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1.
J Parkinsons Dis ; 14(4): 681-692, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38578903

RESUMO

Background: Alpha-synuclein (aSyn) is a key player in neurodegenerative diseases such as Parkinson's disease (PD), dementia with Lewy bodies, or multiple system atrophy. aSyn is expressed throughout the brain, and can also be detected in various peripheral tissues. In fact, initial symptoms of PD are non-motoric and include autonomic dysfunction, suggesting that the periphery might play an important role in early development of the disease. aSyn is expressed at relatively low levels in non-central tissues, which brings challenges for its detection and quantification in different tissues. Objective: Our goal was to assess the sensitivity of aSyn detection in central and peripheral mouse tissues through capillary electrophoresis (CE) immunoblot, considering the traditional SDS-PAGE immunoblot as the current standard. Methods: Tissues from central and non-central origin from wild type mice were extracted, and included midbrain, inner ear, and esophagus/stomach. aSyn detection was assessed through immunoblotting using Simple Western size-based CE and SDS-PAGE. Results: CE immunoblots show a consistent detection of aSyn in central and peripheral tissues. Through SDS-PAGE, immunoblots revealed a reliable signal corresponding to aSyn, particularly following membrane fixation. Conclusion: Our results suggest a reliable detection of aSyn in central and peripheral tissues using the CE Simple Western immunoblot system. These observations can serve as preliminary datasets when aiming to formally compare CE with SDS-PAGE, as well as for further characterization of aSyn using this technique.


Assuntos
Eletroforese Capilar , alfa-Sinucleína , Animais , alfa-Sinucleína/metabolismo , alfa-Sinucleína/análise , Camundongos , Eletroforese Capilar/métodos , Camundongos Endogâmicos C57BL , Immunoblotting/métodos , Esôfago/metabolismo , Mesencéfalo/metabolismo
2.
J Neurochem ; 160(5): 578-589, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34984682

RESUMO

Neurochemical biomarkers can support the diagnosis of Alzheimer's disease and may facilitate clinical trials. In blood plasma, the ratio of the amyloid-ß (Aß) peptides Aß-3-40/Aß1-42 can predict cerebral amyloid-ß pathology with high accuracy (Nakamura et al., 2018). Whether or not Aß-3-40 (aka. amyloid precursor protein (APP) 669-711) is also present in cerebrospinal fluid (CSF) is not clear. Here, we investigated whether Aß-3-40 can be detected in CSF and to what extent the CSF Aß-3-40/Aß42 ratio is able to differentiate between individuals with or without amyloid-ß positron emission tomography (PET) evidence of brain amyloid. The occurrence of Aß-3-40 in human CSF was assessed by immunoprecipitation followed by mass spectrometry. For quantifying the CSF concentrations of Aß-3-40 in 23 amyloid PET-negative and 17 amyloid PET-positive subjects, we applied a sandwich-type immunoassay. Our findings provide clear evidence of the presence of Aß-3-40 and Aß-3-38 in human CSF. While there was no statistically significant difference in the CSF concentration of Aß-3-40 between the two diagnostic groups, the CSF Aß-3-40/Aß42 ratio was increased in the amyloid PET-positive individuals. We conclude that Aß-3-40 appears to be a regular constituent of CSF and may potentially serve to accentuate the selective decrease in CSF Aß42 in Alzheimer's disease.


Assuntos
Doença de Alzheimer , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/diagnóstico por imagem , Amiloide , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Biomarcadores/líquido cefalorraquidiano , Humanos , Fragmentos de Peptídeos/líquido cefalorraquidiano , Tomografia por Emissão de Pósitrons , Proteínas tau/líquido cefalorraquidiano
3.
Int J Mol Sci ; 21(18)2020 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-32911706

RESUMO

The ratio of amyloid precursor protein (APP)669-711 (Aß-3-40)/Aß1-42 in blood plasma was reported to represent a novel Alzheimer's disease biomarker. Here, we describe the characterization of two antibodies against the N-terminus of Aß-3-x and the development and "fit-for-purpose" technical validation of a sandwich immunoassay for the measurement of Aß-3-40. Antibody selectivity was assessed by capillary isoelectric focusing immunoassay, Western blot analysis, and immunohistochemistry. The analytical validation addressed assay range, repeatability, specificity, between-run variability, impact of pre-analytical sample handling procedures, assay interference, and analytical spike recoveries. Blood plasma was analyzed after Aß immunoprecipitation by a two-step immunoassay procedure. Both monoclonal antibodies detected Aß-3-40 with no appreciable cross reactivity with Aß1-40 or N-terminally truncated Aß variants. However, the amyloid precursor protein was also recognized. The immunoassay showed high selectivity for Aß-3-40 with a quantitative assay range of 22 pg/mL-7.5 ng/mL. Acceptable intermediate imprecision of the complete two-step immunoassay was reached after normalization. In a small clinical sample, the measured Aß42/Aß-3-40 and Aß42/Aß40 ratios were lower in patients with dementia of the Alzheimer's type than in other dementias. In summary, the methodological groundwork for further optimization and future studies addressing the Aß42/Aß-3-40 ratio as a novel biomarker candidate for Alzheimer's disease has been set.


Assuntos
Peptídeos beta-Amiloides/análise , Precursor de Proteína beta-Amiloide/análise , Imunoensaio/métodos , Doença de Alzheimer/metabolismo , Biomarcadores/sangue , Humanos , Testes Imunológicos , Imunoprecipitação , Fragmentos de Peptídeos/análise
4.
Photodermatol Photoimmunol Photomed ; 32(5-6): 276-283, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27387384

RESUMO

BACKGROUND: The nucleotide excision repair (NER) pathway, defective in xeroderma pigmentosum (XP) patients, removes DNA photolesions in order to prevent carcinogenesis. Complementation group C (XP-C) is the most frequent group of XP patients worldwide. METHODS: We analyzed seven XP-C patients clinically and molecular-genetically applying: post-UV cell survival (MTT-assay), quantitative Real-time PCR, sequencing on chromosomal as well as cDNA level, and in silico interpretation of sequencing data. RESULTS: All cases displayed diminished post-UV cell survival as well as reduced XPC mRNA levels. Five homozygous and two heterozygous disease causing mutations were identified. A large chromosomal deletion of ~5.8 kb identified in XP174MA leads to an unique in frame deletion of XPC exon 2 and exon 3. In silico analysis revealed the deletion of 102 amino acids in the N-terminal part of XPC while leaving the C-terminal domain intact. The novel c.361delA mutation in XP168MA leads to a frameshift in exon 3 resulting in a premature stop codon 27 codons downstream of the deleted adenine. CONCLUSION: Our analysis confirms that XP-C patients without increased sun sensitivity develop non-melanoma skin cancers earlier than sun-sensitive XP-C patients. Reduced cellular mRNA levels are characteristic for XP complementation group C and qRT-PCR represents a rapid diagnostic tool.


Assuntos
Deleção Cromossômica , Cromossomos Humanos/genética , Proteínas de Ligação a DNA , RNA Mensageiro , Neoplasias Cutâneas , Raios Ultravioleta , Xeroderma Pigmentoso , Linhagem Celular , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Masculino , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/metabolismo , Xeroderma Pigmentoso/patologia
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