RESUMEN
Among others, lectin-mediated drug delivery is currently discussed as a promising strategy towards improved bioavailability of biotech drugs. For quantitative determination of the lectin from wheat germ (WGA), a sandwich ELISA relying on capture of the lectin by pig gastric mucin coated wells and detection of bound WGA by a lectin specific first antibody followed by peroxidase-labelled second antibody was elaborated. The stepwise optimised protocol allows quantification over the range from 10 to 1000 ng/ml WGA with a coefficient of determination of 0.9991. The day to day variation was +/-0.09 OD at 500 ng/ml WGA. Additionally, the presented ELISA-protocol allows determination of WGA in serum with the same sensitivity and reliability as in buffer. Lectins with different carbohydrate specificity such as those from jack beans and peanuts exhibited no cross-reactivity. Among the lectins with the same carbohydrate specificity that from potatoes interfered with the assay, whereas that from tomatoes was not recognised by the first antibody. Since the potato lectin is fully degraded in the intestine, no cross-reactivity with WGA is expected in serum samples. Following on from these results, the absorption rate of WGA in biologically active form might be determined as a basis for further steps towards improved drug delivery systems.
Asunto(s)
Aglutininas del Germen de Trigo/análisis , Animales , Anticuerpos/inmunología , Reacciones Cruzadas/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Mucinas Gástricas/química , Peróxido de Hidrógeno/química , Concentración de Iones de Hidrógeno , Lectinas/inmunología , Plásticos/química , Reproducibilidad de los Resultados , Albúmina Sérica Bovina/química , Porcinos , Factores de Tiempo , Aglutininas del Germen de Trigo/sangre , Aglutininas del Germen de Trigo/químicaRESUMEN
Congenital disorders of glycosylation (CDG) type I are mostly due to a deficient phosphomannomutase activity, called CDG Ia. CDG IIa (mutations in the MGAT2 gene) results from a deficient activity of the Golgi enzyme N-acetylglucosaminyltransferase II. CDG Ia patients predominantly have a thrombotic tendency, whereas our CDG IIa patient has an increased bleeding tendency, despite similar coagulation factor abnormalities in both types. We have investigated whether abnormally glycosylated platelet membrane glycoproteins are involved in the haemostatic complications of both CDG groups. In flow cytometry, the binding of Ricinus communis lectin (reactive with beta-galactose primarily) to control platelets increased after neuraminidase treatment: this increase was smaller (p < 0.01) in CDG Ia patients (3.1 +/- 0.08 times) than in control platelets (8.5 +/- 1.8 times) and did not occur in the CDG IIa patient. Platelet-rich plasma from CDG Ia patients, but not a CDG IIa patient. aggregated spontaneously and gel-filtered platelets from CDG Ia patients agglutinated at very low concentrations of ristocetin, independently of von Willebrand factor (vWF). Accordingly, in stirred whole blood, the rate of single platelet disappearance of CDG Ia patients was twice that of control platelets. In contrast, perfusion of whole anticoagulated blood of the CDG IIa patient over collagen yielded markedly decreased platelet adherence to collagen at shear rates involving glycoprotein (GP) Ib-vWF interactions. Thus, abnormal glycosylation of platelet glycoproteins in CDG Ia enhances nonspecific platelet interactions, in agreement with a thrombotic tendency. The reduced GP Ib-mediated platelet reactivity with vessel wall components in the CDG IIa patient under flow conditions provides a basis for his bleeding tendency.
Asunto(s)
Plaquetas/fisiología , Hemostasis , N-Acetilglucosaminiltransferasas/genética , Fosfotransferasas (Fosfomutasas)/genética , Plaquetas/química , Plaquetas/ultraestructura , Membrana Celular/química , Concanavalina A/sangre , Glicosilación , Hemorreología , Humanos , Lectinas/sangre , Mutación , N-Acetilglucosaminiltransferasas/deficiencia , Neuraminidasa/farmacología , Perfusión , Fosfotransferasas (Fosfomutasas)/deficiencia , Agregación Plaquetaria , Complejo GPIb-IX de Glicoproteína Plaquetaria/análisis , Glicoproteínas de Membrana Plaquetaria/análisis , Aglutininas del Germen de Trigo/sangreRESUMEN
With a new ektacytometry, we studied the relation between the microstructure of red blood cell (RBC) membrane and the rheological properties of RBCs in a shear flow field of low viscosity. The main contributions of this paper are as follows: 1. The hemorheological meanings of the orientation index (DI)or and the small deformation index (DI)d were explored. (DI)or is an overall rheological index depending on the deformability and morphology of RBCs. The better the physiological shape of RBCs is maintained, the greater the (DI)or is. (DI)d can be used to describe the lipid fluidity of RBC membrane. Such an explanation for the meaning of (DI)d has been forcefully supported by our experiments using electron spin resonance (ESR) and fluorescence polarization. 2. The influence of wheat germ agglutinin (WGA) of different concentrations on the lipid fluidity of membrane is different from that of concanavalin A (ConA). The lipid fluidity of membrane changes with WGA concentration treating RBCs and there is a maximum value for the membrane fluidity at a specific concentration of WGA. However, the deformability of membrane described by the integrate deformation index (IDI) monotonically decreased with the increase in WGA concentration treating RBCs. 3. It is concluded that the increase in the lipid fluidity of red cell membrane is not necessarily associated with the improvement of RBC deformability.
Asunto(s)
Concanavalina A/farmacología , Deformación Eritrocítica/efectos de los fármacos , Membrana Eritrocítica/metabolismo , Receptores de Concanavalina A/efectos de los fármacos , Receptores Mitogénicos/efectos de los fármacos , Aglutininas del Germen de Trigo/farmacología , Animales , Concanavalina A/sangre , Lípidos de la Membrana/sangre , Conejos , Receptores de Concanavalina A/sangre , Receptores Mitogénicos/sangre , Aglutininas del Germen de Trigo/sangreRESUMEN
Native horseradish peroxidase (HRP) and the lectin wheat germ agglutinin (WGA) conjugated to HRP are protein probes represented in the blood-brain barrier (BBB) literature for elucidating morphological routes of passage between blood and brain. We report the application of established pharmacokinetic methods, e.g., multiple-time regression analysis and capillary depletion technique, to measure and compare bidirectional rates of passage between blood and brain for radioactive iodine-labeled HRP (I-HRP), WGA-HRP (I-WGA-HRP), and the serum protein albumin (I-ALB) following administration of the probes intravenously (i.v.) or by intracerebroventricular (i.c.v.) injection in mice. The pharmacokinetic data are supplemented with light and electron microscopic analyses of HRP and WGA-HRP delivered i.v. or by i.c.v. injection. The rates of bidirectional movement between blood and brain are the same for coinjected I-HRP and I-ALB. Blood-borne HRP, unlike WGA-HRP, has unimpeded access to the CNS extracellularly through sites deficient in a BBB, such as the circumventricular organs and subarachnoid space/pial surface. Nevertheless, blood-borne I-WGA-HRP enters the brain approximately 10 times more rapidly than I-HRP and I-ALB. Separation of blood vessels from the neocortical parenchyma confirms the entry of blood-borne I-WGA-HRP to the brain and sequestration of I-WGA-HRP by cerebral endothelial cells. Nearly half the I-WGA-HRP radioactivity associated with cortical vessels is judged to be subcellular. Light microscopic results suggest the extracellular pathways into the brain available to blood-borne native HRP do not represent predominant routes of entry for blood-borne WGA-HRP. Ultrastructural analysis further suggests WGA-HRP is likely to undergo adsorptive transcytosis through cerebral endothelia from blood to brain via specific subcellular compartments within the endothelium. Entry of blood-borne I-WGA-HRP, but not of I-ALB, is stimulated with coinjected unlabeled WGA-HRP, suggesting the latter may enhance the adsorptive endocytosis of blood-borne I-WGA-HRP. With i.c.v. coinjection of I-WGA-HRP and I-ALB, I-WGA-HRP exists the brain more slowly than I-ALB. The brain to blood passage of I-WGA-HRP is nil with inclusion of unlabeled WGA-HRP, which does not alter the exist of I-ALB. Adsorptive endocytosis of i.c.v. injected WGA-HRP appears restricted largely to cells lining the ventricular cavities, e.g., ependymal and choroid plexus epithelia. In summary, the data suggest that the bidirectional rates of passage between brain and blood for native HRP are comparable to those for albumin.
Asunto(s)
Encéfalo/metabolismo , Peroxidasa de Rábano Silvestre/sangre , Peroxidasa de Rábano Silvestre/farmacocinética , Albúmina Sérica/farmacocinética , Aglutininas del Germen de Trigo/sangre , Aglutininas del Germen de Trigo/farmacocinética , Animales , Barrera Hematoencefálica , Encéfalo/citología , Endocitosis , Endotelio Vascular/metabolismo , Histocitoquímica , Inyecciones Intravenosas , Inyecciones Intraventriculares , Masculino , Ratones , Ratones Endogámicos ICR , Aglutinina del Germen de Trigo-Peroxidasa de Rábano Silvestre ConjugadaRESUMEN
Morphological evidence of the potential for adsorptive transcytosis of protein through the mammalian blood-brain fluid barriers, first reported from this laboratory in the mouse, has been confirmed and expanded upon in rats injected intravenously or into the lateral cerebral ventricle/subarachnoid space with with exogenous lectin wheatgerm agglutinin (WGA) conjugated to horseradish peroxidase (HRP). Blood-borne WGA-HRP rapidly enters cerebral endothelia by the process of adsorptive endocytosis and labels the vascular tree throughout the CNS. At 3 h post-injection and longer, WGA-HRP occupies the perivascular clefts and labels perivascular cells and basal lamina; this suspected transendothelial transfer of the lectin conjugate from blood to brain involves specific constituents of the endothelial endomembrane system of organelles (e.g., plasmalemma, vesicles, endosomes, Golgi complex). Within 6 h, reaction product is evident in extracellular clefts beyond the perivascular basal lamina and labels endocytic vesicles, endosomes, and dense bodies within cells and processes of the neuropil. Exposure of the abluminal surface of blood-brain barrier endothelia for 1-18 h to WGA-HRP delivered into the cerebral ventricles or subarachnoid space indicates blood-brain barrier endothelia do not engage in demonstrable adsorptive endocytosis at the abluminal surface. In this preparation, no endothelial organelles comparable to those sequestering blood-borne WGA-HRP are labelled with the lectin conjugate; hence, significant adsorptive transcytosis of WGA-HRP through cerebral endothelia from brain to blood is unlikely. The demonstrable difference in membrane internalization of the luminal versus abluminal plasmalemma of blood-brain barrier endothelia suggests the blood-brain barrier is polarized regarding adsorptive endocytosis of WGA-HRP. If adsorptive transcytosis of macromolecules through the blood-brain barrier does occur, the process appears unidirectional, from blood to brain but not from brain to blood. Absence of demonstrable endocytosis at the abluminal front is an enigma in the scheme of transcytosis through the blood-brain barrier from blood to brain insofar as exocytosis and endocytosis are complementary events in the cellular secretory process. This unconventional membrane behavior associated with the abluminal plasmalemma argues against a significant transcytosis of blood-borne protein through blood-brain barrier endothelia. The potential for transcytosis of macromolecules through the blood-cerebrospinal fluid barrier of choroid plexus epithelia is not as problemmatic as that through blood-brain barrier endothelia; additional evidence is provided to suggest choroid plexus epithelia participate in adsorptive endocytosis circumferentially and adsorptive transcytosis of WGA-HRP bidirectionally between the blood and cerebrospinal fluid.
Asunto(s)
Barrera Hematoencefálica , Encéfalo/metabolismo , Peroxidasa de Rábano Silvestre/metabolismo , Aglutininas del Germen de Trigo/metabolismo , Adsorción , Animales , Transporte Biológico , Encéfalo/ultraestructura , Endocitosis , Endotelio/metabolismo , Epitelio/metabolismo , Femenino , Aparato de Golgi/metabolismo , Peroxidasa de Rábano Silvestre/sangre , Peroxidasa de Rábano Silvestre/líquido cefalorraquídeo , Cinética , Masculino , Microscopía Electrónica , Orgánulos/metabolismo , Ratas , Ratas Sprague-Dawley , Aglutinina del Germen de Trigo-Peroxidasa de Rábano Silvestre Conjugada , Aglutininas del Germen de Trigo/sangre , Aglutininas del Germen de Trigo/líquido cefalorraquídeoRESUMEN
In the first paper of a series (Gutowski, et al., 1991) we discussed the use of flow cytometry to follow at the cellular level the aging of red blood cells (RBC) in circulation, using fluorescently labelled lectins and goat anti-human-IgG and -IgM. The Coulter Epics 541 was used for those studies. In this report we describe more extensive experiments using the Becton-Dickinson FACScan flow cytometer, and compare the results with those obtained with the Coulter Epics 541. By changing sample conditions from isotonic to hypotonic, compensation for differences of the two instruments was accomplished. We confirmed our previous observations that RBC react very strongly with fluorescein isothiocyanate labelled wheat germ agglutinin (FITC-WGA) and that there is little change in the intensity of fluorescence given by RBC of all sizes with the exception of the smallest. Reactivity with FITC-WGA is markedly decreased in the presence of competitive inhibitors of sialic acid or upon enzymatic removal of sialic acid from RBC. Removal of sialic acid is accompanied by increased reaction with peanut agglutinin (FITC-PNA). Flow cytometry was also used to monitor the enrichment of a population of smallest RBC (less than 0.05%), isolated from both counterflow centrifugation and the interface obtained from Histopaque separation. These smallest RBC showed low reactivity with FITC-WGA and higher binding of FITC-goat-anti-human-IgG, and -IgM, and therefore represent the most senescent RBC, just prior to their clearance from circulation by the reticuloendothelial system. These observations are in compliance with the hypothesis that physiological desialylation of glycophorin is responsible for clearance of senescent RBC from circulation (Aminoff, 1988).
Asunto(s)
Eritrocitos/fisiología , Citometría de Flujo , Senescencia Celular , Centrifugación , Fraccionamiento Químico , Eritrocitos/metabolismo , Fluoresceína-5-Isotiocianato , Glicoforinas/farmacología , Humanos , Lectinas/sangre , Luz , Neuraminidasa/farmacología , Concentración Osmolar , Aglutinina de Mani , Reproducibilidad de los Resultados , Dispersión de Radiación , Trisacáridos/farmacología , Aglutininas del Germen de Trigo/sangreRESUMEN
Studies were performed to examine the lateral organization of the NADPH oxidase system in the plasma membrane of human neutrophils. Analysis of the subcellular fractionation of human neutrophils by isopycnic sedimentation of cavitated cell lysates suggested that there may be more than one population of plasma membrane vesicles formed upon cell disruption. One population (30-32% sucrose) contained surface accessible wheat germ agglutinin binding sites, alkaline phosphatase activity, and cytochrome b. Another population (34-36% sucrose) contained membrane-bound flavin and, when the cells were prestimulated with phorbol myristate acetate (PMA), NADPH-dependent superoxide generating activity. Approximately 25% of the neutrophil cytochrome b cosedimented with the heavy population, confirming our previous hypothesis (Parkos et al. (1985) J. Biol. Chem. 260, 6541-6547) that only a fraction of the total cellular cytochrome b is involved in superoxide production. The heavy plasma membrane fraction was also enriched in membrane associated actin and fodrin as detected by Western blot analysis. After extraction of the plasma membrane vesicles with detergent cocktails, the majority of superoxide generating activity remained associated with the detergent insoluble pellet. Western blot analysis demonstrated that the pellets were also enriched in actin. Further analysis of these pellets using rate-zonal detergent-containing sucrose density gradients indicated that the superoxide generating complex had an approximate sedimentation coefficient of 80 S, suggesting that the neutrophil superoxide generating system may form a complex on the plasma membrane which is associated with or somehow organized by the membrane skeletal matrix. This organization may be of functional relevance not only to the actual production of superoxide, but also to the targeting of microbicidal oxidants.
Asunto(s)
Membrana Celular/metabolismo , NADH NADPH Oxidorreductasas/sangre , Neutrófilos/metabolismo , Superóxidos/sangre , Actinas/sangre , Fosfatasa Alcalina/sangre , Sitios de Unión , Western Blotting , Proteínas Portadoras/sangre , Fraccionamiento Celular , Membrana Celular/análisis , Membrana Celular/ultraestructura , Centrifugación por Gradiente de Densidad , Grupo Citocromo b/sangre , Detergentes , Humanos , Proteínas de Microfilamentos/sangre , NADPH Oxidasas , Neutrófilos/ultraestructura , Orgánulos/análisis , Orgánulos/metabolismo , Solubilidad , Acetato de Tetradecanoilforbol/farmacología , Aglutininas del Germen de Trigo/sangreRESUMEN
Using the label-fracture technique, an ultrastructural comparison was made of the number and distribution of wheat germ agglutinin (WGA)-binding sites between human normal and sickle red blood cells. The WGA was adsorbed to colloidal gold, and quantitative analysis of the electron micrographs revealed that more binding sites were present on the sickle erythrocytes than on the normal erythrocytes. Moreover, the sites were more clustered on the sickle red cells than on the normal red cells. Use of another lectin, Bandieraea simplicifolia-II, revealed that it did not bind to normal or sickle red cells. Because of the affinity of the WGA for sialic acid residues, it is probable that the WGA is binding to a transmembrane sialoglycoprotein, glycophorin A. The conformation and/or distribution of the glycophorin A molecules may be altered by the sickle hemoglobin that binds to the red cell membrane. Hence, as detected by WGA, new surface receptors, which could play a role in the adhesion of sickle cells to endothelium may be exposed.