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1.
Pharmacol Res ; 141: 32-45, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30553823

RESUMO

Uridine adenosine tetraphosphate (Up4A), biosynthesized by activation of vascular endothelial growth factor receptor (VEGFR) 2, was initially identified as a potent endothelium-derived vasoconstrictor in perfused rat kidney. Subsequently, the effect of Up4A on vascular tone regulation was intensively investigated in arteries isolated from different vascular beds in rodents including rat pulmonary arteries, aortas, mesenteric and renal arteries as well as mouse aortas, in which Up4A produces vascular contraction. In contrast, Up4A produces vascular relaxation in porcine coronary small arteries and rat aortas. Intravenous infusion of Up4A into conscious rats or mice decreases blood pressure, and intravenous bolus injection of Up4A into anesthetized mice increases coronary blood flow, indicating an overall vasodilator influence in vivo. Although Up4A is the first dinucleotide described that contains both purine and pyrimidine moieties, its cardiovascular effects are exerted mainly through activation of purinergic receptors. These effects not only encompass regulation of vascular tone, but also endothelial angiogenesis, smooth muscle cell proliferation and migration, and vascular calcification. Furthermore, this review discusses a potential role for Up4A in cardiovascular pathophysiology, as plasma levels of Up4A are elevated in juvenile hypertensive patients and Up4A-mediated vascular purinergic signaling changes in cardiovascular disease such as hypertension, diabetes, atherosclerosis and myocardial infarction. Better understanding the vascular effect of the novel dinucleotide Up4A and the purinergic signaling mechanisms mediating its effects will enhance its potential as target for treatment of cardiovascular disease.


Assuntos
Fenômenos Fisiológicos Cardiovasculares , Fosfatos de Dinucleosídeos/fisiologia , Receptores Purinérgicos/fisiologia , Animais , Sistema Cardiovascular , Humanos , Transdução de Sinais
2.
J Endod ; 44(9): 1381-1388.e2, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30054101

RESUMO

INTRODUCTION: Enterococcus faecalis is correlated with oral diseases including recurrent root canal treatment failure because of its biofilm formation ability and various virulence factors. Cyclic di-AMP (c-di-AMP) is an omnipresent second messenger involved in many crucial cellular physiological processes, including biofilm formation. ST056083 is a small molecule working as an inhibitor of the c-di-AMP synthetase DNA integrity scanning protein (DisA) in vitro. In this study, the impact of ST056083 on E. faecalis DisA activity, bacterial growth, and biofilm formation was tested. METHODS: The binding affinity between the protein and ligand was evaluated using the Amber score, and the binding mode was analyzed and visualized using UCSF Chimera (Resource for Biocomputing, Visualization, and Informatics, University of California, San Francisco, San Francisco, CA). The effect of ST056083 on E. faecalis DisA was evaluated using the coralyne assay. The effect of ST056083 on E. faecalis biofilm formation was determined by the biofilm quantification assay, scanning electron microscopic examination, and 3-dimensional confocal laser scanning microscopic assay. The effect of ST056083 on E. faecalis exopolysaccharide synthesis was measured by the anthrone-sulfuric method. RESULTS: We expressed and purified E. faecalis DisA in vitro and confirmed the inhibitory effect of ST056083 on its biological activity. In addition, we showed the inhibitory effect of ST056083 on E. faecalis growth, biofilm formation, and exopolysaccharide synthesis. CONCLUSIONS: Our findings enhance the understanding of the physiological role of c-di-AMP in E. faecalis and represent a preliminary study on the ST056083 inhibitory effect and mechanism.


Assuntos
Inibidores de Adenilil Ciclases/farmacologia , Biofilmes/efeitos dos fármacos , Fosfatos de Dinucleosídeos/fisiologia , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/enzimologia , Enterococcus faecalis/fisiologia
3.
Sheng Wu Gong Cheng Xue Bao ; 33(9): 1369-1375, 2017 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-28956388

RESUMO

Bacterial biofilm plays an important role in persistent microbial infection. Delineation of the formation and development of bacterial biofilm would provide a promising strategy to treat recalcitrant infection. c-di-AMP (Cyclic diadenosine monophosphate) is a recently identified second messenger of bacteria and involved in plethora of bacterial activities, including cell growth, cell wall homeostasis, biofilm formation and microbial pathogenicity. Here we review the recent literature pertinent to the role and molecular mechanisms of c-di-AMP in regulating biofilm formation of bacteria. The potential application of c-di-AMP and its related proteins in the development of novel antimicrobial therapeutics has also been discussed.


Assuntos
Bactérias/crescimento & desenvolvimento , Biofilmes , Fosfatos de Dinucleosídeos/fisiologia , Sistemas do Segundo Mensageiro
4.
Proc Natl Acad Sci U S A ; 114(35): E7226-E7235, 2017 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-28808024

RESUMO

Cyclic di-3',5'-adenosine monophosphate (c-di-AMP) is a broadly conserved bacterial second messenger that has been implicated in a wide range of cellular processes. Our earlier studies showed that c-di-AMP regulates central metabolism in Listeria monocytogenes by inhibiting its pyruvate carboxylase (LmPC), a biotin-dependent enzyme with biotin carboxylase (BC) and carboxyltransferase (CT) activities. We report here structural, biochemical, and functional studies on the inhibition of Lactococcus lactis PC (LlPC) by c-di-AMP. The compound is bound at the dimer interface of the CT domain, at a site equivalent to that in LmPC, although it has a distinct binding mode in the LlPC complex. This binding site is not well conserved among PCs, and only a subset of these bacterial enzymes are sensitive to c-di-AMP. Conformational changes in the CT dimer induced by c-di-AMP binding may be the molecular mechanism for its inhibitory activity. Mutations of residues in the binding site can abolish c-di-AMP inhibition. In L. lactis, LlPC is required for efficient milk acidification through its essential role in aspartate biosynthesis. The aspartate pool in L. lactis is negatively regulated by c-di-AMP, and high aspartate levels can be restored by expression of a c-di-AMP-insensitive LlPC. LlPC has high intrinsic catalytic activity and is not sensitive to acetyl-CoA activation, in contrast to other PC enzymes.


Assuntos
Fosfatos de Dinucleosídeos/metabolismo , Piruvato Carboxilase/metabolismo , Piruvato Carboxilase/fisiologia , Monofosfato de Adenosina/metabolismo , Ácido Aspártico/biossíntese , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X/métodos , AMP Cíclico/metabolismo , Fosfatos de Dinucleosídeos/fisiologia , Lactobacillales/metabolismo , Lactococcus lactis/metabolismo , Conformação Proteica , Sistemas do Segundo Mensageiro/fisiologia , Relação Estrutura-Atividade
5.
Mol Microbiol ; 104(2): 212-233, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28097715

RESUMO

Cyclic diadenosine monophosphate (c-di-AMP) is a conserved nucleotide second messenger critical for bacterial growth and resistance to cell wall-active antibiotics. In Listeria monocytogenes, the sole diadenylate cyclase, DacA, is essential in rich, but not synthetic media and ΔdacA mutants are highly sensitive to the ß-lactam antibiotic cefuroxime. In this study, loss of function mutations in the oligopeptide importer (oppABCDF) and glycine betaine importer (gbuABC) allowed ΔdacA mutants to grow in rich medium. Since oligopeptides were sufficient to inhibit growth of the ΔdacA mutant we hypothesized that oligopeptides act as osmolytes, similar to glycine betaine, to disrupt intracellular osmotic pressure. Supplementation with salt stabilized the ΔdacA mutant in rich medium and restored cefuroxime resistance. Additional suppressor mutations in the acetyl-CoA binding site of pyruvate carboxylase (PycA) rescued cefuroxime resistance and resulted in a 100-fold increase in virulence of the ΔdacA mutant. PycA is inhibited by c-di-AMP and these mutations prompted us to examine the role of TCA cycle enzymes. Inactivation of citrate synthase, but not down-stream enzymes suppressed ΔdacA phenotypes. These data suggested that c-di-AMP modulates central metabolism at the pyruvate node to moderate citrate production and indeed, the ΔdacA mutant accumulated six times the concentration of citrate present in wild-type bacteria.


Assuntos
Fosfatos de Dinucleosídeos/metabolismo , Listeria monocytogenes/metabolismo , Acetilcoenzima A/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Fosfatos de Dinucleosídeos/genética , Fosfatos de Dinucleosídeos/fisiologia , Resistência Microbiana a Medicamentos , Regulação Bacteriana da Expressão Gênica/genética , Listeria monocytogenes/crescimento & desenvolvimento , Osmorregulação/fisiologia , Pressão Osmótica , Fósforo-Oxigênio Liases/metabolismo , Piruvato Carboxilase/metabolismo , Sistemas do Segundo Mensageiro , Supressão Genética
6.
Crit Rev Eukaryot Gene Expr ; 26(4): 309-316, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27910745

RESUMO

Nucleotide-based second messengers transduce signals originating from both outside and inside the cell to adaptive responses accordingly. c-di-AMP is a newly established second messenger employed by many organisms. We summarize recent advances in bacterial c-di-AMP-mediated signaling, especially the interaction between c-di-AMP signaling and the host.


Assuntos
Bactérias/metabolismo , Fosfatos de Dinucleosídeos/fisiologia , Sistemas do Segundo Mensageiro
7.
Prog Retin Eye Res ; 55: 182-205, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27421962

RESUMO

Dinucleoside polyphosphates comprises a group of dinucleotides formed by two nucleosides linked by a variable number of phosphates, abbreviated NpnN (where n represents the number of phosphates). These compounds are naturally occurring substances present in tears, aqueous humour and in the retina. As the consequence of their presence, these dinucleotides contribute to many ocular physiological processes. On the ocular surface, dinucleoside polyphosphates can stimulate tear secretion, mucin release from goblet cells and they help epithelial wound healing by accelerating cell migration rate. These dinucleotides can also stimulate the presence of proteins known to protect the ocular surface against microorganisms, such as lysozyme and lactoferrin. One of the latest discoveries is the ability of some dinucleotides to facilitate the paracellular way on the cornea, therefore allowing the delivery of compounds, such as antiglaucomatous ones, more easily within the eye. The compound Ap4A has been described being abnormally elevated in patient's tears suffering of dry eye, Sjogren syndrome, congenital aniridia, or after refractive surgery, suggesting this molecule as biomarker for dry eye condition. At the intraocular level, some diadenosine polyphosphates are abnormally elevated in glaucoma patients, and this can be related to the stimulation of a P2Y2 receptor that increases the chloride efflux and water movement in the ciliary epithelium. In the retina, the dinucleotide dCp4U, has been proven to be useful to help in the recovery of retinal detachments. Altogether, dinucleoside polyphosphates are a group of compounds which present relevant physiological actions but which also can perform promising therapeutic benefits.


Assuntos
Humor Aquoso/metabolismo , Córnea/metabolismo , Fosfatos de Dinucleosídeos/fisiologia , Lágrimas/química , Distribuição Tecidual/fisiologia , Animais , Humanos , Sistemas do Segundo Mensageiro
8.
Br J Pharmacol ; 172(16): 3980-4001, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26031319

RESUMO

Vascular dysfunction plays a pivotal role in the development of systemic complications associated with arterial hypertension and diabetes. The endothelium, or more specifically, various factors derived from endothelial cells tightly regulate vascular function, including vascular tone. In physiological conditions, there is a balance between endothelium-derived factors, that is, relaxing factors (endothelium-derived relaxing factors; EDRFs) and contracting factors (endothelium-derived contracting factors; EDCFs), which mediate vascular homeostasis. However, in disease states, such as diabetes and arterial hypertension, there is an imbalance between EDRF and EDCF, with a reduction of EDRF signalling and an increase of EDCF signalling. Among EDCFs, COX-derived vasoconstrictor prostanoids play an important role in the development of vascular dysfunction associated with hypertension and diabetes. Moreover, uridine adenosine tetraphosphate (Up4 A), identified as an EDCF in 2005, also modulates vascular function. However, the role of Up4 A in hypertension- and diabetes-associated vascular dysfunction is unclear. In the present review, we focused on experimental and clinical evidence that implicate these two EDCFs (vasoconstrictor prostanoids and Up4 A) in vascular dysfunction associated with hypertension and diabetes.


Assuntos
Diabetes Mellitus/metabolismo , Fosfatos de Dinucleosídeos/metabolismo , Hipertensão/metabolismo , Prostaglandinas/metabolismo , Animais , Diabetes Mellitus/fisiopatologia , Fosfatos de Dinucleosídeos/fisiologia , Endotelinas/metabolismo , Endotelinas/fisiologia , Hormônios Esteroides Gonadais/metabolismo , Humanos , Hipertensão/fisiopatologia , Miócitos de Músculo Liso/metabolismo , Prostaglandina-Endoperóxido Sintases/fisiologia , Prostaglandinas/fisiologia , Vasoconstrição/fisiologia
9.
PLoS One ; 10(4): e0124358, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25884619

RESUMO

Here, we have developed an extremely efficient in vivo Tn5-based mutagenesis procedure to construct a Deinococcus radiodurans insertion mutant library subsequently screened for sensitivity to genotoxic agents such as γ and UV radiations or mitomycin C. The genes inactivated in radiosensitive mutants belong to various functional categories, including DNA repair functions, stress responses, signal transduction, membrane transport, several metabolic pathways, and genes of unknown function. Interestingly, preliminary characterization of previously undescribed radiosensitive mutants suggests the contribution of cyclic di-AMP signaling in the recovery of D. radiodurans cells from genotoxic stresses, probably by modulating several pathways involved in the overall cell response. Our analyses also point out a new transcriptional regulator belonging to the GntR family, encoded by DR0265, and a predicted RNase belonging to the newly described Y family, both contributing to the extreme radioresistance of D. radiodurans. Altogether, this work has revealed new cell responses involved either directly or indirectly in repair of various cell damage and confirmed that D. radiodurans extreme radiation resistance is determined by a multiplicity of pathways acting as a complex network.


Assuntos
Deinococcus/genética , Genes Bacterianos , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Dano ao DNA , Reparo do DNA/genética , Elementos de DNA Transponíveis , DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/genética , DNA Bacteriano/efeitos da radiação , Deinococcus/efeitos dos fármacos , Deinococcus/efeitos da radiação , Fosfatos de Dinucleosídeos/fisiologia , Raios gama , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/genética , Biblioteca Gênica , Redes Reguladoras de Genes , Teste de Complementação Genética , Peróxido de Hidrogênio/farmacologia , Mitomicina/farmacologia , Mutagênese Insercional , Mutação , Fases de Leitura Aberta/genética , Estresse Oxidativo , Tolerância a Radiação/genética , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Transposases/genética , Raios Ultravioleta
10.
Nucleic Acids Res ; 42(7): 4527-45, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24470146

RESUMO

Most RNA viruses infecting mammals and other vertebrates show profound suppression of CpG and UpA dinucleotide frequencies. To investigate this functionally, mutants of the picornavirus, echovirus 7 (E7), were constructed with altered CpG and UpA compositions in two 1.1-1.3 Kbase regions. Those with increased frequencies of CpG and UpA showed impaired replication kinetics and higher RNA/infectivity ratios compared with wild-type virus. Remarkably, mutants with CpGs and UpAs removed showed enhanced replication, larger plaques and rapidly outcompeted wild-type virus on co-infections. Luciferase-expressing E7 sub-genomic replicons with CpGs and UpAs removed from the reporter gene showed 100-fold greater luminescence. E7 and mutants were equivalently sensitive to exogenously added interferon-ß, showed no evidence for differential recognition by ADAR1 or pattern recognition receptors RIG-I, MDA5 or PKR. However, kinase inhibitors roscovitine and C16 partially or entirely reversed the attenuated phenotype of high CpG and UpA mutants, potentially through inhibition of currently uncharacterized pattern recognition receptors that respond to RNA composition. Generating viruses with enhanced replication kinetics has applications in vaccine production and reporter gene construction. More fundamentally, the findings introduce a new evolutionary paradigm where dinucleotide composition of viral genomes is subjected to selection pressures independently of coding capacity and profoundly influences host-pathogen interactions.


Assuntos
Fosfatos de Dinucleosídeos/fisiologia , Enterovirus Humano B/fisiologia , Sequência Rica em GC/fisiologia , RNA Viral/química , Replicação Viral , Composição de Bases , Linhagem Celular , Enterovirus Humano B/genética , Mutação
11.
IUBMB Life ; 65(11): 897-903, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24136904

RESUMO

Cyclic (c-di-GMP) is the prevalent intracellular signaling intermediate in bacteria. It triggers a spectrum of responses that cause bacteria to shift from a swarming motile phase to sessile biofilm formation. However, additional functions for c-di-GMP and roles for related molecules, such as c-di-AMP and c-AMP-GMP continue to be uncovered. The first usage of cyclic-di-nucleotide (c-di-NMP) signaling in the eukaryote domain emerged only recently. In dictyostelid social amoebas, c-di-GMP is a secreted signal that induces motile amoebas to differentiate into sessile stalk cells. In humans, c-di-NMPs, which are either produced endogenously in response to foreign DNA or by invading bacterial pathogens, trigger the innate immune system by activating the expression of interferon genes. STING, the human c-di-NMP receptor, is conserved throughout metazoa and their closest unicellular relatives, suggesting protist origins for human c-di-NMP signaling. Compared to the limited number of conserved protein domains that detect the second messengers cAMP and cGMP, the domains that detect the c-di-NMPs are surprisingly varied.


Assuntos
GMP Cíclico/análogos & derivados , Fosfatos de Dinucleosídeos/fisiologia , Transdução de Sinais/fisiologia , Proteínas de Bactérias/metabolismo , Biofilmes , GMP Cíclico/fisiologia , Dictyostelium/fisiologia , Humanos , Imunidade Inata/fisiologia , Proteínas de Membrana/genética , Nucleotídeos Cíclicos/biossíntese , Nucleotidiltransferases/genética , Filogenia , Estrutura Terciária de Proteína , Sistemas do Segundo Mensageiro
12.
Biochem Biophys Res Commun ; 417(3): 1035-40, 2012 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-22214933

RESUMO

The recently discovered dinucleotide uridine adenosine tetraphosphate (Up(4)A) was found in human plasma and characterized as endothelium-derived vasoconstrictive factor (EDCF). A further study revealed a positive correlation between Up(4)A and vascular smooth muscle cell (VSMC) proliferation. Due to the dominant role of migration in the formation of atherosclerotic lesions our aim was to investigate the migration stimulating potential of Up(4)A. Indeed, we found a strong chemoattractant effect of Up(4)A on VSMC by using a modified Boyden chamber. This migration dramatically depends on osteopontin secretion (OPN) revealed by the reduction of the migration signal down to 23% during simultaneous incubation with an OPN-blocking antibody. Due to inhibitory patterns using specific and unspecific purinoreceptor inhibitors, Up(4)A mediates it's migratory signal mainly via the P2Y(2). The signaling behind the receptor was investigated with luminex technique and revealed an activation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway. By use of the specific PDGF receptor (PDGFR) inhibitor AG1296 and siRNA technique against PDGFR-ß we found a strongly reduced migration signal after Up(4)A stimulation in the PDGFR-ß knockdown cells compared to control cells. In this study, we present substantiate data that Up(4)A exhibits migration stimulating potential probably involving the signaling cascade of MEK1 and ERK1/2 as well as the matrix protein OPN. We further suggest that the initiation of the migration process occurs predominant through direct activation of the P2Y(2) by Up(4)A and via transactivation of the PDGFR.


Assuntos
Movimento Celular , Fosfatos de Dinucleosídeos/fisiologia , Miócitos de Músculo Liso/fisiologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Animais , Células Cultivadas , Fosfatos de Dinucleosídeos/farmacologia , MAP Quinase Quinase 1/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , RNA Interferente Pequeno/genética , Ratos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Tirfostinas/farmacologia
13.
Kidney Int ; 81(3): 256-65, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21956191

RESUMO

Purinergic signaling has a crucial role in different vascular processes. The endothelial-derived vasoconstrictor uridine adenosine tetraphosphate (Up(4)A) is a potent activator of the purinoceptor P2Y and is released under pathological conditions. Here we sought to measure purinergic effects on vascular calcification and initially found that Up(4)A plasma concentrations are increased in patients with chronic kidney disease. Exploring this further we found that exogenous Up(4)A enhanced mineral deposition under calcifying conditions ex vivo in rat and mouse aortic rings and in vitro in rat vascular smooth muscle cells. The addition of Up(4)A increased the expression of different genes specific for osteochondrogenic vascular smooth muscle cells such as Cbfa1, while decreasing the expression of SM22α, a marker specific for vascular smooth muscle cells. The influence of different P2Y antagonists on Up(4)A-mediated process indicated that P2Y(2/6) receptors may be involved. Mechanisms downstream of P2Y signaling involved phosphorylation of the mitogen-activated kinases MEK and ERK1/2. Thus, Up(4)A activation of P2Y influences phenotypic transdifferentiation of vascular smooth muscle cells to osteochondrogenic cells, suggesting that purinergic signaling may be involved in vascular calcification.


Assuntos
Fosfatos de Dinucleosídeos/fisiologia , Receptores Purinérgicos P2Y/fisiologia , Calcificação Vascular/etiologia , Idoso , Idoso de 80 Anos ou mais , Animais , Transdiferenciação Celular , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/fisiologia , Fosfatos de Dinucleosídeos/sangue , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Músculo Liso Vascular/patologia , Osteocalcina/fisiologia , Osteopontina/fisiologia , Ratos , Ratos Endogâmicos WKY , Transdução de Sinais
14.
PLoS Pathog ; 7(9): e1002217, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21909268

RESUMO

The cell wall is a vital and multi-functional part of bacterial cells. For Staphylococcus aureus, an important human bacterial pathogen, surface proteins and cell wall polymers are essential for adhesion, colonization and during the infection process. One such cell wall polymer, lipoteichoic acid (LTA), is crucial for normal bacterial growth and cell division. Upon depletion of this polymer bacteria increase in size and a misplacement of division septa and eventual cell lysis is observed. In this work, we describe the isolation and characterization of LTA-deficient S. aureus suppressor strains that regained the ability to grow almost normally in the absence of this cell wall polymer. Using a whole genome sequencing approach, compensatory mutations were identified and revealed that mutations within one gene, gdpP (GGDEF domain protein containing phosphodiesterase), allow both laboratory and clinical isolates of S. aureus to grow without LTA. It was determined that GdpP has phosphodiesterase activity in vitro and uses the cyclic dinucleotide c-di-AMP as a substrate. Furthermore, we show for the first time that c-di-AMP is produced in S. aureus presumably by the S. aureus DacA protein, which has diadenylate cyclase activity. We also demonstrate that GdpP functions in vivo as a c-di-AMP-specific phosphodiesterase, as intracellular c-di-AMP levels increase drastically in gdpP deletion strains and in an LTA-deficient suppressor strain. An increased amount of cross-linked peptidoglycan was observed in the gdpP mutant strain, a cell wall alteration that could help bacteria compensate for the lack of LTA. Lastly, microscopic analysis of wild-type and gdpP mutant strains revealed a 13-22% reduction in the cell size of bacteria with increased c-di-AMP levels. Taken together, these data suggest a function for this novel secondary messenger in controlling cell size of S. aureus and in helping bacteria to cope with extreme membrane and cell wall stress.


Assuntos
Lipopolissacarídeos/deficiência , Diester Fosfórico Hidrolases/genética , Fósforo-Oxigênio Liases/genética , Sistemas do Segundo Mensageiro/genética , Staphylococcus aureus/crescimento & desenvolvimento , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Tamanho Celular , Parede Celular/química , Parede Celular/efeitos dos fármacos , Fosfatos de Dinucleosídeos/metabolismo , Fosfatos de Dinucleosídeos/fisiologia , Staphylococcus aureus Resistente à Meticilina , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Diester Fosfórico Hidrolases/fisiologia , Fósforo-Oxigênio Liases/fisiologia , Staphylococcus aureus/genética , Ácidos Teicoicos
15.
Biosystems ; 105(3): 295-9, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21723367

RESUMO

10-11bp repeating patterns of the particular dinucleotides were suggested in positioning nucleosomes in eukaryotes. In order to assess the role, the 10-11bp periodicities of the dinucleotides were examined both in human well-positioned nucleosome DNA sequences and in promoter DNA sequences of eight species. Our results indicated that the periodical occurrence of the particular dinucleotides correlates to positioning nucleosome. The signals of 10-11bp periodicities are more pronounced in the nucleosome DNA sequences than in the linker DNA sequences. Near the transcription start site, the signals reveal a similar feature that the nucleosome organization exhibits. But, it seems that the species do not share the same dinucleotides patterns. Furthermore, the dinucleotides patterns are dominant at the specific region of genome, indicating their diverse roles in forming and organizing nucleosomes. Moreover, the 10-11bp periodicities signals near the translation start negatively correlate with gene expression, demonstrating a vital role in transcription regulation. Our study reveals some details about the roles of DNA sequence in positioning nucleosomes.


Assuntos
Linfócitos T CD4-Positivos/química , Fosfatos de Dinucleosídeos/fisiologia , Eucariotos/genética , Nucleossomos/química , Animais , Sequência de Bases , Análise de Fourier , Expressão Gênica , Humanos , Sítio de Iniciação de Transcrição
16.
Bioorg Med Chem ; 19(16): 5053-60, 2011 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21757356

RESUMO

Fragile histidine triad (Fhit) protein encoded by tumour suppressor FHIT gene is a proapoptotic protein with diadenosine polyphosphate (Ap(n)A, n=2-6) hydrolase activity. It has been hypothesised that formation of Fhit-substrate complex results in an apoptosis initiation signal while subsequent hydrolysis of Ap(n)A terminates this action. A series of Ap(n)A analogues have been identified in vitro as strong Fhit ligands [Varnum, J. M.; Baraniak, J.; Kaczmarek, R.; Stec, W. J.; Brenner, C. BMC Chem. Biol.2001, 1, 3]. We assumed that in Fhit-positive cells these compounds might preferentially bind to Fhit and inhibit its hydrolytic activity what would prolong the lifetime of apoptosis initiation signalling complex. Therefore, several Fhit inhibitors were tested for their cytotoxicity and ability to induce apoptosis in Fhit-positive HEK293T cells. These experiments have shown that Ap(4)A analogue, containing a glycerol residue instead of the central pyrophosphate and two terminal phosphorothioates [A(PS)-CH(2)CH(OH)CH(2)-(PS)A (1)], is the most cytotoxic among test compounds (IC(50)=17.5±4.2 µM) and triggers caspase-dependent cell apoptosis. The Fhit-negative HEK293T cells (in which Fhit was silenced by RNAi) were not sensitive to compound 1. These results indicate that the Ap(4)A analogue 1 induces Fhit-dependent apoptosis and therefore, it can be considered as a drug candidate for anticancer therapy in Fhit-positive cancer cells and in Fhit-negative cancer cells, in which re-expression of Fhit was accomplished by gene therapy.


Assuntos
Hidrolases Anidrido Ácido/fisiologia , Antineoplásicos/síntese química , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fosfatos de Dinucleosídeos/farmacologia , Proteínas de Neoplasias/fisiologia , Neoplasias/tratamento farmacológico , Hidrolases Anidrido Ácido/química , Hidrolases Anidrido Ácido/metabolismo , Anexina A5/análise , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/genética , Caspases/análise , Caspases/efeitos dos fármacos , Linhagem Celular Tumoral , Citotoxinas , Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/fisiologia , Fosfatos de Dinucleosídeos/uso terapêutico , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Corantes Fluorescentes/análise , Genes Supressores de Tumor/efeitos dos fármacos , Células HEK293 , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , RNA Interferente Pequeno/metabolismo
17.
BMC Genomics ; 12(1): 273, 2011 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-21627783

RESUMO

BACKGROUND: The periodical occurrence of dinucleotides with a period of 10.4 bases now is undeniably a hallmark of nucleosome positioning. Whereas many eukaryotic genomes contain visible and even strong signals for periodic distribution of dinucleotides, the human genome is rather featureless in this respect. The exact sequence features in the human genome that govern the nucleosome positioning remain largely unknown. RESULTS: When analyzing the human genome sequence with the positional autocorrelation method, we found that only the dinucleotide CG shows the 10.4 base periodicity, which is indicative of the presence of nucleosomes. There is a high occurrence of CG dinucleotides that are either 31 (10.4 × 3) or 62 (10.4 × 6) base pairs apart from one another - a sequence bias known to be characteristic of Alu-sequences. In a similar analysis with repetitive sequences removed, peaks of repeating CG motifs can be seen at positions 10, 21 and 31, the nearest integers of multiples of 10.4. CONCLUSIONS: Although the CG dinucleotides are dominant, other elements of the standard nucleosome positioning pattern are present in the human genome as well.The positional autocorrelation analysis of the human genome demonstrates that the CG dinucleotide is, indeed, one visible element of the human nucleosome positioning pattern, which appears both in Alu sequences and in sequences without repeats. The dominant role that CG dinucleotides play in organizing human chromatin is to indicate the involvement of human nucleosomes in tuning the regulation of gene expression and chromatin structure, which is very likely due to cytosine-methylation/-demethylation in CG dinucleotides contained in the human nucleosomes. This is further confirmed by the positions of CG-periodical nucleosomes on Alu sequences. Alu repeats appear as monomers, dimers and trimers, harboring two to six nucleosomes in a run. Considering the exceptional role CG dinucleotides play in the nucleosome positioning, we hypothesize that Alu-nucleosomes, especially, those that form tightly positioned runs, could serve as "anchors" in organizing the chromatin in human cells.


Assuntos
Elementos Alu/fisiologia , Fosfatos de Dinucleosídeos/fisiologia , Nucleossomos/fisiologia , Montagem e Desmontagem da Cromatina , Biologia Computacional , Humanos , Análise de Sequência de DNA
19.
Br J Pharmacol ; 161(3): 527-9, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20880393

RESUMO

In this Commentary, the roles of uridine adenosine tetraphosphate as an endothelium-derived contracting or relaxing factor described in the paper by Tölle et al. are considered and put into the wider context of the mechanisms of control of vascular tone by purinergic signalling via receptors located on both smooth muscle and endothelial cells.


Assuntos
Vasos Sanguíneos/fisiologia , Fosfatos de Dinucleosídeos/fisiologia , Receptores Purinérgicos/fisiologia , Sistema Vasomotor/fisiologia , Animais , Vasos Sanguíneos/efeitos dos fármacos , Fosfatos de Dinucleosídeos/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Modelos Cardiovasculares , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Ratos , Sistema Vasomotor/efeitos dos fármacos
20.
Br J Pharmacol ; 161(3): 530-40, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20880394

RESUMO

BACKGROUND AND PURPOSE: Purinergic signalling plays an important role in vascular tone regulation in humans. We have identified uridine adenosine tetraphosphate (Up(4)A) as a novel and highly potent endothelial-derived contracting factor. Up(4)A induces strong vasoconstrictive effects in the renal vascular system mainly by P2X(1) receptor activation. However, other purinoceptors are also involved and were analysed here. EXPERIMENTAL APPROACH: The rat isolated perfused kidney was used to characterize vasoactive actions of Up(4)A. KEY RESULTS: After desensitization of the P2X(1) receptor by α,ß-methylene ATP (α,ß-meATP), Up(4)A showed dose-dependent P2Y(2)-mediated vasoconstriction. Continuous perfusion with Up(4)A evoked a biphasic vasoconstrictor effect: there was a strong and rapidly desensitizing vasoconstriction, inhibited by P2X(1) receptor desensitization. In addition, there is a long-lasting P2Y(2)-mediated vasoconstriction. This vasoconstriction could be blocked by suramin, but not by PPADS or reactive blue 2. In preparations of the rat isolated perfused kidney model with an elevated vascular tone, bolus application of Up(4)A showed a dose-dependent vasoconstriction that was followed by a dose-dependent vasodilation. The vasoconstriction was in part sensitive to P2X(1) receptor desensitization by α,ß-meATP, and the remaining P2Y(2)-mediated vasoconstriction was only inhibited by suramin. The Up(4)A-induced vasodilation depended on activation of nitric oxide synthases, and was mediated by P2Y(1) and P2Y(2) receptor activation. CONCLUSIONS AND IMPLICATIONS: Up(4)A activated P2X(1) and P2Y(2) receptors to act as a vasoconstrictor, whereas endothelium-dependent vasodilation was induced by P2Y(1/2) receptor activation. Up(4)A might be of relevance in the physiology and pathophysiology of vascular tone regulation.


Assuntos
Fosfatos de Dinucleosídeos/fisiologia , Rim/fisiologia , Receptores Purinérgicos/fisiologia , Vasodilatação/fisiologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Fosfatos de Dinucleosídeos/farmacologia , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Rim/efeitos dos fármacos , Masculino , Óxido Nítrico Sintase/metabolismo , Agonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Agonistas do Receptor Purinérgico P2Y/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , Ratos , Ratos Endogâmicos WKY , Suramina/farmacologia , Triazinas/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasoconstrição/fisiologia , Vasodilatação/efeitos dos fármacos
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