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Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2 总被引:2,自引:1,他引:1
The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens. 相似文献
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Thirty-two patients with congenital neurosensory hearing loss demonstrated dilatation of the peripheral limb of the vestibular aqueduct as visualized on pluridirectional tomograms. Multiple additional anomalies involving most frequently the cochlea (15 patients) but also the ossicles (three patients) or vestibule (one patient) were associated. The axial-pyramidal (Poschl) projection was of great value in evaluation of the vestibular aqueduct and cochlea. The technique, clinical correlation, and surgical implications are discussed. 相似文献
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Crocenzi FA Basiglio CL Pérez LM Portesio MS Pozzi EJ Roma MG 《Biochemical pharmacology》2005,69(7):1113-1120
Estradiol-17beta-d-glucuronide (E(2)17G) and taurolithocholate (TLC) induce acute cholestasis-associated with retrieval of the bile salt export pump (Bsep), which parallels with alteration in transport activity. cAMP stimulates the apically directed vesicular trafficking of transporters, partially preventing these alterations. The hepatoprotector, silymarin, which inhibits cAMP-phosphodiesterase, prevents the cholestasis induced in vivo by both estrogens and TLC. We aimed to assess the ability of silibinin (Sil), the silymarin active component, to prevent the retrieval of Bsep induced by TLC and E(2)17G, and the associated alteration in its transport function. The possible involvement of cAMP as a second messenger and the intracellular signalling pathways implicated were also evaluated. Functional studies were performed analysing the proportion of isolated rat hepatocyte couplets (IRHC) accumulating the fluorescent bile salt analogue, cholyl-lysylfluorescein (CLF), into their sealed canalicular vacuoles. Cellular localisation of Bsep was assessed by immunofluorescent staining. Intracellular levels of cAMP were measured by ELISA. Sil (2.5microM) elevated by 40+/-3% intracellular cAMP, and mimicked the ability of dibutyryl-cAMP (10microM) to prevent internalisation of Bsep and the TLC (2.5microM)- and E(2)17G (50microM)-induced impairment in the capacity of IRHC to accumulate CLF apically. Preventive effects of Sil and dibutyryl-cAMP were not abolished by the specific protein kinase A inhibitors, KT5720 and H89. Contrarily, the intracellular Ca(2+) chelator, BAPTA/AM, significantly blocked the protective effect of both compounds. We conclude that Sil prevented TLC- and E(2)17G-induced bile salt secretory failure, at least in part, by avoiding redistribution of Bsep, by a mechanism probably involving cAMP-induced cytosolic Ca(2+) elevations. 相似文献