RESUMEN
We have isolated a genomic clone from Nicotiana tabacum, designated Nt-PHYB-1, encoding a type-II, "green tissue" phytochrome apoprotein. Recombinant genes, consisting of the 3319-bp promoter of the Nt-PHYB-1 gene (including the entire 5[prime] untranslated sequence but not the ATG) or its deletion derivatives and the bacterial [beta]-glucuronidase reporter gene, were constructed and transferred into tobacco. The expression patterns and levels of the endogenous Nt-PHYB-1, as well as those of the transgenes, were determined by RNase protection assays and by [beta]-glucuronidase histochemical staining. We show that (a) the PHYB-1 gene has three transcription start sites, (b) the abundance of the three PHYB-1-specific mRNAs is different, and that (c) it is not regulated by light. However, we do demonstrate that transcription of the endogenous PHYB-1 gene and that of the recombinant genes exhibit a well-defined organ and tissue specificity. This tobacco PHYB gene is relatively highly expressed in leaf, stem, and different floral organs but not in root. Deletion analysis of the Nt-PHYB-1 promoter indicates that a 382-bp region, located between -1472 and -1089, is required for high-level expression of this gene.
RESUMEN
We have measured the ability of pancreatic duct epithelial cells (DEC) from Syrian hamsters and humans and CK cells, immortalized hamster DEC, to metabolize chemical carcinogens to species that were mutagenic in S. typhimurium TA98 and in V79 cells. The chemicals were N-nitrosobis(2-oxopropyl)amine (BOP), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The ability of ethanol (EtOH) to modify the metabolizing efficiency was also measured. When an S9 preparation from EtOH-treated CK cells was used to metabolize NNK the number of revertants was 271 +/- 73 compared with 17 +/- 2 when the S9 from control CK cells was used. When hamster DEC were used there was no increase in the mutation frequency for BOP in V79 cells (64 +/- 20 mutants/10(6) survivors per mumol) when EtOH-DEC were used. However, the mutation frequencies of NNK and PhIP rose when the EtOH-treated DEC were used from 62 +/- 31 to 198 +/- 28 mutants/10(6) survivors per mumol for NNK and from 94 +/- 25 to 166 +/- 25 mutants/10(6) survivors per mumol for PhIP. A similar result was obtained when human DEC were used, i.e. no change in BOP mutagenicity and a slight increase in PhIP mutagenicity, from 34 +/- 14 to 65 +/- 12 mutants/10(6) survivors per mumol. There were large increases in the mutagenicity of NNK with each of the three samples of human DEC that were used, from 75 +/- 0 to 213 +/- 38, 75 +/- 13 to 175 +/- 25 and 38 +/- 13 to 285 +/- 25 mutants/10(6) survivors per mumol. The EtOH treatment regimen that was used more closely mimicked chronic exposure at low concentrations in vivo. These data show that hamster DEC are capable of metabolizing NNK, which is carcinogenic in these cells in vivo. Furthermore, human DEC metabolized NNK as efficiently as hamster DEC.
Asunto(s)
Carcinógenos , Mutágenos , Nitrosaminas , Conductos Pancreáticos/metabolismo , Adolescente , Adulto , Animales , Biotransformación , Células Cultivadas , Niño , Cocarcinogénesis , Cricetinae , Células Epiteliales/metabolismo , Etanol/farmacología , Femenino , Humanos , Imidazoles/farmacología , Masculino , Mesocricetus , Pruebas de Mutagenicidad , Conductos Pancreáticos/citología , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genéticaRESUMEN
The presence of P-glycoprotein (P-gp) and multiple drug resistance-associated protein (MRP) was examined in four human pancreatic adenocarcinoma cell lines (PANC-1, BxPC-3, AsPC-1, and Capan-1). Cellular accumulation of rhodamine 123 and [3H]vincristine were used to determine functional activity of P-gp and MRP, respectively. None of the cells showed any evidence of P-gp in the rhodamine 123 cellular accumulation assays. In contrast, PANC-1, BxPC-3 and AsPC-1 did display an increased accumulation of [3H]vincristine following treatment with either cyclosporin A or verapamil. Western blot analysis confirmed the expression of MRP, and little, if any, measurable P-gp in the cell lysates. These studies suggest that intrinsic drug resistance in pancreatic duct cancer may be due in part to the presence of MRP.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenocarcinoma/metabolismo , Resistencia a Múltiples Medicamentos , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Antineoplásicos Fitogénicos/farmacocinética , Resistencia a Antineoplásicos , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Células Tumorales Cultivadas , Vincristina/farmacocinéticaRESUMEN
The activation of 3H-labeled N-nitrosobis(2-oxopropyl)amine [( 3H]BOP) by pancreas acinar and duct tissue from Syrian hamsters and MRC-Wistar rats in vitro was measured as DNA alkylation. Hamster tissue was incubated with [3H]BOP (0.1 mM; 20 microCi/ml) for 2 h. Initial levels of alkylation were similar, 41.7 +/- 3.7 (acinar) and 51.5 +/- 7.8 (duct) dpm/micrograms DNA. Alkylation persisted for longer in duct (t/2 greater than 46 h) than in acinar tissue (t/2 = 6 h). The faster repair of alkylation in acinar tissue was not due to acinar cell death. In rat duct tissue the level of alkylation 2 h after incubation (38.9 +/- 4.5 dpm/micrograms DNA) was similar to that in hamster ducts but declined more rapidly (t/2 = 27 h). Hamster and rat acinar and duct tissue was incubated with BOP followed by [3H]thymidine to measure DNA synthesis. BOP stimulated DNA synthesis in hamster but not in rat duct tissue or hamster acinar tissue. These data support the hypothesis that the duct tissue is the target tissue for BOP in Syrian hamsters.
Asunto(s)
Carcinógenos , Nitrosaminas , Páncreas/citología , Alquilación , Animales , Cricetinae , ADN/biosíntesis , Técnicas In Vitro , Masculino , Mesocricetus , Páncreas/metabolismo , Conductos Pancreáticos/citología , Conductos Pancreáticos/metabolismo , Ratas , Ratas Endogámicas , TritioRESUMEN
A procedure is described for the isolation and growth in vitro of epithelial cells from the duct network of human pancreas, referred to as DEC. A significant advantage of our procedure over previously published procedures is that it enables the isolation of DEC from small pieces of pancreas tissue (< 5 g) and, also, from the digest remaining after the isolation of islet cells from human pancreas, material that would normally be discarded. These were the only reliable sources for pancreas tissue available to us. This procedure shows that some of the techniques that have been successfully used for the isolation of rodent DEC are also valuable in the isolation of human DEC. In particular, the use of cholera toxin to prevent fibroblast growth and contamination obviates the need for the time-consuming procedure of physically removing fibroblasts or the use of expensive fibroblast-specific monoclonal antibodies. The use of sieving to separate the digest immediately achieves a partial purification, which, coupled with that of allowing duct cysts to form, adds to the purity of the final preparation. The ductal system of the intact pancreas tissue and the DEC derived from it expressed cytokeratins 7, 8/18, and 19 and markers for the presence of MUC1, CFTR, and carbonic anhydrase II, which are specific for ductal epithelial cells or for pancreatic ductal functions. This study showed that it is possible to obtain selectively viable DEC from small ducts in otherwise waste pieces of human pancreas. It showed that these cells retained all of the epithelial characteristics that were examined and, in combination with data from an earlier study, showed that the cultured DEC retain the metabolic functions of duct epithelial cells in vivo.
Asunto(s)
Conductos Pancreáticos/citología , Anticuerpos Monoclonales , Anhidrasas Carbónicas/análisis , Diferenciación Celular , Separación Celular/métodos , Células Cultivadas , Células Epiteliales/citología , Humanos , Inmunohistoquímica , Insulina/análisis , Queratinas/análisis , Mucina-1/análisis , Mucinas/análisis , Páncreas/química , Trasplante de Páncreas , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ADN Polimerasa Dirigida por ARNRESUMEN
The heterocyclic amines (HA) 2-aminodipyrido[1,2-a:3',2-d]imidazole (Glu-P-2), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were mutagenic in V79 cells (Chinese hamster lung fibroblasts) using 6-thioguanine resistance as the marker of mutagenicity. Pancreas duct epithelial cells (DEC) from untreated hamsters, homogenates of pancreas ducts from untreated hamsters and those fed a high fat diet and human DEC were used to activate the heterocyclic amines. When hamster cells and tissues were used the optimum mutation frequencies (mutants/10(6) survivors) measured were: Glu-P-2, 10 +/- 1; MeIQ, 28 +/- 2 (DEC), 12 +/- 2 (control, duct homogenate), and 21 +/- 2 (high fat diet fed, duct homogenate); PhIP, 61 +/- 5. When human DEC were used the optimum mutation frequencies were: MeIQ, 32 +/- 4; PhIP, 35 +/- 3. 3,8-Dimethylimidazo[4,5-f]quinoxaline, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole and 3-amino-1-methyl-5H-pyrido[4,3-b]indole were not mutagenic in this assay.
Asunto(s)
Aminas/farmacología , Compuestos Heterocíclicos/farmacología , Mutágenos/farmacología , Conductos Pancreáticos/efectos de los fármacos , Adolescente , Adulto , Animales , Carbolinas/farmacología , Células Cultivadas , Cricetinae , Humanos , Imidazoles/farmacología , Pulmón/citología , Masculino , Conductos Pancreáticos/citología , Quinolinas/farmacologíaRESUMEN
Pancreas duct epithelial cells (DEC), isolated from hamsters and cultured for up to 25 days, were able to metabolize N-nitrosobis(2-oxopropyl)amine (BOP) to species that were mutagenic in V79 cells. There was no decline in the nitrosamine-activating ability of DEC over the period of observation (25 d). DEC activated N-nitrosobis(2-hydroxypropyl)amine (BHP), N-nitrosodiethylamine (DEN), N-nitrosodimethylamine (DMN) and N-nitrosomethyl(2-oxopropyl)amine (MOP) and BOP in the same assay, although the mutation frequencies for BHP, DEN and DMN were barely different from that for the controls (4 +/- 1 mutants/10(6) cells). The mutation frequencies for a dose of 0.1 mM were BHP, 2 +/- 1; BOP, 113 +/- 7; DEN, 8 +/- 1; DMN, 5 +/- 2; and MOP, 18 +/- 3 (mutants/10(6) cells; means +/- SE). When hepatocytes were used the mutation frequencies were BHP, 3 +/- 1; BOP, 60 +/- 3; DEN, 8 +/- 2; DMN, 8 +/- 2; and MOP, 121 +/- 10. BOP was toxic to the DEC at doses above 0.1 mM. Experiments in which co-factors were omitted from the medium suggested that an isoform(s) of the cytochrome P-450 IIIA family was involved, directly or indirectly, in BOP activation.
Asunto(s)
Mutágenos/toxicidad , Nitrosaminas/toxicidad , Conductos Pancreáticos/metabolismo , Análisis de Varianza , Animales , Biotransformación , Línea Celular , Cricetinae , Medios de Cultivo , Dexametasona/farmacología , Dietilnitrosamina/toxicidad , Dimetilnitrosamina/toxicidad , Hígado/citología , Hígado/metabolismo , Masculino , Mesocricetus , Pruebas de Mutagenicidad , Mutágenos/farmacocinética , Nitrosaminas/farmacocinética , Conductos Pancreáticos/citologíaRESUMEN
The mutagenicity of N-nitrosobis(2-oxopropyl)amine was measured in the V79 assay using homogenates of acinar cells and duct tissue from the pancreases of Syrian hamsters and MRC-Wistar rats as the activating systems. Mutations at the sodium/potassium ATPase and hypoxanthine:guanine phosphoribosyltransferase loci were measured by resistance to ouabain and 6-thioguanine (TG). The order of effectiveness in generating mutagens from BOP was hamster duct, hamster acinar, rat duct, rat acinar. These data show extensive differences in BOP activation by hamster acinar and duct tissue.
Asunto(s)
Mutación , Nitrosaminas/toxicidad , Páncreas/metabolismo , Conductos Pancreáticos/metabolismo , Animales , Biotransformación , Cricetinae , Resistencia a Medicamentos/genética , Hipoxantina Fosforribosiltransferasa/genética , Hipoxantina Fosforribosiltransferasa/metabolismo , Masculino , Mesocricetus , Pruebas de Mutagenicidad , Nitrosaminas/farmacocinética , Ouabaína/farmacología , Páncreas/citología , Ratas , Ratas Endogámicas , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Tioguanina/farmacologíaRESUMEN
We have shown a role for glutathione (GSH) in the detoxification of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) using mutagenicity in V79 cells as the end-point. Immortalized hamster pancreas duct epithelial cells (CK cells) were used to metabolize PhIP in this assay. Intracellular GSH concentrations were lowered by treatment with buthionine sulfoximine (BSO) and were raised by treatment with sodium sulfite. BSO treatment (10 mM, 4 h) reduced the GSH concentration in V79 cells from 18 +/- 1 to 6 +/- 1 nmol/mg protein, 4 h after treatment. The mutation frequency of PhIP in these V79 cells rose from 15 +/- 2 to 34 +/- 4 mutants/10(6) survivors in BSO-treated V79 cells. In a related experiment both CK and V79 cells were treated with sulfite. Sulfite treatment (2 mM, 4 h) produced a greater reduction in PhIP mutagenicity when the V79 cells were treated with sulfite (from 15 +/- 2 to 3 +/- 1 mutants/10(6) survivors) than when the CK cells were treated (from 15 +/- 2 to 7 +/- 2 mutants/10(6) survivors). These data show a relationship between intracellular GSH concentration and the mutagenicity of PhIP.
Asunto(s)
Glutatión/fisiología , Imidazoles/toxicidad , Conductos Pancreáticos/citología , Conductos Pancreáticos/metabolismo , Animales , Butionina Sulfoximina/farmacología , Células Cultivadas , Cricetinae , Inhibidores Enzimáticos/farmacología , Células Epiteliales , Epitelio/efectos de los fármacos , Glutatión/metabolismo , Imidazoles/farmacocinética , Pruebas de Mutagenicidad , Mutágenos/farmacocinética , Mutágenos/toxicidad , Conductos Pancreáticos/efectos de los fármacos , Sulfitos/farmacologíaRESUMEN
Synthesis of 3-O-acetyl-2-benzyloxycarbonylamino-2-deoxy-4,6-O-ethylidene- alpha-(7 alpha) and-beta-D-glucopyranose (7 beta) and their 3-O-chloroacetyl analogues (11 alpha and 11 beta) are described. Condensation (BF3-etherate, ethyl acetate, -20 degrees) of 7 alpha with 4'-O-benzyloxycarbonyl-4'-O-demethyl-4-epipodophyllotoxin (8) afforded mainly the beta-glycoside 9 beta (alpha, beta-ratio 1:9). Condensation of 11 alpha beta with 8 or the 4'-O-chloroacetyl analogue 13 gave mainly the 4-O-(2-benzyloxycarbonylamino-3-O-chloroacetyl-2-deoxy-4,6-O-ethyl idene-beta-D- glucopyranosyl)-epipodophyllotoxin 12 beta or 15 beta. Glycosidation of podophyllotoxin (14) with 11 alpha beta (during which the aglycon epimerized at C-4 under the action of BF3-etherate) afforded alpha- (16 alpha) and beta-glycoside (16 beta) in the ratio 1:5. Removal of the chloroacetyl groups from 12 beta, its alpha analogue 12 alpha, and 15 beta gave the 4-O-(2-benzyloxycarbonylamino-2-deoxy-4,6-O-ethylidene-alpha-(17 alpha) and -beta-D-glucopyranosyl)-4'-O-demethyl-epipodophyllotoxins (17 beta and 20 beta), respectively. Hydrogenolysis of the benzyloxycarbonyl groups then gave 4-O-(2-amino-2-deoxy-4,6-O-ethylidene-alpha- (18 alpha) and -beta-D-glucopyranosyl)-4'-O-demethyl-4-epipodophyllotoxin (18 beta). Reductive alkylation of 18 beta and 18 alpha afforded the 2"-deoxy-2"-dimethylamino-etoposide 3 and its alpha analogue 19 alpha.
Asunto(s)
Etopósido/síntesis química , Conformación de Carbohidratos , Etopósido/química , Espectroscopía de Resonancia Magnética , Métodos , Estructura Molecular , Rotación ÓpticaRESUMEN
Syntheses and structure-activity relationships of 7-O-(3-amino-2,3,6-trideoxy-a-L-lyxo- (18), -L-arabino- (20) and -L-ribo- hexopyranosyl)-epsilon-isorhodomycins (25) and their 3'-dimethylamino derivatives 22, 23 and 26 are described. Condensation (trimethylsilyl triflate, molecular sieves 4 A, 10:1 dichloromethane-acetone, -15 degrees) of epsilon-isorhodomycinone (epsilon-isoRMN, 6) with 1,5-anhydro-4-O-p-nitrobenzoyl-3-trifluoroacetamido-L-lyxo- (5) -L-arabino- (9) or -L-ribo-hex-l-enitols (10) afforded mainly the 7-O-a-glycosyl-epsilon-isoRMNs 7, 11, and 12. Similar glycosylation of 6 with 1,5-anhydro-3-azido-4-O-p-nitrobenzoyl-2,3,6-trideoxy-L-arabino-hex-1-++ +enitol (15) yielded a-glycoside 16. Removal (M NaOH) of the p-nitrobenzoyl and trifluoroacetyl groups from 7, 11, and 12 gave the 7-O-(3-amino-2,3,6-trideoxy-a-L-hexopyranosyl)-epsilon-isoRMNs 18, 20, and 25. Reductive alkylation (CH2O, NaCNBH3) of these products afforded the 3'-N,N-dimethyl analogues 22, 23, and 26. The cytotoxic effect (IC50) of the semisynthetic epsilon-isorhodomycins was tested in vitro in leukemia cell line L1210.
Asunto(s)
Antraciclinas , Antibióticos Antineoplásicos/síntesis química , Animales , Secuencia de Carbohidratos , Glicosilación , Metilación , Datos de Secuencia Molecular , Estructura Molecular , Oxidación-Reducción , Relación Estructura-Actividad , Ensayo de Tumor de Célula MadreRESUMEN
Glycosylation of benzyl-2,3,6-trideoxy-3-trifluoroacetamido-alpha-L-lyxo-h exopyranoside (6) with 3,4-di-O-acetyl-1,5-anhydro-2,6-dideoxy-L-lyxo-hex-1-enitol (1) or 4-O-acetyl-1,5-anhydro-3-O-benzyl-2,6-dideoxy-L-lyxo-hex-1-enit ol (2) in the presence of trimethylsilyl triflate/triethylamine gave alpha-(1----4)-linked disaccharide derivatives 7 and 8, respectively. In the presence of trimethylsilyl triflate only, 3,4-di-O-acetyl-1-O-tert-butyldimethylsilyl-2,6-dideoxy-beta-L-lyxo++ +-hexopyranose (3) and 6 gave mainly 7. Condensation of 1 or 2 with 9, obtained by O-deacylation of 8, afforded benzyl [O-(3,4-di-O-acetyl-2,6-dideoxy-alpha-L-lyso-hexopyranosyl)-(1----4)-O-( 3-O-benzyl-2,6-dideoxy-2,6-alpha-L-hexopyranosyl)-(1----4)-(2,3,6-tri deo xy-3-trifluoroacetamido-alpha-L-lyxo-hexopyranoside)] (11) and its 3''-benzyl analogue 12, respectively.
Asunto(s)
Antibióticos Antineoplásicos/síntesis química , Oligosacáridos/síntesis química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Disacáridos/síntesis química , Glicosilación , Datos de Secuencia Molecular , Estructura Molecular , Trisacáridos/síntesis químicaRESUMEN
Synthesis of 7-O-(3-amino-2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)-epsilon-(i so)rhodomycinones 16 and 17, and their 3'-morpholino derivatives are described. Glycosylation (trimethylsilyl triflate, 10:1 dichloro-methane-acetone, -35 degrees) of 1-O-tert-butyldimethylsilyl-2,3-6-trideoxy-4-O-p-nitrobenzoyl-3-trifl uoroacetamido-beta-L-lyxo-hexopyranose (4) with epsilon-rhodomycinone (epsilon-RMN, 5) or epsilon-isorhodomycinone (epsilon-isoRMN, 6) afforded 7-O-alpha-glycosyl-epsilon-RMN (9) and -epsilon-isoRMN (12) in high yield. The glycosyl donors 2,3,6-trideoxy-4-O-p-nitrobenzoyl-3-trifluoroacetamido-L-lyxo++ +-hexopyrano se (2) or its 1-O-trimethylsilylated alpha-anomer 3 were less suitable for the glycosylation of these aglycons. Sapinification of 9 and 12 provided 16 and 17, respectively, which reacted with various 2,2'-oxydiacetaldehydes under conditions of reductive alkylation to give 3'-morpholinyl-epsilon-(iso)rhodomycins.
Asunto(s)
Antibióticos Antineoplásicos/síntesis química , Antraciclinas , Antibióticos Antineoplásicos/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Glicosilación , Datos de Secuencia Molecular , Estructura MolecularRESUMEN
CONTEXT: We have isolated five stable clones from a primary culture of Syrian golden hamster pancreatic duct epithelial cells and have designated them as CK1 through CK5. DESIGN: Here we describe the ability of two of these, CK1 and CK5, to metabolize the pancreas carcinogen N-nitrosobis(2-oxopropyl)amine. The metabolism was assessed as the production of mutated V79 cells in a CK cell/V79 co-culture set up. RESULTS: At a dose of 0.1 mM N-nitrosobis(2-oxopropyl)amine, the CK1 cells produced 82.3 +/- 17.2 mutants/1,000,000 survivors while the CK5 cells produced only 33.2 +/- 10.8 mutants/1,000,000 survivors, both are mean +/- SD (n = 8). Furthermore, both cell types responded differently to two inducers of cytochrome P450 activity, namely Arochlor 1254 and EtOH. Arochlor 1254 treatment did not affect the metabolizing ability of CK1 cells while EtOH treatment resulted in a twofold increase in the mutation frequency. Arochlor and EtOH treatment inhibited the ability of CK5 cells to metabolize N-nitrosobis(2-oxopropyl)amine. CONCLUSIONS: These data show that the duct epithelium of the pancreas is a multi-cellular tissue and the different cell types within the epithelium have different abilities to metabolize xenobiotic chemicals.
Asunto(s)
Carcinógenos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Nitrosaminas/metabolismo , Páncreas/metabolismo , Animales , Línea Celular , Células Cultivadas , Células Clonales/efectos de los fármacos , Células Clonales/enzimología , Células Clonales/metabolismo , Cricetinae , Cricetulus , Sistema Enzimático del Citocromo P-450/biosíntesis , Inducción Enzimática , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Masculino , Mesocricetus , Páncreas/citología , Páncreas/enzimología , Conductos Pancreáticos/citología , Conductos Pancreáticos/enzimología , Conductos Pancreáticos/metabolismoRESUMEN
Predicting which species are probable invaders has been a long-standing goal of ecologists, but only recently have quantitative methods been used to achieve such a goal. Although restricted to few taxa, these studies reveal clear relationships between the characteristics of releases and the species involved, and the successful establishment and spread of invaders. For example, the probability of bird establishment increases with the number of individuals released and the number of release events. Also, the probability of plant invasiveness increases if the species has a history of invasion and reproduces vegetatively. These promising quantitative approaches should be more widely applied to allow us to predict patterns of invading species more successfully.
RESUMEN
The carbohydrate structures determining the ABO blood group system have been almost completely characterized. Recent advances in the stereoselective synthesis of such complex structures have made it possible to prepare pure oligosaccharides in large quantities and to study their possible uses for diagnostics and pharmaceutical processing techniques. In the experiments described here A and B blood group specific determinants were synthesized and bound to solid phases by means of suitable spacers in order to study their use as immunoadsorbents. The objective was to adsorb blood group specific antibodies from positive sera and from immunoglobulin preparations. It was shown that the anti-A and anti-B antibodies bound to the immunoadsorbents with high affinity could be removed effectively. The effects were achieved both using affinity chromatography and "batch processing".
Asunto(s)
Sistema del Grupo Sanguíneo ABO/inmunología , Complejo Antígeno-Anticuerpo/síntesis química , Inmunoadsorbentes/síntesis química , Especificidad de Anticuerpos , Fenómenos Químicos , Química , HumanosRESUMEN
Light-induced expression of genes encoding the light-harvesting chlorophyll a/b binding proteins of photosystem II (Cab) was shown to be controlled by a circadian oscillator coupled to the red-light-absorbing plant photoreceptor phytochrome. Here we show that a red-light-insensitive oscillator is also involved in regulating the expression of the Cab genes. We provide evidence that germination leads, in a light-independent manner, to the setting and/or synchronization of endogenous oscillators and that it induces the expression of Cab genes in a circadian fashion. This circadian oscillator is not coupled to phytochrome, as it cannot be reset by red light for at least 44 h after sowing. Short red light pulses given between 12 and 44 h after sowing, however, induce new rhythms without perturbing the already free-running red-light-independent circadian oscillation. At this stage of development, the phytochrome-coupled and uncoupled circadian rhythms coexist. Both circadian rhythms are expressed and exhibit period lengths close to 24 h but are phased differently. At later stages of development (60 h or later after sowing), red light treatments synchronized these free-running rhythms and led to the appearance of a single new circadian oscillation. These data indicate that during early development the expression of single tobacco Cab genes, particularly expression of the Cab21 and Cab40 genes, is controlled in a developmentally dependent manner by two circadian oscillators.
Asunto(s)
Ritmo Circadiano , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Nicotiana/metabolismo , Proteínas del Complejo del Centro de Reacción Fotosintética/biosíntesis , Plantas Tóxicas , Secuencia de Bases , Luz , Complejos de Proteína Captadores de Luz , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Oscilometría , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Complejo de Proteína del Fotosistema II , Fitocromo/metabolismo , ARN Mensajero/biosíntesis , Factores de Tiempo , Nicotiana/genética , Nicotiana/crecimiento & desarrollo , Transcripción GenéticaRESUMEN
Monosaccharides, disaccharides, and trisaccharides were tested as inhibitors of the in vitro growth of Plasmodium falciparum (strain FCB). While certain monosaccharides (N-acetyl-D-glucosamine, D-mannose, and 3-O-methyl-D-glucose) proved to exhibit a toxic or reversibly retarding effect on the intraerythrocytic development of the parasite, the corresponding alpha- or beta-methylglycosides did not. Several methylglycosides, synthetic di- and tri-saccharides, and artificial blood group antigens were further tested for inhibitory effects on invasion of host red blood cells in vitro. The synthetic disaccharides beta DGlcNAc(1----4) alpha DManOMe and beta DGlcNAc(1----4) DGlcNAc (chitobiose) were good inhibitors of invasion at 10 mM concentration, whereas beta DGal(1----4)beta DGlcNAcOMe was negligibly inhibitory. The inhibition rate of N-acetyl-D-glucosamine, beta-glycosidically linked to bovine serum albumin (BSA) by an alipathic spacer, -(CH2)8CO-, was not enhanced, compared to the corresponding hapten, beta DGlcNAcO(CH2)8COOCH3. The inhibition rates of blood group A- and B-trisaccharide haptens, which were inhibitors of invasion, were also not significantly enhanced when coupled to BSA by way of the corresponding amide spacer, -(CH2)2NHCO(CH2)7CO-. A remarkable enhancement of the inhibition rate was, however, observed when beta DGal(1----3) alpha DGalNAcO(CH2)2NHCO(CH2)7COOCH3 (T-hapten) was coupled to BSA. A clear-cut decrease in the inhibition rates of different beta-glycosides of N-acetyl-D-glucosamine, beta DGlcNAcOR, was observed, depending on the nature of the aglycon R(p-nitrophenyl greater than -(CH2)8COOCH3 greater than -(CH2)2NHCO(CH2)2COOCH3 greater than -CH3). Also, p-nitrophenyl-alpha-D-glucopyranoside was a much better inhibitor of invasion than the corresponding methyl glycoside, alpha DGlcOMe, which was not inhibitory. The properties of the aglycon spacer, used for the covalent attachment of the carbohydrate to the carrier protein, may thus be crucial for the outcome of the inhibition rate.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Eritrocitos/parasitología , Plasmodium falciparum/metabolismo , Animales , Antígenos de Grupos Sanguíneos , Carbohidratos/farmacología , Disacáridos/farmacología , Haptenos , Humanos , Metilglicósidos/farmacología , Monosacáridos/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/crecimiento & desarrollo , Trisacáridos/farmacologíaRESUMEN
The lectin from jackfruit (Artocarpus integrifolia) reacts with the peanut-specific lectin receptor represented by the T-disaccharide, which is also part of the Thomsen (= T)-Friedenreich antigen, namely O-beta-D-galactosyl-(1----3)-N-acetyl-D-galactosamine. The difference between the jackfruit lectin and peanut lectin has been shown to be mainly due to the further linkage of the T-disaccharide: Whereas the peanut lectin reacts with both anomeric forms, jackfruit lectin and the closely - with respect to its specificity - related Agaricus bisporus mushroom lectin react predominantly with the alpha-linked T-disaccharide, so that they cannot detect the lipid-linked T-disaccharide, as the peanut lectin does.