RESUMO
CASE REPORT: We report the case of a 77-year-old man who was admitted to the intensive care unit with a serum digoxin level of 45.9 ng/mL. The patient was hemodynamically stable throughout his hospital course and did not require antidigoxin antibody fragments. The elevated digoxin level was determined by subsequent testing to be falsely elevated by interference from human antimouse antibodies in his serum. A repeat digoxin measurement using an assay not affected by human antimouse antibodies indicated a level of 1.3 ng/mL. Newer digoxin assays are not affected by human antimouse antibody interference, but clinicians should be aware of possible human antimouse antibody interference with older digoxin assays and in other tests utilizing mouse monoclonal antibody reagents.
Assuntos
Anticorpos Heterófilos/sangue , Anticorpos Monoclonais/sangue , Digoxina/sangue , Idoso , Animais , Reações Cruzadas , Interações Medicamentosas , Reações Falso-Positivas , Humanos , Masculino , CamundongosRESUMO
A purine-rich splicing enhancer from a constitutive exon has been shown to shift the alternative splicing of calcitonin/CGRP pre-mRNA in vivo. Here, we demonstrate that the native repetitive GAA sequence comprises the optimal enhancer element and specifically binds a saturable complex of proteins required for general splicing in vitro. This complex contains a 37-kDa protein that directly binds the repetitive GAA sequence and SRp40, a member of the SR family of non-snRNP splicing factors. While purified SR proteins do not stably bind the repetitive GAA element, exogenous SR proteins become associated with the GAA element in the presence of nuclear extracts and stimulate GAA-dependent splicing. These results suggest that repetitive GAA sequences enhance splicing by binding a protein complex containing a sequence-specific RNA binding protein and a general splicing activator that, in turn, recruit additional SR proteins. This type of mechanism resembles the tra/tra-2-dependent recruitment of SR proteins to the Drosophila doublesex alternative splicing regulatory element.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/biossíntese , Calcitonina/biossíntese , Elementos Facilitadores Genéticos , Globinas/biossíntese , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Éxons , Glutationa Transferase/biossíntese , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares/isolamento & purificação , Fosfoproteínas/isolamento & purificação , RNA Mensageiro/biossíntese , Proteínas de Ligação a RNA , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Fatores de Processamento de Serina-Arginina , Transfecção , Repetições de TrinucleotídeosRESUMO
The calcitonin/calcitonin gene-related peptide (CGRP) primary transcript is alternatively spliced in thyroid C cells and neurons, resulting in the tissue-specific production of calcitonin and CGRP mRNAs. Analyses of mutated calcitonin/CGRP transcription units in permanently transfected cell lines have indicated that alternative splicing is regulated by a differential capacity to utilize the calcitonin-specific splice acceptor. The analysis of an extensive series of mutations suggests that tissue-specific regulation of calcitonin mRNA production does not depend on the presence of a single, unique cis-active element but instead appears to be a consequence of suboptimal constitutive splicing signals. While only those mutations that altered constitutive splicing signals affected splice choices, the action of multiple regulatory sequences cannot be formally excluded. Further, we have identified a 13-nucleotide purine-rich element from a constitutive exon that, when placed in exon 4, entirely switches splice site usage in CGRP-producing cells. These data suggest that specific exon recruitment sequences, in combination with other constitutive elements, serve an important function in exon recognition. These results are consistent with the hypothesis that tissue-specific alternative splicing of the calcitonin/CGRP primary transcript is mediated by cell-specific differences in components of the constitutive splicing machinery.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina/genética , Calcitonina/genética , Éxons , Íntrons , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Processamento Alternativo , Animais , Sequência de Bases , Calcitonina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Linhagem Celular , DNA , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese , Especificidade de Órgãos/genética , Ratos , Sequências Reguladoras de Ácido Nucleico , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico , TransfecçãoRESUMO
A regulatory region involved in both autoinhibition and calmodulin (CaM) binding has previously been identified in the multifunctional Ca2+/CaM-dependent protein kinase (CaM kinase II). We have tested the role of various segments of the regulatory region in autoinhibition by the analysis of a series of truncation, substitution, and deletion mutants of the CaM kinase II alpha subunit (CaM kinase II alpha). Unexpectedly, the sequence Lys-Lys-Phe-Asn at positions 291-294, adjacent to the CaM binding domain, was found to be sufficient to maintain an inhibited state in a truncated form of the kinase. However, these residues are not essential in the context of the full-length protein, indicating the importance of additional residues from the overlapping CaM binding domain. We propose here a molecular model for CaM kinase II alpha based on the three-dimensional structure of the cAPK-PKI-(5-24) (protein kinase inhibitor fragment) complex. It is predicted from this model that autoinhibition is of the pseudosubstrate variety and that autophosphorylation of Thr-286 could occur by an intersubunit reaction in the holoenzyme complex.
