RESUMEN
UNLABELLED: Viruses exploit molecules on the target membrane as receptors for attachment and entry into host cells. Thus, receptor expression patterns can define viral tissue tropism and might to some extent predict the susceptibility of a host to a particular virus. Previously, others and we have shown that respiratory pathogens of the genus Gammacoronavirus, including chicken infectious bronchitis virus (IBV), require specific α2,3-linked sialylated glycans for attachment and entry. Here, we studied determinants of binding of enterotropic avian gammacoronaviruses, including turkey coronavirus (TCoV), guineafowl coronavirus (GfCoV), and quail coronavirus (QCoV), which are evolutionarily distant from respiratory avian coronaviruses based on the viral attachment protein spike (S1). We profiled the binding of recombinantly expressed S1 proteins of TCoV, GfCoV, and QCoV to tissues of their respective hosts. Protein histochemistry showed that the tissue binding specificity of S1 proteins of turkey, quail, and guineafowl CoVs was limited to intestinal tissues of each particular host, in accordance with the reported pathogenicity of these viruses in vivo. Glycan array analyses revealed that, in contrast to the S1 protein of IBV, S1 proteins of enteric gammacoronaviruses recognize a unique set of nonsialylated type 2 poly-N-acetyl-lactosamines. Lectin histochemistry as well as tissue binding patterns of TCoV S1 further indicated that these complex N-glycans are prominently expressed on the intestinal tract of various avian species. In conclusion, our data demonstrate not only that enteric gammacoronaviruses recognize a novel glycan receptor but also that enterotropism may be correlated with the high specificity of spike proteins for such glycans expressed in the intestines of the avian host. IMPORTANCE: Avian coronaviruses are economically important viruses for the poultry industry. While infectious bronchitis virus (IBV), a respiratory pathogen of chickens, is rather well known, other viruses of the genus Gammacoronavirus, including those causing enteric disease, are hardly studied. In turkey, guineafowl, and quail, coronaviruses have been reported to be the major causative agent of enteric diseases. Specifically, turkey coronavirus outbreaks have been reported in North America, Europe, and Australia for several decades. Recently, a gammacoronavirus was isolated from guineafowl with fulminating disease. To date, it is not clear why these avian coronaviruses are enteropathogenic, whereas other closely related avian coronaviruses like IBV cause respiratory disease. A comprehensive understanding of the tropism and pathogenicity of these viruses explained by their receptor specificity and receptor expression on tissues was therefore needed. Here, we identify a novel glycan receptor for enteric avian coronaviruses, which will further support the development of vaccines.
Asunto(s)
Infecciones por Coronavirus/veterinaria , Coronavirus del Pavo/metabolismo , Receptores Virales/metabolismo , Tropismo Viral/genética , Animales , Pollos/virología , Infecciones por Coronavirus/virología , Enteritis/virología , Galactanos/metabolismo , Virus de la Bronquitis Infecciosa/metabolismo , Intestinos/virología , Enfermedades de las Aves de Corral/virología , Unión Proteica/genética , Pavos/virologíaRESUMEN
The objectives of this study were to evaluate activity, rumination time, and their association with 3 kinds of pasture flies for organic dairy cows (n=57) fed 3 grain supplementation strategies during the grazing season from May to September 2013. Cows were assigned to 1 of 3 replicate supplementation groups: (1) no corn-grain supplementation (100% pasture, PAS, n=19); (2) low corn-grain (2.72kg/cow per day, LG, n=19); and (3) high corn-grain (5.44kg/cow per day, HG, n=19). Cows calved during 2 seasons (fall and spring) at the University of Minnesota West Central Research and Outreach Center, Morris, from October to December 2012 and March to May 2013. Supplement (corn-grain and minerals) was fed in a total mixed ration of corn silage and alfalfa silage, and at least 30% of diet dry matter intake for LG and HG cows consisted of pasture. Activity and rumination time (daily and 2-h blocks of time) were monitored electronically using HR-LD tags (SCR Engineers Ltd., Netanya, Israel) for 125d. Activity (cow body movement and head movement) was reported in activity units from SCR DataFlow II software, and rumination times were reported in minutes per day. PROC HPMIXED in SAS (SAS Institute Inc., Cary, NC) was used for statistical analysis, and independent variables were season of calving (fall or spring), month of grazing (June to September), supplementation group, and interactions of month of grazing and supplementation group. Replicate was a random effect with repeated measures. Daily activity was higher for PAS cows (1,138 activity units) than for HG cows (1,001 activity units), and LG cows (1,019 activity units). Daily activity was highest in July (1,258 activity units) and lowest in September (819 activity units). Rumination was not different for PAS (397min/d), LG (384min/d), or HG (370min/d) cows. Daily rumination was greater in September (402min/d) than in July (361min/d). Daily activity increased rapidly between 0600-0800h and 1600-1800h. From 1800 to 2000h, cows had a rapid decline in activity until 0600h the next day. All supplementation groups had the greatest rumination activity from 0200 to 0400h and the least between 1000 and 1200h. Greater activity of cows on a herd basis was moderately correlated with increased fly populations. Monthly activity patterns of grazing cows were associated with fly populations on cows.
Asunto(s)
Bovinos/fisiología , Digestión , Actividad Motora , Muscidae/fisiología , Ensilaje/análisis , Zea mays/química , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Industria Lechera/métodos , Dieta/veterinaria , Suplementos Dietéticos/análisis , Grano Comestible/química , Femenino , Control de Insectos , Minnesota , Densidad de PoblaciónRESUMEN
Pharmacological evidence is presented for the involvement of microtubules in the process of fast axoplasmic transport. A quantitative measure of the inhibition of axoplasmic transport in an in vitro preparation of rat sciatic nerve is described. The alkaloids colchicine, podophyllotoxin, and vinblastine, which are known both to disrupt microtubules and to bind to the protein subunit of microtubules, are inhibitors of axoplasmic transport. Lumicolchine and picropodophyllin, unlike their respective isomers colchicine and podophyllotoxin, are poor inhibitors of axoplasmic transport. The dissociation constants for the binding of colchicine, lumicolchicine, podophyllotoxin, and picropodophyllin to purified microtubule protein from rat brain have been measured. Inhibition of axoplasmic transport by these drugs correlates favorably with their affinities of microtubule protein.
Asunto(s)
Transporte Axonal/efectos de los fármacos , Glicoproteínas/metabolismo , Podofilotoxina/farmacología , Tubulina (Proteína)/metabolismo , Animales , Colchicina/análogos & derivados , Colchicina/metabolismo , Colchicina/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Microtúbulos/efectos de los fármacos , Podofilino/análogos & derivados , Podofilotoxina/metabolismo , Ratas , Nervio Ciático/efectos de los fármacos , Nervio Ciático/fisiología , Vinblastina/metabolismo , Vinblastina/farmacologíaRESUMEN
Recruitment of neutrophils to sites of inflammation is mediated in part by endothelial leukocyte adhesion molecule-1 (ELAM-1), which is expressed on activated endothelial cells of the blood vessel walls. ELAM-1 is a member of the LEC-CAM or selectin family of adhesion molecules that contain a lectin motif thought to recognize carbohydrate ligands. In this report, cell adhesion by ELAM-1 is shown to be mediated by a carbohydrate ligand, sialyl-Lewis X (SLex; NeuAc alpha 2,3Gal beta 1,4(Fuc alpha 1,3)-GlcNAc-), a terminal structure found on cell-surface glycoprotein and glycolipid carbohydrate groups of neutrophils.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Adhesión Celular/fisiología , Antígeno Lewis X/fisiología , Neutrófilos/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Conformación de Carbohidratos , Secuencia de Carbohidratos , Moléculas de Adhesión Celular/inmunología , Línea Celular , Cricetinae , Selectina E , Glicosilación , Humanos , Antígeno Lewis X/química , Ligandos , Datos de Secuencia Molecular , Neuraminidasa/farmacologíaRESUMEN
For many glycoproteins, the carbohydrate groups confer important physical properties such as conformational stability, protease resistance, charge and water-binding capacity. Equally important, however, are the roles of carbohydrate groups in biological recognition, where sequence diversity provides signals for protein targeting and cell-cell interactions.
Asunto(s)
Glicoproteínas/fisiología , Animales , Secuencia de Carbohidratos , Humanos , Datos de Secuencia Molecular , Estructura Molecular , Oligosacáridos/análisisRESUMEN
Rapid translocation of P-selectin (GMP-140) from cytoplasmic granules to the cell membrane of endothelial cells promotes adhesive interactions with neutrophils which, when activated, damage the endothelium. The role of P-selectin in lung vascular endothelial injury in rats after systemic activation of complement by intravenous infusion of cobra venom factor has been assessed. Within 5-10 min after cobra venom factor infusion, the pulmonary vasculature demonstrated immunohistochemical expression of an epitope that reacts with anti-human P-selectin. Monoclonal antibody to human P-selectin blocked in vitro adherence of rat or human platelets (activated with thrombin) to neutrophils and was demonstrated to react with thrombin-activated rat platelets. The antibody did not react with rat neutrophils. In vivo, the antibody had strongly protective effects against cobra venom factor-induced pulmonary vascular injury as determined by permeability changes and hemorrhage. In parallel, lung myeloperoxidase content was greatly reduced and, by transmission electron microscopy, there was markedly diminished adherence of neutrophils to the pulmonary vascular endothelium and much diminished injury of endothelial cells, as defined by hemorrhage. These data indicate that anti-human P-selectin reacts with a pulmonary vascular antigen in rats and that this antigen is essential for the full expression of lung injury.
Asunto(s)
Neutrófilos/fisiología , Glicoproteínas de Membrana Plaquetaria/fisiología , Neumonía/etiología , Enfermedad Aguda , Animales , Anticuerpos Monoclonales/inmunología , Venenos Elapídicos/toxicidad , Epítopos , Pulmón/enzimología , Pulmón/patología , Masculino , Ratones , Selectina-P , Peroxidasa/análisisRESUMEN
We have previously reported a newly discovered congenital disorder of neutrophil adhesion, leukocyte adhesion deficiency syndrome type 2 (LAD II). The clinical manifestations of this syndrome are similar to those seen in the classic leukocyte adhesion deficiency syndrome, now designated type 1 (LAD I), but the two syndromes differ in the molecular basis of their adhesion defects. LAD I is caused by a deficiency in the CD18 integrin adhesion molecules while LAD II patients are deficient in expression of sialyl-Lewis X (SLeX), a carbohydrate ligand for selectins. In this report we demonstrate that neutrophils from a LAD II patient bind minimally or not at all to recombinant E-selectin, purified platelet P-selectin, or P-selectin expressed on histamine-activated human umbilical vein endothelial cells, but have normal levels of L-selectin and CD11b/CD18 integrin, and adhere to and migrate across endothelium when CD11b/CD18 is activated. We compare LAD I and LAD II patient neutrophil function in vitro, demonstrating that integrin and selectin adhesion molecules have distinct but interdependent roles in neutrophil adhesion during an inflammatory response.
Asunto(s)
Síndrome de Deficiencia de Adhesión del Leucocito/sangre , Neutrófilos/fisiología , Antígenos CD11/fisiología , Antígenos CD18/fisiología , Adhesión Celular , Células Cultivadas , Quimiotaxis de Leucocito , Selectina E/fisiología , Endotelio Vascular/fisiología , Femenino , Citometría de Flujo , Humanos , Técnicas In Vitro , Síndrome de Deficiencia de Adhesión del Leucocito/clasificación , Masculino , Selectina-P/fisiología , Probabilidad , Valores de Referencia , Venas UmbilicalesRESUMEN
Neutrophil adherence and/or aggregation has been implicated in ischemia reperfusion injuries. We examined the role of P-selectin in PMN-mediated injury after reperfusion of the rabbit ear. The ear was partially amputated, and then reattached leaving the central artery and vein intact. To induce ischemia the central artery was then occluded. Treatment was at reperfusion with either saline or one of two murine P-selectin mAbs, designated PB1.3 and PNB1.6 mAb PB1.3 cross-reacts with rabbit P-selectin and prevents histamine-induced leukocyte rolling, whereas PNB1.6 does not. Using a peroxidase-antiperoxidase system P-selectin was detected in the ischemic ear, but not in the nonischemic ear. Ear volume increased to 5.3 times baseline in the saline-treated animals (n = 8), 6.6 times baseline in the nonblocking mAb PNB1.6-treated animals (n = 2), and 3.7 times baseline in the blocking mAb PB1.3-treated animals (n = 8). Estimated tissue necrosis of the combined saline- and PNB1.6-treated animals was 46 vs. 2.7% for the mAb PB1.3-treated animals. We conclude that: (a) P-selectin is expressed in ischemia reperfusion; (b) P-selectin participates in PMN-endothelial cell interactions in ischemia reperfusion; and (c) inhibiting P-selectin adhesion significantly reduces reperfusion injury.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Oído/irrigación sanguínea , Isquemia/fisiopatología , Neutrófilos/fisiología , Glicoproteínas de Membrana Plaquetaria/fisiología , Daño por Reperfusión/prevención & control , Animales , Moléculas de Adhesión Celular/fisiología , Oído/patología , Técnicas para Inmunoenzimas , Isquemia/patología , Necrosis , Selectina-P , Glicoproteínas de Membrana Plaquetaria/análisis , Glicoproteínas de Membrana Plaquetaria/inmunología , ConejosRESUMEN
The objective of this study was to define the nature, magnitude, and mechanisms of histamine-induced leukocyte-endothelial cell interactions in postcapillary venules of the rat mesentery using intravital microscopic techniques. Superfusion of the mesentery with histamine (10(-7)-10(-5) M) resulted in a dose-related increase in the number of rolling leukocytes, a reduction in rolling velocity, and an increased clearance of FITC-labeled rat albumin from blood to superfusate. The histamine-induced recruitment of rolling leukocytes and increased albumin clearance were prevented by histamine H1 (hydroxyzine, diphenhydramine) but not H2 (cimetidine) receptor antagonists. Because histamine induces expression of the adhesion molecule P-selectin in cultured endothelial cells, a monoclonal antibody directed against rat P-selectin and soluble sialyl-LewisX oligosaccharide (the carbohydrate ligand to P-selectin) were also tested as inhibitors. Both were effective in preventing the histamine-induced recruitment of rolling leukocytes, but neither agent attenuated the increased albumin clearance. These observations suggest that (a) histamine recruits rolling leukocytes and increases albumin leakage in postcapillary venules via H1 receptor activation, (b) histamine-induced recruitment of rolling leukocytes is mediated in part by P-selectin expressed on the endothelial cell surface, and (c) the histamine-induced vascular albumin leakage is unrelated to leukocyte-endothelial cell adhesion. Our results are consistent with the view that histamine may act as a mediator of acute inflammatory reactions.
Asunto(s)
Endotelio Vascular/citología , Histamina/farmacología , Leucocitos/fisiología , Glicoproteínas de Membrana Plaquetaria/fisiología , Receptores Histamínicos H1/fisiología , Animales , Permeabilidad Capilar/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Endotelio Vascular/fisiología , Masculino , Selectina-P , Ratas , Ratas Sprague-Dawley , Vénulas/citologíaRESUMEN
The selectins and the beta 2-integrins (CD11/CD18) mediate distinct adhesive interactions between neutrophils and endothelial cells. Selectins are believed to initiate binding by mediating neutrophil rolling, whereas beta 2-integrins are required for subsequent activation-induced firm sticking and emigration. In vitro evidence suggests that two endothelial cell selectins, P- and E-selectin, can mediate rolling by binding to the carbohydrate ligand sialyl-Lewisx (sLex) on neutrophil surface glycoconjugates. To test the relative contribution of selectins and beta 2-integrins in vivo we used intravital microscopy to study the behavior of neutrophils from two patients with distinct inherited leukocyte adhesion deficiency syndromes. Neutrophils from a patient suffering from CD18 deficiency showed normal rolling behavior but were incapable of sticking or emigrating upon chemotactic stimulation. Neutrophils from a second patient with a newly described adhesion deficiency had normal CD18 but did not express sLex. These neutrophils rolled poorly and also failed to stick in venules under shear force. Under static conditions, however, chemoattractant-induced sticking and emigration could be observed. This demonstrates that both selectin-carbohydrate-mediated initiation of adhesion and subsequent activation-induced beta 2-integrin engagement are essential for the normal function of human neutrophils in vivo.
Asunto(s)
Adhesión Celular/genética , Adhesión Celular/fisiología , Neutrófilos/fisiología , Animales , Antígenos CD/análisis , Antígenos CD18 , Secuencia de Carbohidratos , Moléculas de Adhesión Celular/análisis , Citometría de Flujo , Gangliósidos/análisis , Humanos , Selectina L , Antígeno Lewis X , Mesenterio/irrigación sanguínea , Mesenterio/fisiología , Datos de Secuencia Molecular , Conejos , SíndromeRESUMEN
Clostridium difficile toxin A (Tx-A) mediates secretion and inflammation in experimental enterocolitis. Intravital video microscopy was used to define the mechanisms that underlie the inflammatory reactions elicited by direct exposure of the microvasculature to Tx-A. Leukocyte adherence and emigration, leukocyte-platelet aggregation, and extravasation of FITC-albumin were monitored in rat mesenteric venules exposed to Tx-A. Significant increases in leukocyte adherence and emigration (LAE) and albumin leakage were noted within 15-30 min of Tx-A exposure. These responses were accompanied by mast cell degranulation and the formation of platelet-leukocyte aggregates. The Tx-A-induced increases in LAE and albumin leakage were significantly attenuated by pretreatment with either monoclonal antibodies (mAbs) directed against the leukocyte adhesion glycoproteins, CD11/CD18, intercellular adhesion molecule-1, and P-selectin (but not E-selectin) or with sialyl Lewis x, a counter-receptor for P-selectin. The mast cell stabilizer, lodoxamide, an H1- (but not an H2-) receptor antagonist, and diamine oxidase (histaminase) were also effective in reducing the LAE and albumin leakage elicited by Tx-A. The platelet-leukocyte aggregation response was blunted by an mAb against P-selectin, sialyl Lewis x, and the H1-receptor antagonist. These observations indicate that Tx-A induces a leukocyte-dependent leakage of albumin from postcapillary venules. Mast cell-derived histamine appears to mediate at least part of the leukocyte-endothelial cell adhesion and platelet-leukocyte aggregation by engaging H1-receptors on endothelial cells and platelets to increase the expression of P-selectin. The adhesion glycoproteins CD11/CD18 and intercellular adhesion molecule-1 also contribute to the inflammatory responses elicited by toxin A.
Asunto(s)
Toxinas Bacterianas/toxicidad , Clostridioides difficile/patogenicidad , Enterotoxinas/toxicidad , Histamina/fisiología , Vénulas/efectos de los fármacos , Animales , Adhesión Celular/efectos de los fármacos , Moléculas de Adhesión Celular/análisis , Moléculas de Adhesión Celular/fisiología , Leucocitos/efectos de los fármacos , Leucocitos/fisiología , Masculino , Ratas , Ratas Sprague-Dawley , Albúmina Sérica/metabolismo , Vénulas/fisiologíaRESUMEN
Ganglioside GD2 is expressed on membranes of human melanoma cells and induces antibody responses in patients with melanoma. In this study a new enzyme-linked immunosorbent assay was developed for detection of human antibody against GD2. A human IgM monoclonal anti-GD2 antibody was used for development of the assay. The antigen, GD2, was prepared from human brain. Four micrograms of GD2 was required to coat a well in a polystyrene microtiter plate. As little as 10 ng of antibody could be detected. The applicability of the assay for detection of serum anti-GD2 antibody was demonstrated with sera from patients immunized with GD2-positive melanoma cell vaccine.
Asunto(s)
Anticuerpos Monoclonales , Gangliósidos/análisis , Neoplasias/inmunología , Complejo Antígeno-Anticuerpo , Secuencia de Carbohidratos , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Epítopos/análisis , Gangliósidos/inmunología , Humanos , Leucemia Linfoide , Relación Estructura-ActividadRESUMEN
The anomeric specificity of six sialidases (Vibrio cholerae, Arthrobacter ureafaciens, Clostridium perfringens, Newcastle disease virus, fowl plague virus and influenza A2 virus sialidases) was assessed with sialylated antifreeze glycoprotein, ovine submandibular gland glycoprotein and alpha 1-acid glycoprotein, resialylated specifically in alpha(2-3) or alpha(2-6) linkage with N-acetylneuraminic acid or N-glycolylneuraminic acid using highly purified sialyltransferases. The rate of release of sialic acid from these substrates was found to correlate well with the specificity observed earlier with the same sialidases using small oligosaccharide substrates, i.e., alpha(2-3) glycosidic linkages are hydrolyzed faster than alpha(2-6) linkages, with the exception of the enzyme from A. ureafaciens. Sialidase activity was higher with N-acetylneuraminic acid when compared with N-glycolylneuraminic acid. The studies also showed that the core oligosaccharide and protein structure in glycoproteins may influence the rate of release for different glycosidic linkages.
Asunto(s)
Glicoproteínas/metabolismo , Neuraminidasa/metabolismo , Ácidos Siálicos/metabolismo , Arthrobacter/enzimología , Clostridium perfringens/enzimología , Virus de la Influenza A/enzimología , Virus de la Enfermedad de Newcastle/enzimología , Relación Estructura-Actividad , Especificidad por Sustrato , Vibrio cholerae/enzimologíaRESUMEN
OBJECTIVES: Selectins are important adhesion molecules which utilize a carbohydrate ligand such as sialyl Lewisx (SLex). Our objective was to study the effects of a liposome-conjugated SLex (Lipo-SLex) in myocardial ischaemia (MI) and reperfusion (R) injury in order to further clarify the actions of this carbohydrate. METHODS: We studied the efficacy of Lipo-SLex in a feline model of MI (90 min) and R (270 min) injury in vivo. Lipo-SLex (400 micrograms SLex/kg, iv) was administered intravenously 10 min prior to R. We also utilized an in vitro system of neutrophil adherence to thrombin-stimulated coronary endothelium to validate the efficacy of Lipo-SLex. RESULTS: Lipo-SLex significantly attenuated myocardial necrosis (8.6 +/- 1.2 vs. 29.5 +/- 3.1% of area-at-risk, P < 0.01) and plasma creatine kinase activities (P < 0.01) compared to vehicle (liposome alone). Moreover, endothelium-dependent relaxation to acetylcholine and A23187 in ischaemic-reperfused coronary rings obtained from cats treated with Lipo-SLex was significantly preserved compared to cats given liposomes without SLex (P < 0.01). After reperfusion, ex vivo PMN adherence to ischaemic-reperfused coronary endothelium was significantly increased in vehicle-treated cats, however, this was significantly attenuated in Lipo-SLex-treated cats (82 +/- 7 vs. 28 +/- 3 PMNs/mm2, P < 0.01). Myeloperoxidase activity in the ischaemic myocardium, a marker of PMN accumulation, was also significantly attenuated in Lipo-SLex-treated cats compared to liposomes without SLex (P < 0.01). CONCLUSIONS: Liposome-conjugated SLex-oligosaccharide attenuates myocardial necrosis and preserves coronary endothelial function following MI/R in vivo. The mechanism appears to be mediated by inhibition of the initial PMN-endothelial interaction and eventual accumulation into the ischaemic cardiac tissue. The liposome-SLex complex may be an efficient drug formulation for acute inflammatory diseases.
Asunto(s)
Isquemia Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/prevención & control , Oligosacáridos/administración & dosificación , Acetilcolina/farmacología , Animales , Calcimicina/farmacología , Gatos , Adhesión Celular/efectos de los fármacos , Creatina Quinasa/sangre , Portadores de Fármacos , Endotelio Vascular/efectos de los fármacos , Técnicas In Vitro , Liposomas , Masculino , Isquemia Miocárdica/enzimología , Daño por Reperfusión Miocárdica/enzimología , Miocardio/enzimología , Neutrófilos/fisiología , Peroxidasa/metabolismo , Antígeno Sialil Lewis X , Trombina/farmacología , Vasodilatación/efectos de los fármacosRESUMEN
The intracellular distribution of galactosyl- and sialyltransferase was investigated in rat hepatocytes of intact liver, primary monolayer cultures of freshly isolated hepatocytes, in a nontumorigenic hepatocyte cell line and in a hepatoma cell line. The two glycosyltransferases were detected by immunofluorescence using affinity-purified rabbit antibodies. Indirect double immunofluorescence showed that both terminal glycosyltransferases were identically codistributed in the same cell. This codistribution was always observed regardless of the cell type investigated, and in both stationary and migrating cells. The immunofluorescence pattern for both galactosyl- and sialyltransferase was found to be different in hepatocytes in vivo compared to hepatocytes grown in vitro. In hepatocytes of intact liver a spot-like cytoplasmic fluorescence was observed, whereas in cultured normal hepatocytes a perinuclear fluorescence from which an extensive tubular network radiated far into the cytoplasm existed. Cultured hepatoma cells also exhibited an extensive cytoplasmic fluorescence, which in contrast to the normal hepatocytes was rather diffuse. We conclude that (a) galactosyl- and sialyltransferase are codistributed in rat hepatocytes, and (b) a reorganization of (trans) Golgi apparatus elements containing both terminal glycosyltransferases occurs under conditions of in vitro growth and malignant transformation.
Asunto(s)
Galactosiltransferasas/análisis , Aparato de Golgi/enzimología , Hígado/enzimología , Sialiltransferasas/análisis , Animales , Línea Celular , Transformación Celular Neoplásica , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Galactosiltransferasas/inmunología , Hígado/ultraestructura , Ratas , Ratas Endogámicas , Sialiltransferasas/inmunologíaRESUMEN
Influenza viruses of contrasting receptor specificity have been examined for their ability to infect receptor-modified MDCK cells containing sialyloligosaccharide receptor determinants of defined sequence. Cells were treated with sialidase to remove sialic acid and render them resistant to infection and were then incubated with sialyltransferase and CMP-sialic acid to restore sialic acid in the SA alpha 2,6Gal or SA alpha 2,3Gal linkages. The viruses A/RI/5 + /57 and A/duck/Ukraine/1/63, previously shown to exhibit preferential binding of SA alpha 2,6Gal and SA alpha 2,3Gal linkages, respectively, were found to exhibit differential infection of the receptor-modified cells in accord with their receptor specificity. Coinfection of SA alpha 2,3Gal derivatized cells with a mixture of the two viruses resulted in selective propagation of the SA alpha 2,3Gal-specific A/duck/Ukraine/1/63 virus. The results demonstrate the potential for cell surface receptors to mediate selection of receptor-specific variants of influenza virus.
Asunto(s)
Virus de la Influenza A/metabolismo , Oligosacáridos/metabolismo , Receptores Virales/metabolismo , Adsorción , Animales , Anticuerpos Antivirales/análisis , Sitios de Unión , Línea Celular , Perros , Eritrocitos/microbiología , Hemaglutininas Virales , Virus de la Influenza A/inmunología , Riñón , Neuraminidasa/metabolismo , Receptores Virales/genética , Receptores Virales/inmunología , Ácidos Siálicos/metabolismo , Sialiltransferasas/metabolismo , Especificidad de la Especie , Proteínas Virales/análisis , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
The complement of sialyloligosaccharides present on the surface of human tracheal epithelium has been implicated as an important factor in the selection of hemagglutinin receptor specificity of human influenza A virus. Human strains of influenza A virus preferentially recognize host cell receptors bearing SA alpha 2,6Gal sequences, a sequence which is found on the surface of ciliated tracheal epithelium. A fluorescently-labelled H3 human virus strain bound avidly to the apical surface of human tracheal epithelium, while a fluorescently-labelled receptor variant strain, which preferentially binds SA alpha 2,3Gal sequences, showed little binding to the epithelial surface and localized primarily to intracellular mucin droplets. Extracts of human bronchial mucin, which is known to contain sialic acid primarily in the SA alpha 2,3Gal linkage, was a potent inhibitor of the binding of the receptor variant strain to trachea sections, while the binding of the parent strain was unaffected by the presence of mucin. Human bronchial mucin also inhibited the binding of the receptor variant strains, but not the parent virus strains, to human erythrocytes derivatized to contain SA alpha 2,6Gal sequences. These results suggest that a combination of selection pressures present in the respiratory tract environment have resulted in the evolution of a hemagglutinin receptor specificity in human influenza A virus strains which optimizes recognition of, binding to and infection of host cells.
Asunto(s)
Hemaglutininas Virales/metabolismo , Oligosacáridos/metabolismo , Orthomyxoviridae/metabolismo , Receptores Virales/metabolismo , Tráquea/metabolismo , Secuencia de Carbohidratos , Epitelio/metabolismo , Epitelio/microbiología , Hemaglutinación/efectos de los fármacos , Glicoproteínas Hemaglutininas del Virus de la Influenza , Histocitoquímica , Humanos , Virus de la Influenza A/crecimiento & desarrollo , Virus de la Influenza A/metabolismo , Lectinas/metabolismo , Datos de Secuencia Molecular , Mucinas/farmacología , Orthomyxoviridae/crecimiento & desarrollo , Tráquea/microbiologíaRESUMEN
Neuraminidase substrates suitable for analysis of linkage specificity were enzymically synthesized in good yield by linking N-acetylneuraminic acid (Neup5Ac) to O-6 and O-3 of 4-nitrophenyl beta-D-galactopyranoside with beta-D-galactoside-alpha-(2----6)-sialyltransferase and beta-D-galactoside-alpha-(2----3)-sialyltransferase, respectively. By use of these substrates, a convenient colorimetric assay method was developed for the determination of linkage specificity of bacterial and viral neuraminidases. The substrates are incubated with viral or bacterial neuraminidase and subsequently treated with beta-D-galactosidase to convert the liberated 4-nitrophenyl beta-D-galactopyranoside to 4-nitrophenol. The amount of liberated 4-nitrophenol is equivalent to the amount of Neup5Ac released from the substrate, thus allowing measurement of neuraminidase activity. The results showed that bacterial and viral neuraminidases can discriminate between these two compounds, making them useful substrates for the rapid determination of neuraminidase linkage specificity.
Asunto(s)
Colorimetría/métodos , Neuraminidasa/análisis , Ácidos Siálicos/química , Sialiltransferasas/metabolismo , Bacterias/enzimología , Ácidos Siálicos/metabolismo , Especificidad por Sustrato , Virus/enzimología , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
The conformational properties of several sialyloligosaccharides present as terminal sequences in N- and O-linked carbohydrate groups of glycoproteins, have been analyzed based on the n.m.r. data of selected sialosides. The compounds examined include representatives of the alpha-D-NeuAc-(2----6)-beta-D-Gal-(1----4)-beta-D-GlcNAc, alpha-D-NeuAc-(2----3)-beta-D-Gal-(1----4)-beta-D-GlcNAc, alpha-D-NeuAc-(2----3)-beta-D-Gal-(1----3)-beta-D-GlcNAc, and alpha-D-NeuAc-(2----3)-beta-D-Gal-(1----3)-beta-D-GalNAc series. Two deuterated sialosides were prepared by enzymic sialylation of 6-deuterated galactose derivatives of methyl beta-D-galactopyranoside and lactoside. These were useful for the unambiguous establishment of the "gt" orientation of the flexible C-6 methylene unit of the galactose through 1H-1H coupling constants. Of all the (2----6) sialosides examined, only the deuterated di- and tri-saccharide afforded useful nuclear Overhauser enhancement data that could be used to evaluate the global minimum-energy conformations. Hard-sphere exoanomeric effect calculations estimated the glycosidic torsion angles for the global minimum-energy conformer of alpha-D-NeuAc-(2----6)-beta-D-Gal linkages to be -163/-132/61 degrees (theta, psi, and omega, respectively). However, the potential energy well surrounding this global minimum was very shallow and indicated a broad population distribution of conformers. These are illustrated by the isoenergy contour maps. The observation of n.O.e. between the H-3ax and H-6R of the galactose in two deuterated (2----6) sialosides, indeed supported the presence of one of the global minimum-energy conformers. The conformational analysis carried out for the di- and trisaccharide [alpha-D-NeuAc-(2----6)-beta-D-Gal-OMe and alpha-D-NeuAc-(2----6)-beta-D-Gal-(1----4)-beta-D-Glc-OMe respectively] was then extended to sialoside linkages of other tri- and penta-saccharides by comparison of their 1H- and 13C-n.m.r. chemical shifts. HSEA calculations for the (2----3) sialosides indicated the potential energy well containing the global minimum energy-conformer (theta, psi = -160 +/- 4, -11 +/- 2 degrees) was deeper than the one estimated for the (2----6) sialosides. The n.O.e. data are consistent with the distribution of the majority of conformers around the lowest-energy one in solution. CPK models highlighting the topographical differences between the lowest-energy conformations of alpha-(2----6) and alpha-(2----3) sialosides are presented.
Asunto(s)
Oligosacáridos/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Hidrógeno/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Nitrógeno/químicaRESUMEN
Linear and branched glycopeptides containing multiple sialyl-N-acetyllactosamine side chains have been synthesized using a combined chemical and enzymatic approach. Peptide backbones in which beta-GlcNAc-Asn residues were incorporated were obtained in good yields by optimized solid-phase synthesis following the Boc strategy. The resulting multivalent glycopeptides were galactosylated in near-quantitative yields using bovine galactosyltransferase, UDP-galactose, and calf alkaline phosphatase that destroys the inhibiting side product UDP. Subsequent enzymatic sialylation yielded the desired glycopeptides containing asparagine-linked sialyl-N-acetyllactosamine side chains. The compounds were characterized by 1H NMR and FABMS. Recombinant sialyltransferase and CMP-sialate synthetase were used for the enzymatic synthesis of sialosides on a preparative scale. The synthetic glycopeptides were tested as inhibitors of influenza virus to cells, revealing that most of the multivalent sialoglycopeptides exhibit increased binding that depends on the spacing when compared to monovalent compounds. A possible mechanism for increased binding is proposed.