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1.
Physiol Rep ; 10(18): e15464, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36117383

RESUMO

Nucleotidases contribute to the regulation of inflammation, coagulation, and cardiovascular activity. Exercise promotes biological adaptations, but its effects on nucleotidase activities and expression are unclear. The objective of this study was to review systematically the effects of exercise on nucleotidase functionality in healthy and unhealthy subjects. The MEDLINE, EMBASE, Cochrane Library, and Web of Science databases were searched to identify, randomized clinical trials, non-randomized clinical trials, uncontrolled clinical trials, quasi-experimental, pre-, and post-interventional studies that evaluated the effects of exercise on nucleotidases in humans, and was not limited by language and date. Two independent reviewers performed the study selection, data extraction, and assessment of risk of bias. Of the 203 articles identified, 12 were included in this review. Eight studies reported that acute exercise, in healthy and unhealthy subjects, elevated the activities or expression of nucleotidases. Four studies evaluated the effects of chronic training on nucleotidase activities in the platelets and lymphocytes of patients with metabolic syndrome, chronic kidney disease, and hypertension and found a decrease in nucleotidase activities in these conditions. Acute and chronic exercise was able to modify the blood plasma and serum levels of nucleotides and nucleosides. Our results suggest that short- and long-term exercise modulate nucleotidase functionality. As such, purinergic signaling may represent a novel molecular adaptation in inflammatory, thrombotic, and vascular responses to exercise.


Assuntos
Exercício Físico , Hipertensão , Terapia por Exercício , Humanos , Nucleotidases , Nucleotídeos
2.
Bioengineered ; 13(6): 14204-14214, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35754345

RESUMO

Colorectal cancer (CRC) is a common gastrointestinal cancer with high incidence and mortality rates. CRC may be associated with regulation of circulating nucleotides. This study aimed to evaluate the serum levels of nucleotide-metabolizing enzymes (ATPase and AMPase) in patients with CRC and to explore the clinical diagnostic value of these enzymes. The gene set variation analysis (GSVA) score of the ATP-adenosine signature was calculated using tumor samples from The Cancer Genome Atlas (TCGA). ATP-adenosine signaling plays a central role in CRC progression. A total of 135 subjects, including 87 patients with CRC and 48 healthy controls, were included. The serum levels of ATPase and AMPase in the CRC group were significantly higher than those in the control group (P < 0.05). Furthermore, ATP and AMP hydrolysis levels significantly increased in the advanced CRC group (P < 0.05). ATP and AMP hydrolysis was decreased by the ENTPDase inhibitors (POM-1 and ARL67156) and CD73 inhibitor (APCP). The sensitivities of ATPase and AMPase were 95.4% and 75.9%, respectively, which were higher than those of CEA (67.8%) and CA19-9 (72.4%). The specificities of ATPase and AMPase were 69.9% and 73.9%, respectively, which were higher than that of CA19-9 (47.8%). The combination of CEA, ATPase, and AMPase demonstrated high sensitivity (92.0%) and specificity (87.0%). Collectively, ATPase and AMPase activities are upregulated in CRC with considerable diagnostic significance. The combination of CEA, ATPase, and AMPase may provide a novel approach for CRC screening.


Assuntos
Monofosfato de Adenosina , Adenosina Trifosfatases , Trifosfato de Adenosina , Neoplasias Colorretais , Nucleotidases , Monofosfato de Adenosina/sangue , Adenosina Trifosfatases/sangue , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/sangue , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Antígeno CA-19-9/sangue , Antígeno Carcinoembrionário/metabolismo , Neoplasias Colorretais/sangue , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Humanos , Nucleotidases/sangue , Nucleotidases/genética
3.
Arq Neuropsiquiatr ; 80(3): 289-295, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35416841

RESUMO

BACKGROUND: Acupuncture is a treatment for neuropathic pain, but its mechanism remains unclear. Previous studies showed that analgesia was induced in rats with neuropathic pain when their spinal cord adenosine content increased after electroacupuncture (EA); however, the mechanism behind this electroacupuncture-induced increase has not been clarified. OBJECTIVE: This study aimed to determine the role that ecto-5'-nucleotidase plays in EA-induced analgesia for neuropathic pain. METHODS: We performed electroacupuncture at the Zusanli acupoint on the seventh day after establishing a rat model of neuropathic pain induced through chronic constriction injuries. We observed the mechanical withdrawal threshold and thermal pain threshold and detected the expression of ecto-5'-nucleotidase in the spinal cord using Western blot. Chronic constriction injury rat models were intraperitoneally injected with α,ß-methyleneadenosine 5'-diphosphate, an ecto-5'-nucleotidase inhibitor, 30 min before electroacupuncture. The adenosine content of the spinal cord was detected using high-performance liquid chromatography. Lastly, the adenosine A1 receptor agonist N6-cyclopentyladenosine was intrathecally injected into the lumbar swelling of the rats, and the mechanical withdrawal and thermal pain thresholds were reevaluated. RESULTS: Analgesia and increased ecto-5'-nucleotidase expression and adenosine content in the spinal cord were observed 1 h after electroacupuncture. α,ß-methyleneadenosine 5'-diphosphate was able to inhibit upregulation of adenosine content and electroacupuncture-induced analgesia. After administration of N6-cyclopentyladenosine, electroacupuncture-induced analgesia was restored. CONCLUSIONS: Our results suggest that electroacupuncture at Zusanli can produce analgesia in chronic constriction injury rat models, possibly via the increased ecto-5'-nucleotidase expression induced through electroacupuncture, thus leading to increased adenosine expression in the spinal cord.


Assuntos
Analgesia , Eletroacupuntura , Neuralgia , 5'-Nucleotidase/metabolismo , Adenosina , Animais , Neuralgia/terapia , Nucleotidases , Ratos , Ratos Sprague-Dawley , Medula Espinal/metabolismo
4.
Sci Rep ; 12(1): 2852, 2022 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-35181715

RESUMO

Hair loss is one of the most common skin problems experienced by more than half of the world's population. In East Asia, medicinal herbs have been used widely in clinical practice to treat hair loss. Recent studies, including systematic literature reviews, indicate that medicinal herbs may demonstrate potential effects for hair loss treatment. In a previous study, we identified medical herbs used frequently for alopecia treatment. Herein, we explored the potential novel therapeutic mechanisms of 20 vital medicinal herbs for alopecia treatment that could distinguish them from known mechanisms of conventional drugs using network pharmacology analysis methods. We determined the herb-ingredient-target protein networks and ingredient-associated protein (gene)-associated pathway networks and calculated the weighted degree centrality to define the strength of the connections. Data showed that 20 vital medicinal herbs could exert therapeutic effects on alopecia mainly mediated via regulation of various target genes and proteins, including acetylcholinesterase (AChE), phospholipase A2 (PLA2) subtypes, ecto-5-nucleotidase (NTE5), folate receptor (FR), nicotinamide N-methyltransferase (NNMT), and quinolinate phosphoribosyltransferase (QPRT). Findings regarding target genes/proteins and pathways of medicinal herbs associated with alopecia treatment offer insights for further research to better understand the pathogenesis and therapeutic mechanism of medicinal herbs for alopecia treatment with traditional herbal medicine.


Assuntos
Alopecia/tratamento farmacológico , Medicamentos de Ervas Chinesas/uso terapêutico , Farmacologia em Rede , Plantas Medicinais , Acetilcolinesterase/genética , Alopecia/genética , Alopecia/prevenção & controle , Receptor 1 de Folato/genética , Humanos , Medicina Tradicional Chinesa , Nicotinamida N-Metiltransferase/genética , Nucleotidases/genética , Pentosiltransferases/genética , Fosfolipases A2/genética , Fitoterapia , Preparações de Plantas/química , Preparações de Plantas/uso terapêutico
5.
J Mol Biol ; 434(7): 167478, 2022 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-35123996

RESUMO

Despite decades of research and the availability of the full genomic sequence of the baker's yeast Saccharomyces cerevisiae, still a large fraction of its genome is not functionally annotated. This hinders our ability to fully understand cellular activity and suggests that many additional processes await discovery. The recent years have shown an explosion of high-quality genomic and structural data from multiple organisms, ranging from bacteria to mammals. New computational methods now allow us to integrate these data and extract meaningful insights into the functional identity of uncharacterized proteins in yeast. Here, we created a database of sensitive sequence similarity predictions for all yeast proteins. We use this information to identify candidate enzymes for known biochemical reactions whose enzymes are unidentified, and show how this provides a powerful basis for experimental validation. Using one pathway as a test case we pair a new function for the previously uncharacterized enzyme Yhr202w, as an extra-cellular AMP hydrolase in the NAD degradation pathway. Yhr202w, which we now term Smn1 for Scavenger MonoNucleotidase 1, is a highly conserved protein that is similar to the human protein E5NT/CD73, which is associated with multiple cancers. Hence, our new methodology provides a paradigm, that can be adopted to other organisms, for uncovering new enzymatic functions of uncharacterized proteins.


Assuntos
Monofosfato de Adenosina , Nucleotidases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Monofosfato de Adenosina/química , Humanos , Nucleotidases/química , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Análise de Sequência de Proteína/métodos
6.
Bioengineered ; 13(2): 3183-3193, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35068336

RESUMO

Dysregulation of calcium-activated nucleotides 1 (CANT1) has been observed in different organs. Thus, its biological function in cancer has increasingly attracted researchers. The current work aims to study the CANT1 role in lung cancer and understand the underlying pathological mechanisms. High amplification of CANT1 was observed in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) tissues compared to normal tissues. The high-CANT1 patients showed a dismal prognosis in comparison with the low-CANT1 patients. Highly expressed CANT1 was significantly associated with the N stage of LUSC patients. Ectopic expression of CANT1 conspicuously increased the proliferation and viability of A549 cells. Conversely, CANT1 depletion resulted in adverse effects in H1299 cells. CANT1 depletion also resulted in the retardation of tumor growth in vivo. Mechanically, we found that CANT1 could elevate NF-ĸB (nuclear factor-k-gene binding) transcriptional activity in a concentration-dependent manner. This regulatory relationship was also established by the Western blot technique. Inhibiting NF-ĸB can significantly blunt the increased NF-κ-B Inhibitor-α (IκBα) expression caused by CANT1 overexpression in A549 cells. In conclusion, highly amplified CANT1 promotes the proliferation and viability of lung cancer cells. We also elucidate a new signaling axis of CANT1-NF-ĸB in lung cancer. This approach might be a promising strategy for lung cancer treatment.


Assuntos
Neoplasias Pulmonares/metabolismo , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/metabolismo , Nucleotidases/metabolismo , Transdução de Sinais , Células A549 , Animais , Humanos , Neoplasias Pulmonares/genética , Masculino , Camundongos , Camundongos Nus , NF-kappa B/genética , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Nucleotidases/genética
7.
BMC Cancer ; 22(1): 117, 2022 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-35090419

RESUMO

BACKGROUND: Calcium-activated nucleotidase 1 (CANT1), functions as a calcium-dependent nucleotidase with a preference for UDP. However, the potential clinical value of CANT1 in lung adenocarcinoma (LA) has not been fully clarified. Thus, we sought to identify its potential biological function and mechanism through bioinformatics analysis and in vitro experiments in LA. METHODS: In the present study, we comprehensively investigated the prognostic role of CANT1 in LA patients through bioinformatics analysis and in vitro experiments. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were utilized to analyze the expression of CANT1 in LA patients and their clinical-prognostic value. The immunohistochemistry staining was obtained from the Human Protein Atlas (HPA). A Cox regression model was used to evaluate prognostic factors. Gene ontology (GO) and Gene set enrichment analysis (GSEA) was performed to explore the potential regulatory mechanism of CANT1 in the development of LA. Moreover, we also examined the relationship between CANT1 expression and DNA methylation. Finally, we did in vitro experiments to evaluate the biological behavior and role of CANT1 in LA cells (LACs). RESULTS: Our study showed that the CANT1 expression was significantly elevated in the LA tissues compared with the normal lung tissues. Increased CANT1 expression was significantly associated with the TN stage. A univariate Cox analysis indicated that high CANT1 expression levels were correlated with poor overall survival (OS) in LA. Besides, CANT1 expression was independently associated with OS in multivariate analysis. GO and GSEA analysis showed the enrichment of mitotic nuclear division, DNA methylation, and DNA damage. Then we found that the high expression of CANT1 is positively correlated with hypomethylation. The methylation level was associated with prognosis in LA patients. Finally, in vitro experiments indicated that knockdown of CANT1 resulted in decreased cell proliferation, invasion, and G1 phase cell-cycle arrest in LACs. CONCLUSION: The present study suggested that CANT1 may serve as a potential prognosis biomarker in patients with LA. High CANT1 expression and promoter demethylation was associated with worse outcome. Finally, in vitro experiments verified the biological functions and behaviors of CANT1 in LA.


Assuntos
Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/mortalidade , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Nucleotidases/metabolismo , Idoso , Biomarcadores Tumorais/genética , Proliferação de Células/genética , Dano ao DNA/genética , Metilação de DNA/genética , Feminino , Ontologia Genética , Humanos , Masculino , Prognóstico
8.
Artigo em Inglês | MEDLINE | ID: mdl-32954967

RESUMO

Lactococcus lactis has been reported unable to directly incorporate mononucleotides but instead requires their external dephosphorylation by nucleotidases to the corresponding nucleosides prior to their incorporation. Although Lactobacillus gasseri PA-3 (PA-3), a strain of lactic acid bacteria, has been found to incorporate purine mononucleotides such as adenosine 5'-monophosphate (AMP), it remains unclear whether these bacteria directly incorporate these mononucleotides or incorporate them after dephosphorylation to the corresponding nucleosides. This study evaluated whether PA-3 incorporated radioactively-labeled mononucleotides in the presence or absence of the 5'-nucleotidase inhibitor α,ß-methylene ADP (APCP). PA-3 took up 14C-AMP in the presence of APCP, as well as incorporating 32P-AMP. Furthermore, radioactivity was detected in the RNA/DNA of bacterial cells cultured in the presence of 32P-AMP. Taken together, these findings indicated that PA-3 incorporated purine mononucleotides directly rather than after their dephosphorylation to purine nucleosides and that PA-3 utilizes these purine mononucleotides in the synthesis of RNA and DNA. Although additional studies are required to identify purine mononucleotide transporters in PA-3, this study is the first to show that some lactic acid bacteria directly incorporate purine mononucleotides and use them for growth.


Assuntos
Lactobacillus gasseri , Monofosfato de Adenosina/metabolismo , Lactobacillus gasseri/metabolismo , Nucleotidases/metabolismo , Nucleosídeos de Purina/metabolismo
9.
J Neurochem ; 160(3): 305-324, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34905223

RESUMO

Extracellular adenosine plays prominent roles in the brain in both physiological and pathological conditions. Adenosine can be generated following the degradation of extracellular nucleotides by various types of ectonucleotidases. Several ectonucleotidases are present in the brain parenchyma: ecto-nucleotide triphosphate diphosphohydrolases 1 and 3 (NTPDase 1 and 3), ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP 1), ecto-5'-nucleotidase (eN), and tissue non-specific alkaline phosphatase (TNAP, whose function in the brain has received little attention). Here we examined, in a living brain preparation, the role of these ectonucleotidases in generating extracellular adenosine. We recorded local field potentials evoked by electrical stimulation of the lateral olfactory tract in the mouse piriform cortex in vitro. Variations in adenosine level were evaluated by measuring changes in presynaptic inhibition generated by adenosine A1 receptors (A1Rs) activation. A1R-mediated presynaptic inhibition was present endogenously and was enhanced by bath-applied AMP and ATP. We hypothesized that inhibiting ectonucleotidases would reduce extracellular adenosine concentration, which would result in a weakening of presynaptic inhibition. However, inhibiting TNAP had no effect in controlling endogenous adenosine action and no effect on presynaptic inhibition induced by bath-applied AMP. Furthermore, contrary to our expectation, inhibiting TNAP reinforced, rather than reduced, presynaptic inhibition induced by bath-applied ATP. Similarly, inhibition of NTPDase 1 and 3, NPP1, and eN induced stronger, rather than weaker, presynaptic inhibition, both in endogenous condition and with bath-applied ATP and AMP. Consequently, attempts to suppress the functions of extracellular adenosine by blocking its extracellular synthesis in living brain tissue could have functional impacts opposite to those anticipated.


Assuntos
Córtex Cerebral/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Nucleotidases/antagonistas & inibidores , Transmissão Sináptica/efeitos dos fármacos , 5'-Nucleotidase/antagonistas & inibidores , Adenosina/metabolismo , Agonistas do Receptor A1 de Adenosina/farmacologia , Monofosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Fosfatase Alcalina/antagonistas & inibidores , Animais , Estimulação Elétrica , Potenciais Evocados/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Bulbo Olfatório/efeitos dos fármacos , Receptor A1 de Adenosina/efeitos dos fármacos , Receptor A1 de Adenosina/metabolismo
10.
Adv Biol Regul ; 83: 100858, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34920982

RESUMO

Bisphosphate nucleotidase 2 (BPNT2) is a member of a family of phosphatases that are directly inhibited by lithium, the first-line medication for bipolar disorder. BPNT2 is localized to the Golgi, where it metabolizes the by-products of glycosaminoglycan sulfation reactions. BPNT2-knockout mice exhibit impairments in total-body chondroitin-4-sulfation which lead to abnormal skeletal development (chondrodysplasia). These mice die in the perinatal period, which has previously prevented the investigation of BPNT2 in the adult nervous system. Previous work has demonstrated the importance of chondroitin sulfation in the brain, as chondroitin-4-sulfate is a major component of perineuronal nets (PNNs), a specialized neuronal extracellular matrix which mediates synaptic plasticity and regulates certain behaviors. We hypothesized that the loss of BPNT2 in the nervous system would decrease chondroitin-4-sulfation and PNNs in the brain, which would coincide with behavioral abnormalities. We used Cre-lox breeding to knockout Bpnt2 specifically in the nervous system using Bpnt2 floxed (fl/fl) animals and a Nestin-driven Cre recombinase. These mice are viable into adulthood, and do not display gross physical abnormalities. We identified decreases in total glycosaminoglycan sulfation across selected brain regions, and specifically show decreases in chondroitin-4-sulfation which correspond with increases in chondroitin-6-sulfation. Interestingly, these changes were not correlated with gross alterations in PNNs. We also subjected these mice to a selection of neurobehavioral assessments and did not identify significant behavioral abnormalities. In summary, this work demonstrates that BPNT2, a known target of lithium, is important for glycosaminoglycan sulfation in the brain, suggesting that lithium-mediated inhibition of BPNT2 in the nervous system warrants further investigation.


Assuntos
Córtex Cerebral , Sulfatos de Condroitina , Hipocampo , Animais , Córtex Cerebral/metabolismo , Sulfatos de Condroitina/metabolismo , Hipocampo/metabolismo , Compostos de Lítio/farmacologia , Camundongos , Nucleotidases/metabolismo
11.
J. venom. anim. toxins incl. trop. dis ; 28: e20210040, 2022. tab, graf, ilus
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1365074

RESUMO

Background: Naja atra is a venomous snake species medically relevant in China. In the current study, we evaluated the composition and toxicological profile of venom collected from farm-raised N. atra. Methods: Venom was collected from third-generation captive bred N. atra on a snake farm in Hunan Province, China. The venom was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and nano-liquid chromatography with electrospray ionization tandem mass spectrometry. In addition, hemolytic activity, median lethal dose, serum biochemical and histopathological parameters were accessed. Results: N. atra venom proteome was dominated by phospholipase A2 (46.5%) and three-finger toxins (41.4 %), and a set of common low relative abundance proteins, including cysteine-rich secretory proteins (4.7%), NGF-beta (2.4%), snake venom metalloproteinase (1.5%), glutathione peroxidase (0.6%), vespryn (0.3%), and 5ʹ-nucleotidases (0.2%) were also found. Furthermore, the venom exhibited direct hemolytic activity, neurotoxicity, myotoxicity, and high lethal potency in mice, with a subcutaneous median lethal dose of 1.02 mg/kg. Histopathological analysis and serum biochemical tests revealed that venom caused acute hepatic, pulmonary and renal injury in mice. Conclusion: This study revealed the composition and toxicity of venom collected from farm-raised N. atra, thereby providing a reference for the analysis of venom samples collected from captive-born venomous snakes in the future.(AU)


Assuntos
Animais , Venenos de Serpentes/toxicidade , Fosfolipases A2 , Naja naja , Miotoxicidade , Nucleotidases
12.
Int J Mol Sci ; 22(21)2021 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-34768881

RESUMO

Although the enhanced responses against serum cell-free DNA (cfDNA) in cases of sepsis-a life-threatening organ dysfunction due to systemic infection-are understood, the influence of the cytosolic DNA receptor cGAS (cyclic guanosine monophosphate-adenosine monophosphate (GMP-AMP) synthase) on sepsis is still unclear. Here, experiments on cGAS deficient (cGAS-/-) mice were conducted using cecal ligation and puncture (CLP) and lipopolysaccharide (LPS) injection sepsis models and macrophages. Severity of CLP in cGAS-/- mice was less severe than in wildtype (WT) mice, as indicated by mortality, serum LPS, cfDNA, leukopenia, cytokines (TNF-α, IL-6 and IL-10), organ histology (lung, liver and kidney) and spleen apoptosis. With the LPS injection model, serum cytokines in cGAS-/- mice were lower than in WT mice, despite the similar serum cfDNA level. Likewise, in LPS-activated WT macrophages, the expression of several mitochondria-associated genes (as revealed by RNA sequencing analysis) and a profound reduction in mitochondrial parameters, including maximal respiration (determined by extracellular flux analysis), DNA (mtDNA) and mitochondrial abundance (revealed by fluorescent staining), were demonstrated. These data implied the impact of cfDNA resulting from LPS-induced cell injury. In parallel, an additive effect of bacterial DNA on LPS, seen in comparison with LPS alone, was demonstrated in WT macrophages, but not in cGAS-/- cells, as indicated by supernatant cytokines (TNF-α and IL-6), M1 proinflammatory polarization (iNOS and IL-1ß), cGAS, IFN-γ and supernatant cyclic GMP-AMP (cGAMP). In conclusion, cGAS activation by cfDNA from hosts (especially mtDNA) and bacteria was found to induce an additive proinflammatory effect on LPS-activated macrophages which was perhaps responsible for the more pronounced sepsis hyperinflammation observed in WT mice compared with the cGAS-/- group.


Assuntos
Nucleotidiltransferases/metabolismo , Sepse/metabolismo , Animais , Ceco/metabolismo , Citocinas/metabolismo , DNA/metabolismo , Interleucina-10/metabolismo , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/farmacologia , Fígado/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nucleotidases/metabolismo , Nucleotídeos Cíclicos , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/genética , Sepse/prevenção & controle , Índice de Gravidade de Doença , Fator de Necrose Tumoral alfa/metabolismo
13.
Bioengineered ; 12(1): 8953-8964, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34652259

RESUMO

Apoptosis of lens epithelial cells contributed to the formation of age-related cataract (ARC), and previous data revealed that circular RNA (circRNA) was responsible for the underneath mechanism. The study was organized to explore the role of circular RNA erythrocyte membrane protein band 4.1 (circ_EPB41) in ultraviolet (UV) irradiation-induced apoptosis of lens epithelial cells. SRA01/04 cells were irradiated with UV to mimic the ARC cell model. The RNA levels of circ_EPB41, microRNA-24-3p (miR-24-3p), and 3'(2'), 5'-bisphosphate nucleotidase 1 (BPNT1) were detected by quantitative real-time polymerase chain reaction. Protein expression was checked by western blot. 5-Ethynyl-29-deoxyuridine, 3-(4,5-Dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide and DNA content quantitation assays were performed to investigate cell proliferation. Flow cytometry was conducted to analyze cell apoptosis. Dual-luciferase reporter assay was implemented to confirm the interaction among circ_EPB41, miR-24-3p, and BPNT1. Our data showed that circ_EPB41 and BPNT1 expression were downregulated in ARC tissues and UV-irradiated SRA01/04 cells as compared with normal anterior lens capsules and untreated SRA01/04 cells. Circ_EPB41 overexpression ameliorated the effects of UV irradiation on the proliferation and apoptosis of SRA01/04 cells. Besides, miR-24-3p, a target miRNA of circ_EPB41, attenuated circ_EPB41 introduction-mediated proliferation, and apoptosis of UV-irradiated SRA01/04 cells. MiR-24-3p regulated UV irradiation-induced effects by targeting BPNT1. Importantly, it was found that circ_EPB41 stimulated BPNT1 production by miR-24-3p. Taken together, the enforced expression of circ_EPB41 ameliorated UV irradiation-induced apoptosis of lens epithelial cells by miR-24-3p/BPNT1 pathway, providing us with a potential target for the therapy of UV-caused ARC.


Assuntos
Catarata/patologia , Proteínas do Citoesqueleto/genética , Células Epiteliais/patologia , Cristalino/patologia , Proteínas de Membrana/genética , MicroRNAs/genética , Nucleotidases/metabolismo , RNA Circular/genética , Idoso , Apoptose , Catarata/genética , Catarata/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Humanos , Cristalino/metabolismo , Cristalino/efeitos da radiação , Pessoa de Meia-Idade , Nucleotidases/genética , Raios Ultravioleta
14.
J Virol ; 95(19): e0110421, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34232734

RESUMO

Modified vaccinia virus Ankara (MVA) was derived by repeated passaging in chick fibroblasts, during which deletions and mutations rendered the virus unable to replicate in most mammalian cells. Marker rescue experiments demonstrated that the host range defect could be overcome by replacing DNA that had been deleted from near the left end of the genome. One virus isolate, however, recovered the ability to replicate in monkey BS-C-1 cells but not human cells without added DNA, suggesting that it arose from a spontaneous mutation. Here, we showed that variants with enhanced ability to replicate in BS-C-1 cells could be isolated by blind passaging of MVA and that in each there was a point mutation leading to an amino acid substitution in the D10 decapping enzyme. The sufficiency of these single mutations to enhance host range was confirmed by constructing recombinant viruses. The D10 mutations occurred at N- or C-terminal locations distal to the active site, suggesting an indirect effect on decapping or on another previously unknown role of D10. Although increased amounts of viral mRNA and proteins were found in BS-C-1 cells infected with the mutants compared to those with parental MVA, the increases were much less than the 1- to 2-log-higher virus yields. Nevertheless, a contributing role for diminished decapping in overcoming the host range defect was consistent with increased replication and viral protein synthesis in BS-C-1 cells infected with an MVA engineered to have active-site mutations that abrogate decapping activity entirely. Optimal decapping may vary depending on the biological context. IMPORTANCE Modified vaccinia virus Ankara (MVA) is an attenuated virus that is approved as a smallpox vaccine and is in clinical trials as a vector for other pathogens. The safety of MVA is due in large part to its inability to replicate in mammalian cells. Although host range restriction is considered a stable feature of the virus, we describe the occurrence of spontaneous mutations in MVA that increase replication considerably in monkey BS-C-1 cells but only slightly in human cells. The mutants contain single nucleotide changes that lead to amino acid substitutions in one of the two decapping enzymes. Although the spontaneous mutations are distant from the decapping enzyme active site, engineered active-site mutations also increased virus replication in BS-C-1 cells. The effects of these mutations on the immunogenicity of MVA vectors remain to be determined.


Assuntos
Nucleotidases/genética , Nucleotidases/metabolismo , Vírus Vaccinia/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Animais , Domínio Catalítico , Linhagem Celular , Embrião de Galinha , Chlorocebus aethiops , Recombinação Homóloga , Especificidade de Hospedeiro , Humanos , Nucleotidases/química , Fases de Leitura Aberta , Mutação Puntual , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Deleção de Sequência , Vírus Vaccinia/genética , Ensaio de Placa Viral , Proteínas Virais/química , Replicação Viral
15.
Neurochem Int ; 148: 105111, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34171414

RESUMO

Early life stressors, such as social isolation (SI), can disrupt brain development contributing to behavioral and neurochemical alterations in adulthood. Purinergic receptors and ectonucleotidases are key regulators of brain development in embryonic and postnatal periods, and they are involved in several psychiatric disorders, including schizophrenia. The extracellular ATP drives purinergic signaling by activating P2X and P2Y receptors and it is hydrolyzed by ectonucleotidases in adenosine, which activates P1 receptors. The purpose of this study was to investigate if SI, a rodent model used to replicate abnormal behavior relevant to schizophrenia, impacts purinergic signaling. Male Wistar rats were reared from weaning in group-housed or SI conditions for 8 weeks. SI rats exhibited impairment in prepulse inhibition and social interaction. SI presented increased ADP levels in cerebrospinal fluid and ADP hydrolysis in the hippocampus and striatum synaptosomes. Purinergic receptor expressions were upregulated in the prefrontal cortex and downregulated in the hippocampus and striatum. A2A receptors were differentially expressed in SI prefrontal cortex and the striatum, suggesting distinct roles in these brain structures. SI also presented decreased ADP, adenosine, and guanosine levels in the cerebrospinal fluid in response to D-amphetamine. Like patients with schizophrenia, uric acid levels were prominently increased in SI rats after D-amphetamine challenge. We suggest that the SI-induced deficits in prepulse inhibition might be related to the SI-induced changes in purinergic signaling. We provide new evidence that purinergic signaling is markedly affected in a rat model relevant to schizophrenia, pointing out the importance of purinergic system in psychiatry conditions.


Assuntos
Receptores Purinérgicos , Transdução de Sinais , Isolamento Social , Difosfato de Adenosina/líquido cefalorraquidiano , Animais , Comportamento Animal , Estimulantes do Sistema Nervoso Central/farmacologia , Dextroanfetamina/farmacologia , Masculino , Nucleotidases/metabolismo , Ratos , Ratos Wistar , Receptor A2A de Adenosina/metabolismo , Receptores Purinérgicos P2X/metabolismo , Receptores Purinérgicos P2Y/metabolismo , Reflexo de Sobressalto , Psicologia do Esquizofrênico , Comportamento Social , Isolamento Social/psicologia , Desmame
16.
Molecules ; 26(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923386

RESUMO

The 5'-nucleotidase UshA and the 3'-nucleotidase CpdB from Escherichia coli are broad-specificity phosphohydrolases with similar two-domain structures. Their N-terminal domains (UshA_Ndom and CpdB_Ndom) contain the catalytic site, and their C-terminal domains (UshA_Cdom and CpdB_Cdom) contain a substrate-binding site responsible for specificity. Both enzymes show only partial overlap in their substrate specificities. So, it was decided to investigate the catalytic behavior of chimeras bearing the UshA catalytic domain and the CpdB specificity domain, or vice versa. UshA_Ndom-CpdB_Cdom and CpdB_Ndom-UshA_Cdom were constructed and tested on substrates specific to UshA (5'-AMP, CDP-choline, UDP-glucose) or to CpdB (3'-AMP), as well as on 2',3'-cAMP and on the common phosphodiester substrate bis-4-NPP (bis-4-nitrophenylphosphate). The chimeras did show neither 5'-nucleotidase nor 3'-nucleotidase activity. When compared to UshA, UshA_Ndom-CpdB_Cdom conserved high activity on bis-4-NPP, some on CDP-choline and UDP-glucose, and displayed activity on 2',3'-cAMP. When compared to CpdB, CpdB_Ndom-UshA_Cdom conserved phosphodiesterase activities on 2',3'-cAMP and bis-4-NPP, and gained activity on the phosphoanhydride CDP-choline. Therefore, the non-nucleotidase activities of UshA and CpdB are not fully dependent on the interplay between domains. The specificity domains may confer the chimeras some of the phosphodiester or phosphoanhydride selectivity displayed when associated with their native partners. Contrarily, the nucleotidase activity of UshA and CpdB depends strictly on the interplay between their native catalytic and specificity domains.


Assuntos
Nucleotidases/metabolismo , Sítios de Ligação , Catálise , Diester Fosfórico Hidrolases/metabolismo , Especificidade por Substrato
17.
Anticancer Drugs ; 32(7): 693-702, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-33675611

RESUMO

Lung squamous carcinoma (LUSC) is the second most frequent subtype of non-small cell lung cancer. Rarely gene alterations are identified in LUSC. Therefore, identifying LUSC-related genes to explain the relevant molecular mechanism is urgently needed. A potential biomarker, calcium-activated nucleotidase 1 (CANT1), was elevated in tissues of LUSC patients relative to normal cases based on the TCGA and/or GTEx database. CCK-8 and transwell tests were then implemented to measure the proliferative, invasive and migratory capacities, and showed that knockdown of CANT1 blocked LUSC cells proliferation. miR-607, predicted as an upstream factor for CANT1, was declined in LUSC using TargetScan analysis and luciferase activity test. Low miR-607 expression was related with unfavorable outcomes of LUSC patients. Moreover, miR-607 downregulation elevated cell viability, invasion and migration in LUSC cells, which was antagonized by si-CANT1. GEPIA website was accessed to estimate the relevance between CANT1 and epithelial-mesenchymal transition (EMT)-related positive factors. The protein levels of Fibronectin, Vimentin, Snail and ß-catenin were altered due to the abnormal CANT1 and miR-607 expression. Together, these data unveiled that miR-607/CANT1 pair may exert a vital role in the progression of LUSC through mediating EMT process, which would furnish an available therapeutic therapy for LUSC.


Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias Pulmonares/patologia , MicroRNAs/metabolismo , Nucleotidases/metabolismo , Biomarcadores Tumorais , Linhagem Celular Tumoral , Sobrevivência Celular , Regulação para Baixo , Fibronectinas/biossíntese , Regulação Neoplásica da Expressão Gênica , Humanos , Vimentina/biossíntese
18.
J Phys Chem Lett ; 12(39): 9463-9469, 2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34558897

RESUMO

A better understanding of the mechanism of oxidative DNA damage requires obtaining a molecular level description of nucleotides in various charge states. Herein, we report a systematic photoelectron spectroscopy and theoretical investigation of the electronic and geometric structures of four doubly deprotonated 2'-deoxynucleoside 5'-monophosphate dianions, the smallest quintessential DNA building block. These dianions are intrinsically stable with their adiabatic/vertical detachment energies (ADE/VDE) ranging from 0.85/1.07 (A) and 1.05/1.30 (G) to 1.20/1.50 (C) and 1.80/2.10 eV (T). The repulsive Coulomb barrier against electron detachment is 2.0 eV for purines and 2.5 eV for pyrimidines. Dianions are deprotonated at the phosphate group and the amino group of a nucleobase. The π-type HOMO orbital resides on the nucleobase moiety for each dianion. This spatial distribution of HOMO suggests that the most loosely bound electron is detached along the direction perpendicular to the nucleobase. When combined with the previous results, this work makes complete the depiction of basic building blocks of DNA at the molecular level.


Assuntos
Nucleotidases/química , Espectroscopia Fotoeletrônica , Ânions/química , Dano ao DNA , Gases/química , Conformação Molecular , Teoria Quântica
19.
J Dev Orig Health Dis ; 12(1): 124-131, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32241331

RESUMO

Prenatal exposure to mercury in utero causes abnormal foetal growth and adverse outcomes. DNA methylation is currently considered a possible mechanism through which this occurs. However, few studies have investigated the association between prenatal exposure to mercury and DNA methylation in detail. This study aimed to clarify the relationship between prenatal exposure to total mercury (Hg) and DNA methylation and its associations with sex-specific characteristics in male and female offspring. In a birth cohort study known as the Chiba study of Mother and Child Health, the DNA methylation status in cord tissue and Hg concentrations in cord serum were examined. A total of 67 participants (27 males and 40 females) were analysed based on Spearman's correlations, adjusted by a false discovery rate of the sex of each offspring. Only one methylated locus was positively correlated with Hg concentrations in cord serum in male offspring, but not in female offspring, and was annotated to the haloacid dehalogenase-like hydrolase domain-containing protein 1 (HDHD1) gene on chromosome X. This locus was located in the intron of the HDHD1 gene body and is a binding site for the zinc finger protein CCCTC-binding factor. One of the other loci, located in HDHD1, was highly methylated in the group with higher mercury concentrations, and this locus was in the gene body of HDHD1. Our results suggest that prenatal exposure to Hg might affect the epigenetic status of male foetuses.


Assuntos
Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Exposição Materna/efeitos adversos , Mercúrio/efeitos adversos , Efeitos Tardios da Exposição Pré-Natal/genética , Adulto , Cromossomos Humanos X/efeitos dos fármacos , Cromossomos Humanos X/genética , Exposição Ambiental/efeitos adversos , Exposição Ambiental/prevenção & controle , Poluentes Ambientais/efeitos adversos , Poluentes Ambientais/sangue , Feminino , Sangue Fetal/química , Feto/irrigação sanguínea , Feto/efeitos dos fármacos , Loci Gênicos/efeitos dos fármacos , Humanos , Masculino , Idade Materna , Exposição Materna/prevenção & controle , Mercúrio/sangue , Nucleotidases/efeitos dos fármacos , Nucleotidases/genética , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/prevenção & controle , Fatores Sexuais , Cordão Umbilical/efeitos dos fármacos
20.
Angew Chem Int Ed Engl ; 60(19): 10775-10783, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33325148

RESUMO

Recent demonstrations of RNA-DNA chimeras (RDNA) enabling RNA and DNA replication, coupled with prebiotic co-synthesis of deoxyribo- and ribo-nucleotides, have resurrected the hypothesis of co-emergence of RNA and DNA. As further support, we show that diamidophosphate (DAP) with 2-aminoimidazole (amido)phosphorylates and oligomerizes deoxynucleosides to form DNA-under conditions similar to those of ribonucleosides. The pyrimidine deoxynucleoside 5'-O-amidophosphates are formed in good (≈60 %) yields. Intriguingly, the presence of pyrimidine deoxynucleos(t)ides increased the yields of purine deoxynucleotides (≈20 %). Concomitantly, oligomerization (≈18-31 %) is observed with predominantly 3',5'-phosphodiester DNA linkages, and some (<5 %) pyrophosphates. Combined with previous observations of DAP-mediated chemistries and the constructive role of RDNA chimeras, the results reported here help set the stage for systematic investigation of a systems chemistry approach of RNA-DNA coevolution.


Assuntos
DNA/química , Nucleotidases/síntese química , Estrutura Molecular , Nucleotidases/química , Fosforilação
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