RESUMEN
During apoptosis, proapoptotic factors are released from mitochondria by as yet undefined mechanisms. Patch-clamping of mitochondria and proteoliposomes formed from mitochondrial outer membranes of mammalian (FL5.12) cells has uncovered a novel ion channel whose activity correlates with onset of apoptosis. The pore diameter inferred from the largest conductance state of this channel is approximately 4 nm, sufficient to allow diffusion of cytochrome c and even larger proteins. The activity of the channel is affected by Bcl-2 family proteins in a manner consistent with their pro- or antiapoptotic properties. Thus, the channel activity correlates with presence of proapoptotic Bax in the mitochondrial outer membrane and is absent in mitochondria from cells overexpressing antiapoptotic Bcl-2. Also, a similar channel activity is found in mitochondrial outer membranes of yeast expressing human Bax. These findings implicate this channel, named mitochondrial apoptosis-induced channel, as a candidate for the outer-membrane pore through which cytochrome c and possibly other factors exit mitochondria during apoptosis.
Asunto(s)
Apoptosis/fisiología , Canales Iónicos/metabolismo , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Humanos , Membranas Intracelulares/metabolismo , Liposomas/química , Liposomas/metabolismo , Técnicas de Placa-Clamp , Porinas/metabolismo , Proteolípidos/química , Proteolípidos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Canales Aniónicos Dependientes del Voltaje , Levaduras/fisiología , Proteína X Asociada a bcl-2RESUMEN
Multiple death signals influence mitochondria during apoptosis, yet the critical initiating event for mitochondrial dysfunction in vivo has been unclear. tBID, the caspase-activated form of a "BH3-domain-only" BCL-2 family member, triggers the homooligomerization of "multidomain" conserved proapoptotic family members BAK or BAX, resulting in the release of cytochrome c from mitochondria. We find that cells lacking both Bax and Bak, but not cells lacking only one of these components, are completely resistant to tBID-induced cytochrome c release and apoptosis. Moreover, doubly deficient cells are resistant to multiple apoptotic stimuli that act through disruption of mitochondrial function: staurosporine, ultraviolet radiation, growth factor deprivation, etoposide, and the endoplasmic reticulum stress stimuli thapsigargin and tunicamycin. Thus, activation of a "multidomain" proapoptotic member, BAX or BAK, appears to be an essential gateway to mitochondrial dysfunction required for cell death in response to diverse stimuli.
Asunto(s)
Apoptosis/fisiología , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/metabolismo , Animales , Anticuerpos , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Biopolímeros , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Grupo Citocromo c/metabolismo , Retículo Endoplásmico/metabolismo , Etopósido/farmacología , Hepatocitos/citología , Hepatocitos/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/genética , Ratones , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética , Transducción de Señal , Estaurosporina/farmacología , Transfección , Rayos Ultravioleta , Proteína Destructora del Antagonista Homólogo bcl-2 , Proteína X Asociada a bcl-2 , Receptor fas/inmunología , Receptor fas/fisiologíaRESUMEN
Caspases are a family of cysteine proteases implicated in the biochemical and morphological changes that occur during apoptosis (programmed cell death). The loop domain of Bcl-2 is cleaved at Asp34 by caspase-3 (CPP32) in vitro, in cells overexpressing caspase-3, and after induction of apoptosis by Fas ligation and interleukin-3 withdrawal. The carboxyl-terminal Bcl-2 cleavage product triggered cell death and accelerated Sindbis virus-induced apoptosis, which was dependent on the BH3 homology and transmembrane domains of Bcl-2. Inhibitor studies indicated that cleavage of Bcl-2 may further activate downstream caspases and contribute to amplification of the caspase cascade. Cleavage-resistant mutants of Bcl-2 had increased protection from interleukin-3 withdrawal and Sindbis virus-induced apoptosis. Thus, cleavage of Bcl-2 by caspases may ensure the inevitability of cell death.
Asunto(s)
Apoptosis , Caspasas , Cisteína Endopeptidasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Células COS , Caspasa 3 , Línea Celular , Inhibidores de Cisteína Proteinasa/farmacología , Activación Enzimática , Humanos , Interleucina-3/fisiología , Células Jurkat , Mutación , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Recombinantes/metabolismo , Virus Sindbis/fisiología , Transfección , Proteína X Asociada a bcl-2 , Receptor fas/fisiologíaRESUMEN
Despite the importance of intestinal stem cells (ISCs) for epithelial maintenance, there is limited understanding of how immune-mediated damage affects ISCs and their niche. We found that stem cell compartment injury is a shared feature of both alloreactive and autoreactive intestinal immunopathology, reducing ISCs and impairing their recovery in T cell-mediated injury models. Although imaging revealed few T cells near the stem cell compartment in healthy mice, donor T cells infiltrating the intestinal mucosa after allogeneic bone marrow transplantation (BMT) primarily localized to the crypt region lamina propria. Further modeling with ex vivo epithelial cultures indicated ISC depletion and impaired human as well as murine organoid survival upon coculture with activated T cells, and screening of effector pathways identified interferon-γ (IFNγ) as a principal mediator of ISC compartment damage. IFNγ induced JAK1- and STAT1-dependent toxicity, initiating a proapoptotic gene expression program and stem cell death. BMT with IFNγ-deficient donor T cells, with recipients lacking the IFNγ receptor (IFNγR) specifically in the intestinal epithelium, and with pharmacologic inhibition of JAK signaling all resulted in protection of the stem cell compartment. In addition, epithelial cultures with Paneth cell-deficient organoids, IFNγR-deficient Paneth cells, IFNγR-deficient ISCs, and purified stem cell colonies all indicated direct targeting of the ISCs that was not dependent on injury to the Paneth cell niche. Dysregulated T cell activation and IFNγ production are thus potent mediators of ISC injury, and blockade of JAK/STAT signaling within target tissue stem cells can prevent this T cell-mediated pathology.
Asunto(s)
Interferón gamma/inmunología , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Células Madre/inmunología , Linfocitos T/inmunología , Animales , Muerte Celular , Mucosa Intestinal/patología , RatonesRESUMEN
Pretreatment of L5178Y murine leukemia cells with uracil arabinoside (ara-U) enhances the cytotoxicity of cytosine arabinoside (ara-C). This effect is mediated by the cytostatic effect of ara-U, which causes a delay of cell progression through S-phase. Consequently, the specific activity of enzymes that peak during S-phase increases, and deoxycytidine kinase increases 3.6-fold over untreated controls. This allows enhanced anabolism of ara-C to nucleotides, as well as increased incorporation into DNA with ultimate synergistic cytotoxicity. It is postulated that the systemic metabolism of high-dose ara-C to sustained high levels of ara-U in patients with acute leukemia may enhance the activity of subsequent doses of ara-C, and thus contribute to a means for pharmacologic self-potentiation, contributing to the unique therapeutic activity of high-dose ara-C.
Asunto(s)
Arabinofuranosil Uracilo/farmacología , Citarabina/uso terapéutico , Leucemia L5178/tratamiento farmacológico , Leucemia Experimental/tratamiento farmacológico , Uridina/análogos & derivados , Animales , Arabinofuranosil Uracilo/sangre , Citarabina/inmunología , Citarabina/metabolismo , Citotoxicidad Inmunológica/efectos de los fármacos , Interacciones Farmacológicas , Humanos , Cinética , Leucemia L5178/sangre , Ensayo de Tumor de Célula MadreRESUMEN
Because the defect in Cl- secretion exhibited by cystic fibrosis (CF) epithelia reflects regulatory rather than conductive abnormalities of an apical membrane Cl- channel, we investigated the role of different regulatory pathways in the activation of Cl- secretion in freshly excised normal and CF nasal epithelia mounted in Ussing chambers. A beta agonist (isoproterenol [ISO]), a Ca2+ ionophore (A23187), and a phorbol ester (PMA) were all effective Cl- secretagogues in normal human nasal epithelia. Agonist addition studies indicated that ISO and PMA but not A23187 may share a common regulatory pathway. In contrast, only A23187 induced Cl- secretion in CF epithelia. Bradykinin raised cytosolic Ca2+ and induced Cl- secretion in both normal and CF tissues, indicating that receptor gated Ca2+ dependent Cl- secretory mechanisms were preserved in CF. The defective Cl- secretory response in CF epithelia to ISO and PMA did not reflect abnormalities in cAMP-dependent (A) and phospholipid Ca2+-dependent (C) kinase activities. We conclude that (a) a Ca2+-sensitive mechanism for regulating Cl- secretion is maintained in CF airway epithelia, and (b) a regulatory pathway shared by two distinct protein kinases is defective in CF, indicating that the CF genetic lesion is not tightly coupled to a single (e.g., cAMP dependent) regulatory mechanism.
Asunto(s)
Calcio/fisiología , Cloruros/metabolismo , Fibrosis Quística/fisiopatología , Mucosa Nasal/metabolismo , Proteína Quinasa C/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Adolescente , Adulto , Amilorida/farmacología , Bradiquinina/farmacología , Calcimicina/farmacología , Niño , AMP Cíclico/biosíntesis , Conductividad Eléctrica , Epitelio/metabolismo , Femenino , Humanos , Isoproterenol/farmacología , Masculino , Mucosa Nasal/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
We previously reported that polyinosinic-polycytidylic acid, a potent interferon inducer, inhibits the growth of B16 malignant melanoma in the C57BL/6 mouse. Two experiments were done to evaluate the effectiveness of interferon in tumor inhibition in vivo. In the first, mice were implanted with melanoma and divided into four groups, according to treatment: interferon preparation; interferon control preparation ("breakthrough fraction"); phosphate-buffered saline control; and murine serum albumin control. Daily, each mouse was given i.p. injections of 200,000 NIH reference units (hereafter called units) of interferon or of one of the control substances. The second experiment was similar to the first, except that bovine serum albumin was an additional control. In both experiments, the average tumor volume in interferon-treated mice was statistically significantly smaller than that of each control group. Mouse interferon preparations also inhibited the multiplication of B16 malignant melanoma cells in culture. This inhibition was statistically significant from interferon levels as low as 5 to as high as 5000 units/ml. The degree of inhibition markedly increased from 5 up to 500 units, the inhibition reaching its maximum at this concentration. The inhibitory effect of interferon was abrogated by anti-murine interferon serum produced in a rabbit. These findings suggest that the in vivo inhibition of the growth of B16 melanoma demonstrated with polyinosinicpolycytidylic acid and with exogenous interferon probably results, at least in part, from a direct effect of interferon on the tumor cells themselves.
Asunto(s)
Interferones/uso terapéutico , Melanoma/terapia , Animales , Adhesión Celular , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Melanoma/patología , Ratones , Neoplasias Experimentales/terapiaRESUMEN
Certain calmodulin (CaM)-dependent protein kinases phosphorylate substrates have been implicated in regulating cellular proliferation. In this study, CaM-dependent phosphorylation has been examined in normal and tumor tissue from rat brain to determine whether differences exist. Using in vitro phosphorylation reactions, we compared endogenous substrates for Ca2+/CaM-dependent protein kinases in rat brain white matter (RBWM), a tissue rich in normal glia, to those of C6 rat glioma cells. A major phosphoprotein having a M(r) of 100,000 was observed in proliferating C6 cells that was not present in RBWM or in nonproliferating cells. Phosphorylation was stimulated by Ca2+ and CaM and inhibited by trifluoperazine. An antibody to elongation factor 2 (EF-2) immunoprecipitated the M(r) 100,000 protein from C6 cells. EF-2 was present in RBWM but was not phosphorylated. Homogenates of RBWM did not phosphorylate exogenous EF-2, which suggested the absence of CaM kinase III activity in normal glial tissue. Furthermore, the addition of purified, exogenous CaM kinase III to homogenates of RBWM resulted in EF-2 phosphorylation. These data demonstrate that a basal level of EF-2 phosphorylation exists in proliferating glioma cells that is markedly diminished or absent in normal glial tissue and is due to the activity of CaM kinase III.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina , Glioma/metabolismo , Neuroglía/metabolismo , Factores de Elongación de Péptidos/metabolismo , Animales , Calcio/farmacología , Calmodulina/metabolismo , Calmodulina/farmacología , División Celular/fisiología , Células Cultivadas , Quinasa del Factor 2 de Elongación , Glioma/enzimología , Masculino , Neuroglía/enzimología , Factor 2 de Elongación Peptídica , Fosforilación , Pruebas de Precipitina , Proteínas Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Trifluoperazina/farmacología , Células Tumorales CultivadasRESUMEN
The relationship between the presence of Campylobacter pylori and esophagitis was studied in patients undergoing paired biopsies of distal esophagus and gastric antrum during esophagogastroduodenoscopy. Biopsy specimens were examined for urease activity and for the presence of C pylori by culture and by histologic examination of hematoxylin-eosin- and Warthin-Starry-stained sections. Sixty-two patients were entered into the study. All esophageal biopsy specimens, regardless of histologic findings, were negative for the presence of C pylori by urease test, culture, and histologic examination. Of 35 patients with normal esophageal biopsy specimens, 11 (31%) had antral specimens that were positive for C pylori, while 11 (41%) of the 27 patients with esophagitis had antral specimens that were positive for the organism. Campylobacter pylori was detected in 14 (70%) of 20 patients with chronic gastritis, in 8 (67%) of 12 patients with endoscopically documented duodenal ulcers and erosions, but in only 3 (33%) of 9 patients with endoscopically defined duodenitis. We conclude that histologic esophagitis is not associated with increased prevalence of either gastric or esophageal C pylori. The well-described association of chronic gastritis and duodenal ulcers with C pylori was present in our study population.
Asunto(s)
Infecciones por Campylobacter/epidemiología , Enfermedades Gastrointestinales/microbiología , Adulto , Anciano , Anciano de 80 o más Años , Campylobacter/enzimología , Úlcera Duodenal/microbiología , Esofagitis/microbiología , Femenino , Gastritis/microbiología , Humanos , Masculino , Persona de Mediana Edad , Ureasa/metabolismoRESUMEN
Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.
Asunto(s)
Apoptosis , Transducción de Señal , Animales , Humanos , Terminología como AsuntoRESUMEN
Studies on the protein specificity of the intestinal antibody response to rotavirus infection have been hampered by lack of antigenically conserved isolated proteins to serve as antigens in immunochemical assays. In this report, the use of an antigenically conserved baculovirus-expressed rotavirus protein (VP4) as a capture antigen in the ELISPOT assay is described. Anti-VP4 antibody-secreting hybridoma cells are used as a test population to show that expressed VP4 as the capture antigen detects numbers of antibody secreting cells comparable to intact rotavirus particles. Hybridoma cells specific for other rotavirus proteins are used to ensure the specificity of the expressed VP4 in the assay. The flexibility and ease of use of a recombinant expressed protein product as a capture antigen in this assay dramatically enhances the ability to quantitate intestinal antibody responses to specific viral proteins.
Asunto(s)
Anticuerpos Antivirales/análisis , Células Productoras de Anticuerpos/inmunología , Antígenos Virales/inmunología , Proteínas de la Cápside , Cápside/inmunología , Ensayo de Inmunoadsorción Enzimática , Immunoblotting , Proteínas Recombinantes de Fusión/inmunología , Rotavirus/inmunología , Animales , Antígenos Virales/biosíntesis , Antígenos Virales/genética , Baculoviridae , Cápside/biosíntesis , Cápside/genética , Hibridomas/inmunología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Simultaneous occurrence of gastrointestinal diverticula at multiple sites in a single patient is unusual. We report a patient with a Zenker's, a mid-esophageal, a gastric, and multiple colonic diverticula. The presence of multifocal gastrointestinal diverticula suggests as one possible etiology congenital weaknesses in the bowel wall, since esophageal manometry and histology of a resected diverticulum did not reveal any abnormalities other than gastroesophageal reflux.
Asunto(s)
Divertículo/diagnóstico por imagen , Enfermedades Gastrointestinales/diagnóstico por imagen , Anciano , Divertículo del Colon/complicaciones , Divertículo del Colon/diagnóstico por imagen , Divertículo Esofágico/complicaciones , Divertículo Esofágico/diagnóstico por imagen , Divertículo Gástrico/complicaciones , Divertículo Gástrico/diagnóstico por imagen , Humanos , Masculino , RadiografíaRESUMEN
The Cumberland County CMHC in Fayetteville, North Carolina, developed a series of computer programs to produce the information that is required by area regional utilization review committees. There is a total of eight programs. The first establishes a master file and prints out all the information that has been gathered on all the clients. The other seven programs retrieve various portions of the data stored in the master file. The system does not require elaborate in-house computer equipment. It is easy to operate and benefits clients by enabling clinical staff to spend less time on documentation and more time on direct treatment.
Asunto(s)
Servicios Comunitarios de Salud Mental , Computadores , Sistemas de Información , Revisión de Utilización de Recursos , Confidencialidad , Atención a la Salud , Humanos , Medicaid , Registros Médicos , North Carolina , Planificación de Atención al Paciente , Estados UnidosRESUMEN
Phytobezoars are frequently associated with partial gastrectomy and are generally considered benign. However, we report two patients who developed a gastric bezoar due to malignancy at the anastomotic site. Development of intragastric bezoar may be an indication of neoplastic growth sufficient to obstruct a small gastric outlet. Consequently, we conclude that endoscopic or surgical evaluation of the anastomotic site is necessary in patients who develop bezoars after gastric surgery.
Asunto(s)
Bezoares/terapia , Gastrectomía , Complicaciones Posoperatorias/terapia , Neoplasias Gástricas/complicaciones , Adulto , Bezoares/etiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/complicaciones , Complicaciones Posoperatorias/etiología , Neoplasias Gástricas/cirugíaRESUMEN
The Ca(2+)-mobilizing action of bradykinin (BK) was investigated in ciliated human nasal epithelial (HNE) cells utilizing fura-2 fluorescence and microspectrofluorimetry. In ciliated cells, basal intracellular Ca2+ concentration ([Ca2+]i) was 123 +/- 3 nM (n = 142). BK caused [Ca2+]i to increase (spike) rapidly (within 6 s) to greater than 550 nM by releasing Ca2+ from intracellular pools. The mean effective dose for the process was 1.5 x 10(-7) M BK. The spike was due to the activation of a beta 2-receptor. The spike was unaffected by inhibitors of either cyclooxygenase or voltagegated Ca2+ channels. After the spike, [Ca2+]i decreased to a plateau level (120-250 nM). This plateau persisted (up to 10 min) until the addition of La3+ (0.3 x 10(-3) M) or until the removal of either extracellular Ca2+ or the agonist. No changes in adenosine 3',5'-cyclic monophosphate (cAMP) levels were detected after BK exposure. Additional studies revealed that indomethacin (10(-6) M), isoproterenol (10(-5) M), forskolin (10(-5) M), and dibutyryl cAMP (1 mM) had no effect on [Ca2+]i in ciliated HNE cells. In summary, these data suggest that 1) BK mediates both Ca2+ release from internal pools and Ca2+ entry into the cytoplasm from the extracellular space, and 2) unlike the response to cultured dog airway epithelia, the release of [Ca2+]i in response to BK does not appear to be mediated by either cyclooxygenase pathway or adenylate cyclase-cAMP systems.
Asunto(s)
Bradiquinina/farmacología , Calcio/metabolismo , Membranas Intracelulares/metabolismo , Mucosa Nasal/metabolismo , Adulto , Bradiquinina/análogos & derivados , Bucladesina/farmacología , Cilios , Colforsina/farmacología , AMP Cíclico/metabolismo , Femenino , Humanos , Indometacina/farmacología , Isoproterenol/farmacología , Masculino , Persona de Mediana Edad , Concentración OsmolarRESUMEN
We used an ELISPOT (enzyme-linked immunosorbent spot) assay to quantitate the long-term rotavirus-specific intestinal antibody response in a murine model. The frequency of murine intestinal antibody-secreting cells (ASCs) was followed for a period of 1 year after a single dose of rhesus rotavirus (10(6) PFU) was administered at 10 days of age. Some animals were boosted at that time with a second dose. One year after infection, virus-specific ASCs declined from acute-phase levels, but they were still present at significant levels (1.32 x 10(4) virus-specific ASCs per 10(6) intestinal mononuclear cells; approximately 17% of the previously reported response at 1 month after infection). A booster dose 1 year after the primary infection produced a 100% increase in virus-specific ASCs but did not restore the response to that of the primary infection.
Asunto(s)
Anticuerpos Antivirales/biosíntesis , Infecciones por Rotavirus/inmunología , Rotavirus/inmunología , Animales , Anticuerpos Antivirales/análisis , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulina A Secretora/análisis , Inmunoglobulina A Secretora/biosíntesis , Intestino Delgado/inmunología , Ratones , Factores de TiempoRESUMEN
Rotavirus is the most important worldwide cause of severe gastroenteritis. Extensive efforts have been devoted to the design of a vaccine that will prevent disease, but development of a more effective vaccine strategy may require progress in the understanding of the mucosal immune response to replicating viral antigens. In this article, we report the characterization of the intestinal antibody response of a murine model to heterologous infection with the rhesus rotavirus vaccine strain. We have adapted the enzyme-linked immunospot assay to measure this response without the difficulties associated with measurement of antibodies in intestinal contents or the artifacts associated with culturing of lymphocytes. The predominant response in terms of antibody-secreting cells (ASC) is seen in the small intestine lamina propria, which can be measured within 4 days of infection, peaks 3 weeks after infection, and remains near that level for longer than 8 weeks. The magnitude of the immunoglobulin A (IgA) cell response is approximately 10 times greater than the intestinal IgG cell response, and IgM cells are rare. Virus-specific ASC constitute approximately 50% of all ASC in the gut at the peak of the virus-specific response. This response is considerably greater than responses to nonreplicating mucosal antigens measured by similar techniques. Enteral infection engenders minimal virus-specific ASC response in the spleen. Rhesus rotavirus-specific enzyme-linked immunosorbent assay and neutralization assays of serum and intestinal contents did not correlate with virus-specific ASC response.
Asunto(s)
Mucosa Intestinal/inmunología , Intestino Delgado/inmunología , Infecciones por Rotavirus/inmunología , Animales , Formación de Anticuerpos , Células Productoras de Anticuerpos/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Vida Libre de Gérmenes , Inmunoglobulina A/biosíntesis , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Leucocitos Mononucleares/inmunología , Mesenterio/inmunología , Ratones , Pruebas de Neutralización , Ganglios Linfáticos Agregados/inmunología , Embarazo , Bazo/inmunología , Factores de TiempoRESUMEN
Bcl-x(L), an antiapoptotic Bcl-2 family member, is postulated to function at multiple stages in the cell death pathway. The possibility that Bcl-x(L) inhibits cell death at a late (postmitochondrial) step in the death pathway is supported by this report of a novel apoptosis inhibitor, Aven, which binds to both Bcl-x(L) and the caspase regulator, Apaf-1. Identified in a yeast two-hybrid screen, Aven is broadly expressed and is conserved in other mammalian species. Only those mutants of Bcl-x(L)that retain their antiapoptotic activity are capable of binding Aven. Aven interferes with the ability of Apaf-1 to self-associate, suggesting that Aven impairs Apaf-1-mediated activation of caspases. Consistent with this idea, Aven inhibited the proteolytic activation of caspases in a cell-free extract and suppressed apoptosis induced by Apaf-1 plus caspase-9. Thus, Aven represents a new class of cell death regulator.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Caspasas/metabolismo , Proteínas de la Membrana , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis/fisiología , Proteínas Reguladoras de la Apoptosis , Factor Apoptótico 1 Activador de Proteasas , Proteínas Portadoras/química , Proteínas Portadoras/genética , Caspasa 9 , Línea Celular , Dimerización , Activación Enzimática , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Cuaternaria de Proteína , Proteínas/química , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Distribución Tisular , Transfección , Proteína bcl-XRESUMEN
The Bcl-2-related protein, Bcl-XL, has been shown to block apoptosis induced by a variety of stimuli and to be a stronger protector against apoptosis than Bcl-2 under certain circumstances. Using site-specific mutagenesis, we show here that the amino-acid residues critical for protection of cells by Bcl-XL against Sindbis virus-induced apoptosis are clustered within the Bcl-2-homology regions 1 and 2 (BH1 and BH2 regions). The residues necessary for Bcl-XL function are not identical to those required for Bcl-2 function. Although it has been suggested that heterodimerization between Bcl-XL and Bax is essential for the anti-death activity of Bcl-XL (refs 7,8), our results suggest that the interaction with Bax is not required for Bcl-XL to exert its death-repressing activity. Specific mutations that disrupt the ability of Bcl-XL to interact with Bax or Bak still preserve 70-80% of the anti-death activity of wild-type Bcl-XL.
Asunto(s)
Apoptosis/fisiología , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas/fisiología , Secuencia de Aminoácidos , Apoptosis/genética , Sitios de Unión , Biopolímeros , Línea Celular , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Proto-Oncogénicas/genética , Virus Sindbis/fisiología , Transfección , Proteína X Asociada a bcl-2 , Proteína bcl-XRESUMEN
We have adapted a murine model of heterotypic rotavirus infection for the purpose of evaluating the intestinal antibody response to an infection that mimics human vaccination. Neonatal mice were infected with the rhesus rotavirus (RRV). The enzyme-linked immunospot assay was used in order to avoid common artifacts in the quantitation of intestinal immune responses inherent in measurements of luminal or serum immunoglobulins and to obtain easily quantifiable data in a flexible and convenient format. Functionally active lymphocytes were harvested from the spleen, small intestinal lamina propria, Peyer's patches, and mesenteric lymph nodes and processed into single-cell suspensions. Antibody-secreting cells (ASC) were quantitated from 5 to 50 days after infection for total, RRV-specific, baculovirus-expressed VP4-specific, and single-shell RRV-specific ASC secreting either immunoglobulin G (IgG), IgM, or IgA. The response to VP4 constituted less than 1.5% of the total virus-specific response, which was located almost exclusively in the gut and was 90% IgA. Intestinal ASC were directed overwhelmingly toward proteins incorporated in the single-shell particle, predominantly VP2 and VP6. We conclude that the antibody response to VP4, thought to be the site of the important neutralization sites conserved among several rotavirus serotypes, is an extremely small portion of the overall antibody response in the intestinal tract.