RESUMEN
Recently, we cloned two Na(+)-coupled lactate transporters from mouse kidney, a high-affinity transporter (SMCT1 or slc5a8) and a low-affinity transporter (SMCT2 or slc5a12). Here we report on the cloning and functional characterization of human SMCT2 (SLC5A12) and compare the immunolocalization patterns of slc5a12 and slc5a8 in mouse kidney. The human SMCT2 cDNA codes for a protein consisting of 618 amino acids. When expressed in mammalian cells or Xenopus oocytes, human SMCT2 mediates Na(+) -coupled transport of lactate, pyruvate and nicotinate. The affinities of the transporter for these substrates are lower than those reported for human SMCT1. Several non-steroidal anti-inflammatory drugs inhibit human SMCT2-mediated nicotinate transport, suggesting that NSAIDs interact with the transporter as they do with human SMCT1. Immunofluorescence microscopy of mouse kidney sections with an antibody specific for SMCT2 shows that the transporter is expressed predominantly in the cortex. Similar studies with an anti-SMCT1 antibody demonstrate that SMCT1 is also expressed mostly in the cortex. Dual-labeling of SMCT1 and SMCT2 with 4F2hc (CD98), a marker for basolateral membrane of proximal tubular cells in the S1 and S2 segments of the nephron, shows that both SMCT1 and SMCT2 are expressed in the apical membrane of the tubular cells. These studies also show that while SMCT2 is broadly expressed along the entire length of the proximal tubule (S1/S2/S3 segments), the expression of SMCT1 is mostly limited to the S3 segment. These studies suggest that the low-affinity transporter SMCT2 initiates lactate absorption in the early parts of the proximal tubule followed by the participation of the high-affinity transporter SMCT1 in the latter parts of the proximal tubule.
Asunto(s)
Riñón/química , Transportadores de Ácidos Monocarboxílicos/análisis , Transportadores de Ácidos Monocarboxílicos/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/química , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Transportadores de Ácidos Monocarboxílicos/fisiología , Simportadores , Xenopus laevisRESUMEN
NaS2 is a Na+-coupled transporter for sulfate that belongs to the SLC13 gene family. This transporter was originally cloned from high endothelial venule endothelial cells, but nothing is known about the functional characteristics of this transporter except that it transports sulfate in a Na+-coupled manner. Northern blot analysis indicates that NaS2 is expressed most robustly in placenta. In the present study, we cloned NaS2 from rat placenta and characterized its transport function in detail using the Xenopus laevis oocyte expression system. Rat NaS2 consists of 629 amino acids and is highly similar to human NaS2. In situ hybridization studies with mouse placental sections show that NaS2 transcripts are expressed primarily in trophoblasts of the labyrinth zone. The expression of the transporter is confirmed in primary cultures of trophoblasts isolated from human placenta. When expressed in X. laevis oocytes, rat NaS2 mediates Na+-coupled transport of sulfate. The transport of sulfate is inhibited by oxyanions of selenium, chromium, arsenic, molybdenum, and phosphorous, suggesting that the transporter may mediate the transport of these oxyanions in addition to sulfate. The Kt for sulfate is 153+/-30 microM and the Na+:sulfate stoichiometry is 3:1. The transport process is electrogenic as evidenced from the inhibition of the uptake process by K+-induced depolarization. We conclude that NaS2 is a placenta-specific Na+-coupled, electrogenic, transporter for sulfate expressed in trophoblasts and that it is also responsible for the transport of oxyanions of the micronutrients selenium and chromium.
Asunto(s)
Aniones/metabolismo , Cromo/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Selenio/metabolismo , Sulfatos/metabolismo , Simportadores/metabolismo , Trofoblastos/metabolismo , Secuencia de Aminoácidos , Animales , Biblioteca de Genes , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Ratas , Homología de Secuencia de Aminoácido , Transportadores de Sulfato , Xenopus laevisRESUMEN
The transient exposure of immature rodents to ethanol during postnatal day 7 (P7), comparable to a time point within the third trimester of human pregnancy, induces neurodegeneration. However, the molecular mechanisms underlying the deleterious effects of ethanol on the developing brain are poorly understood. In our previous study, we showed that a high dose administration of ethanol at P7 enhances G9a and leads to caspase-3-mediated degradation of dimethylated H3 on lysine 9 (H3K9me2). In this study, we investigated the potential role of epigenetic changes at G9a exon1, G9a-mediated H3 dimethylation on neurodegeneration and G9a-associated proteins in the P7 brain following exposure to a low dose of ethanol. We found that a low dose of ethanol induces mild neurodegeneration in P7 mice, enhances specific acetylation of H3 on lysine 14 (H3K14ace) at G9a exon1, G9a protein levels, augments the dimethylation of H3K9 and H3 lysine 27 (H3K27me2). However, neither dimethylated H3K9 nor K27 underwent degradation. Pharmacological inhibition of G9a activity prior to ethanol treatment prevented H3 dimethylation and neurodegeneration. Further, our immunoprecipitation data suggest that G9a directly associates with DNA methyltransferase (DNMT3A) and methyl-CpG-binding protein 2 (MeCP2). In addition, DNMT3A and MeCP2 protein levels were enhanced by a low dose of ethanol that was shown to induce mild neurodegeneration. Collectively, these epigenetic alterations lead to association of G9a, DNMT3A and MeCP2 to form a larger repressive complex and have a significant role in low-dose ethanol-induced neurodegeneration in the developing brain.
Asunto(s)
Depresores del Sistema Nervioso Central/toxicidad , Etanol/toxicidad , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Enfermedades Neurodegenerativas/inducido químicamente , Enfermedades Neurodegenerativas/metabolismo , Acetilación/efectos de los fármacos , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Regulación de la Expresión Génica/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Metilación/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Activación Transcripcional/efectos de los fármacosRESUMEN
We have developed a novel, high-throughput scintillation proximity assay to measure the membrane-associated steps (stages 2 and 3) of peptidoglycan synthesis in Escherichia coli. At least five enzymes are involved in these two stages, all of which are thought to be essential for the survival of the cell. The individual enzymes are difficult to assay since the substrates are lipidic and difficult to isolate in large quantities and analysis is done by paper chromatography. We have assayed all five enzymes in a single mixture by monitoring synthesis of cross-linked peptidoglycan, which is the final product of the pathway. E. coli membranes are incubated with the two sugar precursors, UDP-N-acetyl muramylpentapeptide and UDP-[(3)H]-N-acetylglucosamine. The radiolabel is incorporated into peptidoglycan, which is captured using wheat germ agglutinin-coated scintillation proximity assay beads. The assay monitors the activity of the translocase (MraY), the transferase (MurG), the lipid pyrophosphorylase, and the transglycosylase and transpeptidase activities of the penicillin-binding proteins. Vancomyin, tunicamycin, nisin, moenomycin, bacitracin, and penicillin inhibit the assay, and these inhibitors have been used to validate the assay. The search for new antimicrobial agents that act via the late stages of peptidoglycan biosynthesis can now be performed in high throughput in a microtiter plate.