RESUMO
For the first time soluble, full-length, recombinant, human thyroid-stimulating hormone (TSH) receptor (TSHR) has been expressed in a prokaryotic system. The full-length TSHR cDNA, obtained from normal human thyroid, was cloned into a pQE-9 vector, sequenced, and confirmed to be identical to the published sequence, to be full length, and to be in frame. Expression of the receptor was as a fusion protein with a hexahistidine tail at the amino terminal, in an Escherichia coli expression system. Approximately 2.5 mg of protein per liter of bacterial culture was recovered from the cell homogenate, after a single passage through a nickel-nitrilotriacetic acid resin column. An estimated 60% increase in purity of a band of expected size, 87 kDa, was observed upon gel electrophoresis and staining with Coomassie blue, after the single purification step. Immunoreactivity of the 87-kDa protein with Graves' sera was confirmed by Western blotting.
Assuntos
Escherichia coli/genética , Receptores da Tireotropina/genética , Sequência de Aminoácidos , Autoanticorpos/imunologia , Western Blotting , Cromatografia de Afinidade , Expressão Gênica , Doença de Graves/imunologia , Histidina/metabolismo , Humanos , Receptores da Tireotropina/imunologia , Receptores da Tireotropina/isolamento & purificação , Receptores da Tireotropina/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , SolubilidadeRESUMO
The existence of an autoantigen common to Graves' disease and its associated extrathyroidal manifestations has been proposed. As the TSH receptor (TSHR) is the primary target for autoimmunity against the thyroid in Graves' disease, much effort has gone into investigating the role of TSHR messenger ribonucleic acid (mRNA) transcripts in extrathyroidal tissue, particularly in the orbit. A high stringency RT-PCR technique had previously been employed by our laboratory to demonstrate the presence of full-length and splice variant TSHR mRNA in extraocular muscle (EOM). This technique demonstrated selective amplification of TSHR mRNA transcripts in thyroid and EOM, but not in abdominal muscle, kidney, or brain tissue. We used this technique in the present study to investigate the reported presence of TSHR mRNA transcripts in cardiac tissue. After removal of all visible fat from muscle tissues, high stringency RT-PCR was performed, and we found no TSHR transcripts in the muscle of any chamber of the normal human heart. In addition, in situ hybridization on fresh-frozen and paraffin-embedded sections confirmed the presence of TSHR transcripts in thyroid and EOM, but not in abdominal or cardiac muscle. These findings support the hypothesis that the TSHR is a shared antigen in the thyroid and EOM, but not in cardiac muscle.
Assuntos
Doença de Graves/metabolismo , Músculos/metabolismo , Miocárdio/metabolismo , RNA Mensageiro/análise , Receptores da Tireotropina/genética , Idoso , Autorradiografia , Southern Blotting , Doença de Graves/complicações , Humanos , Hibridização In Situ , Masculino , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The authors analyzed the association between APOE epsilon4 genotype and clinical and MRI findings in 43 refractory temporal lobe epilepsy patients. The distribution of the alleles were normal. Ten patients (23%) had an APOE epsilon 4 allele and had an earlier onset of habitual seizures (with epsilon4 5 +/- 5 years; without epsilon4 15 +/- 10 years). Quantitative MRI findings were not influenced by the APOE epsilon4 genotype. APOE epsilon4 may shorten the latency between an initial injury and seizure onset.
