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1.
Food Chem ; 368: 130841, 2022 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-34425335

RESUMO

This study aimed to optimize an ion-exchange chromatography method for an accurate quantification of phosphates. Coelution of nucleotides was detected following the quantification of small amounts of di- and trimetaphosphates in hake fillets. In some samples, triphosphates were added to the glaze ice, therefore, defrosting waters (glaze ice + drip loss of fillets) should be analyzed for an effective assessment of the use of phosphates in seafood. Even low amounts of adenosine 5'-diphosphate (ADP) and inosine 5'-monophosphate (IMP) triggered false positive results for P2O74- and P3O93-. Keeping the initial isocratic conditions for longer was crucial for the separation of ADP from P2O74- and of IMP from P3O93-. The improved method showed that in general, hake fillets did not have diphosphates, and trimetaphosphates were residual. Triphosphates were clearly added in only four defrosting waters, and in most of the remaining ones, diphosphates and trimetaphosphates contents corresponded to false positive results.


Assuntos
Gadiformes , Fosfatos , Nucleotídeos de Adenina , Difosfato de Adenosina , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Difosfatos
2.
Cell Mol Life Sci ; 79(1): 51, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34910247

RESUMO

Bacteriophage genomes are the richest source of modified nucleobases of any life form. Of these, 2,6 diaminopurine, which pairs with thymine by forming three hydrogen bonds violates Watson and Crick's base pairing. 2,6 diaminopurine initially found in the cyanophage S-2L is more widespread than expected and has also been detected in phage infecting Gram-negative and Gram-positive bacteria. The biosynthetic pathway for aminoadenine containing DNA as well as the exclusion of adenine are now elucidated. This example of a natural deviation from the genetic code represents only one of the possibilities explored by nature and provides a proof of concept for the synthetic biology of non-canonical nucleic acids.


Assuntos
Nucleotídeos de Adenina/química , Adenina/química , Bacteriófagos/genética , DNA Viral/química , Genoma Viral , Pareamento de Bases , Ligação de Hidrogênio
3.
Molecules ; 26(20)2021 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-34684903

RESUMO

A new HPLC method for the simultaneous quantitative analysis of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) was developed and validated. ATP, ADP, and AMP were extracted from human bronchial epithelial cells with a rapid extraction procedure and separated with a C18 column (3 × 150 mm, 2.7 µm) using isocratic elution with a mobile phase consisting of 50 mM of potassium hydrogen phosphate (pH 6.80). The absorbance was monitored at 254 nm. The calibration curves were linear in 0.2 to 10 µM, selective, precise, and accurate. This method allowed us to quantify the nucleotides from two cell models: differentiated NHBE primary cells grown at the air-liquid interface (ALI) and BEAS-2B cell line. Our study highlighted the development of a sensitive, simple, and green analytical method that is faster and less expensive than other existing methods to measure ATP, ADP, and AMP and can be carried out on 2D and 3D cell models.


Assuntos
Nucleotídeos de Adenina/metabolismo , Brônquios/metabolismo , Células Epiteliais/metabolismo , Linhagem Celular , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Indicadores e Reagentes/metabolismo
4.
Sheng Li Xue Bao ; 73(5): 707-722, 2021 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-34708228

RESUMO

Glucose and lipid metabolism is the most fundamental metabolic activity of higher organisms. This process is affected by both genetic polymorphisms and environmental factors. Excessive uptake and accumulation of lipids lead to obesity and disorder of glucose metabolic homeostasis characterized by insulin resistance and hyperglycemia, suggesting that the cross-regulation between lipid and glucose metabolism happens precisely at organ, cellular and molecular levels by known mechanisms. Adenine nucleotides and their metabolites have emerged as mediators in the mutual regulation of glucose and lipid metabolism. This review summarizes the roles of purinergic signaling induced by fatty acids in glucose metabolism and the development of type 2 diabetes.


Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Nucleotídeos de Adenina , Glucose , Homeostase , Humanos , Metabolismo dos Lipídeos
5.
ACS Chem Biol ; 16(9): 1680-1691, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34477366

RESUMO

While alarmone nucleotides guanosine-3',5'-bisdiphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3',5'-bisdiphosphate) and pppApp (adenosine-5'-triphosphate-3'-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNuNpp analogues starting from 3'-azido-3'-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppGNpp as a molecular tool, we show that (i) as an HD substrate mimic, ppGNpp competes with ppGpp to inhibit the enzymatic activity of human MESH1 Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppGNpp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and RelA. Finally, by solving the structure of the N-terminal domain region (NTD) of T. thermophilus Rel complexed with pppGNpp, we show that as an HD substrate mimic, the analogue serves as a bona fide orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate.


Assuntos
Nucleotídeos de Adenina/metabolismo , Proteínas de Bactérias/metabolismo , Ligases/metabolismo , Nucleotídeos de Adenina/síntese química , Sítio Alostérico , Bacillus subtilis , Desoxirribonucleotídeos , Escherichia coli , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Ligação Proteica , Conformação Proteica , Pirofosfatases/metabolismo
6.
mBio ; 12(4): e0178121, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34372695

RESUMO

The 2',5'-oligoadenylate (2-5A)-dependent endoribonuclease, RNase L, is a principal mediator of the interferon (IFN) antiviral response. Therefore, the regulation of cellular levels of 2-5A is a key point of control in antiviral innate immunity. Cellular 2-5A levels are determined by IFN-inducible 2',5'-oligoadenylate synthetases (OASs) and by enzymes that degrade 2-5A. Importantly, many coronaviruses (CoVs) and rotaviruses encode 2-5A-degrading enzymes, thereby antagonizing RNase L and its antiviral effects. A-kinase-anchoring protein 7 (AKAP7), a mammalian counterpart, could possibly limit tissue damage from excessive or prolonged RNase L activation during viral infections or from self-double-stranded RNAs that activate OAS. We show that these enzymes, members of the two-histidine phosphoesterase (2H-PE) superfamily, constitute a subfamily referred here as 2',5'-PEs. 2',5'-PEs from the mouse CoV mouse hepatitis virus (MHV) (NS2), Middle East respiratory syndrome coronavirus (MERS-CoV) (NS4b), group A rotavirus (VP3), and mouse (AKAP7) were investigated for their evolutionary relationships and activities. While there was no activity against 3',5'-oligoribonucleotides, they all cleaved 2',5'-oligoadenylates efficiently but with variable activity against other 2',5'-oligonucleotides. The 2',5'-PEs are shown to be metal ion-independent enzymes that cleave trimer 2-5A (2',5'-p3A3) producing mono- or diadenylates with 2',3'-cyclic phosphate termini. Our results suggest that the elimination of 2-5A might be the sole function of viral 2',5'-PEs, thereby promoting viral escape from innate immunity by preventing or limiting the activation of RNase L. IMPORTANCE Viruses often encode accessory proteins that antagonize the host antiviral immune response. Here, we probed the evolutionary relationships and biochemical activities of two-histidine phosphoesterases (2H-PEs) that allow some coronaviruses and rotaviruses to counteract antiviral innate immunity. In addition, we investigated the mammalian enzyme AKAP7, which has homology and shared activities with the viral enzymes and might reduce self-injury. These viral and host enzymes, which we refer to as 2',5'-PEs, specifically degrade 2',5'-oligoadenylate activators of the antiviral enzyme RNase L. We show that the host and viral enzymes are metal ion independent and exclusively cleave 2',5'- and not 3',5'-phosphodiester bonds, producing cleavage products with cyclic 2',3'-phosphate termini. Our study defines 2',5'-PEs as enzymes that share characteristic conserved features with the 2H-PE superfamily but have specific and distinct biochemical cleavage activities. These findings may eventually lead to pharmacological strategies for developing antiviral drugs against coronaviruses, rotaviruses, and other viruses.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Nucleotídeos de Adenina/metabolismo , Endorribonucleases/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Vírus da Hepatite Murina/enzimologia , Oligorribonucleotídeos/metabolismo , Rotavirus/enzimologia , Animais , Humanos , Imunidade Inata/imunologia , Interferons/imunologia , Camundongos
7.
Nat Commun ; 12(1): 5033, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34413302

RESUMO

Characteristic properties of type III CRISPR-Cas systems include recognition of target RNA and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of the target RNA and production of cyclic oligoadenylate (cOA) molecules. Here we report that an exposed seed region at the 3' end of the crRNA is essential for target RNA binding and cleavage, whereas cOA production requires base pairing at the 5' end of the crRNA. Moreover, we uncover that the variation in the size and composition of type III complexes within a single host results in variable seed regions. This may prevent escape by invading genetic elements, while controlling cOA production tightly to prevent unnecessary damage to the host. Lastly, we use these findings to develop a new diagnostic tool, SCOPE, for the specific detection of SARS-CoV-2 from human nasal swab samples, revealing sensitivities in the atto-molar range.


Assuntos
Nucleotídeos de Adenina/química , COVID-19/diagnóstico , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Oligorribonucleotídeos/química , RNA Bacteriano/genética , Ribonucleases/metabolismo , SARS-CoV-2/genética , COVID-19/genética , COVID-19/metabolismo , COVID-19/virologia , Testes Diagnósticos de Rotina/métodos , Humanos , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/patogenicidade
8.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445322

RESUMO

The hydrolysis of nucleotides is of paramount importance as an energy source for cellular processes. In addition, the transfer of phosphates from nucleotides onto proteins is important as a post-translational protein modification. Monitoring the enzymatic turnover of nucleotides therefore offers great potential as a tool to follow enzymatic activity. While a number of fluorescence sensors are known, so far, there are no methods available for the real-time monitoring of ATP hydrolysis inside live cells. We present the synthesis and application of a novel fluorogenic adenosine 5'-tetraphosphate (Ap4) analog suited for this task. Upon enzymatic hydrolysis, the molecule displays an increase in fluorescence intensity, which provides a readout of its turnover. We demonstrate how this can be used for monitoring cellular processes involving Ap4 hydrolysis. To this end, we visualized the enzymatic activity in live cells using confocal fluorescence microscopy of the Ap4 analog. Our results demonstrate that the Ap4 analog is hydrolyzed in lysosomes. We show that this approach is suited to visualize the lysosome distribution profiles within the live cell and discuss how it can be employed to gather information regarding autophagic flux.


Assuntos
Nucleotídeos de Adenina/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Células HEK293 , Células HeLa , Humanos , Hidrólise
9.
Sci Rep ; 11(1): 13317, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172767

RESUMO

All medically important unicellular protozoans cannot synthesize purines de novo and they entirely rely on the purine salvage pathway (PSP) for their nucleotide generation. Therefore, purine derivatives have been considered as a promising source of anti-parasitic compounds since they can act as inhibitors of the PSP enzymes or as toxic products upon their activation inside of the cell. Here, we characterized a Trypanosoma brucei enzyme involved in the salvage of adenine, the adenine phosphoribosyl transferase (APRT). We showed that its two isoforms (APRT1 and APRT2) localize partly in the cytosol and partly in the glycosomes of the bloodstream form (BSF) of the parasite. RNAi silencing of both APRT enzymes showed no major effect on the growth of BSF parasites unless grown in artificial medium with adenine as sole purine source. To add into the portfolio of inhibitors for various PSP enzymes, we designed three types of acyclic nucleotide analogs as potential APRT inhibitors. Out of fifteen inhibitors, four compounds inhibited the activity of the recombinant APRT1 with Ki in single µM values. The ANP phosphoramidate membrane-permeable prodrugs showed pronounced anti-trypanosomal activity in a cell-based assay, despite the fact that APRT enzymes are dispensable for T. brucei growth in vitro. While this suggests that the tested ANP prodrugs exert their toxicity by other means in T. brucei, the newly designed inhibitors can be further improved and explored to identify their actual target(s).


Assuntos
Nucleotídeos de Adenina/metabolismo , Adenina Fosforribosiltransferase/metabolismo , Nucleosídeos/metabolismo , Organofosfonatos/metabolismo , Trypanosoma brucei brucei/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células HeLa , Humanos , Purinas/metabolismo
10.
J Biol Chem ; 297(1): 100846, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34058194

RESUMO

Hepatic gluconeogenesis is the major contributor to the hyperglycemia observed in both patients and animals with type 2 diabetes. The transcription factor FOXO1 plays a dominant role in stimulating hepatic gluconeogenesis. FOXO1 is mainly regulated by insulin under physiological conditions, but liver-specific disruption of Foxo1 transcription restores normal gluconeogenesis in mice in which insulin signaling has been blocked, suggesting that additional regulatory mechanisms exist. Understanding the transcriptional regulation of Foxo1 may be conducive to the development of insulin-independent strategies for the control of hepatic gluconeogenesis. Here, we found that elevated plasma levels of adenine nucleotide in type 2 diabetes are the major regulators of Foxo1 transcription. We treated lean mice with 5'-AMP and examined their transcriptional profiles using RNA-seq. KEGG analysis revealed that the 5'-AMP treatment led to shifted profiles that were similar to db/db mice. Many of the upregulated genes were in pathways associated with the pathology of type 2 diabetes including Foxo1 signaling. As observed in diabetic db/db mice, lean mice treated with 5'-AMP displayed enhanced Foxo1 transcription, involving an increase in cellular adenosine levels and a decrease in the S-adenosylmethionine to S-adenosylhomocysteine ratio. This reduced methylation potential resulted in declining histone H3K9 methylation in the promoters of Foxo1, G6Pc, and Pepck. In mouse livers and cultured cells, 5'-AMP induced expression of more FOXO1 protein, which was found to be localized in the nucleus, where it could promote gluconeogenesis. Our results revealed that adenine nucleotide-driven Foxo1 transcription is crucial for excessive glucose production in type 2 diabetic mice.


Assuntos
Diabetes Mellitus Tipo 2/genética , Proteína Forkhead Box O1/genética , Hiperglicemia/genética , Transcrição Genética/genética , Nucleotídeos de Adenina/sangue , Animais , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/patologia , Regulação da Expressão Gênica/genética , Gluconeogênese/genética , Glucose/metabolismo , Humanos , Hiperglicemia/sangue , Hiperglicemia/patologia , Insulina/genética , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos NOD
11.
J Biol Chem ; 297(1): 100829, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34048711

RESUMO

Brr2 is an essential Ski2-like RNA helicase that exhibits a unique structure among the spliceosomal helicases. Brr2 harbors a catalytically active N-terminal helicase cassette and a structurally similar but enzymatically inactive C-terminal helicase cassette connected by a linker region. Both cassettes contain a nucleotide-binding pocket, but it is unclear whether nucleotide binding in these two pockets is related. Here we use biophysical and computational methods to delineate the functional connectivity between the cassettes and determine whether occupancy of one nucleotide-binding site may influence nucleotide binding at the other cassette. Our results show that Brr2 exhibits high specificity for adenine nucleotides, with both cassettes binding ADP tighter than ATP. Adenine nucleotide affinity for the inactive C-terminal cassette is more than two orders of magnitude higher than that of the active N-terminal cassette, as determined by slow nucleotide release. Mutations at the intercassette surfaces and in the connecting linker diminish the affinity of adenine nucleotides for both cassettes. Moreover, we found that abrogation of nucleotide binding at the C-terminal cassette reduces nucleotide binding at the N-terminal cassette 70 Å away. Molecular dynamics simulations identified structural communication lines that likely mediate these long-range allosteric effects, predominantly across the intercassette interface. Together, our results reveal intricate networks of intramolecular interactions in the complex Brr2 RNA helicase, which fine-tune its nucleotide affinities and which could be exploited to regulate enzymatic activity during splicing.


Assuntos
Nucleotídeos de Adenina/metabolismo , RNA Helicases/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Aminoácidos/metabolismo , Sítios de Ligação , Humanos , Cinética , Simulação de Dinâmica Molecular , Mutação/genética , Domínios Proteicos , Ribonucleoproteínas Nucleares Pequenas/química , Especificidade por Substrato
12.
Klin Lab Diagn ; 66(3): 172-176, 2021 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-33793117

RESUMO

Adenine nucleotides (ATP, ADP and AMP) play a central role in the regulation of metabolism and energy: they provide the energy balance of the cell, determine its redox state, act as allosteric effectors of a number of enzymes, modulate signaling and transcription factors and activate oxidation or biosynthesis substrates. A large number of methods have been developed to determine the level of ATP, ADP and AMP, but the most universal and effective method for the separation and analysis of complex mixtures is the reversed-phase high-performance liquid chromatography method (RP-HPLC). The aim of this study is to determine the optimal conditions for the qualitative separation and quantitative determination of standard solutions of ATP (1 mmol/l), ADP (0,5 mmol/l) and AMP (0,1 mmol/l) by RP-HPLC. The degree of separation of adenine nucleotides was estimated by the time of peak output in the chromatogram. To achieve the goal, the following tasks were set: assess the effect of the temperature of the analysis on the separation and change of the release time of the analytes in the chromatogram; determine the most optimal composition of the mobile phase for the separation of ATP, ADP and AMP in the chromatogram (the content of the organic solvent in the solution); to identify the effect of pH of the mobile phase on the separation of standard solutions of adenine nucleotides; set the optimal molarity of the mobile phase for the separation of ATP, ADP and AMP in the chromatogram. It was found that the temperature of the analysis does not affect the quality of peak separation, while the composition and pH of the mobile phase have a significant effect on the complete and clear separation of the studied nucleotides in the chromatogram. It was determined that the analysis temperature of 37°C and the mobile phase of 0.05 M KH2PO4 (pH 6.0) are optimal for separating the peaks of adenine nucleotides.


Assuntos
Nucleotídeos de Adenina , Trifosfato de Adenosina , Cromatografia Líquida de Alta Pressão , Indicadores e Reagentes
13.
Clin Sci (Lond) ; 135(9): 1103-1126, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33899910

RESUMO

Poor maternal nutrition in pregnancy affects fetal development, predisposing offspring to cardiometabolic diseases. The role of mitochondria during fetal development on later-life cardiac dysfunction caused by maternal nutrient reduction (MNR) remains unexplored. We hypothesized that MNR during gestation causes fetal cardiac bioenergetic deficits, compromising cardiac mitochondrial metabolism and reserve capacity. To enable human translation, we developed a primate baboon model (Papio spp.) of moderate MNR in which mothers receive 70% of control nutrition during pregnancy, resulting in intrauterine growth restriction (IUGR) offspring and later exhibiting myocardial remodeling and heart failure at human equivalent ∼25 years. Term control and MNR baboon offspring were necropsied following cesarean-section, and left ventricle (LV) samples were collected. MNR adversely impacted fetal cardiac LV mitochondria in a sex-dependent fashion. Increased maternal plasma aspartate aminotransferase, creatine phosphokinase (CPK), and elevated cortisol levels in MNR concomitant with decreased blood insulin in male fetal MNR were measured. MNR resulted in a two-fold increase in fetal LV mitochondrial DNA (mtDNA). MNR resulted in increased transcripts for several respiratory chain (NDUFB8, UQCRC1, and cytochrome c) and adenosine triphosphate (ATP) synthase proteins. However, MNR fetal LV mitochondrial complex I and complex II/III activities were significantly decreased, possibly contributing to the 73% decreased ATP content and increased lipid peroxidation. MNR fetal LV showed mitochondria with sparse and disarranged cristae dysmorphology. Conclusion: MNR disruption of fetal cardiac mitochondrial fitness likely contributes to the documented developmental programming of adult cardiac dysfunction, indicating a programmed mitochondrial inability to deliver sufficient energy to cardiac tissues as a chronic mechanism for later-life heart failure.


Assuntos
Transtornos da Nutrição Fetal/metabolismo , Fenômenos Fisiológicos da Nutrição Materna , Mitocôndrias Cardíacas/metabolismo , Nucleotídeos de Adenina/metabolismo , Animais , Feminino , Transtornos da Nutrição Fetal/patologia , Mitocôndrias Cardíacas/ultraestrutura , Estresse Oxidativo , Papio , Gravidez
14.
Biomolecules ; 11(4)2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923845

RESUMO

Multi-enzyme cascade reactions for the synthesis of complex products have gained importance in recent decades. Their advantages compared to single biotransformations include the possibility to synthesize complex molecules without purification of reaction intermediates, easier handling of unstable intermediates, and dealing with unfavorable thermodynamics by coupled equilibria. In this study, a four-enzyme cascade consisting of ScADK, AjPPK2, and SmPPK2 for ATP synthesis from adenosine coupled to the cyclic GMP-AMP synthase (cGAS) catalyzing cyclic GMP-AMP (2'3'-cGAMP) formation was successfully developed. The 2'3'-cGAMP synthesis rates were comparable to the maximal reaction rate achieved in single-step reactions. An iterative optimization of substrate, cofactor, and enzyme concentrations led to an overall yield of 0.08 mole 2'3'-cGAMP per mole adenosine, which is comparable to chemical synthesis. The established enzyme cascade enabled the synthesis of 2'3'-cGAMP from GTP and inexpensive adenosine as well as polyphosphate in a biocatalytic one-pot reaction, demonstrating the performance capabilities of multi-enzyme cascades for the synthesis of pharmaceutically relevant products.


Assuntos
Adenosina Quinase/metabolismo , Proteínas de Bactérias/metabolismo , Nucleotídeos Cíclicos/síntese química , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Acinetobacter/enzimologia , Nucleotídeos de Adenina/metabolismo , Biocatálise , Biotecnologia/métodos , Saccharomyces cerevisiae/enzimologia , Sinorhizobium meliloti/enzimologia
15.
J Biomed Nanotechnol ; 17(3): 477-486, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33875082

RESUMO

Nano-titanium dioxide (nano-TiO2) has been shown to inhibit testosterone synthesis in male mice or rats; however, the mechanisms underlying these effects have yet to be elucidated. In this study, we investigated whether the inhibition of testosterone synthesis by nano-TiO2 on Leydig cells (LCs) was related to the dysfunction of the cAMP/CGMP/EGFR/MMP signaling pathway in primary cultures of LCs prepared from rat testis exposed to nano-TiO2. We found that the early apoptotic rate of LCs increased by 4.34 and 4.94 times, respectively, after exposure to 20 g/mL and 40 g/mL nano-TiO2 ; we also found that NO increased by 1.1 and 2.86 times, respectively. ROS increased by times of 0.71, 3.15 and 3.43; RNS increased by 0.62, 1.34 and 1.14 times; and SOD activity decreased by 18.3%, 28.16%, and 67.6%, respectively, when the concentration of nano-TiO2 was 10, 20 and 40 g/mL. These results indicated that nano-TiO2 treatment resulted caused damage to the LCs, including an imbalance of oxidation and antioxidation. Following nano-TiO2 treatment, the cAMP content had decreased by 48%, 48% and 47.6%; cGMP content had decreased by 18.7%, 52.2% and 56.7%; the levels of ATP in the LCs had decreased by 15.15%, 45.75% and 66.67%; the expression of HCGR protein had decreased by 26.7%, 45.07% and 74.64%; the expression of LHR protein had decreased by 18.3%, 28.16% and 67.6%; and the levels of T had decreased by 34.48%, 46.62% and 44.12%. Collectively, our results indicated that the inhibition of testosterone production by nano-TiO2 is related to the dysfunction of the cAMP/CGMP/EGFR/MMP signaling pathway.


Assuntos
Células Intersticiais do Testículo , Testosterona , Nucleotídeos de Adenina , Animais , Células Cultivadas , Receptores ErbB , Nucleotídeos de Guanina , Guanosina , Masculino , Metaloproteinases da Matriz , Camundongos , Ratos , Transdução de Sinais , Testículo , Titânio
16.
Fa Yi Xue Za Zhi ; 37(1): 7-10, 2021 Feb.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-33780177

RESUMO

Abstract: Objective To determine the purine adenylate [adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP)] content in the muscles of both hind limbs of rats at different postmortem interval (PMI), calculate the changes in the total adenine nucleotide (TAN) content and the adenylic-acid energy charge (AEC), and explore their relationship with PMI. Methods Healthy rats were sacrificed by cervical dislocation and kept at 20 ℃. The muscles of their hind limbs were extracted at 0, 24, 48, 72, 96, 120, 144, and 168 h after death. Reversed-phase high performance liquid chromatography was used to determine the content of purine adenylates, the TAN and AEC of the muscles of the both hind limbs were calculated, and the related regression equations of their relationship with PMI were established. Results Within 168 h of death of rats, the trend of ATP change was different from ADP, and the content of AMP continuously increased. The TAN value gradually increased with the extension of PMI, and the AEC showed a downward trend within 168 h after death. Among them, the patterns of AEC changes with PMI were obvious, the correlation coefficient was high ( R2=0.903), and the curve fitting relationship was good; the fitting relationship between ATP, ADP, AMP, TAN and PMI was poor ( R2=0.198-0.754). Conclusion The postmortem change patterns of AEC provide new research ideas for PMI estimation in the forensic field.


Assuntos
Nucleotídeos de Adenina , Músculos , Monofosfato de Adenosina , Animais , Patologia Legal , Ratos , Ratos Sprague-Dawley , Fatores de Tempo
17.
Int J Mol Sci ; 22(4)2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33671212

RESUMO

Lysophosphatidic acid (LPA) species are a family of bioactive lipids that transmit signals via six cognate G protein-coupled receptors, which are required for brain development and function of the nervous system. LPA affects the function of all cell types in the brain and can display beneficial or detrimental effects on microglia function. During earlier studies we reported that LPA treatment of microglia induces polarization towards a neurotoxic phenotype. In the present study we investigated whether these alterations are accompanied by the induction of a specific immunometabolic phenotype in LPA-treated BV-2 microglia. In response to LPA (1 µM) we observed slightly decreased mitochondrial respiration, increased lactate secretion and reduced ATP/ADP ratios indicating a switch towards aerobic glycolysis. Pathway analyses demonstrated induction of the Akt-mTOR-Hif1α axis under normoxic conditions. LPA treatment resulted in dephosphorylation of AMP-activated kinase, de-repression of acetyl-CoA-carboxylase and increased fatty acid content in the phospholipid and triacylglycerol fraction of BV-2 microglia lipid extracts, indicating de novo lipogenesis. LPA led to increased intracellular amino acid content at one or more time points. Finally, we observed LPA-dependent generation of reactive oxygen species (ROS), phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2), upregulated protein expression of the Nrf2 target regulatory subunit of glutamate-cysteine ligase and increased glutathione synthesis. Our observations suggest that LPA, as a bioactive lipid, induces subtle alterations of the immunometabolic program in BV-2 microglia.


Assuntos
Aminoácidos/metabolismo , Glicólise/efeitos dos fármacos , Lipogênese/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Microglia/metabolismo , Nucleotídeos de Adenina/metabolismo , Aerobiose/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Linhagem Celular , Respiração Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Ácidos Graxos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ácido Láctico/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Camundongos , Microglia/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Fator 2 Relacionado a NF-E2/metabolismo , Fosfocreatina/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo
18.
Poult Sci ; 100(5): 101067, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33752069

RESUMO

The enzyme 2'-5' oligoadenylate synthase (OAS) is one of the key interferon-induced antiviral factors that act through inhibition of viral replication. In chickens, there is a single well-characterized OAS gene, oligoadenylate synthase-like (OASL) that has been shown to be upregulated after infection with various viruses. However, a deeper understanding of how chicken OASL acts against viral infection is still necessary. In this study, we tested the hypothesis that OASL short interfering RNA (siRNA)-mediated knockdown would decrease the host gene expression response to the Newcastle disease virus (NDV) by impacting antiviral pathways. To assess our hypothesis, a chicken fibroblast cell line (DF-1) was infected with the NDV (LaSota strain) and OASL expression was knocked down using a specific siRNA. The level of NDV viral RNA in the cells and the expression of interferon response- and apoptosis-related genes were evaluated by quantitative PCR at 4, 8, and 24 h postinfection (hpi). Knockdown of OASL increased the level of NDV viral RNA at 4, 8, and 24 hpi (P < 0.05) and eliminated the difference between NDV-infected and noninfected cells for expression of interferon response- and apoptosis-related genes (P > 0.05). The lack of differential expression suggests that knockdown of OASL resulted in a decreased response to NDV infection. Within NDV-infected cells, OASL knockdown reduced expression of signal transducer and activator of transcription 1, interferon alfa receptor subunit 1, eukaryotic translation initiation factor 2 alpha kinase 2, ribonuclease L, caspase 8 (CASP8) and caspase 9 (CASP9) at 4 hpi, CASP9 at 8 hpi, and caspase 3, CASP8, and CASP9 at 24 hpi (P < 0.05). We suggest that the increased NDV viral load in DF-1 cells after OASL knockdown was the result of a complex interaction between OASL and interferon response- and apoptosis-related genes that decreased host response to the NDV. Our results provide comprehensive information on the role played by OASL during NDV infection in vitro. Targeting this mechanism could aid in future prophylactic and therapeutic treatments for Newcastle disease in poultry.


Assuntos
Doença de Newcastle , Vírus da Doença de Newcastle , Nucleotídeos de Adenina , Animais , Galinhas/genética , Doença de Newcastle/genética , Oligorribonucleotídeos , Replicação Viral
19.
Plant Cell Physiol ; 62(3): 401-410, 2021 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-33416847

RESUMO

Various proteins in plant chloroplasts are subject to thiol-based redox regulation, allowing light-responsive control of chloroplast functions. Most redox-regulated proteins are known to be reductively activated in the light in a thioredoxin (Trx)-dependent manner, but its regulatory network remains incompletely understood. Using a biochemical procedure, we here show that a specific form of phosphofructokinase (PFK) is a novel redox-regulated protein whose activity is suppressed upon reduction. PFK is a key enzyme in the glycolytic pathway. In Arabidopsis thaliana, PFK5 is targeted to chloroplasts and uniquely contains an insertion sequence harboring two Cys residues (Cys152 and Cys157) in the N-terminal region. Redox shift assays using a thiol-modifying reagent indicated that PFK5 is efficiently reduced by a specific type of Trx, namely, Trx-f. PFK5 enzyme activity was lowered with the Trx-f-dependent reduction. PFK5 redox regulation was bidirectional; PFK5 was also oxidized and activated by the recently identified Trx-like2/2-Cys peroxiredoxin pathway. Mass spectrometry-based peptide mapping analysis revealed that Cys152 and Cys157 are critical for the intramolecular disulfide bond formation in PFK5. The involvement of Cys152 and Cys157 in PFK5 redox regulation was further supported by a site-directed mutagenesis study. PFK5 catalyzes the reverse reaction of fructose 1,6-bisphosphatase (FBPase), which is reduced and activated specifically by Trx-f. Our data suggest that PFK5 redox regulation, together with that of FBPase, constitutes a checkpoint for switching light/dark metabolism in chloroplasts.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Oxirredução , Fosfofrutoquinases/metabolismo , Nucleotídeos de Adenina/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Cloroplastos/enzimologia , Cisteína/metabolismo , Redes e Vias Metabólicas , Peroxirredoxinas/metabolismo , Fosfofrutoquinases/genética
20.
Analyst ; 146(2): 463-470, 2021 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-33491016

RESUMO

Selective detection of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) which are less charged molecules than adenosine triphosphate (ATP) or pyrophosphate (PPi) in aqueous solution has been considered challenging because AMP and ADP have relatively low binding affinity for phosphate receptors. In this study, colorimetric discrimination of nucleoside phosphates was achieved based on catalytic signal amplification through the activation of artificial peroxidase. This method showed high selectivity for AMP and ADP over ATP and PPi, unlike previous phosphate sensors that use Zn2+-dipicolylamine-based receptors. High selectivity of the suggested method allowed discrimination of AMP in aqueous solution by the naked eye, and the detection limit was estimated to be 0.5 µM. Mechanism analysis revealed AMP acted as activators in the peroxidation cycle of the Mn2(bpmp)/ABTS/H2O2 system despite having relatively low binding affinity. Additionally, high selectivity and quantitative signal amplification allowed for the development of colorimetric phosphodiesterase and a small molecule kinase assay method. The newly proposed method offers direct, real-time, and quantitative analysis of enzyme activities and inhibition, and is expected to be further applied to high-throughput screening of inhibitors.


Assuntos
Nucleotídeos de Adenina/análise , Colorimetria/métodos , Benzotiazóis/química , Materiais Biomiméticos/química , Catálise , Complexos de Coordenação/química , Ensaios Enzimáticos , Peróxido de Hidrogênio/química , Cinética , Manganês/química , Ácidos Sulfônicos/química
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