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Repercussão funcional da expressão dos receptores A2A, NK1 e M3 em várias subpopulações celulares no plexo mioentérico do íleo de rato

Silva, Isabel Sofia Dias da
Fonte: Universidade de Trás-os-Montes e Alto Douro Publicador: Universidade de Trás-os-Montes e Alto Douro
Tipo: Dissertação de Mestrado
POR
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Dissertação de Mestrado em Genética Molecular Comparativa e Tecnológica; O sistema nervoso entérico (SNE) é formado por uma extensa rede neuronal intrínseca que acompanha todo o tracto gastrointestinal e que se organiza em dois grandes plexos nervosos; o plexo submucoso ou de Meissner (mais interno, localizado na submucosa) e o plexo mioentérico ou de Auerbach (mais externo, localizado entre as duas camadas musculares circular e longitudinal). O SNE possui numerosas células gliais e intersticiais de Cajal (ICC), que em conjunto com os neurónios controlam funções essenciais como a motilidade, secreção e fluxo sanguíneo intestinal. A motilidade gastrointestinal é regulada pela libertação de acetilcolina (ACh), de purinas (e.g. ATP, adenosina) e de taquicininas (e.g. Substância P) a partir de neurónios entéricos excitatórios (Furness, 2000). Por outro lado, tanto as células gliais como as intersticiais de Cajal estão envolvidas no metabolismo dos neurotransmissores e são capazes de libertar substâncias neuromoduladoras. O equilíbrio entre a activação de neurónios excitatórios e/ou inibitórios, das células não-neuronais adjacentes e das células efectoras (musculares, glandulares ou vasculares) determina a actividade funcional do intestino em cada momento. Substâncias irritantes como a capsaicina...

Prolonged derangements of canine myocardial purine metabolism after a brief coronary artery occlusion not associated with anatomic evidence of necrosis

DeBoer, Laurence W. V.; Ingwall, Joanne S.; Kloner, Robert A.; Braunwald, Eugene
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1980 EN
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Changes in myocardial purine metabolism were studied after temporary coronary artery occlusion and subsequent reperfusion in the dog. Sequential myocardial biopsies were performed to allow for measurements of ATP, adenine nucleotide, nucleoside, and base concentrations after 15 min of ischemia, and after 90 min and 72 hr of reperfusion following this period of ischemia. Control, nonischemic sites were also sampled. After 15 min of coronary occlusion, subendocardial ATP concentrations (reported in nmol/mg of protein; mean ± SEM) were depressed in the ischemic zone at 19.9 ± 3.5 compared to 38.1 ± 2.8 in the nonischemic zone (P < 0.001). Subepicardial ATP concentrations also were depressed at 27.0 ± 2.2 in ischemic sites compared to subepicardial nonischemic sites (40.0 ± 4.0, P < 0.005). After 90 min of reperfusion ATP concentrations remained depressed in the previously ischemic subendocardium 26.8 ± 4.2 (P < 0.025 vs. nonischemic sites). After 72 hr of reperfusion, ATP was still depressed in the previously ischemic subendocardium at 29.2 ± 2.5 (P < 0.025 vs. nonischemic) and subepicardium (27.9 ± 3.3, P < 0.05 vs. nonischemic). Total purines were determined as the sum of ATP, ADP, AMP, adenosine, inosine, and hypoxanthine. After 15 min of occlusion...

Oligonucleotide studies. Optical rotatory dispersion of five homodinucleotides

Inoue, Yasuo; Satoh, Kimihiko
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1969 EN
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1. The optical rotatory dispersion (ORD) of five homodinucleotides, ApAp(3′), CpCp(3′), GpGp(3′), IpIp(3′) and UpUp(3′) (where A, C, G, I and U represent adenosine, cytidine, guanosine, inosine and uridine respectively, and p to the left of a nucleoside symbol indicates a 5′-phosphate and to the right it indicates a 3′-phosphate), were measured as a function of pH, ionic strength and Mg2+ concentration. 2. The ORD titrations of ApAp(3′) and CpCp(3′), which were made by measuring the ORD curves at closely spaced pH intervals, exhibit a maximum at approx. pH5·0 and 5·7 for ApAp(3′) and CpCp(3′) respectively in the profile of the magnitude of the first Cotton effect versus pH. The results indicate that the conformational rigidity of these dinucleotides depends on the ionization state of a 3′-terminal phosphate group. 3. ApAp(3′) was shown to exist as an approximately 1:1 equilibrium mixture of the two major ionic species represented by Ap(−1)Ap(−1) and Ap(−1)Ap(−2) at pH6·16, whereas at pH7·5 it exists exclusively as a form of Ap(−1)Ap(−2). 4. To ascertain the effects of the presence of a terminal phosphate group and of the ionization of the secondary phosphate on the conformation of adenylate dimer...

Reduction of equilibrative nitrobenzylthioinosine-sensitive nucleoside transporter in tamoxifen-treated MCF-7 cells: an oestrogen-reversible phenomenon.

Goh, L B; Lee, C W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/10/1997 EN
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MCF-7 cells display both nitrobenzylthioinosine (NBMPR)-sensitive (es) and NBMPR-insensitive (ei) equilibrative, but not the Na+-dependent, nucleoside transport. Transport of uridine by es is more sensitive to inhibition by purine nucleosides, whereas the ei component is more sensitive to nucleosides without an amino side group, such as inosine and thymidine. When exposed to 10 microM tamoxifen for 5 days, MCF-7 cells displayed a 44% decrease in the total number of NBMPR-binding sites [Bmax from 245000+/-18000 to 136000+/-25000 sites per cell (mean+/-S.E.M.; n=5; P<0.05)], and a 57% decrease in cell growth with no significant change in binding affinities [Kd from 0.37+/-0.05 to 0.45+/-0.08 nM (n=5; P>0.05)]. Kinetic studies of [3H]uridine transport showed a decrease in the Vmax of the es component from 21.7+/-0.3 (n=8) to 8.4 +/- 2.2 microM/s (n=4; P < 0.05), whereas the Vmax of the ei component [from 4.7 +/- 0.5 (n=8) to 5.8 +/- 1.6 microM/s (n=4; P > 0.05)] and Km values for both components [es from 460 +/- 80 to 630 +/- 280 microM (n>/=4; P > 0.05) and ei from 355 +/- 115 to 440 +/- 220 microM (n>/=4; P > 0.05)] did not change significantly. Oestradiol at 100 nM reversed almost completely the NBMPR-binding site decrease and growth retardation in tamoxifen-treated cells. Thus tamoxifen is shown to cause an oestrogen-reversible decrease of es nucleoside transporters in MCF-7 cells.

Dysregulated Editing of Serotonin 2C Receptor mRNAs Results in Energy Dissipation and Loss of Fat Mass

Kawahara, Yukio; Grimberg, Adda; Teegarden, Sarah; Mombereau, Cedric; Liu, Sui; Bale, Tracy L.; Blendy, Julie A.; Nishikura, Kazuko
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 26/11/2008 EN
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RNA editing that converts adenosine to inosine replaces the gene-encoded Ile, Asn, and Ile (INI) of serotonin (5-hydroxytryptamine, 5-HT) receptor 2C (5-HT2CR) with Val, Gly, and Val (VGV). Up to 24 different 5-HT2CR isoforms are detected in different brain regions (Burns et al., 1997; Fitzgerald et al., 1999; Wang et al., 2000). To elucidate the physiological significance of 5-HT2CR mRNA editing, we derived mutant mouse lines harboring a knock-in INI or VGV allele, resulting in sole expression of one of two extremely different editing isoforms 5-HT2CR-INI (editing blocked) or -VGV (fully edited). Although INI mice grew normally, VGV mice had a severely reduced fat mass, in spite of compensatory hyperphagia, due to constitutive activation of the sympathetic nervous system and increased energy expenditure. Furthermore, serotonergic neurotransmission was oversensitized in VGV mice, most likely due to the increased cell surface expression of VGV receptors. Melanocortin 4 receptor (MC4R) regulates energy homeostasis (Balthasar et al., 2005; Heisler et al., 2006; Lam et al., 2008), and Mc4r−/− mice are obese due to hyperphagia and reduced energy expenditure (Huszar et al., 1997). However, the elevated energy expenditure of VGV mice could not be rescued in the Mc4r−/− background...

Assessing serotonin receptor mRNA editing frequency by a novel ultra high-throughput sequencing method

Abbas, Atheir I.; Urban, Daniel J.; Jensen, Niels H.; Farrell, Martilias S.; Kroeze, Wesley K.; Mieczkowski, Piotr; Wang, Zefeng; Roth, Bryan L.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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RNA editing is a post-transcriptional modification of pre-mRNA that results in increased diversity in transcriptomes and proteomes. It occurs in a wide variety of eukaryotic organisms and in some viruses. One of the most common forms of pre-mRNA editing is A-to-I editing, in which adenosine is deaminated to inosine, which is read as guanosine during translation. This phenomenon has been observed in numerous transcripts, including the mammalian 5-HT2C receptor, which can be edited at five distinct sites. Methods used to date to quantify 5-HT2C receptor editing are labor-intensive, expensive and provide limited information regarding the relative abundance of 5-HT2C receptor editing variants. Here, we present a novel, ultra high-throughput method to quantify 5-HT2C receptor editing, compare it to a more conventional method, and use it to assess the effect of a range of genetic and pharmacologic manipulations on 5-HT2C editing. We conclude that this new method is powerful and economical, and we provide evidence that alterations in 5-HT2C editing appear to be a result of regional changes in brain activity, rather than a mechanism to normalize 5-HT2C signaling.

An Improved Isolation Procedure for Adult Mouse Cardiomyocytes

Pinz, Ilka; Zhu, Ming; Mende, Ulrike; Ingwall, Joanne S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/2011 EN
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Isolated adult mouse cardiomyocytes are an important tool in cardiovascular research, but are challenging to prepare. Because the energy supply determines cell function and viability, we compared total creatine ([Cr]) and [ATP] in isolated cardiomyocytes with the intact mouse heart. Isolated myocytes suffered severe losses of Cr (−70%) and ATP (−53%). Myocytes were not able to replete [Cr] during a 5 h incubation period in medium supplemented with 1 mM Cr. In contrast, adding 20 mM Cr to the digestion buffers was sufficient to maintain normal [Cr]. Supplementing buffers with 5 mM of inosine (Ino) and adenosine (Ado) to prevent loss of cellular nucleosides partially protected against loss of ATP. To test whether maintaining [ATP] and [Cr] improves contractile function, myocytes were challenged by varying pacing rate from 0.5 to 10 Hz and by adding isoproterenol (Iso) at 5 and 10 Hz. All groups performed well up to 5 Hz, showing a positive cell shortening–frequency relationship; however, only 16% of myocytes isolated under standard conditions were able to sustain pacing with Iso challenge at 10 Hz. In contrast, 30–50% of the myocytes with normal Cr levels were able to contract and maintain low diastolic [Ca2+]. Cell yield also improved in Cr and the Cr/Ino/Ado-treated groups (85–90% vs. 70–75% rod shaped in untreated myocytes). These data suggest that viability and performance of isolated myocytes are improved when they are protected from the severe loss of Cr and ATP during the isolation...

The Brain In Vivo Expresses the 2′,3′-cAMP-Adenosine Pathway

Verrier, Jonathan D.; Jackson, Travis C.; Bansal, Rashmi; Kochanek, Patrick M.; Puccio, Ava M.; Okonkwo, David O.; Jackson, Edwin K.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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Although multiple biochemical pathways produce adenosine, studies suggest that the 2′,3′-cAMP-adenosine pathway (2′,3′-cAMP → 2′-AMP/3′-AMP → adenosine) contributes to adenosine production in some cells/tissues/organs. To determine whether the 2′,3′-cAMP-adenosine pathway exists in vivo in the brain, we delivered to the brain (gray matter and white matter separately) via the inflow perfusate of a microdialysis probe either 2′,3′-cAMP, 3′,5′-cAMP, 2′-AMP, 3′-AMP, or 5′-AMP and measured the recovered metabolites in the microdialysis outflow perfusate with mass spectrometry. In both gray and white matter, 2′,3′-cAMP increased 2′-AMP, 3′-AMP and adenosine, and 3′,5′-cAMP increased 5′-AMP and adenosine. In both brain regions, 2′-AMP, 3-AMP and 5′-AMP were converted to adenosine. Microdialysis experiments in 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) wild-type mice demonstrated that traumatic brain injury (TBI; controlled cortical impact model) activated the brain 2,3′-cAMP-adenosine pathway; similar experiments in CNPase knockout mice indicated that CNPase was involved in the metabolism of endogenous 2′,3′-cAMP to 2′-AMP and to adenosine. In CSF from TBI patients...

Mapeamento do padrão de expressão tecidual dos genes relacionados à adenosina deaminase e caracterização cinética da atividade de desaminação da adenosina em cérebro de zebrafish (Danio rerio)

Rosemberg, Denis Broock
Fonte: Universidade Federal do Rio Grande do Sul Publicador: Universidade Federal do Rio Grande do Sul
Tipo: Dissertação Formato: application/pdf
POR
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O zebrafish é um modelo experimental consolidado em diversas áreas, tais como genética e neurociências. Estudos têm demonstrado que muitos genes deste peixe são evolutivamente conservados e similares aos de mamíferos, incluindo a espécie humana. Com relação ao sistema purinérgico, já foi demonstrado que o zebrafish apresenta diferentes membros da família das NTPDases (nucleosídeo trifosfato difosfoidrolases) e uma ecto-5’-nucleotidase, capazes de clivar o ATP até adenosina, que atua através de purinoreceptores P1. A adenosina deaminase (ADA) é responsável pela clivagem da adenosina à inosina. Dois membros da família da ADA, conhecidos como ADA1 e ADA2, foram descritos e evidências recentes demonstraram a existência de um outro grupo similar de proteína, denominado ADAL (adenosina deaminase “like”). Portanto, no primeiro capítulo desta Dissertação, foram identificados distintos genes relacionados à ADA (ADA1, ADAL e dois genes ortólogos da ADA2) através de uma análise filogenética. Primers específicos para cada membro da ADA foram desenhados, experimentos otimizados de RT-PCR foram conduzidos e a quantidade relativa de transcritos determinada em diversos tecidos. Os resultados demonstraram que os genes da ADA1...

Efeito do estresse crônico imprevisível no metabolismo de nucleotídeos e nucleosídeos em encéfalo de zebrafish (Danio rerio)

Zimmermann, Fernanda Francine
Fonte: Pontifícia Universidade Católica do Rio Grande do Sul; Porto Alegre Publicador: Pontifícia Universidade Católica do Rio Grande do Sul; Porto Alegre
Tipo: Dissertação de Mestrado
PORTUGUêS
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De acordo com dados da Organização Mundial de Saúde (OMS), atualmente o estresse afeta mais de 90% da população mundial, sendo considerado uma epidemia global. O estresse tornou-se parte integrante da vida humana e os organismos são constantemente submetidos a estímulos estressantes que alteram vários processos fisiológicos, sendo um fator de risco para diversas patologias, como câncer, doenças cardiovasculares, metabólicas, infecciosas, gastrointestinais e depressão. O peixe-zebra (Danio rerio) tem muitas semelhanças com mamíferos em termos de organização geral e o funcionamento dos respectivos sistemas reguladores do estresse. O eixo hipotálamo-pituitária-interrenal (HPI), um sistema análogo ao eixo hipotálamo-pituitária-adrenal (HPA), foi descrito em zebrafish e a ativação desta via é essencial para a manutenção das funções vitais como resposta a eventos estressantes. Apesar do amplo conhecimento das respostas do estresse em mamíferos, os dados sobre a relação entre o estresse crônico imprevisível e seus efeitos na sinalização purinérgica são limitados. A sinalização purinérgica é mediada por ATP e adenosina por meio da ativação dos receptores P2 e P1, respectivamente. O catabolismo de ATP extracelular até adenosina é promovido pelas ectonucleotidases...

Avaliação dos efeitos de fármacos benzodiazepínicos sobre o catabolismo de nucleotídeos, nucleosídeos e acetilcolina em encéfalo de zebrafish adulto: (Danio rerio)

Altenhofen, Stefani
Fonte: Pontifícia Universidade Católica do Rio Grande do Sul; Porto Alegre Publicador: Pontifícia Universidade Católica do Rio Grande do Sul; Porto Alegre
Tipo: Dissertação de Mestrado
PORTUGUêS
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Fármacos benzodiazepínicos, como diazepam e midazolam, são muito usados na prática clínica para o tratamento da ansiedade, possuindo propriedades ansiolíticas, hipnóticas e anticonvulsivantes. O uso do zebrafish (Danio rerio) como modelo para avaliar mecanismos farmacológicos tem ganhado grande importância devido ao rápido desenvolvimento e alta sensibilidade a drogas que essa espécie possui. Estudos têm demonstrado que parâmetros comportamentais mostraram-se alterados em zebrafish após tratamento com benzodiazepínicos. Muitos sistemas de neurotransmissão foram identificados nessa espécie, incluindo os sistemas purinérgico e colinérgico. O sistema purinérgico é caracterizado pela ação do ATP e adenosina (ADO) nos purinoreceptores P2 e P1, respectivamente. Os níveis dessas moléculas são regulados pela ação das ectonucleotidases, especialmente as nucleosídeo trifosfato difosfoidrolases (NTPDases) e a ecto-5’-nucleotidase, que catalisam a hidrólise do ATP a adenosina. A adenosina pode ser desaminada a inosina pela ação da adenosina desaminase (ADA). O ATP é coliberado com outros neurotransmissores, entre eles a acetilcolina, e tem sido demonstrado que a adenosina pode controlar a liberação de acetilcolina. O sistema colinérgico é caracterizado pela ação da acetilcolina (ACh) nos receptores muscarínicos e nicotínicos. O nível dessa molécula é regulado pela acetilcolinesterase (AChE)...

Efeito de antidepressivos sobre as enzimas envolvidas no controle da sinalização purinérgica e colinérgica em cérebro de peixe-zebra (Danio rerio)

Oliveira, Renata da Luz
Fonte: Pontifícia Universidade Católica do Rio Grande do Sul; Porto Alegre Publicador: Pontifícia Universidade Católica do Rio Grande do Sul; Porto Alegre
Tipo: Dissertação de Mestrado
PORTUGUêS
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O tratamento clínico da depressão enfrenta sérios obstáculos já que o mecanismo da doença não é totalmente elucidado. Além disso, não existem meios eficazes para prever e prevenir a depressão bem como nenhum método biológico de diagnóstico. O uso de fármacos antidepressivos ainda é a base dos tratamentos para depressão. O lítio tem sido usado clinicamente como fármaco eficaz para tratar todas as fases do transtorno bipolar, incluindo depressão aguda. Os inibidores seletivos da recaptação de serotonina (ISRSs), como fluoxetina e citalopram, e os antidepressivos tricíclicos (TCA), como a clomipramina, são fármacos constantemente utilizados para o tratamento da depressão. Evidências recentes mostram o envolvimento da adenosina e seus receptores na patofisiologia da depressão. O ATP pode ser armazenado e co-liberado, juntamente com outros neurotransmissores, como serotonina e noradrenalina, e pode ser hidrolisado até adenosina por uma família de enzimas de superfície celular, conhecidas como ectonucleotidases. Dentre elas, destacam-se as Nucleosídeo Trifosfato Difosfoidrolases (NTPDases) e a ecto-5´-nucleotidase, capazes de controlar a disponibilidade de ligantes como ATP e adenosina aos seus receptores específicos. A Adenosina desaminase (ADA) pode promover a desaminação hidrolítica da adenosina em inosina...

Priming and inhibitory activities of RNAs for the influenza viral transcriptase do not require base pairing with the virion template RNA.

Krug, R M; Broni, B A; LaFiandra, A J; Morgan, M A; Shatkin, A J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1980 EN
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Capped ribopolymers lacking a sequence complementary to the common 3' end of the influenza virion RNA segments effectively stimulated transcription of these RNAs by the virion-associated transcriptase. Thus, stimulation of transcription results not from hydrogen bonding between the capped RNA and the 3' end of the virion RNA but presumably from a specific interaction of the capped RNA with protein(s) in the transcriptase complex. Although no specific nucleotide sequence was required for priming activity, capped mRNAs with diminished secondary structure were preferred as primers. Inosine-substituted or bisulfite-modified capped reovirus mRNAs were at least 3- to 5-fold more effective as primers than were the native capped mRNAs. On the other hand, inosine substitution or bisulfite treatment of the uncapped form of reovirus mRNAs converted them from essentially inactive species to potent inhibitors of the transcriptase reaction primed by either ApG or globin mRNA. These effects of reduced secondary structure also most probably reflect an interaction of the exogenous RNAs with transcriptase protein(s). The results obtained from screening a series of native uncapped ribopolymers were consistent with inhibitory activity requiring the absence of most hydrogen bonding in the ribopolymer and also suggested that specific structural feature(s) of the nucleotides in the chain were important.

Effects of pyrimidines on the guinea-pig coronary vasculature.

Vials, A. J.; Burnstock, G.
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
Publicado em /11/1993 EN
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1. The effects of the pyrimidines, uridine 5'-triphosphate (UTP), thymidine 5'-triphosphate (TTP) and cytidine 5'-triphosphate (CTP), were examined in the guinea-pig coronary bed, by use of a Langendorff technique. Comparisons were made with the actions of the purines adenosine 5'-triphosphate (ATP), inosine 5'-triphosphate (ITP) and guanosine 5'-triphosphate (GTP). The effect of, the nitric oxide synthase inhibitor, L-NG-nitroarginine methyl ester (L-NAME) and, the prostaglandin synthesis inhibitor, indomethacin on the vasodilator response to these purines and pyrimidines was examined. The effects of these inhibitors were assessed on their ability to inhibit both the amplitude and the area of the vasodilator response. 2. The relative order of potency of the purines and pyrimidines studied was ATP > UTP > ITP >> GTP, TTP, CTP. 3. The maximum amplitude and area of the vasodilator response to the pyrimidines, UTP (5 x 10(-10)-5 x 10(-7) mol), TTP (5 x 10(-8)-5 x 10(-7) mol) and CTP (5 x 10(-7) mol), and purines, ITP (5 x 10(-9)-5 x 10(-7) mol) and GTP (5 x 10(-8)-5 x 10(-7) mol), were significantly reduced by L-NAME (3 x 10(-5) and 10(-4) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of hypoxanthine transport in Neurospora crassa.

Sabina, R L; Magill, J M; Magill, C W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1976 EN
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Hypoxanthine uptake and hypoxanthine phosphoribosyltransferase activity (EC 2.4.2.8) were determined in germinated conidia from the adenine auxotrophic strains ad-1 and ad-8 and the double mutant strain ad-1 ad-8. The mutant strain ad-1 appears to lack aminoimidazolecarboximide ribonucleotide formyltransferase (EC 2.1.2.3) or inosine 5'monophosphate cyclohydrolase (EC 3.5.1.10) activities, or both, whereas the ad-8 strain lacks adenylosuccinate synthase activity (EC 6.3.4.4). Normal (or wild-type) hypoxanthine transport capacity was found to the ad-1 conidia, whereas the ad-8 strains failed to take up any hypoxanthine. The double mutant strains showed intermediate transport capacities. Similar results were obtained for hypoxanthine phosphoribosyl-transferase activity assayed in germinated conidia. The ad-1 strain showed greatest activity, the ad-8 strain showed the least activity, and the double mutant strain showed intermediate activity levels. Ion-exchange chromatography of the growth media revealed that in the presence of NH+/4, the ad-8 strain excreted hypoxanthine or inosine, the ad-1 strain did not excrete any purines, and the ad-1 ad-8 double mutant strain excreted uric acid. In the absence of NH+/4, none of the strains excreted any detectable purine compounds.

Comparison of the base pairing properties of a series of nitroazole nucleobase analogs in the oligodeoxyribonucleotide sequence 5'-d(CGCXAATTYGCG)-3'.

Bergstrom, D E; Zhang, P; Johnson, W T
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/05/1997 EN
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The nucleoside analogs 1-(2'-deoxy-beta-D-ribofuranosyl)- 3-nitropyrrole (9), 1-(2'-deoxy-beta-D-ribofuranosyl)-4-nitropyrazole (10), 1-(2'-deoxy-beta-D-ribofuranosyl)-4-nitroimidazole (11) and 1-(2'-deoxy-beta-D-ribofuranosyl)-5-nitroindole (21) were incorporated into the oligonucleotide 5'-d(CGCXAATTYGCG)-3'in the fourth position from the 5'-end. Procedures for synthesis of two of the nitroazole nucleosides, 10 and 11, were developed for this study. Each of the nitroazoles was converted into a 3'-phosphoramidite for oligonucleotide synthesis by conventional automated protocols. Four oligonucleotides were synthesized for each modified nucleoside in order to obtain duplexes in which each of the four natural bases was placed opposite (position 9) the nitroazole. In order to assess the role of the nitro group on base stacking interaction, sequences were also synthesized in which the fourth base was 1-(2'-deoxy-beta-D-ribofuranosyl)pyrazole. Corresponding sequences containing an abasic site, as well as sequences containing inosine, were synthesized for comparison. Thermal melting studies yielded T m values and thermodynamic parameters. Each nucleoside analog displayed a unique pattern of base pairing preferences. The least discriminating analog was 3-nitropyrrole...

Role of A1 receptors in renal sympathetic neurotransmission in the mouse kidney

Jackson, Edwin K.; Cheng, Dongmei; Mi, Zaichuan; Verrier, Jonathan D.; Janesko-Feldman, Keri; Kochanek, Patrick M.
Fonte: American Physiological Society Publicador: American Physiological Society
Tipo: Artigo de Revista Científica
EN
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A1 receptors may participate in renal sympathetic neurotransmission by enhancing the postjunctional effects of norepinephrine. The purpose of this study was to test this concept using A1 receptor knockout (A1AR−/−) mice. In isolated kidneys from nontransgenic mice perfused with Tyrode's solution at a constant rate, renal sympathetic nerve stimulation (RSNS) increased (P < 0.0001) renal venous perfusate levels of inosine (adenosine metabolite) from 23.9 ± 3.7 to 32.7 ± 5.1, 68.2 ± 12.4, and 94.0 ± 14.3 ng/ml at 3, 5, and 7 Hz, respectively (n = 28), suggesting frequency-dependent production of adenosine. Conversely, RSNS decreased (P < 0.0001) renal venous perfusate levels of 5′-AMP (adenosine precursor) from 1.4 ± 0.3 to 1.1 ± 0.3, 0.80 ± 0.2, and 0.6 ± 0.2 ng/ml at 3, 5, and 7 Hz, respectively (n = 28), suggesting frequency-dependent increased metabolism of 5′-AMP. In kidneys from nontransgenic mice, blockade of adenosine receptors with 1,3-dipropyl-8-p-sulfophenylxanthine attenuated (P = 0.0130) vasoconstrictor responses to RSNS at 3, 5, and 7 Hz [control (n = 29): 22 ± 4, 34 ± 6, 42 ± 6 mmHg, respectively; 1,3-dipropyl-8-p-sulfophenylxanthine-treated (n = 11): 6 ± 1, 12 ± 3, 15 ± 3 mmHg, respectively]. In A1AR−/− kidneys (n = 10)...

Demonstration of adenosine deaminase activity in human fibroblast lysosomes.

Lindley, E R; Pisoni, R L
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/03/1993 EN
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Human fibroblast lysosomes, purified on Percoll density gradients, contain an adenosine deaminase (ADA) activity that accounts for approximately 10% of the total ADA activity in GM0010A human fibroblasts. In assays of lysosomal ADA, the conversion of [3H]adenosine into [3H]inosine was proportional to incubation time and the amount of lysosomal material added to reaction mixtures. Maximal activity was observed between pH 7 and 8, and lysosomal ADA displayed a Km of 37 microM for adenosine at 25 degrees C and pH 5.5. Lysosomal ADA was completely inhibited by 2.5 mM Cu2+ or Hg2+ salts, but not by other bivalent cations (Ba2+, Cd2+, Ca2+, Fe2+, Mg2+, Mn2+ and Zn2+). Coformycin (2.5 mM), deoxycoformycin (0.02 mM), 2'-deoxyadenosine (2.5 mM), 6-methylaminopurine riboside (2.5 mM), 2'-3'-isopropylidene-adenosine (2.5 mM) and erythro-9-(2-hydroxy-3-nonyl)adenine (0.2 mM) inhibited lysosomal ADA by > 97%. In contrast, 2.5 mM S-adenosyl-L-homocysteine and cytosine were poor inhibitors. Nearly all lysosomal ADA activity is eluted as a high-molecular-mass protein (> 200 kDa) just after the void volume on a Sephacryl S-200 column, and is very heat-stable, retaining 70% of its activity after incubation at 65 degrees C for 80 min. We speculate that compartmentalization of ADA within lysosomes would allow deamination of adenosine to occur without competition by adenosine kinase...

Síntesis quimio-enzimática de nucleósidos naturales y modificados

Taverna Porro, Marisa Lía
Fonte: Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires Publicador: Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires
Tipo: info:eu-repo/semantics/doctoralThesis; tesis doctoral; info:eu-repo/semantics/publishedVersion Formato: application/pdf
Publicado em //2010 SPA
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Este trabajo de tesis presenta una estrategia versátil y económica para la síntesis de nucleósidos tanto naturales como modificados partiendo de azúcares naturales. La aproximación quimio-enzimática de la síntesis de nucleósidos que se exploró, se encuentra resumida en la Figura 1. En primer lugar, se desarrolló una estrategia general de obtención de furanosas-5- fosfato utilizando un esquema de protección química y desprotección selectiva con lipasas, cornbinado con una fosforilación quimica. De esta forrna se sintetizaron D-ribosa 5-fosfato, 2- desoxi-D-ribosa 5-fosfato y D-arabinosa 5-fosfato. Luego se exploró la reacción de glicosidación enzimática de nucleósidos mediante el acoplamiento de nucleósido fosforilasas (NPs) con la fosfopentomutasa (PPM). Esta última, al no ser una enzima comercial, debió ser sobreexpresada y purificada, y su actividad debió ser optimizada para ser utilizada en las reacciones de síntesis de nucleósidos. De esta forrna se han obtenido con altos rendimientos (70-100%) los nucleósidos: adenosina, inosina, 6- metcaptopurín ribonucleósido, 1,2,4-triazol-3-carboxamida ribonucleósido (Rivabirina), timidina, 2'-desoxiuridina, 5-fluoro-2'-desoxiuridina y 5-bromo-2'-desoxiuridina. Asimismo se han obtenido diversos nucleósidos con moderados rendimientos (30-50%)...

In Silico Investigation of Flavonoids as Potential Trypanosomal Nucleoside Hydrolase Inhibitors

Ha, Christina Hung Hung; Fatima, Ayesha; Gaurav, Anand
Fonte: Hindawi Publishing Corporation Publicador: Hindawi Publishing Corporation
Tipo: Artigo de Revista Científica
EN
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Human African Trypanosomiasis is endemic to 37 countries of sub-Saharan Africa. It is caused by two related species of Trypanosoma brucei. Current therapies suffer from resistance and public accessibility of expensive medicines. Finding safer and effective therapies of natural origin is being extensively explored worldwide. Pentamidine is the only available therapy for inhibiting the P2 adenosine transporter involved in the purine salvage pathway of the trypanosomatids. The objective of the present study is to use computational studies for the investigation of the probable trypanocidal mechanism of flavonoids. Docking experiments were carried out on eight flavonoids of varying level of hydroxylation, namely, flavone, 5-hydroxyflavone, 7-hydroxyflavone, chrysin, apigenin, kaempferol, fisetin, and quercetin. Using AutoDock 4.2, these compounds were tested for their affinity towards inosine-adenosine-guanosine nucleoside hydrolase and the inosine-guanosine nucleoside hydrolase, the major enzymes of the purine salvage pathway. Our results showed that all of the eight tested flavonoids showed high affinities for both hydrolases (lowest free binding energy ranging from −10.23 to −7.14 kcal/mol). These compounds, especially the hydroxylated derivatives...