We describe the molecular etiology of β(+)-thalassemia that is caused by the insertion of the full-length transposable element LINE-1 (L1) into the intron-2 of the β-globin gene (HBB). The transcript level of the affected β-globin gene was severely reduced. The remaining transcripts consisted of full-length, correctly processed β-globin mRNA and a minute amount of three aberrantly spliced transcripts with a decreased half-life due to activation of the nonsense-mediated decay pathway. The lower steady-state amount of mRNA produced by the β-globin(L1) allele also resulted from a reduced rate of transcription and decreased production of full-length β-globin primary transcripts. The promoter and enhancer sequences of the β-globin(L1) allele were hypermethylated; however, treatment with a demethylating agent did not restore the impaired transcription. A histone deacetylase inhibitor partially reactivated the β-globin(L1) transcription despite permanent β-globin(L1) promoter CpG methylation. This result indicates that the decreased rate of transcription from the β-globin(L1) allele is associated with an altered chromatin structure. Therefore, the molecular defect caused by intronic L1 insertion in the β-globin gene represents a novel etiology of β-thalassemia.

• MeSH
• alely MeSH
• alternativní sestřih MeSH
• beta-globiny genetika   MeSH
• beta-talasemie genetika   MeSH
• CpG ostrůvky MeSH
• dlouhé rozptýlené jaderné elementy * MeSH
• dospělí MeSH
• genetická transkripce MeSH
• introny * MeSH
• inzerční mutageneze * MeSH
• lidé MeSH
• metylace DNA MeSH
• pořadí genů MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese MeSH
• stabilita RNA MeSH
• umlčování genů MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH

Premature termination codon (PTC) mutations in the ATP-Binding Cassette, Sub-Family A, Member 7 gene (ABCA7) have recently been identified as intermediate-to-high penetrant risk factor for late-onset Alzheimer's disease (LOAD). High variability, however, is observed in downstream ABCA7 mRNA and protein expression, disease penetrance, and onset age, indicative of unknown modifying factors. Here, we investigated the prevalence and disease penetrance of ABCA7 PTC mutations in a large early onset AD (EOAD)-control cohort, and examined the effect on transcript level with comprehensive third-generation long-read sequencing. We characterized the ABCA7 coding sequence with next-generation sequencing in 928 EOAD patients and 980 matched control individuals. With MetaSKAT rare variant association analysis, we observed a fivefold enrichment (p = 0.0004) of PTC mutations in EOAD patients (3%) versus controls (0.6%). Ten novel PTC mutations were only observed in patients, and PTC mutation carriers in general had an increased familial AD load. In addition, we observed nominal risk reducing trends for three common coding variants. Seven PTC mutations were further analyzed using targeted long-read cDNA sequencing on an Oxford Nanopore MinION platform. PTC-containing transcripts for each investigated PTC mutation were observed at varying proportion (5-41% of the total read count), implying incomplete nonsense-mediated mRNA decay (NMD). Furthermore, we distinguished and phased several previously unknown alternative splicing events (up to 30% of transcripts). In conjunction with PTC mutations, several of these novel ABCA7 isoforms have the potential to rescue deleterious PTC effects. In conclusion, ABCA7 PTC mutations play a substantial role in EOAD, warranting genetic screening of ABCA7 in genetically unexplained patients. Long-read cDNA sequencing revealed both varying degrees of NMD and transcript-modifying events, which may influence ABCA7 dosage, disease severity, and may create opportunities for therapeutic interventions in AD.

• MeSH
• ABC transportéry genetika   MeSH
• Alzheimerova nemoc genetika   MeSH
• dospělí MeSH
• genetická predispozice k nemoci * MeSH
• genetické asociační studie MeSH
• jednonukleotidový polymorfismus * MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace * MeSH
• senioři MeSH
• věk při počátku nemoci MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

CART (cocaine- and amphetamine-regulated transcript) peptides are neuropeptides abundant in the central nervous system and periphery found to be involved in the regulation of food intake behavior and other physiological processes. Recently, we reported specific binding of (125)I-CART(61-102) to the rat adrenal pheochromocytoma cell line PC12, both intact cells and cell membranes. In this study, several fragments of CART(61-102) corresponding to its structural loops were synthesized and tested for their potency in binding experiments using PC12 intact cells and cell membranes and in feeding test with fasted mice. From all shorter peptides tested, only CART(74-86) and CART(62-86) containing disulfide bridges kept partial binding potency of the original molecule with K(i) in 10(-5) and 10(-4)M range. However, these fragments were not able to inhibit food intake after their central administration up to a dose of 4 nmol/mouse. The results showed that a compact structure containing three disulfide bridges is necessary for preservation of full biological activity of CART peptides.

• MeSH
• buňky PC12 MeSH
• financování organizované MeSH
• krysa rodu rattus MeSH
• molekulární sekvence - údaje MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• peptidové fragmenty farmakologie   MeSH
• proteiny nervové tkáně farmakologie   chemie   MeSH
• sekvence aminokyselin MeSH
• stravovací zvyklosti účinky léků   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH

The human p73 protein is essential for normal morphogenesis and maintenance of neural tissue. Recently, several TP73 transcripts have been revealed in medulloblastoma (MB), the most common malignant brain tumor in children. Here, we performed immunohistochemical analysis on 29 MB specimens using anti-p73alpha and anti-DeltaNp73 antibodies. Real-time PCR quantification was performed to assess TAp73 and DeltaNp73 transcripts in a subset of 13 MB samples. Normal cerebellar tissues and RNA were used for comparison. Pilot clinical-pathological correlations were also provided. We report significant differences for TAp73 and DeltaNp73 mRNA expression between tumor tissues and reference (P = 0.013, P = 0.028). Immunohistochemically, 52 and 29% MB samples were positive for p73alpha and DeltaNp73, respectively. p73alpha expression was found to be in both the nucleus and cytoplasm, whereas DeltaNp73 was localized predominantly in the cytoplasm. In normal cerebellum, positive staining for p73alpha and DeltaNp73 was observed in the Purkinje cells of newborns, not adult samples, which supports the developmental role of TP73 during organogenesis of the human cerebellum. Survival analysis has shown negative relationship of DeltaNp73-immunoreactivity with overall survival (OS) and event free survival (EFS) (P = 0.026 and P = 0.127, respectively). For p73alpha-positive cases, the negative trend in OS (P = 0.149) and EFS (P = 0.216) was also apparent. Our results indicate the involvement of p73 protein in MB tumorigenesis and define TP73 as a potential prognostic and therapeutic target for medulloblastom.

• MeSH
• dítě MeSH
• DNA vazebné proteiny genetika   imunologie   metabolismus   MeSH
• dospělí MeSH
• financování organizované MeSH
• imunohistochemie MeSH
• lidé MeSH
• meduloblastom genetika   imunologie   metabolismus   MeSH
• messenger RNA analýza   metabolismus   MeSH
• míra přežití MeSH
• mladiství MeSH
• mozek metabolismus   patofyziologie   patologie   MeSH
• nádorová transformace buněk genetika   metabolismus   MeSH
• nádorové biomarkery analýza   genetika   metabolismus   MeSH
• nádorové supresorové proteiny genetika   imunologie   metabolismus   MeSH
• nádory mozku genetika   metabolismus   patofyziologie   MeSH
• předškolní dítě MeSH
• prognóza MeSH
• protein - isoformy genetika   izolace a purifikace   metabolismus   MeSH
• regulace genové exprese u nádorů genetika   MeSH
• retrospektivní studie MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH

• MeSH
• eukaryotické buňky fyziologie   MeSH
• genetická transkripce * fyziologie   MeSH
• posttranskripční úpravy RNA fyziologie   MeSH
• prekurzory RNA fyziologie   MeSH
• prokaryotické buňky fyziologie   MeSH
• RNA heterogenní jaderná fyziologie   MeSH

Úvod: Komplexy cyklin-dependentních kináz (CDK) s cyklinem spouští různá stadia buněčného cyklu eukaryotické buňky tím, že fosforylují specifické cílové proteiny. Syntetické inhibitory cyklin-dependentních kináz (CDKI) jako je olomoucin (OC), bohemin (BOH), R-roskovitin (ROSC, seliciclib, CYC202) atd., jsou typickými představiteli léčiv, které můžeme využít ke kontrole buněčné proliferace a ke klinické léčbě nádorů. Účel studie: Záměrem studie bylo stanovit cytotoxickou aktivitu syntetického CDKI boheminu na lidských primárních nádorových buňkách a porovnat ji s klasickými protinádorovými léčivy. Metody: Nádorové buňky byly získány gradientovou centrifugací po mechanickém rozmělnění vzorku tumoru a natrávení enzymy. Nádorová populace byla inkubována se vzestupnou koncentrací BOH a tradičních protinádorových léčiv s cílem zjistit koncentraci cytostatika, jež usmrtí 50 % nádorové populace (TCS50). Hodnoty TCS50 byly vzájemně korelovány pro zjištění podobnosti/rozdílu v mechanizmu účinku a/nebo lékové rezistence. Výsledky: V průběhu studie bylo úspěšně vyšetřeno 112 nádorů různého histogenetického původu. Naše studie naznačuje tendenci vyšší senzitivity maligních ve srovnání s benigními tumory (20.94/51.57 µM, p = 0.162). Hodnota mediánu TCS50 pro BOH u hematologických/solidních nádorů (16.7/23.05 µM) poukazuje na vyšší sensitivitu hematologických malignit (p = 0,0159). Závěr: Pouze 20 pacientů se ukázalo rezistentních vůči BOH ve srovnání s tradičními protinádorovými léčivy. Porovnání mechanizmu účinku/rezistence s ostatními cytostatiky prokázalo: 1. asociaci in vitro účinnosti BOH s účinností antimetabolitů pravděpodobně v důsledku společného mechanizmu rezistence s ohledem na jejich strukturální podobnost, 2. korelaci efektivity BOH s látkami vyvolávající DNA poškození (topotekan, cisplatina, daunorubicin, mitoxantron) a/nebo inhibitory RNA transkripce (aktinomycin D), která zjevně souvisí s inhibicí DNA reparace a aktivační fosforylace RNA polymerázy II cyklin-dependentními kinázami 7 a 9, které jsou BOH přímo inhibovány.

Background: Cyclin dependent kinases complexed with cyclins trigger eukaryotic cell cycle proliferation by phosphorylation of specific target protein. Synthetic inhibitors of cyclin dependent kinases (CDKIs) as olomoucine (OC), bohemine (BOH), R-roscovitine (ROSC, seliciclib, CYC202) etc., are typical representatives of drugs, which can be used for control of cellular proliferation and clinical treatment of tumours. Aim: The purpose of this study was to assess the cytotoxic activity of the synthetic CDKI, bohemine, on human primary tumour cells and compare its potency with classic anti-cancer drugs. Methods: Malignant cells were isolated, after mechanical and enzymatic digestion of tumour tissues, by gradient centrifugation. The cells were then incubated with ascendant concentrations of BOH or classical anti-cancer drugs in order to determine the concentrations lethal to 50 % of tumour cells (TCS50). TCS50 values were then mutually correlated in order to determine similarities/dissimilarities in the mechanism of action and/or drug resistance. Results: In the course of study we have successfully analyzed 112 primary tumour samples for drug sensitivity to BOH. Our study showed a tendency for a higher sensitivity of the malignant versus the benignant tumours (20.94/51.57 µM, p = 0.162). The median value of BOH TCS50 in haematological/solid tumours (16.7/23.05 μM) indicates higher sensitivity of haematological malignancies (p = 0.0159). Conclusion: There were only 20 BOH resistant patients compared to the resistance of classical anti-cancer drugs. Comparison of mechanism of action/resistance with other anti-cancer drugs revealed: 1. an association of BOH activity with antimetabolites, probably signifying common mechanisms of resistance due to their structural similarity, 2. a correlation of efficiency of BOH with drugs targeting RNA transcription (actinomycin D) and/or compounds causing DNA damage (topotecan, cisplatin, daunorubicin, mitoxantrone), which is evidently connected with BOH mediated inhibition of DNA repair and/or activating fosforylation of RNA polymerase II mediated by CDK7 and CDK9.

• MeSH
• cyklin-dependentní kinasy antagonisté a inhibitory   biosyntéza   terapeutické užití   MeSH
• finanční podpora výzkumu jako téma MeSH
• inhibitory proteinkinas terapeutické užití   toxicita   MeSH
• lidé MeSH
• nádorové buňky kultivované cytologie   enzymologie   imunologie   MeSH
• protinádorové látky terapeutické užití   toxicita   MeSH
• puriny MeSH
• techniky in vitro MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• srovnávací studie MeSH

OBJECTIVES: MicroRNAs (miRNAs) are short single-stranded RNAs that play a role in the post-transcriptional regulation of gene expression. Their deregulation can be associated with various diseases, such as cancer, neurodegenerative, and immune-related diseases. The aim of our study was to compare miRNA levels in plasma that could potentially influence the progression of hyperuricemia to gout, since the mechanism of progression is still unclear. METHODS: Total RNA, including miRNA, was isolated from the plasma of 45 patients with asymptomatic hyperuricemia, 131 patients with primary gout (including 16 patients having a gout attack), and 130 normouricemic controls. The expression of 18 selected miRNAs (cel-miR-39 and cel-miR-54 as spike-in controls, hsa-miR-16-5p and hsa-miR-25-3p as endogenous controls, hsa-miR-17-5p, hsa-miR-18a-5p, hsa-miR-30a-3p, hsa-miR-30c-5p, hsa-miR-126-3p, hsa-miR-133a-3p, hsa-miR-142-3p, hsa-miR-143-3p, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-miR-222-3p, hsa-miR-223-3p, hsa-miR-488-3p and hsa-miR-920) was measured using qPCR. RESULTS: We found that hsa-miR-17-5p, hsa-miR-18a-5p, hsa-miR-30c-5p, hsa-miR-142-3p, and hsa-miR-223-3p were significantly upregulated (p < 0.001) in the plasma of hyperuricemia and gout patients compared to normouricemic individuals. As part of the follow-up of our previous study, we found a negative correlation between hsa-miR-17-5p, hsa-miR-30c-5p, hsa-miR-126-3p, hsa-miR-142-3p, and hsa-miR-223-3p with plasma levels of chemokine MCP-1. Additionally, we found a positive correlation between CRP and plasma levels of hsa-miR-17-5p, hsa-miR-18a-5p, hsa-miR-30c-5p, hsa-miR-126-3p, hsa-miR-142-3p, hsa-miR-146a-5p, hsa-miR-155-5p, hsa-miR-222-3p, and hsa-miR-223-3p. Five of those miRNAs (hsa-miR-126-3p, hsa-miR-142-3p, hsa-miR-146a-5p, hsa-miR-155-5p, and hsa-miR-222-3p) also had a positive correlation with serum creatinine and therefore a negative correlation with eGFR. CONCLUSION: Five miRNAs were significantly upregulated in the plasma of patients with hyperuricemia and gout (and those during a gout attack) compared to normouricemic controls. We also found a correlation between the plasma levels of several miRNA and plasma levels of MCP-1, CRP, serum creatinine, and eGFR.

• MeSH
• cirkulující mikroRNA * MeSH
• dna (nemoc) * genetika   MeSH
• hyperurikemie * genetika   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• mikro RNA * genetika   MeSH
• regulace genové exprese MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Breast cancer is a leading cause of cancer-related death in women worldwide. Despite extensive studies in all areas of basic, clinical and applied research, accurate prognosis remains elusive, thus leading to overtreatment of many patients. Diagnosis could be improved by introducing multigene molecular scores in standard clinical practice. Several tests that work with formalin-fixed tissue have become routine. Molecular scores usually include several genes representing processes, response to oestrogens, progestogens and human epidermal growth factor receptor 2 (Her2), respectively, which are combined additively in single values. These multi-gene scores have the advantage of being more robust and reproducible than single-gene scores. Their utility may be further enhanced by combining them with classical diagnostic parameters. Here, we present an exploratory study comparing the RISK and research versions of Oncotype DX recurrence score (RS), Prosigna Risk of Recurrence (ROR) and EndoPredict (EP) with respect to their prognostic potential for ipsilateral recurrence and/or distant relapse in brain, and we compared the scores to the intrinsic subtypes based on PAM50. METHODS: RNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue cores of primary tumours, local recurrences and brain metastases. Gene expression was measured on a NanoString nCounter Analysis System. Intrinsic subtypes and molecular scores were computed according to published literature and RISK, RS, ROR and EP were compared against each other and to the intrinsic subtypes Luminal A (lumA), Luminal B (lumB), Her2-enriched (Her2↑), Basal-like (basal), and Normal-like (normal) of PAM50. Local recurrences and brain metastases were compared to their corresponding primary tumours. RESULTS: All four molecular scores were highly correlated. Highest correlations were observed among genes related to proliferation while lower correlations were found among oestrogen-related genes. The scores were significantly higher in primary tumours progressing to brain metastases as compared to recurrence-free primary tumours and primary tumours that relapsed as local recurrences. CONCLUSIONS: RISK and ROR-P are prognostic for primary tumours metastasizing to the brain. All four scores, RISK, RS, EP and ROR-P failed to discriminate between primary tumours that remained recurrence-free and primary tumours relapsing as local recurrences.

• MeSH
• dospělí MeSH
• imunohistochemie MeSH
• lidé středního věku MeSH
• lidé MeSH
• lokální recidiva nádoru MeSH
• nádorové biomarkery MeSH
• nádory mozku diagnóza   sekundární   MeSH
• nádory prsu genetika   patologie   MeSH
• následné studie MeSH
• prognóza MeSH
• regulace genové exprese u nádorů MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• stanovení celkové genové exprese MeSH
• stupeň nádoru MeSH
• transkriptom * MeSH
• výpočetní biologie metody   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Organic cation transporter 1 (OCT1, SLC22A1) is a membrane transporter that is important for therapeutic effect of the antidiabetic drug metformin. Its liver-specific expression in hepatocytes is strongly controlled by hepatocyte nuclear factor-4α (HNF4α). HNF4α expression and transcriptional activity have been demonstrated to be augmented by glucocorticoid receptor (GR) in human hepatocytes and rodent livers. METHODS: It was examined whether GR activation indirectly induces OCT1 gene expression via HNF4α up-regulation in primary human hepatocytes. We also examined which other transcription factors are involved in OCT1 gene expression and whether they are regulated by dexamethasone using qRT-PCR and gene reporter assays. RESULTS: We found that dexamethasone significantly up-regulates OCT1 mRNA and protein in normal primary human hepatocytes, but not in hepatocyte-derived tumor cell lines HepG2 and MZ-Hep1. Consistently, we observed that HNF4α is induced by dexamethasone in primary human hepatocytes, but not in hepatocyte tumor-derived cell lines. Viral transduction of MZ-Hep1 cells with the expression constructs for HNF4α, CCAAT/enhancer binding proteins β (C/EBPβ) and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) demonstrated significant roles of the transcription factors in OCT1 gene regulation. We found that expression of OCT1 mRNA in human livers significantly correlates with C/EBPβ and HNF4α mRNAs expression and that C/EBPβ co-transfection stimulates OCT1 gene reporter construct in HepG2 cells. Nevertheless, neither C/EBPβ nor PGC1α were upregulated in human hepatocytes by dexamethasone. CONCLUSION: We can conclude that GR-induced expression of HNF4α may contribute to indirect OCT1 gene up-regulation by dexamethasone in primary human hepatocytes, but not in hepatocyte-derived tumor cell lines.

• MeSH
• buňky Hep G2 MeSH
• časové faktory MeSH
• dexamethason farmakologie   MeSH
• glukokortikoidy farmakologie   MeSH
• hepatocytární jaderný faktor 4 genetika   metabolismus   MeSH
• hepatocyty účinky léků   metabolismus   MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• přenašeč organických kationtů 1 genetika   metabolismus   MeSH
• primární buněčná kultura MeSH
• protein beta vázající zesilovač transkripce CCAAT genetika   metabolismus   MeSH
• receptory glukokortikoidů agonisté   metabolismus   MeSH
• transdukce genetická MeSH
• transfekce MeSH
• transkripční faktory genetika   metabolismus   MeSH
• upregulace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The human glucocorticoid receptor (hGR) plays a pivotal role in cellular processes such as development, differentiation, homeostasis, immune response and in regulation of xenobiotic metabolism. It has been demonstrated recently that colchicine inhibits hGR transcriptional activity in primary cultures of human hepatocytes by a mechanism involving impairment of hGR nucleo-cytoplasmic shuttling. In the present work, we investigated the role of the nuclear factor kappa B (NFkappaB) and c-jun-N-terminal kinase (JNK), the functional hGR antagonists, in this process. We found that microtubule disarray caused by colchicine, vincristine or nocodazole does not activate NFkappaB in human hepatocytes as revealed by p50 and p65 subunits nuclear translocation. On the other hand, we demonstrate that JNK mediates hGR transcriptional inhibition by microtubules disarray, because a specific inhibitor of JNK, 1,9-pyrazoloanthrone (SP600125), partially blocked tyrosine aminotransferase mRNA suppression due to colchicine treatment. In conclusion, JNK is at least partly involved in hGR transcriptional inhibition by colchicine in human hepatocytes, while NFkappaB involvement is doubtful.

• MeSH
• aktivace enzymů MeSH
• aktivace transkripce účinky léků   MeSH
• anthraceny farmakologie   MeSH
• hepatocyty metabolismus   ultrastruktura   MeSH
• JNK mitogenem aktivované proteinkinasy antagonisté a inhibitory   metabolismus   MeSH
• kolchicin farmakologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mikrotubuly účinky léků   ultrastruktura   MeSH
• NF-kappa B metabolismus   farmakologie   MeSH
• receptory glukokortikoidů antagonisté a inhibitory   fyziologie   MeSH
• tyrosinaminotransferasa metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH