RESUMEN
Reduction of a mesoscopic dynamical theory to equilibrium thermodynamics brings to the latter theory the fundamental thermodynamic relation (i.e. entropy as a function of the thermodynamic state variables). The reduction is made by following the mesoscopic time evolution to its conclusion, i.e. to fixed points at which the time evolution ceases to continue. The approach to fixed points is driven by entropy, that, if evaluated at the fixed points, becomes the thermodynamic entropy. Since the fixed points are parametrized by the thermodynamic state variables (by constants of motion), the thermodynamic entropy arises as a function of the thermodynamic state variables and thus the final outcome of the reduction is the fundamental thermodynamic relation. This reduction process extends also to reductions in which the reduced theory still involves the time evolution (e.g. reduction of kinetic theory to hydrodynamics). The essence of the extension is the replacement of the mesoscopic time evolution of the state variables with the corresponding mesoscopic time evolution of the vector field (i.e. of the fluxes). The fixed point in this flux time evolution is the vector field generating the reduced mesoscopic time evolution. The flux-entropy driving the flux time evolution becomes, if evaluated at the fixed point, the flux fundamental thermodynamic relation in the reduced dynamical theory. We show that the flux-entropy is a potential related to the entropy production. This article is part of the theme issue 'Fundamental aspects of nonequilibrium thermodynamics'.
RESUMEN
BACKGROUND: Recent data obtained in mouse models have initiated a controversy whether basophils are the key antigen-presenting cells (APCs) in allergy. Here, we investigate whether basophils are of importance for the presentation of allergen and the induction of T cell proliferation in allergic patients. METHODS: T cells, basophils, and APCs depleted of basophils were purified from allergic patients. Co-culture systems based on purified major allergens were established to study allergen-specific T cell responses using proliferation assays. RESULTS: Only co-cultures of T cells with APCs depleted of basophils but not with basophils proliferated in response to allergen. Even addition of IL-3 to T cell-basophil co-cultures failed to induce allergen-specific T cell proliferation. CONCLUSIONS: Our data demonstrate by classical in vitro proliferation assays that basophils are not key antigen-presenting cells that promote T cell proliferation in secondary immune responses to allergen in allergic patients.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Basófilos/inmunología , Hipersensibilidad/inmunología , Alérgenos/inmunología , Antígenos de Plantas/inmunología , Basófilos/metabolismo , Epítopos/inmunología , Humanos , Inmunofenotipificación , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Colchicine administered to adult rats at a dosage of 0.5 mg/100 g of body weight effected a disorganization of the Golgi apparatus in pancreatic acinar cells. The results obtained after various periods of treatment (10 min to 6 h) showed (a) changes in all components of the Golgi complex, and (b) occurrence of large vacuoles that predominated in cytoplasmic areas outside the Golgi region. The alterations in Golgi stacks concerned elements of the proximal and distal side: (a) accumulation of transport vesicles, (b) formation of small, polymorphic secretion granules, and (c) alterations in the cytochemical localization of enzymes and reaction product after osmification. Transport vesicles accumulated and accompanied short, dilated cisternae, which lack mostly the reaction products of thiamine pyrophosphatase, inosine diphosphatase, and acid phosphatase, and osmium deposits after prolonged osmification. After 4 to 6 h of treatment, accumulated transport vesicles occupied extensive cellular areas; stacked cisternae were not demonstrable in these regions. The changes on the distal Golgi side included GERL elements: condensing vacuoles were diminished; they were substituted by small, polymorphic zymogen granules, which appeared to be formed by distal Golgi cisternae and by rigid lamellae. Unusually extended coated regions covered condensing vacuoles, rigid lamellae, and polymorphic secretion granules. A cytochemical distinction between Golgi components and GERL was possible neither in controls nor after colchicine treatment. The cytochemical alterations in Golgi components were demonstrable 20-30 min following administration of colchicine; at 45 min, initial morphological changes--augmentation of transport vesicles and formation of polymorphic zymogen granules--became apparent. 20 min after administration of colchicine, conspicuous groups of large vacuoles occurred. They were located mostly in distinct fields between cisternae of the endoplasmic reticulum, and were accompanied by small osmium--reactive vesicles. Stacked cisternae were not demonstrable in these fields. Vacuoles and vesicles were devoid of reaction products of thiamine pyrophosphatase, inosine diphosphatase, and acid phosphatase. The results provide evidence that formation of stacked Golgi cisternae is impaired after colchicine treatment. The colchicine--induced disintegration of the Golgi complex suggests a regulatory function of microtubules in the organization of the Golgi apparatus.
Asunto(s)
Ácido Anhídrido Hidrolasas , Colchicina/farmacología , Aparato de Golgi/efectos de los fármacos , Páncreas/efectos de los fármacos , Fosfatasa Ácida/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Gránulos Citoplasmáticos/ultraestructura , Femenino , Aparato de Golgi/ultraestructura , Microscopía Electrónica , Páncreas/citología , Monoéster Fosfórico Hidrolasas/metabolismo , Ratas , Tiamina Pirofosfatasa/metabolismo , Factores de TiempoRESUMEN
Chromatin condensation paralleled by DNA fragmentation is one of the most important criteria which are used to identify apoptotic cells. However, comparable changes are also observed in interphase nuclei which have been treated with cell extracts from mitotic cells. In this respect it is known that in mitosis, the lamina structure is broken down as a result of lamin solubilization and it is possible that a similar process is happening in apoptotic cells. The experiments described in this study have used confluent cultures of an embryonic fibroblast cell line which can be induced to undergo either apoptosis at low serum conditions or mitosis. Solubilization of lamin A+B was analyzed by immunoblotting and indirect immunofluorescence. These studies showed that in mitotic cells lamina breakdown is accompanied by lamin solubilization. In apoptotic cells, a small amount of lamin is solubilized before the onset of apoptosis, thereafter, chromatin condensation is accompanied by degradation of lamin A+B to a 46-kD fragment. Analysis of cellular lysates by probing blots with anti-PSTAIR followed by anti-phosphotyrosine showed that in contrast to mitosis, dephosphorylation on tyrosine residues did not occur in apoptotic cells. At all timepoints after the onset of apoptosis there was no significant increase in the activation of p34cdc2 as determined in the histone H1 kinase assay. Coinduction of apoptosis and mitosis after release of cells from aphidicolin block showed that apoptosis could be induced in parallel with S-phase. The sudden breakdown of chromatin structure may be the result of detachment of the chromatin loops from their anchorage at the nuclear matrix, as bands of 50 kbp and corresponding multimers were detectable by field inversion gel electrophoresis (FIGE). In apoptotic cells all of the DNA was fragmented, but only 14% of the DNA was smaller than 50 kbp. DNA strand breaks were detected at the periphery of the condensed chromatin by in situ tailing (ISTAIL). Chromatin condensation during apoptosis appears to be due to a rapid proteolysis of nuclear matrix proteins which does not involve the p34cdc2 kinase.
Asunto(s)
Apoptosis/fisiología , Proteína Quinasa CDC2/metabolismo , Cromatina/fisiología , ADN/metabolismo , Proteínas Nucleares/metabolismo , Animales , Línea Celular , Cromatina/ultraestructura , ADN/análisis , Embrión de Mamíferos , Activación Enzimática , Genes ras , Cinética , Lamina Tipo A , Laminas , Mitosis , Papillomaviridae/genética , Hipófisis , Protamina Quinasa/metabolismo , Ratas , Factores de Tiempo , TransfecciónRESUMEN
The combination of the capabilities of light microscopical techniques with the power of resolution of electron microscopy along with technical advances has led to a gradual decline of the gap between classical light and electron microscopy. Among the correlative techniques using the synergistic opportunities, photooxidation methods have been established as valuable tools for visualizing cell structures at both light and electron microscopic level. Fluorescent dyes are used to oxidize the substrate diaminobenzidine, which in its oxidized state forms fine granular precipitates. Stained with osmium, the diaminobenzidine precipitates are well discernible in the electron microscope, thus labelling and defining the cellular structures, which at light microscopy level are recorded by fluorescent probes. The underlying photooxidation reaction is based on the excitation of free oxygen radicals that form upon illumination of fluorochromes; this is a central step in the procedure, which mainly influences the success of the method. This article summarizes basic steps of the technology and progresses, shows efforts and elaborated pathways, and focuses on methodical solutions as to the applicability of different fluorochromes, as well as conditions for fine structural localizations of the reaction products.
Asunto(s)
Microscopía Electrónica/métodos , Microscopía/métodos , Coloración y Etiquetado/métodos , 3,3'-Diaminobencidina/metabolismo , Colorantes Fluorescentes/metabolismo , Osmio/metabolismo , Oxidación-ReducciónRESUMEN
The aim of this work was to modify the method of Ladd and Buttler (1972), by substituting Tris-HCl buffer (pH 8.52) with demineralized water (DEMI H(2)O), in order to assess its suitability for measurement of casein-protease activity at pH levels close to those of real soil in H(2)O. Measurements were undertaken over a range of incubation temperatures from 3 to 49 degrees C. Testing was performed on one organic soil and two different mineral soils. The substitution of Tris-HCl buffer by DEMI H(2)O at 49 degrees C decreased casein-protease activity to 67.25% in mineral soil and to 53.76% in organic soil. With decreasing temperature casein-protease activity decreased the most in organic soil, i.e., 0.07% of original its value at 3 degrees C. The incubation period was extended to maximally 336 h at 3 degrees C to totally obtain >10.0% of L-tyrosine equivalents released at optimum or close to optimum temperature and pH conditions. The Q(10) values of casein-protease activity measured after substituting Tris-HCl buffer with DEMI H(2)O were unexpectedly high. Between the temperatures of 3 and 49 degrees C Q(10) ranged from 3.46 to 4.25, whereas between 3 and 25 degrees C Q(10) ranged from 6.78 to 11.08. Therefore, the modified method of Ladd and Buttler (1972) presented can be used for measurement of soil casein-protease activity under pH conditions close to that of real soil pH and at an averaged soil temperatures measured in the field. This modification makes possible an expression of soil casein-protease activity potential - when being combined with measurements of casein-protease activity under optimum or close to optimum temperature and pH conditions, if high concentration of casein is present.
Asunto(s)
Caseínas/química , Péptido Hidrolasas/química , Suelo/análisis , Temperatura , Trometamina/química , Agua/química , Tampones (Química) , Concentración de Iones de Hidrógeno , Péptido Hidrolasas/análisis , Sensibilidad y EspecificidadRESUMEN
Adult T-cell leukemia is associated with high levels of neopterin, released in large amounts from human macrophages upon stimulation with interferon-gamma. Recent data suggested a potential role of neopterin-derivatives in oxygen radical-mediated processes, and evidence accumulates that oxidative stress is involved in the pathogenesis of viral diseases. We now report that increased concentrations of 7,8-dihydroneopterin may lead to enhanced apoptosis and disturbance of the redox-balance of human leukemic Jurkat T cells. Additionally, we demonstrate that 7,8-dihydroneopterin and hydrogen peroxide activate the type 1 human T-cell leukemia virus (HTLV-1) long terminal repeat (LTR). Furthermore, we found that the activity of the HTLV-1 transactivator protein Tax is amplified by an elevated concentration of 7,8-dihydroneopterin. Tax did not significantly augment 7,8-dihydroneopterin mediated apoptosis. Based on our data we propose that 7,8-dihydroneopterin may be involved in the progression to higher stages of HTLV-1 associated disease.
Asunto(s)
Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Virus Linfotrópico T Tipo 1 Humano/genética , Pteridinas/farmacología , Secuencias Repetitivas de Ácidos Nucleicos/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Apoptosis/fisiología , Productos del Gen tax/genética , Productos del Gen tax/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Neopterin/análogos & derivados , Oxidación-Reducción , Transfección , Células Tumorales CultivadasRESUMEN
The tumor suppressor p53 has been implicated in gamma irradiation-induced apoptosis. To investigate possible consequences of wild-type p53 loss in leukemia, we studied the effect of a single dose of gamma irradiation upon p53-deficient human T-ALL (acute lymphoblastic leukemia) CCRF - CEM cells. Exposure to 3 - 96 Gy caused p53-independent cell death in a dose and time-dependent fashion. By electron microscopic and other criteria, this cell death was classified as apoptosis. At low to intermediate levels of irradiation, apoptosis was preceded by accumulation of cells in the G2/M phase of the cell division cycle. Expression of Bcl-2 and Bax were not detectably altered after irradiation. Expression of the temperature sensitive mouse p53 V135 mutant induced apoptosis on its own but only slightly increased the sensitivity of CCRF - CEM cells to gamma irradiation. Thus, in these, and perhaps other leukemia cells, a p53- and Bcl-2/Bax-independent mechanism is operative that efficiently senses irradiation effects and translates this signal into arrest in the G2/M phase of the cell cycle and subsequent apoptosis.
Asunto(s)
Apoptosis , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína p53 Supresora de Tumor/fisiología , Ciclo Celular , División Celular , Fase G2 , Rayos gamma , Mitosis , Leucemia-Linfoma Linfoblástico de Células Precursoras , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteína X Asociada a bcl-2RESUMEN
We have determined the subcellular distribution of fucosyl residues in rat duodenal absorptive enterocytes and goblet cells, using the binding affinity of the lectin I of Ulex europaeus (UEA I). In absorptive enterocytes, UEA I-lectin gold complexes were detected at the brush border and at the basolateral plasma membrane; pits of the plasma membrane were labeled, as were small vesicles, multivesicular bodies, lysosomes, and the Golgi apparatus. In the Golgi stacks, about half of the cisternae showed gold marker particles: accessible fucosyl residues were sparse in the cis subcompartment, the cismost cisterna mostly remaining negative; more intense label was found in medial cisternae; reactions were concentrated in the trans and transmost Golgi subcompartments. Cisternae, tubules and vesicles located at the trans Golgi side were the most constantly and intensely stained Golgi elements. In goblet cells, mucin granules and trans Golgi cisternae were labeled. Rarely, UEA I-gold bound to cisternae of the medial subcompartment; the cis subcompartment remained unstained. In part, UEA I-gold particles were restricted to dilated portions of the transmost Golgi cisterna and to secretory granules.
Asunto(s)
Residuos de Medicamentos/análisis , Duodeno/citología , Fucosa/análisis , Absorción Intestinal , Lectinas de Plantas , Animales , Duodeno/metabolismo , Duodeno/ultraestructura , Femenino , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Inmunohistoquímica , Lectinas/metabolismo , Microscopía Electrónica , Ratas , Ratas Endogámicas , Coloración y Etiquetado/métodosRESUMEN
The distribution in the Golgi apparatus of binding sites for the galactose-specific Ricinus communis I lectin (RCA I) was studied in differently specialized cells, including goblet cells and absorptive enterocytes of the rat small intestine as well as acinar cells of the rat embryonic pancreas and submandibular gland. For the purpose of localizing the binding reactions, a pre-embedment method using horseradish peroxidase for electron microscopic visualization, and a post-embedding technique making use of the colloidal gold system were employed. The reactions obtained, localizing cell constituents which contain saccharides with terminal or internal beta-D-galactosyl residues, labeled diverse Golgi subcompartments. The goblet cells showed intense RCA I staining of the cisternae of the trans side of the Golgi stacks. The reaction was weak in the medial cisternae and the cis side of the stacks mostly was devoid of label. In the absorptive cells, in addition to the RCA I reaction of trans Golgi elements, binding sites for this lectin were concentrated in the stacks' medial section. In the embryonic acinar cells, accessible galactosyl residues were either confined to the trans and/or medial cisternae, or distributed across elements of all the stacked saccules. In the latter stacks, the reactions mostly were weak in the cis cisternae and increased in intensity towards the trans side. As regards the respective labeling patterns, similar percentages were calculated for the early and late stages of development: they were approximately 62% for the pattern which showed RCA I label limited to trans/medial cisternae and approximately 38% for the "cis-to-trans"-distributed RCA I reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Duodeno/inmunología , Aparato de Golgi/inmunología , Lectinas/metabolismo , Páncreas/embriología , Lectinas de Plantas , Receptores Mitogénicos/metabolismo , Animales , Duodeno/ultraestructura , Femenino , Aparato de Golgi/ultraestructura , Microscopía Electrónica , Páncreas/citología , Páncreas/inmunología , Embarazo , RatasRESUMEN
Ultrastructural localization of thiamine pyrophosphatase (TTP) and acid phosphatase (AcPase) activity was performed on jejunal absorptive cells of rats pretreated with the antimicrotubular agent colchicine and of control animals. Demonstration of TPP activity showed that most of the dislocated Golgi stacks after colchicine application lacked positively staining cisternae of the mature side. This cytochemical finding is in agreement with the morphologically demonstrable changes of the Golgi stacks resulting in a loss of polarity and give evidence for a colchicine-induced deficiency of the Golgi apparatus. The cytochemical localization of AcPase activity showed deposits of reaction product over lysosomes and GERL and demonstrated a dislocation of GERL occurring concomitantly with the changes of the Golgi apparatus. The antimicrotubular effect of colchicine is well documented; thus the morphological and cytochemical changes of the Golgi apparatus and of GERL might be due to a disturbed microtubular function after application of this agent suggesting an influence of microtubules in the maintenance of the integrity of these organelles. This hypothesis includes the possibility of an involvement of microtubules in formation and differentiation of Golgi stacks and GERL as well as a kind of "skeletal"function being responsible for their characteristic structure and fashion.
Asunto(s)
Fosfatasa Ácida/metabolismo , Colchicina/farmacología , Aparato de Golgi/efectos de los fármacos , Yeyuno/efectos de los fármacos , Pirofosfatasas/metabolismo , Tiamina Pirofosfatasa/metabolismo , Animales , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/enzimología , Femenino , Aparato de Golgi/enzimología , Yeyuno/enzimología , Lisosomas/efectos de los fármacos , Lisosomas/enzimología , Microscopía Electrónica , RatasRESUMEN
In the small intestine cell differentiation from immature crypt cells to mature absorptive cells localized along the villi is accompanied by alterations in the organization of the trans Golgi side. In immature crypt cells the transmost Golgi cisterna is usually located closely adjacent to the other cisternae thus being a component of the stack. Concomitantly with cell differentiation the transmost cisterna of an increasing number of Golgi stacks sets off from the other cisternae being then located at various distances to the stacks. This transmost cisterna has, as in several other cell types, been interpreted as "GERL" (Golgi associated endoplasmic reticulum lysosomes [20, 28]) and thus, has been postulated to represent a specialized region of the endoplasmic reticulum. Our results, however, have shown that the cytochemical staining pattern which has been used as a basis for the differentiation of GERL from Golgi components is not present in crypt cells nor in mature absorptive cells of the proximal small intestine: identical cisternae react for thiamine pyrophosphatase, inosine diphosphatase, and acid phosphatase. Thiamine pyrophosphatase and inosine diphosphatase--enzymes characteristic for Golgi cisternae--are apparent over transmost cisternae defined as GERL, too, and in addition, acid phosphatase--postulated as GERL-marker--is demonstrable over stacked Golgi cisternae. This overlapping cytochemical reaction, as well as the alterations during cell differentiation, indicate that those structures which have been described as GERL are to be interpreted as Golgi components rather than as endoplasmic reticulum. On the other hand, endoplasmic reticulum is a constant component of the trans Golgi face in undifferentiated crypt-base cells and in maturing cells of the crypt-top region. From its localization closely adjacent to trans Golgi cisternae it may be termed "Golgi-associated endoplasmic reticulum"; however, these cisternae of endoplasmic reticulum are constantly devoid of acid phosphatase. No indications exist for continuities with the thiamine pyrophosphatase-, inosine diphosphatase-, and acid phosphatase-positive transmost Golgi cisternae, and for an engagement in production of lysosomes.
Asunto(s)
Ácido Anhídrido Hidrolasas , Aparato de Golgi/ultraestructura , Intestino Delgado/ultraestructura , Fosfatasa Ácida/metabolismo , Animales , Diferenciación Celular , Retículo Endoplásmico/ultraestructura , Femenino , Aparato de Golgi/enzimología , Histocitoquímica , Intestino Delgado/enzimología , Microscopía Electrónica , Monoéster Fosfórico Hidrolasas/metabolismo , Ratas , Tiamina Pirofosfatasa/metabolismoRESUMEN
Atopy is a genetically determined disorder that affects 10%-20% of the population. Many symptoms of patients with atopy (allergic rhinitis, conjunctivitis, asthma, and anaphylaxis) result from events occurring after crosslinking of cell-bound IgE by per se innocuous environmental antigens. The frequently raised hypothesis that autosensitization can also be a pathogenetic factor in atopy, gained support by our recent demonstration of IgE antibodies against human proteins in atopic dermatitis patients. To unravel the molecular nature of IgE-defined autoantigens, we used serum IgE from atopic dermatitis patients to screen a human epithelial cDNA expression library. One of the cDNA-encoding IgE-reactive products contained 1501 bp of a 2274 bp open-reading frame finally identified by sequence analysis of two additional cDNA clones resulting from oligonucleotide screening. The IgE-defined autoantigen, designated Hom s 1, exhibited an almost complete sequence identity with a recently described antigen recognized by cytotoxic T cells of a squamous cell carcinoma patient. Purified recombinant Hom s 1 specifically bound IgE from patients with severe atopy. When used as immunogen in rabbits, recombinant Hom s 1 gave rise to an anti-serum that reacted with a cytoplasmic protein exhibiting a broad cellular and tissue reactivity (skin, lung >> gastrointestinal tract >> muscle, brain) and identified a 55 kDa protein in blotted serum IgE preparations. The attractive possibility remains that the Hom s 1-triggered IgE response contributes to the events resulting in allergic tissue inflammation. If so, the respective recombinant molecule may serve as a paradigmatic tool for the diagnosis and treatment of patients with "intrinsic" atopy.
Asunto(s)
Alérgenos/inmunología , Alérgenos/aislamiento & purificación , Autoantígenos/química , Autoantígenos/aislamiento & purificación , Dermatitis Atópica/inmunología , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Alérgenos/química , Secuencia de Aminoácidos , Antígenos de Plantas , Autoantígenos/sangre , Secuencia de Bases , Proteínas de Unión al Calcio , Proteínas de Transporte de Catión , Dermatitis Atópica/sangre , Epítopos , Humanos , Proteínas de Transporte de Membrana Mitocondrial , Datos de Secuencia Molecular , Fracciones Subcelulares/química , Fracciones Subcelulares/inmunologíaRESUMEN
We have previously demonstrated that soluble amyloid beta protein (A beta) induces IL-2 receptor expression and proliferation in peripheral T cells from young and old healthy individuals, but not from patients with Alzheimer's disease (AD). It seemed of interest to examine how the immune system would react upon stimulation with A beta in its aggregated form. It was the aim of this study to define interactions between the spontaneously aggregating A beta (25-35) and antigen-presenting cells. Human dendritic cells (DC), propagated from the peripheral blood of young healthy individuals, were incubated with A beta (25-35) and its effects on DC survival, cytokine release, and surface marker expression were monitored. The question whether DC could present amyloid to T cells was also addressed. We demonstrated that A beta (25-35) does not induce DC apoptosis or necrosis. This was shown by electron microscopy as well as by nuclear staining with propidium iodide. Some peptide aggregates were found in intracellular vacuoles of DC. This process did not increase production of TNF alpha and did not change the surface expression of CD18, CD11a or CD11b. A decreased surface expression of MHC class II molecules was, however, noted. DC pulsed with A beta aggregates were unable to stimulate T cells in an autologous coculture system. The results demonstrate that amyloid may escape immune recognition by its failure to activate antigen-presenting cells and by inhibiting MHC class II surface expression.
Asunto(s)
Enfermedad de Alzheimer/inmunología , Péptidos beta-Amiloides/inmunología , Células Dendríticas/inmunología , Fragmentos de Péptidos/inmunología , Adulto , Muerte Celular , Células Dendríticas/efectos de los fármacos , Endocitosis , Femenino , Humanos , Leucocitos Mononucleares/inmunología , MasculinoRESUMEN
Binding sites for concanvalin A (Con A), Ricinus communis I agglutinin (RCA I), and Helix pomatia lectin (HPA) were localized in the Golgi apparatus of rat small intestinal absorptive cells. A preembedment technique, a modification of the one originally used by Bernhard and Avrameas (Exp Cell Res 64:232, 1971), was employed, with horse-radish peroxidase being used for cytochemical visualization. Incubations were performed on 10 microns thick cryosections of duodenal segments that were fixed in a mixture of 4% formaldehyde and 0.5% glutaraldehyde; fixation was preceded by a 2-min rinse in 0.1 M sodium cacodylate and followed by storage in the same buffer for up to 7 days. Incubation with Con A, which binds preferably to alpha-D-mannose and alpha-D-glucose residues, caused intense reaction of the dilated cisternae of the cis Golgi side; staining was variable in intermediate and trans cisternae. RCA I, recognizing beta-D-galactose residues, could only be demonstrated in intermediate cisternae. Reaction for HPA, which indicates alpha-N-acetyl-D-galactosamine residues, stained intensely 1 to 2 cisternae of the cis Golgi side, as well as being localized in the peripheral regions of the cisternae of the intermediate compartment of the stacks. Deposits of reaction product covered the luminal surface of the cisternal membranes, but usually left the lumen itself, as well as lipid particles, devoid of reaction. The differences in Con A, RCA I, and HPA reactivity between cis, intermediate, and trans cisternae suggest compositional and structural differences of the carbohydrates in the respective compartments; they may reflect conversion processes that are known to occur in the oligosaccharide side chains of glycoconjugates at the Golgi complex level.
Asunto(s)
Concanavalina A/metabolismo , Aparato de Golgi/metabolismo , Intestino Delgado/ultraestructura , Lectinas/metabolismo , Lectinas de Plantas , Receptores Mitogénicos/metabolismo , Absorción , Animales , Femenino , Aparato de Golgi/ultraestructura , Histocitoquímica , Microscopía Electrónica , RatasRESUMEN
Using lectin binding, we characterized subdomains of the rough endoplasmic reticulum (rER) in goblet cells of the rat colon. In this cell type, special rER regions can be differentiated on the basis of their content of low electron density and dilated cisternal spaces in conventional transmission electron microscopic preparations. The fine fibrillar content of these cisternal regions demonstrated high-affinity binding with lectins from wheat germ, Helix pomatia, Griffonia simplicifolia I-A4 and -B4, and Ricinus communis I, although not with the sialic acid-specific Limax flavus lectin and the fucose-binding Ulex europaeus I lectin. Sugar-inhibitory experiments indicated that glycoconjugates packed within these regions bound the lectins with higher affinity than molecules present in the Golgi apparatus and secretory granules. Furthermore, the lectin binding patterns of the rER subdomains differed from those of the Golgi apparatus and mucin granules: the terminal sugar residues sialic acid and fucose were demonstrable in the Golgi apparatus and mucin granules and were absent from the rER, while galactose-recognizing lectins bound intensely at these rER regions, weakly to Golgi elements, and were almost absent from mucin granules.
Asunto(s)
Colon/química , Retículo Endoplásmico/química , Glicoconjugados/análisis , Aparato de Golgi/química , Lectinas , Animales , Secuencia de Carbohidratos , Lectinas/química , Datos de Secuencia Molecular , Mucinas/análisis , Ratas , Ratas EndogámicasRESUMEN
We studied the subcellular localization of glycoconjugates recognized by the garden pea and lentil lectins (Pisum sativum, PSA; Lens culinaris, LCA) in mature absorptive cells of duodenum and jejunum of fasted rats. PSA and LCA are mannose-, glucose-, and N-acetyl-glucosamine-recognizing lectins that bind with high affinity to fucosylated core regions of N-glycosidically linked glycans. The binding reactions were cytochemically demonstrated in a pre-embedment incubation system using peroxidase-labeled lectins. Both pea and lentil lectins bound with constituents of nuclear envelope and endoplasmic reticulum, cisternae of the Golgi apparatus, several Golgi-associated vesicles, lysosomes, and portions of the plasma membrane. PSA and LCA label was non-homogeneous in the endoplasmic reticulum; in the Golgi apparatus the reactions were most intense in the cis and medial cisternae of the stacks. For inhibition of the intense reactions apparent in the Golgi apparatus, in lysosomes, and at the plasma membrane, considerably higher concentrations of competitive sugars were necessary than for abolition of the endoplasmic reticulum label. This indicates that endoplasmic reticulum glycoconjugates bind at low affinities with pea and lentil lectins, and that high-affinity PSA/LCA-binding glycoconjugates, which may correspond to corefucosylated N-linked glycans, predominate in cis and medial Golgi cisternae, lysosomes, and at the plasma membrane.
Asunto(s)
Marcadores de Afinidad/metabolismo , Glicoconjugados/metabolismo , Intestino Delgado/citología , Lectinas/metabolismo , Lectinas de Plantas , Acetilglucosamina/metabolismo , Animales , Unión Competitiva , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Femenino , Glucosa/metabolismo , Aparato de Golgi/metabolismo , Aparato de Golgi/fisiología , Aparato de Golgi/ultraestructura , Histocitoquímica/métodos , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/ultraestructura , Intestino Delgado/metabolismo , Intestino Delgado/ultraestructura , Lisosomas/metabolismo , Lisosomas/ultraestructura , Manosa/metabolismo , Microscopía Electrónica , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestructura , Polisacáridos/biosíntesis , Ratas , Ratas EndogámicasRESUMEN
We investigated the early and late changes of the Golgi region under the influence of the fungal metabolite brefeldin A (BFA) by electron microscopy and lectin cytochemistry using the beta-galactose-specific lectin from the beetle Allomyrina dichotoma (Allo A). In control cells, Allo-A reactions were confined to trans-Golgi elements, the trans-Golgi network, and to endosomes and lysosomes; the nuclear envelope and endoplasmic reticulum were consistently free of Allo A reactions. Our findings with cells from three different lines (i.e., HepG2 hepatoma cells, WI38 fibroblasts, and L132 embryonic lung cells) showed tubular-reticular transformations of the Golgi stacks as early as 30 sec after application of BFA. The transformations started at the cis side and proceeded rapidly; after only a few minutes the Golgi apparatus was no longer apparent as an individual entity. Simultaneously, giant tubules grew out of the Golgi region and traversed the cytoplasm over micrometer-long distances. In part, they were reactive for Allo A. Reactions for beta-galactose occurred in cisternae of the endoplasmic reticulum after 4-5 min of BFA treatment; after 30 min the entire endoplasmic reticulum was intensely reactive for Allo A. At 5 min and later, the tubular-reticular transformations appeared more compact, forming glomerulus-like structures (glomerulini). These were closely associated with cisternae of the endoplasmic reticulum. Initially, glomerulini were mostly Allo A negative or showed peripheral Allo A-positive segments. The number of Allo A-positive glomerulini increased with the duration of treatment. Our findings identify the glomerulini as bipolar structures forming a link between the endoplasmic reticulum and the dissociating Golgi stacks.
Asunto(s)
Ciclopentanos/farmacología , Aparato de Golgi/efectos de los fármacos , Micotoxinas/farmacología , Brefeldino A , Línea Celular , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/ultraestructura , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Histocitoquímica , Humanos , Lectinas , Microscopía Electrónica , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Currently, there is little doubt that the immune system plays a role in the neurodegenerative process in Alzheimer's disease (AD). Inflammatory proteins such as complement components, enzymes, eicosanoids, and cytokines are found in association with cerebral amyloid plaques and may exacerbate the fundamental pathology of AD, by stimulating Amyloid beta (A beta) production, supporting its aggregation and increasing its cytotoxicity. Activated microglia and astrocytes are the main source of these proteins, and A beta may trigger their release. Interestingly, there are also indications that the immune system may play a protective role against the development of AD. Microglial cells have been shown to degrade A beta, and recent evidence suggests that autoreactive A beta-specific T cells may be relevant to the elimination of the peptide. This mechanism seems, however, impaired in the majority of patients with AD. The immune system seems thus to represent a natural line of defense against the accumulation of dangerous amyloidogenic substances. Impairment of this specific immunological defense mechanism and the failure to eliminate a toxic metabolite can be the basis for a chronic nonspecific inflammatory process in the brain, as described above. AD is a good example how an immune response initially aiming at maintaining the integrity of the body may fail and consequently lead to tissue destruction and neuronal loss.
Asunto(s)
Enfermedad de Alzheimer/inmunología , Sistema Inmunológico/fisiopatología , Enfermedad de Alzheimer/metabolismo , Amiloide/fisiología , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/fisiología , Precursor de Proteína beta-Amiloide/metabolismo , Humanos , Placa Amiloide/patologíaRESUMEN
Damage to vascular endothelium may play an important role during metastasis. We used a three-dimensional model of tumour cell extravasation to test the hypothesis that certain types of tumour cells are able to induce vascular endothelial cell injury. Multicellular tumour spheroids (MCTS) of 14 human cancer cell lines and spheroids from two benign cell lines were transferred onto confluent monolayers of human endothelial cells (EC). MCTS from 4 of 7 melanoma cell lines induced damage of the endothelium which was closely associated with tumour cell attachment. Endothelial cell injury became evident morphologically by loss of cell membrane integrity and sensitivity to shear stress. Similar results were obtained with EC derived from human umbilical veins, umbilical arteries and saphenous veins. Addition of the oxygen radical scavenger catalase showed a dose- and time-dependent inhibition (up to 48 h) of EC damage in the case of the melanoma cell lines ST-ML-11, ST-ML-14 and SK-MEL-28. The scavenging enzyme superoxide dismutase proved to be protective (up to 12 h) in ST-ML-12 MCTS. In contrast, allopurinol, deferoxamine mesylate, ibuprofen, nor-dihydroguaretic acid, soybean trypsin inhibitor or aprotinin had no protective effect. None of the non-melanoma cancer cell lines or benign cells induced endothelial cell damage. Endothelial injury has been shown to enhance the process of metastasis. Our results suggest that free-radical-mediated endothelial cell damage may be one of the mechanisms contributing to the devastating metastatic potential of melanoma.