Receptor adenylate cyclases (RACs) on the surface of trypanosomatids are important players in the host-parasite interface. They detect still unidentified environmental signals that affect the parasites' responses to host immune challenge, coordination of social motility, and regulation of cell division. A lesser known class of oxygen-sensing adenylate cyclases (OACs) related to RACs has been lost in trypanosomes and expanded mostly in Leishmania species and related insect-dwelling trypanosomatids. In this work, we have undertaken a large-scale phylogenetic analysis of both classes of adenylate cyclases (ACs) in trypanosomatids and the free-living Bodo saltans. We observe that the expanded RAC repertoire in trypanosomatids with a two-host life cycle is not only associated with an extracellular lifestyle within the vertebrate host, but also with a complex path through the insect vector involving several life cycle stages. In Trypanosoma brucei, RACs are split into two major clades, which significantly differ in their expression profiles in the mammalian host and the insect vector. RACs of the closely related Trypanosoma congolense are intermingled within these two clades, supporting early RAC diversification. Subspecies of T. brucei that have lost the capacity to infect insects exhibit high numbers of pseudogenized RACs, suggesting many of these proteins have become redundant upon the acquisition of a single-host life cycle. OACs appear to be an innovation occurring after the expansion of RACs in trypanosomatids. Endosymbiont-harboring trypanosomatids exhibit a diversification of OACs, whereas these proteins are pseudogenized in Leishmania subgenus Viannia. This analysis sheds light on how ACs have evolved to allow diverse trypanosomatids to occupy multifarious niches and assume various lifestyles.

• Klíčová slova
• Kinetoplastida, adenylate cyclase, oxygen, phylogenetics, receptor, trypanosomatid,
• MeSH
• adenylátcyklasy genetika   MeSH
• duplikace genu MeSH
• fylogeneze * MeSH
• genom protozoální MeSH
• interakce hostitele a patogenu genetika   MeSH
• molekulární evoluce * MeSH
• Trypanosomatina enzymologie   genetika   MeSH
• upregulace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasy MeSH

Combined and complex dystonias are heterogeneous movement disorders combining dystonia with other motor and/or systemic signs. Although we are beginning to understand the diverse molecular causes of these disease entities, clinical pattern recognition and conventional genetic workup achieve an etiological diagnosis only in a minority of cases. Our goal was to provide a window into the variable genetic origins and distinct clinical patterns of combined/complex dystonia more broadly. Between August 2016 and January 2017, we applied whole-exome sequencing to a cohort of nine patients with varied combined and/or complex dystonic presentations, being on a diagnostic odyssey. Bioinformatics analyses, co-segregation studies, and sequence-interpretation algorithms were employed to detect causative mutations. Comprehensive clinical review was undertaken to define the phenotypic spectra and optimal management strategies. On average, we observed a delay in diagnosis of 23 years before whole-exome analysis enabled determination of each patient's genetic defect. Whereas mutations in ACTB, ATP1A3, ADCY5, and SGCE were associated with particular phenotypic clues, trait manifestations arising from mutations in PINK1, MRE11A, KMT2B, ATM, and SLC6A1 were different from those previously reported in association with these genes. Apart from improving counseling for our entire cohort, genetic findings had actionable consequences on preventative measures and therapeutic interventions for five patients. Our investigation confirms unique genetic diagnoses, highlights key clinical features and phenotypic expansions, and suggests whole-exome sequencing as a first-tier diagnostic for combined/complex dystonia. These results might stimulate independent teams to extend the scope of agnostic genetic screening to this particular phenotypic group that remains poorly characterized through existing studies.

• Klíčová slova
• Combined dystonia, Complex dystonia, Exome, Genetic heterogeneity, Mutation,
• MeSH
• adenylátcyklasy genetika   MeSH
• dospělí MeSH
• dystonické poruchy diagnóza   genetika   MeSH
• dystonie diagnóza   genetika   MeSH
• exom genetika   MeSH
• fenotyp MeSH
• genetické testování metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace genetika   MeSH
• proteiny přenášející GABA přes plazmatickou membránu genetika   MeSH
• sodíko-draslíková ATPasa genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenylátcyklasy MeSH
• adenylyl cyclase type V MeSH Prohlížeč
• ATP1A3 protein, human MeSH Prohlížeč
• proteiny přenášející GABA přes plazmatickou membránu MeSH
• SLC6A1 protein, human MeSH Prohlížeč
• sodíko-draslíková ATPasa MeSH

β-Adrenergic signaling plays an important role in regulating diverse brain functions and alterations in this signaling have been observed in different neuropathological conditions. In this study, we investigated the effect of a 10-day treatment with high doses of morphine (10 mg/kg per day) on major components and functional state of the β-adrenergic receptor (β-AR) signaling system in the rat cerebral cortex. β-ARs were characterized by radioligand binding assays and amounts of various G protein subunits, adenylyl cyclase (AC) isoforms, G protein-coupled receptor kinases (GRKs), and β-arrestin were examined by Western blot analysis. AC activity was determined as a measure of functionality of the signaling system. We also assessed the partitioning of selected signaling proteins between the lipid raft and non-raft fractions prepared from cerebrocortical plasma membranes. Morphine treatment resulted in a significant upregulation of β-ARs, GRK3, and some AC isoforms (AC-I, -II, and -III). There was no change in quantity of G proteins and some other signaling molecules (AC-IV, AC-V/VI, GRK2, GRK5, GRK6, and β-arrestin) compared with controls. Interestingly, morphine exposure caused a partial redistribution of β-ARs, Gsα, Goα, and GRK2 between lipid rafts and bulk plasma membranes. Spatial localization of other signaling molecules within the plasma membrane was not changed. Basal as well as fluoride- and forskolin-stimulated AC activities were not significantly different in membrane preparations from control and morphine-treated animals. However, AC activity stimulated by the beta-AR agonist isoprenaline was markedly increased. This is the first study to demonstrate lipid raft association of key components of the cortical β-AR system and its sensitivity to morphine.

• Klíčová slova
• Adenylyl cyclase, Beta-adrenergic receptors, G proteins, Lipid rafts, Morphine, Rat brain cortex,
• MeSH
• adenylátcyklasy genetika   metabolismus   MeSH
• beta arrestiny genetika   metabolismus   MeSH
• beta-adrenergní receptory metabolismus   MeSH
• kinasa 3 receptorů spřažených s G-proteiny genetika   metabolismus   MeSH
• krysa rodu Rattus MeSH
• membránové mikrodomény metabolismus   MeSH
• morfin farmakologie   MeSH
• mozková kůra účinky léků   metabolismus   MeSH
• narkotika farmakologie   MeSH
• potkani Wistar MeSH
• signální transdukce * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenylátcyklasy MeSH
• beta arrestiny MeSH
• beta-adrenergní receptory MeSH
• Grk3 protein, rat MeSH Prohlížeč
• kinasa 3 receptorů spřažených s G-proteiny MeSH
• morfin MeSH
• narkotika MeSH

The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA) that plays a crucial role in host respiratory tract colonization. CyaA targets CR3-expressing cells and disrupts their bactericidal functions by delivering into their cytosol an adenylate cyclase enzyme that converts intracellular ATP to cAMP. In parallel, the hydrophobic domain of CyaA forms cation-selective pores that permeabilize cell membrane. The invasive AC and pore-forming domains of CyaA are linked by a segment that is unique in the RTX cytolysin family. We used mass spectrometry and circular dichroism to show that the linker segment forms α-helical structures that penetrate into lipid bilayer. Replacement of the positively charged arginine residues, proposed to be involved in target membrane destabilization by the linker segment, reduced the capacity of the toxin to translocate the AC domain across cell membrane. Substitutions of negatively charged residues then revealed that two clusters of negative charges within the linker segment control the size and the propensity of CyaA pore formation, thereby restricting the cell-permeabilizing capacity of CyaA. The 'AC to Hly-linking segment' thus appears to account for the smaller size and modest cell-permeabilizing capacity of CyaA pores, as compared to typical RTX hemolysins.

• MeSH
• adenylátcyklasový toxin chemie   genetika   metabolismus   MeSH
• adenylátcyklasy chemie   genetika   MeSH
• AMP cyklický metabolismus   MeSH
• Bordetella pertussis chemie   patogenita   MeSH
• hemolyziny genetika   MeSH
• lidé MeSH
• lipidové dvojvrstvy chemie   metabolismus   MeSH
• perforin chemie   MeSH
• permeabilita buněčné membrány účinky léků   MeSH
• pertuse genetika   mikrobiologie   patologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• adenylátcyklasy MeSH
• AMP cyklický MeSH
• hemolyziny MeSH
• lipidové dvojvrstvy MeSH
• perforin MeSH

All organisms have the capacity to sense and respond to environmental changes. These signals often involve the use of second messengers such as cyclic adenosine monophosphate (cAMP). This second messenger is widely distributed among organisms and coordinates gene expression related with pathogenesis, virulence, and environmental adaptation. Genomic analysis in Mycobacterium tuberculosis has identified 16 adenylyl cyclases (AC) and one phosphodiesterase, which produce and degrade cAMP, respectively. To date, ten AC have been biochemically characterized and only one (Rv0386) has been found to be important during murine infection with M. tuberculosis. Here, we investigated the impact of hsp60-driven Rv2212 gene expression in Mycobacterium bovis Bacillus Calmette-Guerin (BCG) during growth in vitro, and during macrophage and mice infection. We found that hsp60-driven expression of Rv2212 resulted in an increased capacity of replication in murine macrophages but an attenuated phenotype in lungs and spleen when administered intravenously in mice. Furthermore, this strain displayed an altered proteome mainly affecting proteins associated with stress conditions (bfrB, groEL-2, DnaK) that could contribute to the attenuated phenotype observed in mice.

• MeSH
• adenylátcyklasy genetika   metabolismus   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• buněčné linie MeSH
• chaperon hsp60 genetika   metabolismus   MeSH
• lidé MeSH
• makrofágy mikrobiologie   MeSH
• Mycobacterium bovis genetika   metabolismus   patogenita   MeSH
• Mycobacterium tuberculosis enzymologie   genetika   patogenita   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• plíce mikrobiologie   MeSH
• proteom genetika   metabolismus   MeSH
• slezina mikrobiologie   MeSH
• tuberkulóza mikrobiologie   MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasy MeSH
• bakteriální proteiny MeSH
• chaperon hsp60 MeSH
• proteom MeSH

The adenylyl cyclase (AC) signaling system plays a crucial role in the regulation of cardiac contractility. Here we analyzed the key components of myocardial AC signaling in the developing chick embryo and assessed the impact of selected β-blocking agents on this system. Application of metoprolol and carvedilol, two commonly used β-blockers, at embryonic day (ED) 8 significantly downregulated (by about 40%) expression levels of AC5, the dominant cardiac AC isoform, and the amount of Gsα protein at ED9. Activity of AC stimulated by forskolin was also significantly reduced under these conditions. Interestingly, when administered at ED4, these drugs did not produce such profound changes in the myocardial AC signaling system, except for markedly increased expression of Giα protein. These data indicate that β-blocking agents can strongly derange AC signaling during the first half of embryonic heart development.

• MeSH
• adenylátcyklasy biosyntéza   genetika   MeSH
• antiarytmika aplikace a dávkování   MeSH
• karbazoly aplikace a dávkování   MeSH
• karvedilol MeSH
• kur domácí genetika   růst a vývoj   MeSH
• kuřecí embryo MeSH
• metoprolol aplikace a dávkování   MeSH
• myokard enzymologie   MeSH
• propanolaminy aplikace a dávkování   MeSH
• regulace genové exprese účinky léků   MeSH
• signální transdukce účinky léků   MeSH
• srdce účinky léků   růst a vývoj   MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasy MeSH
• adenylyl cyclase type V MeSH Prohlížeč
• antiarytmika MeSH
• karbazoly MeSH
• karvedilol MeSH
• metoprolol MeSH
• propanolaminy MeSH

Dysfunction or abnormalities in the regulation of fatty acid translocase Cd36, a multifunctional membrane protein participating in uptake of long-chain fatty acids, has been linked to the development of heart diseases both in animals and humans. We have previously shown that the Cd36 transgenic spontaneously hypertensive rat (SHR-Cd36), with a wild type Cd36, has higher susceptibility to ischemic ventricular arrhythmias when compared to spontaneously hypertensive rat (SHR) carrying a mutant Cd36 gene, which may have been related to increased β-adrenergic responsiveness of these animals (Neckar et al., 2012 Physiol. Genomics 44:173-182). The present study aimed to determine whether the insertion of the wild type Cd36 into SHR would affect the function of myocardial G protein-regulated adenylyl cyclase (AC) signaling. β-Adrenergic receptors (β-ARs) were characterized by radioligand-binding experiments and the expression of selected G protein subunits, AC, and protein kinase A (PKA) was determined by RT-PCR and Western blot analyses. There was no significant difference in the amount of trimeric G proteins, but the number of β-ARs was higher (by about 35 %) in myocardial preparations from SHR-Cd36 as compared to SHR. Besides that, transgenic rats expressed increased amount (by about 20 %) of the dominant myocardial isoforms AC5/6 and contained higher levels of both nonphosphorylated (by 11 %) and phosphorylated (by 45 %) PKA. Differently stimulated AC activity in SHR-Cd36 significantly exceeded (by about 18-30 %) the enzyme activity in SHR. Changes at the molecular level were reflected by higher contractile responses to stimulation by the adrenergic agonist dobutamine. In summary, it can be concluded that the increased susceptibility to ischemic arrhythmias of SHR-Cd36 is attributable to upregulation of some components of the β-AR signaling pathway, which leads to enhanced sensitization of AC and increased cardiac adrenergic responsiveness.

• MeSH
• adenylátcyklasy genetika   metabolismus   MeSH
• agonisté beta-1-adrenergních receptorů farmakologie   MeSH
• antigeny CD36 genetika   metabolismus   MeSH
• beta-adrenergní receptory metabolismus   MeSH
• dobutamin farmakologie   MeSH
• kontrakce myokardu MeSH
• krysa rodu Rattus MeSH
• myokard metabolismus   MeSH
• potkani inbrední SHR MeSH
• potkani transgenní MeSH
• proteinkinasy závislé na cyklickém AMP metabolismus   MeSH
• proteiny vázající GTP metabolismus   MeSH
• signální transdukce * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasy MeSH
• agonisté beta-1-adrenergních receptorů MeSH
• antigeny CD36 MeSH
• beta-adrenergní receptory MeSH
• dobutamin MeSH
• proteinkinasy závislé na cyklickém AMP MeSH
• proteiny vázající GTP MeSH

Human CMV infects between 50-85% of healthy individuals and can cause live-threatening infections in immunocompromised patients. Therefore, peptide vaccination is being developed as a promising immunotherapeutic approach for treatment of patients at risk of CMV disease. The enzymatically inactive toxoid of Bordetella adenylate cyclase (CyaA-AC(-)) was shown to be an efficient tool for delivery of peptide epitopes and stimulation of Ag-specific T-cell immune responses. We investigated here the capacity of two CyaA-AC(-) constructs to deliver epitopes derived from the CMV phosphoprotein pp65 for activation of human T cells in vitro. Expansion of γ-IFN-secreting CMV-specific CD8(+) T cells, as well as increase of total IFN-γ and TNF-α production by PBMCs from CMV-seropositive donors were observed after in vitro stimulation with CyaA-AC(-) constructs carrying CMV epitopes, whereas limited activation of immune response occurred with free peptides. The activation of immune response was confirmed by expansion of CMV-specific T-cell clones and anti-CMV cytotoxic effect of stimulated PBMCs. These data open the way to clinical evaluation of CyaA-AC(-) constructs as tools for detection and expansion of CMV-specific T-cell immune responses for diagnostic and immunotherapeutic applications against CMV-associated diseases.

• MeSH
• adenylátcyklasy genetika   imunologie   MeSH
• aktivace lymfocytů MeSH
• CD8-pozitivní T-lymfocyty imunologie   MeSH
• Cytomegalovirus imunologie   MeSH
• epitopy T-lymfocytární imunologie   MeSH
• fosfoproteiny imunologie   MeSH
• lidé MeSH
• peptidové fragmenty genetika   imunologie   MeSH
• proteiny virové matrix imunologie   MeSH
• sekvence aminokyselin MeSH
• vakcíny proti cytomegalovirové infekci genetika   imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasy MeSH
• cytomegalovirus matrix protein 65kDa MeSH Prohlížeč
• epitopy T-lymfocytární MeSH
• fosfoproteiny MeSH
• peptidové fragmenty MeSH
• proteiny virové matrix MeSH
• vakcíny proti cytomegalovirové infekci MeSH

G proteins-coupled signaling pathways appear to play a role in the development of cardiac hypertrophy and its progression to heart failure. The present study aimed to investigate trimeric G proteins and adenylyl cyclase signaling in immature as well as in adult rat myocardium during this process caused by pressure overload. Pressure overload was induced in newborn (2-day-old) rats by abdominal aortic banding and myocardial preparations from left ventricular myocardium of immature (10-day-old) and adult (90-day-old) animals were analyzed for the relative content of different G protein subunits and adenylyl cyclase (AC) by immunoblotting with specific antibodies. A functional status of the AC signaling system was also evaluated. Normal maturation of rat heart was accompanied by increased expression of AC type V/VI and VII and of the long isoform (G(s)alphaL) of G(s)alpha protein. In parallel, the amounts of myocardial G(i)alpha/G(o)alpha proteins tended to decrease, and G(q)alpha/G(11)alpha and Gbeta did not change. Interestingly, whereas fluoride-stimulated AC activity increased in the course of maturation, activity of AC measured under other experimental conditions (stimulation by Mn2+, forskolin or isoproterenol) was lower in adult than in young rat myocardium. Pressure overload did not influence distribution of G proteins in immature myocardium, but considerably decreased the content of G(s)alphaL and increased G(o)alpha proteins in hearts of 90-day-old rats. These hearts exhibited worsened functional reserve as compared to age-matched controls and activity of AC was also markedly lower. A considerable reduction in Mn(2+)-stimulated AC activity together with similar decrease in AC activity determined under other stimulation conditions suggests that it is a function of AC catalytic subunit that is primarily impaired in this model of pressure overload.

• MeSH
• adenylátcyklasy genetika   metabolismus   MeSH
• exprese genu MeSH
• fluoridy farmakologie   MeSH
• hypertrofie levé komory srdeční metabolismus   MeSH
• isoprenalin farmakologie   MeSH
• kardiomegalie genetika   metabolismus   patologie   MeSH
• kardiotonika farmakologie   MeSH
• kolforsin farmakologie   MeSH
• krysa rodu Rattus MeSH
• mangan farmakologie   MeSH
• myokard metabolismus   MeSH
• novorozená zvířata * MeSH
• potkani Wistar MeSH
• protein - isoformy genetika   metabolismus   MeSH
• proteiny vázající GTP metabolismus   MeSH
• signální transdukce MeSH
• stárnutí MeSH
• tlak * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• adenylátcyklasy MeSH
• fluoridy MeSH
• isoprenalin MeSH
• kardiotonika MeSH
• kolforsin MeSH
• mangan MeSH
• protein - isoformy MeSH
• proteiny vázající GTP MeSH

Recombinant adenylate cyclase toxoids are shown to deliver inserted foreign CD4(+)-T-cell epitopes into the major histocompatibility complex class II presentation pathway, inducing a specific CD4(+)-T-cell response in vivo and yielding in vitro stimulation of specific CD4(+) T cells at a 100-times-higher molar efficiency than the free peptide containing the epitope.

• MeSH
• adenylátcyklasový toxin MeSH
• adenylátcyklasy genetika   imunologie   MeSH
• bakteriální proteiny genetika   imunologie   MeSH
• Bordetella pertussis enzymologie   MeSH
• CD4-pozitivní T-lymfocyty cytologie   imunologie   MeSH
• epitopy T-lymfocytární genetika   imunologie   MeSH
• faktory virulence rodu Bordetella genetika   imunologie   MeSH
• H-2 antigeny imunologie   MeSH
• histokompatibilita - antigeny třídy II imunologie   MeSH
• interleukin-2 biosyntéza   MeSH
• kultivované buňky MeSH
• molekulární sekvence - údaje MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buňky kultivované MeSH
• prezentace antigenu imunologie   MeSH
• proteinové prekurzory genetika   imunologie   MeSH
• proteiny vázající maltosu MeSH
• sekvence aminokyselin MeSH
• transportní proteiny genetika   imunologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• adenylátcyklasy MeSH
• bakteriální proteiny MeSH
• epitopy T-lymfocytární MeSH
• faktory virulence rodu Bordetella MeSH
• H-2 antigeny MeSH
• H-2K(K) antigen MeSH Prohlížeč
• H-2Kb protein, mouse MeSH Prohlížeč
• histokompatibilita - antigeny třídy II MeSH
• interleukin-2 MeSH
• proteinové prekurzory MeSH
• proteiny vázající maltosu MeSH
• transportní proteiny MeSH