RESUMEN
Inflammatory responses of myeloid cells to LPS are mediated through CD14, a glycosylphosphatidylinositol-anchored receptor that binds LPS. Since CD14 does not traverse the plasma membrane and alternatively anchored forms of CD14 still enable LPS-induced cellular activation, the precise role of CD14 in mediating these responses remains unknown. To address this, we created a transmembrane and a glycosylphosphatidylinositol-anchored form of LPS-binding protein (LBP), a component of serum that binds and transfers LPS to other molecules. Stably transfected Chinese hamster ovary (CHO) fibroblast and U373 astrocytoma cell lines expressing membrane-anchored LBP (mLBP), as well as separate CHO and U373 cell lines expressing membrane CD14 (mCD14), were subsequently generated. Under serum-free conditions, CHO and U373 cells expressing mCD14 responded to as little as 0.1 ng/ml of LPS, as measured by NF-kappaB activation as well as ICAM and IL-6 production. Conversely, the vector control and mLBP-expressing cell lines did not respond under serum-free conditions even in the presence of more than 100 ng/ml of LPS. All the cell lines exhibited responses to less than 1 ng/ml of LPS in the presence of the soluble form of CD14, demonstrating that they are still capable of LPS-induced activation. Taken together, these results demonstrate that mLBP, a protein that brings LPS to the cell surface, does not mediate cellular responses to LPS independently of CD14. These findings suggest that CD14 performs a more specific role in mediating responses to LPS than that of simply bringing LPS to the cell surface.
Asunto(s)
Proteínas de Fase Aguda , Proteínas Portadoras/fisiología , Receptores de Lipopolisacáridos/fisiología , Lipopolisacáridos/inmunología , Lipopolisacáridos/metabolismo , Glicoproteínas de Membrana , Secuencia de Aminoácidos , Animales , Astrocitoma , Secuencia de Bases , Células CHO , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/metabolismo , Membrana Celular/química , Membrana Celular/inmunología , Membrana Celular/metabolismo , Cricetinae , Relación Dosis-Respuesta Inmunológica , Humanos , Inmunidad Celular , Molécula 1 de Adhesión Intercelular/biosíntesis , Interleucina-6/biosíntesis , Receptores de Lipopolisacáridos/genética , Receptores de Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Proteínas Recombinantes/inmunología , Células Tumorales CultivadasRESUMEN
BACKGROUND: Calretinin and calbindin-D28k are similar Ca(2+)-binding proteins previously described in specific central neurons and other cells. METHODS: The immunocytochemical distribution of these two proteins was studied in the human gastrointestinal tract. RESULTS: In gastric and small intestinal endocrine cells, calbindin-D28k immunoreactivity was confirmed, but calretinin immunoreactivity was not found. Nerve cell bodies in both submucous and myenteric ganglia were immunoreactive for calbindin (13% and 38% of total cells, respectively) or calretinin (23% and 21%), some containing both proteins. In nerve processes, calretinin was generally more abundant than calbindin and was found particularly around blood vessels. Calretinin co-localized with immunoreactive vasoactive intestinal peptide, neuropeptide Y, galanin, or substance P in submucous ganglion cells and with substance P in myenteric cells. Calbindin-D28k colocalized with fewer peptides, specifically vasoactive intestinal peptide or galanin in submucous cells. By 8 weeks of fetal development, discrete neuronal localizations for both proteins and for calbindin-D28k in endocrine cells were apparent. CONCLUSIONS: In the enteric neuroendocrine system, calretinin and calbindin-D28k are useful markers that may help elucidate Ca(2+)-mediated functions in health and disease.
Asunto(s)
Sistema Digestivo/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Calbindina 1 , Calbindina 2 , Calbindinas , Sistema Digestivo/citología , Sistema Digestivo/inervación , Glándulas Endocrinas/citología , Glándulas Endocrinas/metabolismo , Feto/metabolismo , Humanos , Inmunohistoquímica , Intestinos/embriología , Persona de Mediana Edad , Peso Molecular , Sistema Nervioso/metabolismo , Péptidos/metabolismo , Proteína G de Unión al Calcio S100/química , Distribución TisularRESUMEN
Pigeon retina has been mapped immunocytochemically for vitamin D-dependent calcium-binding protein (D-CaBP). Immunoreactivity was found in the cones of the yellow field, but not in photoreceptors of the red field. The D-CaBP-containing cones were a subpopulation of those in the yellow field having straight fibres leading to their synaptic terminals. D-CaBP immunoreactivity was also found in horizontal cells, the amount present varying according to position along the retina, and in some amacrine cells. Immunoblots of pigeon retinal proteins separated by SDS-polyacrylamide gel electrophoresis indicated two D-CaBP forms, having apparent molecular weights of 27000 and 29000. Both these forms of D-CaBP have been found previously in rat and pigeon brain.
Asunto(s)
Columbidae/anatomía & histología , Retina/análisis , Proteína G de Unión al Calcio S100/análisis , Animales , Columbidae/metabolismo , Electroforesis en Gel de Poliacrilamida , Histocitoquímica , Técnicas para Inmunoenzimas , Peso Molecular , Células Fotorreceptoras/análisis , Retina/citologíaRESUMEN
Vitamin D-dependent calcium binding protein (D-CaBP) was localized in the brains of high frequency gymnotid fish. In birds and mammals this protein is seen in a variety of cell types including Purkinje cells, inferior olivary cells and CA1 pyramids of the hippocampus. This distribution has led us to speculate that D-CaBP may be important in buffering intracellular calcium, perhaps more specifically that calcium which enters the cell during dendritic calcium spikes. In the gymnotid fish D-CaBP was found in many of the same cell types in which it is also seen in birds and mammals. In addition, D-CaBP is specifically present in neurons which drive the electric organ (pacemaker and relay cells) and neurons within the electrosensory system which are phase-locked to the electric organ discharge (spherical and giant cells). Relay cells and giant cells have exceptionally high concentrations of D-CaBP. These cells do not exhibit calcium spikes and the role of their D-CaBP may be to regulate calcium released from intracellular stores.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Órgano Eléctrico/inervación , Canales Iónicos/metabolismo , Bulbo Raquídeo/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Células Receptoras Sensoriales/metabolismo , Médula Espinal/metabolismo , Vías Aferentes/metabolismo , Animales , Calcio/metabolismo , Cerebelo/metabolismo , Peces , Mesencéfalo/metabolismo , Neuronas/metabolismo , Núcleo Olivar/metabolismo , Células de Purkinje/metabolismoRESUMEN
1. The rapid stimulation of intestinal Ca(2+) transport observed in vitamin D-deficient chicks after receiving 1,25-dihydroxycholecalciferol has necessitated a re-evaluation of the correlation hitherto observed between this stimulation and the induction of calcium-binding protein synthesis. By 1h after a dose of 125ng of 1,25-dihydroxycholecalciferol, Ca(2+) transport is increased. This is at least 2h before calcium-binding protein can be detected immunologically and 1h before synthesis of the protein begins on polyribosomes, and thus the hormone stimulates Ca(2+) transport before calcium-binding-protein biosynthesis is induced. 2. The maximum increase in Ca(2+) transport observed after this dose of 1,25-dihydroxycholecalciferol (attained by 8h) is similar to that observed after 1.25-25mug of cholecalciferol, but the stimulation is only short-lived, in contrast with the effect observed after the vitamin. At later times after the hormone, however, when Ca(2+) transport has declined to its basal rate, the cellular content of calcium-binding protein remains elevated. 3. Calcium-binding protein is synthesized on free rather than membrane-bound polyribosomes, which implies that it is an intracellular protein. 4. Rachitic chicks require the presence of dietary calcium for maximum stimulation of calcium-binding protein production by cholecalciferol. 5. These results suggest that calcium-binding protein is an intracellular protein, and that its synthesis may be a consequence of the raised intracellular calcium content of the intestinal epithelial cells resulting from 1,25-dihydroxycholecalciferol-stimulated Ca(2+) transport. We propose that calcium-binding-protein synthesis is necessary for maintaining the stimulated rate of Ca(2+) transport, which is initiated by other factors.
Asunto(s)
Calcio/metabolismo , Proteínas Portadoras/metabolismo , Mucosa Intestinal/metabolismo , Vitamina D/farmacología , Animales , Transporte Biológico , Pollos , Colecalciferol/farmacología , Dihidroxicolecalciferoles/farmacología , Técnicas In Vitro , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Polirribosomas/metabolismo , Deficiencia de Vitamina D/metabolismoRESUMEN
1. The synthesis of calcium-binding protein, a protein produced in the small intestine in response to vitamin D, was investigated with a view to determining whether calcium-binding-protein production could be correlated with the stimulation of calcium absorption by vitamin D. 2. A radioimmunological assay, which can quantitatively estimate calcium-binding-protein concentrations as low as 1mug/g wet wt., was used to detect the synthesis of soluble calcium-binding protein. 3. When used on intestinal supernatants from chicks dosed with vitamin D, calcium-binding protein was not detectable at 8h but was present after 12h at a concentration of 8.6mug/g wet wt.; in agreement with this an increase in calcium absorption due to vitamin D was detected at 12h but not at 8h. 4. The synthesis of calcium-binding protein was also monitored directly by making use of the ability of the iodinated antiserum to bind specifically to nascent calcium-binding protein chains on intestinal polyribosomes; in this way calcium-binding-protein synthesis could be detected 8h after dosage with vitamin D. Further, the binding reaction indicated a near linear increase in the calcium-binding-protein-synthesizing capacity over a 16h period. 5. From the amount of calcium-binding protein present 12 and 24h after vitamin D administration it is calculated that calcium-binding-protein mRNA is produced at approx. 1mol/min per intestinal cell. 6. It is concluded that the high correlation between the initiation of calcium-binding-protein synthesis and the stimulation of calcium absorption by vitamin D strengthens the proposal that calcium-binding protein plays an important role in calcium transport.