Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 335
Filtrar
1.
Biomarkers ; 24(8): 739-749, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31617777

RESUMO

An improved amperometric creatinine biosensor was fabricated that dependent on covalent immobilisation of nanoparticles of creatininase (CANPs), creatinase (CINPs) and sarcosine oxidase (SOxNPs) onto gold electrode (AuE). The CANPs/CINPs/SOxNPs/AuE was characterised by scanning electron microscopy and cyclic voltammetry at various stages. The working electrode exhibited optimal response within 2 s at a potential of 0.6 V, against Ag/AgCl, pH 6.5 and 30 °C. A linear relationship was observed between creatinine concentration range, 0.1-200µM and biosensor response i.e. current in mA, under optimum conditions. Biosensor offered a low detection limit of 0.1 µM with long storage stability. Analytical recoveries of added creatinine in blood sera at 0.5 mM and at 1.0 mM concentrations, were 92.0% and 79.20% respectively. The precision i.e. within and between-batch coefficients of variation were 2.04% and 3.06% respectively. There was a good correlation (R2 = 0.99) between level of creatinine in sera, as calculated by the colorimetric method and present electrode. The CANPs/CINPs/SOxNPs/Au electrode was reused 200 times during the period of 180 days, with just 10% loss in its initial activity, while being stored at 4 °C, when not in use.HighlightsPrepared and characterised creatininase (CA), creatinase (CI) sarcosine oxidase (SOx) nanoparticles and immobilised them onto gold electrode (AuE) for fabrication of an improved amperometric creatinine biosensor.The biosensor displayed a limit of detection (LOD) of 0.1 µM with a linear working range of 0.1 µM-200 µM.The biosensor was evaluated and applied to measure elevated creatinine levels in sera from whom suffering from kidney and muscular disorders.The working electrode retained 90% of its initial activity, while being stored dry at 4 ˚C for 180 days.


Assuntos
Técnicas Biossensoriais/instrumentação , Creatinina/sangue , Ouro/metabolismo , Amidoidrolases/metabolismo , Técnicas Biossensoriais/normas , Eletrodos , Humanos , Nefropatias/diagnóstico , Limite de Detecção , Doenças Musculares/diagnóstico , Nanopartículas , Sarcosina Oxidase/metabolismo , Ureo-Hidrolases/metabolismo
2.
Int J Mol Sci ; 20(15)2019 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-31357701

RESUMO

The thermophilic fungus Humicola insolens produces cellulolytic enzymes that are of great scientific and commercial interest; however, few reports have focused on its cellulase expression regulation mechanism. In this study, we constructed a creA gene (carbon catabolite repressor gene) disruption mutant strain of H. insolens that exhibited a reduced radial growth rate and stouter hyphae compared to the wild-type (WT) strain. The creA disruption mutant also expressed elevated pNPCase (cellobiohydrolase activities), pNPGase (ß-glucosidase activities), and xylanase levels in non-inducing fermentation with glucose. Unlike other fungi, the H. insolens creA disruption mutant displayed lower FPase (filter paper activity), CMCase (carboxymethyl cellulose activity), pNPCase, and pNPGase activity than observed in the WT strain when fermentation was induced using Avicel, whereas its xylanase activity was higher than that of the parental strain. These results indicate that CreA acts as a crucial regulator of hyphal growth and is part of a unique cellulase expression regulation mechanism in H. insolens. These findings provide a new perspective to improve the understanding of carbon catabolite repression regulation mechanisms in cellulase expression, and enrich the knowledge of metabolism diversity and molecular regulation of carbon metabolism in thermophilic fungi.


Assuntos
Carbono/metabolismo , Repressão Catabólica/genética , Sordariales/enzimologia , Ureo-Hidrolases/genética , Carbono/química , Carboximetilcelulose Sódica/metabolismo , Celulase/química , Celulase/genética , Celulase/metabolismo , Celulose/farmacologia , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Fermentação , Regulação Fúngica da Expressão Gênica/genética , Glucose/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Mutação/genética , Sordariales/metabolismo , Ureo-Hidrolases/química , beta-Glucosidase/química , beta-Glucosidase/metabolismo
3.
Int J Psychiatry Clin Pract ; 23(2): 128-133, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31081413

RESUMO

Objectives: Agmatine is a cationic amine resulting from the decarboxylation of l-arginine. Agmatine has neuroprotective, anti-inflammatory, anti-stress, and anti-depressant properties. In this study, plasma agmatine, arginine decarboxylase, and agmatinase levels were measured during manic episode and remission period in patients with bipolar disorder. Methods: Thirty healthy volunteers and 30 patients who meet Bipolar Disorder Manic Episode diagnostic criteria were included in the study. Additionally, the changes in the patient group between manic episode and remission period were examined. We evaluated the relationship between levels of l-arginine and arginine decarboxylase in the agmatine synthesis pathway, and level of agmatinase that degrades agmatine. Results: Levels of agmatine and l-arginine were significantly increased than control group during manic episode (p < .01). All parameters were increased during manic episode compared to remission period (p < .05). Agmatinase was significantly decreased both during manic episode (p < .01) and remission period (p < .05) in comparison to the control group. Arginine decarboxylase levels did not show a significant difference between the groups (p > .05). Conclusions: This study indicate that there may be a relationship between bipolar disorder and agmatine and its metabolic pathway. Nonetheless, we believe more comprehensive studies are needed in order to reveal the role of agmatine in etiology of bipolar disorder. Key points Agmantine, agmatinase, l-arginine and arginine decarboxylase levels in BD have not been explored before. Various neuro-chemical mechanisms act to increase agmatine in BD; however, agmatine could have elevated to compensate agmatine deficit prior to the manifestation of the disease as in schizophrenia. Elevated agmatine degradation resulting from excess expression of agmatinase which is suggested to be effective in pathogenesis of mood disorders was compensated by this way. Elevated agmatine may be one of the causes which play a role in mania development. Elevated agmatine levels are also suggested to trigger psychosis and be related with the etiology of manic episode and lead to BD.


Assuntos
Agmatina/sangue , Transtorno Bipolar/sangue , Transtorno Bipolar/fisiopatologia , Carboxiliases/sangue , Redes e Vias Metabólicas , Ureo-Hidrolases/sangue , Adulto , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Indução de Remissão
4.
Microbiology ; 165(4): 396-410, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30806615

RESUMO

Arginase is the only fungal ureohydrolase that is well documented in the literature. More recently, a novel route for agmatine catabolism in Aspergillus niger involving another ureohydrolase, 4-guanidinobutyrase (GBase), was reported. We present here a detailed characterization of A. niger GBase - the first fungal (and eukaryotic) enzyme to be studied in detail. A. niger GBase is a homohexamer with a native molecular weight of 336 kDa and an optimal pH of 7.5. Unlike arginase, the Mn2+ enzyme from the same fungus, purified GBase protein is associated with Zn2+ ions. A sensitive fluorescence assay was used to determine its kinetic parameters. GBase acted 25 times more efficiently on 4-guanidinobutyrate (GB) than 3-guanidinopropionic acid (GP). The Km for GB was 2.7±0.4 mM, whereas for GP it was 53.7±0.8 mM. While GB was an efficient nitrogen source, A. niger grew very poorly on GP. Constitutive expression of GBase favoured fungal growth on GP, indicating that GP catabolism is limited by intracellular GBase levels in A. niger. The absence of a specific GPase and the inability of GP to induce GBase expression confine the fungal growth on GP. That GP is a poor substrate for GBase and a very poor nitrogen source for A. niger offers an opportunity to select GBase specificity mutations. Further, it is now possible to compare two distinct ureohydrolases, namely arginase and GBase, from the same organism.


Assuntos
Aspergillus niger/enzimologia , Butiratos/metabolismo , Proteínas Fúngicas/metabolismo , Guanidinas/metabolismo , Ureo-Hidrolases/metabolismo , Agmatina/metabolismo , Arginase/metabolismo , Aspergillus niger/genética , Aspergillus niger/metabolismo , Cátions/química , Meios de Cultura/química , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Expressão Gênica , Cinética , Peso Molecular , Mutação , Propionatos/metabolismo , Multimerização Proteica , Especificidade por Substrato , Ureo-Hidrolases/antagonistas & inibidores , Ureo-Hidrolases/química , Ureo-Hidrolases/genética
5.
Talanta ; 195: 62-68, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30625593

RESUMO

A new disposable microfluidic electrochemical paper-based device (ePAD) consisting of two spot sensors in the same working electrode for the simultaneous determination of uric acid and creatinine was developed. The spot 1 surface was modified with graphene quantum dots for direct uric acid oxidation and spot 2 surface modified with graphene quantum dots, creatininase and a ruthenium electrochemical mediator for creatinine oxidation. The ePAD was employed to construct an electrochemical sensor (based on square wave voltammetry analysis) for the simultaneous determination of uric acid and creatinine in the 0.010-3.0 µmol L-1 range. The device showed excellent analytical performance with a very low simultaneous detection limit of 8.4 nmol L-1 to uric acid and 3.7 nmol L-1 to creatinine and high selectivity. The ePAD was applied to the rapid and successful determination of those clinical biomarkers in human urine samples.


Assuntos
Creatinina/urina , Técnicas Eletroquímicas/instrumentação , Dispositivos Lab-On-A-Chip , Ácido Úrico/urina , Biomarcadores/química , Biomarcadores/urina , Creatinina/química , Eletrodos , Grafite/química , Humanos , Oxirredução , Papel , Pontos Quânticos/química , Rutênio/química , Ureo-Hidrolases/química , Ácido Úrico/química
7.
BMC Plant Biol ; 18(1): 287, 2018 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-30458716

RESUMO

BACKGROUND: The ureides allantoin and allantoate are major metabolic intermediates of purine catabolism with high nitrogen-to-carbon ratios. Ureides play a key role in nitrogen utilization in ureide-type legumes, but their effects on growth and development in non-legume plants are poorly understood. Here, we examined the effects of knocking out genes encoding ureide-degrading enzymes, allantoinase (ALN) and allantoate amidohydrolase (AAH), on the vegetative-to-reproductive transition and subsequent growth of Arabidopsis plants. RESULTS: The ureide-degradation mutants (aln and aah) showed symptoms similar to those of nitrogen deficiency: early flowering, reduced size at maturity, and decreased fertility. Consistent with these phenotypes, carbon-to-nitrogen ratios and nitrogen-use efficiencies were significantly decreased in ureide-degradation mutants; however, adding nitrogen to irrigation water did not alleviate the reduced growth of these mutants. In addition to nitrogen status, levels of indole-3-acetic acid and gibberellin in five-week-old plants were also affected by the aln mutations. To test the possibility that ureides are remobilized from source to sink organs, we measured ureide levels in various organs. In wild-type plants, allantoate accumulated predominantly in inflorescence stems and siliques; this accumulation was augmented by disruption of its catabolism. Mutants lacking ureide transporters, ureide permeases 1 and 2 (UPS1 and UPS2), exhibited phenotypes similar to those of the ureide-degradation mutants, but had decreased allantoate levels in the reproductive organs. Transcript analysis in wild-type plants suggested that genes involved in allantoate synthesis and ureide transport were coordinately upregulated in senescing leaves. CONCLUSIONS: This study demonstrates that ureide degradation plays an important role in supporting healthy growth and development in non-legume Arabidopsis during and after transition from vegetative to reproductive stages.


Assuntos
Alantoína/metabolismo , Arabidopsis/metabolismo , Amidoidrolases/genética , Amidoidrolases/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Mutação , Nitrogênio/metabolismo , Ureo-Hidrolases/genética , Ureo-Hidrolases/metabolismo
8.
Plant Physiol ; 178(3): 1027-1044, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30190419

RESUMO

The nitrogen (N)-rich ureides allantoin and allantoate, which are products of purine catabolism, play a role in N delivery in Leguminosae. Here, we examined their role as an N source in nonlegume plants using Arabidopsis (Arabidopsis thaliana) plants mutated in XANTHINE DEHYDROGENASE1 (AtXDH1), a catalytic bottleneck in purine catabolism. Older leaves of the Atxdh1 mutant exhibited early senescence, lower soluble protein, and lower organic N levels as compared with wild-type older leaves when grown with 1 mm nitrate but were comparable to the wild type under 5 mm nitrate. Similar nitrate-dependent senescence phenotypes were evident in the older leaves of allantoinase (Ataln) and allantoate amidohydrolase (Ataah) mutants, which also are impaired in purine catabolism. Under low-nitrate conditions, xanthine accumulated in older leaves of Atxdh1, whereas allantoin accumulated in both older and younger leaves of Ataln but not in wild-type leaves, indicating the remobilization of xanthine-degraded products from older to younger leaves. Supporting this notion, ureide transporter expression was enhanced in older leaves of the wild type in low-nitrate as compared with high-nitrate conditions. Elevated transcripts and proteins of AtXDH and AtAAH were detected in low-nitrate-grown wild-type plants, indicating regulation at protein and transcript levels. The higher nitrate reductase activity in Atxdh1 leaves compared with wild-type leaves indicated a need for nitrate assimilation products. Together, these results indicate that the absence of remobilized purine-degraded N from older leaves of Atxdh1 caused senescence symptoms, a result of higher chloroplastic protein degradation in older leaves of low-nitrate-grown plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Nitratos/metabolismo , Nitrogênio/metabolismo , Purinas/metabolismo , Xantina Desidrogenase/metabolismo , Alantoína/metabolismo , Amidoidrolases/genética , Amidoidrolases/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Mutação , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/fisiologia , Fatores de Tempo , Ureo-Hidrolases/genética , Ureo-Hidrolases/metabolismo , Xantina Desidrogenase/genética
9.
Transl Psychiatry ; 8(1): 201, 2018 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-30250120

RESUMO

The dorsal diencephalic conduction system connects limbic forebrain structures to monaminergic mesencephalic nuclei via a distinct relay station, the habenular complexes. Both habenular nuclei, the lateral as well as the medial nucleus, are considered to play a prominent role in mental disorders like major depression. Herein, we investigate the effect of the polyamine agmatine on the electrical activity of neurons within the medial habenula in rat. We present evidence that agmatine strongly decreases spontaneous action potential firing of medial habenular neurons by activating I1-type imidazoline receptors. Additionally, we compare the expression patterns of agmatinase, an enzyme capable of inactivating agmatine, in rat and human habenula. In the medial habenula of both species, agmatinase is similarly distributed and observed in neurons and, in particular, in distinct neuropil areas. The putative relevance of these findings in the context of depression is discussed. It is concluded that increased activity of the agmatinergic system in the medial habenula may strengthen midbrain dopaminergic activity. Consequently, the habenular-interpeduncular axis may be dysregulated in patients with major depression.


Assuntos
Agmatina/farmacologia , Depressão/fisiopatologia , Habenula/efeitos dos fármacos , Habenula/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Benzofuranos/farmacologia , Depressão/prevenção & controle , Feminino , Humanos , Idazoxano/farmacologia , Imidazóis/farmacologia , Receptores de Imidazolinas/agonistas , Receptores de Imidazolinas/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Ratos Wistar , Ureo-Hidrolases/metabolismo
10.
Mol Microbiol ; 109(6): 763-780, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29923645

RESUMO

Polyamines are primordial, small organic polycations present in almost all cells, but their roles in bacteria are poorly understood. sym-Homospermidine is the dominant polyamine in the filamentous, N2 -fixing, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Synthesis of homospermidine was dependent on speA (encoding arginine decarboxylase), speB (agmatinase) and speY (deoxyhypusine synthase homologue), which in bacteria is an unprecedented pathway. Inactivation of any of these genes impaired diazotrophic growth. Heterocyst differentiation in the speA mutant was blocked at an early step, after induction of the regulatory gene hetR but before production of heterocyst-specific glycolipids (HGL). In contrast, the speY mutant produced HGL and showed slow diazotrophic growth. Analysis of fusions to green fluorescent protein revealed that SpeA (like SpeB previously described) accumulates at higher levels in vegetative cells than in heterocysts, and that SpeY accumulates in vegetative cells but also at significant levels in heterocysts. The homospermidine biosynthetic pathway is therefore active primarily in vegetative cells but the last step can be completed in heterocysts. Our findings indicate an important role for polyamines in the diazotrophic biology of Anabaena. Furthermore, inactivation of a gene cluster (potADB) encoding a polyamine ABC transporter disrupted diazotrophic growth, corroborating the importance of polyamine homeostasis in Anabaena.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Anabaena/metabolismo , Carboxiliases/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Espermidina/análogos & derivados , Espermidina/biossíntese , Ureo-Hidrolases/genética , Anabaena/crescimento & desenvolvimento , Carboxiliases/metabolismo , Fixação de Nitrogênio/fisiologia , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Ureo-Hidrolases/metabolismo
11.
Reprod Toxicol ; 78: 90-96, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29635046

RESUMO

This study evaluated the effects of bisphenol A (BPA) on proliferation of ovine trophectoderm (oTr1) cells, as well as expression of genes for transport of arginine and synthesis of polyamines. BPA reduced proliferation of oTr1 cells at concentrations of 1 × 10-6, 1 × 10-5, 1 × 10-4 M compared to concentrations of 0, 1 × 10-9, and 1 × 10-8 M at 24 and 96 h of culture. Lower concentrations of BPA significantly increased expression of mRNAs for agmatinase (AGMAT), arginine decarboxylase (ADC), ornithine decarboxylase (ODC1) and solute carrier family 7 member 1 (SLC7A1). Similarly, synthesis of polyamines by oTr1 cells was greatest at lower concentrations of BPA and decreased as the dose of BPA increased. Expression of mRNAs for interferon tau (IFNT) and insulin-like growth factor 2 (IGF2) by oTr1 cells was greater than for controls at 1 × 10-9 M BPA. Overall, the effects of BPA on proliferation and gene expression by oTr1 cells were highly dose-dependent.


Assuntos
Compostos Benzidrílicos/toxicidade , Disruptores Endócrinos/toxicidade , Fenóis/toxicidade , Trofoblastos/efeitos dos fármacos , Animais , Arginina/metabolismo , Carboxiliases/genética , Transportador 1 de Aminoácidos Catiônicos/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento Insulin-Like II/genética , Interferon Tipo I/genética , Ornitina Descarboxilase/genética , Poliaminas/metabolismo , Proteínas da Gravidez/genética , RNA Mensageiro/metabolismo , Ovinos , Trofoblastos/metabolismo , Ureo-Hidrolases/genética
12.
Fungal Genet Biol ; 115: 41-51, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29655909

RESUMO

Carbon catabolite repression (CCR) is a very important mechanism employed in the utilization of carbon as an energy source, required for the regulation of growth, development and secondary metabolite production in fungi. Despite the wide study of this mechanism in fungi, little is known about the major CCR gene creA in A. flavus. Hence, we report identification of A. flavus carbon catabolite repression gene creA, which is responsible for the repression of secondary carbon sources. Gene deletion and over-expression was employed to explicate the role of creA in the morphology, pathogenicity, and secondary metabolite production in A. flavus. We investigated these factors using three carbon sources including glucose, sucrose and maltose. Gene deletion mutant (ΔcreA) had a significant growth defect on complete medium and minimal medium containing maltose. Conidia production in ΔcreA was significantly impaired irrespective of the carbon source available, while sclerotia production was significantly increased, compared to wild type (WT) and over-expression strain (OE::creA). Importantly, ΔcreA produced insignificant amount of aflatoxin in complete medium, and its ability to colonize hosts was also impaired. Concisely, we showed that creA played an important role in the morphology, pathogenicity and secondary metabolite production of A. flavus.


Assuntos
Aflatoxinas/biossíntese , Aspergillus flavus/genética , Repressão Catabólica/genética , Ureo-Hidrolases/genética , Aflatoxinas/genética , Aspergillus flavus/patogenicidade , Proteínas Fúngicas/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Virulência/genética
13.
Transpl Immunol ; 46: 23-28, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29157597

RESUMO

The virtual crossmatch, which is based on single antigen bead technology, is used in the prediction of crossmatch results. However, this assay differs in sensitivity and specificity from crossmatch methods. In our study, the results of physical crossmatches, performed with three different methods, were assessed against virtual crossmatch results. The aim was to determine the potential cut-off values for donor specific antibodies (DSA) that would predict the crossmatch results obtained by different methods. The results of different crossmatch techniques were correlated with the virtual crossmatch. The receiver operating characteristic (ROC) analysis revealed the Flow cytometric crossmatch (FCXM) and Luminex crossmatch (LXM) to be the most accurate, with area under curve (AUC) values of 0.861 and 0.805, respectively. While we found that the virtual crossmatch correlated well with all the crossmatch results, FCXM produced the best results (83% of the DSA detected). LXM outperformed the other tests in terms of the accuracy in separating class II DSA.


Assuntos
Tipagem e Reações Cruzadas Sanguíneas/métodos , Rejeição de Enxerto/diagnóstico , Antígenos HLA/imunologia , Transplante de Rim , Ureo-Hidrolases/sangue , Citometria de Fluxo , Rejeição de Enxerto/prevenção & controle , Teste de Histocompatibilidade , Humanos , Microesferas , Valor Preditivo dos Testes , Prognóstico , Curva ROC , Sensibilidade e Especificidade , Doadores de Tecidos
14.
Metabolism ; 81: 35-44, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29162499

RESUMO

Agmatine (1-amino-4-guanidinobutane), a precursor for polyamine biosynthesis, has been identified as an important neuromodulator with anticonvulsant, antineurotoxic and antidepressant actions in the brain. In this context it has emerged as an important mediator of addiction/satiety pathways associated with alcohol misuse. Consequently, the regulation of the activity of key enzymes in agmatine metabolism is an attractive strategy to combat alcoholism and related addiction disorders. Agmatine results from the decarboxylation of L-arginine in a reaction catalyzed by arginine decarboxylase (ADC), and can be converted to either guanidine butyraldehyde by diamine oxidase (DAO) or putrescine and urea by the enzyme agmatinase (AGM) or the more recently identified AGM-like protein (ALP). In rat brain, agmatine, AGM and ALP are predominantly localised in areas associated with roles in appetitive and craving (drug-reinstatement) behaviors. Thus, inhibitors of AGM or ALP are promising agents for the treatment of addictions. In this review, the properties of DAO, AGM and ALP are discussed with a view to their role in the agmatine metabolism in mammals.


Assuntos
Agmatina/metabolismo , Neurotransmissores/metabolismo , Amina Oxidase (contendo Cobre)/fisiologia , Animais , Carboxiliases/fisiologia , Humanos , Ureo-Hidrolases/fisiologia
15.
Mol Biotechnol ; 60(1): 55-61, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29214500

RESUMO

The role of the transcription factor creA-mediating carbon catabolite repression in Trichoderma orientalis EU7-22 was investigated for cellulase and hemicellulase production. The binary vector pUR5750G/creA::hph was constructed to knock out creA by homologous integration, generating the ΔcreA mutant Trichoderma orientalis CF1D. For strain CF1D, the filter paper activities (FPA), endoglucanase activities (CMC), cellobiohydrolase activity(CBH), ß-glucosidase activity (BG), xylanase activity (XYN), and extracellular protein concentration were 1.45-, 1.15-, 1.71-, 2.51-, 2.72, and 1.95-fold higher in inducing medium and were 6.41-, 7.50-, 10.27-, 11.79-, 9.25-, and 3.77-fold higher in glucose repressing medium, respectively, than those in the parent strain after 4 days. SDS-PAGE demonstrated that the extracellular proteins were largely secreted in the mutant CF1D. Quantitative reverse-transcription polymerase chain reaction indicated that the expressions of cbh1, cbh2, eg1, eg2, bgl1, xyn1, and xyn2 were significantly increasing for the mutant CF1D not only in the inducing medium but also in the repressing medium. Those results indicated that creA was a valid target gene in strain engineering for improved enzyme production in T. orientalis.


Assuntos
Celulase/metabolismo , Glicosídeo Hidrolases/metabolismo , Trichoderma/genética , Trichoderma/metabolismo , Ureo-Hidrolases/genética , Deleção de Genes , Regulação Enzimológica da Expressão Gênica , Técnicas de Introdução de Genes , Engenharia Genética/métodos , Glucose/metabolismo , Mutação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Trichoderma/enzimologia , Ureo-Hidrolases/metabolismo
16.
Methods Mol Biol ; 1694: 163-172, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29080167

RESUMO

The enzyme 4-guanidinobutyrase (GBase) catalyzes the hydrolysis of 4-guanidinobutyric acid (GB) to 4-aminobutyric acid (GABA) and urea. Here we describe methods to estimate urea and GABA that were suitably adapted from the published literature. The urea is determined by colorimetric assay using modified Archibald's method. However, the low sensitivity of this method often renders it impractical to perform fine kinetic analysis. To overcome this limitation, a high sensitive method for detecting GABA is exploited that can even detect 1 µM of GABA in the assay mixture. The samples are deproteinized by perchloric acid (PCA) and potassium hydroxide treatment prior to HPLC analysis of GABA. The method involves a pre-column derivatization with o-phthalaldehyde (OPA) in combination with the thiol 3-mercaptopropionic acid (MPA). The fluorescent GABA derivative is then detected after reversed phase high performance liquid chromatography (RP-HPLC) using isocratic elution. The protocols described here are broadly applicable to other biological samples involving urea and GABA as metabolites.


Assuntos
Agmatina/metabolismo , Aspergillus niger/metabolismo , Agmatina/química , Bioensaio , Cromatografia Líquida de Alta Pressão , Ureia/química , Ureo-Hidrolases/química , Ureo-Hidrolases/metabolismo , Ácido gama-Aminobutírico/química , Ácido gama-Aminobutírico/metabolismo , o-Ftalaldeído/química , o-Ftalaldeído/metabolismo
17.
Amino Acids ; 50(2): 293-308, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29196820

RESUMO

This study investigated the effect of agmatine (Agm) in proliferation of ovine trophecdoderm cells (oTr1) as well as the importance of the arginine decarboxylase (ADC) and agmatinase (AGMAT) alternative pathway for synthesis of polyamines in ovine conceptuses during the peri-implantation period of pregnancy. Morpholino antisense oligonucleotides (MAOs) were used to inhibit translation of mRNAs for ODC1 alone, AGMAT alone, and their combination. Rambouillet ewes (N = 50) were assigned randomly to the following treatments on Day 8 of pregnancy: MAO control (n = 10); MAO-ODC1 (n = 8); MAO-ADC (n = 6); MAO-ODC1:MAO-ADC (n = 9); or MAO-ODC1:MAO-AGMAT (n = 9). Ewes were ovario-hysterectomized on Day 16 of pregnancy to obtain uterine flushings, uterine endometrium, and conceptus tissues. Inhibition of translation of both ODC1 and AGMAT resulted in 22% of ewes having morphologically and functionally normal (elongated and healthy) conceptuses designated MAO-ODC1:MAO-AGMAT (A). But, 78% of the MAO-ODC1:MAO-AGMAT ewes had morphologically and functionally abnormal (not elongated and fragmented) conceptuses designated MAO-ODC1:MAO-AGMAT (B). The pregnancy rate was less (22%; P < 0.05) for MAO-ODC1:MAO-AGMAT ewes than for MAO-control (80%), MAO-ODC1 (75%), MAO-ADC (84%), and MAO-ODC1:MAO-ADC (44%) ewes. Moreover, inhibition of translational of both ODC1 and AGMAT mRNAs increased expression of ADC, SLC22A1, SLC22A2, and SLC22A3 mRNAs, as well as abundances of agmatine, putrescine, spermindine, and spermine in conceptus tissue. However, MAO-ODC1:AGMAT(B) ewes had greater abundances of agmatine, putrescine, and spermidine and reduced amounts of spermine in uterine flushes. Thus, in vivo knockdown of translation of ODC1 and AGMAT mRNAs increased expression of genes for the synthesis and transport of polyamines in ovine conceptuses during the peri-implantation period of pregnancy.


Assuntos
Agmatina/metabolismo , Implantação do Embrião/fisiologia , Desenvolvimento Embrionário/fisiologia , Poliaminas/metabolismo , Prenhez/fisiologia , Ovinos , Ureo-Hidrolases/metabolismo , Agmatina/análise , Agmatina/farmacologia , Sistemas de Transporte de Aminoácidos Básicos/genética , Animais , Carboxiliases/genética , Carboxiliases/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Interferon Tipo I/genética , Modelos Animais , Oligonucleotídeos Antissenso , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/metabolismo , Poliaminas/análise , Gravidez , Proteínas da Gravidez/genética , Somatomedinas/genética , Ureo-Hidrolases/genética
18.
Anal Biochem ; 537: 41-49, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-28870830

RESUMO

An improved amperometric biosensor for detection of creatinine was developed based on immobilization of nanoparticles (NPs) of creatininase (CA), creatinase (CI), and sarcosine oxidase (SOx) onto glassy carbon (GC) electrode. Transmission electron microscopy (TEM) and fourier transform infrared spectroscopy (FTIR) were employed for characterization of enzyme nanoparticles (ENPs). The GC electrode was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectra (EIS) at different stages of its amendment. The biosensor showed optimum response within 2s at pH 6.0 in 0.1 M sodium phosphate buffer and 25 °C, when operated at 1.0 V against Ag/AgCl. Biosensor exhibited wider linear range from 0.01 µM to 12 µM with a limit of detection (LOD) of 0.01 µM. The analytical recoveries of added creatinine in sera were 97.97 ± 0.1% for 0.1 mM and 98.76 ± 0.2% for 0.15 mM, within and between batch coefficients of variation (CV) were 2.06% and 3.09% respectively. A good correlation (R2 = 0.99) was observed between sera creatinine values obtained by standard enzymic colorimetric method and the present biosensor. This biosensor measured creatinine level in sera of apparently healthy subjects and persons suffering from renal and muscular dysfunction. The ENPs electrode lost 10% of its initial activity within 240 days of its regular uses, when stored at 4 °C.


Assuntos
Amidoidrolases/metabolismo , Técnicas Biossensoriais/instrumentação , Creatinina/sangue , Técnicas Eletroquímicas/instrumentação , Nanopartículas Metálicas/química , Sarcosina Oxidase/metabolismo , Ureo-Hidrolases/metabolismo , Amidoidrolases/química , Ácido Ascórbico/química , Espectroscopia Dielétrica , Eletrodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Ouro/química , Humanos , Limite de Detecção , Microscopia Eletrônica de Varredura , Sarcosina Oxidase/química , Ureo-Hidrolases/química , Ácido Úrico/química
19.
Physiol Behav ; 171: 207-215, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28093218

RESUMO

Classical music has been shown to reduce stress in kennelled dogs; however, rapid habituation of dogs to this form of auditory enrichment has also been demonstrated. The current study investigated the physiological and behavioural response of kennelled dogs (n=38) to medium-term (5days) auditory enrichment with five different genres of music including Soft Rock, Motown, Pop, Reggae and Classical, to determine whether increasing the variety of auditory stimulation reduces the level of habituation to auditory enrichment. Dogs were found to spend significantly more time lying and significantly less time standing when music was played, regardless of genre. There was no observable effect of music on barking, however, dogs were significantly (z=2.2, P<0.05) more likely to bark following cessation of auditory enrichment. Heart Rate Variability (HRV) was significantly higher, indicative of decreased stress, when dogs were played Soft Rock and Reggae, with a lesser effect observed when Motown, Pop and Classical genres were played. Relative to the silent period prior to auditory enrichment, urinary cortisol:creatanine (UCCR) values were significantly higher during Soft Rock (t=2.781, P<0.01) and the second silent control period following auditory enrichment (t=2.46, P<0.05). Despite the mixed response to different genres, the physiological and behavioural changes observed remained constant over the 5d of enrichment suggesting that the effect of habituation may be reduced by increasing the variety of auditory enrichment provided.


Assuntos
Comportamento Animal/fisiologia , Musicoterapia/métodos , Música , Nível de Percepção Sonora/fisiologia , Estresse Psicológico/reabilitação , Estimulação Acústica , Animais , Estudos de Coortes , Cães , Feminino , Frequência Cardíaca/fisiologia , Hidrocortisona/urina , Masculino , Fatores de Tempo , Ureo-Hidrolases/urina
20.
J Biol Chem ; 292(10): 4371-4381, 2017 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-28077628

RESUMO

Most ribosomally synthesized and post-translationally modified peptide (RiPP) natural products are processed by tailoring enzymes to create complex natural products that are still recognizably peptide-based. However, some tailoring enzymes dismantle the peptide en route to synthesis of small molecules. A small molecule natural product of as yet unknown structure, mycofactocin, is thought to be synthesized in this way via the mft gene cluster found in many strains of mycobacteria. This cluster harbors at least six genes, which appear to be conserved across species. We have previously shown that one enzyme from this cluster, MftC, catalyzes the oxidative decarboxylation of the C-terminal Tyr of the substrate peptide MftA in a reaction that requires the MftB protein. Herein we show that mftE encodes a creatininase homolog that catalyzes cleavage of the oxidatively decarboxylated MftA peptide to liberate its final two residues, including the C-terminal decarboxylated Tyr (VY*). Unlike MftC, which requires MftB for function, MftE catalyzes the cleavage reaction in the absence of MftB. The identification of this novel metabolite, VY*, supports the notion that the mft cluster is involved in generating a small molecule from the MftA peptide. The ability to produce VY* from MftA by in vitro reconstitution of the activities of MftB, MftC, and MftE sets the stage for identification of the novel metabolite that results from the proteins encoded by the mft cluster.


Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/metabolismo , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Ribossomos/metabolismo , Ureo-Hidrolases/metabolismo , Sequência de Aminoácidos , Catálise , Cristalografia por Raios X , Mycobacterium smegmatis/crescimento & desenvolvimento , Oxirredução , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA