RESUMEN
Insulin-dependent diabetes mellitus (IDDM) is characterized by the T cell-mediated destruction of insulin-producing beta cells. Accordingly, APCs, such as macrophage, have also been shown to be important in the disease process. However, the role(s) of dendritic cells (DCs) that exhibit potent APC function remains undefined in IDDM. Here we demonstrate that DCs derived from nonobese diabetic (NOD) mice, a model for IDDM, are more sensitive to various forms of stimulation compared with those from C57BL/6 and BALB/c mice, resulting in increased IL-12 secretion. This property is a consequence of hyperactivation of NF-kappaB, a transcription factor known to regulate IL-12 gene expression. Specifically, NOD DCs exhibit persistent hyperactivation of both IkappaB kinase and NF-kappaB in response to stimuli, in addition to selective degradation of IkappaBepsilon. Transfection of NOD DCs with a modified form of IkappaBalpha significantly reduced IL-12 secretion, suggesting that hyperactivation of NF-kappaB was in part responsible for increased IL-12 production. An enhanced capacity of NOD DCs to secrete IL-12 would be expected to contribute to the development of pathogenic Th1 (Tc1) cells during the diabetogenic response.
Asunto(s)
Células Dendríticas/enzimología , Células Dendríticas/inmunología , Proteínas I-kappa B , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte Activo de Núcleo Celular/inmunología , Animales , Núcleo Celular/metabolismo , Células Cultivadas , Células Dendríticas/metabolismo , Activación Enzimática/inmunología , Femenino , Quinasa I-kappa B , Interleucina-12/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , FN-kappa B/fisiología , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Proteínas Proto-Oncogénicas/metabolismo , Activación Transcripcional/inmunologíaRESUMEN
We previously demonstrated that administration of plasmid DNAs (pDNAs) encoding IL-4 and a fragment of glutamic acid decarboxylase 65 (GAD65) fused to IgGFc induces GAD65-specific Th2 cells and prevents insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. To assess the general applicability of pDNA vaccination to mediate Ag-specific immune deviation, we examined the immunotherapeutic efficacy of recombinants encoding murine insulin A and B chains fused to IgGFc. Insulin was chosen based on studies demonstrating that administration of insulin or insulin B chain by a variety of strategies prevents IDDM in NOD mice. Surprisingly, young NOD mice receiving i.m. injections of pDNA encoding insulin B chain-IgGFc with or without IL-4 exhibited an accelerated progression of insulitis and developed early diabetes. Exacerbation of IDDM correlated with an increased frequency of IFN-gamma-secreting CD4(+) and CD8(+) T cells in response to insulin B chain-specific peptides compared with untreated mice. In contrast, treatment with pDNAs encoding insulin A chain-IgGFc and IL-4 elicited a low frequency of IL-4-secreting Th cells and had no effect on the progression of IDDM. Vaccination with pDNAs encoding GAD65-IgGFc and IL-4, however, prevented IDDM. These results demonstrate that insulin- and GAD65-specific T cell reactivity induced by pDNA vaccination has distinct effects on the progression of IDDM.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/prevención & control , Glutamato Descarboxilasa/genética , Insulina/genética , Isoenzimas/genética , Plásmidos/inmunología , Vacunas de ADN/inmunología , Envejecimiento/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Diabetes Mellitus Tipo 1/etiología , Diabetes Mellitus Tipo 1/patología , Progresión de la Enfermedad , Epítopos de Linfocito T/inmunología , Femenino , Glutamato Descarboxilasa/administración & dosificación , Glutamato Descarboxilasa/inmunología , Fragmentos Fc de Inmunoglobulinas/administración & dosificación , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/genética , Inyecciones Intramusculares , Insulina/administración & dosificación , Insulina/inmunología , Interleucina-4/administración & dosificación , Interleucina-4/genética , Isoenzimas/administración & dosificación , Isoenzimas/inmunología , Activación de Linfocitos/genética , Recuento de Linfocitos , Ratones , Ratones Endogámicos NOD , Plásmidos/administración & dosificación , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/inmunología , Células TH1/patología , Vacunas de ADN/administración & dosificaciónRESUMEN
In this study, we have investigated the use of plasmid DNA (pDNA) vaccination to elicit Th2 effector cell function in an Ag-specific manner and in turn prevent insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. pDNA recombinants were engineered encoding a secreted fusion protein consisting of a fragment of glutamic acid decarboxylase 65 (GAD65) linked to IgGFc, and IL-4. Intramuscular injection of pDNA encoding GAD65-IgGFc and IL-4 effectively prevented diabetes in NOD mice treated at early or late preclinical stages of IDDM. This protection was GAD65-specific since NOD mice immunized with pDNA encoding hen egg lysozyme-IgGFc and IL-4 continued to develop diabetes. Furthermore, disease prevention correlated with suppression of insulitis and induction of GAD65-specific regulatory Th2 cells. Importantly, GAD65-specific immune deviation was dependent on pDNA-encoded IL-4. In fact, GAD65-specific Th1 cell reactivity was significantly enhanced in animals immunized with pDNA encoding only GAD65-IgGFc. Finally, NOD.IL4(null) mice treated with pDNA encoding GAD65-IgGFc and IL-4 continued to develop diabetes, indicating that endogenous IL-4 was also required for disease prevention. These results demonstrate that pDNA vaccination is an effective strategy to elicit beta cell-specific Th2 regulatory cell function for the purpose of preventing IDDM even at a late stage of disease development.
Asunto(s)
Autoantígenos/inmunología , Epítopos de Linfocito T/inmunología , Inmunosupresores/inmunología , Islotes Pancreáticos/inmunología , Plásmidos/inmunología , Vacunas de ADN/inmunología , Animales , Bovinos , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/prevención & control , Epítopos de Linfocito T/administración & dosificación , Femenino , Glutamato Descarboxilasa/administración & dosificación , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/inmunología , Fragmentos Fc de Inmunoglobulinas/administración & dosificación , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/genética , Inmunosupresores/administración & dosificación , Inmunosupresores/uso terapéutico , Inyecciones Intramusculares , Interleucina-4/administración & dosificación , Interleucina-4/genética , Interleucina-4/fisiología , Islotes Pancreáticos/patología , Isoenzimas/administración & dosificación , Isoenzimas/genética , Isoenzimas/inmunología , Ganglios Linfáticos/enzimología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos NOD , Páncreas/enzimología , Páncreas/inmunología , Páncreas/patología , Plásmidos/administración & dosificación , Células Th2/enzimología , Células Th2/inmunología , Vacunas de ADN/administración & dosificaciónRESUMEN
Extensive documentation shows that macrophage efficiently present antigen to CD 4(+) T-cells in conjunction with the MHC II molecule. Previously, a novel fluorescent probe, FITC-BSA, was developed to analyze intracellular antigen processing and presentation pathways within viable peritoneal murine macrophage. The studies revealed fluorescein's accessibility to antibody binding when associated with peptides bound within the MHC II cleft. To determine if MHC II-fluoresceinated-peptide complexes on the surface of macrophage were also sufficient to stimulate antigen-specific B-cells, nylon wool-purified splenic B-cells from FITC-KLH injected BALB/c mice (H-2(d)) were co-cultured with antigen-pulsed macrophage. B-cell stimulation and antibody production was observed in the presence of FITC-BSA-pulsed macrophage, whereas, macrophage incubated in the presence of unlabeled BSA were not stimulated. Compared with control cells, similar levels of stimulation were detected following depletion of Thy 1.2(+) cells from nylon wool-based spleen cell preparations. Stimulation was inhibited upon preincubation with anti-fluorescein IgG antibodies. Stimulation was not measurable using B-cells derived from the naive mice. The interaction was inhibited upon addition of MHC II specific antibodies and leupeptin, a microbial product that inhibits MHC II-peptide complex formation. Importantly, antibody production was not observed in the presence of antigen-pulsed macrophage from H-2(b) mice. Moreover, B-cell stimulation via this pathway was dependent upon antigen concentration as well as the cell to cell ratio.
Asunto(s)
Presentación de Antígeno , Linfocitos B/inmunología , Macrófagos/inmunología , Animales , Formación de Anticuerpos , Antígenos/administración & dosificación , Linfocitos B/efectos de los fármacos , Bovinos , Membrana Celular/inmunología , Fluoresceína-5-Isotiocianato/análogos & derivados , Colorantes Fluorescentes , Antígenos H-2/metabolismo , Técnicas In Vitro , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Mitomicina/farmacología , Albúmina Sérica BovinaRESUMEN
A fluorescent antigen, FITC10BSA, that is sensitive to several of the biochemical processes involved in antigen processing was constructed. In combination with both flow cytometry and subcellular fractionation, the unique probe provided new details regarding the kinetics and intracellular pathways involved in antigen processing in murine macrophage. These studies suggested that macrophage utilized multiple vesicles as opposed to a few specific organelles for major histocompatibility complex (MHC) type II-peptide loading and transport. Although newly formed MHC II-peptide complexes were detected in cathepsin D-positive, lysosomal associated membrane glycoprotein (LAMP-1)-positive lysosomes, MHC II-peptide loading also occurred in transferrin receptor-positive endosomes. Interestingly, MHC II-fluoresceinated complexes were only observed in transferrin receptor-positive organelles as opposed to MHC II-unlabelled peptide complexes which were detected in traditional early lysosomal compartments. More importantly, MHC II-peptide complexes were monitored in light transferrin receptor-positive fractions following their initial appearance in dense endosomal/lysosomal fractions. Control experiments suggested that these complexes represented intermediates in the process of migrating to the cell surface through a retrograde pathway within the macrophage.
Asunto(s)
Presentación de Antígeno/inmunología , Haptenos/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Macrófagos Peritoneales/inmunología , Péptidos/metabolismo , Animales , Técnicas de Cultivo de Célula , Fraccionamiento Celular , Electroforesis en Gel de Poliacrilamida , Endosomas/inmunología , Fluoresceína-5-Isotiocianato , Lisosomas/inmunología , Ratones , Albúmina Sérica Bovina/inmunología , Transducción de Señal/inmunologíaRESUMEN
A receptor possessing specificity for fluorescein was previously identified on murine macrophage. The goal of the present study was to determine if this receptor influenced MHC II-peptide loading and surface expression of a hapten-protein conjugate within murine macrophage. Although inhibition of fluid-phase pinocytosis had no detectable effect, lower levels of intracellular MHC II-peptide complexes were observed upon inhibition of receptor-mediated endocytosis. Moreover, lower levels of surface expressed MHC II-fluoresceinated peptide complexes were also detected. Following subcellular fractionation experiments, it was revealed that the receptor altered the endocytic trafficking of the antigen within the cell. Namely, degraded antigen and MHC II-peptide complexes were not observed in dense transferrin receptor positive, cathepsin D positive, LAMP-1 positive organelles upon inhibition of the receptor. Previous studies also suggested that this receptor enhanced MHC II-peptide loading by concentrating high levels of antigen to endocytic organelles. The implications of these findings on subsequent development of the immune response were also discussed.
Asunto(s)
Presentación de Antígeno , Haptenos/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Macrófagos Peritoneales/inmunología , Fragmentos de Péptidos/inmunología , Receptores Inmunológicos/inmunología , Amilorida/farmacología , Animales , Biomarcadores , Endocitosis , Fluoresceína-5-Isotiocianato/análogos & derivados , Haptenos/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Fragmentos de Péptidos/metabolismo , Fenilalanina/farmacología , Potasio/fisiología , Receptores Inmunológicos/metabolismo , Albúmina Sérica Bovina , Fracciones Subcelulares/inmunologíaRESUMEN
In a cross-sectional study in Dar es Salaam, Tanzania, we determined the seroprevalence of markers for hepatitis A, B, C and E viruses and examined associated risk markers. Among 403 healthy adults, the seroprevalence of antibodies to hepatitis A virus was 99.0% (95% confidence interval: 97.5-99.7). Prior exposure to hepatitis C and E viruses was rare (hepatitis C: 0.7% (0.2-2.1); hepatitis E: 0.2% (< 0.1-1.4)). The prevalence of all markers of hepatitis B was 70.7% (66.0-75.1). Hepatitis B surface antigen was identified in 6.0% (3.9-8.7) of subjects. Independent predictors of hepatitis B infection identified by logistic regression included older age, male gender, Muslim religion and type of abode. Given the high prevalence of hepatitis B and the low prevalence of hepatitis C, the majority of chronic viral hepatitis is likely to be associated with hepatitis B. Control efforts should focus primarily on hepatitis B.
Asunto(s)
Hepatitis Viral Humana/epidemiología , Hepatitis Viral Humana/virología , Adulto , Distribución por Edad , Biomarcadores/sangre , Estudios Transversales , Femenino , Hepatitis Viral Humana/inmunología , Hepatitis Viral Humana/prevención & control , Humanos , Modelos Logísticos , Masculino , Vigilancia de la Población , Valor Predictivo de las Pruebas , Prevalencia , Factores de Riesgo , Estudios Seroepidemiológicos , Distribución por Sexo , Factores Socioeconómicos , Tanzanía/epidemiologíaRESUMEN
A novel fluorescent hapten-protein conjugate was constructed to monitor the events required for CD 4+ lymphocyte recognition of antigenic proteins. Previous studies utilizing the probe demonstrated that the hapten-protein was localized to an acidic endocytic compartment within the macrophage and that the hapten-protein was sensitive to multiple intracellular events including enzymatic degradation, acidification, and disulfide bond reduction. More importantly, recent experiments indicated that efficient internalization of the probe was dependent upon specific recognition of the hapten. Therefore, the present report addressed the effect of receptor-mediated endocytosis upon the processing of the hapten-protein within murine peritoneal macrophage. These studies determined that the rate of endocytosis was significantly faster than the rate of exocytosis. Specifically, the rate of exocytosis was estimated to be 3.4 x 10(4)s-1 based on a unimolecular rate constant. Although at higher concentrations, a slightly slower rate was observed (1.9 x 10(4)s-1). This study also represented one of the first efforts to measure the intracellular concentration effect typically associated with receptor-mediated endocytosis. Experiments involving a radioactively labeled hapten-protein conjugate revealed that the probe was at 100-fold higher concentration within the endocytic vesicles when compared to the extracellular media. The intracellular mechanism involved in this phenomenon was discussed as well as the implications of these findings upon MHC II-peptide binding.
Asunto(s)
Antígenos/metabolismo , Endocitosis , Exocitosis , Macrófagos Peritoneales/inmunología , Receptores Inmunológicos/fisiología , Albúmina Sérica Bovina/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Compartimento Celular , Fluoresceína-5-Isotiocianato/metabolismo , Colorantes Fluorescentes/metabolismo , Haptenos/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Cinética , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/ultraestructura , Ratones , Ratones Endogámicos BALB C , Albúmina Sérica Bovina/inmunologíaRESUMEN
LLC-PK1, an epithelial cell line derived from the kidney proximal tubule, was used to study the ability of the G protein alpha-subunit, G alpha q, to regulate cell differentiation. A constitutively active mutant protein, alpha qQ209L, was expressed using the LacSwitch-inducible mammalian expression system. Induction of alpha qQ209L expression with isopropyl-beta-D-thiogalactopyranoside (IPTG) enhanced phospholipase C activity maximally by 6- to 7.5-fold. Increasing concentrations of IPTG progressively inhibited the activity of two differentiation markers, Na(+)-dependent hexose transport and alkaline phosphatase activity. Induction of alpha qQ209L expression also caused a change from an epithelial to a spindle-shaped morphology. The effects of alpha qQ209L expression on cell differentiation were similar to those observed with 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment. However, protein kinase C (PKC) levels were downregulated in TPA-treated cells but not in alpha qQ209L-expressing cells, suggesting that the regulation of PKC by G alpha q may be different from regulation by TPA. Interestingly, the PKC inhibitor GF-109203X did not inhibit the effect of IPTG on the development of Na(+)-dependent hexose transport in alpha qQ209L-expressing cells. These data implicate PKC delta and PKC epsilon in the pathway used by G alpha q to block the development of Na(+)-dependent hexose transport in IPTG-treated cells.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Riñón/metabolismo , Fosfatasa Alcalina/antagonistas & inhibidores , Fosfatasa Alcalina/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Diferenciación Celular/fisiología , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Células Epiteliales/metabolismo , Proteínas de Unión al GTP/genética , Hexosas/metabolismo , Riñón/citología , Células LLC-PK1/citología , Mutación , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Sodio/fisiología , Porcinos , Fosfolipasas de Tipo C/metabolismoRESUMEN
Previous studies indicated that fluorescein derivatized bovine serum albumin was an ideal probe to monitor the time-dependent kinetics of antigen processing in the murine macrophage cell line J774. Whereas previous work focused on fluorescence intensity measurements, the present study relied on fluorescence polarization to dissect the local environment of the fluorescent hapten-protein within the endocytic system of the cell. A steady increase in both fluorescence intensity and fluorescence polarization of the cell population was detected for the first 100 min. However, at 100 min, a plateau in both fluorescence intensity and polarization was observed and was followed by a decrease in fluorescence polarization and a corresponding increase in fluorescence intensity. Western blot analyses revealed that the decrease in fluorescence polarization was due to proteolytic degradation of the probe within the cell. Using a combination of in vitro experiments and an additional fluorescent probe, it was determined that the initial increase in fluorescence polarization was due to movement of the probe through a pH gradient within the cell, suggestive of transport through the endocytic system. By combining fluorescence polarization, flow cytometry, and a unique fluorescent enhancement substrate, these studies represented a novel approach for monitoring intracellular trafficking and processing of proteins within macrophages.
Asunto(s)
Presentación de Antígeno , Polarización de Fluorescencia , Macrófagos/inmunología , Animales , Línea Celular , Endopeptidasas/metabolismo , Citometría de Flujo , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/metabolismo , Haptenos/inmunología , Haptenos/metabolismo , Macrófagos/metabolismo , Ratones , Polilisina/inmunología , Polilisina/metabolismo , Albúmina Sérica Bovina/inmunología , Albúmina Sérica Bovina/metabolismo , Espectrometría de FluorescenciaRESUMEN
Two-photon fluorescence lifetime imaging microscopy was used noninvasively to monitor a fluorescent antigen during macrophage-mediated endocytosis, intracellular vacuolar encapsulation, and protease-dependent processing. Fluorescein-conjugated bovine serum albumin (FITC-BSA) served as the soluble exogenous antigen. As a relatively nonfluorescent probe in the native state, the antigen was designed to reflect sequential intracellular antigen processing events through time-dependent changes in fluorescence properties. Using two-photon lifetime imaging microscopy, antigen processing events were monitored continuously for several hours. During this time, the initial fluorescein fluorescence lifetime of 0.5 ns increased to approximately 3.0 ns. Control experiments using fluorescein conjugated poly-L-lysine and poly-D-lysine demonstrated that the increase in fluorescence parameters observed with FITC-BSA were due to intracellular proteolysis since addition of the inert D-isomer did not promote an increase in fluorescence lifetime or intensity. Comparisons of intravacuolar and extracellular FITC-dextran concentration suggested active localization of dextran in the vacuoles by the macrophage. In addition, the kinetics of degradation observed using two-photon microscopy were similar to results obtained on the flow cytometer, thus validating the use of flow cytometry for future studies.
Asunto(s)
Presentación de Antígeno , Macrófagos/inmunología , Microscopía Fluorescente/métodos , Animales , Western Blotting , Células Cultivadas , Dextranos/inmunología , Dextranos/metabolismo , Endocitosis , Endopeptidasas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Procesamiento de Imagen Asistido por Computador , Ratones , Péptidos/análisis , Péptidos/síntesis química , Polilisina/inmunología , Polilisina/metabolismo , Albúmina Sérica Bovina/inmunología , Albúmina Sérica Bovina/metabolismo , Vacuolas/inmunología , Vacuolas/metabolismoRESUMEN
To elucidate time-dependent pathways and mechanisms involved in antigen processing, a fluorescent probe suitable to monitor several steps within this pathway was developed. Previous studies utilizing two-photon fluorescence microscopy with time resolved and intensity imaging demonstrated that the probe, fluorescein derivatized BSA, was localized to the endocytic system and degraded over an extended period of time. However, an additional method, flow cytometry, was required to monitor the kinetics of these intracellular events and to better assess the total cell population. Flow cytometric studies indicated that the antigen entered an acidic intracellular environment consistent with the endocytic system of the macrophage. Additional experiments suggested that minimal proteolytic degradation began 10 min after addition of the antigenic probe while extensive enzymatic degradation did not occur until 180-200 min. Inhibitor studies indicated that degradation of the probe was dependent upon both acidic pH and ATP synthesis as well as all four classes of proteases. Experiments involving specific protease inhibitors also revealed that various classes of proteases were active at different time points throughout the processing of the probe. By combining these results with additional kinetic data, a model for the sequence of events involved in the processing of FITC10BSA was proposed. More importantly, these studies represented some of the first time-dependent kinetic measurements of antigen processing in living cells.
Asunto(s)
Presentación de Antígeno/inmunología , Macrófagos/inmunología , Adenosina Trifosfato/biosíntesis , Álcalis/farmacología , Cloruro de Amonio/farmacología , Animales , Presentación de Antígeno/efectos de los fármacos , Línea Celular/inmunología , Citometría de Flujo , Fluoresceína-5-Isotiocianato , Polarización de Fluorescencia , Cinética , Macrófagos/citología , Ratones , Polímeros , Inhibidores de Proteasas/farmacología , Albúmina Sérica Bovina/inmunología , Albúmina Sérica Bovina/metabolismo , Factores de TiempoRESUMEN
OBJECTIVE: To find optimal conditions for cryopreservation of human spermatozoa with glycerol as cryoprotectant. MATERIALS AND METHODS: Pooled semen from groups of three normal donors was allowed to liquefy for 30 minutes at 37 degrees C. Samples were subjected to a prefreeze equilibration interval of 0, 30, or 60 minutes, with 5% or 10% glycerol, at 0 degrees C, 22 degrees C, or 37 degrees C. Samples were frozen in liquid nitrogen for 24 hours, thawed, and assessed for post-thaw motility. Uptake and metabolism of glycerol were studied using [3H] glycerol. Thin-layer chromatography of ethanol extracts of washed spermatozoa that had been incubated with the labeled glycerol was used to determine metabolic products. RESULTS: Overall best cryopreservation was obtained at 37 degrees C with 5% glycerol and 30 minutes' prefreeze incubation. Labeled glycerol was incorporated into both polar and nonpolar metabolites. Two chromatographic peaks were found associated with the spermatozoa, and one peak was found in the incubation medium.
Asunto(s)
Criopreservación/métodos , Glicerol/metabolismo , Preservación de Semen/métodos , Espermatozoides/metabolismo , Análisis de Varianza , Cromatografía en Capa Delgada , Frío/efectos adversos , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Factores de TiempoRESUMEN
The purpose of this study was to characterize further a unique protein that is a component of the cytoskeleton of crayfish neurons. This protein, referred to as P600, is unique because it is unusually large (Mr greater than 600 kD), and because it has characteristics in common with both mammalian microtubule-associated proteins and neurofilaments. Immunohistochemical techniques have shown that P600 colocalizes with microtubules and is a component of the fibrous side-arms that extend from microtubules (Weaver and Viancour, Brain Res. 544:49, 1991). We have developed a method for obtaining purified P600 by using gel filtration techniques. When viewed by negative staining electron microscopy, P600 obtained by that method produced 11 nm-wide beaded filaments. The number of filaments was strictly related to the P600 concentration in a column fraction. A small amount of P600 consistently copurified with taxol-stabilized microtubules. The proportion copurifying with microtubules was increased by using apyrase to deplete ATP, or by using a nonhydrolyzable ATP analogue to compete with ATP. Immunogold labeling localized P600 near the ends of a subset of the fibrous side-arms extending from endogenous axonal microtubules. Several polyclonal antibodies against mammalian microtubule-associated proteins were tested for P600 labeling on immunoblots, and positive labeling was obtained with an antiserum directed against a region of microtubule-associated protein 1B that has microtubule binding activity. Epitope homology between P600, mammalian microtubule-associated protein 1B, and the mammalian mid-molecular weight neurofilament subunit is discussed in the context of possible evolutionary relationships among these cytoskeletal proteins.
Asunto(s)
Astacoidea/metabolismo , Citoesqueleto/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Neurofilamentos/metabolismo , Neuronas/metabolismo , Animales , Cromatografía en Gel , Citoesqueleto/ultraestructura , Electroforesis en Gel de Poliacrilamida , Endopeptidasas , Glicoproteínas/análisis , Hidrólisis , Immunoblotting , Inmunohistoquímica , Microscopía Electrónica , Proteínas Asociadas a Microtúbulos/química , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/aislamiento & purificación , Proteínas de Neurofilamentos/química , Neuronas/ultraestructura , Fosforilación , Ratas , Ratas Endogámicas F344 , Ratas Sprague-DawleyRESUMEN
We have evaluated the possibility that proteins similar to mammalian neurofilament proteins (NFPs) are present in crustacean neurons. A panel of monoclonal antibodies (mAbs), raised against mammalian NFP, was used to identify candidate proteins. The degree to which these proteins are similar to mammalian NFPs was further evaluated using the following criteria: tissue specificity, recognition by the neurofilament-specific Bodian silver strain, recognition by the intermediate filament-specific Pruss mAb, and insolubility following detergent-extraction. Three candidate polypeptides were identified by mAb screening: a very high molecular weight polypeptide (Mr greater than 300 kDa), a 40 kDa polypeptide, and a group of 4 bands at Mr = 66-84 kDa. Although all of these polypeptides were recognized by one or more anti-NFP mAb, not one of them was found exclusively in neuronal tissue, not one was stained by the NFP-specific Bodian method, and all were soluble under conditions in which mammalian NFPs are insoluble. As a result of this thorough evaluation, we conclude that crayfish neurons do not contain neurofilament-like proteins. Although not closely related to mammalian neurofilaments, the very high molecular weight crayfish polypeptide which was strongly labeled by a commercially available anti-NF-M mAb (clone NN18) during the mAb screening, may be a novel cytoskeletal protein. The evidence for this conclusion comes from immunocytochemical labeling experiments. Indirect immunofluorescence labeling of this protein differentially labeled axons, such that labeling intensity of specific axons was proportional to the relative concentration of cytoskeletal organelles in those axons. Labeling of neuronal cell bodies delineated a fibrous network throughout the cytoplasm, and intensely labeled microtubule-rich regions of cytoplasm which are characteristic of larger neuronal somata. Immunogold labeling and electron microscopic analysis of the distribution of this protein revealed that the NN18-clone antibody bound to an antigen located on microtubule side-arms.
Asunto(s)
Proteínas del Citoesqueleto/análisis , Citoesqueleto/química , Proteínas de Filamentos Intermediarios/análisis , Neuronas/química , Animales , Anticuerpos Monoclonales , Astacoidea , Axones/ultraestructura , Western Blotting , Citoesqueleto/ultraestructura , Técnica del Anticuerpo Fluorescente , Immunoblotting , Sustancias Macromoleculares , Microscopía Inmunoelectrónica , Sistema Nervioso/química , Proteínas de Neurofilamentos , Neuronas/citología , Neuronas/ultraestructura , Ratas , Médula Espinal/químicaRESUMEN
A total of 96 patients were treated for localized carcinoma of the prostate using combined Iodine-125 (125I) implantation and external beam radiotherapy. The implant was tailored to deliver 10,000 rad to the periphery of the prostate. A significant incidence of serious late rectal complications was observed. Positive pelvic nodes were found in 28 percent of the patients. Disease-free survival at seven years was 76 percent for those with negative nodes and 46 percent for patients with positive nodes.
Asunto(s)
Radioisótopos de Yodo/uso terapéutico , Neoplasias de la Próstata/radioterapia , Fístula/etiología , Estudios de Seguimiento , Humanos , Metástasis Linfática , Masculino , Estadificación de Neoplasias , Proctitis/etiología , Enfermedades de la Próstata/etiología , Neoplasias de la Próstata/mortalidad , Neoplasias de la Próstata/patología , Traumatismos por Radiación , Radioterapia/métodos , Fístula Rectal/etiología , Recurrencia , Tasa de Supervivencia , Incontinencia Urinaria/etiologíaRESUMEN
Renal oncocytomas were tested for loss of alleles at loci on the short arm of chromosome 3, a genetic change characteristic of human renal cell carcinoma. Five renal oncocytomas did not show loss of alleles at loci on 3p supporting the view that renal oncocytoma is a distinct form of renal neoplasia.