of a type 0 cytokine pattern at both population and single cell levels in CD8+ T cells and exclusively at the population level in CD4+ T cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Citocinas/metabolismo , Vacunas contra la Influenza/inmunología , Animales , Técnicas de Cultivo de Célula , Citometría de Flujo , Inmunización , Virus de la Influenza A/inmunología , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Interleucina-4/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Bazo/inmunologíaRESUMEN
Long-term exposure to high altitude has been reported to impair cognitive functions, possibly resulting in an increased risk of mountain accidents. To assess the modification of cognitive functions during acclimatisation to altitude, 17 climbers were studied at 5350 m a.s.l. by means of a neuropsychological learning test. The results clearly show that by extending the period spent at elevations above 5350 m to more than 15 days, the response to a memory task was significantly enhanced. The improvements resulting from acclimatisation were more evident in the organisation of information than in information storage. We suggest that inappropriate acclimatisation has a detrimental effect on cognitive functions and the resulting impairment may particularly affect the more demanding technical tasks.
Asunto(s)
Aclimatación/fisiología , Altitud , Aprendizaje/fisiología , Adulto , Análisis de Varianza , Femenino , Humanos , Masculino , Memoria/fisiología , Pruebas Neuropsicológicas , Factores de TiempoRESUMEN
The accumulation of adriamycin (ADR), daunomycin (DAUNO) and their glutathione (GSH)-conjugates, recently obtained by anaerobic reaction of the parent anthracyclines with reduced GSH, was examined in drug-sensitive and multidrug resistant (MDR) cell lines using confocal laser scanning microscopy. In all drug-sensitive lines used (TVM-A12 and TVM-A197 human melanoma cells, K562 lymphoblastoid cells and MCF-7 human breast cancer cells) ADR and DAUNO were mostly located in the nuclei, while their GSH-conjugates were found only in the cytoplasm, predominantly in the Golgi region. On the contrary, in MDR MCF-7/Dox cells, both conjugated and non conjugated anthracyclines gave fluorescence only in the cytoplasm, mostly in the Golgi region, the intensity of the fluorescence being stronger in cells pretreated with verapamil. Viability assay showed that the GSH-conjugate are significantly less cytotoxic than the parent anthracyclines in sensitive cells and showed the same scarce cytotoxicity in MDR MCF-7/Dox cells. These results demonstrate that conjugation of anthracycline antitumor drugs with GSH prevents their access to the nucleus and decreases their cytotoxicity. Furthermore, the observations on MCF-7/Dox suggest that GSH-conjugation of anthracycline might occur in resistant cells and can be in part responsible for the MDR in breast cancer cells.
Asunto(s)
Antibióticos Antineoplásicos/metabolismo , Citoplasma/metabolismo , Resistencia a Antineoplásicos , Glutatión/metabolismo , Antibióticos Antineoplásicos/farmacología , Resistencia a Múltiples Medicamentos , Aparato de Golgi/metabolismo , Humanos , Microscopía Confocal , Células Tumorales CultivadasRESUMEN
The generalized polarization function of the fluorescent probe 2-dimethylamino-6-lauroylnaphthalene has been used to evaluate the lipid dynamics in Friend erythroleukemia cell membrane. The values of this function varied during the culture growth cycle, showing decreased lipid dynamics 24-48 h from the cell seeding. When the cycle occurred in a solenoid producing a magnetic field of 70 microT at 50 Hz in addition to the 45 microT DC of the earth (short-term 4-day exposure), the membrane lipid dynamics during this same time-period decreased by about 10% (P < .04). After long-term (184 days) or extremely long-term (395 days) exposure of the cells to the magnetic field, little additional variation in the membrane lipid dynamics was observed, suggesting an adaptation phenomenon. A variation of membrane lipid dynamics was also observed due to in vitro cell differentiation (P < .02). Nevertheless, the exposure of both undifferentiating and differentiating cells to a highly attenuated magnetic field in a magnetically shielded room (20 nT DC plus 2.5 pT AC) did not induce any modification of membrane lipid dynamics.
Asunto(s)
Campos Electromagnéticos , Ambiente Controlado , Lípidos de la Membrana/metabolismo , Animales , Ciclo Celular/efectos de la radiación , Diferenciación Celular/efectos de la radiación , División Celular/efectos de la radiación , Membrana Celular/metabolismo , Membrana Celular/efectos de la radiación , Conductividad Eléctrica , Campos Electromagnéticos/efectos adversos , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patología , Lípidos de la Membrana/efectos de la radiación , Ratones , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
The treatment of human peripheral blood mononuclear cells (PBMC) with micromolar concentrations of SV-IV, a major protein secreted from the rat seminal vesicle epithelium, promotes in this cell population a marked cytotoxic activity against the Raji lymphoblastoid cell line. This activity is apparently due to cell-to-cell contact interactions. The expression of HLA DR on Raji cells has a modulatory effect on the SV-IV-induced cytotoxic activity. The experimental evidence strongly suggests that the cytotoxic effector cells are functionally activated NK cells.
Asunto(s)
Citotoxicidad Inmunológica , Linfocitos/inmunología , Proteínas/inmunología , Proteínas de Secreción de la Vesícula Seminal , Vesículas Seminales/metabolismo , Animales , Epitelio/metabolismo , Humanos , Masculino , Proteínas/metabolismo , Ratas , Células Tumorales CultivadasRESUMEN
The solution structure of the complex between 4', 6-diamidino-2-phenylindole (DAPI) and DNA oligomer (d(CGATCG))2 at a 2:1 drug/duplex ratio has been characterized by combined use of proton one- and two-dimensional NMR spectroscopy, molecular mechanics, and molecular dynamics computations. Intermolecular nuclear Overhauser effects (NOEs), DNA structure perturbations, and resonance shifts induced by binding provide evidence that DAPI interacts with DNA hexamer by two different binding mechanisms, in fast exchange on the NMR time scale, without any significant distortion of the B-type conformation of DNA hexamer. The results indicate that the ligand binds into the minor groove of the central 5'-ATC-3' region of the hexamer and on the outside of the oligomer by a pi,pi-stacking interaction with the terminal C1:G6 base pairs. A model for both binding mechanisms that accounts for all experimental data was generated by molecular mechanics and dynamics calculations based on experimental NOEs. In the minor groove binding, N2 amino group of G2 precludes a deep insertion of phenyl ring of DAPI into the groove. Position and orientation of the drug in the external stacking interaction resemble those suggested for intercalation of DAPI between C:G base pairs.
Asunto(s)
ADN/química , Indoles/química , Sustancias Intercalantes/química , Oligodesoxirribonucleótidos/química , Composición de Base , Secuencia de Bases , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/síntesis químicaRESUMEN
The cytokine responses exerted by virus-primed spleen T cells upon in vitro restimulation were studied. Spleen cells obtained from mice injected intraperitoneally with A/PR8 (H1N1) influenza virus (PR8) were restimulated in vitro with UV-inactivated PR8 virus. The percentage of both CD4+ and CD8+ T cells producing IL2, IL4, or IFN-gamma was assayed at the single cell level by flow cytometric analysis of intracytoplasmic cytokine content. In parallel, the levels of the different cytokines in spleen cell culture supernatants were quantitated by enzyme linked immunosorbent assay. The results showed that in vitro virus restimulation of immune spleen cells induced the concurrent increase, in both CD4+ and CD8+ T cells, of the frequency of IL2-, IFN-gamma-, and IL4-producing cells. The frequency of IFN-gamma-producing T cells was found to be significantly higher in CD8+ T cells. Significant levels of the three cytokines were also detected in the culture supernatants. These data suggest that both CD4+ and CD8+ T cells play an important role in cytokine response to virus infection and that the synthesis and secretion of antiviral and regulatory cytokines is not mutually exclusive either between or within the two T cell subsets. The results of the experiments also indicated that the virus restimulation did not induce a dominant type 1 or type 2 cytokine response.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Virus de la Influenza A/inmunología , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Interleucina-4/biosíntesis , Animales , Células Cultivadas , Embrión de Pollo , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Bazo/citologíaRESUMEN
The measurement of fluorescence lifetime distribution of 1,6-diphenyl-1,3,5-hexatriene is used for the detection of oxidative damage produced in phospholipid membranes by ionizing radiation. The recently developed method is based on the linear relationship between the width of the probe lifetime distribution and the logarithm of the dose. The molecular origin of the damage resides in the production of hydroperoxide residues at the level of acyl chains double bonds. A chemiluminescence assay was used to quantitate the amount of produced hydroperoxides. Consequences of the produced damages include an increased disorder in the upper portion of the bilayer, accompanied by the penetration of water molecules. In the presence of the physiological concentration of cholesterol in phopholipid bilayers, the amount of hydroperoxides produced by ionizing radiation is dramatically reduced. The packing effect of cholesterol in phopholipid bilayers is well recognized, as well as its influence on the reduction of water concentration in the bilayer. The dramatic reduction of hydroperoxides concentration observed when irradiation is performed in the presence of cholesterol probably originates from a steric hindrance to the radical chain reaction through the unsaturated lipids due to the presence of cholesterol.
Asunto(s)
Colesterol/farmacología , Membrana Dobles de Lípidos/metabolismo , Membrana Dobles de Lípidos/efectos de la radiación , Estrés Oxidativo , Fosfolípidos/metabolismo , Difenilhexatrieno/química , Fluorescencia , Radicales Libres , Peróxido de Hidrógeno/metabolismo , Mediciones Luminiscentes , Oxidación-Reducción , FosfatidilcolinasRESUMEN
A commercially available selective group A streptococcal agar (ssA) was evaluated for the recovery of group A streptococci (GAS) in comparison with recovery from simultaneous cultures on conventional sheep blood agar (SBA). Both sets of plates were incubated in air, 5% CO2, and anaerobically for 48 h, with a first reading taken at 24 h. A total of 402 (67.0%) GAS were isolated from the 600 specimens that were submitted. Recovery of GAS was significantly greater after 48 h of incubation than after 24 h of incubation for each medium-atmosphere combination. After 48 h of incubation, the sensitivities of GAS detection obtained by each culture technique were as follows: ssA-anaerobic atmosphere, 98.5%; SBA-anaerobic atmosphere, 89.5%; ssA-CO2 atmosphere, 88.0%; SBA-air, 86.5%; SBA-CO2 atmosphere, 82.0%; and ssA-air, 74.6%. There were no cultures positive in air or CO2 which were not positive anaerobically on either medium. The increased sensitivity of detecting positive GAS cultures when incubation was done in an ssA-anaerobic atmosphere for 48 h uncovered patients truly infected with the organisms.