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1.
Nat Commun ; 11(1): 3921, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764564

RESUMO

The vacuolar-type H+-ATPases (V-ATPase) hydrolyze ATP to pump protons across the plasma or intracellular membrane, secreting acids to the lumen or acidifying intracellular compartments. It has been implicated in tumor metastasis, renal tubular acidosis, and osteoporosis. Here, we report two cryo-EM structures of the intact V-ATPase from bovine brain with all the subunits including the subunit H, which is essential for ATPase activity. Two type-I transmembrane proteins, Ac45 and (pro)renin receptor, along with subunit c", constitute the core of the c-ring. Three different conformations of A/B heterodimers suggest a mechanism for ATP hydrolysis that triggers a rotation of subunits DF, inducing spinning of subunit d with respect to the entire c-ring. Moreover, many lipid molecules have been observed in the Vo domain to mediate the interactions between subunit c, c", (pro)renin receptor, and Ac45. These two structures reveal unique features of mammalian V-ATPase and suggest a mechanism of V1-Vo torque transmission.


Assuntos
Encéfalo/enzimologia , ATPases Vacuolares Próton-Translocadoras/química , Trifosfato de Adenosina/metabolismo , Animais , Bovinos , Microscopia Crioeletrônica , Hidrólise , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , Prótons , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/ultraestrutura
2.
Proc Natl Acad Sci U S A ; 117(26): 14694-14702, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32554491

RESUMO

Innate immune cells destroy pathogens within a transient organelle called the phagosome. When pathogen-associated molecular patterns (PAMPs) displayed on the pathogen are recognized by Toll-like receptors (TLRs) on the host cell, it activates inducible nitric oxide synthase (NOS2) which instantly fills the phagosome with nitric oxide (NO) to clear the pathogen. Selected pathogens avoid activating NOS2 by concealing key PAMPs from their cognate TLRs. Thus, the ability to map NOS2 activity triggered by PAMPs can reveal critical mechanisms underlying pathogen susceptibility. Here, we describe DNA-based probes that ratiometrically report phagosomal and endosomal NO, and can be molecularly programmed to display precise stoichiometries of any desired PAMP. By mapping phagosomal NO produced in microglia of live zebrafish brains, we found that single-stranded RNA of bacterial origin acts as a PAMP and activates NOS2 by engaging TLR-7. This technology can be applied to study PAMP-TLR interactions in diverse organisms.


Assuntos
Encéfalo/enzimologia , DNA/química , Corantes Fluorescentes/química , Óxido Nítrico Sintase Tipo II , Animais , Encéfalo/metabolismo , Química Encefálica , DNA/metabolismo , Corantes Fluorescentes/metabolismo , Técnicas de Inativação de Genes , Camundongos , Microglia/química , Microglia/enzimologia , Microglia/metabolismo , Microscopia de Fluorescência , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Óxido Nítrico Sintase Tipo II/análise , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/metabolismo , Fagossomos/química , Fagossomos/metabolismo , Peixe-Zebra
3.
Toxicol Lett ; 331: 143-151, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32525014

RESUMO

Although organotin compounds are known to disturb thyroid signaling and antioxidant defense system, the sex-differences underlying these effects of triphenyltin chloride (TPT) in fish remain unclear. To understand these differences, adult zebrafish (Danio rerio) were exposed to different concentrations of TPT (0, 10, 100, or 1000 ng/L) for 28 days. Female zebrafish exposed to TPT showed significantly increased thyroxine (T4) content and decrease triiodothyronine (T3) content, possibly due to downregulation of deiodinase (dio2) and uridine diphosphate glucuronosyl transferase (ugt1ab). However, decreased T4 and T3 contents in male zebrafish accompanied with upregulation of dio1, dio2 and ugt1ab. TPT exposure can lead to sex-specific thyroid disruption in adult zebrafish via alterations the Hypothalamus-pituitary-thyroid-liver axis. In addition, the gene expression levels of metabolizing enzymes, such as cyp1b, cyp1c, gpx1a, or sult1st1 were also to vary in a sex-dependent manner in adult zebrafish liver. Downregulation of cyp19a and cyp19b and decreased 17ß-estradiol (E2) contents were detected in both female and male zebrafish. Therefore, a sex-specific of thyroid disruption response after TPT exposure was observed in adult zebrafish, possibly due to inherent in female or males detoxifying enzyme capacities.


Assuntos
Disruptores Endócrinos/toxicidade , Compostos Orgânicos de Estanho/toxicidade , Caracteres Sexuais , Glândula Tireoide/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Proteínas de Peixe-Zebra , Peixe-Zebra/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Relação Dose-Resposta a Droga , Feminino , Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , Glândula Tireoide/metabolismo , Hormônios Tireóideos/genética , Hormônios Tireóideos/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
4.
Aquat Toxicol ; 224: 105493, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32408004

RESUMO

Currently, the contamination of water with different insecticides like profenofos (PFF) is a critical concern in the aquatic ecosystem. There are limited studies available on the negative impacts of PFF on common carp fish (Cyprinus carpio L.). Therefore, the existing study was designed to investigate the effect of PFF exposure (1/10 of the 96 h-LC50) on the neurobehavior, growth performance, chemical composition, oxidative status, DNA damage, apoptotic status and histological indices of the brain and gill tissues. In addition, this study seeks to detect the ability of geranium essential oil (GEO) dietary supplementation to mitigate the negative impacts of PFF. Accordingly, a total of 120 healthy fish were divided into four groups: the control group, fed on basal diet only; the other groups were fed on a basal diet supplemented with 400 mg kg-1 GEO, basal diet and PFF in water (PFF group), and supplemented diet with GEO and PFF in water (GEO + PFF), respectively, for 60 days. The results showed that PFF significantly reduced fish growth performance, crude protein, and lipid contents. It caused several behavioral alterations including spiral movement, decreased activeness, and changes in feeding behavior. Moreover, PFF increased the DNA tail length, tail moment, and the level of 8-hydroxy-2'-deoxyguanosine. Histologically, PFF induced a wide array of circulatory, inflammatory, regressive and progressive alterations in the brain and gill tissues. PFF significantly downregulated Bcl-2 and upregulated caspase-3 immuno-expression in both organs. Further, it considerably depleted the antioxidant enzymes, including superoxide dismutase, catalase, and glutathione peroxidase. The GEO supplementation did not reach the respective control values but markedly improved most of the behavioral, physical, biochemical, oxidative, apoptotic, and inflammatory markers, altered by PFF exposure. It also protected the gill and brain tissues from the branchial and encephalopathic effects of PFF. These findings suggest that GEO dietary supplements could be advantageous for mitigating PFF negative impacts and presenting a promising feed additive for common carp in aquaculture.


Assuntos
Apoptose/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Carpas , Dano ao DNA/efeitos dos fármacos , Geranium/química , Óleos Voláteis/farmacologia , Organotiofosfatos/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Antioxidantes/metabolismo , Aquicultura , Encéfalo/enzimologia , Encéfalo/patologia , Carpas/genética , Carpas/metabolismo , Dieta , Suplementos Nutricionais , Ecossistema , Brânquias/efeitos dos fármacos , Brânquias/enzimologia , Brânquias/patologia , Óleos Voláteis/isolamento & purificação
5.
Infect Dis Poverty ; 9(1): 45, 2020 Apr 28.
Artigo em Inglês | MEDLINE | ID: covidwho-133403

RESUMO

BACKGROUND: Since its discovery in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 2 180 000 people worldwide and has caused more than 150 000 deaths as of April 16, 2020. SARS-CoV-2, which is the virus causing coronavirus disease 2019 (COVID-19), uses the angiotensin-converting enzyme 2 (ACE2) as a cell receptor to invade human cells. Thus, ACE2 is the key to understanding the mechanism of SARS-CoV-2 infection. This study is to investigate the ACE2 expression in various human tissues in order to provide insights into the mechanism of SARS-CoV-2 infection. METHODS: We compared ACE2 expression levels across 31 normal human tissues between males and females and between younger (ages ≤ 49 years) and older (ages > 49 years) persons using two-sided Student's t test. We also investigated the correlations between ACE2 expression and immune signatures in various tissues using Pearson's correlation test. RESULTS: ACE2 expression levels were the highest in the small intestine, testis, kidneys, heart, thyroid, and adipose tissue, and were the lowest in the blood, spleen, bone marrow, brain, blood vessels, and muscle. ACE2 showed medium expression levels in the lungs, colon, liver, bladder, and adrenal gland. ACE2 was not differentially expressed between males and females or between younger and older persons in any tissue. In the skin, digestive system, brain, and blood vessels, ACE2 expression levels were positively associated with immune signatures in both males and females. In the thyroid and lungs, ACE2 expression levels were positively and negatively associated with immune signatures in males and females, respectively, and in the lungs they had a positive and a negative correlation in the older and younger groups, respectively. CONCLUSIONS: Our data indicate that SARS-CoV-2 may infect other tissues aside from the lungs and infect persons with different sexes, ages, and races equally. The different host immune responses to SARS-CoV-2 infection may partially explain why males and females, young and old persons infected with this virus have markedly distinct disease severity. This study provides new insights into the role of ACE2 in the SARS-CoV-2 pandemic.


Assuntos
Betacoronavirus , Peptidil Dipeptidase A/genética , Receptores Virais/genética , Adulto , Fatores Etários , Idoso , Encéfalo/enzimologia , Sistema Cardiovascular/enzimologia , Sistema Cardiovascular/imunologia , Sistema Digestório/enzimologia , Sistema Digestório/imunologia , Glândulas Endócrinas/enzimologia , Glândulas Endócrinas/imunologia , Feminino , Perfilação da Expressão Gênica , Humanos , Sistema Imunitário/enzimologia , Interferons/imunologia , Pulmão/enzimologia , Pulmão/imunologia , Linfócitos/imunologia , Masculino , Pessoa de Meia-Idade , Especificidade de Órgãos , Peptidil Dipeptidase A/sangue , RNA-Seq , Receptores Virais/sangue , Fatores Sexuais , Sistema Urogenital/enzimologia
6.
J Stroke Cerebrovasc Dis ; 29(6): 104801, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32249206

RESUMO

BACKGROUND: Ischemic stroke is the leading cause of disability and death globally. Micro-RNAs (miRNAs) have been reported to play important roles in the development and pathogenesis of the nervous system. However, the exact function and mechanism of miRNAs have not been fully elucidated about brain damage caused by cerebral ischemia/reperfusion (I/R). METHODS: In this study, we explored the neuroprotective effects of miR-219a-5p on brain using an in vitro ischemia model (mouse neuroblastoma N2a cells treated with oxyglucose deprivation and reperfusion), and in vivo cerebral I/R model in mice. Western blot assay and Reverse Transcription-Polymerase Chain Reaction were used to check the expression of molecules involved. Flow cytometry and cholecystokinin were used to examine cell apoptosis, respectively. RESULTS: Our research shows that miR-219a-5p gradually decreases in cerebral I/R models in vivo and in vitro. In vitro I/R, we find that miR-219a-5p mimics provided evidently protection for cerebral I/R damage, as shown by increased cell viability and decreased the release of LDH and cell apoptosis. Mechanically, our findings indicate that miR-219a-5p binds to cAMP specific 3', 5'-cyclic phosphodiesterase 4D (PDE4D) mRNA in the 3'-UTR region, which subsequently leads to a decrease in Pde4d expression in I/R N2a cells. CONCLUSIONS: Our results provide new ideas for the study of the mechanism of cerebral ischemia/reperfusion injury, and lay the foundation for further research on the treatment of brain I/R injury. Upregulation of miR-219a-5p decreases cerebral ischemia/reperfusion injury by targeting Pde4d in vitro.


Assuntos
Apoptose , Encéfalo/enzimologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Infarto da Artéria Cerebral Média/enzimologia , MicroRNAs/metabolismo , Neurônios/enzimologia , Traumatismo por Reperfusão/enzimologia , Regiões 3' não Traduzidas , Animais , Sítios de Ligação , Encéfalo/patologia , Linhagem Celular Tumoral , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/patologia , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neurônios/patologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/prevenção & controle , Transdução de Sinais
7.
PLoS One ; 15(4): e0231310, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32282847

RESUMO

Shrimps can be used as indicators of the quality of aquatic systems exposed to a variety of pollutants. Chlorpyrifos is one of the most common pesticides found in environmental samples. In order to evaluate the effects of chlorpyrifos, adult organisms of Litopenaeus vannamei were exposed to two sublethal concentrations of the pesticide (0.7 and 1.3 µg/L) for four days. The LC50 (96-hours) value was determined and Lipid oxidation levels (LPO) and the activities of catalase (CAT), glutathion peroxidase (GPx), glutathion-S-transferase (GST) were assessed on the muscle, hepatopancreas and gills from the exposed organisms. In addition, inhibition of acetylcholinesterase (AChE) was determined in the brain. LC50 (96-hours) was 2.10 µg/L of chlorpyrifos. Catalase activity and LPO were elevated in the three tissues, whereas a decrease of AChE activities in the brain and an increase of GST activity in the hepatopancreas were observed.


Assuntos
Clorpirifos/toxicidade , Monitoramento Ambiental/métodos , Penaeidae/efeitos dos fármacos , Praguicidas/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Biomarcadores Ambientais , Brânquias/efeitos dos fármacos , Brânquias/enzimologia , Hepatopâncreas/efeitos dos fármacos , Hepatopâncreas/enzimologia , Dose Letal Mediana , Penaeidae/enzimologia
8.
Biochim Biophys Acta Proteins Proteom ; 1868(8): 140428, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32305689

RESUMO

Investigations of Alzheimer's disease (AD), traumatic brain injury (TBI), and related brain disorders have provided extensive evidence for involvement of cathepsin B, a lysosomal cysteine protease, in mediating the behavioral deficits and neuropathology of these neurodegenerative diseases. This review integrates findings of cathepsin B regulation in clinical biomarker studies, animal model genetic and inhibitor evaluations, structural studies, and lysosomal cell biological mechanisms in AD, TBI, and related brain disorders. The results together indicate the role of cathepsin B in the behavioral deficits and neuropathology of these disorders. Lysosomal leakage occurs in AD and TBI, and related neurodegeneration, which leads to the hypothesis that cathepsin B is redistributed from the lysosome to the cytosol where it initiates cell death and inflammation processes associated with neurodegeneration. These results together implicate cathepsin B as a major contributor to these neuropathological changes and behavioral deficits. These findings support the investigation of cathepsin B as a potential drug target for therapeutic discovery and treatment of AD, TBI, and TBI-related brain disorders.


Assuntos
Doença de Alzheimer/enzimologia , Lesões Encefálicas Traumáticas/enzimologia , Encéfalo/enzimologia , Catepsina B/genética , Transtornos Neurocognitivos/enzimologia , Neurônios/enzimologia , Adulto , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Lesões Encefálicas Traumáticas/tratamento farmacológico , Lesões Encefálicas Traumáticas/genética , Lesões Encefálicas Traumáticas/patologia , Catepsina B/antagonistas & inibidores , Catepsina B/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Criança , Citosol/efeitos dos fármacos , Citosol/enzimologia , Modelos Animais de Doenças , Feto , Regulação da Expressão Gênica , Humanos , Lactente , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Terapia de Alvo Molecular , Transtornos Neurocognitivos/tratamento farmacológico , Transtornos Neurocognitivos/genética , Transtornos Neurocognitivos/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/uso terapêutico , Transdução de Sinais
9.
Infect Dis Poverty ; 9(1): 45, 2020 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345362

RESUMO

BACKGROUND: Since its discovery in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 2 180 000 people worldwide and has caused more than 150 000 deaths as of April 16, 2020. SARS-CoV-2, which is the virus causing coronavirus disease 2019 (COVID-19), uses the angiotensin-converting enzyme 2 (ACE2) as a cell receptor to invade human cells. Thus, ACE2 is the key to understanding the mechanism of SARS-CoV-2 infection. This study is to investigate the ACE2 expression in various human tissues in order to provide insights into the mechanism of SARS-CoV-2 infection. METHODS: We compared ACE2 expression levels across 31 normal human tissues between males and females and between younger (ages ≤ 49 years) and older (ages > 49 years) persons using two-sided Student's t test. We also investigated the correlations between ACE2 expression and immune signatures in various tissues using Pearson's correlation test. RESULTS: ACE2 expression levels were the highest in the small intestine, testis, kidneys, heart, thyroid, and adipose tissue, and were the lowest in the blood, spleen, bone marrow, brain, blood vessels, and muscle. ACE2 showed medium expression levels in the lungs, colon, liver, bladder, and adrenal gland. ACE2 was not differentially expressed between males and females or between younger and older persons in any tissue. In the skin, digestive system, brain, and blood vessels, ACE2 expression levels were positively associated with immune signatures in both males and females. In the thyroid and lungs, ACE2 expression levels were positively and negatively associated with immune signatures in males and females, respectively, and in the lungs they had a positive and a negative correlation in the older and younger groups, respectively. CONCLUSIONS: Our data indicate that SARS-CoV-2 may infect other tissues aside from the lungs and infect persons with different sexes, ages, and races equally. The different host immune responses to SARS-CoV-2 infection may partially explain why males and females, young and old persons infected with this virus have markedly distinct disease severity. This study provides new insights into the role of ACE2 in the SARS-CoV-2 pandemic.


Assuntos
Betacoronavirus , Peptidil Dipeptidase A/genética , Receptores Virais/genética , Adulto , Fatores Etários , Idoso , Encéfalo/enzimologia , Sistema Cardiovascular/enzimologia , Sistema Cardiovascular/imunologia , Sistema Digestório/enzimologia , Sistema Digestório/imunologia , Glândulas Endócrinas/enzimologia , Glândulas Endócrinas/imunologia , Feminino , Perfilação da Expressão Gênica , Humanos , Sistema Imunitário/enzimologia , Interferons/imunologia , Pulmão/enzimologia , Pulmão/imunologia , Linfócitos/imunologia , Masculino , Pessoa de Meia-Idade , Especificidade de Órgãos , Peptidil Dipeptidase A/sangue , RNA-Seq , Receptores Virais/sangue , Fatores Sexuais , Sistema Urogenital/enzimologia
10.
J Stroke Cerebrovasc Dis ; 29(5): 104743, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32127256

RESUMO

BACKGROUND: Bone marrow stromal cell (BMSC) transplantation is a promising therapeutic approach for cerebral ischemia, as it elicits multiple neuroprotective effects. However, it remains unclear how BMSC transplantation modulates the ubiquitin-proteasome system (UPS) and autophagy under cerebral ischemia. METHODS: In the present study, an intermediate level of cerebral ischemia (30 minutes) was chosen to examine the effect of BMSC transplantation on the molecular switch regulating UPS and autophagy. BMSC or vehicle was stereotactically injected into the penumbra 15 minutes after sham operation or transient middle cerebral artery occlusion (tMCAO). RESULTS: Thirty minutes of tMCAO artery occlusion significantly increased TUNEL-, ubiquitin-, and p62-positive cells (which peaked at 72 hours, 2 hours, and 2 hours after reperfusion, respectively) and ratios of both BAG3/BAG1 and LC3-II/LC3-I at 24 hours after reperfusion. However, intracerebral injection of BMSCs significantly reduced infarct volume and numbers of TUNEL- and p62-positive cells, and improved BAG3/BAG1 and LC3-II/LC3-I ratios. In addition, observed increases in ubiquitin-positive cells 2 hours after reperfusion were slightly suppressed by BMSC transplantation. CONCLUSIONS: These data suggest a protective role of BMSC transplantation, which drove the molecular switch from autophagy to UPS in a murine model of ischemic stroke.


Assuntos
Autofagia , Encéfalo/enzimologia , Infarto da Artéria Cerebral Média/cirurgia , Transplante de Células-Tronco Mesenquimais , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Encéfalo/patologia , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/enzimologia , Infarto da Artéria Cerebral Média/patologia , Masculino , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição/metabolismo , Ubiquitinação
11.
Science ; 367(6483): 1240-1246, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32165585

RESUMO

In neurons, the loading of neurotransmitters into synaptic vesicles uses energy from proton-pumping vesicular- or vacuolar-type adenosine triphosphatases (V-ATPases). These membrane protein complexes possess numerous subunit isoforms, which complicates their analysis. We isolated homogeneous rat brain V-ATPase through its interaction with SidK, a Legionella pneumophila effector protein. Cryo-electron microscopy allowed the construction of an atomic model, defining the enzyme's ATP:proton ratio as 3:10 and revealing a homolog of yeast subunit f in the membrane region, which we tentatively identify as RNAseK. The c ring encloses the transmembrane anchors for cleaved ATP6AP1/Ac45 and ATP6AP2/PRR, the latter of which is the (pro)renin receptor that, in other contexts, is involved in both Wnt signaling and the renin-angiotensin system that regulates blood pressure. This structure shows how ATP6AP1/Ac45 and ATP6AP2/PRR enable assembly of the enzyme's catalytic and membrane regions.


Assuntos
Biomarcadores/química , Encéfalo/enzimologia , Receptores de Superfície Celular/química , ATPases Vacuolares Próton-Translocadoras/química , Animais , Proteínas de Bactérias/química , Biocatálise , Membrana Celular/enzimologia , Microscopia Crioeletrônica , Modelos Químicos , Domínios Proteicos , Ratos , Sistema Renina-Angiotensina , Via de Sinalização Wnt
12.
Mol Biol (Mosk) ; 54(1): 60-68, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32163389

RESUMO

A decrease in the light in autumn and winter causes depression like seasonal affective disorders (SAD) in sensitive patients, in which the serotonin (5-HT) and dopamine (DA) brain mediator systems are involved. We studied the interaction of the 5-HT and DA brain systems in an experimental SAD model in sexually mature male mice of the congenic B6-1473C and B6-1473G lines with high and low activity of tryptophan hydroxylase 2, a key enzyme of 5-HT synthesis in the brain. Mice of each line (divided into two groups of eight individuals) were kept for 30 days in standard (14 h light/10 h dark) and short (4 h light/20 h dark) daylight. The presence of the C1473G variant in the tryptophan hydroxylase 2 gene did not affect the expression of key genes of DA system: Drd1, Drd2, Scl6a3, Th, and Comt, that encode the D1 and D2 receptors, dopamine transporter, tyrosine hydroxylase, and catechol-o-methyltransferase, respectively. A decrease in the level of DA in the midbrain, as well as of its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum, was detected in B6-1473G mice. Keeping mice in short daylight did not affect expression of the Drd1 gene in all brain structures nor the expression of the Slc6a3 and Th genes in the midbrain. Drd2 expression increased in the midbrain and decreased in the hippocampus, where Comt expression increased. An increase in DA level in the midbrain and DOPAC in the striatum was detected in mice kept in short daylight. This indicates the involvement of the brain's DA system in the reaction to a decrease in daylight duration. No statistically significant effect of the interaction between the presence of the C1473G variant and daylight length on indicators of the activity of DA system was detected. No reasons were found to assert that this polymorphism determines the observed reaction of the brain DA system in keeping of animals under short daylight conditions.


Assuntos
Encéfalo/metabolismo , Dopamina/metabolismo , Fotoperíodo , Polimorfismo Genético , Triptofano Hidroxilase/genética , Animais , Encéfalo/enzimologia , Masculino , Camundongos , Triptofano Hidroxilase/metabolismo
13.
Analyst ; 145(4): 1389-1395, 2020 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-32009140

RESUMO

Nitroreductase (NTR) with a high expression level in tumors has been considered as a biomarker of highly aggressive hypoxia tumors. Thus, it is important to develop powerful tools for tumor hypoxia detection. Here, we developed a two-photon fluorescent probe hTP-NNO2 for NTR detection. The probe with one-step synthesis exhibited high yield. hTP-NNO2 showed high selectivity and sensitivity for NTR and the detection limit was as low as 43 ng mL-1. hTP-NNO2 also showed low cytotoxicity and high stability, indicating that hTP-NNO2 is suitable for NTR detection in real-time and in situ under physiological conditions. hTP-NNO2 was used for NTR imaging in hypoxia cells and the fluorescence intensity of hTP-NNO2 increased with decreasing oxygen concentration. Benefiting from the advantages of two-photon fluorescent probes, we performed NTR detection in deep brain tissue with an imaging depth of up to 100 µm. hTP-NNO2 was further successfully applied for NTR detection in zebrafish and tumors. These results indicated that we developed a promising fluorescence imaging tool for NTR detection in vitro and in vivo.


Assuntos
Encéfalo/enzimologia , Corantes Fluorescentes/química , Nitrorredutases/análise , Imagem Óptica/métodos , Hipóxia Tumoral , Células A549 , Animais , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/toxicidade , Humanos , Limite de Detecção , Camundongos , Camundongos Nus , Microscopia Confocal , Nitrorredutases/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra
14.
Biochemistry (Mosc) ; 85(1): 27-39, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32079515

RESUMO

To study the mechanisms of the non-coenzyme action of thiamine and its diphosphate (ThDP) on brain proteins, proteins of acetone extract of bovine brain synaptosomes or the homogenate of rat brain cortex were subjected to affinity chromatography on thiamine-modified Sepharose. In the step-wise eluates by thiamine (at pH 7.4 or 5.6), NaCl, and urea, the occurrence of glutamate dehydrogenase (GDH) and isoenzymes of malate dehydrogenase (MDH) along with the influence of thiamine and/or ThDP on the enzymatic activities were characterized using mass spectrometry and kinetic experiments. Maximal activation of the malate dehydrogenase reaction by thiamine is observed after the protein elution with the acidic thiamine solution, which does not elute the MDH1 isoenzyme. Effects of exogenous thiamine or ThDP on the GDH activity may depend on endogenous enzyme regulators. For example, thiamine and/or ThDP activate the brain GDH in eluates from thiamine-Sepharose but inhibit the enzyme in the crude preparations applied to the sorbent. Inhibition of GDH by ThDP is observed using the ADP-activated enzyme. Compared to the affinity chromatography employing the elution by thiamine at pH 7.4, the procedure at pH 5.6 decreases the activation of GDH by thiamine (but not ThDP) in the eluates with NaCl and urea. Simultaneously, the MDH2 content and total GDH activity are higher after the affinity elution at pH 5.6 than at pH 7.4, suggesting the role of the known interaction of GDH with MDH2 in stabilizing the activity of GDH and in the regulation of GDH by thiamine. The biological potential of thiamine-dependent regulation of the brain GDH is confirmed in vivo by demonstration of changes in regulatory properties of GDH after administration of a high dose of thiamine to rats. Bioinformatics analysis of the thiamine-eluted brain proteins shows a specific enrichment of their annotation terms with "phosphoprotein", "acetylation", and "methylation". The relationship between thiamine and the posttranslational modifications in brain may contribute to the neuroprotective effects of high doses of thiamine, including the regulation of oxidation of the major excitatory neurotransmitter in brain - glutamate.


Assuntos
Encéfalo/enzimologia , Glutamato Desidrogenase/metabolismo , Malato Desidrogenase/metabolismo , Tiamina Pirofosfato/farmacologia , Tiamina/farmacologia , Animais , Bovinos , Ativação Enzimática , Oxirredução , Ratos , Ratos Wistar
15.
Sci Adv ; 6(2): eaaw6284, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31950075

RESUMO

Zika virus (ZIKV) infection during pregnancy is associated with a spectrum of developmental impairments known as congenital Zika syndrome (CZS). The prevalence of this syndrome varies across ZIKV endemic regions, suggesting that its occurrence could depend on cofactors. Here, we evaluate the relevance of protein malnutrition for the emergence of CZS. Epidemiological data from the ZIKV outbreak in the Americas suggest a relationship between undernutrition and cases of microcephaly. To experimentally examine this relationship, we use immunocompetent pregnant mice, which were subjected to protein malnutrition and infected with a Brazilian ZIKV strain. We found that the combination of protein restriction and ZIKV infection leads to severe alterations of placental structure and embryonic body growth, with offspring displaying a reduction in neurogenesis and postnatal brain size. RNA-seq analysis reveals gene expression deregulation required for brain development in infected low-protein progeny. These results suggest that maternal protein malnutrition increases susceptibility to CZS.


Assuntos
Desnutrição/complicações , Infecção por Zika virus/congênito , Infecção por Zika virus/complicações , Animais , Animais Recém-Nascidos , Peso Corporal , Encéfalo/enzimologia , Encéfalo/patologia , Brasil/epidemiologia , Dieta com Restrição de Proteínas , Surtos de Doenças , Embrião de Mamíferos/patologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Desnutrição/virologia , Camundongos Endogâmicos C57BL , Microcefalia/complicações , Microcefalia/virologia , Neurogênese , Tamanho do Órgão , Gravidez , Síndrome , Carga Viral , Infecção por Zika virus/virologia
16.
Microvasc Res ; 129: 103973, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31891716

RESUMO

Ischemic stroke represents a major cause of adult death and severe neurological disability worldwide. Reperfusion following brain ischemia produces an inflammatory cascade that increases brain damage. In this context, matrix metalloproteinases (MMPs) play an important role as pro-inflammatory mediators. The MMP 2 up-regulation seems to promote matrix degradation, blood-brain barrier (BBB) disruption and facilitates the influx of peripheral inflammatory cells to the brain after stroke. However, there are not studies about MMP-1 in this condition. The aim of this study is to evaluate the association of brain damage, inflammatory response and the immunostaining profile of matrix metalloproteinases 1 and 2 after transient global cerebral ischemia. Mice were submitted to bilateral common carotid arterial occlusion (BCCAo) during 25 min. After three days of reperfusion, the neurological deficit score was evaluated and the animals were euthanized. Brain samples were collected in order to analyze the histopathological damage, MMPs 1 and 2 immunostaining and cytokines and chemokines levels. Ischemic group showed neurological deficits associated with brain lesions, characterized by necrotic core and penumbra zone three days after reperfusion. Higher brain immunostaining of MMP-1 and MMP-2 was observed in BCCAo samples than in sham samples. Ischemic group also exhibited increased brain levels of the cytokines tumoral necrosis factor (TNF) and interleukin 1ß (IL-1ß), chemokine (C-X-C motif) ligand 1 (CXCL1), and chemokine (C-C motif) ligand 5 (CCL5) in comparison to sham group. Our results suggest that the MMP-1 and MMP-2 raise, associated with the up-regulation of inflammatory mediators, contributes to brain damage and neurological deficits after global brain ischemia followed by three days of reperfusion in mice.


Assuntos
Encéfalo/enzimologia , Citocinas/metabolismo , Ataque Isquêmico Transitório/enzimologia , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Quimiocina CCL5/metabolismo , Quimiocina CXCL1/metabolismo , Modelos Animais de Doenças , Interleucina-1beta/metabolismo , Ataque Isquêmico Transitório/patologia , Ataque Isquêmico Transitório/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Necrose , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima
17.
Bull Exp Biol Med ; 168(3): 326-329, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31940129

RESUMO

Thermostability of rat brain lactate dehydrogenase (LDH) was studied in intact animals and animals subjected to moderate short-term hypothermia. Two exponential stages, rapid and slow, were distinguished in the thermodenaturation kinetics. The contribution of the rapid phase to the lactate dehydrogenase denaturation kinetics was more significant: the energy of activation for this phase was 2.33 times lower than that for the slow phase. Moderate shortterm hypothermia led to a significant decrease of lactate dehydrogenase thermostability: thermodenaturation rate constants for the rapid (k1) and slow (k2) phases increased. Significant changes in parameters a and b reflecting the initial proportion of the two native forms of the enzyme developed only at 40°C. As hypothermia caused no appreciable changes in the energy of activation of lactate dehydrogenase denaturation, a significant contribution of the entropic factor to the decrease of free energy of enzyme denaturation was hypothesized. The data indicated significant labilization of lactate dehydrogenase structure under conditions of moderate hypothermia.


Assuntos
Encéfalo/enzimologia , L-Lactato Desidrogenase/metabolismo , Animais , Estabilidade Enzimática/fisiologia , Hipotermia Induzida , Masculino , Ratos , Temperatura
18.
J Agric Food Chem ; 68(8): 2547-2553, 2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-31995978

RESUMO

Anticholinergic organophosphate (OP) agents act on the diverse serine hydrolases, thereby revealing unexpected biological effects. Epidemiological studies indicate a relationship between the OP exposure and development of attention-deficit/hyperactivity disorder (ADHD)-like symptoms, whereas no plausible mechanism for the OP-induced ADHD has been established. The present investigation employs ethyl octylphosphonofluoridate (EOPF) as an OP-probe, which is an extremely potent inhibitor of endocannabinoid (EC, anandamide and 2-arachidonoylglycerol)-hydrolyzing enzymes: that is, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). An ex vivo experiment shows that EOPF treatment decreases FAAH and MAGL activities and conversely increases EC levels in the rat brain. Subsequently, EOPF (treated intraperitoneally once at 0, 1, 2, or 3 mg/kg) clearly induces ADHD-like behaviors (in elevated plus-maze test) in both Wistar and spontaneously hypertensive rats. The EOPF-induced behaviors are reduced by a concomitant administration of cannabinoid receptor inverse agonist SLV-319. Accordingly, the EC system is a feasible target for OP-caused ADHD-like behaviors in adolescent rats.


Assuntos
Amidoidrolases/antagonistas & inibidores , Transtorno do Deficit de Atenção com Hiperatividade/enzimologia , Encéfalo/enzimologia , Endocanabinoides/metabolismo , Inibidores Enzimáticos/efeitos adversos , Monoacilglicerol Lipases/antagonistas & inibidores , Compostos Organofosforados/efeitos adversos , Amidoidrolases/metabolismo , Animais , Transtorno do Deficit de Atenção com Hiperatividade/induzido quimicamente , Transtorno do Deficit de Atenção com Hiperatividade/metabolismo , Transtorno do Deficit de Atenção com Hiperatividade/psicologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Humanos , Masculino , Monoacilglicerol Lipases/metabolismo , Ratos , Ratos Wistar
19.
Nat Commun ; 11(1): 78, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911626

RESUMO

The SLC12A cation-Cl- cotransporters (CCC), including NKCC1 and the KCCs, are important determinants of brain ionic homeostasis. SPAK kinase (STK39) is the CCC master regulator, which stimulates NKCC1 ionic influx and inhibits KCC-mediated efflux via phosphorylation at conserved, shared motifs. Upregulation of SPAK-dependent CCC phosphorylation has been implicated in several neurological diseases. Using a scaffold-hybrid strategy, we develop a novel potent and selective SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide ("ZT-1a"). ZT-1a inhibits NKCC1 and stimulates KCCs by decreasing their SPAK-dependent phosphorylation. Intracerebroventricular delivery of ZT-1a decreases inflammation-induced CCC phosphorylation in the choroid plexus and reduces cerebrospinal fluid (CSF) hypersecretion in a model of post-hemorrhagic hydrocephalus. Systemically administered ZT-1a reduces ischemia-induced CCC phosphorylation, attenuates cerebral edema, protects against brain damage, and improves outcomes in a model of stroke. These results suggest ZT-1a or related compounds may be effective CCC modulators with therapeutic potential for brain disorders associated with impaired ionic homeostasis.


Assuntos
Encéfalo/metabolismo , Inibidores Enzimáticos/administração & dosagem , Hidrocarbonetos Clorados/administração & dosagem , Nitrilos/administração & dosagem , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/genética , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/metabolismo
20.
Adv Exp Med Biol ; 1232: 339-345, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31893429

RESUMO

We used a miniature broadband NIRS system to monitor concentration changes in brain oxygenation (oxy- and deoxy- haemoglobin [HbO2], [HHb]) and oxidised cytochrome-c-oxidase ([oxCCO]) during a high +Gz acceleration, induced by a human centrifuge, on two healthy experienced volunteers (2 male, 34 and 37 years). We performed a sequence of several +Gz exposures that were terminated at the onset of visual symptoms (loss of peripheral vision). Systemic parameters were recorded (i.e. heart rate, blood pressure and arterial saturation), and brain tissue blood volume changes ([HbT] = [HbO2] + [HHb]) and oxygen delivery ([HbDiff] = [HbO2] - [HHb]) were calculated. Volunteer 1 demonstrated a decrease in [HbT] of -3.49 ± 0.02 µMol and [HbDiff] of -3.23 ± 0.44 µMol, and an increase of [oxCCO] of 0.42 ± 0.01µMol. Volunteer 2 demonstrated a decrease in [HbDiff] of -4.37 ± 0.23 µMol, and no significant change in [HbT] (0.53 ± 0.06 µMol) and [oxCCO] (0.09 ± 0.06 µMol). The variability of the brain metabolic response was related to the level of ischaemia, suggesting that suppression of metabolism was due to lack of glucose substrate delivery rather than oxygen availability.


Assuntos
Aceleração , Complexo IV da Cadeia de Transporte de Elétrons , Hemodinâmica , Espectroscopia de Luz Próxima ao Infravermelho , Adulto , Encéfalo/enzimologia , Encéfalo/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Voluntários Saudáveis , Humanos , Masculino , Estresse Oxidativo , Oximetria/instrumentação , Oxigênio/metabolismo
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