RESUMO
Recent studies have suggested that dogs were domesticated during the Last Glacial Maximum (LGM) in Siberia, which contrasts with previous proposed domestication centers (e.g. Europe, the Middle East, and East Asia). Ancient DNA provides a powerful resource for the study of mammalian evolution and has been widely used to understand the genetic history of domestic animals. To understand the maternal genetic history of East Asian dogs, we have made a complete mitogenome dataset of 120 East Asian canids from 38 archaeological sites, including 102 newly sequenced from 12.9 to 1â ka BP (1,000â years before present). The majority (112/119, 94.12%) belonged to haplogroup A, and half of these (55/112, 49.11%) belonged to sub-haplogroup A1b. Most existing mitochondrial haplogroups were present in ancient East Asian dogs. However, mitochondrial lineages in ancient northern dogs (northeastern Eurasia and northern East Asia) were deeper and older than those in southern East Asian dogs. Results suggests that East Asian dogs originated from northeastern Eurasian populations after the LGM, dispersing in two possible directions after domestication. Western Eurasian (Europe and the Middle East) dog maternal ancestries genetically influenced East Asian dogs from approximately 4â ka BP, dramatically increasing after 3â ka BP, and afterwards largely replaced most primary maternal lineages in northern East Asia. Additionally, at least three major mitogenome sub-haplogroups of haplogroup A (A1a, A1b, and A3) reveal at least two major dispersal waves onto the Qinghai-Tibet Plateau in ancient times, indicating eastern (A1b and A3) and western (A1a) Eurasian origins.
Assuntos
Genoma Mitocondrial , Animais , Cães , Animais Domésticos/genética , Ásia Oriental , DNA Mitocondrial/genética , Variação Genética , Haplótipos , Mamíferos/genética , FilogeniaRESUMO
An asymptomatic infant of Ghanaian descent had hemoglobin F only detected on newborn screening. ß-globin gene sequencing identified the intervening sequence (IVS)-II-849 (A â G) mutation with no normal ß-globin gene. ß-globin/δ-globin gene sequencing showed that both parents were heterozygous for the IVS-II-849 (A â G) mutation. The mother was heterozygous for the HbA2' δ-globin mutation (δ16 (A13) Gly â Arg), thus ß-thalassemia trait was unrecognized due to coinheritance of HbA2'. The infant developed anemia, splenomegaly, and began transfusion therapy by the age 6 of months. This is the first report of ß-thalassemia major with homozygous IVS-II-849 (A â G) mutations. This case highlights the importance of δ-globin gene mutations in prenatal testing.
Assuntos
Hemoglobina Fetal , Talassemia beta/diagnóstico , Talassemia beta/genética , Cromatografia Líquida de Alta Pressão , Hemoglobina A2/genética , Humanos , Recém-Nascido , Focalização Isoelétrica , MasculinoRESUMO
Of the 1570 reported hemoglobin (Hb) variants detected to date, 390 are α2-globin chain (some variants have yet to be identified by DNA analyses and are therefore presumed) and 827 are the result of mutations of the ß-globin chain. Due to their location on the Hb structure, only a minority of these variants result in a clinical phenotype; most are silent and are detected during routine surveillance, are found incidentally during other disease-related investigations or following newborn screening programs. In this report we discuss phenotype/genotype and molecular characteristics of two new Hb variants, both of which were clinically silent. One is an α2-globin chain variant located at codon 3 [α3(A1)SerâTyr; HBA2: c.11C > A] named Hb Tallahassee and the other is a ß-globin chain variant located at codon 119 [ß119(GH2)GlyâSer; HBB: c.358G > A] called Hb Madison-NC.
Assuntos
Códon/genética , Hemoglobinas Anormais/genética , Mutação , Fenótipo , alfa-Globinas/genética , Criança , Humanos , Lactente , MasculinoRESUMO
The total number of hemoglobin (Hb) variants so far reported to the HbVar database is 1598 (April 9 2014) and 130 of them are fetal Hb variants. Fetal Hb are categorized as two different subunits, (G)γ- and (A)γ-globin chains, and γ chain variants can be observed in both subunits. There are 72 (G)γ- and 58 (A)γ-globin chain variants. Most of them are clinically silent and detected during newborn screening programs in the USA and outside the USA. In this report, we discuss the molecular characteristics and diagnostic difficulties of two new γ-globin chain variants found in an African American baby with no clinical symptoms. One is a new (G)γ-globin chain variant, Hb F-Augusta GA [(G)γ59(E3)Lys â Arg; HBG2: c.179A > G] and the other one is Hb F-Port Royal-II [(A)γ125(H3)Glu â Ala; HBG1: c.377A > C].
Assuntos
Hemoglobina Fetal/genética , Hemoglobinopatias/genética , Mutação , gama-Globinas/genética , Negro ou Afro-Americano , Substituição de Aminoácidos , Pai , Feminino , Hemoglobina Fetal/química , Georgia , Hemoglobinopatias/sangue , Hemoglobinopatias/diagnóstico , Hemoglobinopatias/fisiopatologia , Heterozigoto , Homozigoto , Humanos , Recém-Nascido , Mães , Triagem Neonatal , Mutação Puntual , Índice de Gravidade de Doença , gama-Globinas/químicaRESUMO
We report a novel hemoglobin (Hb) variant that we named Hb Fulton-Georgia, caused by a point mutation in exon 1/codon 20 of the α-globin gene [α20(B1)HisâPro; HBA1: c.62A>C]. This α chain variant was identified in an adult African-American female with Hb SC disease who was also heterozygous for the α-thalassemia-2 (α-thal-2) (3.7 kb deletion or αα/-α(3.7)). The Hb Fulton-Georgia mutation was located on the intact α1-globin gene not involved by α-thal-2. Molecular models indicated that the α20 residue of Hb Fulton-Georgia was the first amino acid of the B helix, and was not involved in α1/ß1 or α1/ß2 contacts in Hb S [ß6(A3)GluâVal; HBB: c.20A>T] or Hb C [ß6(A3)GluâLys; HBB: c.19G>A] tetramers. Furthermore, the histidineâproline substitution at α20 did not disrupt the helical structure. High performance liquid chromatography (HPLC) detected Hb Fulton-Georgia in 16.0% of total Hb, consistent with inheritance on the α1 gene. Coinheritance of Hb Fulton-Georgia, heterozygous α-thal-2 and Hb SC disease was associated with a mild phenotype, consisting of microcytosis and anisocytosis, but no anemia or other hematological abnormality.
Assuntos
Hemoglobinas Glicadas/genética , Hemoglobinas Anormais/genética , Mutação Puntual , Adulto , Análise Mutacional de DNA , Feminino , Doença da Hemoglobina SC/complicações , Doença da Hemoglobina SC/genética , Humanos , Deleção de Sequência , Talassemia alfa/complicações , Talassemia alfa/genéticaRESUMO
Multi-shot coded aperture snapshot spectral imaging (CASSI) uses multiple measurement snapshots to encode the three-dimensional hyperspectral image (HSI). Increasing the number of snapshots will multiply the number of measurements, making CASSI system more appropriate for detailed spatial or spectrally rich scenes. However, the reconstruction algorithms still face the challenge of being ineffective or inflexible. In this paper, we propose a plug-and-play (PnP) method that uses denoiser as priors for multi-shot CASSI. Specifically, the proposed PnP method is based on the primal-dual algorithm with linesearch (PDAL), which makes it flexible and can be used for any multi-shot CASSI mechanisms. Furthermore, a new subspaced-based nonlocal reweighted low-rank (SNRL) denoiser is presented to utilize the global spectral correlation and nonlocal self-similarity priors of HSI. By integrating the SNRL denoiser into PnP-PDAL, we show the balloons ( 512×512×31 ) in CAVE dataset recovered from two snapshots compressive measurements with MPSNR above 50 dB. Experimental results demonstrate that our proposed method leads to significant improvements compared to the current state-of-the-art methods.
RESUMO
The decrease in the widths of spectral bands in hyperspectral imaging leads to a decrease in signal-to-noise ratio (SNR) of measurements. The decreased SNR reduces the reliability of measured features or information extracted from hyperspectral images (HSIs). Furthermore, the image degradations linked with various mechanisms also result in different types of noise, such as Gaussian noise, impulse noise, deadlines, and stripes. This article introduces a fast and parameter-free hyperspectral image mixed noise removal method (termed FastHyMix), which characterizes the complex distribution of mixed noise by using a Gaussian mixture model and exploits two main characteristics of hyperspectral data, namely, low rankness in the spectral domain and high correlation in the spatial domain. The Gaussian mixture model enables us to make a good estimation of Gaussian noise intensity and the locations of sparse noise. The proposed method takes advantage of the low rankness using subspace representation and the spatial correlation of HSIs by adding a powerful deep image prior, which is extracted from a neural denoising network. An exhaustive array of experiments and comparisons with state-of-the-art denoisers was carried out. The experimental results show significant improvement in both synthetic and real datasets. A MATLAB demo of this work is available at https://github.com/LinaZhuang for the sake of reproducibility.
RESUMO
The performance of deep learning-based denoisers highly depends on the quantity and quality of training data. However, paired noisy-clean training images are generally unavailable in hyperspectral remote sensing areas. To solve this problem, this work resorts to the self-supervised learning technique, where our proposed model can train itself to learn one part of noisy input from another part of noisy input. We study a general hyperspectral image (HSI) denoising framework, called Eigenimage2Eigenimage (E2E), which turns the HSI denoising problem into an eigenimage (i.e., the subspace representation coefficients of the HSI) denoising problem and proposes a learning strategy to generate noisy-noisy paired training eigenimages from noisy eigenimages. Consequently, the E2E denoising framework can be trained without clean data and applied to denoise HSIs without the constraint with the number of frequency bands. Experimental results are provided to demonstrate the performance of the proposed method that is better than the other existing deep learning methods for denoising HSIs. A MATLAB demo of this work is available at https://github.com/LinaZhuang/HSI-denoiser-Eigenimage2Eigenimagehttps://github.com/LinaZhuang/HSI-denoiser-Eigenimage2Eigenimage for the sake of reproducibility.
RESUMO
The occurrence of multiple abnormalities of α, ß, δ, and γ globin genes may lead to unusual and complex phenotypes when they arise simultaneously in the same individual. Here, we report the findings of an African American boy who coinherited 3 heterozygous globin gene abnormalities: the unstable ß-globin chain variant; hemoglobin (Hb) Showa-Yakushiji [ß110(G12) LeuâPro], the δ-globin chain variant; HbB2 [δ16(A13) GlyâArg] and α-thalassemia (α-thal); (α-/αα). Hb Showa-Yakushiji had been previously described in Japanese, Indian, and European populations. We report its first occurrence in a child of African ancestry who presented with anemia not responsive to iron and an incomplete ß-thalassemia minor phenotype. Although the clinical and laboratory features of Hb Showa-Yakushiji mimic those of a ß-thalassemia, the coinheritance of the δ-globin chain variant Hb B2 suppressed the relative increase in Hb A2 usually observed in heterozygotes for the Hb Showa-Yakushiji mutation. Protein-based methods detected only a trace amount of HbB2 and failed to reveal presence of Hb Showa-Yakushiji and α-thal. The latter were only identified through DNA analyses. The diagnostic difficulties, molecular characteristics, and genotype/phenotype correlations of this novel complex hemoglobinopathy syndrome are reviewed.
Assuntos
Anemia/genética , Hemoglobinas Anormais/genética , Talassemia alfa/genética , Talassemia beta/genética , Talassemia delta/genética , Negro ou Afro-Americano/genética , Anemia/etiologia , Pré-Escolar , Heterozigoto , Humanos , Masculino , Fenótipo , Talassemia alfa/sangue , Talassemia alfa/complicações , Talassemia beta/sangue , Talassemia beta/complicações , Talassemia delta/complicaçõesRESUMO
SMCT1 is a Na(+)-coupled monocarboxylate transporter expressed in a variety of tissues including kidney, thyroid, small intestine, colon, brain, and retina. We found recently that several non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the activity of SMCT1. Here we evaluated the effect of diclofenac, also a NSAID, on SMCT1. SMCT1 cDNA was expressed heterologously in the human retinal pigment epithelial cell lines HRPE and ARPE-19, the human mammary epithelial cell line MCF7, and in Xenopus laevis oocytes. Transport was monitored by substrate uptake and substrate-induced currents. Na(+)-dependent uptake/current was considered as SMCT1 activity. The effect of diclofenac was evaluated for specificity, dose-response, and influence on transport kinetics. To study the specificity of the diclofenac effect, we evaluated the influence of this NSAID on the activity of several other cloned transporters in mammalian cells under identical conditions. In contrast to several NSAIDs that inhibited SMCT1, diclofenac stimulated SMCT1 when expressed in HRPE and ARPE-19 cells. The stimulation was marked, ranging from 2- to 5-fold depending on the concentration of diclofenac. The stimulation was associated with an increase in the maximal velocity of the transport system as well as with an increase in substrate affinity. The observed effect on SMCT1 was selective because the activity of several other cloned transporters, when expressed in HRPE cells and studied under identical conditions, was not affected by diclofenac. Interestingly, the stimulatory effect on SMCT1 observed in HRPE and ARPE-19 cells was not evident in MCF7 cells nor in the X. laevis expression system, indicating that SMCT1 was not the direct target for diclofenac. The RPE-specific effect suggests that the target of diclofenac that mediates the stimulatory effect is expressed in RPE cells but not in MCF7 cells or in X. laevis oocytes. Since SMCT1 is a concentrative transporter for metabolically important compounds such as pyruvate, lactate, beta-hydroxybutyrate, and nicotinate, the stimulation of its activity by diclofenac in RPE cells has biological and clinical significance.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Proteínas de Transporte de Cátions/agonistas , Diclofenaco/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Animais , Proteínas de Transporte de Cátions/biossíntese , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Transportadores de Ácidos Monocarboxílicos , Oócitos , Epitélio Pigmentado da Retina/metabolismo , Xenopus laevisRESUMO
Taurine and its transporter (TauT) are expressed in preimplantation embryos, but their role in embryogenesis is not known. To investigate the role of TauT during embryonic development, we cloned and functionally characterized the zebrafish TauT. The zebrafish TauT cDNA codes for a protein of 625 amino acids which is highly homologous to mammalian TauT. When expressed in mammalian cells, zebrafish TauT mediates taurine uptake in a Na(+)/Cl(-)-dependent manner with a Na(+):Cl(-):taurine stoichiometry of 2:1:1. In the zebrafish embryo, taurine and TauT mRNA are present during early cleavage stages, indicating that both the transporter and its substrate are maternally derived. During embryogenesis, zygotic expression of TauT mRNA is evident in the retina, brain, heart, kidney, and blood vessels. Knockdown of TauT by antisense morpholino oligonucleotides leads to cell death in the central nervous system and increased mortality. These findings suggest a specific role for TauT during development in vertebrates.
Assuntos
Desenvolvimento Embrionário , Glicoproteínas de Membrana/biossíntese , Proteínas de Membrana Transportadoras/biossíntese , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Clonagem Molecular , Desenvolvimento Embrionário/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Hibridização In Situ , Glicoproteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/farmacologia , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Peixe-Zebra/metabolismoRESUMO
Neonatal cyanosis can result from a multitude of acquired and inherited causes. Cyanosis resulting from fetal M hemoglobin (Hb) variants is very rare. Only two (G)gamma variants causing methemoglobinemia and cyanosis in the newborn have been reported to date. Here we describe a novel fetal Hb variant, Hb F-Circleville [Ggamma63(E7)His-->Leu], associated with methemoglobinemia and cyanosis in the newborn. The patient's sister also had neonatal cyanosis at birth.
Assuntos
Cianose/genética , Hemoglobina Fetal/genética , Globinas/genética , Substituição de Aminoácidos , Sequência de Bases , Cianose/etiologia , Feminino , Hemoglobinas Anormais , Histidina/química , Histidina/genética , Humanos , Recém-Nascido , Leucina/química , Leucina/genética , Masculino , Dados de Sequência Molecular , Mutação/genéticaRESUMO
SMCT (sodium-coupled monocarboxylate transporter; slc5a8) is a Na+-coupled transporter for lactate, pyruvate and short-chain fatty acids. Similar to these already known substrates of SMCT, the water-soluble B-complex vitamin nicotinic acid also exists as a monocarboxylate anion (nicotinate) under physiological conditions. Therefore we evaluated the ability of SMCT to mediate the uptake of nicotinate. In mammalian cells, the cloned mouse SMCT (slc5a8) induced the uptake of nicotinate. The SMCT-induced uptake was Na+-dependent. The Michaelis constant for the uptake process was 296+/-88 microM. The Na+-activation kinetics indicated that at least two Na+ ions are involved in the process. Among the various structural analogues tested, nicotinate was the most effective substrate. Nicotinamide and methylnicotinate were not recognized by the transporter. 2-pyrazine carboxylate and isonicotinate interacted with the transporter to a moderate extent. SMCT-mediated uptake of nicotinate was inhibited by lactate and pyruvate. In the Xenopus laevis oocyte expression system, SMCT-mediated nicotinate transport was electrogenic, as evident from the nicotinate-induced inward currents under voltage-clamp conditions. Substrate-induced currents in this expression system corroborated the substrate specificity determined in the mammalian cell expression system. The kinetic parameters with regard to the affinity of the transporter for nicotinate and the Hill coefficient for Na+ activation, determined by using the oocyte expression system, were also similar to those obtained from the mammalian cell expression system. We conclude that SMCT functions not only as a Na+-coupled transporter for short-chain fatty acids and lactate but also as a Na+-coupled transporter for the water-soluble vitamin nicotinic acid.
Assuntos
Transporte Biológico Ativo , Proteínas de Transporte de Cátions/metabolismo , Eletrofisiologia , Niacina/metabolismo , Sódio/fisiologia , Animais , Células Cultivadas , Células Epiteliais/metabolismo , Expressão Gênica , Humanos , Estrutura Molecular , Transportadores de Ácidos Monocarboxílicos , Niacina/química , Oócitos , Epitélio Pigmentado da Retina/citologia , Especificidade por Substrato , Xenopus laevisRESUMO
We report in the present paper, on the isolation and functional characterization of slc5a12, the twelfth member of the SLC5 gene family, from mouse kidney. The slc5a12 cDNA codes for a protein of 619 amino acids. Heterologous expression of slc5a12 cDNA in mammalian cells induces Na+-dependent transport of lactate and nicotinate. Several other short-chain monocarboxylates compete with nicotinate for the cDNA-induced transport process. Expression of slc5a12 in Xenopus oocytes induces electrogenic and Na+-dependent transport of lactate, nicotinate, propionate and butyrate. The substrate specificity of slc5a12 is similar to that of slc5a8, an Na+-coupled transporter for monocarboxylates. However, the substrate affinities of slc5a12 were much lower than those of slc5a8. slc5a12 mRNA is expressed in kidney, small intestine and skeletal muscle. In situ hybridization with sagittal sections of mouse kidney showed predominant expression of slc5a12 in the outer cortex. This is in contrast with slc5a8, which is expressed in the cortex as well as in the medulla. The physiological function of slc5a12 in the kidney is likely to mediate the reabsorption of lactate. In the intestinal tract, slc5a12 is expressed in the proximal parts, whereas slc5a8 is expressed in the distal parts. The expression of slc5a12 in the proximal parts of the intestinal tract, where there is minimal bacterial colonization, suggests that the physiological function of slc5a12 is not to mediate the absorption of short-chain monocarboxylates derived from bacterial fermentation but rather to mediate the absorption of diet-derived short-chain monocarboxylates. Based on the functional and structural similarities between slc5a8 and slc5a12, we suggest that the two transporters be designated as SMCT1 (sodium-coupled monocarboxylate transporter 1) and SMCT2 respectively.
Assuntos
Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas de Transporte de Sódio-Glucose/genética , Proteínas de Transporte de Sódio-Glucose/metabolismo , Sódio/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular , Clonagem Molecular , Humanos , Intestino Delgado/metabolismo , Rim/metabolismo , Camundongos , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Oócitos , Especificidade de Órgãos , Filogenia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Xenopus laevisRESUMO
We have cloned and functionally characterized an Na+-coupled citrate transporter from Caenorhabditis elegans (ceNAC-2). This transporter shows significant sequence homology to Drosophila Indy and the mammalian Na+-coupled citrate transporter NaCT (now known as NaC2). When heterologously expressed in a mammalian cell line or in Xenopus oocytes, the cloned ceNAC-2 mediates the Na+-coupled transport of various intermediates of the citric acid cycle. However, it transports the tricarboxylate citrate more efficiently than dicarboxylates such as succinate, a feature different from that of ceNAC-1 (formerly known as ceNaDC1) and ceNAC-3 (formerly known as ceNaDC2). The transport process is electrogenic, as evidenced from the substrate-induced inward currents in oocytes expressing the transporter under voltage-clamp conditions. Expression studies using a reporter-gene fusion method in transgenic C. elegans show that the gene is expressed in the intestinal tract, the organ responsible for not only the digestion and absorption of nutrients but also for the storage of energy in this organism. Functional knockdown of the transporter by RNAi (RNA interference) not only leads to a significant increase in life span, but also causes a significant decrease in body size and fat content. The substrates of ceNAC-2 play a critical role in metabolic energy production and in the biosynthesis of cholesterol and fatty acids. The present studies suggest that the knockdown of these metabolic functions by RNAi is linked to an extension of life span and a decrease in fat content and body size.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Ácido Cítrico/metabolismo , Transportadores de Ânions Orgânicos/fisiologia , Sódio/metabolismo , Tecido Adiposo/anatomia & histologia , Animais , Animais Geneticamente Modificados , Transporte Biológico , Composição Corporal/genética , Constituição Corporal/genética , Caenorhabditis elegans/anatomia & histologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/isolamento & purificação , Linhagem Celular , Ciclo do Ácido Cítrico , Clonagem Molecular , Genes de Helmintos , Genes Reporter , Humanos , Mucosa Intestinal/metabolismo , Longevidade/genética , Dados de Sequência Molecular , Oócitos , Especificidade de Órgãos , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/isolamento & purificação , Técnicas de Patch-Clamp , Epitélio Pigmentado Ocular/citologia , Interferência de RNA , Proteínas Recombinantes de Fusão/metabolismo , Xenopus laevisRESUMO
In the present study, we report on the molecular cloning and functional characterization of mouse NaCT (Na+-coupled citrate transporter), the mouse orthologue of Drosophila Indy. Mouse NaCT consists of 572 amino acids and is highly similar to rat and human NaCTs in primary sequence. The mouse nact gene coding for the transporter is approx. 23 kb long and consists of 12 exons. When expressed in mammalian cells, the cloned transporter mediates the Na+-coupled transport of citrate and succinate. Competition experiments reveal that mouse NaCT also recognizes other tricarboxylic acid cycle intermediates such as malate, fumarate and 2-oxo-glutarate as excellent substrates. The Michaelis-Menten constant for the transport process is 38+/-5 mM for citrate and 37+/-6 mM for succinate at pH 7.5. The transport process is electrogenic and exhibits an obligatory requirement for Na+. Na+-activation kinetics indicates that multiple Na+ ions are involved in the activation process. Extracellular pH has a differential effect on the transport function of mouse NaCT depending on whether the transported substrate is citrate or succinate. The Michaelis-Menten constants for these substrates are also influenced markedly by pH. When examined in the Xenopus laevis oocyte expression system with the two-microelectrode voltage-clamp technique, the transport process mediated by mouse NaCT is electrogenic. The charge-to-substrate ratio is 1 for citrate and 2 for succinate. The most probable transport mechanism predicted by these studies involves the transport of citrate as a tervalent anion and succinate as a bivalent anion with a fixed Na+/substrate stoichiometry of 4:1. The present study provides the first unequivocal evidence for the electrogenic nature of mammalian NaCT.
Assuntos
Ciclo do Ácido Cítrico , Simportadores/genética , Simportadores/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Ácido Cítrico/metabolismo , Clonagem Molecular , Transportadores de Ácidos Dicarboxílicos , Éxons , Genoma , Humanos , Íntrons , Camundongos , Dados de Sequência Molecular , Oócitos/metabolismo , Sódio/farmacologia , Especificidade por Substrato , Ácido Succínico/metabolismo , Simportadores/química , Xenopus laevisRESUMO
PURPOSE: Sodium-coupled monocarboxylate transporter 1 (SMCT1) is a Na+-coupled transporter for monocarboxylates. Many nonsteroidal anti-inflammatory drugs (NSAIDs) are monocarboxylates. Therefore, we investigated the interaction of these drugs with human SMCT1 (hSMCT1). METHODS: We expressed hSMCT1 in a mammalian cell line and in Xenopus laevis oocytes and used the uptake of nicotinate and propionate-induced currents to monitor its transport function, respectively. We also used [14C]-nicotinate and [3H]-ibuprofen for direct measurements of uptake in oocytes. RESULTS: In mammalian cells, hSMCT1-mediated nicotinate uptake was inhibited by ibuprofen and other structurally related NSAIDs. The inhibition was Na+ dependent. With ibuprofen, the concentration necessary for 50% inhibition was 64 +/- 16 microM. In oocytes, the transport function of hSMCT1 was associated with inward currents in the presence of propionate. Under identical conditions, ibuprofen and other structurally related NSAIDs failed to induce inward currents. However, these compounds blocked propionate-induced currents. With ibuprofen, the blockade was dose dependent, Na+ dependent, and competitive. However, there was no uptake of [3H]-ibuprofen into oocytes expressing hSMCT1, although the uptake of [14C]-nicotinate was demonstrable under identical conditions. CONCLUSIONS: Ibuprofen and other structurally related NSAIDs interact with hSMCT1 as blockers of its transport function rather than as its transportable substrates.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Proteínas de Transporte de Cátions/antagonistas & inibidores , Fenoprofeno/farmacologia , Ibuprofeno/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Fenoprofeno/química , Humanos , Ibuprofeno/química , Potenciais da Membrana , Estrutura Molecular , Transportadores de Ácidos Monocarboxílicos , Niacina/metabolismo , Oócitos/metabolismo , Propionatos , Sódio/metabolismo , Relação Estrutura-Atividade , Transfecção , Xenopus laevisRESUMO
SMCT1 is a sodium-coupled (Na(+)-coupled) transporter for l-lactate and short-chain fatty acids. Here, we show that the ketone bodies, beta-d-hydroxybutyrate and acetoacetate, and the branched-chain ketoacid, alpha-ketoisocaproate, are also substrates for the transporter. The transport of these compounds via human SMCT1 is Na(+)-coupled and electrogenic. The Michaelis constant is 1.4 +/- 0.1 mm for beta-d-hydroxybutyrate, 0.21 +/- 0.04 mm for acetoacetate and 0.21 +/- 0.03 mm for alpha-ketoisocaproate. The Na(+) : substrate stoichiometry is 2 : 1. As l-lactate and ketone bodies constitute primary energy substrates for neurons, we investigated the expression pattern of this transporter in the brain. In situ hybridization studies demonstrate widespread expression of SMCT1 mRNA in mouse brain. Immunofluorescence analysis shows that SMCT1 protein is expressed exclusively in neurons. SMCT1 protein co-localizes with MCT2, a neuron-specific Na(+)-independent monocarboxylate transporter. In contrast, there was no overlap of signals for SMCT1 and MCT1, the latter being expressed only in non-neuronal cells. We also demonstrate the neuron-specific expression of SMCT1 in mixed cultures of rat cortical neurons and astrocytes. This represents the first report of an Na(+)-coupled transport system for a major group of energy substrates in neurons. These findings suggest that SMCT1 may play a critical role in the entry of l-lactate and ketone bodies into neurons by a process driven by an electrochemical Na(+) gradient and hence, contribute to the maintenance of the energy status and function of neurons.
Assuntos
Encéfalo/citologia , Proteínas de Transporte de Cátions/fisiologia , Corpos Cetônicos/metabolismo , Lactatos/metabolismo , Neurônios/fisiologia , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Encéfalo/metabolismo , Isótopos de Carbono/farmacocinética , Linhagem Celular , Estimulação Elétrica/métodos , Agonistas de Aminoácidos Excitatórios , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Hidroxibutiratos/farmacocinética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Camundongos , Transportadores de Ácidos Monocarboxílicos , Neurônios/efeitos dos fármacos , Oócitos , Técnicas de Patch-Clamp/métodos , Fosfopiruvato Hidratase/metabolismo , Epitélio Pigmentado Ocular/citologia , Sódio/metabolismo , Transfecção/métodos , XenopusRESUMO
Reabsorption of amino acids is an important function of the renal proximal tubule. pH-dependent amino acid transport has been measured previously using rabbit renal brush-border membrane vesicles (BBMV). The purpose of this investigation was to determine whether this pH-dependent uptake represents H(+)/amino acid cotransport via a PAT1-like transport system. The rabbit PAT1 cDNA was isolated (2296bp including both 5' and 3' untranslated regions and poly(A) tail) and the open reading frame codes for a protein of 475 amino acids (92% identity to human PAT1). Rabbit PAT1 mRNA was found in all tissues investigated including kidney. When expressed heterologously in a mammalian cell line, rabbit PAT1 mediates pH-dependent, Na(+)-independent uptake of proline, glycine, l-alanine and alpha-(methylamino)isobutyric acid. Proline uptake was maximal at pH 5.0 (K(m) 2.2+/-0.7 mM). A transport system with identical characteristics (ion dependency, substrate specificity) was detected in rabbit renal BBMV where an overshoot was observed in the absence of Na+ but in the presence of an inwardly directed H+ gradient. In the presence of Na+ and under conditions in which PAT1 transport function was suppressed, a second proline uptake system was detected that exhibited functional characteristics similar to those of the IMINO system. The functional characteristics of rabbit PAT1 in either mammalian cells or renal BBMV suggest that PAT1 is the low-affinity transporter of proline, glycine and hydroxyproline believed to be defective in patients with iminoglycinuria.