Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.229
Filtrar
1.
Life Sci ; 232: 116643, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31299237

RESUMO

AIMS: Increased plasma soluble endoglin concentrations (sEng) are frequently detected in metabolic disorders accompanied with hypercholesterolemia in serum, but effect of sEng on the cholesterol biochemistry is unknown. Cholesterol and bile acids (BA) are important products of liver metabolism with numerous functions within the organism. Turnover of these substances requires precise regulation due to potential toxicities during their cumulation. In this study, we hypothesized that high sEng levels affect cholesterol homeostasis and BA turnover in mice liver. MAIN METHODS: Nine-month-old transgenic male mice overexpressing human sEng and wild-type mice underwent plasma, bile, stool, and organ samples analysis by analytical, qRT-PCT and Western blot methods. KEY FINDINGS: sEng mice demonstrated decreased plasma total and LDL cholesterol concentrations due to upregulation of hepatic Sr-b1 and Ldlr receptors, increased liver cholesterol content, and increased Abcg8-mediated cholesterol efflux into bile. sEng also increased conversion of cholesterol into bile acids (BA) via upregulation of Cyp7a1 and increased Mdr1 expression. Plasma concentrations of BA were increased in sEng mice due to their enhanced reabsorption via ileum. Increased hepatic disposition of BA led to their increased biliary excretion coupled with choleretic activity. SIGNIFICANCE: For the first time, we have shown that high sEng plasma levels affect cholesterol and BA homeostasis on the basis of complex liver and intestinal effects. The significance of these findings for pathophysiology of diseases associated with increased sEng concentrations remains to be elucidated in prospective studies.


Assuntos
Ácidos e Sais Biliares/metabolismo , Colesterol/metabolismo , Endoglina/sangue , Endoglina/fisiologia , Homeostase , Fígado/metabolismo , Animais , Ácidos e Sais Biliares/sangue , Colesterol/sangue , Fezes , Inflamação/sangue , Masculino , Camundongos , Camundongos Transgênicos , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Estresse Oxidativo , Receptores de LDL/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Simportadores/metabolismo , Regulação para Cima
2.
Emerg Microbes Infect ; 8(1): 879-894, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31179847

RESUMO

Hepatocyte proliferation could result in the loss of covalently closed circular DNA (cccDNA) and the emergence of cccDNA-cleared nascent hepatocytes, which appear refractory to hepatitis B virus (HBV) reinfection with unknown mechanism(s). Sodium taurocholate cotransporting polypeptide (NTCP) is the functional receptor for HBV entry. In this study, down-regulation of cell membrane localized NTCP expression in proliferating hepatocytes was found to prevent HBV infection in HepG2-NTCP-tet cells and in liver-humanized mice. In patients, lower NTCP protein expression was correlated well with higher levels of hepatocyte proliferation and less HBsAg expression in HBV-related focal nodular hyperplasia (FNH) tissues. Clinically, significantly lower NTCP protein expression was correlated with more active hepatocyte proliferation in CHB patients with severe active necroinflammation and better antiviral treatment outcome. Mechanistically, the activation of cell cycle regulatory genes p53, S-phase kinase-associated protein 2 (SKP2) and cyclin D1 during cell proliferation, as well as proliferative and inflammatory cytokine Interleukin-6 (IL-6) could transcriptionally down-regulate NTCP expression. From these aspects, we conclude that within the milieu of hepatocyte proliferation, down-regulation of cell membrane localized NTCP expression level renders nascent hepatocytes resistant to HBV reinfection. This may accelerate virus clearance during immune-mediated cell death and compensatory proliferation of survival hepatocytes.


Assuntos
Membrana Celular/metabolismo , Regulação para Baixo , Vírus da Hepatite B/fisiologia , Hepatite B/metabolismo , Hepatócitos/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Simportadores/genética , Animais , Membrana Celular/genética , Proliferação de Células , Feminino , Células Hep G2 , Hepatite B/genética , Hepatite B/fisiopatologia , Hepatite B/virologia , Vírus da Hepatite B/genética , Hepatócitos/citologia , Hepatócitos/virologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Simportadores/metabolismo
3.
Ideggyogy Sz ; 72(5-6): 181-186, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31241262

RESUMO

Background and purpose: Methylation is a key epigenetic modification of DNA and regarding its impact on epilepsy, it is argued that "DNA methylation may play an important role in seizure susceptibility and maintenance of the disorder". DNA methylation status of KCC2 (SCL12A5) and NKCC1 (SCL12A2) associated with refractory temporal lobe epilepsy was investigated in our study. Methods: Thirty-eight patients with temporal lobe epilepsy (TLE) who were diagnosed by video EEG monitoring and 32 healthy control subjects were included in the study. Twenty-three patients in TLE group were men and the remaining 15 were women. Among them, 27 had unilateral temporal focus (9 with right; 18 with left) and 11 patients had bilateral TLE. We analyzed promoter region methylation status of the KCC2 (SCL12A5) and NKCC1 (SCL12A2) genes in the case and control groups. Gene regions of interest were amplified through PCR and sequencing was accomplished with pyro-sequencing. Results: We found a significant relationship between TLE and methylation on the NKCC1. However, there was no association between TLE and methylation on the KCC2 gene. Also, we found no association between right or left and unilateral or bilateral foci of TLE. There was no relationship between TLE and methylation on the NKCC1and KCC2 genes in terms of mesial temporal sclerosis in cranial MRI, head trauma or febrile convulsions. Conclusion: The methylation of NKCC1 can be a mecha-nism of refractory temporal lobe epilepsy. There are limited findings about DNA methylation in TLE. Therefore, further studies with large sample sizes are necessary.


Assuntos
Metilação de DNA , Epilepsia do Lobo Temporal/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/metabolismo , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Estudos de Casos e Controles , Eletroencefalografia , Epilepsia do Lobo Temporal/diagnóstico , Epilepsia do Lobo Temporal/genética , Feminino , Humanos , Imagem por Ressonância Magnética , Masculino , Regiões Promotoras Genéticas , Membro 2 da Família 12 de Carreador de Soluto/genética , Simportadores/genética
4.
Life Sci ; 229: 173-179, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31103606

RESUMO

AIMS: The innate immune response induced by bacterial peptidoglycan peptides, such as γ-d-glutamyl-meso-diaminopimelic acid (iE-DAP), is an important host defense system. However, little is known about the innate immune response in the lung alveolar region. In this study, we examined induction of the innate immune response by iE-DAP in human alveolar epithelial cell lines, NCI-H441 (H441) and A549. MAIN METHODS: Induction of the innate immune response was evaluated by measuring the mRNA expression of cytokines and their release into the culture medium. KEY FINDINGS: iE-DAP treatment increased the mRNA expression of interleukin (IL)-6 and IL-8, and increased release of these pro-inflammatory cytokines into the culture medium in H441 cells, but not in A549 cells. Lack of release of these cytokines in A549 cells may have been due to lack of peptide transporter 2 (PEPT2) function. Intracellular nucleotide-binding oligomerization domain 1 (NOD1) recognizes iE-DAP and activates downstream signaling pathways to initiate the immune response. Therefore, the role of mitogen-activated protein kinase (MAPK) signaling pathways was examined in H441 cells. As a result of inhibition studies, receptor-interacting serine/threonine-protein kinase 2 and MAPK signaling pathways, such as p38 MAPK and extracellular signal-regulated kinase, but not c-Jun N-terminal kinase, were determined to be involved in the innate immune response in H441 cells. In addition, the nuclear factor κB pathway also played a role in the innate immune response. SIGNIFICANCE: These findings indicated that the innate immune response induced by bacterial peptides could occur in a PEPT2- and NOD1-dependent manner in alveolar epithelial cells.


Assuntos
Células Epiteliais Alveolares/imunologia , Ácido Diaminopimélico/análogos & derivados , Imunidade Inata/imunologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Simportadores/metabolismo , Células A549 , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Citocinas/metabolismo , Ácido Diaminopimélico/farmacologia , Humanos , Imunidade Inata/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Transdução de Sinais , Simportadores/genética
5.
Cell Physiol Biochem ; 52(6): 1427-1445, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31088037

RESUMO

BACKGROUND/AIMS: Hydrophobic bile salts, such as glycochenodeoxycholate (GCDC) can trigger hepatocyte apoptosis, which is prevented by tauroursodesoxycholate (TUDC), but the effects of GCDC and TUDC on sinusoidal bile salt uptake via the Na⁺-taurocholate transporting polypeptide (Ntcp) are unclear. METHODS: The effects of GCDC and TUDC on the plasma membrane localization of Ntcp were studied in perfused rat liver by means of immunofluorescence analysis and super-resolution microscopy. The underlying signaling events were investigated by Western blotting and inhibitor studies. RESULTS: GCDC (20 µmol/l) induced within 60 min a retrieval of Ntcp from the basolateral membrane into the cytosol, which was accompanied by an activating phosphorylation of the Src kinases Fyn and Yes. Both, Fyn activation and the GCDC-induced Ntcp retrieval from the plasma membrane were sensitive to the NADPH oxidase inhibitor apocynin, the antioxidant N-acetylcysteine and the Src family kinase inhibitors SU6656 and PP-2, whereas PP-2 did not inhibit GCDC-induced Yes activation. Internalization of Ntcp by GCDC was also prevented by the protein kinase C (PKC) inhibitor Gö6850. TUDC (20 µmol/l) reversed the GCDC-induced retrieval of Ntcp from the plasma membrane and prevented the activation of Fyn and Yes in GCDC-perfused rat livers. Reinsertion of Ntcp into the basolateral membrane in GCDC-perfused livers by TUDC was sensitive to the protein kinase A (PKA) inhibitor H89 and the integrin-inhibitory peptide GRGDSP, whereas the control peptide GRADSP was ineffective. Ex posure of cultured rat hepatocytes to GCDC (50 µmol/l, 15min) increased the fluorescence intensity of the reactive oxygen fluorescent indicator DCF to about 1.6-fold of untreated controls in a TUDC (50 µmol/l)-sensitive way. GCDC caused a TUDC-sensitive canalicular dilatation without evidence for Bsep retrieval from the canalicular membrane. CONCLUSION: The present study suggests that GCDC triggers the retrieval of Ntcp from the basolateral membrane into the cytosol through an oxidative stress-dependent activation of Fyn. TUDC prevents the GCDC-induced Fyn activation and Ntcp retrieval through integrin-dependent activation of PKA.


Assuntos
Membrana Celular/metabolismo , Ácido Glicoquenodesoxicólico , Fígado/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Ácido Taurocólico , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Ácido Glicoquenodesoxicólico/metabolismo , Ácido Glicoquenodesoxicólico/farmacologia , Masculino , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Ácido Taurocólico/metabolismo , Ácido Taurocólico/farmacologia
6.
Mol Biotechnol ; 61(6): 442-450, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30980224

RESUMO

Soil salinity imposes a serious threat to the productivity of agricultural crops. Among several other transporters, high-affinity K+ transporter (HKT)'s play an important role in reducing the phytotoxicity of Na+. Expression of Eutrema salsugineum (a halophyte) HKT1;2 is induced upon salt exposure. To elucidate the role of its promoter, we compared the sequences of HKT1;2 promoters from E. salsugineum (1822 bp) and E. botschantzevii (1811 bp) with Arabidopsis thaliana HKT1;1 (846 bp) promoter. In silico analysis predicted several cis-acting regulatory elements (GT-1 elements, core motifs of DRE/CRT, MYC/MYB-recognition sites and ACGT elements). Activities of the three promoters were analyzed by measuring HKT1;1 and/or HKT1;2 transcript level in the Athkt1;1 mutant plants. NaCl tolerance of the transgenics was also assessed. Our results depicted that expressing either AtHKT1;1 or EsHKT1;2 coding regions under the control of AtHKT1;1 promoter, almost reversed the hypersensitivity of the mutant for salt, on contrarily, when AtHKT1;1 coding sequence expressed under either Es or EbHKT1;2 promoters did not. Changes in shoot Na+/K+ concentrations under salt exposure is significantly consistent with the complementation ability of the mutant. The transcript concentration for genes under the control of either of Eutrema promoters, at control level was very less. This may suggest that either an important upstream response motif is missed or that A. thaliana misses a transcriptional regulator that is essential for salt-inducible HKT1 expression in Eutrema.


Assuntos
Arabidopsis/genética , Brassicaceae/genética , Proteínas de Transporte de Cátions/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Tolerância ao Sal/genética , Simportadores/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Sequência de Bases , Brassicaceae/efeitos dos fármacos , Brassicaceae/crescimento & desenvolvimento , Brassicaceae/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Teste de Complementação Genética , Transporte de Íons/efeitos dos fármacos , Mutação , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Potássio/metabolismo , Regiões Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Sódio/metabolismo , Cloreto de Sódio/farmacologia , Especificidade da Espécie , Estresse Fisiológico/genética , Simportadores/metabolismo
7.
Phytomedicine ; 59: 152916, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30978651

RESUMO

BACKGROUND: Shengmai Formula (SMF) is widely used to treat cardiovascular disease such as chronic heart disease, coronary atherosclerotic heart disease, viral myocarditis, and others. Our previous studies have shown that OATP1B1/1B3 mediates the interactions between ophiopogon D and ginsenoside Rb1/Rd, which are the major active components in SMF. The herb-drug interactions that involve sodium taurocholate co-transporting polypeptide (NTCP) have been drawing increasing amounts of attention. PURPOSE: The aim of the present study was to investigate the interactions of the major effective components in SMF mediated by NTCP. METHODS: By using NTCP-overexpressing HEK293T cells and liquid chromatograph-mass spectrometer (LC-MS) analytical methods, we investigated the impact of the four main effective fractions and the 12 main effective components in SMF on NTCP-mediated sodium taurocholate (TCNa) uptake. The interactions of these effective components in SMF mediated by NTCP were further studied. RESULTS: The main effective fractions, ginseng total saponins (GTS), ophiopogon total saponins (OTS), ophiopogon total flavonoids (OTF), and fructus schisandrae total lignans (STL), all exhibited a certain inhibitory effect on the uptake of TCNa. Among the 12 main effective components, only ginsenoside Rg1, ophiopogon D', and schizandrin A showed inhibition of TCNa uptake, with IC50 values of 50.49 ± 4.24 µM, 6.71 ± 0.70 µM, and 45.80 ± 3.10 µM, respectively. Additionally, we found that ginsenoside Re and schizandrin B could be transported by NTCP-overexpressing HEK293T cells, and that the uptake of ginsenoside Re was significantly inhibited by OTS, OTF, STL, ginsenoside Rg1, ophiopogon D', and schizandrin A. The uptake of schizandrin B was significantly inhibited by GTS, OTS, OTF, and ophiopogon D'. CONCLUSION: Ginsenoside Rg1, ophiopogon D', and schizandrin A are potential inhibitors of NTCP and may interact with clinical drugs mediated by NTCP. Ginsenoside Re and schizandrin B are also potential substrates of NTCP, and their uptake mediated by NTCP was inhibited by the other components in SMF. The interaction of complex components based on NTCP may be one of the important compatibility mechanisms in SMF.


Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Cromatografia Líquida , Células HEK293 , Humanos , Espectrometria de Massas
8.
PLoS Genet ; 15(4): e1007786, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30946740

RESUMO

At the molecular level, the evolution of new traits can be broadly divided between changes in gene expression and changes in protein-coding sequence. For proteins, the evolution of novel functions is generally thought to proceed through sequential point mutations or recombination of whole functional units. In Saccharomyces, the uptake of the sugar maltotriose into the cell is the primary limiting factor in its utilization, but maltotriose transporters are relatively rare, except in brewing strains. No known wild strains of Saccharomyces eubayanus, the cold-tolerant parent of hybrid lager-brewing yeasts (Saccharomyces cerevisiae x S. eubayanus), are able to consume maltotriose, which limits their ability to fully ferment malt extract. In one strain of S. eubayanus, we found a gene closely related to a known maltotriose transporter and were able to confer maltotriose consumption by overexpressing this gene or by passaging the strain on maltose. Even so, most wild strains of S. eubayanus lack native maltotriose transporters. To determine how this rare trait could evolve in naive genetic backgrounds, we performed an adaptive evolution experiment for maltotriose consumption, which yielded a single strain of S. eubayanus able to grow on maltotriose. We mapped the causative locus to a gene encoding a novel chimeric transporter that was formed by an ectopic recombination event between two genes encoding transporters that are unable to import maltotriose. In contrast to classic models of the evolution of novel protein functions, the recombination breakpoints occurred within a single functional domain. Thus, the ability of the new protein to carry maltotriose was likely acquired through epistatic interactions between independently evolved substitutions. By acquiring multiple mutations at once, the transporter rapidly gained a novel function, while bypassing potentially deleterious intermediate steps. This study provides an illuminating example of how recombination between paralogs can establish novel interactions among substitutions to create adaptive functions.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces/genética , Saccharomyces/metabolismo , Trissacarídeos/metabolismo , Sequência de Aminoácidos , Cerveja/microbiologia , Proteínas de Transporte/química , Evolução Molecular Direcionada , Fermentação , Proteínas Fúngicas/química , Conversão Gênica , Genes Fúngicos , Hibridização Genética , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Filogenia , Proteínas Recombinantes de Fusão/química , Saccharomyces/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Simportadores/química , Simportadores/genética , Simportadores/metabolismo
9.
Mol Med Rep ; 19(5): 4514-4522, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30942410

RESUMO

Thyroid stimulating hormone (TSH) consists of an α­subunit and a unique ß­subunit. The first in­frame TSHß splice variant produced by the cells of immune system was identified in 2009. The TSHß splice variant and native TSHß exhibit different expression profiles, and research has been conducted to elucidate the role of the TSHß splice variant in different diseases. However, understanding of the fundamental physiological characteristics of the TSHß splice variant is currently limited. To verify whether the TSHß splice variant has the potential to induce thyroid follicular cells to synthesize thyroid hormone, in vivo and in vitro stimulation experiments were conducted in the present study. A total of 60 C57BL/6 mice were divided into control­, 5 and 10 µg TSHß splice variant­treated groups at random. Mice were sacrificed at 0.5, 1 and 4 h after intraperitoneal injection, and serum levels of tri­iodothyronine (T3) and thyroxine (T4) were determined using a radioimmunoassay. Thyroid follicular cells were isolated from the thyroids of mice, and stimulated with 2 µg/ml TSHß splice variant. Supernatants were collected, and the levels of T3 and T4 were detected. The protein expression levels of the sodium­iodide symporter, thyroperoxidase and thyroglobulin in thyroid follicular cells were quantified using western blot analysis. To verify whether the TSHß splice variant expression was regulated by the hypothalamus­pituitary­thyroid (HPT) axis, similar to native TSHß, a total of 60 C57BL/6 mice were equally divided into control, 2 mg/kg T3 intraperitoneal injection and 0.05 mg/kg thyroid­releasing hormone intraperitoneal injection groups at random. Mice were sacrificed at 1 and 4 h after injection. Alterations in the expression of the TSHß splice variant in the pituitary, thyroid, peripheral blood leukocytes and spleen tissues were detected using western blot analysis. The present study demonstrated that the TSHß splice variant is not regulated by the HPT axis and may affect thyroid hormone synthesis. Modifications in the expression of the TSHß splice variant may occur in a uniquely regulated manner to provide peripheral immunological compartments with a source of activated cells, particularly under immune stress.


Assuntos
Hormônios Tireóideos/biossíntese , Tireotropina Subunidade beta/genética , Animais , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Processamento de RNA , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Simportadores/metabolismo , Tireoglobulina/metabolismo , Glândula Tireoide/citologia , Glândula Tireoide/metabolismo , Tireotropina Subunidade beta/metabolismo , Tiroxina/sangue , Tri-Iodotironina/sangue
10.
J Agric Food Chem ; 67(16): 4493-4504, 2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-30938528

RESUMO

Expression of sodium-iodide symporter (NIS) is stimulated by sterol-regulatory-element-binding transcription factors (SREBFs) in mammary epithelial MCF-7 cells. Because conjugated linoleic acid (CLA) isomers have been shown to inhibit transcriptional activity of SREBFs in the mammary gland, the hypothesis was tested that CLA isomers inhibit NIS expression induced by all- trans retinoic acid (ATRA) in MCF-7 cells through inhibiting SREBF activity. c9t11-CLA and t10c12-CLA decreased ATRA-induced NIS-mRNA expression from 1.00 (ATRA alone) to 0.80 ± 0.12 (200 µM c9t11-CLA, P < 0.05) and 0.62 ± 0.10 (200 µM t10c12-CLA, P < 0.05), NIS-protein expression from 1.00 (ATRA alone) to 0.77 ± 0.08 (200 µM c9t11-CLA, P < 0.05) and 0.63 ± 0.05 (200 µM t10c12-CLA, P < 0.05), and NIS-promoter activity from 1.00 (ATRA alone) to 0.74 ± 0.13 (200 µM c9t11-CLA, P < 0.05) and 0.76 ± 0.13 (200 µM t10c12-CLA, P < 0.05); however, c9t11-CLA and t10c12-CLA increased the mRNA levels of SREBF isoforms and their target genes. In contrast, the mRNA expression of peroxisome-proliferator-activated receptor γ (PPARG) was strongly induced by ATRA alone but decreased by CLA isomers from 1.00 (ATRA alone) to 0.80 ± 0.06 (200 µM c9t11-CLA, P < 0.05) and 0.86 ± 0.06 (200 µM t10c12-CLA, P < 0.05). Overexpression of PPARγ in MCF-7 cells increased basal NIS-promoter activity, and treatment with the PPARγ ligand troglitazone stimulated ATRA-induced NIS-promoter activity. In conclusion, the results suggest that CLA isomers exert their effect on the expression of NIS by decreasing PPARG expression in MCF-7 cells.


Assuntos
Células Epiteliais/efeitos dos fármacos , Ácidos Linoleicos Conjugados/farmacologia , Glândulas Mamárias Humanas/metabolismo , Simportadores/genética , Tretinoína/farmacologia , Células Epiteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Isomerismo , Células MCF-7 , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/efeitos dos fármacos , PPAR gama/genética , PPAR gama/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Iodeto de Sódio/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Simportadores/metabolismo
11.
Chem Asian J ; 14(11): 1926-1931, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-30969484

RESUMO

We prepared an amphiphile with a penta-phenylene lipophilic group and a branched trimaltoside head group. This new agent, designated penta-phenylene maltoside (PPM), showed a marked tendency to self-assembly into micelles via strong aromatic-aromatic interactions in aqueous media, as evidenced by 1 H NMR spectroscopy and fluorescence studies. When utilized for membrane protein studies, this new agent was superior to DDM, a gold standard conventional detergent, in stabilizing multiple proteins long term. The ability of this agent to form aromatic-aromatic interactions is likely responsible for enhanced protein stabilization when associated with a target membrane protein.


Assuntos
Detergentes/química , Maltose/química , Proteínas de Membrana/química , Espectroscopia de Ressonância Magnética , Maltose/análise , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Micelas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Salmonella typhimurium/enzimologia , Simportadores/química , Simportadores/genética , Simportadores/metabolismo , Temperatura Ambiente
12.
Br J Anaesth ; 122(4): 490-499, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30857605

RESUMO

BACKGROUND: The factors determining peak susceptibility of the developing brain to anaesthetics are unclear. It is unknown why postnatal day 7 (P7) male rats are more vulnerable to anaesthesia-induced memory deficits than littermate females. Given the precocious development of certain regions in the female brain during the neonatal critical period, we hypothesised that females are susceptible to anaesthetic brain injury at an earlier time point than previously tested. METHODS: Female rats were exposed to isoflurane (Iso) 1 minimum alveolar concentration or sham anaesthesia at P4 or P7. Starting at P35, rats underwent a series of behavioural tasks to test their spatial and recognition memory. Cell death immediately after anaesthesia was quantified by Fluoro-Jade C staining in select brain regions, and developmental expression of the chloride transporters KCC2 and NKCC1 was analysed by immunoblotting in male and female rats at P4 and P7. RESULTS: Female rats exposed to Iso at P4 displayed impaired spatial, object-place, -context, and social recognition memory, and increased cell death in the hippocampus and laterodorsal thalamus. Female rats exposed at P7 exhibited only decreased performance in object-context compared with control. The ratio of NKCC1/KCC2 expression in cerebral cortex was higher in P4 females than in P7 females, and similar to that in P7 males. CONCLUSIONS: Female rats exposed to Iso at P4 are sensitive to anaesthetic injury historically observed in P7 males. This is consistent with a comparably immature developmental state in P4 females and P7 males. The window of anaesthetic vulnerability correlates with sex-specific cortical expression of chloride transporters NKCC1 and KCC2. These findings suggest that both sex and developmental age play important roles in determining the outcome after early anaesthesia exposure.


Assuntos
Anestésicos Inalatórios/toxicidade , Disfunção Cognitiva/induzido quimicamente , Isoflurano/toxicidade , Fatores Etários , Anestésicos Inalatórios/administração & dosagem , Animais , Animais Recém-Nascidos , Comportamento Animal/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Córtex Cerebral/metabolismo , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/patologia , Esquema de Medicação , Feminino , Isoflurano/administração & dosagem , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Ratos Sprague-Dawley , Recognição (Psicologia)/efeitos dos fármacos , Fatores Sexuais , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/metabolismo
13.
Biol Pharm Bull ; 42(3): 501-506, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30828081

RESUMO

The mechanism underlying the increased pharmacological effects of phenobarbital in rats with glycerol-induced acute renal failure (ARF) was examined. In the experiments, a surgical cannula was inserted in the lateral ventricle of the rats for phenobarbital infusion, and the ARF induction was performed by intramuscular administration of 50% glycerol. The onset time of anesthesia by phenobarbital was determined with the tail flick method. In addition, cerebral microsomes were prepared from excised cerebral cortices of sham and ARF rats, and the cerebral expression of the γ-aminobutyric acid (GABA)A receptor and two cation-chloride transporters, KCC2 and NKCC1, was evaluated by Western blotting, as their functions are involved in the anesthetic effects of phenobarbital. When phenobarbital was infused in the ventricle, anesthesia was induced 2.2-times faster in ARF rats than in sham rats, and there was no detectable increase in the cerebral expression of the GABAA receptor in ARF rats. It was additionally noted that the cerebral expression of KCC2 decreased, whereas that of NKCC1 was unaltered in ARF rats. These findings indicated that the anesthetic effects of phenobarbital are potentiated in ARF rats, probably due to imbalanced cerebral expression of KCC2 and NKCC1, suggesting that altered cation-chloride handling in nerve cells is associated.


Assuntos
Lesão Renal Aguda/induzido quimicamente , Glicerol/toxicidade , Fenobarbital/farmacologia , Lesão Renal Aguda/metabolismo , Anestésicos Intravenosos/farmacologia , Animais , Bumetanida/farmacologia , Diuréticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hipnóticos e Sedativos/farmacologia , Masculino , Distribuição Aleatória , Ratos , Ratos Wistar , Receptores de GABA-A/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/genética , Simportadores/metabolismo , Uretana/farmacologia
14.
Int J Nanomedicine ; 14: 1779-1787, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30880979

RESUMO

Background: This study was designed to explore a novel approach for transferring NIS protein to cells using extracellular vesicle (EV) and enhancing iodine avidity in hepatocellular carcinoma (HCC) cells. Methods: We transfected the HCC cells (Huh7) with NIS gene, designated as Huh7/NIS, and isolated the EVs from them. Presence of NIS protein in EVs and EV-mediated transport of NIS protein to recipient Huh7 cells were tested using Western blotting. We also examined radioiodine uptake in Huh7 cells treated with EV-Huh7/NIS. Results: Successful transfer of NIS protein into Huh7 cells was confirmed by WB and microscopy. EVs showed high levels of NIS protein in them. Treatment of Huh7 cells with EV-Huh7/NIS increased the NIS protein level and enhanced 125I uptake in recipient Huh7 cells. In addition, EV-huh7/NIS pre-treatment enhanced the cytotoxicity of 131I therapy against Huh7 cells by inducing increased DNA damage/increased γH2A.X foci formation. Conclusion: This is the first-of-its-kind demonstration of successful transportation of the NIS protein to cells via EVs, which increased radioiodine uptake. This approach can revert radioiodine-resistant cancers into radioiodine-sensitive cancers.


Assuntos
Vesículas Extracelulares/metabolismo , Iodo/metabolismo , Simportadores/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Morte Celular , Linhagem Celular Tumoral , Dano ao DNA , Vesículas Extracelulares/ultraestrutura , Regulação Neoplásica da Expressão Gênica , Humanos , Radioisótopos do Iodo/química , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia
15.
Nat Commun ; 10(1): 1225, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30874549

RESUMO

Although cortical interneurons are apparently well-placed to suppress seizures, several recent reports have highlighted a paradoxical role of perisomatic-targeting parvalbumin-positive (PV+) interneurons in ictogenesis. Here, we use an acute in vivo model of focal cortical seizures in awake behaving mice, together with closed-loop optogenetic manipulation of PV+ interneurons, to investigate their function during seizures. We show that photo-depolarization of PV+ interneurons rapidly switches from an anti-ictal to a pro-ictal effect within a few seconds of seizure initiation. The pro-ictal effect of delayed photostimulation of PV+ interneurons was not shared with dendrite-targeting somatostatin-positive (SOM+) interneurons. We also show that this switch can be prevented by overexpression of the neuronal potassium-chloride co-transporter KCC2 in principal cortical neurons. These results suggest that strategies aimed at improving the ability of principal neurons to maintain a trans-membrane chloride gradient in the face of excessive network activity can prevent interneurons from contributing to seizure perpetuation.


Assuntos
Interneurônios/fisiologia , Neocórtex/fisiologia , Inibição Neural/fisiologia , Convulsões/fisiopatologia , Simportadores/metabolismo , Animais , Cloretos/metabolismo , Modelos Animais de Doenças , Eletrocorticografia , Eletrodos , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Masculino , Camundongos , Neocórtex/citologia , Vias Neurais/fisiologia , Optogenética/instrumentação , Optogenética/métodos , Parvalbuminas/metabolismo , Técnicas de Patch-Clamp , Estimulação Luminosa , Convulsões/diagnóstico , Somatostatina/metabolismo , Simportadores/genética
16.
Zhonghua Zhong Liu Za Zhi ; 41(3): 208-213, 2019 Mar 23.
Artigo em Chinês | MEDLINE | ID: mdl-30917457

RESUMO

Objective: To investigate the molecular mechanism of down-regulation of monocarboxylic acid transporter 1 (MCT1) on the proliferation inhibition of glioma cell. Methods: siMCT1, siMCT4 and negative control siRNA were transfected into glioma cell lines including U-251 and U-87. The proliferation activities of U-251 and U-87 cells were detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay and clonogenic assay. Glucose consumption and lactic acid efflux of U-251 and U-87 cells were determined by spectrophotometry.Western blot was used to detect the expressions of MCT1, MCT4, human glucose transporter 1 (GLUT1), GLUT4, tuberous sclerosis associated protein (TSC2), p-TSC2, 4E binding protein 1 (4EBP1), p-4EBP1, ribosomal S6 protein kinase (S6) and p-S6 protein in U-251 and U-87 cells. Results: Compared with negative control group, siMCT1 and siMCT4 significantly inhibited the expressions of MCT1 and MCT4 protein in U-251 and U-87 cells (both P<0.05). However, only knockdown of MCT1, the proliferation activities of U-251 and U-87 cells significantly decreased (P<0.05). The clone formation rates of U-251 and U-87 cells decreased to (55.20±3.27)% and (68.33±4.58) %, respectively (P<0.05). The glucose consumption of U-251 and U-87 cells in the negative control group at 72 hours were (82.65±6.66) pmol/L and (63.33±5.27) pmol/L, respectively, significantly higher than (31.70±3.17) pmol/L and (26.41±3.19) pmol/L of the siMCT1 transfected group (P<0.05). The extracellular lactate flow of U-251 and U-87 cells in negative control group at 72 h were (155.49±8.15) mmol/L and (135.37±8.21) mmol/L, respectively, significantly higher than (42.69±4.66) mmol/L and (38.91±4.83) mmol/L of the siMCT1 transfected group (P<0.05). Western blot analysis showed that knockdown of MCT1 significantly decreased the protein levels of GLUT1 p-TSC2, p-4EBP1 and p-S6 in U-251 and U-87 cells. Conclusions: Downregulation of MCT1 expression can inhibit the proliferation of glioma cells. Deletion of MCT1 inhibits the glycolysis and metabolism of glioma cells through regulating the mTOR signaling pathway.


Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proliferação de Células , Regulação para Baixo , Glioma/metabolismo , Glioma/patologia , Transportadores de Ácidos Monocarboxílicos/metabolismo , Simportadores/metabolismo , Linhagem Celular , Técnicas de Silenciamento de Genes , Glicólise , Humanos , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Musculares/metabolismo , Simportadores/genética , Serina-Treonina Quinases TOR/metabolismo
17.
Int J Mol Sci ; 20(5)2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30823627

RESUMO

Abiotic stresses generally cause a series of morphological, biochemical and molecular changes that unfavorably affect plant growth and productivity. Among these stresses, soil salinity is a major threat that can seriously impair crop yield. To cope with the effects of high salinity on plants, it is important to understand the mechanisms that plants use to deal with it, including those activated in response to disturbed Na⁺ and K⁺ homeostasis at cellular and molecular levels. HKT1-type transporters are key determinants of Na⁺ and K⁺ homeostasis under salt stress and they contribute to reduce Na⁺-specific toxicity in plants. In this review, we provide a brief overview of the function of HKT1-type transporters and their importance in different plant species under salt stress. Comparison between HKT1 homologs in different plant species will shed light on different approaches plants may use to cope with salinity.


Assuntos
Proteínas de Transporte de Cátions/genética , Proteínas de Plantas/genética , Estresse Salino , Plantas Tolerantes a Sal/genética , Simportadores/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Simportadores/química , Simportadores/metabolismo
18.
Int J Mol Sci ; 20(5)2019 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-30832374

RESUMO

HKT1 and SOS1 are two key Na⁺ transporters that modulate salt tolerance in plants. Although much is known about the respective functions of HKT1 and SOS1 under salt conditions, few studies have examined the effects of HKT1 and SOS1 mutations on the expression of other important Na⁺ and K⁺ transporter genes. This study investigated the physiological parameters and expression profiles of AtHKT1;1, AtSOS1, AtHAK5, AtAKT1, AtSKOR, AtNHX1, and AtAVP1 in wild-type (WT) and athkt1;1 and atsos1 mutants of Arabidopsis thaliana under 25 mM NaCl. We found that AtSOS1 mutation induced a significant decrease in transcripts of AtHKT1;1 (by 56⁻62% at 6⁻24 h), AtSKOR (by 36⁻78% at 6⁻24 h), and AtAKT1 (by 31⁻53% at 6⁻24 h) in the roots compared with WT. This led to an increase in Na⁺ accumulation in the roots, a decrease in K⁺ uptake and transportation, and finally resulted in suppression of plant growth. AtHKT1;1 loss induced a 39⁻76% (6⁻24 h) decrease and a 27⁻32% (6⁻24 h) increase in transcripts of AtSKOR and AtHAK5, respectively, in the roots compared with WT. At the same time, 25 mM NaCl decreased the net selective transport capacity for K⁺ over Na⁺ by 92% in the athkt1;1 roots compared with the WT roots. Consequently, Na⁺ was loaded into the xylem and delivered to the shoots, whereas K⁺ transport was restricted. The results indicate that AtHKT1;1 and AtSOS1 not only mediate Na⁺ transport but also control ion uptake and the spatial distribution of Na⁺ and K⁺ by cooperatively regulating the expression levels of relevant Na⁺ and K⁺ transporter genes, ultimately regulating plant growth under salt stress.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Transporte de Cátions/genética , Homeostase , Estresse Salino , Trocadores de Sódio-Hidrogênio/genética , Simportadores/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação , Canais de Potássio/genética , Canais de Potássio/metabolismo , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Simportadores/metabolismo
19.
Int J Mol Sci ; 20(6)2019 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-30917504

RESUMO

Na-amino acid co-transporters (NaAAcT) are uniquely affected in rabbit intestinal villus cell brush border membrane (BBM) during chronic intestinal inflammation. Specifically, Na-alanine co-transport (ASCT1) is inhibited secondary to a reduction in the affinity of the co-transporter for alanine, whereas Na-glutamine co-transport (B0AT1) is inhibited secondary to a reduction in BBM co-transporter numbers. During chronic intestinal inflammation, there is abundant production of the potent oxidant peroxynitrite (OONO). However, whether OONO mediates the unique alteration in NaAAcT in intestinal epithelial cells during chronic intestinal inflammation is unknown. In this study, ASCT1 and B0AT1 were inhibited by OONO in vitro. The mechanism of inhibition of ASCT1 by OONO was secondary to a reduction in the affinity of the co-transporter for alanine, and secondary to a reduction in the number of co-transporters for B0AT1, which were further confirmed by Western blot analyses. In conclusion, peroxynitrite inhibited both BBM ASCT1 and B0AT1 in intestinal epithelial cells but by different mechanisms. These alterations in the villus cells are similar to those seen in the rabbit model of chronic enteritis. Therefore, this study indicates that peroxynitrite may mediate the inhibition of ASCT1 and B0AT1 during inflammation, when OONO levels are known to be elevated in the mucosa.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Enterócitos/metabolismo , Proteínas de Transporte de Glutamato da Membrana Plasmática/metabolismo , Microvilosidades/metabolismo , Simportadores/metabolismo , Animais , Linhagem Celular , Enterócitos/efeitos dos fármacos , Enterócitos/patologia , Inflamação/metabolismo , Ácido Peroxinitroso/toxicidade , Ratos
20.
Proc Natl Acad Sci U S A ; 116(11): 4934-4939, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30792352

RESUMO

Lactose permease is a paradigm for the major facilitator superfamily, the largest family of ion-coupled membrane transport proteins known at present. LacY carries out the coupled stoichiometric symport of a galactoside with an H+, using the free energy released from downhill translocation of H+ to drive accumulation of galactosides against a concentration gradient. In neutrophilic Escherichia coli, internal pH is kept at ∼7.6 over the physiological range, but the apparent pK (pKapp) for galactoside binding is 10.5. Surface-enhanced infrared absorption spectroscopy (SEIRAS) demonstrates that the high pKa is due to Glu325 (helix X), which must be protonated for LacY to bind galactoside effectively. Deprotonation is also obligatory for turnover, however. Here, we utilize SEIRAS to study the effect of mutating residues in the immediate vicinity of Glu325 on its pKa The results are consistent with the idea that Arg302 (helix IX) is important for deprotonation of Glu325.


Assuntos
Arginina/metabolismo , Proteínas de Escherichia coli/metabolismo , Ácido Glutâmico/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Simportadores/metabolismo , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutação/genética , Espectrofotometria Infravermelho
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA