BACKGROUND: Apoptosis plays a critical role in cancer cell survival and tumor development. We provide a hypothesis-generating screen for further research by exploring the expression profile and genetic variability of caspases (2, 3, 7, 8, 9, and 10) in breast carcinoma patients. This study addressed isoform-specific caspase transcript expression and genetic variability in regulatory sequences of caspases 2 and 9. METHODS: Gene expression profiling was performed by quantitative real-time PCR in tumor and paired non-malignant tissues of two independent groups of patients. Genetic variability was determined by high resolution melting, allelic discrimination, and sequencing analysis in tumor and peripheral blood lymphocyte DNA of the patients. RESULTS: CASP3 A+B and S isoforms were over-expressed in tumors of both patient groups. The CASP9 transcript was down-regulated in tumors of both groups of patients and significantly associated with expression of hormonal receptors and with the presence of rs4645978-rs2020903-rs4646034 haplotype in the CASP9 gene. Patients with a low intratumoral CASP9A/B isoform expression ratio (predicted to shift equilibrium towards anti-apoptotic isoform) subsequently treated with adjuvant chemotherapy had a significantly shorter disease-free survival than those with the high ratio (p=0.04). Inheritance of CC genotype of rs2020903 in CASP9 was associated with progesterone receptor expression in tumors (p=0.003). CONCLUSIONS: Genetic variability in CASP9 and expression of its splicing variants present targets for further study.

• MeSH
• cílená molekulární terapie * MeSH
• genetická transkripce * MeSH
• genetická variace * genetika   MeSH
• kaspasa 9 genetika   metabolismus   MeSH
• kaspasy * genetika   metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádory prsu * enzymologie   genetika   MeSH
• regulace genové exprese u nádorů * MeSH
• stanovení celkové genové exprese MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH

Background: Microarray technologies are used to measure the simultaneous expression of a certain set of thousands of genes based on ribonucleic acid (RNA) obtained from a biological sample. We are interested in several statistical analyses such as 1) finding differentially expressed genes between or among several experimental groups, 2) finding a small number of genes allowing for the correct classification of a sample in a certain group, and 3) finding relations among genes. Objectives: Gene expression data are high dimensional, and this fact complicates their analysis because we are able to perform only a few samples (e.g. the peripheral blood from a limited number of patients) for a certain set of thousands of genes. The main purpose of this paper is to present the shrinkage estimator and show its application in different statistical analyses. Methods: The shrinkage approach relates to the shift of a certain value of a classic estimator towards a certain value of a specified target estimator. More precisely, the shrinkage estimator is the weighted average of the classic estimator and the target estimator. Results: The benefit of the shrinkage estimator is that it improves the mean squared error (MSE) as compared to a classic estimator. The MSE combines the measure of an estimator’s bias away from its true unknown value and the measure of the estimator’s variability. The shrinkage estimator is a biased estimator but has a lower variability. Conclusions: The shrinkage estimator can be considered as a promising estimator for analyzing high dimensional gene expression data.

• MeSH
• exprese genu * genetika   MeSH
• lidé MeSH
• mikročipová analýza * metody   statistika a číselné údaje   MeSH
• RNA * genetika   MeSH
• statistické modely MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Among computationally predicted and experimentally validated plant miRNAs, several are conserved across species boundaries in the plant kingdom. In this study, a combined experimental-in silico computational based approach was adopted for the identification and characterization of miRNAs in Humulus lupulus (hop), which is widely cultivated for use by the brewing industry and apart from, used as a medicinal herb. A total of 22 miRNAs belonging to 17 miRNA families were identified in hop following comparative computational approach and EST-based homology search according to a series of filtering criteria. Selected miRNAs were validated by end-point PCR and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), confirmed the existence of conserved miRNAs in hop. Based on the characteristic that miRNAs exhibit perfect or nearly perfect complementarity with their targeted mRNA sequences, a total of 47 potential miRNA targets were identified in hop. Strikingly, the majority of predicted targets were belong to transcriptional factors which could regulate hop growth and development, including leaf, root and even cone development. Moreover, the identified miRNAs may also be involved in other cellular and metabolic processes, such as stress response, signal transduction, and other physiological processes. The cis-regulatory elements relevant to biotic and abiotic stress, plant hormone response, flavonoid biosynthesis were identified in the promoter regions of those miRNA genes. Overall, findings from this study will accelerate the way for further researches of miRNAs, their functions in hop and shows a path for the prediction and analysis of miRNAs to those species whose genomes are not available.

• MeSH
• databáze genetické MeSH
• exprimované sekvenční adresy * MeSH
• genové regulační sítě genetika   MeSH
• Humulus genetika   MeSH
• mikro RNA genetika   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• regulace genové exprese u rostlin genetika   MeSH
• rostlinné geny genetika   MeSH
• software * MeSH
• stanovení celkové genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Poor semen quality is one of the main causes of infertility. We have generated a set of monoclonal antibodies to human sperm and used them to investigate sperm quality. Some of these antibodies found differences in the expression of proteins between normal sperm and pathological sperm displaying severe defects. One of them was the Hs-14 antibody. The aim of this paper was to determine the target protein of the Hs-14 monoclonal antibody and to investigate the expression of the Hs-14-reacting protein on the sperm of asthenozoospermic men with sperm motility defect and of healthy normozoospermic men. METHODS: Indirect immunofluorescence, one-dimensional and two-dimensional polyacrylamide gel electrophoresis, immunoblotting and mass spectrometry. RESULTS: The Hs-14 antibody binds fibronectin, β-tubulin and valosin-containing protein - new name for this protein is transitional endoplasmic reticulum ATPase (TERA). Since the Hs-14 reaction with TERA remained the strongest at the highest antibody dilution, and Hs-14 consistently labelled the same spot or band as the monospecific anti-TERA antibody on immunoblots, we assume that TERA is an Hs-14-specific protein. Binding of fibronectin and β-tubulin might represent nonspecific cross-reactivity or Hs-14 reaction with similar epitopes of these proteins. A significant difference (P < 0.001) in immunofluorescence staining with Hs-14 was found between the normozoospermic and asthenozoospermic men. CONCLUSION: The Hs-14 antibody enables discrimination between sterile or subfertile asthenozoospermic and fertile normozoospermic men. Decreased levels of TERA in men can be used as a biomarker of reduced fertility.

• Publikační typ
• časopisecké články MeSH

BACKGROUND: Cbf11 and Cbf12, the fission yeast CSL transcription factors, have been implicated in the regulation of cell-cycle progression, but no specific roles have been described and their target genes have been only partially mapped. METHODOLOGY/PRINCIPAL FINDINGS: Using a combination of transcriptome profiling under various conditions and genome-wide analysis of CSL-DNA interactions, we identify genes regulated directly and indirectly by CSL proteins in fission yeast. We show that the expression of stress-response genes and genes that are expressed periodically during the cell cycle is deregulated upon genetic manipulation of cbf11 and/or cbf12. Accordingly, the coordination of mitosis and cytokinesis is perturbed in cells with genetically manipulated CSL protein levels, together with other specific defects in cell-cycle progression. Cbf11 activity is nutrient-dependent and Δcbf11-associated defects are mitigated by inactivation of the protein kinase A (Pka1) and stress-activated MAP kinase (Sty1p38) pathways. Furthermore, Cbf11 directly regulates a set of lipid metabolism genes and Δcbf11 cells feature a stark decrease in the number of storage lipid droplets. CONCLUSIONS/SIGNIFICANCE: Our results provide a framework for a more detailed understanding of the role of CSL proteins in the regulation of cell-cycle progression in fission yeast.

• MeSH
• cytokineze MeSH
• fyziologický stres MeSH
• mitogenem aktivované proteinkinasy genetika   MeSH
• mitóza MeSH
• proteinkinasy závislé na cyklickém AMP genetika   MeSH
• regulace genové exprese u hub MeSH
• Schizosaccharomyces pombe - proteiny genetika   metabolismus   MeSH
• Schizosaccharomyces genetika   metabolismus   MeSH
• stanovení celkové genové exprese metody   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Ionotropní glutamátové receptory NMDA typu hrají podstatnou roli v procesu synaptické plasticity, která je základem učení a paměti. Změna spojení mezi neurony je důsledkem působení jednak lokálních faktorů v synapsi a jednak celkových faktorů, které zahrnují aktivaci proteinkináz a přenos signálu do buněčného jádra, kde proteinkinázy fosforylují konstitutivně exprimované transkripční faktory, které řídí expresi tzv. genů časné odpovědi, z nichž některé jsou samy transkripční faktory a kontrolují expresi dalších cílových genů. Článek prezentuje dosud známé poznatky o mechanizmu přenosu signálu od NMDA receptoru k indukci modelového genu časné odpovědi c-fos, jehož exprese je pod kontrolou několika konstitutivních faktorů, mezi jinými CREB (cAMP-response element binding protein), který musí být fosforylován na kritickém Ser133, aby aktivoval transkripci. Rychlým důsledkem aktivace NMDA receptoru je translokace komplexu Ca2+-kalmodulin do jádra a aktivace Ca2+/kalmodulin dependentní kinázy IV. S pomalejší kinetikou dochází k aktivaci Ras-MAPK (mitogen-activated protein kinase) kinázové kaskády, která je zřejmě v signalizaci do jádra neuronu nejdůležitější a přes jiné specifické signální molekuly integruje i signalizaci využívající Ca2+ nebo cyklický AMP jako druhého posla.

Ionotropic NMDA glutamate receptors play a central role in the process of synaptic plasticity underlying learning and memory. Alteration of neuronal connections results from local effects in the target synapse as well as from the whole-cell factors that encompass activation of protein kinases and signal transduction to the cell nucleus, where the kinases phosphorylate constitutively expressed transcription factors controlling expression of a set of immediate-early genes; some of them, in turn, are themselves transcription factors and regulate expression of further target genes. The article presents some of knowledge accumulated to date, on the mechanism of signal transduction from NMDA receptor to induction of the model immediate-early gene c-fos, whose expression is controlled by several constitutive factors, among them by the CREB (cAMP-response element binding protein), that must be phosphorylated at a critical Ser133, in order to activate transcription. One fast event in response to NMDA receptor activation is translocation of the Ca2+-calmodulin complex to the cell nucleus and activation of Ca2+/calmodulin-dependent protein kinase IV. With slower kinetics, the Ras-MAPK (mitogen-activated protein kinase) kinase cascade is activated, emerging in the signalling to the neuronal nucleus as the most significant pathway, through other specific signalling molecules integrating also signals originally using Ca2+ or cyclic AMP as the second messengers.

• MeSH
• buněčné jádro MeSH
• exprese genu MeSH
• glutamátové receptory MeSH
• kalmodulin MeSH
• neuroplasticita MeSH
• signální transdukce MeSH

V našem sdělení chceme informovat o použití dvou metodik ke studiu molekulární genetické problematiky mnohočetného myelomu (MM). První zmíníme využití DNA čipů (microarrays) a ve druhé části se zaměříme na chromatinovou imunoprecipitaci v analýzách epigenetických změn genů. Obě metodiky náš výzkumný tým používá, a proto představíme i první výstupy. Využití obou metodik je u MM stejné jako u všech ostatních nádorů. Představují možnost studia patogeneze maligních onemocnění na molekulární úrovni, klasifikaci jinak neodlišitelných prognostických skupin choroby, predikci léčebné odpovědi na daný terapeutický zásah a identifikaci možných molekulárních cílů protinádorové terapie.

This review informs about utilization of two methods used in molecular examination in multiple myeloma on genomic level: DNA microarrays and chromatin immunoprecipitation. The profit of both methods is very similar in myeloma as well as in other cancers. They allow to study disease pathogenesis, generate new disease classification, and try to predict effect of therapy. Also, they are used to find the new potential target of therapy in multiple myeloma.

• MeSH
• chromatinová imunoprecipitace metody   využití   MeSH
• DNA fingerprinting metody   využití   MeSH
• exprese genu genetika   MeSH
• financování organizované MeSH
• hematologické nádory diagnóza   etiologie   genetika   MeSH
• lékařská onkologie metody   trendy   MeSH
• lékové transportní systémy metody   využití   MeSH
• lidé MeSH
• mnohočetný myelom diagnóza   etiologie   genetika   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů metody   využití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

Metody exprese a purifikace rekombinantních proteinů umožňují produkci a detailní charakterizaci proteinů v základním výzkumu během in vitro experimentů, ale také přípravu proteinů s terapeutickým využitím. Publikace shrnuje základní postupy od přípravy expresních vektorů až po techniku afinitní purifikace. Dále pojednává o vlastnostech různých prokaryotických a eukaryotických expresních systémů a možnostech jejich využití. Molekulární klonování, které slouží k přípravě expresních vektorů pro rekombinantní proteiny, umožňuje cíleně modifikovat vlastnosti těchto proteinů tak, aby byla usnadněna jejich purifikace a také pozměněna jejich stabilita, aktivita nebo funkce. V současné době je k dispozici široká škála metodických přístupů, jež umožňují rychlou a efektivní přípravu expresních vektorů. Zvolený produkční organizmus a způsob purifikace rekombinantního proteinu určují výběr expresního vektoru. První volbou často bývá expresní systém využívající bakterii Escherichia coli, jehož přednostmi jsou zejména technická, časová i finanční nenáročnost. Tento expresní systém není příliš vhodný pro produkci komplexních savčích proteinů, pro které jsou optimální expresní systémy založené na využití eukaryotických organizmů (kvasinky, hmyzí buňky nebo savčí buňky). Kultivace hmyzích a savčích buněk je však technicky i finančně náročná. Rekombinantní proteiny jsou purifikovány nejčastěji metodou afinitní chromatografie využívající specifickou interakci peptidu nebo proteinu s afinitní matricí. Tyto peptidy či proteiny jsou fúzovány s N‑ nebo C‑koncem purifikovaného proteinu. Purifikace probíhá ve třech krocích, kdy je rekombinantní protein prostřednictvím afinitních značek specificky zachycen na matrici chromatografické kolony, dále následuje promývací krok, po kterém je uvolněn z kolony čistý protein.

Production of recombinant proteins is essential for many applications in both basic research and also in medicine, where recombinant proteins are used as pharmaceuticals. This review sum­marizes procedures involved in recombinant protein expression and purification, including molecular cloning of target genes into expression vectors, selection of the appropriate expres­sion system, and protein purification techniques. Recombinant DNA technology allows protein engineering to modify protein stability, activity and function or to facilitate protein purification by affinity tag fusions. A wide range of cloning systems enabling fast and effective design of expression vectors is currently available. A first choice of protein expression system is usually the bacteria Escherichia coli. The main advantages of this prokaryotic expression system are low cost and simplicity; on the other hand this system is often unsuitable for production of complex mam­malian proteins. Protein expression mediated by eukaryotic cells (yeast, insect and mammalian cells) usually produces properly folded and posttranslationally modified proteins. How­ever, cultivation of insect and, especially, mammalian cells is time consuming and expensive. Affinity tagged recombinant proteins are purified efficiently using affinity chromatography. An affinity tag is a protein or peptide that mediates specific binding to a chromatography column, unbound proteins are removed during a washing step and pure protein is subsequently eluted. Key words: recombinant protein – molecular cloning – purification – expression system This work was supported by the European Regional Development Fund and the State Budget of the Czech Republic (RECAMO, CZ.1.05/2.1.00/03.0101) and by MH CZ – DRO (MMCI, 00209805). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for bio­medical papers. Submitted: 22. 1. 2014 Accepted: 20. 3. 2014

• Klíčová slova
• purifikace, expresní systém, expresní vektor,
• MeSH
• chromatografie afinitní MeSH
• Escherichia coli genetika   MeSH
• eukaryotické buňky MeSH
• exprese genu * MeSH
• genetická transkripce MeSH
• genetické vektory * MeSH
• klonování DNA * MeSH
• kultivační média MeSH
• kvasinky genetika   MeSH
• prokaryotické buňky MeSH
• proteinové inženýrství MeSH
• rekombinantní proteiny * genetika   chemická syntéza   MeSH
• virové proteiny genetika   MeSH
• Publikační typ
• práce podpořená grantem MeSH

One recently discussed general mechanism affecting gene expression is 3'-untranslated region (3'UTR) length. Events such as shortening, translocation or loss of 3'UTRs are observed within oncogenes and are proposed to associate with increased expression. Thus, increased efforts are being made to understand constitutive and differential transcript 3'end formation. Investigation of AGR2 mRNA revealed a direct impact of its 3'UTR length on AGR2 expression. In silico analyses identified several regulatory sequences within the distal part of AGR2 mRNA that may regulate 3'UTR length and associated protein levels. Short 3'UTRs were observed in a panel of AGR2-positive cancer cell lines and in human breast cancer specimens, in which more extensive 3'UTR shortening correlated with increased AGR2 protein levels. AGR2 is an important member of PI3K/AKT signalling pathway, which along with the proposed involvement of mTOR in the regulation of alternative polyadenylation, prompted us to study the role of mTOR in relation to AGR2 mRNA 3'UTR shortening. A direct impact of mTOR signalling on AGR2 3'UTR shortening associated with increased protein synthesis was found, which led to the identification of a novel molecular mechanism involved in upregulation of AGR2 levels in mTOR-activated cells via modulating the 3'UTR length of AGR2 mRNA.

• MeSH
• 3' nepřekládaná oblast genetika   MeSH
• buňky A549 MeSH
• HCT116 buňky MeSH
• HEK293 buňky MeSH
• klonování DNA MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• MFC-7 buňky MeSH
• multiproteinové komplexy genetika   MeSH
• nádorové buněčné linie MeSH
• nádory prsu genetika   MeSH
• polyadenylace genetika   MeSH
• proteiny genetika   metabolismus   MeSH
• proteosyntéza genetika   MeSH
• regulace genové exprese genetika   MeSH
• regulační sekvence ribonukleových kyselin genetika   MeSH
• signální transdukce genetika   MeSH
• TOR serin-threoninkinasy genetika   metabolismus   MeSH
• upregulace genetika   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Polycythemia vera is a clonal hematopoietic stem cell disorder in which the JAK2 V617F mutation is observed in >95% of patients, but an as yet unidentified process appears to initiate the clonal expansion of hematopoiesis. Because microRNA regulate hematopoietic differentiation, we hypothesized that dysregulated expression of microRNA may contribute to the pathophysiology of polycythemia vera. DESIGN AND METHODS: We performed gene expression profiling in five patients with polycythemia vera and in five controls using CombiMatrix MicroRNA CustomArray. ANOVA identified deregulated microRNA in polycythemia vera, and their expression was studied in a larger set of samples by quantitative reverse transcriptase polymerase chain reaction. The expression of these microRNA was also analyzed in other myeloproliferative disorders. RESULTS: We observed down-regulation of let-7a and up-regulation of miR-182 in polycythemia vera granulocytes, up-regulation of miR-143, miR-145 and miR-223 in polycythemia vera mononuclear cells, up-regulation of miR-26b in polycythemia vera platelets, and down-regulation of miR-30b, miR-30c and miR-150 in polycythemia vera reticulocytes. JAK2 V617F frequency was positively correlated with miR-143 expression and inversely correlated with let-7a, miR-30c, miR-342 and miR-150. Transcript level of predicted target genes was determined, and overexpression of IRAK2 was detected in all granulocytes from patients with myeloproliferative disorders and in polycythemia vera reticulocytes. Abnormally high HMGA2 microRNA was found in myelofibrosis granulocytes. CONCLUSIONS: Our study demonstrates that peripheral blood cells from patients with polycythemia vera have microRNA signatures distinct from those of controls. Our findings of aberrant microRNA expression underline the complexity of the molecular basis of polycythemia vera.

• MeSH
• buněčná diferenciace MeSH
• buněčný rodokmen MeSH
• granulocyty cytologie   MeSH
• hematopoéza MeSH
• Janus kinasa 2 genetika   MeSH
• leukocyty mononukleární cytologie   MeSH
• lidé MeSH
• mikro RNA * metabolismus   MeSH
• mutace * MeSH
• polycythaemia vera * genetika   metabolismus   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• primární myelofibróza metabolismus   MeSH
• regulace genové exprese * MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• stanovení celkové genové exprese * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH