High-resolution ribosome fractionation and low-input ribosome profiling of bovine oocytes and preimplantation embryos has enabled us to define the translational landscapes of early embryo development at an unprecedented level. We analyzed the transcriptome and the polysome- and non-polysome-bound RNA profiles of bovine oocytes (germinal vesicle and metaphase II stages) and early embryos at the two-cell, eight-cell, morula and blastocyst stages, and revealed four modes of translational selectivity: (1) selective translation of non-abundant mRNAs; (2) active, but modest translation of a selection of highly expressed mRNAs; (3) translationally suppressed abundant to moderately abundant mRNAs; and (4) mRNAs associated specifically with monosomes. A strong translational selection of low-abundance transcripts involved in metabolic pathways and lysosomes was found throughout bovine embryonic development. Notably, genes involved in mitochondrial function were prioritized for translation. We found that translation largely reflected transcription in oocytes and two-cell embryos, but observed a marked shift in the translational control in eight-cell embryos that was associated with the main phase of embryonic genome activation. Subsequently, transcription and translation become more synchronized in morulae and blastocysts. Taken together, these data reveal a unique spatiotemporal translational regulation that accompanies bovine preimplantation development.

• Klíčová slova
• Bovine, Preimplantation embryo development, Ribosome profiling, Transcription, Translation, Translational selectivity,
• MeSH
• blastocysta * metabolismus   MeSH
• embryonální vývoj * genetika   MeSH
• morula metabolismus   MeSH
• oocyty metabolismus   MeSH
• ribozomy genetika   MeSH
• skot MeSH
• těhotenství MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Post-translational modifications (PTMs) of biomacromolecules can be useful for understanding the processes by which a relatively small number of individual genes in a particular genome can generate enormous biological complexity in different organisms. The proteomes of barley and the brewing process were investigated by different techniques. However, their diverse and complex PTMs remain understudied. As standard analytical approaches have limitations, innovative analytical approaches need to be developed and applied in PTM studies. To make further progress in this field, it is necessary to specify the sites of modification, as well as to characterize individual isoforms with increased selectivity and sensitivity. This review summarizes advances in the PTM analysis of barley proteins, particularly those involving mass spectrometric detection. Our focus is on monitoring phosphorylation, glycation, and glycosylation, which critically influence functional behavior in metabolism and regulation in organisms.

• Klíčová slova
• barley, mass spectrometry, post-translational modification, protein,
• MeSH
• fosforylace MeSH
• glykosylace MeSH
• ječmen (rod) * genetika   MeSH
• posttranslační úpravy proteinů MeSH
• proteom chemie   MeSH
• proteomika metody   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• proteom MeSH

There are two strategies applicable to revealing non-enzymatic post-translational modifications of proteins; while assaying of the hydrolytically stable adducts was the subject of our previous communication [1], here we attempted to review separation technologies for the unfragmented modified proteins. There are a few standard procedures used for this purpose, namely Laemmli gel electrophoresis, different modes of gel permeation chromatography and boronate affinity chromatography. The latter approach makes use of the vicinal hydroxy groups present in glycated proteins. Some (but not all) arising adducts exhibit typical fluorescence which can be exploited for detection. In most cases fluorescence is measured at 370/440 nm for the so-called advanced glycation products or at 335/385 nm for the only so far well characterized glycation marker (pentosidine). Some indication exists that, e.g., synchronous fluorescence detection will probably in the future add to the selectivity and allow the distinction of the different adducts arising during non-enzymatic post-translational modifications (glycation). The proteins reviewed are serum albumin, collagen and lens proteins while glycation of hemoglobin is the subject of another review within the present volume.

• MeSH
• chromatografie kapalinová metody   MeSH
• elektroforéza metody   MeSH
• glykosylace MeSH
• kolagen chemie   MeSH
• krystaliny chemie   MeSH
• lipidové peroxidy chemie   MeSH
• posttranslační úpravy proteinů * MeSH
• proteiny chemie   metabolismus   MeSH
• sérový albumin chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• kolagen MeSH
• krystaliny MeSH
• lipidové peroxidy MeSH
• proteiny MeSH
• sérový albumin MeSH

Translational control targeting the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast translation complex profile sequencing (TCP-seq), related to ribosome profiling, and adapted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ss) in addition to 80S ribosomes (80Ss), revealed that mammalian and yeast 40Ss distribute similarly across 5'TRs, indicating considerable evolutionary conservation. We further developed yeast and human selective TCP-seq (Sel-TCP-seq), enabling selection of 40Ss and 80Ss associated with immuno-targeted factors. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5' UTRs with scanning 40Ss to successively dissociate upon AUG recognition; notably, a proportion of eIF3 lingers on during the initial elongation cycles. Highlighting Sel-TCP-seq versatility, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes.

• Klíčová slova
• ATF4, GCN4, Ribo-seq, TCP-seq, UTR, co-translational assembly, eIF2, eIF3, gene expression, mRNA, ribosome, ribosome profiling, translational control,
• MeSH
• 5' nepřekládaná oblast MeSH
• eukaryotický iniciační faktor 2 genetika   metabolismus   MeSH
• eukaryotický iniciační faktor 3 genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• iniciační faktory genetika   metabolismus   MeSH
• kodon iniciační MeSH
• lidé MeSH
• malé podjednotky ribozomu eukaryotické genetika   metabolismus   MeSH
• multiproteinové komplexy genetika   metabolismus   MeSH
• proteosyntéza * MeSH
• ribozomy genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• transkripční faktor ATF4 genetika   metabolismus   MeSH
• transkripční faktory bZIP genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 5' nepřekládaná oblast MeSH
• ATF4 protein, human MeSH Prohlížeč
• eukaryotický iniciační faktor 2 MeSH
• eukaryotický iniciační faktor 3 MeSH
• GCN4 protein, S cerevisiae MeSH Prohlížeč
• iniciační faktory MeSH
• kodon iniciační MeSH
• multiproteinové komplexy MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• transkripční faktor ATF4 MeSH
• transkripční faktory bZIP MeSH

Histone deacetylase 6 (HDAC6) is a unique member of the HDAC family of enzymes due to its complex domain organization and cytosolic localization. Experimental data point toward the therapeutic use of HDAC6-selective inhibitors (HDAC6is) for use in both neurological and psychiatric disorders. In this article, we provide side-by-side comparisons of hydroxamate-based HDAC6is frequently used in the field and a novel HDAC6 inhibitor containing the difluoromethyl-1,3,4-oxadiazole function as an alternative zinc-binding group (compound 7). In vitro isotype selectivity screening uncovered HDAC10 as a primary off-target for the hydroxamate-based HDAC6is, while compound 7 features exquisite 10,000-fold selectivity over all other HDAC isoforms. Complementary cell-based assays using tubulin acetylation as a surrogate readout revealed approximately 100-fold lower apparent potency for all compounds. Finally, the limited selectivity of a number of these HDAC6is is shown to be linked to cytotoxicity in RPMI-8226 cells. Our results clearly show that off-target effects of HDAC6is must be considered before attributing observed physiological readouts solely to HDAC6 inhibition. Moreover, given their unparalleled specificity, the oxadiazole-based inhibitors would best be employed either as research tools in further probing HDAC6 biology or as leads in the development of truly HDAC6-specific compounds in the treatment of human disease states.

• Klíčová slova
• histone deacetylase, inhibitor profiling, metallohydrolase, nanoBRET, tubulin/histone acetylation,
• MeSH
• acetylace MeSH
• histondeacetylasa 6 * antagonisté a inhibitory   MeSH
• histondeacetylasy * metabolismus   MeSH
• inhibitory histondeacetylas * chemie   farmakologie   MeSH
• kyseliny hydroxamové * chemie   farmakologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• oxadiazoly * chemie   farmakologie   MeSH
• posttranslační úpravy proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• HDAC10 protein, human MeSH Prohlížeč
• histondeacetylasa 6 * MeSH
• histondeacetylasy * MeSH
• inhibitory histondeacetylas * MeSH
• kyseliny hydroxamové * MeSH
• oxadiazoly * MeSH

Extracorporeal life support is a treatment modality that provides prolonged blood circulation, gas exchange and can substitute functions of heart and lungs to provide urgent cardio-respiratory stabilization in patients with severe but potentially reversible cardiopulmonary failure refractory to conventional therapy. Generally, the therapy targets blood pressure, volume status, and end-organs perfusion. As there are significant differences in hemodynamic efficacy among different percutaneous circulatory support systems, it should be carefully considered when selecting the most appropriate circulatory support for specific medical conditions in individual patients. Despite severe metabolic and hemodynamic deterioration during prolonged cardiac arrest, venoarterial extracorporeal membrane oxygenation (VA ECMO) can rapidly revert otherwise fatal prognosis, thus carrying a potential for improvement in survival rate, which can be even improved by introduction of mild therapeutic hypothermia. In order to allow a rapid transfer of knowledge to clinical medicine two porcine models were developed for studying efficiency of the VA ECMO in treatments of acute cardiogenic shock and progressive chronic heart failure. These models allowed also an intensive research of adverse events accompanying a clinical use of VA ECMO and their possible compensations. The results indicated that in order to weaken the negative effects of increased afterload on the left ventricular function the optimal VA ECMO flow in cardiogenic shock should be as low as possible to allow adequate tissue perfusion. The left ventricle can be also unloaded by an ECG-synchronized pulsatile flow if using a novel pulsatile ECMO system. Thus, pulsatility of VA ECMO flow may improve coronary perfusion even under conditions of high ECMO blood flows. And last but not least, also the percutaneous balloon atrial septostomy is a very perspective method how to passively decompress overloaded left heart.

• MeSH
• kardiogenní šok terapie   MeSH
• kardiovaskulární systém * MeSH
• mimotělní membránová oxygenace * škodlivé účinky   MeSH
• prasata MeSH
• srdeční selhání * terapie   MeSH
• translační biomedicínský výzkum MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

BACKGROUND AND OBJECTIVES: Diamond-Blackfan anemia (DBA) is a rare congenital pure red cell aplasia characterized by normochromic macrocytic anemia, reticulocytopenia, and normocellular bone marrow with a selective deficiency of erythroid precursors. Ribosomal protein S19 (RPS19), currently the only gene associated with DBA, is mutated in 25% of DBA patients, but its role in erythropoiesis is unknown. We attempted to elucidate the importance of RPS19 in translation in relation to the pathogenesis of DBA. DESIGN AND METHODS: We measured translation and proliferation rates in unstimulated and phytohemagglutinin (PHA)-stimulated lymphocytes isolated from DBA patients, as well as in K562 cells expressing several RPS19 mutants to directly test the effect of RPS19 mutations on translation. The effect of leucine on overall translation was also studied. RESULTS: We found that the level of translation was on average 48-73% of controls in both unstimulated and PHA-activated DBA lymphocytes irrespective of mutations in RPS19. The addition of leucine increased the translational level in RPS19-non-mutated DBA cells, but not in cells with an RPS19 mutation. In unstimulated DBA cells, proliferation was significantly impaired in both RPS19-mutated and non-mutated cells, but in both groups could be efficiently activated by PHA. Studies on K562 cells showed that RPS19 mutations affecting RPS19 conserved arginines R56Q and R62Q could significantly inhibit the rate of protein synthesis, indicating the importance of RPS19 in translation. INTERPRETATION AND CONCLUSIONS: Our results indicate that inefficient translation may be the main cause of DBA, and administration of leucine may be beneficial for at least some DBA patients.

• MeSH
• arginin genetika   MeSH
• buňky K562 MeSH
• Diamondova-Blackfanova anemie krev   genetika   patologie   MeSH
• kojenec MeSH
• krevní proteiny antagonisté a inhibitory   biosyntéza   MeSH
• lidé MeSH
• lymfocyty metabolismus   patologie   MeSH
• mutace MeSH
• novorozenec MeSH
• proteosyntéza * MeSH
• regulace genové exprese MeSH
• ribozomální proteiny biosyntéza   krev   genetika   MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• novorozenec MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• arginin MeSH
• krevní proteiny MeSH
• ribosomal protein S19 MeSH Prohlížeč
• ribozomální proteiny MeSH

Translational regulation is pivotal during preimplantation development. However, how mRNAs are selected for temporal regulation and their dynamic utilization and fate during this period are still unknown. Using a high-resolution ribosome profiling approach, we analyzed the transcriptome, as well as monosome- and polysome-bound RNAs of mouse oocytes and embryos, defining an unprecedented extent of spatiotemporal translational landscapes during this rapid developmental phase. We observed previously unknown mechanisms of translational selectivity, i.e., stage-wise deferral of loading monosome-bound mRNAs to polysome for active translation, continuous translation of both monosome and polysome-bound mRNAs at the same developmental stage, and priming to monosomes after initial activation. We showed that a eukaryotic initiation factor Eif1ad3, which is exclusively translated in the 2-Cell embryo, is required for ribosome biogenesis post embryonic genome activation. Our study thus provides genome-wide datasets and analyses of spatiotemporal translational dynamics accompanying mammalian germ cell and embryonic development and reveals the contribution of a novel translation initiation factor to mammalian pre-implantation development.

• Publikační typ
• časopisecké články MeSH
• preprinty MeSH

The capacity of adenylate cyclase toxin (ACT) to penetrate into target cells depends on post-translational fatty-acylation by the acyltransferase CyaC, which can palmitoylate the conserved lysines 983 and 860 of ACT. Here, the in vivo acylating capacity of a set of mutated CyaC acyltransferases was characterized by two-dimensional gel electrophoresis and mass spectrometric analyses of the ACT product. Substitutions of the potentially catalytic serine 20 and histidine 33 residues ablated acylating activity of CyaC. Conservative replacements of alanine 140 by glycine (A140G) and valine (A140V) residues, however, affected selectivity of CyaC for the two acylation sites on ACT. Activation by the A140G variant of CyaC generated a mixture of bi- and monoacylated ACT molecules, modified either at both Lys-860 and Lys-983, or only at Lys-860, respectively. In contrast, the A140V CyaC produced a nearly 1:1 mixture of nonacylated pro-ACT with ACT monoacylated almost exclusively at Lys-983. The respective proportion of toxin molecules acylated at Lys-983 correlated well with the cell-invasive activity of both ACT mixtures, which was about half of that of ACT fully acylated on Lys-983 by intact CyaC. These results show that acylation of Lys-860 alone does not confer cell-invasive activity on ACT, whereas acylation of Lys-983 is necessary and sufficient.

• MeSH
• 2D gelová elektroforéza MeSH
• acylace MeSH
• acyltransferasy chemie   genetika   metabolismus   MeSH
• adenylátcyklasový toxin * MeSH
• Bordetella pertussis enzymologie   MeSH
• erytrocyty účinky léků   metabolismus   patologie   MeSH
• faktory virulence rodu Bordetella chemie   genetika   metabolismus   toxicita   MeSH
• hemolýza účinky léků   MeSH
• histidin genetika   metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• kyselina palmitová metabolismus   MeSH
• lysin genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• ovce MeSH
• peptidové fragmenty chemie   metabolismus   MeSH
• posttranslační úpravy proteinů * MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• serin genetika   metabolismus   MeSH
• substituce aminokyselin MeSH
• substrátová specifita MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• acyltransferasy MeSH
• adenylátcyklasový toxin * MeSH
• faktory virulence rodu Bordetella MeSH
• histidin MeSH
• kyselina palmitová MeSH
• lysin MeSH
• peptidové fragmenty MeSH
• serin MeSH

To investigate the influence of sequence context of translation initiation codon on translation efficiency in Kinetoplastida, we constructed a library of expression plasmids randomized in the three nucleotides prefacing ATG of a reporter gene encoding enhanced green fluorescent protein (EGFP). All 64 possible combinations of pre-ATG triplets were individually stably integrated into the rDNA locus of Leishmania tarentolae and the resulting cell lines were assessed for EGFP expression. The expression levels were quantified directly by measuring the fluorescence of EGFP protein in living cells and confirmed by Western blotting. We observed a strong influence of the pre-ATG triplet on the level of protein expression over a 20-fold range. To understand the degree of evolutionary conservation of the observed effect, we transformed Phytomonas serpens, a trypanosomatid parasite of plants, with a subset of the constructs. The pattern of translational efficiency mediated by individual pre-ATG triplets in this species was similar to that observed in L. tarentolae. However, the pattern of translational efficiency of two other proteins (red fluorescent protein and tetracycline repressor) containing selected pre-ATG triplets did not correlate with either EGFP or each other. Thus, we conclude that a conserved mechanism of translation initiation site selection exists in kinetoplastids that is strongly influenced not only by the pre-ATG sequences but also by the coding region of the gene.

• MeSH
• 5' nepřekládaná oblast genetika   MeSH
• červený fluorescenční protein MeSH
• kodon iniciační * MeSH
• Leishmania genetika   růst a vývoj   metabolismus   MeSH
• luminescentní proteiny genetika   metabolismus   MeSH
• proteosyntéza * MeSH
• regulace genové exprese * MeSH
• reportérové geny MeSH
• rezistence na tetracyklin genetika   MeSH
• transfekce MeSH
• Trypanosomatina genetika   růst a vývoj   metabolismus   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 5' nepřekládaná oblast MeSH
• kodon iniciační * MeSH
• luminescentní proteiny MeSH
• zelené fluorescenční proteiny MeSH