The universal nine-amino-acid transactivation domains (9aaTADs) have been identified in numerous transcription activators. Here, we identified the conserved 9aaTAD motif in all nine members of the specificity protein (SP) family. Previously, the Sp1 transcription factor has been defined as a glutamine-rich activator. We showed by amino acid substitutions that the glutamine residues are completely dispensable for 9aaTAD function and are not conserved in the SP family. We described the origin and evolutionary history of 9aaTADs. The 9aaTADs of the ancestral Sp2 gene became inactivated in early chordates. We next discovered that an accumulation of valines in 9aaTADs inactivated their transactivation function and enabled their strict conservation during evolution. Subsequently, in chordates, Sp2 has duplicated and created new paralogs, Sp1, Sp3, and Sp4 (the SP1-4 clade). During chordate evolution, the dormancy of the Sp2 activation domain lasted over 100 million years. The dormant but still intact ancestral Sp2 activation domains allowed diversification of the SP1-4 clade into activators and repressors. By valine substitution in the 9aaTADs, Sp1 and Sp3 regained their original activator function found in ancestral lower metazoan sea sponges. Therefore, the vertebrate SP1-4 clade could include both repressors and activators. Furthermore, we identified secondary 9aaTADs in Sp2 introns present from fish to primates, including humans. In the gibbon genome, introns containing 9aaTADs were used as exons, which turned the Sp2 gene into an activator. Similarly, we identified introns containing 9aaTADs used conditionally as exons in the (SP family-unrelated) transcription factor SREBP1, suggesting that the intron-9aaTAD reservoir is a general phenomenon.
- MeSH
- aktivace transkripce MeSH
- duplikace genu MeSH
- fylogeneze MeSH
- introny * genetika MeSH
- lidé MeSH
- molekulární evoluce * MeSH
- regulace genové exprese * MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie MeSH
- transkripční faktor Sp2 * antagonisté a inhibitory genetika metabolismus MeSH
- valin genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
The universal nine-amino-acid transactivation domains (9aaTADs) have been identified in numerous transcription activators. Here, we identified the conserved 9aaTAD motif in all nine members of the specificity protein (SP) family. Previously, the Sp1 transcription factor has been defined as a glutamine-rich activator. We showed by amino acid substitutions that the glutamine residues are completely dispensable for 9aaTAD function and are not conserved in the SP family. We described the origin and evolutionary history of 9aaTADs. The 9aaTADs of the ancestral Sp2 gene became inactivated in early chordates. We next discovered that an accumulation of valines in 9aaTADs inactivated their transactivation function and enabled their strict conservation during evolution. Subsequently, in chordates, Sp2 has duplicated and created new paralogs, Sp1, Sp3, and Sp4 (the SP1-4 clade). During chordate evolution, the dormancy of the Sp2 activation domain lasted over 100 million years. The dormant but still intact ancestral Sp2 activation domains allowed diversification of the SP1-4 clade into activators and repressors. By valine substitution in the 9aaTADs, Sp1 and Sp3 regained their original activator function found in ancestral lower metazoan sea sponges. Therefore, the vertebrate SP1-4 clade could include both repressors and activators. Furthermore, we identified secondary 9aaTADs in Sp2 introns present from fish to primates, including humans. In the gibbon genome, introns containing 9aaTADs were used as exons, which turned the Sp2 gene into an activator. Similarly, we identified introns containing 9aaTADs used conditionally as exons in the (SP family-unrelated) transcription factor SREBP1, suggesting that the intron-9aaTAD reservoir is a general phenomenon.
- MeSH
- aktivace transkripce MeSH
- duplikace genu MeSH
- fylogeneze MeSH
- introny genetika MeSH
- lidé MeSH
- molekulární evoluce * MeSH
- regulace genové exprese * MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie MeSH
- transkripční faktor Sp2 antagonisté a inhibitory genetika metabolismus MeSH
- valin genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The success of bottom-up proteomic analysis frequently depends on the efficient removal of contaminants from protein or peptide samples before LC-MS/MS. For a peptide clean-up workflow, single-pot solid-phase-enhanced peptide sample preparation on carboxylate-modified paramagnetic beads (termed SP2) was evaluated for sodium dodecyl sulfate or polyethylene glycol removal from Arabidopsis thaliana tryptic peptides. The robust and efficient 40-min SP2 protocol, tested for 10-ng, 250-ng, and 10-μg peptide samples, was proposed and benchmarked thoroughly against the ethyl acetate extraction protocol. The SP2 protocol on carboxylated magnetic beads proved to be the most robust approach, even for the simultaneous removal of massive sodium dodecyl sulfate (SDS) and polyethylene glycol (PEG) contaminations from AT peptide samples in respect of the LC-MS/MS data outperforming ethyl acetate extraction.
This study tested whether emotional contagion occurs when piglets directly observe a penmate in distress (restraint) and whether there is an effect of previous experience on the response to subsequent restraint or exposure to conspecific distress. Piglets (49.7 ± 0.7 days) were exposed in pairs to two stress phases (SP1 and SP2) in an arena divided into two pens by a wire mesh wall. During SP1, one of the pigs of a pair was either restrained (Stress treatment) or sham-restrained (Control treatment), while the other pig was considered observer. During SP2, the previous observer was restrained, while its penmate took the observer role. Heart rate variability, locomotion, vocalizations, body/head/ear and tail postures were monitored. During SP1, observer pigs responded to conspecific distress with increased indicators of attention (looking at, proximity to and snout contacts with the distressed pigs) and increased indicators of fear (reduced locomotion, increased freezing). During SP2, the observer pigs that had been restrained previously reacted more strongly (through higher proximity, decreased locomotion, increased freezing) to observing the penmate in restraint than pigs without the previous negative experience. This study suggests that young pigs are susceptible to emotional contagion and that this contagion is potentiated by previous exposure to the same stressor. These findings have implications for pig welfare in practical animal husbandry systems.
- MeSH
- emoce fyziologie MeSH
- empatie MeSH
- lokomoce MeSH
- postura těla MeSH
- psychický stres patofyziologie MeSH
- srdeční frekvence fyziologie MeSH
- Sus scrofa fyziologie MeSH
- vokalizace zvířat MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Autoři hodnotí klinické a elektrofyziologické nálezy při neinvazivním a invazivním videoEEG monitorování u 3 epileptických pacientů, u kterých byl zaznamenán iktální vomitus (ictus emeticus). Hodnoceno bylo celkem 14 záchvatů s tímto příznakem, 12 při neinvazivním a 2 při invazivním videoEEG nionitorování. U všech pacientů se jednalo o sekundární epilepsii temporálního laloku vpravo, u 2 ze 3 pacientů, kteří podstoupili invazivní EEG vyšetření, byl prokázán počátek záchvatů v pravém hipokampu. Iktální vomitus při neinvazivním videoEEG monitorování byl asociován s iktálním výbojem v pravé sfenoidální elektrodě (Sp2) a temporálních a paracentrálních skalpových kontaktech vpravo (T4, C4, T2). Při invazivním videoEEG u obou záchvatů je v průběhu iktálního vomitu přítomna paroxysmální epileptická aktivita v amygdale a hipokampu vpravo, laterálních neokortikálních strukturách vpravo (gyrus temporalis superior, gyrus temporalis medius). U jednoho záchvatu je přítomna v průběhu tohoto fenoménu iktální aktivita mimo uvedené struktury i v gyrus temporalis inferior a fronto-orbitálním kortexu vpravo. Přední cingulum a laterální prefi'ontální koriex (area 9) nejsou v průběhu iktálního vomitu zavzaty do iktálního výboje. Iktální vomitus byl pozorován u 2,6 % ze všech monitorovaných epileptických pacientů a 3,5 % ze všech pacientů s epilepsií temporálního laloku. U studovaných pacientů byl zachycen v téměř 42 % parciálních epileptických záchvatů. Iktální vomitus lze považovat za spolehlivý lateralizační příznak u epilepsie temporálního laloku, který odpovídá postižení pravostranného, respektive nedominantního temporálního laloku. Pravděpodobně neexistuje v kortexu kritické místo, které by zodpovídalo za zvracivý reflex. Spolu s literárními prameny autoři poukazují na úlohu pravostranného fronto-orbito-inzulo-operkulo-temporálního okruhu při genezi tohoto vzácného iktálního fenoménu. Uvedené výsledky tak mohou demonstrovat určitou funkční hemisferální asymetrii v řízení vegetativních funkcí.
The authors evaluate the clinical and electrophysiological findings of non-invasive and invasive video EEG monitoring in three epileptic patients with ictus emeticus. They evaluated a total of 14 seizures with this symptom, 12 with non-invasive and two with invasive video EEGmonitoring. In all patients secondary epilepsy of the temporal lobe on the right side was involved. In two of three patients who had invasive EEG examination the onset of seizures in the right hippocampus was proved. Ictus emeticus in non-invasive videoEEG monitoring was associated with ictal discharge in the right sphenoidal electrode (Sp2) and temporal and paracentral scalp contacts on the right (T4, C4, T2). In invasive videoEEG in both seizures during ictus emeticus paroxysmal epileptic activity in the amygdaloid nucleus and hippocampus on the right is present as well as in the lateral neocortical structures on the right (gyrus temporalis superior, gjnrus temporalis medius). In one seizure during this phenomenon ictal activity beyond the mentioned structures is present also in the gyrus temporalis inferior and in the fŕonto-orbital cortex on the right side. The anterior cingulum and lateral prefrontal cortex (area 9) are not included in the ictal discharge during the ictus emeticus. Ictus emeticus was observed in 2.6% of all monitored epileptic patients and 3.5% of all patients with epilepsy of the temporal lobe. It was recorded in the investigated patients in almost 42% partial epileptic seizures. Ictus emeticus can oe considered a reliable lateralizing symptom in epilepsy of the temporal lobe which corresponds with affection of the dextrolateral or non-dominant temporal lobe resp. Probably there is no critical site in the cortex responsible for the vomiting reflex. Consistent with data in the literature the authors draw attention to the role of the dextrolateral fronto-orbito-insulo-operculo-temporal circle in the genesis of this rare ictal phenomenon. The presented results thus may demonstrate a certain functional hemispheric asymmetry in the control of vegetative functions.
- MeSH
- audiovizuální záznam MeSH
- dospělí MeSH
- elektroencefalografie metody MeSH
- epilepsie temporálního laloku patologie MeSH
- lidé MeSH
- svalové křeče MeSH
- zvracení patologie MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
The synthesis of axially chiral benzimidazoles with a peri-substituted naphthalene and a dimethylamino group at positions 1 and 2, respectively, was developed. We evaluated these compounds in the desymmetrization reaction of cis-tetrahydrophthalic anhydride with benzyl alcohol, as the nucleophilic sp2 imidazole nitrogen atom is able to catalyze the acyl-transfer reaction. The prepared benzimidazoles demonstrated catalytic activity and showed that their axial chirality impacts stereoselectivity.
- Publikační typ
- časopisecké články MeSH
The catalytic versatility of cytochrome P450 monooxygenases is remarkable. Here, we present mechanistic and structural characterizations of TleB from Streptomyces blastmyceticus and its homolog HinD from Streptoalloteichus hindustanus, which catalyze unusual intramolecular C-N bond formation to generate indolactam V from the dipeptide N-methylvalyl-tryptophanol. In vitro analyses demonstrated that both P450s exhibit promiscuous substrate specificity, and modification of the N13-methyl group resulted in the formation of indole-fused 6/5/6 tricyclic products. Furthermore, X-ray crystal structures in complex with substrates and structure-based mutagenesis revealed the intimate structural details of the enzyme reactions. We propose that the generation of a diradical species is critical for the indolactam formation, and that the intramolecular C(sp2)-H amination is initiated by the abstraction of the N1 indole hydrogen. After indole radical repositioning and subsequent removal of the N13 hydrogen, the coupling of the properly-folded diradical leads to the formation of the C4-N13 bond of indolactam.
Although several scaling bioreactor models of mammalian cell cultures are suggested and described in the literature, they mostly lack a significant validation at pilot or manufacturing scale. The aim of this study is to validate an oscillating hydrodynamic stress loop system developed earlier by our group for the evaluation of the maximum operating range for stirring, based on a maximum tolerable hydrodynamic stress. A 300-L pilot-scale bioreactor for cultivation of a Sp2/0 cell line was used for this purpose. Prior to cultivations, a stress-sensitive particulate system was applied to determine the stress values generated by stirring and sparging. Pilot-scale data, collected from 7- to 28-Pa maximum stress conditions, were compared with data from classical 3-L cultivations and cultivations from the oscillating stress loop system. Results for the growth behavior, analyzed metabolites, productivity, and product quality showed a dependency on the different environmental stress conditions but not on reactor size. Pilot-scale conditions were very similar to those generated in the oscillating stress loop model confirming its predictive capability, including conditions at the edge of failure.
Stimuli-responsive copolymers are of great interest for targeted drug delivery. This study reports on a controllable post-polymerization quaternization with 2-bromomethyl-4-fluorophenylboronic acid of the poly(4-vinyl pyridine) (P4VP) block of a common poly(styrene)-b-poly(4-vinyl pyridine)-b-poly(ethylene oxide) (SVE) triblock terpolymer in order to achieve a selective responsivity to various diols. For this purpose, a reproducible method was established for P4VP block quaternization at a defined ratio, confirming the reaction yield by 11B, 1H NMR. Then, a reproducible self-assembly protocol is designed for preparing stable micelles from functionalized stimuli-responsive triblock terpolymers, which are characterized by light scattering and by cryogenic transmission electron microscopy. In addition, UV-Vis spectroscopy is used to monitor the boron-ester bonding and hydrolysis with alizarin as a model drug and to study encapsulation and release of this drug, induced by sensing with three geminal diols: fructose, galactose and ascorbic acid. The obtained results show that only the latter, with the vicinal diol group on sp2-hybridized carbons, was efficient for alizarin release. Therefore, the post-polymerization method for triblock terpolymer functionalization presented in this study allows for preparation of specific stimuli-responsive systems with a high potential for targeted drug delivery, especially for cancer treatment.
- Publikační typ
- časopisecké články MeSH
A straightforward methodology of fluorine substitution by tritium/deuterium is reported. The described method is selective towards the F─C (sp3 ) group and leaves both the aromatic F─C (sp2 ) and F2 ─C (sp3 ) moieties unaffected. Alkylfluorides, readily synthesized from appropriate alcohols by treatment with diethylaminosulfur trifluoride (DAST) reagent in an overall yield up to 76%, undergoes activation with the boron-based Lewis acid B(C6 F5 )3 , and stoichiometric in situ reduction with a tritide/deuteride reagent-the [TMP2(3) H][2(3) HB(C6 F5 )3 ] system of frustrated Lewis pair. This methodology provides an isolated yield of up to 93% of regio-specifically labeled small organic compounds with superior 2 H-enrichment of over 95%. The specific activity of prepared 1-(2-[3 H]-ethyl)naphthalene was determined at 29.0 Ci/mmol. The site selectivity of the Lewis acid/ [TMP2(3) H][2(3) HB(C6 F5 )3 ] approach is orthogonal to currently used methods and allows for isotopic labeling of complementary positions in molecules. Reported labeling methodology proceeds well at ultra-mild reaction conditions (220 mbar of T2 ), allowing very low consumption of the radioactive source (4.2 Ci/156 GBq), and producing limited amount of radioactive waste.
