The aim of the present work was to develop a chromatographic system for the separation of individual fluorophores extracted from neuronal ceroid lipofuscinosis (NCL) brain and isolated storage bodies. Extracts from gray matter were best resolved on silica-gel HPTLC plates using a mixture of chloroform/methanol/water (55:45:10 by vol.). Two other chromatographic systems were tested which gave poorer separation. Corrected fluorescence spectra were obtained on the original extract and fluorescence intensity, especially at longer wavelengths was increased in both samples. Yellow and blue fluorophores were detected on HPTLC plates using a primary violet and secondary yellow filter with cut-off levels of 400 and 520 nm, respectively. Plates were photographed at 20 min, 2 h and 1 week after chromatography. With this filter system, up to 12 yellow bands of differing intensity were observed at 20 min but with time, some of these changed to blue as a result of autoxidation. NCL tissues emit yellow fluorescence when viewed under light microscopy, however extracted material did not demonstrate a distinct peak in this region of the spectrum which should be around 575 nm. HPTLC confirmed this observation and time studies revealed that autoxidation changes occur and must be carefully controlled to reduce artifacts. The discrepancy between extracted and non-extracted observations may be the result of superposition of multiple fluorophores with differing maxima and/or a self-absorption phenomenon. The combination of chromatographic separation and spectral analysis as described in this study, may be a valuable technique to further clarify the characteristics of compound fluorescent lipopigments. It is suggested that NCL fluorophores of human brain differ in their properties from other models.
- MeSH
- ceroid izolace a purifikace MeSH
- chromatografie na tenké vrstvě metody MeSH
- fluorescenční barviva * izolace a purifikace MeSH
- fluorescenční spektrometrie MeSH
- lidé MeSH
- lipidy * izolace a purifikace MeSH
- mozek - chemie * MeSH
- neuronální ceroidlipofuscinózy * metabolismus MeSH
- oxidace-redukce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- MeSH
- dospělí MeSH
- drenáž MeSH
- intrakraniální krvácení terapie MeSH
- lidé středního věku MeSH
- lidé MeSH
- lumbální punkce MeSH
- nucleus caudatus patologie MeSH
- péče o pacienty v kritickém stavu metody MeSH
- výsledek terapie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
Adult-onset neuronal ceroid lipofuscinoses (ANCL, Kufs disease) are rare hereditary neuropsychiatric disorders characterized by intralysosomal accumulation of ceroid in tissues. The ceroid accumulation primarily affects the brain, leading to neuronal loss and progressive neurodegeneration. Although several causative genes have been identified (DNAJC5, CLN6, CTSF, GRN, CLN1, CLN5, ATP13A2), the genetic underpinnings of ANCL in some families remain unknown. Here we report one family with autosomal dominant (AD) Kufs disease caused by a 30 bp in-frame duplication in DNAJC5, encoding the cysteine-string protein alpha (CSPα). This variant leads to a duplication of the central core motif of the cysteine-string domain of CSPα and affects palmitoylation-dependent CSPα sorting in cultured neuronal cells similarly to two previously described CSPα variants, p.(Leu115Arg) and p.(Leu116del). Interestingly, the duplication was not detected initially by standard Sanger sequencing due to a preferential PCR amplification of the shorter wild-type allele and allelic dropout of the mutated DNAJC5 allele. It was also missed by subsequent whole-exome sequencing (WES). Its identification was facilitated by reanalysis of original WES data and modification of the PCR and Sanger sequencing protocols. Independently occurring variants in the genomic sequence of DNAJC5 encoding the cysteine-string domain of CSPα suggest that this region may be more prone to DNA replication errors and that insertions or duplications within this domain should be considered in unsolved ANCL cases.
- MeSH
- buněčné linie MeSH
- dospělí MeSH
- duplikace genu * MeSH
- falešně negativní reakce MeSH
- genetické testování normy MeSH
- lidé středního věku MeSH
- lidé MeSH
- membránové proteiny genetika metabolismus MeSH
- myši MeSH
- neuronální ceroidlipofuscinózy genetika patologie MeSH
- neurony metabolismus MeSH
- posttranslační úpravy proteinů MeSH
- proteiny tepelného šoku HSP40 genetika metabolismus MeSH
- sekvenování celého genomu normy MeSH
- transport proteinů MeSH
- zvířata MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- molekulární biologie MeSH
- neuronální ceroidlipofuscinózy etiologie klasifikace patofyziologie MeSH
- Publikační typ
- kongresy MeSH
Non-small cell lung cancer (NSCLC) results in high mortality and has gained increasing attention. C-Phycocyanin (C-PC) has been identified as a potential therapeutic inhibitor for NSCLC, but its underlying mechanism remains obscure. The gene expression of the long noncoding RNA neighbour of BRCAI RNA 2 (NBR2) in NSCLC cells was evaluated by quantitative reverse transcription-PCR. The cell capacity for proliferation and migration was examined by EdU and wound-healing assays. Furthermore, the viability and apoptosis of cells was measured with CCK-8 and annexin V/PI, respectively. Next, the protein level of activation of adenosine monophosphate- activated protein kinase and the rapamycin kinase (mTOR) signalling pathway-associated molecules was evaluated by western blotting. H292 cells were pre-treated with C-PC or transfected with plasmids encoding NBR2 or the shNBR2 plasmid, to over-express or knock down NBR2 expression, respectively. NBR2 expression was robustly down-regulated in NSCLC cell lines compared with a normal cell line (BEAS-2B). NBR2 over-expression inhibited migration and promoted apoptosis of H292 cells. Treatment of H292 cells with C-PC enhanced NBR2 levels in a dose- and time-dependent manner. Downregulation of NBR2 in H292 cells inhibited the activity of C-PC on cell proliferation, viability and clone formation. Further mechanistic investigation showed that the down-regulation of NBR2 abolished the modulatory effects of C-PC on the AMPK/mTOR signalling pathway. In conclusion, C-PC inhibits H292 cell growth by enhancing the NBR2/AMPK signalling pathway.
- MeSH
- adenosinmonofosfát farmakologie terapeutické užití MeSH
- annexin A5 farmakologie terapeutické užití MeSH
- apoptóza MeSH
- fykocyanin metabolismus farmakologie terapeutické užití MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory plic * farmakoterapie genetika metabolismus MeSH
- nemalobuněčný karcinom plic * farmakoterapie genetika metabolismus MeSH
- pohyb buněk MeSH
- proliferace buněk MeSH
- proteinkinasy aktivované AMP genetika metabolismus farmakologie MeSH
- RNA dlouhá nekódující * genetika MeSH
- sinkalid metabolismus farmakologie terapeutické užití MeSH
- sirolimus farmakologie terapeutické užití MeSH
- TOR serin-threoninkinasy metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
