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2.
Anim Genet ; 51(1): 95-100, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31633210

RESUMO

In previous GWAS carried out in a Duroc commercial line (Lipgen population), we detected on pig chromosomes 3, 4 and 14 several QTL for gluteus medius muscle redness (GM a*), electric conductivity in the longissimus dorsi muscle (LD CE) and vaccenic acid content in the LD muscle (LD C18:1 n - 7), respectively. We have genotyped, in the Lipgen population, 19 SNPs mapping to 14 genes located within these QTL. Subsequently, association analyses have been performed. After correction for multiple testing, two SNPs in the TGFBRAP1 (rs321173745) and SELENOI (rs330820437) genes were associated with GM a*, whereas ACADSB (rs81449951) and GPR26 (rs343087568) genotypes displayed significant associations with LD vaccenic content. Moreover, the polymorphisms located at the ATP1A2 (rs344748241), ATP8B2 (rs81382410) and CREB3L4 (rs321278469 and rs330133789) genes showed significant associations with LD CE. We made a second round of association analyses including the SNPs mentioned above as well as other SNPs located in the chromosomes to which they map. After performing a correction for multiple testing, the only association that remained significant at the chromosome-wide level was that between the ATP1A2 genotype and LD CE. From a functional point of view, this association is meaningful because this locus encodes a subunit of the Na+ /K+ -ATPase responsible for maintaining an electrochemical gradient across the plasma membrane.


Assuntos
Condutividade Elétrica , Músculo Esquelético/fisiologia , Locos de Características Quantitativas , Carne Vermelha , ATPase Trocadora de Sódio-Potássio/genética , Sus scrofa/genética , Animais , Estudos de Associação Genética/veterinária , Genótipo
3.
Cell Prolif ; 53(1): e12732, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31746080

RESUMO

OBJECTIVES: Temozolomide (TMZ) is one of the most commonly used clinical drugs for glioblastoma (GBM) treatment, but its drug sensitivity needs to be improved. Gamabufotalin (CS-6), the primary component of the traditional Chinese medicine "ChanSu," was shown to have strong anti-cancer activity. However, more efforts should be directed towards reducing its toxicity or effective treatment doses. METHODS: Target fishing experiment, Western blotting, PCR, confocal immunofluorescence and molecular cloning techniques were performed to search for possible downstream signalling pathways. In addition, GBM xenografts were used to further determine the potential molecular mechanisms of the synergistic effects of CS-6 and TMZ in vivo. RESULTS: Mechanistic research revealed a negative feedback loop between ATP1A3 and AQP4 through which CS-6 inhibited GBM growth and mediated the synergistic treatment effect of CS-6 and TMZ. In addition, by mutating potential amino acid residues of ATP1A3, which were predicted by modelling and docking to interact with CS-6, we demonstrated that abrogating hydrogen bonding of the amino acid Thr794 interferes with the activation of ATP1A3 by CS-6 and that the Thr794Ala mutation directly affects the synergistic treatment efficacy of CS-6 and TMZ. CONCLUSIONS: As the main potential target of CS-6, ATP1A3 activation critically depends on the hydrogen bonding of Thr794 with CS-6. The combination of CS-6 and TMZ could significantly reduce the therapeutic doses and promote the anti-cancer efficacy of CS-6/TMZ monotherapy.


Assuntos
Aquaporina 4/metabolismo , Bufanolídeos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Proteínas de Neoplasias/biossíntese , ATPase Trocadora de Sódio-Potássio/biossíntese , Temozolomida/farmacologia , Animais , Aquaporina 4/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Camundongos , Proteínas de Neoplasias/genética , ATPase Trocadora de Sódio-Potássio/genética , Ensaios Antitumorais Modelo de Xenoenxerto
4.
BMJ Case Rep ; 12(10)2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31586957

RESUMO

Hemiplegic migraine (HM) is a rare subtype of migraine with aura in which attacks include transient motor weakness or hemiparesis that can last several days. HM is linked to mutations in three different genes, CACNA1A, ATP1A2 and SCN1A, which encode for ion transporters. The clinical spectrum includes atypical symptoms such as impaired consciousness, epileptic seizures, permanent cerebellar ataxia or mental retardation. We describe a novel mutation found in the ATP1A2 gene in a patient with late-onset HM. His attacks were characterised by motor weakness associated with altered mental status, diplopia and ataxia. He also showed up MRI abnormalities and incomplete response to prophylactic therapy with verapamil. Late-onset HM should be considered among the possible causes of focal neurological deficits even in older patients with cerebrovascular risk factors when a stroke appears to be more likely.


Assuntos
Enxaqueca com Aura/diagnóstico , ATPase Trocadora de Sódio-Potássio/genética , Idoso , Diagnóstico Diferencial , Humanos , Imagem por Ressonância Magnética , Masculino , Enxaqueca com Aura/diagnóstico por imagem , Enxaqueca com Aura/genética , Mutação de Sentido Incorreto , Exame Neurológico , Linhagem
5.
Arch Anim Nutr ; 73(6): 505-516, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31617788

RESUMO

A growth trial was performed to optimise the inclusion of potassium (K) in feeds of Heteropneustes fossilis (body weight [BW] 6.92 ± 0.1 g). Eight isonitrogenous and isoenergetic diets with varying dietary K levels were prepared by supplementing 0, 1.91, 3.82, 5.73, 7.64, 9.55, 11.46 and 13.37 g KCl/kg basal diet. Analysed dietary K levels were 0.16, 1.12, 2.08, 3.19, 4.18, 5.16, 6.11, 7.14 and 8.16 g/kg dry matter. BW gain, feed conversion ratio (FCR), protein gain (PG) and gill Na+/K+-ATPase activity were best in fish fed 4.18 g K/kg diet. The K concentrations in the whole body and vertebrae increased linearly with the increase up to 5.16 g K/kg diet and reached then a plateau. The K-retention [%] was highest in fish fed the basal diet and decreased with the further inclusion of dietary K up to 2.08 g/kg followed by no change up to diet containing 4.18 g K/kg and then declined further in fish fed higher levels of dietary K. Serum alkaline phosphatase activity was found to increase up to 4.18 g K/kg diet. Regression of BW gain, PG, gill Na+/K+-ATPase and vertebrae K concentration against varying levels of dietary K using broken-line model indicated that an inclusion of 5.44 g K/kg diet is the optimum for maximising growth and mineralisation of H. fossilis.


Assuntos
Peixes-Gato/fisiologia , Proteínas de Peixes/genética , Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal/efeitos dos fármacos , Animais , Composição Corporal/efeitos dos fármacos , Peixes-Gato/crescimento & desenvolvimento , Dieta/veterinária , Suplementos Nutricionais/análise , Relação Dose-Resposta a Droga , Proteínas de Peixes/metabolismo , Potássio/administração & dosagem , ATPase Trocadora de Sódio-Potássio/metabolismo
6.
Int J Mol Sci ; 20(18)2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31540261

RESUMO

The Na+, K+-ATPase transports Na+ and K+ across the membrane of all animal cells. In addition to its ion transporting function, the Na+, K+-ATPase acts as a homotypic epithelial cell adhesion molecule via its ß1 subunit. The extracellular region of the Na+, K+-ATPase ß1 subunit includes a single globular immunoglobulin-like domain. We performed Molecular Dynamics simulations of the ectodomain of the ß1 subunit and a refined protein-protein docking prediction. Our results show that the ß1 subunit Ig-like domain maintains an independent structure and dimerizes in an antiparallel fashion. Analysis of the putative interface identified segment Lys221-Tyr229. We generated triple mutations on YFP-ß1 subunit fusion proteins to assess the contribution of these residues. CHO fibroblasts transfected with mutant ß1 subunits showed a significantly decreased cell-cell adhesion. Association of ß1 subunits in vitro was also reduced, as determined by pull-down assays. Altogether, we conclude that two Na+, K+-ATPase molecules recognize each other by a large interface spanning residues 221-229 and 198-207 on their ß1 subunits.


Assuntos
Mutagênese Sítio-Dirigida , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Motivos de Aminoácidos , Animais , Células CHO , Cricetulus , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , ATPase Trocadora de Sódio-Potássio/genética
7.
PLoS Pathog ; 15(8): e1007963, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31381610

RESUMO

Human respiratory syncytial virus (RSV) is the leading viral cause of acute pediatric lower respiratory tract infections worldwide, with no available vaccine or effective antiviral drug. To gain insight into virus-host interactions, we performed a genome-wide siRNA screen. The expression of over 20,000 cellular genes was individually knocked down in human airway epithelial A549 cells, followed by infection with RSV expressing green fluorescent protein (GFP). Knockdown of expression of the cellular ATP1A1 protein, which is the major subunit of the Na+,K+-ATPase of the plasma membrane, had one of the strongest inhibitory effects on GFP expression and viral titer. Inhibition was not observed for vesicular stomatitis virus, indicating that it was RSV-specific rather than a general effect. ATP1A1 formed clusters in the plasma membrane very early following RSV infection, which was independent of replication but dependent on the attachment glycoprotein G. RSV also triggered activation of ATP1A1, resulting in signaling by c-Src-kinase activity that transactivated epidermal growth factor receptor (EGFR) by Tyr845 phosphorylation. ATP1A1 signaling and activation of both c-Src and EGFR were found to be required for efficient RSV uptake. Signaling events downstream of EGFR culminated in the formation of macropinosomes. There was extensive uptake of RSV virions into macropinosomes at the beginning of infection, suggesting that this is a major route of RSV uptake, with fusion presumably occurring in the macropinosomes rather than at the plasma membrane. Important findings were validated in primary human small airway epithelial cells (HSAEC). In A549 cells and HSAEC, RSV uptake could be inhibited by the cardiotonic steroid ouabain and the digitoxigenin derivative PST2238 (rostafuroxin) that bind specifically to the ATP1A1 extracellular domain and block RSV-triggered EGFR Tyr845 phosphorylation. In conclusion, we identified ATP1A1 as a host protein essential for macropinocytic entry of RSV into respiratory epithelial cells, and identified PST2238 as a potential anti-RSV drug.


Assuntos
Pinocitose , Infecções por Vírus Respiratório Sincicial/complicações , Vírus Sincicial Respiratório Humano/patogenicidade , Infecções Respiratórias/prevenção & controle , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Proteínas Virais/metabolismo , Internalização do Vírus , Células A549 , Cardiotônicos/farmacologia , Digitoxigenina/química , Digitoxigenina/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/virologia , Receptores ErbB/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Ouabaína/farmacologia , RNA Interferente Pequeno/genética , Infecções por Vírus Respiratório Sincicial/virologia , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/enzimologia , Sistema Respiratório/virologia , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/virologia , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/genética , Proteínas Virais/genética
8.
Mol Biol Rep ; 46(5): 5273-5286, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31414310

RESUMO

The investigation was carried out to analyse the genetic polymorphism and gene expression of ATP1A1 gene in four different Indian goat breeds by using high resolution melting (HRM) and real time-PCR. ATPase is electro-genic ion pump which is maintains the balance of sodium and potassium ions in animal cells. The transport of Na+& K+ is variable at cellular level during extreme hot period. Therefore, susceptible and tolerant animals were selected based on the physiological responses during hot period. Blood samples were collected from individuals, DNA was isolated. The 300 bp fragment of ATP1A1 gene was amplified by PCR and HRM genotyping was performed. The melting curves were analysed, differential temperature-shift plot showed three different genotypes in all the analysed samples. Out of the 135 samples, the distribution percentages were 55.56% (AA/blue), 33.33% (AC/red) and 11.11% (CC/green). The sequence variation revealed a SNP at 143rd position (A>C). The nucleotide diversity was 0.695 ± 0.403, 0.732 ± 0.424, 0.662 ± 0.433 and 0.687 ± 0.398 in Barbari, Jamunapari, Jakharna and Sirohi, respectively. The respiration rate (RR) was significantly different (P < 0.05) between AA and AC (t = 1.875, df = 38) genotype and heart rate (HR) was significantly different (P < 0.05) between AA and CC genotype. The relative expression pattern of ATP1A1 in SNP variants and non-variants animal tissues showed 19.09 and 6.93 fold higher than control (non-variant), respectively. Jamunapari showed higher fold value of ATP1A1 gene in comparison to Barbari, Jakharna and Sirohi. However, the heat stress-susceptible phenotype had significantly higher gene expression than stress-tolerant in all the breeds. The variation may be used as a marker for selection on the basis of physiological parameters and expression of ATP1A1 gene in goats indicating the specificity of expression in each tissue.


Assuntos
Técnicas de Genotipagem/veterinária , Cabras/genética , Polimorfismo de Nucleotídeo Único , ATPase Trocadora de Sódio-Potássio/genética , Estresse Fisiológico , Animais , Cruzamento , Éxons , Feminino , Regulação da Expressão Gênica , Cabras/classificação , Temperatura Alta , Masculino , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
Nat Commun ; 10(1): 3765, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31434886

RESUMO

Of the 1.8 billion people worldwide infected with Mycobacterium tuberculosis, 5-15% will develop active tuberculosis (TB). Approximately half will progress to active TB within the first 18 months after infection, presumably because they fail to mount an effective initial immune response. Here, in a genome-wide genetic study of early TB progression, we genotype 4002 active TB cases and their household contacts in Peru. We quantify genetic heritability ([Formula: see text]) of early TB progression to be 21.2% (standard error 0.08). This suggests TB progression has a strong genetic basis, and is comparable to traits with well-established genetic bases. We identify a novel association between early TB progression and variants located in a putative enhancer region on chromosome 3q23 (rs73226617, OR = 1.18; P = 3.93 × 10-8). With in silico and in vitro analyses we identify rs73226617 or rs148722713 as the likely functional variant and ATP1B3 as a potential causal target gene with monocyte specific function.


Assuntos
Progressão da Doença , Mycobacterium tuberculosis/patogenicidade , Tuberculose/genética , Adulto , Feminino , Expressão Gênica , Loci Gênicos , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Masculino , Monócitos , Mycobacterium tuberculosis/genética , Peru , ATPase Trocadora de Sódio-Potássio/genética
10.
Zygote ; 27(5): 337-346, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31405390

RESUMO

The aim of this study was to evaluate the effects of different timing for frozen-thawed bovine ampullary epithelial cell (BAEC) and bovine oviductal epithelial cell (BOEC) co-culture on the development and quality of bovine embryos produced in vitro. Embryo development was assessed by day 8 blastocyst yield, whereas embryo quality was determined using blastocyst differential cell count, cryotolerance and the expression of selected genes related to embryo quality. The results showed that the presence of BAECs during the last 6 h of in vitro maturation (IVM) increased blastocyst yield and survival of the vitrified-warmed blastocysts. In addition, embryos produced in the presence of BAECs during the last 6 h of IVM or in the presence of BOECs during the first 4 days of in vitro culture (IVC) showed a greater number of trophectoderm cells and a greater inner cell mass. In terms of gene expression, IFN-T was downregulated and PLAC8, AQP3 and ATP1A1 were upregulated in the presence of the BAECs during the last 6 h of the IVM and/or in the presence of BOECs during the first 4 days of IVC. In conclusion, co-culturing bovine oocytes with a frozen-thawed ampullary cell monolayer during the last 6 h of maturation increased blastocyst yield and quality.


Assuntos
Blastocisto/citologia , Blastocisto/fisiologia , Criopreservação , Técnicas de Cultura Embrionária/métodos , Oviductos/citologia , Animais , Aquaporina 3/genética , Bovinos , Técnicas de Cocultura , Células Epiteliais , Feminino , Fertilização In Vitro , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Maturação in Vitro de Oócitos/métodos , Masculino , ATPase Trocadora de Sódio-Potássio/genética , Proteína X Associada a bcl-2/genética
11.
Zhonghua Er Ke Za Zhi ; 57(7): 543-547, 2019 Jul 02.
Artigo em Chinês | MEDLINE | ID: mdl-31269555

RESUMO

Objective: To characterize fever-induced paroxysmal weakness and encephalopathy (FIPWE) caused by ATP1A3 gene pathogenic variant. Methods: Phenotypic and genotypic characteristics of 4 FIPWE patients (3 boys and 1 girl), who were ascertained from October 2016 to March 2018 in Beijing Children's Hospital due to ATP1A3 heterozygous variants, were retrospectively analyzed. The whole exsome sequencing was used for genetic testing. Results: The onset ages of 4 patients were 2 years and 9 months, 2 years and 4 months, 8 months, 2 years and 5 months respectively. The episode ranged from 1 to 3 times, and at 3 months to 2 years and 10 months intervals. All 4 patients had symptoms of limb weakness and encephalopathy, accompanied with mild to severe ataxia or athetosis. The tendon reflex was absent in all patients, and the Babinski's sign was positive. Three patients had dysphagia and 3 patients had slurred speech. Three patients had abnormal eye movements, including strabismus and opsoclonus. None of the 4 patients exhibited visual impairment, auditory impairment or talipes cavus. The duration of acute phase ranged from 1 week to 3 months. In 3 relapsing patients, symptoms became progressively worse, with relapses occurring frequently and recovery being more difficult, and various sequelae were found after the last relapse. All patients carried heterozygous variant in ATP1A3 gene. The missense variants result in the substitution of an arginine residue at position 756. Three variants were identified, including C. 2267G > T (p. R756L) (1 case), C. 2266C > T (p. R756C) (2 cases), and C. 2267G > A (p. R756H) (1 case). Three were de novo and one inherited from his father, but the grandparents did not carry the variant. All variants were reported as pathogenic. Conclusions: FIPWE is one of new clinical phenotypes of ATP1A3 spectrum disease and most cases are sporadic. The missense variants result in the substitution of an arginine residue at position 756. This report provided insights into the phenotype-genotype association in patients with FIPWE caused by pathogenic variants of ATP1A3.


Assuntos
Encefalopatias/etiologia , Febre/complicações , Febre/genética , Debilidade Muscular/complicações , Mutação/genética , ATPase Trocadora de Sódio-Potássio/genética , Criança , Pré-Escolar , Feminino , Testes Genéticos , Genótipo , Humanos , Masculino , Fenótipo , Estudos Retrospectivos
12.
Int J Mol Sci ; 20(13)2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261667

RESUMO

The plasma membrane of sperm contains highly dynamic lipid microdomains (rafts), which house signaling proteins with a role in regulating capacitation. We reported that ATP1A4, the testis-specific isoform of Na/K-ATPase, interacted with caveolin-1, Src, epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinases 1/2 (ERK1/2) in raft and non-raft domains of the plasma membrane of bovine sperm during capacitation. The objective of the present study was to use a proteomic approach to characterize the ATP1A4 interactome in rafts and non-rafts from capacitated bovine sperm. The non-raft interactome included hexokinase 1, plakophilin 1, desmoglein 1, 14-3-3 protein ζ/δ, cathepsin D and heat shock protein beta1 proteins exclusively, whereas glutathione S-transferase and annexin A2 were unique to raft interactome. However, a disintegrin and metalloprotease 32 (ADAM 32), histone H4, actin, acrosin, serum albumin and plakoglobin were identified in both raft and non-raft fractions of capacitated sperm. Based on gene ontology studies, these differentially interacted proteins were implicated in cell-cell adhesion, signal transduction, fertilization, metabolism, proteolysis and DNA replication, in addition to acting as transport/carrier and cytoskeletal proteins. Overall, we identified proteins not previously reported to interact with ATP1A4; furthermore, we inferred that ATP1A4 may have a role in sperm capacitation.


Assuntos
Microdomínios da Membrana/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Espermatozoides/metabolismo , Animais , Anexina A2/metabolismo , Catepsina D/metabolismo , Bovinos , Desmogleínas/metabolismo , Glutationa Transferase/metabolismo , Proteínas de Choque Térmico/metabolismo , Hexoquinase/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Placofilinas/metabolismo , Ligação Proteica , ATPase Trocadora de Sódio-Potássio/genética
14.
J Therm Biol ; 83: 165-171, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31331515

RESUMO

Temperature is a key factor that influences fish metabolism. Therefore, it is important to understand how temperature variation affects lipid metabolism and energy consumption in fish. We determined respiration frequency, Na+/K+-ATPase activity, and lipid metabolism in common carp (Cyprinus carpio) after 18 days of temperature stress (5 °C and 30 °C, with 17 °C as the control). We found that respiratory frequency was positively correlated with water temperature (p < 0.01), whereas red blood cell count and mean corpuscular hemoglobin content and concentration were negatively correlated with temperature. In liver and heart, triglyceride (TG) levels were lower in both stress groups (5 °C and 30 °C) than in the control (17 °C) (p < 0.01), and highest in muscle from the high temperature stress group (p < 0.01). The non-esterified fatty acid concentration was negatively correlated with TG levels. In brain, lipoprotein lipase (LPL) activity decreased with increasing temperature and hepatic lipase (HL) activity was lower in both stress groups than in the control (p < 0.01). In muscle, the activity of LPL and HL was lowest in the high temperature stress group, resulting in a significant increase in TG levels. Na+/K+-ATPase activity in heart was lower in both stress groups than in the control (p < 0.01). The expression of LPL mRNA in heart increased with increasing temperature (p < 0.01), whereas LPL mRNA expression in brain and liver increased in both stress groups (p < 0.01). Our results show that temperature can significantly affect lipid metabolism in common carp, and that different tissues respond differently to changes in temperature.


Assuntos
Carpas/fisiologia , Resposta ao Choque Frio , Proteínas de Peixes/metabolismo , Resposta ao Choque Térmico , Metabolismo dos Lipídeos , Respiração , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Encéfalo/metabolismo , Carpas/genética , Carpas/metabolismo , Fígado/metabolismo , ATPase Trocadora de Sódio-Potássio/genética
15.
Cells ; 8(6)2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-31208048

RESUMO

Na-K-ATPase on the basolateral membrane provides the favorable transcellular Na gradient for the proper functioning of Na-dependent nutrient co-transporters on the brush border membrane (BBM) of enterocytes. As cells mature from crypts to villus, Na-K-ATPase activity doubles, to accommodate for the increased BBM Na-dependent nutrient absorption. However, the mechanism of increased Na-K-ATPase activity during the maturation of enterocytes is not known. Therefore, this study aimed to determine the mechanisms involved in the functional transition of Na-K-ATPase during the maturation of crypts to villus cells. Na-K-ATPase activity gradually increased as IEC-18 cells matured in vitro from day 0 (crypts) through day 4 (villus) of post-confluence. mRNA abundance and Western blot studies showed no change in the levels of Na-K-ATPase subunits α1 and ß1 from 0 to 4 days post-confluent cells. However, Na-K-ATPase α1 phosphorylation levels on serine and tyrosine, but not threonine, residues gradually increased. These data indicate that as enterocytes mature from crypt-like to villus-like in culture, the functional activity of Na-K-ATPase increases secondary to altered affinity of the α1 subunit to extracellular K+, in order to accommodate the functional preference of the intestinal cell type. This altered affinity is likely due to increased phosphorylation of the α1 subunit, specifically at serine and tyrosine residues.


Assuntos
Diferenciação Celular , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Intestinos/citologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Glucose/metabolismo , Cinética , Fosforilação , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/genética
16.
Int J Mol Sci ; 20(9)2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31035668

RESUMO

Adhesion is a crucial characteristic of epithelial cells to form barriers to pathogens and toxic substances from the environment. Epithelial cells attach to each other using intercellular junctions on the lateral membrane, including tight and adherent junctions, as well as the Na+,K+-ATPase. Our group has shown that non-adherent chinese hamster ovary (CHO) cells transfected with the canine ß1 subunit become adhesive, and those homotypic interactions amongst ß1 subunits of the Na+,K+-ATPase occur between neighboring epithelial cells. Ouabain, a cardiotonic steroid, binds to the α subunit of the Na+,K+-ATPase, inhibits the pump activity and induces the detachment of epithelial cells when used at concentrations above 300 nM. At nanomolar non-inhibiting concentrations, ouabain affects the adhesive properties of epithelial cells by inducing the expression of cell adhesion molecules through the activation of signaling pathways associated with the α subunit. In this study, we investigated whether the adhesion between ß1 subunits was also affected by ouabain. We used CHO fibroblasts stably expressing the ß1 subunit of the Na+,K+-ATPase (CHO ß1), and studied the effect of ouabain on cell adhesion. Aggregation assays showed that ouabain increased the adhesion between CHO ß1 cells. Immunofluorescence and biotinylation assays showed that ouabain (50 nM) increases the expression of the ß1 subunit of the Na+,K+-ATPase at the cell membrane. We also examined the effect of ouabain on the activation of signaling pathways in CHO ß1 cells, and their subsequent effect on cell adhesion. We found that cSrc is activated by ouabain and, therefore, that it likely regulates the adhesive properties of CHO ß1 cells. Collectively, our findings suggest that the ß1 subunit adhesion is modulated by the expression levels of the Na+,K+-ATPase at the plasma membrane, which is regulated by ouabain.


Assuntos
Adesão Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Ouabaína/farmacologia , Subunidades Proteicas/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Células CHO , Membrana Celular/metabolismo , Cricetulus , Expressão Gênica , Ligação Proteica , Subunidades Proteicas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genética , Quinases da Família src/metabolismo
17.
Nat Commun ; 10(1): 1993, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31040285

RESUMO

Microbial rhodopsins are photoreceptive membrane proteins that transport various ions using light energy. While they are widely used in optogenetics to optically control neuronal activity, rhodopsins that function with longer-wavelength light are highly demanded because of their low phototoxicity and high tissue penetration. Here, we achieve a 40-nm red-shift in the absorption wavelength of a sodium-pump rhodopsin (KR2) by altering dipole moment of residues around the retinal chromophore (KR2 P219T/S254A) without impairing its ion-transport activity. Structural differences in the chromophore of the red-shifted protein from that of the wildtype are observed by Fourier transform infrared spectroscopy. QM/MM models generated with an automated protocol show that the changes in the electrostatic interaction between protein and chromophore induced by the amino-acid replacements, lowered the energy gap between the ground and the first electronically excited state. Based on these insights, a natural sodium pump with red-shifted absorption is identified from Jannaschia seosinensis.


Assuntos
Luz , Rodopsina/genética , ATPase Trocadora de Sódio-Potássio/genética , Chlamydomonas/metabolismo , Humanos , Mutação/genética , Teoria Quântica , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática
18.
Fish Physiol Biochem ; 45(4): 1217-1232, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31069608

RESUMO

We aim to study the optimum salinity concentration for Nile tilapia, through the assessment of its growth performance and the expression of its related genes (Gh and Igf-1), as well as its salinity adaptation and immune status through the assessment of the gene expression of ion-regulation genes (Na+/K+-ATPase α-1a and α-1b), stress-related genes (GST, HSP27, and HSP70), inflammatory-related genes (IL1, IL8, CC, and CXC chemokine), and immune-related genes (IgMH TLR7, MHC, and MX) at the osmoregulatory organs (gills, liver, and kidney). Based on the least mortality percentage and the physical appearance of the fish, three salt concentrations (6, 16, and 20 ppt) were chosen following a 6-month preliminary study using serial salt concentrations ranged from 6 to 36 ppt, which were obtained by rearing the fish in gradual elevated pond salinity through daily addition of 0.5 ppt saline water. The fish size was 10.2-12 cm and weight was 25.5-26.15 g. No significant differences in the fish weight gain were observed among the studied groups. The group reared at 16-ppt salt showed better performance than that of 20 ppt, as they have lower morality % and higher expression of ion-regulated gene (Na+/K+-ATPase α1-b), stress-related genes (GST, HSP27, and HSP70) of the gills and also GST, inflammatory-related genes (IL-1ß and IL8), and TLR in the liver tissue. Higher expression of kidney-immune-related genes at 20-ppt salt may indicate that higher salinity predispose to fish infection and increased mortality. We concluded that 16-ppt salinity concentration is suitable for rearing O. niloticus as the fish are more adaptive to salinity condition without changes in their growth rate. Also, we indicate the use of immune stimulant feed additive to overcome the immune suppressive effect of hyper-salinity. Additionally, the survival of some fish at higher salinity concentrations (30-34 ppt) increase the chance for selection for salinity resistance in the Nile tilapia.


Assuntos
Ciclídeos/fisiologia , Proteínas de Peixes/genética , Salinidade , Adaptação Fisiológica/genética , Adaptação Fisiológica/imunologia , Animais , Encéfalo/metabolismo , Citocinas/genética , Brânquias/metabolismo , Glutationa Transferase/genética , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP70/genética , Inflamação/genética , Rim/metabolismo , Fígado/metabolismo , RNA Mensageiro/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Receptor 7 Toll-Like/genética
19.
J Steroid Biochem Mol Biol ; 192: 105381, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31128249

RESUMO

Hypothermal stress changes the balance of osmoregulation by affecting Na+, K+-ATPase (Na-K-ATPase) activity or inducing modulation to epithelium permeability in fish. Meanwhile, cellular concentrations of cortisol can be modulated by the pre-receptor enzymes 11ß-hydroxysteroid dehydrogenase 1 and 2 (11ß-Hsd1 and 2). In fish, increasing levels of exogenous cortisol stimulate Na+ uptake via specific interaction with cortisol. This study investigated cortisol effects on expression of Na-K-ATPase subunit proteins and activity in gills of milkfish under hypothermal stress and revealed that the plasma cortisol contents as well as gill 11ß-hsd1l and na-k-atpase ß1 mRNA abundance were decreased in fresh water (FW) milkfish. Meanwhile, in the seawater (SW) milkfish, the plasma cortisol contents and gill 11ß-hsd1l and na-k-atpase ß1 mRNA abundance was increased under hypothermal stress. On the other hand, the abundance of 11ß-hsd2 mRNA increased in both FW and SW. In addition, 11ß-hsd1l expression increased in FW milkfish but decreased in SW milkfish after cortisol injection. Accordingly, the results that gill Na-K-ATPase activity of FW milkfish was affected by environmental temperatures as well as cortisol-dependent Na-K-ATPase ß1-subunit levels might be due to increased expression of 11ß-hsd1l that elevated intracellular cortisol contents. In hypothermal SW milkfish, decreasing abundance of Na-K-ATPase ß1 protein due to reduced expression of 11ß-hsd1l was found after cortisol injection. Thus, under hypothermal stress, 11ß-HSD1L in FW milkfish gills was used to modulate cortisol and the following effects on increasing the transcription of Na-K-ATPase ß1 protein.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Brânquias/fisiologia , Hidrocortisona/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , Animais , Temperatura Baixa , Proteínas de Peixes/genética , Peixes , Água Doce , ATPase Trocadora de Sódio-Potássio/metabolismo , Equilíbrio Hidroeletrolítico
20.
Hypertension ; 74(1): 73-82, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31132948

RESUMO

Cardiotonic steroids (CTSs) are NKA α-1 (Na+/K+-ATPase α-1) ligands that are increased in volume expanded states and associated with cardiac and renal diseases. Although initiation and resolution of inflammation is an important component of cellular injury and repair in renal disease, it is unknown whether CTS activation of NKA α-1 signaling in this setting regulates this inflammatory response. On this background, we hypothesized that CTS signaling through the NKA α-1-Src kinase complex promotes a proinflammatory response in renal epithelial and immune cells. First, we observed that the CTS telocinobufagin activated multiple proinflammatory cytokines/chemokines in renal epithelial cells, and these effects were attenuated after either NKA α-1 knockdown or with a specific inhibitor of the NKA α-1-Src kinase complex (pNaKtide). Similar findings were observed in immune cells, where we demonstrated that while telocinobufagin induced both oxidative burst and enhanced Nuclear factor kappa-light-chain-enhancer of activated B cells activation in macrophages ( P<0.05), the effects were abolished in NKA α-1+/- macrophages or by pretreatment with pNaKtide or the Src inhibitor PP2 ( P<0.01). In a series of in vivo studies, we found that 5/6th partial nephrectomy induced significantly less oxidative stress in the remnant kidney of NKA α-1+/- versus wild-type mice. Similarly, 5/6th partial nephrectomy yielded decreased levels of the urinary oxidative stress marker 8-Oxo-2'-deoxyguanosine in NKA α-1+/- versus wild-type mice. Finally, we found that in vivo inhibition of the NKA α-1-Src kinase complex with pNaKtide significantly inhibited renal proinflammatory gene expression after 5/6th partial nephrectomy. These findings suggest that the NKA α-1-Src kinase complex plays a central role in regulating the renal inflammatory response induced by elevated CTS both in vitro and in vivo.


Assuntos
Bufanolídeos/farmacologia , Glicosídeos Cardíacos/farmacologia , Insuficiência Renal Crônica/patologia , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/genética , Animais , Biópsia por Agulha , Células Cultivadas , Quimiocinas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Imuno-Histoquímica , Inflamação/patologia , Rim/citologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Insuficiência Renal Crônica/tratamento farmacológico , Sensibilidade e Especificidade
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