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1.
Int J Mol Sci ; 22(3)2021 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-33530472

RESUMO

Mammalian oocytes must degrade maternal transcripts through a process called translational mRNA decay, in which maternal mRNA undergoes translational activation, followed by deadenylation and mRNA decay. Once a transcript is translationally activated, it becomes deadenylated by the CCR4-NOT complex. Knockout of CCR4-NOT Transcription Complex Subunit 6 Like (Cnot6l), a deadenylase within the CCR4-NOT complex, results in mRNA decay defects during metaphase I (MI) entry. Knockout of B-cell translocation gene-4 (Btg4), an adaptor protein of the CCR4-NOT complex, results in mRNA decay defects following fertilization. Therefore, mechanisms controlling mRNA turnover have significant impacts on oocyte competence and early embryonic development. Post-transcriptional inosine RNA modifications can impact mRNA stability, possibly through a translation mechanism. Here, we assessed inosine RNA modifications in oocytes, eggs, and embryos from Cnot6l-/- and Btg4-/- mice, which display stabilization of mRNA and over-translation of the stabilized transcripts. If inosine modifications have a role in modulating RNA stability, we hypothesize that in these mutant backgrounds, we would observe changes or a disruption in inosine mRNA modifications. To test this, we used a computational approach to identify inosine RNA modifications in total and polysomal RNA-seq data during meiotic maturation (GV, MI, and MII stages). We observed pronounced depletion of inosine mRNA modifications in samples from Cnot6l-/-, but not in Btg4-/- mice. Additionally, analysis of ribosome-associated RNA revealed clearance of inosine modified mRNA. These observations suggest a novel mechanism of mRNA clearance during oocyte maturation, in which inosine-containing transcripts decay in an independent, but parallel mechanism to CCR4-NOT deadenylation.


Assuntos
Nucleotídeos de Inosina/genética , Nucleotídeos de Inosina/metabolismo , Oócitos/metabolismo , RNA/genética , Ribonucleases/genética , Animais , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Oogênese/genética , Fases de Leitura Aberta , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleases/deficiência , Ribossomos/metabolismo
2.
Artigo em Inglês | MEDLINE | ID: mdl-31997708

RESUMO

A facile, straightforward, reliable, and efficient chemical synthesis of inosine nucleotides such as inosine-5'-monophosphate, inosine-5'-diphosphate, and inosine-5'-triphosphate, starting from inosine is delineated. The inosine-5'-monophosphate is achieved by the highly regioselective monophosphorylation of inosine using the Yoshikawa procedure. The inosine-5'-diphosphate is obtained by the coupling reaction of tributylammonium phosphate with an activated inosine-5'-monophosphate using zinc chloride as a catalyst. The inosine-5'-triphosphate is efficiently achieved by the improved "one-pot, three-step" Ludwig synthetic strategy. In all the cases, the resulting final product is isolated in good yields with high purity (>99.5%).


Assuntos
Nucleotídeos de Inosina/síntese química , Catálise , Cloretos/química , Inosina/química , Conformação Molecular , Fosfatos/química , Fosforilação , Compostos de Amônio Quaternário/química , Solventes/química , Estereoisomerismo , Temperatura , Compostos de Zinco/química
3.
Meat Sci ; 151: 82-88, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30743184

RESUMO

The role of moisture evaporation in the taste attributes of dry- and wet-aged beef was determined in this study. A total of 30 striploins (longissimus lumborum) were dry or wet aged for 28 days and analyzed for moisture content, taste-active compounds [free amino acids (FAAs), inosine 5'-monophophate (IMP), and reducing sugars], and taste attributes by an electronic tongue. After the completion of aging process, higher amounts of FAAs and reducing sugars were found in dry-aged beef (P < .05) in negative correlations with moisture content (r2 = -0.9 and - 0.9, respectively), which were not detected in wet-aged beef. However, the different taste attributes of dry- and wet-aged beef were observed by the electronic tongue from day 14, whereas their moisture content was significantly different only at day 28. Consequently, although the moisture evaporation during dry aging process contributed to the increased flavor of dry-aged beef, there are other factors affecting flavor development including microbial activity on the surface crust.


Assuntos
Carne Vermelha/análise , Paladar , Água , Aminoácidos/análise , Animais , Bovinos , Nariz Eletrônico , Manipulação de Alimentos/métodos , Nucleotídeos de Inosina/análise , Músculo Esquelético/química , Açúcares/análise
4.
Biochem Biophys Res Commun ; 498(4): 764-768, 2018 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-29524424

RESUMO

Damaged tissues and cells release intracellular purine nucleotides, which serve as intercellular signaling factors. We previously showed that exogenously added adenine nucleotide (250 µM ATP) suppressed the activation of murine splenic T lymphocytes. Here, we examined the effects of other purine nucleotides/nucleosides on mouse T cell activation. First, we found that pretreatment of mouse spleen T cells with 250 µM GTP, GDP, GMP, guanosine, ITP, IDP, IMP or inosine significantly reduced the release of stimulus-inducible cytokine IL-2. This suppression of IL-2 release was not caused by induction of cell death. Further studies with GTP, ITP, guanosine and inosine showed that pretreatment with these nucleotides/nucleosides also suppressed release of IL-6. However, these nucleotides/nucleosides did not suppress stimulus-induced phosphorylation of ERK1/2, suggesting that the suppression of the release of inflammatory cytokines does not involve inhibition of ERK1/2 signaling. In contrast to ATP pretreatment at the same concentration, guanine or inosine nucleotides/nucleosides did not attenuate the expression of CD25. Our findings indicate that exogenous guanine or inosine nucleotides/nucleosides can suppress inflammatory cytokine release from T cells, and may be promising candidates for use as supplementary agents in the treatment of T cell-mediated immune diseases.


Assuntos
Anti-Inflamatórios/farmacologia , Nucleotídeos de Guanina/farmacologia , Guanosina/farmacologia , Nucleotídeos de Inosina/farmacologia , Inosina/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Animais , Interleucina-2/antagonistas & inibidores , Interleucina-2/imunologia , Interleucina-6/antagonistas & inibidores , Interleucina-6/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/imunologia
5.
Sci Rep ; 6: 32849, 2016 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-27618981

RESUMO

Deoxyinosine (dI) occurs in DNA either by oxidative deamination of a previously incorporated deoxyadenosine residue or by misincorporation of deoxyinosine triphosphate (dITP) from the nucleotide pool during replication. To exclude dITP from the pool, mammals possess specific hydrolysing enzymes, such as inosine triphosphatase (ITPA). Previous studies have shown that deficiency in ITPA results in cell growth suppression and DNA instability. To explore the mechanisms of these phenotypes, we analysed ITPA-deficient human and mouse cells. We found that both growth suppression and accumulation of single-strand breaks in nuclear DNA of ITPA-deficient cells depended on MLH1/PMS2. The cell growth suppression of ITPA-deficient cells also depended on p53, but not on MPG, ENDOV or MSH2. ITPA deficiency significantly increased the levels of p53 protein and p21 mRNA/protein, a well-known target of p53, in an MLH1-dependent manner. Furthermore, MLH1 may also contribute to cell growth arrest by increasing the basal level of p53 activity.


Assuntos
Proliferação de Células/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Instabilidade Genômica/genética , Inosina/metabolismo , Endonuclease PMS2 de Reparo de Erro de Pareamento/metabolismo , Proteína 1 Homóloga a MutL/metabolismo , Pirofosfatases/genética , Proteína Supressora de Tumor p53/metabolismo , Animais , Linhagem Celular Tumoral , DNA/metabolismo , Células HCT116 , Células HeLa , Humanos , Inosina/análise , Nucleotídeos de Inosina/metabolismo , Camundongos , Camundongos Knockout , Pirofosfatases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética
6.
Neuroscience ; 316: 94-108, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26701297

RESUMO

G-protein-coupled receptors are thought to be involved in the detection of umami and L-amino acid taste. These include the heterodimer taste receptor type 1 member 1 (T1r1)+taste receptor type 1 member 3 (T1r3), taste and brain variants of mGluR4 and mGluR1, and calcium sensors. While several studies suggest T1r1+T1r3 is a broadly tuned lLamino acid receptor, little is known about the function of metabotropic glutamate receptors (mGluRs) in L-amino acid taste transduction. Calcium imaging of isolated taste sensory cells (TSCs) of T1r3-GFP and T1r3 knock-out (T1r3 KO) mice was performed using the ratiometric dye Fura 2 AM to investigate the role of different mGluRs in detecting various L-amino acids and inosine 5' monophosphate (IMP). Using agonists selective for various mGluRs such as (RS)-3,5-dihydroxyphenylglycine (DHPG) (an mGluR1 agonist) and L-(+)-2-amino-4-phosphonobutyric acid (l-AP4) (an mGluR4 agonist), we evaluated TSCs to determine if they might respond to these agonists, IMP, and three L-amino acids (monopotassium L-glutamate, L-serine and L-arginine). Additionally, we used selective antagonists against different mGluRs such as (RS)-L-aminoindan-1,5-dicarboxylic acid (AIDA) (an mGluR1 antagonist), and (RS)-α-methylserine-O-phosphate (MSOP) (an mGluR4 antagonist) to determine if they can block responses elicited by these L-amino acids and IMP. We found that L-amino acid- and IMP-responsive cells also responded to each agonist. Antagonists for mGluR4 and mGluR1 significantly blocked the responses elicited by IMP and each of the L-amino acids. Collectively, these data provide evidence for the involvement of taste and brain variants of mGluR1 and mGluR4 in L-amino acid and IMP taste responses in mice, and support the concept that multiple receptors contribute to IMP and L-amino acid taste.


Assuntos
Aminoácidos/metabolismo , Cálcio/metabolismo , Nucleotídeos de Inosina/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Papilas Gustativas/citologia , Percepção Gustatória/fisiologia , Análise de Variância , Animais , Relação Dose-Resposta a Droga , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Percepção Gustatória/efeitos dos fármacos
7.
J Biomed Mater Res B Appl Biomater ; 102(4): 721-8, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24155207

RESUMO

The aim of this study is to identify a plasticizer that is effective in the suppression of the autohemolysis of the stored blood and can be used to replace di(2-ethylhexyl) phthalate (DEHP) in blood containers. The results of hemolysis test using mannitol-adenine-phosphate/red cell concentrates (MAP/RCC) spiked with plasticizers included phthalate, phthalate-like, trimeliate, citrate, and adipate derivatives revealed that di-isononyl-cyclohexane-1,2-dicarboxylate (Hexamoll(®) DINCH), di(2-ethylhexyl)-1,2,3,6-tetrahydro-phthalate (DOTP), and diisodecyl phthalate (DIDP) exhibited a hemolysis suppression effect almost equal to that of DEHP, but not other plasticizers. This finding suggested that the presence of 2 carboxy-ester groups at the ortho position on a 6-membered ring of carbon atoms may be required to exhibit such an effect. The hemolytic ratios of MAP/RCC-soaked polyvinyl chloride (PVC) sheets containing DEHP or different amounts of DINCH or DOTP were reduced to 10.9%, 9.2-12.4%, and 5.2-7.8%, respectively (MAP/RCC alone, 28.2%) after 10 weeks of incubation. The amount of plasticizer eluted from the PVC sheet was 53.1, 26.1-36.5, and 78.4-150 µg/mL for DEHP, DINCH, and DOTP, respectively. PVC sheets spiked with DIDP did not suppress the hemolysis induced by MAP/RCC because of low leachability (4.8-6.0 µg/mL). These results suggested that a specific structure of the plasticizer and the concentrations of least more than ∼10 µg/mL were required to suppress hemolysis due to MAP/RCC.


Assuntos
Preservação de Sangue/instrumentação , Hemólise/efeitos dos fármacos , Plastificantes/farmacologia , Cloreto de Polivinila , Adenina , Benzoatos/farmacologia , Citratos , Ácidos Cicloexanocarboxílicos/farmacologia , Depressão Química , Ácidos Dicarboxílicos/farmacologia , Dietilexilftalato/farmacologia , Dietilexilftalato/toxicidade , Cromatografia Gasosa-Espectrometria de Massas , Glucose , Heparina , Humanos , Nucleotídeos de Inosina/farmacologia , Manitol , Oxazóis/farmacologia , Plastificantes/química , Pirimidinonas/farmacologia , Relação Estrutura-Atividade
8.
Chem Commun (Camb) ; 49(98): 11503-5, 2013 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-24177564

RESUMO

Inosine derivatives bearing a phosphodiester group at the O(6)-position of the nucleobase were synthesized via phosphitylation of the carbonyl oxygen using phosphoramidites activated by non-nucleophilic acidic activators.


Assuntos
Nucleotídeos de Inosina/química , Aziridinas/química , Ésteres/química , Hipoxantina/química , Mesilatos/química , Oxigênio/química
9.
Arterioscler Thromb Vasc Biol ; 33(3): 629-36, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23288168

RESUMO

OBJECTIVE: Adenosine dilates human coronary arteries by activating potassium channels in an endothelial cell-independent manner. Cell surface ecto-5'-nucleotidase (CD73) rapidly dephosphorylates extracellular adenosine 5'-monophosphate to adenosine. We tested the hypothesis that coronary vasodilation to adenine nucleotides is mediated by an endothelial CD73-dependent, extracellular production of adenosine that acts as an endothelium-derived hyperpolarizing factor. METHODS AND RESULTS: Videomicroscopy showed that adenine nucleotides, but not inosine, potently dilated and hyperpolarized human coronary arteries independent of nitric oxide, prostacyclin, and classical endothelium-derived hyperpolarizing factors, whereas endothelial denudation, adenosine receptor antagonism, adenosine deaminase, or CD73 blockers reduced vasodilations. Liquid chromatography-electrospray ionization-mass spectrometry revealed adenosine accumulation in perfusates from arteries in the presence of adenosine 5'-diphosphate. CD73 was localized on the cell surface of endothelial cells, but not of vascular smooth muscle cells, and its deficiency suppressed vasodilation of mouse coronary arteries to adenine nucleotides and augmented vasodilation to adenosine. Adenosine dose-dependently dilated and hyperpolarized human coronary arteries to a similar extent as adenosine 5'-diphosphate. CONCLUSIONS: Coronary vasodilation to adenine nucleotides is associated with endothelial CD73-dependent production of extracellular adenosine that acts as an endothelium-derived hyperpolarizing factor by relaxing and hyperpolarizing underlying vascular smooth muscle cells via activating adenosine receptors. Thus, CD73 is a novel endothelium-derived hyperpolarizing factor synthase in human and mouse coronary arteries.


Assuntos
5'-Nucleotidase/metabolismo , Nucleotídeos de Adenina/farmacologia , Adenosina/metabolismo , Fatores Biológicos/metabolismo , Vasos Coronários/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , 5'-Nucleotidase/antagonistas & inibidores , 5'-Nucleotidase/deficiência , 5'-Nucleotidase/genética , Nucleotídeos de Adenina/metabolismo , Adenosina Desaminase/metabolismo , Animais , Cromatografia Líquida , Vasos Coronários/enzimologia , Relação Dose-Resposta a Droga , Células Endoteliais/enzimologia , Inibidores Enzimáticos/farmacologia , Feminino , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/deficiência , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Nucleotídeos de Inosina/farmacologia , Masculino , Potenciais da Membrana , Camundongos , Camundongos Knockout , Microscopia de Vídeo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Antagonistas de Receptores Purinérgicos P1/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Vasodilatadores/metabolismo
10.
Mol Cell Biochem ; 366(1-2): 69-80, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22422046

RESUMO

Cyclic ADP-ribose (cADPR), a metabolite of NAD(+), is known to function as a second messenger for intracellular Ca(2+) mobilization in various vertebrate and invertebrate tissues. In this study, we isolated two Xenopus laevis cDNAs (frog cd38 and cd157 cDNAs) homologous to the one encoding the human cADPR-metabolizing enzyme CD38. Frog CD38 and CD157 are 298-amino acid proteins with 35.9 and 27.2 % identity to human CD38 and CD157, respectively. Transfection of expression vectors for frog CD38 and CD157 into COS-7 cells revealed that frog CD38 had NAD(+) glycohydrolase, ADP-ribosyl cyclase (ARC), and cADPR hydrolase activities, and that frog CD157 had no enzymatic activity under physiological conditions. In addition, when recombinant CD38 and frog brain homogenate were electrophoresed on an SDS-polyacrylamide gel, ARC of the brain homogenate migrated to the same position in the gel as that of frog CD38, suggesting that frog CD38 is the major enzyme responsible for cADPR metabolism in amphibian cells. The frog cd38 gene consists of eight exons and is ubiquitously expressed in various tissues. These findings provide evidence for the existence of the CD38-cADPR signaling system in frog cells and suggest that the CD38-cADPR signaling system is conserved during vertebrate evolution.


Assuntos
ADP-Ribosil Ciclase 1/genética , ADP-Ribosil Ciclase/genética , Antígenos CD/genética , ADP-Ribose Cíclica/biossíntese , Proteínas de Xenopus/genética , Xenopus laevis/genética , ADP-Ribosil Ciclase/biossíntese , ADP-Ribosil Ciclase/química , ADP-Ribosil Ciclase 1/biossíntese , ADP-Ribosil Ciclase 1/química , Sequência de Aminoácidos , Animais , Antígenos CD/biossíntese , Antígenos CD/química , Sequência de Bases , Encéfalo/enzimologia , Células COS , Chlorocebus aethiops , Clonagem Molecular , Sequência Conservada , ADP-Ribose Cíclica/metabolismo , Evolução Molecular , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Humanos , Hidrólise , Nucleotídeos de Inosina/química , Cinética , Dados de Sequência Molecular , NAD/análogos & derivados , NAD/química , Especificidade de Órgãos , Filogenia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Análise de Sequência de DNA , Proteínas de Xenopus/biossíntese , Proteínas de Xenopus/química
11.
Naunyn Schmiedebergs Arch Pharmacol ; 383(6): 573-83, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21484439

RESUMO

Membranous adenylyl cyclases play a major role in G-protein-coupled receptor signalling and regulate various cellular responses, such as cardiac contraction. Cardiac apoptosis and development of cardiac dysfunction is prevented in mice lacking AC 5, a predominant isoform in the heart. In the search for a potent and selective AC 5 inhibitor, we recently identified 2'(3')-methylanthraniloyl-inosine-5'-triphosphate(MANT-ITP) as the most potent AC 5 inhibitor with a K ( i ) of 13 nM. Therefore, AC inhibition of MANT-ITP was assessed in ventricular cardiomyocytes and compared to three other MANT-nucleotides to evaluate its effect on cardiac signalling. Basal and isoproterenol-induced L-type calcium currents (I (Ca,L)) in murine ventricular cardiomyocytes were recorded by whole-cell patch-clamp technique, using four different MANT-nucleotides. The effects of the MANT-nucleotides on I (Ca,L) were unexpectedly complex. All MANT-nucleotides exhibited an inhibitory effect on basal I (Ca,L). Additionally, several MANT-nucleotides, i.e., MANT-ITPγS, MANT-ATP, and MANT-ITP, caused a strong initial increase in basal I (Ca,L) within the first 2.5 min that appeared to be unrelated to AC 5 inhibition. However, we detected a significant reduction on isoproterenol-induced I (Ca,L) with MANT-ITP, supporting the notion that AC 5 plays an important role in agonist-stimulated activation of I (Ca,L). Collectively, MANT-nucleotides are useful tools for the characterization of recombinant ACs, for fluorescence studies and crystallography, but in intact cardiomyocytes, caution must be exerted since MANT-nucleotides apparently possess additional effects than AC 5 inhibition, limiting their usefulness as tools for intact cell studies.


Assuntos
Inibidores de Adenilil Ciclases , Canais de Cálcio Tipo L/efeitos dos fármacos , Nucleotídeos de Inosina/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Animais , Canais de Cálcio Tipo L/metabolismo , Inibidores Enzimáticos/farmacologia , Inosina Trifosfato/análogos & derivados , Inosina Trifosfato/farmacologia , Isoproterenol/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Receptores Acoplados a Proteínas G/metabolismo
12.
Mar Biotechnol (NY) ; 13(6): 1140-7, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21519809

RESUMO

The enzyme adenosine 5'-monophosphate deaminase (AMPD, EC 3.5.4.6) catalyzes the conversion of adenosine 5'-monophosphate to inosine 5'-mononucleotide (IMP). IMP is generally known as the compound responsible for the umami taste of the edible red alga Porphyra yezoensis Ueda that is known in Japan as nori. Therefore, we suspect that AMPD plays a key role in providing a favorable nori taste. In this study, we undertake the molecular characterization of nori-derived AMPD. The nori AMPD protein has a molecular mass of 55 kDa as estimated from both gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The calculated molecular mass from the amino acid sequence deduced from cDNA is 57.1 kDa. The isoelectric point is 5.71. The coding region of AMPD consists of 1,566 bp encoding 522 amino acids and possesses a transmembrane domain and two N-glycosylation sites. The sequence identity of nori AMPD in human and yeast AMPDs was found to be less than 50% and 20% in DNA and amino acid sequences, respectively. Proline in the conserved motif of [SA]-[LIVM]-[NGS]-[STA]-D-D-P was found to be converted to glutamate. These results indicate that nori AMPD is a novel type of AMPD.


Assuntos
AMP Desaminase/genética , Nucleotídeos de Inosina/química , Porphyra/enzimologia , Paladar , AMP Desaminase/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Cromatografia em Gel , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência
13.
Zh Evol Biokhim Fiziol ; 46(5): 406-10, 2010.
Artigo em Russo | MEDLINE | ID: mdl-21061651

RESUMO

Activity of the phosphoinositide system of the intracellular signalization was studied in offspring of rats exposed to severe hypobaric hypoxia at the 14-16th (group 1) or the 18-20th day (group 2) of prenatal development. At the age of 15 days, in animals of both experimental groups the basal level of triphosphoinositides in the brain cortex was shown to be elevated as compared with control. In the group 1 this parameters also remains elevated in adult animals. Application of glutamate produces a more pronounced increase of the inositephosphates in brain sections of the 15-day old rats of the group 1 than in sections of animals of the control group. In the 15-day old rats of the group 2, as compared with control, the phosphoinositide response to glutamate application was reduced. No changes in the inositephosphate levels were revealed after application of glutamate upon sections of adult (the 90-day old) control animals and of adult rats of the group 2. In sections of adult rats of the group 1, on the contrary, the glutamate application produced an increase of the inositephosphate content. The obtained data indicate essential changes of the phosphoinositide metabolism in the brain of rats exposed to action of hypoxia at the period of prenatal development. The character and the degree of these changes depend on the period of development when the action of hypoxia occurs.


Assuntos
Encéfalo/metabolismo , Hipóxia/metabolismo , Nucleotídeos de Inosina/metabolismo , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Sistemas do Segundo Mensageiro , Animais , Química Encefálica/efeitos dos fármacos , Feminino , Ácido Glutâmico/farmacologia , Hipóxia/complicações , Gravidez , Ratos
14.
Nucleic Acids Res ; 38(14): 4834-43, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20385596

RESUMO

Nucleotides function in a variety of biological reactions; however, they can undergo various chemical modifications. Such modified nucleotides may be toxic to cells if not eliminated from the nucleotide pools. We performed a screen for modified-nucleotide binding proteins and identified human nucleoside diphosphate linked moiety X-type motif 16 (NUDT16) protein as an inosine triphosphate (ITP)/xanthosine triphosphate (XTP)/GTP-binding protein. Recombinant NUDT16 hydrolyzes purine nucleoside diphosphates to the corresponding nucleoside monophosphates. Among 29 nucleotides examined, the highest k(cat)/K(m) values were for inosine diphosphate (IDP) and deoxyinosine diphosphate (dIDP). Moreover, NUDT16 moderately hydrolyzes (deoxy)inosine triphosphate ([d]ITP). NUDT16 is mostly localized in the nucleus, and especially in the nucleolus. Knockdown of NUDT16 in HeLa MR cells caused cell cycle arrest in S-phase, reduced cell proliferation, increased accumulation of single-strand breaks in nuclear DNA as well as increased levels of inosine in RNA. We thus concluded that NUDT16 is a (deoxy)inosine diphosphatase that may function mainly in the nucleus to protect cells from deleterious effects of (d)ITP.


Assuntos
Hidrolases Anidrido Ácido/metabolismo , Quebras de DNA de Cadeia Simples , Pirofosfatases/metabolismo , Hidrolases Anidrido Ácido/deficiência , Hidrolases Anidrido Ácido/genética , Sequência de Aminoácidos , Núcleo Celular/química , Proliferação de Células , Técnicas de Silenciamento de Genes , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Nucleotídeos de Inosina/metabolismo , Inosina Trifosfato/metabolismo , Dados de Sequência Molecular , Pirofosfatases/deficiência , Pirofosfatases/genética , Ribonucleotídeos/metabolismo
15.
Arch Biochem Biophys ; 498(1): 13-22, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20227382

RESUMO

The human DNA methyltransferase 3A (DNMT3A) is essential for establishing DNA methylation patterns. Knowing the key factors involved in the regulation of mammalian DNA methylation is critical to furthering understanding of embryonic development and designing therapeutic approaches targeting epigenetic mechanisms. We observe substrate inhibition for the full length DNMT3A but not for its isolated catalytic domain, demonstrating that DNMT3A has a second binding site for DNA. Deletion of recognized domains of DNMT3A reveals that the conserved PWWP domain is necessary for substrate inhibition and forms at least part of the allosteric DNA binding site. The PWWP domain is demonstrated here to bind DNA in a cooperative manner with muM affinity. No clear sequence preference was observed, similar to previous observations with the isolated PWWP domain of Dnmt3b but with one order of magnitude weaker affinity. Potential roles for a low affinity, low specificity second DNA binding site are discussed.


Assuntos
Domínio Catalítico , DNA (Citosina-5-)-Metiltransferases/química , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Deleção de Sequência , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Sequência Conservada , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Humanos , Nucleotídeos de Inosina/química , Nucleotídeos de Inosina/farmacologia , Cinética , Dados de Sequência Molecular , Oligonucleotídeos/genética , Oligonucleotídeos/farmacologia , Polímeros/química , Estrutura Terciária de Proteína/genética
16.
Nucleic Acids Res ; 38(9): 2891-903, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20081199

RESUMO

Mammalian inosine triphosphatase encoded by ITPA gene hydrolyzes ITP and dITP to monophosphates, avoiding their deleterious effects. Itpa(-) mice exhibited perinatal lethality, and significantly higher levels of inosine in cellular RNA and deoxyinosine in nuclear DNA were detected in Itpa(-) embryos than in wild-type embryos. Therefore, we examined the effects of ITPA deficiency on mouse embryonic fibroblasts (MEFs). Itpa(-) primary MEFs lacking ITP-hydrolyzing activity exhibited a prolonged doubling time, increased chromosome abnormalities and accumulation of single-strand breaks in nuclear DNA, compared with primary MEFs prepared from wild-type embryos. However, immortalized Itpa(-) MEFs had neither of these phenotypes and had a significantly higher ITP/IDP-hydrolyzing activity than Itpa(-) embryos or primary MEFs. Mammalian NUDT16 proteins exhibit strong dIDP/IDP-hydrolyzing activity and similarly low levels of Nudt16 mRNA and protein were detected in primary MEFs derived from both wild-type and Itpa(-) embryos. However, immortalized Itpa(-) MEFs expressed significantly higher levels of Nudt16 than the wild type. Moreover, introduction of silencing RNAs against Nudt16 into immortalized Itpa(-) MEFs reproduced ITPA-deficient phenotypes. We thus conclude that NUDT16 and ITPA play a dual protective role for eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.


Assuntos
Hidrolases Anidrido Ácido/fisiologia , Instabilidade Cromossômica , Inosina Difosfato/metabolismo , Inosina Trifosfato/metabolismo , Pirofosfatases/fisiologia , Hidrolases Anidrido Ácido/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Nucleotídeos de Inosina/metabolismo , Inosina Trifosfato/análogos & derivados , Camundongos , Camundongos Knockout , Fenótipo , Pirofosfatases/genética , Pirofosfatases/metabolismo
17.
J Biol Chem ; 284(40): 27637-45, 2009 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-19640846

RESUMO

Cyclic ADP-ribose (cADPR) is a universal calcium messenger molecule that regulates many physiological processes. The production and degradation of cADPR are catalyzed by a family of related enzymes, including the ADP-ribosyl cyclase from Aplysia california (ADPRAC) and CD38 from human. Although ADPRC and CD38 share a common evolutionary ancestor, their enzymatic functions toward NAD and cADPR homeostasis have evolved divergently. Thus, ADPRC can only generate cADPR from NAD (cyclase), whereas CD38, in contrast, has multiple activities, i.e. in cADPR production and degradation, as well as NAD hydrolysis (NADase). In this study, we determined a number of ADPRC and CD38 structures bound with various nucleotides. From these complexes, we elucidated the structural features required for the cyclization (cyclase) reaction of ADPRC and the NADase reaction of CD38. Using the structural approach in combination with site-directed mutagenesis, we identified Phe-174 in ADPRC as a critical residue in directing the folding of the substrate during the cyclization reaction. Thus, a point mutation of Phe-174 to glycine can turn ADPRC from a cyclase toward an NADase. The equivalent residue in CD38, Thr-221, is shown to disfavor the cyclizing folding of the substrate, resulting in NADase being the dominant activity. The comprehensive structural comparison of CD38 and APDRC presented in this study thus provides insights into the structural determinants for the functional evolution from a cyclase to a hydrolase.


Assuntos
ADP-Ribosil Ciclase/química , ADP-Ribosil Ciclase/metabolismo , Evolução Molecular , NAD/metabolismo , ADP-Ribosil Ciclase/genética , ADP-Ribosil Ciclase 1/química , ADP-Ribosil Ciclase 1/metabolismo , Animais , Aplysia/enzimologia , Sítios de Ligação , Hidrólise , Nucleotídeos de Inosina/metabolismo , Modelos Moleculares , Mutação , Conformação Proteica , Especificidade por Substrato
18.
Cell Death Differ ; 16(10): 1315-22, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19498443

RESUMO

Inosine triphosphate pyrophosphatase (ITPase), the enzyme that hydrolyzes ITP and other deaminated purine nucleoside triphosphates to the corresponding purine nucleoside monophosphate and pyrophosphate, is encoded by the Itpa gene. In this study, we established Itpa knockout (KO) mice and used them to show that ITPase is required for the normal organization of sarcomeres in the heart. Itpa(-/-) mice died about 2 weeks after birth with features of growth retardation and cardiac myofiber disarray, similar to the phenotype of the cardiac alpha-actin KO mouse. Inosine nucleotides were found to accumulate in both the nucleotide pool and RNA of Itpa(-/-) mice. These data suggest that the role of ITPase in mice is to exclude ITP from the ATP pool, and the main target substrate of this enzyme is rITP. Our data also suggest that cardiomyopathy, which is mainly caused by mutations in sarcomeric protein-encoding genes, is also caused by a defect in maintaining the quality of the ATP pool, which is an essential requirement for sarcomere function.


Assuntos
Cardiomiopatias/enzimologia , Transtornos do Crescimento/enzimologia , Pirofosfatases/fisiologia , Actinas/genética , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Cardiomiopatias/genética , Cardiomiopatias/patologia , Feminino , Genótipo , Transtornos do Crescimento/genética , Transtornos do Crescimento/mortalidade , Nucleotídeos de Inosina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Miocárdio/patologia , Fenótipo , Pirofosfatases/deficiência , Pirofosfatases/genética , RNA Mensageiro/metabolismo , Sarcômeros/metabolismo , Sarcômeros/fisiologia , Desmame
19.
Biochem J ; 422(1): 139-49, 2009 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-19492987

RESUMO

cADPR (cyclic ADP-ribose) is a universal Ca(2+) mobilizing second messenger. In T-cells cADPR is involved in sustained Ca(2+) release and also in Ca(2+) entry. Potential mechanisms for the latter include either capacitative Ca(2+) entry, secondary to store depletion by cADPR, or direct activation of the non-selective cation channel TRPM2 (transient receptor potential cation channel, subfamily melastatin, member 2). Here we characterize the molecular target of the newly-described membrane-permeant cADPR agonist 8-Br-N(1)-cIDPR (8-bromo-cyclic IDP-ribose). 8-Br-N(1)-cIDPR evoked Ca(2+) signalling in the human T-lymphoma cell line Jurkat and in primary rat T-lymphocytes. Ca(2+) signalling induced by 8-Br-N(1)-cIDPR consisted of Ca(2+) release and Ca(2+) entry. Whereas Ca(2+) release was sensitive to both the RyR (ryanodine receptor) blocker RuRed (Ruthenium Red) and the cADPR antagonist 8-Br-cADPR (8-bromo-cyclic ADP-ribose), Ca(2+) entry was inhibited by the Ca(2+) entry blockers Gd(3+) (gadolinium ion) and SKF-96365, as well as by 8-Br-cADPR. To unravel a potential role for TRPM2 in sustained Ca(2+) entry evoked by 8-Br-N(1)-cIDPR, TRPM2 was overexpressed in HEK (human embryonic kidney)-293 cells. However, though activation by H(2)O(2) was enhanced dramatically in those cells, Ca(2+) signalling induced by 8-Br-N(1)-cIDPR was almost unaffected. Similarly, direct analysis of TRPM2 currents did not reveal activation or co-activation of TRPM2 by 8-Br-N(1)-cIDPR. In summary, the sensitivity to the Ca(2+) entry blockers Gd(3+) and SKF-96365 is in favour of the concept of capacitative Ca(2+) entry, secondary to store depletion by 8-Br-N(1)-cIDPR. Taken together, 8-Br-N(1)-cIDPR appears to be the first cADPR agonist affecting Ca(2+) release and secondary Ca(2+) entry, but without effect on TRPM2.


Assuntos
ADP-Ribose Cíclica/análogos & derivados , Nucleotídeos de Inosina/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Gadolínio/farmacologia , Humanos , Imidazóis/farmacologia , Nucleotídeos de Inosina/síntese química , Nucleotídeos de Inosina/química , Ativação do Canal Iônico/efeitos dos fármacos , Células Jurkat , Microinjeções , Ratos , Rutênio Vermelho/farmacologia , Canais de Cátion TRPM/metabolismo
20.
Cell Calcium ; 45(4): 346-57, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19201464

RESUMO

Cellular metabolism is required for cell proliferation. However, the way in which metabolic signals are conveyed to cell cycle decisions is unclear. Cyclic ADP-ribose (cADPR), the NAD(+) metabolite, mobilizes calcium from calcium stores in many cells. We found that dinoflagellate cells with higher metabolic rate underwent multiple fission (MF), a division mode in which cells can exceed twice their sizes at G1. A temperature shift-down experiment suggested that MF involves a commitment point at late G1. In fast-growing cells, cADPR level peaked in G(1) and increased with increasing concentrations of glucose in the medium. Addition of glycolytic poison iodoacetate inhibited cell growth, reduced cADPR levels as well as the commitment of cell cycles in fast-growing cells. Commitment of MF cell cycles was induced by a cell permeant cADPR agonist, but blocked by a specific antagonist of cADPR-induced Ca(2+) release. Our results establish cADPR as a link between cellular metabolism and cell cycle control.


Assuntos
Divisão Celular , ADP-Ribose Cíclica/metabolismo , Dinoflagelados/citologia , ADP-Ribosil Ciclase/metabolismo , Animais , Cálcio/metabolismo , Divisão Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Quelantes/farmacologia , ADP-Ribose Cíclica/análogos & derivados , ADP-Ribose Cíclica/antagonistas & inibidores , ADP-Ribose Cíclica/farmacologia , Dinoflagelados/efeitos dos fármacos , Dinoflagelados/enzimologia , Glucose/farmacologia , Nucleotídeos de Inosina/farmacologia , Iodoacetatos/farmacologia , Ionóforos/farmacologia , Modelos Biológicos , NAD/metabolismo , Temperatura
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