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1.
Comput Biol Chem ; 80: 16-22, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30861403

RESUMO

Prostate cancer (PCa) is the most frequent type of cancer in men. Hypericum perforatum (H. Perforatum) extract (HPE) administration provides remarkable decrease of PCa development. H. perforatum contains 7 conserved miRNAs (Hyp-miR-156a, Hyp-miR-156b, Hyp-miR-166, Hyp-miR-390, Hyp-miR-394, Hyp-miR-396 and Hyp-miR-414) with different targets. In this study, we aimed to investigate cross-kingdom gene regulation via miRNAs of H. perforatum flower dietetically absorbed in manner of an in silico approach to define potential biomarkers for PCa. psRNATarget database was used to find human genes targeted by 7 pre-defined H. perforatum miRNAs. We defined the mostly affected gene families from these miRNAs as ZNF, TMEM, SLC and FAM gene families. GeneMANIA database was used to define the most affected genes (TMEM41B and SLC4A7) from these 7 miRNAs. cBioPortal database was used to define alteration frequencies of TMEM41B and SLC4A7 on different types of PCa and to measure the mutual interaction potency and significance of co-occurence in PCa. This analysis showed that neuroendocrine prostate cancer (NEPC) had the highest total mutation frequency (22%) of TMEM41B and SLC4A7 genes. Also, TMEM41B and SLC4A7 genes had an average 2.1% pathway change potential among all different types of PCa. Moreover, TMEM41B and SLC4A7 gene pair was found significantly co-occurrent in PCa (p < 0.001). Finally, via GEPIA database, we used Spearman correlation analysis to measure the correlation degree of TMEM41B and SLC4A7 genes in PCa and found their significant correlation with PCa (p = 1.2 × 10-12, R = 0.28). All in all, it was proved in silico and supported with previously known clinical data that SLC4A7 and TMEM41B potentially have a significant and critical tumor suppressive role for PCa, and show this effect combinatorily working together. This is the first study correlating SLC4A7 and TMEM41B with PCa significantly.


Assuntos
Flores/genética , Regulação Neoplásica da Expressão Gênica , Hypericum/genética , MicroRNAs/genética , Neoplasias da Próstata/genética , RNA de Plantas/genética , Biomarcadores , Simulação por Computador , Humanos , Masculino , Proteínas de Membrana/genética , Mutação , Simportadores de Sódio-Bicarbonato/genética , Software
2.
Int J Mol Sci ; 20(2)2019 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-30642095

RESUMO

The advancement of bioinformatics and machine learning has facilitated the discovery and validation of omics-based biomarkers. This study employed a novel approach combining multi-platform transcriptomics and cutting-edge algorithms to introduce novel signatures for accurate diagnosis of colorectal cancer (CRC). Different random forests (RF)-based feature selection methods including the area under the curve (AUC)-RF, Boruta, and Vita were used and the diagnostic performance of the proposed biosignatures was benchmarked using RF, logistic regression, naïve Bayes, and k-nearest neighbors models. All models showed satisfactory performance in which RF appeared to be the best. For instance, regarding the RF model, the following were observed: mean accuracy 0.998 (standard deviation (SD) < 0.003), mean specificity 0.999 (SD < 0.003), and mean sensitivity 0.998 (SD < 0.004). Moreover, proposed biomarker signatures were highly associated with multifaceted hallmarks in cancer. Some biomarkers were found to be enriched in epithelial cell signaling in Helicobacter pylori infection and inflammatory processes. The overexpression of TGFBI and S100A2 was associated with poor disease-free survival while the down-regulation of NR5A2, SLC4A4, and CD177 was linked to worse overall survival of the patients. In conclusion, novel transcriptome signatures to improve the diagnostic accuracy in CRC are introduced for further validations in various clinical settings.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/diagnóstico , Biologia Computacional/métodos , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Algoritmos , Área Sob a Curva , Teorema de Bayes , Fatores Quimiotáticos/genética , Neoplasias Colorretais/genética , Feminino , Proteínas Ligadas por GPI/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Isoantígenos/genética , Modelos Logísticos , Aprendizado de Máquina , Prognóstico , Receptores de Superfície Celular/genética , Receptores Citoplasmáticos e Nucleares/genética , Proteínas S100/genética , Sensibilidade e Especificidade , Simportadores de Sódio-Bicarbonato/genética , Análise de Sobrevida , Fator de Crescimento Transformador beta1/genética
3.
Cell Physiol Biochem ; 50(4): 1361-1375, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30355950

RESUMO

BACKGROUND/AIMS: The sodium-dependent bicarbonate transporter Slc4a8 (a.k.a NDCBE) mediates the co-transport of sodium and bicarbonate in exchange for chloride. It is abundantly detected in the brain, with low expression levels in the kidney. The cell distribution and subcellular localization of Slc4a8 in the kidney and its role in acid/base and electrolyte homeostasis has been the subject of conflicting reports. There are no conclusive localization or functional studies to pinpoint the location and demonstrate the function of Slc4a8 in the kidney. METHODS: Molecular techniques, including RT-PCR and in situ hybridization, were performed on kidney sections and tagged epitopes were used to examine the membrane targeting of Slc4a8 in polarized kidney cells. Crispr/Cas9 was used to generate and examine Slc4a8 KO mice. RESULTS: Zonal distribution and in situ hybridization studies showed very little expression for Slc4a8 (NDCBE) in the cortex or in cortical collecting ducts (CCD). Slc4a8 was predominantly detected in the outer and inner medullary collecting ducts (OMCD and IMCD), and was targeted to the basolateral membrane of osmotically tolerant MDCK cells. Slc4a8 KO mice did not show any abnormal salt or bicarbonate wasting under baseline conditions or in response to bicarbonate loading, salt restriction or furosemide-induced diuresis. CONCLUSION: Slc4a8 (NDCBE) is absent in the CCD and is predominantly localized on the basolateral membrane of medullary collecting duct cells. Further, Slc4a8 deletion does not cause significant acid base or electrolyte abnormalities in pathophysiologic states. Additional studies are needed to examine the role of Slc4a8 (NDCBE) in intracellular pH and volume regulation in medullary collecting duct cells.


Assuntos
Rim/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Animais , Bicarbonatos/metabolismo , Sistemas CRISPR-Cas/genética , Diurese/efeitos dos fármacos , Cães , Furosemida/farmacologia , Hibridização In Situ , Túbulos Renais Coletores/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Camundongos Knockout , Oligorribonucleotídeos Antissenso/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Sódio/urina , Cloreto de Sódio/metabolismo , Simportadores de Sódio-Bicarbonato/deficiência , Simportadores de Sódio-Bicarbonato/genética
4.
PLoS One ; 13(4): e0189464, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29642240

RESUMO

RATIONALE: Salt sensitivity of blood pressure affects >30% of the hypertensive and >15% of the normotensive population. Variants of the electrogenic sodium bicarbonate cotransporter NBCe2 gene, SLC4A5, are associated with increased blood pressure in several ethnic groups. SLC4A5 variants are also highly associated with salt sensitivity, independent of hypertension. However, little is known about how NBCe2 contributes to salt sensitivity, although NBCe2 regulates renal tubular sodium bicarbonate transport. We hypothesized that SLC4A5 rs10177833 and rs7571842 increase NBCe2 expression and human renal proximal tubule cell (hRPTC) sodium transport and may be a cause of salt sensitivity of blood pressure. OBJECTIVE: To characterize the hRPTC ion transport of wild-type (WT) and homozygous variants (HV) of SLC4A5. METHODS AND RESULTS: The expressions of NBCe2 mRNA and protein were not different between hRPTCs carrying WT or HV SLC4A5 before or after dopaminergic or angiotensin (II and III) stimulation. However, luminal to basolateral sodium transport, NHE3 protein, and Cl-/HCO3- exchanger activity in hRPTCs were higher in HV than WT SLC4A5. Increasing intracellular sodium enhanced the apical location of NBCe2 in HV hRPTCs (4.24±0.35% to 11.06±1.72% (P<0.05, N = 3, 2-way ANOVA, Holm-Sidak test)) as determined by Total Internal Reflection Fluorescence Microscopy (TIRFM). In hRPTCs isolated from kidney tissue, increasing intracellular sodium enhanced bicarbonate-dependent pH recovery rate and increased NBCe2 mRNA and protein expressions to a greater extent in HV than WT SLC4A5 (+38.00±6.23% vs HV normal salt (P<0.01, N = 4, 2-way ANOVA, Holm-Sidak test)). In hRPTCs isolated from freshly voided urine, bicarbonate-dependent pH recovery was also faster in those from salt-sensitive and carriers of HV SLC4A5 than from salt-resistant and carriers of WT SLC4A5. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was normalized by SLC4A5- but not SLC4A4-shRNA. The binding of purified hepatocyte nuclear factor type 4A (HNF4A) to DNA was increased in hRPTCs carrying HV SLC4A5 rs7571842 but not rs10177833. The faster NBCe2-specific bicarbonate-dependent pH recovery rate in HV SCL4A5 was abolished by HNF4A antagonists. CONCLUSION: NBCe2 activity is stimulated by an increase in intracellular sodium and is hyper-responsive in hRPTCs carrying HV SLC4A5 rs7571842 through an aberrant HNF4A-mediated mechanism.


Assuntos
Bicarbonatos/metabolismo , Túbulos Renais Proximais/citologia , Polimorfismo de Nucleotídeo Único , Simportadores de Sódio-Bicarbonato/genética , Sódio/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/genética , Agonistas de Dopamina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Homozigoto , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Receptores Dopaminérgicos/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo
5.
Genome Biol ; 18(1): 144, 2017 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-28754144

RESUMO

BACKGROUND: Variable expressivity is a well-known phenomenon in which patients with mutations in one gene display varying degrees of clinical severity, potentially displaying only subsets of the clinical manifestations associated with the multisystem disorder linked to the gene. This remains an incompletely understood phenomenon with proposed mechanisms ranging from allele-specific to stochastic. RESULTS: We report three consanguineous families in which an isolated ocular phenotype is linked to a novel 3' UTR mutation in SLC4A4, a gene known to be mutated in a syndromic form of intellectual disability with renal and ocular involvement. Although SLC4A4 is normally devoid of AU-rich elements (AREs), a 3' UTR motif that mediates post-transcriptional control of a subset of genes, the mutation we describe creates a functional ARE. We observe a marked reduction in the transcript level of SLC4A4 in patient cells. Experimental confirmation of the ARE-creating mutation is shown using a post-transcriptional reporter system that reveals consistent reduction in the mRNA-half life and reporter activity. Moreover, the neo-ARE binds and responds to the zinc finger protein ZFP36/TTP, an ARE-mRNA decay-promoting protein. CONCLUSIONS: This novel mutational mechanism for a Mendelian disease expands the potential mechanisms that underlie variable phenotypic expressivity in humans to also include 3' UTR mutations with tissue-specific pathology.


Assuntos
Regiões 3' não Traduzidas , Elementos Ricos em Adenilato e Uridilato , Distrofias Hereditárias da Córnea/genética , Mutação , Fenótipo , Simportadores de Sódio-Bicarbonato/genética , Adulto , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Criança , Consanguinidade , Córnea/metabolismo , Córnea/patologia , Distrofias Hereditárias da Córnea/metabolismo , Distrofias Hereditárias da Córnea/patologia , Feminino , Regulação da Expressão Gênica , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Masculino , Análise da Randomização Mendeliana , Linhagem , Estabilidade de RNA , Simportadores de Sódio-Bicarbonato/metabolismo
6.
Cell Mol Biol (Noisy-le-grand) ; 63(5): 11-18, 2017 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-28719339

RESUMO

Diabetes is known to alter both oxidative and glycolytic pathways in a fiber type-dependent manner. The aim of present study was to investigate the effects of endurance training on muscle NHE1 and NBC1 genes and proteins expression in type 2 diabetic rats. Male wistar rats (n=30), 4 weeks old and 95.7±10.8g, were randomly selected and divided into control, diabetic without training and diabetic with training groups. Diabetes was induced by injection of low dose of streptotozin and feeding with high-fat diet. The Endurance training was performed for 7 weeks that started with relatively low speed and duration of 20 m min-1 for 20 min in the first week and gradually reached to 30 m min-1 for 35min in the last week. NHE1 and NBC1 genes and proteins expression were determined by Real time-PCR and western blotting techniques, respectively, in Soleus as an oxidative and EDL (Extensor digitorum longus) as a glycolytic muscle preparation. NHE1 mRNA and protein expression reduced significantly in EDL and Soleus in the diabetic without training group compared with the control group. However, reduction in the expression of NBC1 gene and protein in the diabetic without training group compared to controls did not significant. Endurance training increased NHE1 and NBC1 genes and proteins expression in both EDL and Soleus in the diabetic training group compared to control groups. In conclusion, endurance training may improve the capacity of pHi regulation in muscles by lactate-independent pathway.


Assuntos
Diabetes Mellitus Experimental/genética , Dieta Hiperlipídica , Regulação da Expressão Gênica , Glicólise , Fibras Musculares de Contração Rápida/metabolismo , Simportadores de Sódio-Bicarbonato/genética , Trocador 1 de Sódio-Hidrogênio/genética , Animais , Biomarcadores/metabolismo , Glicemia/metabolismo , Peso Corporal , Glicólise/genética , Insulina/sangue , Resistência à Insulina , Masculino , Oxirredução , Condicionamento Físico Animal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Simportadores de Sódio-Bicarbonato/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Estreptozocina
7.
Glia ; 65(8): 1361-1375, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28568893

RESUMO

The electrogenic sodium bicarbonate cotransporter NBCe1 (SLC4A4) expressed in astrocytes regulates intracellular and extracellular pH. Here, we introduce transforming growth factor beta (TGF-ß) as a novel regulator of NBCe1 transcription and functional expression. Using hippocampal slices and primary hippocampal and cortical astrocyte cultures, we investigated regulation of NBCe1 and elucidated the underlying signaling pathways by RT-PCR, immunoblotting, immunofluorescence, intracellular H(+ ) recording using the H(+ ) -sensitive dye 2',7'-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein, mink lung epithelial cell (MLEC) assay, and chromatin immunoprecipitation. Activation of TGF-ß signaling significantly upregulated transcript, protein, and surface expression of NBCe1. These effects were TGF-ß receptor-mediated and suppressed following inhibition of JNK and Smad signaling. Moreover, 4-aminopyridine (4AP)-dependent NBCe1 regulation requires TGF-ß. TGF-ß increased the rate and amplitude of intracellular H+ changes upon challenging NBCe1 in wild-type astrocytes but not in cortical astrocytes from Slc4a4-deficient mice. A Smad4 binding sequence was identified in the NBCe1 promoter and Smad4 binding increased after activation of TGF-ß signaling. The data show for the first time that NBCe1 is a direct target of TGF-ß/Smad4 signaling. Through activation of the canonical pathway TGF-ß acts directly on NBCe1 by binding of Smad4 to the NBCe1 promoter and regulating its transcription, followed by increased protein expression and transport activity.


Assuntos
Astrócitos/metabolismo , Regulação da Expressão Gênica/fisiologia , Transdução de Sinais/fisiologia , Simportadores de Sódio-Bicarbonato/metabolismo , Fator de Crescimento Transformador beta/metabolismo , 4-Aminopiridina/farmacologia , Animais , Benzamidas/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Antiportadores de Cloreto-Bicarbonato/farmacologia , Dioxóis/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/citologia , Concentração de Íons de Hidrogênio , Isoenzimas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Bloqueadores dos Canais de Potássio/farmacologia , Retinal Desidrogenase/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Smad4/metabolismo , Simportadores de Sódio-Bicarbonato/antagonistas & inibidores , Simportadores de Sódio-Bicarbonato/genética , Fator de Crescimento Transformador beta/genética
8.
J Am Soc Nephrol ; 28(8): 2311-2321, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28360221

RESUMO

Disorders of water balance, an excess or deficit of total body water relative to body electrolyte content, are common and ascertained by plasma hypo- or hypernatremia, respectively. We performed a two-stage genome-wide association study meta-analysis on plasma sodium concentration in 45,889 individuals of European descent (stage 1 discovery) and 17,637 additional individuals of European descent (stage 2 replication), and a transethnic meta-analysis of replicated single-nucleotide polymorphisms in 79,506 individuals (63,526 individuals of European descent, 8765 individuals of Asian Indian descent, and 7215 individuals of African descent). In stage 1, we identified eight loci associated with plasma sodium concentration at P<5.0 × 10-6 Of these, rs9980 at NFAT5 replicated in stage 2 meta-analysis (P=3.1 × 10-5), with combined stages 1 and 2 genome-wide significance of P=5.6 × 10-10 Transethnic meta-analysis further supported the association at rs9980 (P=5.9 × 10-12). Additionally, rs16846053 at SLC4A10 showed nominally, but not genome-wide, significant association in combined stages 1 and 2 meta-analysis (P=6.7 × 10-8). NFAT5 encodes a ubiquitously expressed transcription factor that coordinates the intracellular response to hypertonic stress but was not previously implicated in the regulation of systemic water balance. SLC4A10 encodes a sodium bicarbonate transporter with a brain-restricted expression pattern, and variant rs16846053 affects a putative intronic NFAT5 DNA binding motif. The lead variants for NFAT5 and SLC4A10 are cis expression quantitative trait loci in tissues of the central nervous system and relevant to transcriptional regulation. Thus, genetic variation in NFAT5 and SLC4A10 expression and function in the central nervous system may affect the regulation of systemic water balance.


Assuntos
Loci Gênicos , Plasma/química , Simportadores de Sódio-Bicarbonato/genética , Sódio/análise , Fatores de Transcrição/genética , Desequilíbrio Hidroeletrolítico/sangue , Desequilíbrio Hidroeletrolítico/genética , Idoso , Grupos de Populações Continentais , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Concentração Osmolar
9.
Acta Physiol (Oxf) ; 221(2): 129-141, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28319329

RESUMO

AIM: The electroneutral Na+ , HCO3- cotransporter NBCn1 and Na+ /H+ exchanger NHE1 regulate acid-base balance in vascular smooth muscle cells (VSMCs) and modify artery function and structure. Pathological conditions - notably ischaemia - can dramatically perturb intracellular (i) and extracellular (o) pH and [Na+ ]. We examined effects of low [Na+ ]o and pHo on NBCn1 and NHE1 activity in VSMCs of small arteries. METHODS: We measured pHi by 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-based fluorescence microscopy of mouse mesenteric arteries and induced intracellular acidification by NH4+ prepulse technique. RESULTS: NBCn1 activity - defined as Na+ -dependent, amiloride-insensitive net base uptake with CO2 /HCO3- present - was inhibited equally when pHo decreased from 7.4 (22 mm HCO3-/5% CO2 ) by metabolic (pHo 7.1/11 mm HCO3-: 22 ± 8%; pHo 6.8/5.5 mm HCO3-: 61 ± 7%) or respiratory (pHo 7.1/10% CO2 : 35 ± 11%; pHo 6.8/20% CO2 : 56 ± 7%) acidosis. Extracellular acidosis more prominently inhibited NHE1 activity - defined as Na+ -dependent net acid extrusion without CO2 /HCO3- present - at both pHo 7.1 (45 ± 9%) and 6.8 (85 ± 5%). Independently of pHo , lowering [Na+ ]o from 140 to 70 mm reduced NBCn1 and NHE1 activity <20% whereas transport activities declined markedly (25-50%) when [Na+ ]o was reduced to 35 mm. Steady-state pHi decreased more during respiratory (ΔpHi /ΔpHo  = 71 ± 4%) than metabolic (ΔpHi /ΔpHo  = 30 ± 7%) acidosis. CONCLUSION: Extracellular acidification inhibits NBCn1 and NHE1 activity in VSMCs. NBCn1 is equivalently inhibited when pCO2 is raised or [HCO3-]o decreased. Lowering [Na+ ]o inhibits NBCn1 and NHE1 markedly only below the typical physiological and pathophysiological range. We propose that inhibition of Na+ -dependent net acid extrusion at low pHo protects against cellular Na+ overload at the cost of intracellular acidification.


Assuntos
Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/fisiologia , Simportadores de Sódio-Bicarbonato/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Sódio/sangue , Acidose , Animais , Transporte Biológico Ativo , Células Cultivadas , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Simportadores de Sódio-Bicarbonato/genética , Trocador 1 de Sódio-Hidrogênio/genética
10.
Proc Natl Acad Sci U S A ; 114(15): 3921-3926, 2017 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-28348216

RESUMO

IRBIT [inositol 1,4,5-trisphosphate receptor (IP3R) binding protein released with inositol 1,4,5-trisphosphate (IP3)] is a multifunctional protein that regulates several target molecules such as ion channels, transporters, polyadenylation complex, and kinases. Through its interaction with multiple targets, IRBIT contributes to calcium signaling, electrolyte transport, mRNA processing, cell cycle, and neuronal function. However, the regulatory mechanism of IRBIT binding to particular targets is poorly understood. Long-IRBIT is an IRBIT homolog with high homology to IRBIT, except for a unique N-terminal appendage. Long-IRBIT splice variants have different N-terminal sequences and a common C-terminal region, which is involved in multimerization of IRBIT and Long-IRBIT. In this study, we characterized IRBIT and Long-IRBIT splice variants (IRBIT family). We determined that the IRBIT family exhibits different mRNA expression patterns in various tissues. The IRBIT family formed homo- and heteromultimers. In addition, N-terminal splicing of Long-IRBIT changed the protein stability and selectivity to target molecules. These results suggest that N-terminal diversity of the IRBIT family and various combinations of multimer formation contribute to the functional diversity of the IRBIT family.


Assuntos
Adenosil-Homocisteinase/metabolismo , Lectinas Tipo C/metabolismo , Proteínas de Membrana/metabolismo , Adenosil-Homocisteinase/genética , Animais , Células COS , Sinalização do Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cercopithecus aethiops , Feminino , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Lectinas Tipo C/genética , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Isoformas de Proteínas , Estabilidade Proteica , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/metabolismo , Trocador 3 de Sódio-Hidrogênio/genética , Trocador 3 de Sódio-Hidrogênio/metabolismo , Xenopus laevis
11.
PLoS One ; 12(3): e0172765, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28253299

RESUMO

Dysregulation of uterine fluid environment could impair successful reproduction and this could be due to the effect of environmental estrogens. Therefore, in this study, effect of quercetin, an environmental estrogen on uterine fluid and electrolytes concentrations were investigated under sex-steroid influence. Ovariectomised adult female Sprague-Dawley rats were given 10, 50 or 100mg/kg/day quercetin subcutaneously with 17-ß estradiol (E) for seven days or three days E, then three days E plus progesterone (P) (E+P) treatment. Uterine fluid secretion rate, Na+, Cl- and HCO3- concentrations were determined by in-vivo perfusion. Following sacrifice, uteri were harvested and levels of the proteins of interest were identified by Western blotting and Realtime PCR. Distribution of these proteins in the uterus was observed by immunofluorescence. Levels of uterine cAMP were measured by enzyme-linked immunoassay (EIA). Administration of quercetin at increasing doses increased uterine fluid secretion rate, Na+, Cl- and HCO3- concentrations, but to the levels lesser than that of E. In concordant, levels of CFTR, SLC4A4, ENaC (α, ß and γ), Na+/K+-ATPase, GPα/ß, AC and cAMP in the uterus increased following increased in the doses of quercetin. Co-administration of quercetin with E caused uterine fluid secretion rate, Na+, Cl- and HCO3- concentrations to decrease. In concordant, uterine CFTR, SLC26A6, SLC4A4, ENaC (α, ß and γ), Na+/K+-ATPase, GPα/ß, AC and cAMP decreased. Greatest effects were observed following co-administration of 10mg/kg/day quercetin with E. Co-administration of quercetin with E+P caused uterine fluid Na+ and HCO3- concentrations to increase but no changes in fluid secretion rate and Cl- concentration were observed. Co-administration of high dose quercetin (100 mg/kg/day) with E+P caused uterine CFTR, SLC26A6, AC, GPα/ß and ENaC (α, ß and γ) to increase. Quercetin-induced changes in the uterine fluid secretion rate and electrolytes concentrations could potentially affect the uterine reproductive functions under female sex-steroid influence.


Assuntos
Líquidos Corporais/efeitos dos fármacos , Líquidos Corporais/metabolismo , Eletrólitos/metabolismo , Hormônios Esteroides Gonadais/farmacologia , Ovariectomia , Quercetina/farmacologia , Útero/efeitos dos fármacos , Inibidores de Adenilil Ciclases , Animais , Antiporters/genética , Antiporters/metabolismo , Bicarbonatos/metabolismo , Cloretos/metabolismo , AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Interações de Medicamentos , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Feminino , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Sódio/metabolismo , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Transportadores de Sulfato , Útero/metabolismo
12.
Fish Shellfish Immunol ; 64: 226-233, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28257848

RESUMO

The sodium bicarbonate cotransporter (NBC) is an integral membrane ion transporter that can transport HCO3- (or a related species, such as CO32-) across the plasma membrane. Previous researches revealed that NBC might play an important role in the regulation of intracellular pH in vertebrates. In the present study, an NBC cDNA was identified from Pacific white shrimp (Litopenaeus vannamei) and designated as Lv-NBC. The full-length Lv-NBC cDNA is 4479 bp in size, containing a 5'-untranslated region (UTR) of 59 bp, a 3'-UTR of 835 bp and an open reading frame (ORF) of 3585 bp that encodes a protein of 1194 amino acids with a deduced molecular weight of 134.34 kDa. The Lv-NBC protein contains two functional domains (Band_3_cyto and HCO3_cotransp) and twelve transmembrane (TM) domains. Expression of the Lv-NBC mRNA was ubiquitously detected in all selected tissues, with the highest level in the gill. By in situ hybridization (ISH) with Digoxigenin-labeled probe, the Lv-NBC positive cells were shown mainly located in the secondary gill filaments. After low or high pH challenge, the transcript levels of Lv-NBC in the gill were found to be up-regulated. After knockdown of the Lv-NBC level by siRNA, the mortality of shrimp significantly increased under pH stress. Our study, as a whole, may provide evidences for the role of NBC in shrimp responding to pH stress, and give a new insight of the acid/base homeostasis mechanism in crustaceans.


Assuntos
Proteínas de Artrópodes/genética , Penaeidae/fisiologia , Simportadores de Sódio-Bicarbonato/genética , Estresse Fisiológico/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/metabolismo , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Brânquias/metabolismo , Concentração de Íons de Hidrogênio , Penaeidae/genética , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Simportadores de Sódio-Bicarbonato/química , Simportadores de Sódio-Bicarbonato/metabolismo , Distribuição Tecidual
13.
Hum Mol Genet ; 26(5): 989-1002, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28087731

RESUMO

Genome-wide association studies have revealed an association between variation at the SLC4A7 locus and blood pressure. SLC4A7 encodes the electroneutral Na+/HCO3- co-transporter NBCn1 which regulates intracellular pH (pHi). We conducted a functional study of variants at this locus in primary cultures of vascular smooth muscle and endothelial cells. In both cell types, we found genotype-dependent differences for rs13082711 in DNA-nuclear protein interactions, where the risk allele is associated with increased SLC4A7 expression level, NBCn1 availability and function as reflected in elevated steady-state pHi and accelerated recovery from intracellular acidosis. However, in the presence of Na+/H+ exchange activity, the SLC4A7 genotypic effect on net base uptake and steady-state pHi persisted only in vascular smooth muscle cells but not endothelial cells. We found no discernable effect of the missense polymorphism resulting in the amino acid substitution Glu326Lys. The finding of a genotypic influence on SLC4A7 expression and pHi regulation in vascular smooth muscle cells provides an insight into the molecular mechanism underlying the association of variation at the SLC4A7 locus with blood pressure.


Assuntos
Hipertensão/metabolismo , Músculo Liso Vascular/metabolismo , Simportadores de Sódio-Bicarbonato/genética , Alelos , Substituição de Aminoácidos/genética , Animais , Pressão Sanguínea/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Concentração de Íons de Hidrogênio , Hipertensão/genética , Hipertensão/patologia , Músculo Liso Vascular/patologia , Mutação , Ratos , Sódio/metabolismo , Simportadores de Sódio-Bicarbonato/biossíntese
14.
Physiol Genomics ; 49(3): 167-176, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28087757

RESUMO

Genome-wide association studies have identified the single nucleotide polymorphism (SNP) rs3278 in the human SLC4A7 gene as one of the marker loci for addiction vulnerability. This marker is located in an intron of the gene, and its genomic role has been unknown. In this study, we examined rs3278 and three adjacent SNPs prevalent in alcoholics for their effects on an alternative promoter that would lead to the production of the NH2-terminally truncated protein NBCn1ΔN450, missing the first 450 amino acids. Analysis of the transcription start site database and a promoter prediction algorithm identified a cluster of three promoters in intron 7 and two short CpG-rich sites in intron 6. The promoter closest to rs3278 showed strong transcription activity in luciferase reporter gene assays. Major-to-minor allele substitution at rs3278 resulted in increased transcription activity. Equivalent substitutions at adjacent rs3772723 (intron 7) and rs13077400 (exon 8) had negligible effect; however, the substitution at nonsynonymous rs3755652 (exon 8) increased the activity by more than twofold. The concomitant substitution at rs3278/rs3755652 produced an additive effect. The rs3755652 had more profound effects on the promoter than the upstream regulatory CpG sites. The amino acid change E326K caused by rs3755652 had negligible effect on transporter function. In HEK 293 cells, NBCn1ΔN450 was expressed in plasma membranes, but at significantly lower levels than the nontruncated NBCn1-E. The pH change mediated by NBCn1ΔN450 was also low. We conclude that rs3278 and rs3755652 stimulate an alternative transcription of the SLC4A7 gene, increasing the production of a defective transporter.


Assuntos
Polimorfismo de Nucleotídeo Único/genética , Simportadores de Sódio-Bicarbonato/genética , Transcrição Genética , Alelos , Substituição de Aminoácidos/genética , Animais , Ilhas de CpG/genética , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Íntrons/genética , Proteínas Mutantes/metabolismo , Regiões Promotoras Genéticas , Simportadores de Sódio-Bicarbonato/metabolismo , Sítio de Iniciação de Transcrição , Xenopus
15.
J Biochem Mol Toxicol ; 31(4)2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27891704

RESUMO

We hypothesized that genistein could affect the chloride (Cl- ) and bicarbonate (HCO3- ) secretory mechanisms in uterus. Ovariectomized female rats were given estradiol or estradiol plus progesterone with 25, 50, or 100 mg/kg/day genistein. Following completion of the treatment, uterine fluid Cl- and HCO3- concentrations were determined by in vivo uterine perfusion. Uteri were subjected for molecular biological analysis (Western blot, qPCR, and immunohistochemistry) to detect levels of expression of Cystic Fibrosis transmembrane regulator (CFTR), Cl- /HCO3- exchanger (SLC26a6), Na+ /HCO3- cotransporter (SLC4a4), and estrogen receptor (ER)-α and ß. Coadministration of genistein resulted in decrease in Cl- and HCO3- concentrations and expression of CFTR, SLC26a6, SLC4a4, and ER-α and ER-ß in the uteri of estradiol-treated rats. In estradiol plus progesterone-treated rats, a significant increase in the above parameters were observed following high-dose genistein treatment except for the SLC24a4 level. In conclusion, genistein-induced changes in the uterus could affect the reproductive processes that might result in infertility.


Assuntos
Bicarbonatos/metabolismo , Cloretos/metabolismo , Estrogênios/farmacologia , Genisteína/farmacologia , Útero/efeitos dos fármacos , Animais , Antiporters/efeitos dos fármacos , Antiporters/genética , Regulador de Condutância Transmembrana em Fibrose Cística/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Regulação da Expressão Gênica , Ratos , Ratos Sprague-Dawley , Receptores Estrogênicos/efeitos dos fármacos , Receptores Estrogênicos/genética , Simportadores de Sódio-Bicarbonato/efeitos dos fármacos , Simportadores de Sódio-Bicarbonato/genética , Transportadores de Sulfato , Útero/metabolismo
16.
Am J Hypertens ; 30(2): 202-208, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27784683

RESUMO

BACKGROUND: The large-scale meta-analysis of genome-wide association study (GWAS) recently identified a genomic locus where the genetic variant at rs820430 was strongly associated with hypertension in Chinese Han population, with its T allele conferred increased risks. However, the biological and disease-relevant mechanisms for this association remain elusive. METHODS: A group of 275 participants from rural district of Shandong Province were enrolled, rs820430 was genotyped using genomic DNA with the fluorogenic 5'-nuclease TaqMan allelic discrimination assay system (Applied Biosystems, CA). In vitro experiments were performed in this study, such as luciferase reporter assays, gel mobility shift assays (electrophoretic mobility shift assay), and chromatin immunoprecipitation. RESULTS: We found the risk T allele of rs820430 was associated with higher SLC4A7 mRNA level in cohort population. Furthermore, we characterized a cis-regulatory mechanism that the T allele of rs820430 distinctively increased c-Fos transcription factor binding, by which leading to increased SLC4A7 expression. CONCLUSIONS: The present study indicated that the disease-associated T allele of a new hypertension risk variant rs820430 linked increased hypertension risk through higher SLC4A7 expression, and rs820430 functioned as an enhancer of SLC4A7 transcription by allele distinctively increased c-Fos transcription factor binding.


Assuntos
DNA/genética , Regulação da Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla/métodos , Hipertensão/genética , Vigilância da População , Simportadores de Sódio-Bicarbonato/genética , Alelos , China/epidemiologia , Eletroforese , Feminino , Variação Genética , Genótipo , Células HEK293 , Humanos , Hipertensão/epidemiologia , Hipertensão/metabolismo , Incidência , Masculino , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Fatores de Risco , População Rural , Simportadores de Sódio-Bicarbonato/metabolismo , Taxa de Sobrevida/tendências
17.
World J Gastroenterol ; 22(43): 9525-9533, 2016 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-27920473

RESUMO

AIM: To determine the expression and localization of the electrogenic Na+/HCO3- cotransporter (NBC1) in rat pancreas during development. METHODS: The rat pancreas from postnatal and embryos removed from the uterus of pregnant rats that had been sacrificed by CO2 asphyxiation were used. Rat pancreas from embryonic day (E) 15.5 and E18.5 rat embryos was isolated under a stereomicroscope. Rat pancreas from postnatal (P) days 0, 7, 14, 21 and adult was directly isolated by the unaided eye. The RT-PCR analysis of the NBC1 specific region on rat pancreas tissues from different developmental stages. The two antibodies which target the NBC1 common COOH-terminal region and NH2-terminal region detected a clear band of about 145 kDa in the Western blot analysis. The localization of NBC1 was examined by immuno-fluorescence detection. RESULTS: The results revealed the first peak of NBC1 expression at E18.5 and the second peak at P14. Meanwhile, the low NBC1 expression occurred at P7 and adult stages. Our results demonstrated, for the first time, the presence of NBC1 in the plasma membrane of ß and α cells, as well as in the basolateral membrane of acinar cells of the rat pancreas at different stages of development. CONCLUSION: The data strongly suggests that NBC1 is diversely expressed in the pancreas at different developmental stages, where it may exert its functions in pancreatic development especially islet cell growth through HCO3- transport and pH regulation.


Assuntos
Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Pâncreas/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Western Blotting , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Morfogênese , Pâncreas/embriologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Simportadores de Sódio-Bicarbonato/genética
18.
Physiol Rep ; 4(23)2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27923977

RESUMO

The major site of fructose metabolism in the kidney is the proximal tubule (PT). To test whether insulin and/or IGF1 signaling in the PT is involved in renal structural/functional responses to dietary fructose, we bred mice with dual knockout (KO) of the insulin receptor (IR) and the IGF1 receptor (IGF1R) in PT by Cre-lox recombination, using a γ-glutamyl transferase promoter. KO mice had slightly (~10%) reduced body and kidney weights, as well as, a reduction in mean protein-to-DNA ratio in kidney cortex suggesting smaller cell size. Under control diet, IR and IGF1R protein band densities were 30-50% (P < 0.05) lower than WT, and the relative difference was greater in male animals. Male, but not female KO, also had significantly reduced band densities for Akt (protein kinase B), phosphorylated AktT308 and IRY1162/1163 A high-fructose diet (1-month) led to a significant increase in kidney weight in WT males (12%), but not in KO males or in either genotype of female mice. Kidney enlargement in the WT males was accompanied by a small, insignificant fall in protein-to-DNA ratio, supporting hyperplasia rather than hypertrophy. Fructose feeding of male WT mice led to significantly higher sodium bicarbonate exchanger (NBCe1), sodium hydrogen exchanger (NHE3), sodium phosphate co-transporter (NaPi-2), and transforming growth factor-ß (TGF-ß) abundances, as compared to male KO, suggesting elevated transport capacity and an early feature of fibrosis may have accompanied the renal enlargement. Overall, IR and/or IGF1R appear to have a role in PT cell size and enlargement in response to high-fructose diet.


Assuntos
Frutose/farmacologia , Túbulos Renais Proximais/metabolismo , Receptor IGF Tipo 1/genética , Receptor de Insulina/genética , Animais , Dieta , Feminino , Frutose/administração & dosagem , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tamanho do Órgão , Receptor IGF Tipo 1/deficiência , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/deficiência , Receptor de Insulina/metabolismo , Fatores Sexuais , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato/genética , Proteínas Cotransportadoras de Sódio-Fosfato/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
19.
J Neurosci ; 36(42): 10750-10758, 2016 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-27798130

RESUMO

Ventral regions of the medulla oblongata of the brainstem are populated by astrocytes sensitive to physiological changes in PCO2/[H+]. These astrocytes respond to decreases in pH with elevations in intracellular Ca2+ and facilitated exocytosis of ATP-containing vesicles. Released ATP propagates Ca2+ excitation among neighboring astrocytes and activates neurons of the brainstem respiratory network triggering adaptive increases in breathing. The mechanisms linking increases in extracellular and/or intracellular PCO2/[H+] with Ca2+ responses in chemosensitive astrocytes remain unknown. Fluorescent imaging of changes in [Na+]i and/or [Ca2+]i in individual astrocytes was performed in organotypic brainstem slice cultures and acute brainstem slices of adult rats. It was found that astroglial [Ca2+]i responses triggered by decreases in pH are preceded by Na+ entry, markedly reduced by inhibition of Na+/HCO3- cotransport (NBC) or Na+/Ca2+ exchange (NCX), and abolished in Na+-free medium or by combined NBC/NCX blockade. Acidification-induced [Ca2+]i responses were also dramatically reduced in brainstem astrocytes of mice deficient in the electrogenic Na+/HCO3- cotransporter NBCe1. Sensitivity of astrocytes to changes in pH was not affected by inhibition of Na+/H+ exchange or blockade of phospholipase C. These results suggest that in pH-sensitive astrocytes, acidification activates NBCe1, which brings Na+ inside the cell. Raising [Na+]i activates NCX to operate in a reverse mode, leading to Ca2+ entry followed by activation of downstream signaling pathways. Coupled NBC and NCX activities are, therefore, suggested to be responsible for functional CO2/H+ sensitivity of astrocytes that contribute to homeostatic regulation of brain parenchymal pH and control of breathing. SIGNIFICANCE STATEMENT: Brainstem astrocytes detect physiological changes in pH, activate neurons of the neighboring respiratory network, and contribute to the development of adaptive respiratory responses to the increases in the level of blood and brain PCO2/[H+]. The mechanisms underlying astroglial pH sensitivity remained unknown and here we show that in brainstem astrocytes acidification activates Na+/HCO3- cotransport, which brings Na+ inside the cell. Raising [Na+]i activates the Na+/Ca2+ exchanger to operate in a reverse mode leading to Ca2+ entry. This identifies a plausible mechanism of functional CO2/H+ sensitivity of brainstem astrocytes, which play an important role in homeostatic regulation of brain pH and control of breathing.


Assuntos
Astrócitos/efeitos dos fármacos , Dióxido de Carbono/farmacologia , Hidrogênio/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Astrócitos/metabolismo , Bicarbonatos/metabolismo , Sinalização do Cálcio , Exocitose , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Ratos , Respiração , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sódio/metabolismo , Simportadores de Sódio-Bicarbonato/antagonistas & inibidores , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/metabolismo , Trocador de Sódio e Cálcio/antagonistas & inibidores , Trocador de Sódio e Cálcio/metabolismo
20.
Am J Hum Genet ; 99(5): 1045-1058, 2016 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-27843122

RESUMO

DNA methylation is globally reprogrammed after fertilization, and as a result, the parental genomes have similar DNA-methylation profiles after implantation except at the germline differentially methylated regions (gDMRs). We and others have previously shown that human blastocysts might contain thousands of transient maternally methylated gDMRs (transient mDMRs), whose maternal methylation is lost in embryonic tissues after implantation. In this study, we performed genome-wide allelic DNA methylation analyses of purified trophoblast cells from human placentas and, surprisingly, found that more than one-quarter of the transient-in-embryo mDMRs maintained their maternally biased DNA methylation. RNA-sequencing-based allelic expression analyses revealed that some of the placenta-specific mDMRs were associated with expression of imprinted genes (e.g., TIGAR, SLC4A7, PROSER2-AS1, and KLHDC10), and three imprinted gene clusters were identified. This approach also identified some X-linked gDMRs. Comparisons of the data with those from other mammals revealed that genomic imprinting in the placenta is highly variable. These findings highlight the incomplete erasure of germline DNA methylation in the human placenta; understanding this erasure is important for understanding normal placental development and the pathogenesis of developmental disorders with imprinting effects.


Assuntos
Alelos , Perfilação da Expressão Gênica , Impressão Genômica , Placenta/metabolismo , Blastocisto/citologia , Blastocisto/metabolismo , Metilação de DNA , Exoma , Feminino , Genes Ligados ao Cromossomo X , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Anotação de Sequência Molecular , Placenta/citologia , Polimorfismo de Nucleotídeo Único , Gravidez , Análise de Sequência de RNA , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/metabolismo , Trofoblastos/citologia , Trofoblastos/metabolismo
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