RESUMO
Prediabetes is a risk state that defines a high chance of developing diabetes and cardiovascular disease. Oxidative stress mediated by hyperglycemia-induced production of reactive species could play a crucial role in this context. In the present study, we investigated whether the anion exchange capability mediated by AE1 (SLC4A1), which is sensitive to oxidative stress, was altered in human red blood cells (RBCs) obtained from prediabetic volunteers. In addition, we assessed the precise composition of bioactive compounds and the potential benefits of finger lime juice extract (Citrus australasica, Faustrime cultivar) in counteracting oxidative stress-related functional alterations. Human RBCs from normal and prediabetic volunteers were incubated with 50 µg/mL juice extract for 2 h at 25°C. Juice extract restored alterations of the anion exchange capability mediated by AE1 and prevented the structural rearrangements of AE1 and α/ß-spectrin in prediabetic RBCs. AE1 functional and structural alterations were not associated with an increase in lipid peroxidation or protein oxidation at the level of the plasma membrane. An increased production of intracellular ROS, which provoked the oxidation of hemoglobin to methemoglobin, both reverted by juice extract, was instead observed. Importantly, juice extract also induced a reduction in glycated hemoglobin levels in prediabetic RBCs. Finally, juice extract blunted the overactivation of the endogenous antioxidant enzymes catalase and superoxide dismutase and prevented glutathione depletion in prediabetic RBCs. These findings contribute to clarifying cellular and molecular mechanisms related to oxidative stress and glycation events that may influence RBC and systemic homeostasis in prediabetes, identify AE1 as a sensitive biomarker of RBC structural and function alterations in prediabetes and propose finger lime juice extract as a natural antioxidant for the treatment and/or prevention of the complications associated with the prediabetic condition.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito , Citrus , Eritrócitos , Estresse Oxidativo , Extratos Vegetais , Estado Pré-Diabético , Humanos , Citrus/química , Eritrócitos/metabolismo , Eritrócitos/efeitos dos fármacos , Estado Pré-Diabético/metabolismo , Estado Pré-Diabético/tratamento farmacológico , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Sucos de Frutas e Vegetais/análise , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Antioxidantes/químicaRESUMO
PURPOSE: Hearing loss (HL) is often monogenic. The clinical importance of genetic testing in HL may further increase when gene therapy products become available. Diagnoses are, however, complicated by a high genetic and allelic heterogeneity, particularly of autosomal dominant (AD) HL. This work aimed to characterize the mutational spectrum of AD HL in Austria. METHODS: In an ongoing prospective study, 27 consecutive index patients clinically diagnosed with non-syndromic AD HL, including 18 previously unpublished cases, were analyzed using whole-exome sequencing (WES) and gene panels. Novel variants were characterized using literature and bioinformatic means. Two additional Austrian medical centers provided AD HL mutational data obtained with in-house pipelines. Other Austrian cases of AD HL were gathered from literature. RESULTS: The solve rate (variants graded as likely pathogenic (LP) or pathogenic (P)) within our cohort amounted to 59.26% (16/27). MYO6 variants were the most common cause. One third of LP/P variants were truncating variants in haploinsufficiency genes. Ten novel variants in HL genes were identified, including six graded as LP or P. In one cohort case and one external case, the analysis uncovered previously unrecognized syndromic presentations. CONCLUSION: More than half of AD HL cases analyzed at our center were solved with WES. Our data demonstrate the importance of genetic testing, especially for the diagnosis of syndromic presentations, enhance the molecular knowledge of genetic HL, and support other laboratories in the interpretation of variants.
Assuntos
Sequenciamento do Exoma , Mutação , Humanos , Áustria , Masculino , Feminino , Estudos Prospectivos , Adulto , Criança , Adolescente , Pré-Escolar , Pessoa de Meia-Idade , Adulto Jovem , Testes Genéticos/métodos , Genes Dominantes , Idoso , Perda Auditiva/genética , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/diagnóstico , LactenteRESUMO
Oxidative stress and immune response play an important role in the development of several cancers, including melanoma. Ion channels are aberrantly expressed in tumour cells and regulate neoplastic transformation, malignant progression, and resistance to therapy. Ion channels are localized in the plasma membrane or other cellular membranes and are targets of oxidative stress, which is particularly elevated in melanoma. At the same time, ion channels are crucial for normal and cancer cell physiology and are subject to multiple layers of regulation, and therefore represent promising targets for therapeutic intervention. In this review, we analyzed the effects of oxidative stress on ion channels on a molecular and cellular level and in the context of melanoma progression and immune evasion. The possible role of ion channels as targets of alternative therapeutic strategies in melanoma was discussed.
Assuntos
Canais Iônicos , Melanoma , Humanos , Canais Iônicos/metabolismo , Melanoma/tratamento farmacológico , Transformação Celular Neoplásica/metabolismo , Imunidade , Estresse OxidativoRESUMO
Aging, a time-dependent multifaceted process, affects both cell structure and function and involves oxidative stress as well as glycation. The present investigation focuses on the role of the band 3 protein (B3p), an anion exchanger essential to red cells homeostasis, in a d-galactose ( d-Gal)-induced aging model. Anion exchange capability, measured by the rate constant of SO4²- uptake through B3p, levels of lipid peroxidation, oxidation of membrane sulfhydryl groups, B3p expression, methemoglobin, glycated hemoglobin (Hb), and the reduced glutathione/oxidized glutathione ratio were determined after exposure of human erythrocytes to 25, 35, 50, and 100 mmol/L d-Gal for 24 h. Our results show that: (i) in vitro application of d-Gal is useful to model early aging in human erythrocytes; (ii) assessment of B3p ion transport function is a sensitive tool to monitor aging development; (iii) d-Gal leads to Hb glycation and produces substantial changes on the endogenous antioxidant system; (iv) the impact of aging on B3p function proceeds through steps, first involving Hb glycation and then oxidative events at the membrane level. These findings offer a useful tool to understand the mechanisms of aging in human erythrocytes and propose B3p as a possible target for new therapeutic strategies to counteract age-related disturbances.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito , Galactose , Envelhecimento , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Eritrócitos/metabolismo , Galactose/metabolismo , Galactose/farmacologia , Humanos , Estresse OxidativoRESUMO
Aging is a multi-factorial process developing through a complex net of interactions between biological and cellular mechanisms and it involves oxidative stress (OS) as well as protein glycation. The aim of the present work was to verify the protective role of Quercetin (Q), a polyphenolic flavonoid compound, in a d-Galactose (d-Gal)-induced model of aging in human erythrocytes. The anion-exchange capability through the Band 3 protein (B3p) measured by the rate constant of the SO42- uptake, thiobarbituric acid reactive substances (TBARS) levels-a marker of lipid peroxidation-total sulfhydryl (-SH) groups, glycated hemoglobin (A1c), and a reduced glutathione/oxidized glutathione (GSH-GSSG) ratio were determined following the exposure of erythrocytes to 100 mM d-Gal for 24 h, with or without pre-incubation with 10 µM Q. The results confirmed that d-Gal activated OS pathways in human erythrocytes, affecting both membrane lipids and proteins, as denoted by increased TBARS levels and decreased total sulfhydryl groups, respectively. In addition, d-Gal led to an acceleration of the rate constant of the SO42- uptake through the B3p. Both the alteration of the B3p function and oxidative damage have been improved by pre-treatment with Q, which preferentially ameliorated lipid peroxidation rather than protein oxidation. Moreover, Q prevented glycated A1c formation, while no protective effect on the endogenous antioxidant system (GSH-GSSG) was observed. These findings suggest that the B3p could be a novel potential target of antioxidant treatments to counteract aging-related disturbances. Further studies are needed to confirm the possible role of Q in pharmacological strategies against aging.
Assuntos
Estresse Oxidativo , Quercetina , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Eritrócitos/metabolismo , Galactose/metabolismo , Galactose/farmacologia , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Hemoglobinas Glicadas/metabolismo , Humanos , Quercetina/metabolismo , Quercetina/farmacologia , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
During their lifespan, red blood cells (RBCs) are exposed to a large number of stressors and are therefore considered as a suitable model to investigate cell response to oxidative stress (OS). This study was conducted to evaluate the potential beneficial effects of the natural antioxidant quercetin (Q) on an OS model represented by human RBCs treated with H2O2. Markers of OS, including % hemolysis, reactive oxygen species (ROS) production, thiobarbituric acid reactive substances (TBARS) levels, oxidation of protein sulfhydryl groups, CD47 and B3p expression, methemoglobin formation (% MetHb), as well as the anion exchange capability through Band 3 protein (B3p) have been analyzed in RBCs treated for 1 h with 20 mM H2O2 with or without pre-treatment for 1 h with 10 µM Q, or in RBCs pre-treated with 20 mM H2O2 and then exposed to 10 µM Q. The results show that pre-treatment with Q is more effective than post-treatment to counteract OS in RBCs. In particular, pre-exposure to Q avoided morphological alterations (formation of acanthocytes), prevented H2O2-induced OS damage, and restored the abnormal distribution of B3p and CD47 expression. Moreover, H2O2 exposure was associated with a decreased rate constant of SO42- uptake via B3p, as well as an increased MetHb formation. Both alterations have been attenuated by pre-treatment with 10 µM Q. These results contribute (1) to elucidate OS-related events in human RBCs, (2) propose Q as natural antioxidant to counteract OS-related alterations, and (3) identify B3p as a possible target for the treatment and prevention of OS-related disease conditions or aging-related complications impacting on RBCs physiology.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito , Antioxidantes , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Proteína 1 de Troca de Ânion do Eritrócito/farmacologia , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Antígeno CD47/metabolismo , Eritrócitos/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , Metemoglobina/metabolismo , Estresse Oxidativo , Quercetina/metabolismo , Quercetina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
The transcriptional regulator peroxisome proliferator activated receptor gamma coactivator 1A (PGC-1α), encoded by PPARGC1A, has been linked to neurodegenerative diseases. Recently discovered CNS-specific PPARGC1A transcripts are initiated far upstream of the reference promoter, spliced to exon 2 of the reference gene, and are more abundant than reference gene transcripts in post-mortem human brain samples. The proteins translated from the CNS and reference transcripts differ only at their N-terminal regions. To dissect functional differences between CNS-specific isoforms and reference proteins, we used clustered regularly interspaced short palindromic repeats transcriptional activation (CRISPRa) for selective endogenous activation of the CNS or the reference promoters in SH-SY5Y cells. Expression and/or exon usage of the targets was ascertained by RNA sequencing. Compared to controls, more differentially expressed genes were observed after activation of the CNS than the reference gene promoter, while the magnitude of alternative exon usage was comparable between activation of the two promoters. Promoter-selective associations were observed with canonical signaling pathways, mitochondrial and nervous system functions and neurological diseases. The distinct N-terminal as well as the shared downstream regions of PGC-1α isoforms affect the exon usage of numerous genes. Furthermore, associations of risk genes of amyotrophic lateral sclerosis and Parkinson's disease were noted with differentially expressed genes resulting from the activation of the CNS and reference gene promoter, respectively. Thus, CNS-specific isoforms markedly amplify the biological functions of PPARGC1A and CNS-specific isoforms and reference proteins have common, complementary and selective functions relevant for neurodegenerative diseases.
Assuntos
Redes Reguladoras de Genes , Doenças Neurodegenerativas/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Regiões Promotoras Genéticas , Ativação Transcricional , Linhagem Celular Tumoral , Éxons , Células HEK293 , Humanos , Neurônios/metabolismo , Motivos de Nucleotídeos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , TranscriptomaRESUMO
Due to development of molecular techniques at hand, the number of genomic sequence variants detected in patient investigations is rising constantly. The number of potentially involved genes in hereditary hearing loss is rising simultaneously.In this overview, current methods for diagnostic workup on a molecular and functional level for variants of the SLC26A4 gene are described. Based on the description of the physiological function of the resulting protein Pendrin, molecular investigations for interpretation of the function are explained. Based on these investigations, the potential clinical consequences of a variant may be predicted more precisely and simplify routine reporting of a proven genotype and a phenotype, at hand. Finally, subsequent clinical investigations necessary, such as perchlorate discharge test, as well as therapeutic options are discussed.
Assuntos
Perda Auditiva Neurossensorial , Aqueduto Vestibular , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/genética , Humanos , Proteínas de Membrana Transportadoras/genética , Mutação , Transportadores de Sulfato/genéticaRESUMO
Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. PGC-1α, encoded by PPARGC1A, is a transcriptional co-activator that has been implicated in the pathogenesis of neurodegenerative disorders. We recently discovered multiple new PPARGC1A transcripts that initiate from a novel promoter located far upstream of the reference gene promoter, are CNS-specific and are more abundant than reference gene transcripts in whole brain. These CNS-specific transcripts encode two main full-length and several truncated isoforms via alternative splicing. Truncated CNS-isoforms include 17â¯kDa proteins that lack the second LXXLL motif serving as an interaction site for several nuclear receptors. We now determined expression levels of CNS- and reference gene transcripts in 5 brain regions of 21, 8, and 13 deceased subjects with idiopathic PD, Lewy body dementia and controls without neurodegenerative disorders, respectively. We observed reductions of CNS-specific transcripts (encoding full-length isoforms) only in the substantia nigra pars compacta of PD and Lewy body dementia. However, in the substantia nigra and globus pallidus of PD cases we found an up-regulation of transcripts encoding the 17â¯kDa proteins that inhibited the co-activation of several transcription factors by full-length PGC-1α proteins in transfection assays. In two established animal models of PD, the PPARGC1A expression profiles differed from the profile in human PD in that the levels of CNS- and reference gene transcripts were decreased in several brain regions. Furthermore, we identified haplotypes in the CNS-specific region of PPARGC1A that appeared protective for PD in a clinical cohort and a post-mortem sample (Pâ¯=â¯.0002). Thus, functional and genetic studies support a role of the CNS-specific PPARGC1A locus in PD.
Assuntos
Encéfalo/metabolismo , Doença de Parkinson/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Loci Gênicos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Isoformas de Proteínas/genéticaRESUMO
Pendrin (SLC26A4), a Cl-/anion exchanger, is expressed at high levels in kidney, thyroid, and inner ear epithelia, where it has an essential role in bicarbonate secretion/chloride reabsorption, iodide accumulation, and endolymph ion balance, respectively. Pendrin is expressed at lower levels in other tissues, such as airways and esophageal epithelia, where it is transcriptionally regulated by the inflammatory cytokines interleukin (IL)-4 and IL-13 through a signal transducer and activator of transcription 6 (STAT6)-mediated pathway. In the airway epithelium, increased pendrin expression during inflammatory diseases leads to imbalances in airway surface liquid thickness and mucin release, while, in the esophageal epithelium, dysregulated pendrin expression is supposed to impact the intracellular pH regulation system. In this review, we discuss some of the recent findings on interleukin-mediated transcriptional regulation of pendrin and how this dysregulation impacts airway and esophagus epithelial homeostasis during inflammatory diseases.
Assuntos
Mucosa Esofágica/metabolismo , Regulação da Expressão Gênica , Interleucinas/metabolismo , Mucosa Respiratória/metabolismo , Transportadores de Sulfato/genética , Transcrição Gênica , Animais , Biomarcadores , Mucosa Esofágica/efeitos dos fármacos , Humanos , Inflamação/tratamento farmacológico , Inflamação/etiologia , Inflamação/metabolismo , Muco/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Transportadores de Sulfato/metabolismo , Transportadores de Sulfato/uso terapêuticoRESUMO
BACKGROUND/AIMS: In the human genome, more than 400 genes encode ion channels, which are ubiquitously expressed and often coexist and participate in almost all physiological processes. Therefore, ion channel blockers represent fundamental tools in discriminating the contribution of individual channel types to a physiological phenomenon. However, unspecific effects of these compounds may represent a confounding factor. Three commonly used chloride channel inhibitors, i.e. 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS), 5-nitro-2-[(3-phenylpropyl) amino]benzoic acid (NPPB) and the anti-inflammatory drug niflumic acid were tested to identify the lowest concentration effective on Cl- channels and ineffective on K+ channels. METHODS: The activity of the above mentioned compounds was tested by whole cell patch-clamp on the swelling-activated Cl- current ICl,swell and on the endogenous voltage-dependent, outwardly rectifying K+ selective current in human kidney cell lines (HEK 293/HEK 293 Phoenix). RESULTS: Micromolar (1-10 µM) concentrations of DIDS and NPPB could not discriminate between the Cl- and K+ selective currents. Specifically, 1 µM DIDS only affected the K+ current and 10 µM NPPB equally affected the Cl- and K+ currents. Only relatively high (0.1-1 mM) concentrations of DIDS and prolonged (5 minutes) exposure to 0.1-1 mM NPPB preferentially suppressed the Cl- current. Niflumic acid preferentially inhibited the Cl- current, but also significantly affected the K+ current. The endogenous voltage-dependent, outwardly rectifying K+ selective current in HEK 293/HEK 293 Phoenix cells was shown to arise from the Kv 3.1 channel, which is extensively expressed in brain and is involved in neurological diseases. CONCLUSION: The results of the present study underscore that sensitivity of a given physiological phenomenon to the Cl- channel inhibitors NPPB, DIDS and niflumic acid may actually arise from an inhibition of Cl- channels but can also result from an inhibition of voltage-dependent K+ channels, including the Kv 3.1 channel. The use of niflumic acid as anti-inflammatory drug in patients with concomitant Kv 3.1 dysfunction may result contraindicated.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Canal de Potássio Kv1.3/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Potássio/metabolismo , Animais , Cloretos/metabolismo , Células Epiteliais/citologia , Células HEK293 , Humanos , Túbulos Renais Proximais/citologia , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/genética , Camundongos , Células NIH 3T3 , Ácido Niflúmico/química , Ácido Niflúmico/farmacologia , Nitrobenzoatos/química , Nitrobenzoatos/farmacologia , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/química , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
The prevalence and spectrum of sequence alterations in the SLC26A4 gene, which codes for the anion exchanger pendrin, are population-specific and account for at least 50% of cases of non-syndromic hearing loss associated with an enlarged vestibular aqueduct. A cohort of nineteen patients from Austria with hearing loss and a radiological alteration of the vestibular aqueduct underwent Sanger sequencing of SLC26A4 and GJB2, coding for connexin 26. The pathogenicity of sequence alterations detected was assessed by determining ion transport and molecular features of the corresponding SLC26A4 protein variants. In this group, four uncharacterized sequence alterations within the SLC26A4 coding region were found. Three of these lead to protein variants with abnormal functional and molecular features, while one should be considered with no pathogenic potential. Pathogenic SLC26A4 sequence alterations were only found in 12% of patients. SLC26A4 sequence alterations commonly found in other Caucasian populations were not detected. This survey represents the first study on the prevalence and spectrum of SLC26A4 sequence alterations in an Austrian cohort and further suggests that genetic testing should always be integrated with functional characterization and determination of the molecular features of protein variants in order to unequivocally identify or exclude a causal link between genotype and phenotype.
Assuntos
Perda Auditiva Neurossensorial/patologia , Proteínas de Membrana Transportadoras/genética , Aqueduto Vestibular/anormalidades , População Branca/genética , Adolescente , Adulto , Idoso , Alelos , Áustria , Criança , Conexina 26/genética , Retículo Endoplasmático/metabolismo , Feminino , Genótipo , Células HEK293 , Perda Auditiva Neurossensorial/genética , Humanos , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Pessoa de Meia-Idade , Fenótipo , Polimorfismo de Nucleotídeo Único , Transportadores de Sulfato , Aqueduto Vestibular/patologia , Adulto JovemRESUMO
O-linked N-acetylglucosamine (O-GlcNAc) is a dynamic post-translational modification of serine and threonine residues on nuclear and cytoplasmic proteins. O-GlcNAc modification influences many cellular mechanisms, including carbohydrate metabolism, signal transduction and protein degradation. Multiple studies also showed that cell cycle might be modulated by O-GlcNAc. Although the role of O-GlcNAc in the regulation of some cell cycle processes such as mitotic spindle organization or histone phosphorylation is well established, the general behaviour of O-GlcNAc regulation during cell cycle is still controversial. In this study, we analysed the dynamic changes of overall O-GlcNAc levels in HeLa cells using double thymidine block. O-GlcNAc levels in G1, S, G2 and M phase were measured. We observed that O-GlcNAc levels are significantly increased during mitosis in comparison to the other cell cycle phases. However, this change could only be detected when mitotic cells were enriched by harvesting round shaped cells from the G2/M fraction of the synchronized cells. Our data verify that O-GlcNAc is elevated during mitosis, but also emphasize that O-GlcNAc levels can significantly change in a short period of time. Thus, selection and collection of cells at specific cell-cycle checkpoints is a challenging, but necessary requirement for O-GlcNAc studies.
Assuntos
Acetilglucosamina/metabolismo , Mitose , Ciclo Celular , Glicosilação , Células HeLa , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Timidina/metabolismoRESUMO
Interleukin-13 (IL13) is a major player in the development of airway hyperresponsiveness in several respiratory disorders. Emerging data suggest that an increased expression of pendrin in airway epithelia is associated with elevated airway hyperreactivity in asthma. Here, we investigate the effect of IL13 on pendrin localization and function using bronchiolar NCI-H292 cells. The data obtained revealed that IL13 increases the cell surface expression of pendrin. This effect was paralleled by a significant increase in the intracellular pH, possibly via indirect stimulation of NHE. IL13 effect on pendrin localization and intracellular pH was reversed by theophylline, a bronchodilator compound used to treat asthma. IL13 upregulated RhoA activity, a crucial protein controlling actin dynamics, via G-alpha-13. Specifically, IL13 stabilized actin cytoskeleton and promoted co-localization and a direct molecular interaction between pendrin and F-actin in the plasma membrane region. These effects were reversed following exposure of cells to theophylline. Selective inhibition of Rho kinase, a downstream effector of Rho, reduced the IL13-dependent cell surface expression of pendrin. Together, these data indicate that IL13 increases pendrin abundance to the cell surface via Rho/actin signaling, an effect reversed by theophylline.
Assuntos
Actinas/metabolismo , Brônquios/metabolismo , Interleucina-13/metabolismo , Transdução de Sinais/fisiologia , Transportadores de Sulfato/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Asma/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Epitélio/metabolismo , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
Pendrin is upregulated in bronchial epithelial cells following IL-4 stimulation via binding of STAT6 to an N4 GAS motif. Basal CpG methylation of the pendrin promoter is cell-specific. We studied if a correlation exists between IL-4 sensitivity and the CpG methylation status of the pendrin promoter in human bronchial epithelial cell models. METHODS: Real-time PCR and pyrosequencing were used to respectively quantify pendrin mRNA levels and methylation of pendrin promoter, with and without IL-4 stimulation, in healthy and diseased primary HBE cells, as well as NCI-H292 cells. RESULTS: Increases in pendrin mRNA after IL-4 stimulation was more robust in NCI-H292 cells than in primary cells. The amount of gDNA methylated varied greatly between the cell types. In particular, CpG site 90 located near the N4 GAS motif was highly methylated in the primary cells. An additional CpG site (90bis), created by a SNP, was found only in the primary cells. IL-4 stimulation resulted in dramatic demethylation of CpG sites 90 and 90bis in the primary cells. CONCLUSIONS: IL-4 induces demethylation of specific CpG sites within the pendrin promoter. These epigenetic alterations are cell type specific, and may in part dictate pendrin mRNA transcription.
Assuntos
Brônquios/metabolismo , Ilhas de CpG , Metilação de DNA , Células Epiteliais/metabolismo , Interleucina-4/metabolismo , Proteínas de Membrana Transportadoras/biossíntese , Elementos de Resposta , Brônquios/citologia , Linhagem Celular , Epigênese Genética , Células Epiteliais/citologia , Feminino , Humanos , Pessoa de Meia-Idade , RNA Mensageiro/biossíntese , Transportadores de SulfatoRESUMO
BACKGROUND/AIM: Accurate genotyping of CYP2D6 is challenging due to its inherent genetic variation, copy number variation (duplications and deletions) and hybrid formation with highly homologous pseudogenes. Because a relatively high percentage (â¼25%) of clinically prescribed drugs are substrates for this enzyme, accurate determination of its genotype for phenotype prediction is essential. METHODS: A cohort of 365 patient samples was genotyped for CYP2D6 using Sanger sequencing (as the gold standard), hydrolysis probe assays or pyrosequencing. RESULTS: A discrepant result between the three genotyping methods for the loss of function CYP2D6*3 (g.2549delA, rs35742686) genetic variant was found in one of the samples. This sample also contained the CYP2D6 g.2470T>C (rs17002852) variation, which had an allele frequency of 2.47% in our cohort. Redesign of the CYP2D6*3 pyrosequencing and hydrolysis probe assays to avoid CYP2D6 g.2470 corrected the anomaly. CONCLUSION: To evidence allele drop out and increase the accuracy of genotyping, intra-patient validation of the same genetic variation with at least two separate methods should be considered.
Assuntos
Citocromo P-450 CYP2D6/genética , Variações do Número de Cópias de DNA , Técnicas de Genotipagem/métodos , Alelos , Estudos de Coortes , DNA/química , DNA/isolamento & purificação , DNA/metabolismo , Análise Discriminante , Frequência do Gene , Genótipo , Haplótipos , Humanos , Fenótipo , Análise de Sequência de DNARESUMO
Sequence alterations in the pendrin gene (SLC26A4) leading to functionally affected protein variants are frequently involved in the pathogenesis of syndromic and nonsyndromic deafness. Considering the high number of SLC26A4 sequence alterations reported to date, discriminating between functionally affected and unaffected pendrin protein variants is essential in contributing to determine the genetic cause of deafness in a given patient. In addition, identifying molecular features common to the functionally affected protein variants can be extremely useful to design future molecule-directed therapeutic approaches. Here we show the functional and molecular characterization of six previously uncharacterized pendrin protein variants found in a cohort of 58 Brazilian deaf patients. Two variants (p.T193I and p.L445W) were undetectable in the plasma membrane, completely retained in the endoplasmic reticulum and showed no transport function; four (p.P142L, p.G149R, p.C282Y and p.Q413R) showed reduced function and significant, although heterogeneous, expression levels in the plasma membrane. Importantly, total expression levels of all of the functionally affected protein variants were significantly reduced with respect to the wild-type and a fully functional variant (p.R776C), regardless of their subcellular localization. Interestingly, reduction of expression may also reduce the transport activity of variants with an intrinsic gain of function (p.Q413R). As reduction of overall cellular abundance was identified as a common molecular feature of pendrin variants with affected function, the identification of strategies to prevent reduction in expression levels may represent a crucial step of potential future therapeutic interventions aimed at restoring the transport activity of dysfunctional pendrin variants.
RESUMO
BACKGROUND/AIMS: Pendrin is a Cl-/I-/HCO3- exchanger playing a fundamental role in controlling blood pressure and airway function, therefore representing an attractive target for the treatment of hypertensive states and respiratory distresses. A review of the literature regarding the ability of some compounds (namely several known inhibitors of ion transport) to block pendrin activity revealed discordant findings. These incongruous findings may be due, in part, to the concentration of compound and/or the nature of the model system used in the study. METHODS: Pendrin activity was evaluated by measuring pendrin-dependent iodide influx following overexpression of the transporter in a human kidney cell line, in the presence of selected test compounds or the respective vehicles. RESULTS: Pendrin activity was significantly hampered by 0.1 mM 5-nitro-2-[(3-phenylpropyl)amino]benzoic acid (NPPB), niflumic acid and tenidap, but was resistant to 0.1 mM 4, 4'-diisothiocyano-2, 2'-stilbene-disulfonic acid (DIDS), furosemide and probenecid. CONCLUSIONS: The results of the present study indicate that clinically effective non-steroidal anti-inflammatory drugs (niflumic acid and tenidap) directly inhibit pendrin activity.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/química , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/metabolismo , Western Blotting , Linhagem Celular , Furosemida/química , Furosemida/metabolismo , Células HEK293 , Humanos , Indóis/química , Indóis/metabolismo , Rim/citologia , Rim/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Ácido Niflúmico/química , Ácido Niflúmico/metabolismo , Nitrobenzoatos/química , Nitrobenzoatos/metabolismo , Oxindóis , Plasmídeos/genética , Plasmídeos/metabolismo , Probenecid/química , Probenecid/metabolismo , Ligação Proteica , Espectrometria de Fluorescência , Transportadores de SulfatoRESUMO
The small hydrophobic (SH) protein is a 64-amino-acid polypeptide encoded by the human respiratory syncytial virus (hRSV). SH protein has a single α-helical transmembrane (TM) domain that forms pentameric ion channels. Herein, we report the first inhibitor of the SH protein channel, pyronin B, and we have mapped its binding site to a conserved surface of the RSV SH pentamer, at the C-terminal end of the transmembrane domain. The validity of the SH protein structural model used has been confirmed by using a bicellar membrane-mimicking environment. However, in bicelles the α-helical stretch of the TM domain extends up to His-51, and by comparison with previous models both His-22 and His-51 adopt an interhelical/lumenal orientation relative to the channel pore. Neither His residue was found to be essential for channel activity although His-51 protonation reduced channel activity at low pH, with His-22 adopting a more structural role. The latter results are in contrast with previous patch clamp data showing channel activation at low pH, which could not be reproduced in the present work. Overall, these results establish a solid ground for future drug development targeting this important viroporin. Importance: The human respiratory syncytial virus (hRSV) is responsible for 64 million reported cases of infection and 160,000 deaths each year. Lack of adequate antivirals fuels the search for new targets for treatment. The small hydrophobic (SH) protein is a 64-amino-acid polypeptide encoded by hRSV and other paramyxoviruses, and its absence leads to viral attenuation in vivo and early apoptosis in infected cells. SH protein forms pentameric ion channels that may constitute novel drug targets, but no inhibitor for this channel activity has been reported so far. A small-molecule inhibitor, pyronin B, can reduce SH channel activity, and its likely binding site on the SH protein channel has been identified. Black lipid membrane (BLM) experiments confirm that protonation of both histidine residues reduces stability and channel activity. These results contrast with previous patch clamp data that showed low-pH activation, which we have not been able to reproduce.
Assuntos
Bicamadas Lipídicas , Vírus Sinciciais Respiratórios/metabolismo , Proteínas Virais/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação , Chlorocebus aethiops , Clonagem Molecular , Genes Virais , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Técnicas de Patch-Clamp , Pironina/análogos & derivados , Pironina/farmacologia , Vírus Sinciciais Respiratórios/genética , Vírus Sinciciais Respiratórios/crescimento & desenvolvimento , Células Vero , Ensaio de Placa Viral , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
BACKGROUND: Pollution of marine ecosystems and, specifically, heavy metals contamination may compromise the physiology of marine animals with events occurring on a cellular and molecular level. The present study focuses on the effect of short-term exposure to heavy metals like Zinc, Cadmium, Cobalt and Lanthanum (2-10 mM) on the homeostasis of Pelagia noctiluca (Cnidaria, Scyphozoa), a jellyfish abundant in the Mediterranean sea. This species possesses stinging organoids, termed nematocysts, whose discharge and concomitant delivery of venom underlie the survival of all Cnidaria. METHODS: Nematocysts discharge response, elicited by combined chemico-physical stimulation, was verified on excised oral arms exposed to heavy metals for 20 min. In addition, the hemolytic activity of toxins, contained in the crude venom extracted from nematocysts isolated from oral arms, was tested on human erythrocytes, in the presence of heavy metals or their mixture. RESULTS: Treatment with heavy metals significantly inhibited both nematocysts discharge response and hemolytic activity of crude venom, in a dose-dependent manner, not involving oxidative events, that was irreversible in the case of Lanthanum. CONCLUSION: Our findings show that the homeostasis of Pelagia noctiluca, in terms of nematocysts discharge capability and effectiveness of venom toxins, is dramatically and rapidly compromised by heavy metals and confirm that this jellyfish is eligible as a model for ecotoxicological investigations.