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Organic molecules that exhibit charge-transfer (CT) excited states are known to play an important role in processes linked to electron transfer properties and molecular conductance. In this article, we present a simple technique based on "Becke's excitation theorem" that offers an accurate picture of these electronic states. It expresses the correlated energy splitting between triplet and its corresponding singlet states by a two-electron integral, which is numerically evaluated by our recently developed strategy on Cartesian grid. We first examine the consistency of our adopted numerical strategy to evaluate the integral with the original prescribed technique. Then we assess the method on weakly bound CT complexes with three different functionals (BLYP, B3LYP, and LC-BLYP). The accuracy on asymptotic limit of CT excitation is also explored. Finally in order to illustrate the strength and feasibility, it is further extended to a few "challenging" molecules. The method, when employed with hybrid B3LYP functional, turns out to be quite accurate to describe CT excitation energy.
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BACKGROUND: Circulating tumor (ct) DNA assays performed in clinical laboratories provide tumor biomarker testing support for biopharmaceutical clinical trials. Yet it is neither practical nor economically feasible for many of these clinical laboratories to internally develop their own liquid biopsy assay. Commercially available ctDNA kits are a potential solution for laboratories seeking to incorporate liquid biopsy into their test menus. However, the scarcity of characterized patient samples and cost of purchasing validation reference standards creates a barrier to entry. In the current study, we evaluated the analytical performance of the AVENIO ctDNA liquid biopsy platform (Roche Sequencing Solutions) for use in our clinical laboratory. METHOD: Intra-laboratory performance evaluation of AVENIO ctDNA Targeted, Expanded, and Surveillance kits (Research Use Only) was performed according to College of American Pathologists (CAP) guidelines for the validation of targeted next generation sequencing assays using purchased reference standards, de-identified human plasma cell-free (cf) DNA samples, and contrived samples derived from commercially purchased normal and cancer human plasma. All samples were sequenced at read depths relevant to clinical settings using the NextSeq High Output kit (Illumina). RESULTS: At the clinically relevant read depth, Avenio ctDNA kits demonstrated 100% sensitivity in detecting single nucleotide variants (SNVs) at ≥0.5% allele frequency (AF) and 50% sensitivity in detecting SNVs at 0.1% AF using 20-40 ng sample input amount. The assay integrated seamlessly into our laboratory's NGS workflow with input DNA mass, target allele frequency (TAF), multiplexing, and number of reads optimized to support a high-throughput assay appropriate for biopharmaceutical trials. CONCLUSIONS: Our study demonstrates that AVENIO ctDNA liquid biopsy platform provides a viable alternative for efficient incorporation of liquid biopsy assays into the clinical laboratory for detecting somatic alterations as low as 0.5%. Accurate detection of variants lower than 0.5% could potentially be achieved by deeper sequencing when clinically indicated and economically feasible.
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Biomarcadores de Tumor/sangre , ADN Tumoral Circulante/sangre , Secuenciación de Nucleótidos de Alto Rendimiento , Neoplasias/sangre , Ácidos Nucleicos Libres de Células/sangre , Ácidos Nucleicos Libres de Células/genética , ADN Tumoral Circulante/genética , Humanos , Biopsia Líquida , Mutación/genética , Neoplasias/genéticaRESUMEN
We present a purely numerical approach in a Cartesian grid, for efficient computation of the Hartree-Fock (HF) exchange contribution in the HF and density functional theory models. This takes inspiration from a recently developed algorithm by Liu et al., in 2017, where the rate-determining step is the accurate evaluation of electrostatic potential. This introduces the Fourier convolution theorem in conjunction with a range-separated Coulomb interaction kernel. The latter is efficiently mapped into a real grid through a simple optimization procedure, giving rise to a constraint in the range-separated parameter. The overall process offers logarithmic scaling with respect to the molecular size. It is then extended toward global hybrid functionals such as B3LYP, PBE0, and BHLYP within pseudopotential Kohn-Sham theory, through an LCAO-MO ansatz in a Cartesian grid, developed earlier in our laboratory. For the sake of comparison, a parallel semi-numerical approach has also been worked out that exploits the familiar Obara-Saika recursion algorithm without any additional techniques. An excellent agreement between these two routes is demonstrated through total energy and orbital energy in a series of atoms and molecules (including 10 π-electron molecules), employing an LANL2DZ-type basis function. A critical analysis of these two algorithms reveals that the proposed numerical scheme could lead to very attractive and competitive scaling. The success of our approach also enables us for further development of optimally tuned range-separated hybrid and hyper functionals.
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Intestinal absorption of the water-soluble vitamins biotin and pantothenic acid is carrier mediated and involves the sodium-dependent multivitamin transporter (SMVT; product of the SLC5A6 gene). We recently observed that intestinal-specific (conditional) knockout of the mouse Slc5a6 gene (SMVT-cKO) is associated with growth retardation, the development of spontaneous and severe inflammation, abnormal histology in the large intestine, altered gut permeability, and early death. Our aim in this study was to examine the possibility that biotin and pantothenic acid oversupplementation (BPS) of the SMVT-cKO mice could reverse the above-described abnormalities. BPS was provided in the drinking water to mice before conception, to dams during pregnancy and lactation, and to the SMVT-cKO mice throughout their life. Our findings showed that such a regimen prevents early death, as well as normalizes the growth rate, intestinal integrity, pathology, and inflammation in SMVT-cKO mice. These findings provide clear evidence for a role for biotin and/or pantothenic acid in the maintenance of normal intestinal integrity and health.
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Biotina/farmacología , Mucosa Intestinal/efectos de los fármacos , Ácido Pantoténico/farmacología , Simportadores/metabolismo , Animales , Femenino , Inflamación/metabolismo , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Lactancia/efectos de los fármacos , Ratones , Ratones Noqueados , EmbarazoRESUMEN
Utilizing a conditional (intestinal-specific) knockout (cKO) mouse model, we have recently shown that the sodium-dependent multivitamin transporter (SMVT) (SLC5A6) is the only biotin uptake system that operates in the gut and that its deletion leads to biotin deficiency. Unexpectedly, we also observed that all SMVT-cKO mice develop chronic active inflammation, especially in the cecum. Our aim here was to examine the role of SMVT in the maintenance of intestinal mucosal integrity [permeability and expression of tight junction (TJ) proteins]. Our results showed that knocking out the mouse intestinal SMVT is associated with a significant increase in gut permeability and with changes in the level of expression of TJ proteins. To determine whether these changes are related to the state of biotin deficiency that develops in SMVT-cKO mice, we induced (by dietary means) biotin deficiency in wild-type mice and examined its effect on the above-mentioned parameters. The results showed that dietary-induced biotin deficiency leads to a similar development of chronic active inflammation in the cecum with an increase in the level of expression of proinflammatory cytokines, as well as an increase in intestinal permeability and changes in the level of expression of TJ proteins. We also examined the effect of chronic biotin deficiency on permeability and expression of TJ proteins in confluent intestinal epithelial Caco-2 monolayers but observed no changes in these parameters. These results show that the intestinal SMVT plays an important role in the maintenance of normal mucosal integrity, most likely via its role in providing biotin to different cells of the gut mucosa.
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Mucosa Intestinal/fisiología , Simportadores/metabolismo , Animales , Biotina/administración & dosificación , Deficiencia de Biotinidasa/metabolismo , Células CACO-2 , Regulación de la Expresión Génica/fisiología , Homeostasis/fisiología , Humanos , Ratones , Ratones Noqueados , Permeabilidad , Simportadores/genética , Proteínas de Uniones Estrechas/genética , Proteínas de Uniones Estrechas/metabolismoRESUMEN
The widely distributed colonization factor (CF) CS6 of enterotoxigenic Escherichia coli (ETEC) has gained importance over the years in terms of its structure and function. CS6 is an afimbrial assembly in contrast to the other ETEC CFs, which are mostly fimbrial. A recent study predicted a linear fibre model for recombinant chimeric CS6 and formation of oligomers in solution. In this study, we characterized the oligomeric assembly of CS6, purified from a clinical ETEC isolate and identified its existence in the WT strain. We found that purified CS6 forms a continuous array of higher order oligomers composed of two tightly associated subunits, CssA and CssB in an equal (1:1) stoichiometry. This oligomerization occurs by formation of (CssA-CssB)n complex where 'n' increases with the concentration. The diameter of CS6 oligomers also proportionally increases with concentration. More significantly, we showed CS6 oligomers to be spherical in shape instead of being linear fibres as predicted earlier and this was further confirmed by electron microscopy. We also showed CS6 assembled on the bacterial surface in the form of an oligomeric complex. This process depends on the expression of properly folded CssA and CssB together, guided by the chaperone CssC and usher CssD. In conclusion, our results provide evidence for the existence of concentration-dependent, spherical oligomers of CS6 comprising both the structural subunits in equal stoichiometry and the CS6 oligomeric complex on the ETEC surface.
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Antígenos Bacterianos/química , Antígenos Bacterianos/metabolismo , Escherichia coli Enterotoxigénica/metabolismo , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Antígenos Bacterianos/genética , Escherichia coli Enterotoxigénica/química , Escherichia coli Enterotoxigénica/genética , Proteínas de Escherichia coli/genética , HumanosRESUMEN
The human riboflavin (RF) transporter-3 (product of the SLC52A3 gene) plays an important role in intestinal RF absorption. Our aims in this study were to identify the minimal 5'-regulatory region of the SLC52A3 gene and the regulatory element(s) involved in its activity in intestinal epithelial cells, as well as to confirm promoter activity and establish physiological relevance in vivo in transgenic mice. With the use of transiently transfected human intestinal epithelial HuTu 80 cells and 5'-deletion analysis, the minimal SLC52A3 promoter was found to be encoded between -199 and +8 bp (using the start of the transcription start site as position 1). Although several putative cis-regulatory elements were predicted in this region, only the stimulating protein-1 (Sp1) binding site (at position -74/-71 bp) was found to play a role in promoter activity, as indicated by mutational analysis. Binding of Sp1 to the minimal SLC52A3 promoter was demonstrated by means of EMSA and supershift assays and by chromatin immunoprecipitation analysis. Studies with Drosophila SL2 cells (which lack Sp activity) confirmed the importance of Sp1 in driving the activity of the SLC52A3 minimal promoter; they further showed that Sp3 can also do the activation. Finally, with the use of luciferase gene fusions, the activity of the cloned SLC52A3 promoter was confirmed in vivo in transgenic mice. These studies report, for the first time, on the identification and characterization of the SLC52A3 promoter and also demonstrate the importance of Sp1 in regulating its activity in intestinal epithelial cells.
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Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Células Cultivadas , Inmunoprecipitación de Cromatina/métodos , Drosophila/genética , Drosophila/metabolismo , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Regiones Promotoras Genéticas/genética , Receptores Acoplados a Proteínas G , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3/genética , Factor de Transcripción Sp3/metabolismoRESUMEN
Infection with the nontyphoidal Salmonella is a common cause of food-borne disease that leads to acute gastroenteritis/diarrhea. Severe/prolonged cases of Salmonella infection could also impact host nutritional status, but little is known about its effect on intestinal absorption of vitamins, including biotin. We examined the effect of Salmonella enterica serovar Typhimurium (S. typhimurium) infection on intestinal biotin uptake using in vivo (streptomycin-pretreated mice) and in vitro [mouse (YAMC) and human (NCM460) colonic epithelial cells, and human intestinal epithelial Caco-2 cells] models. The results showed that infecting mice with wild-type S. typhimurium, but not with its nonpathogenic isogenic invA spiB mutant, leads to a significant inhibition in jejunal/colonic biotin uptake and in level of expression of the biotin transporter, sodium-dependent multivitamin transporter. In contrast, infecting YAMC, NCM460, and Caco-2 cells with S. typhimurium did not affect biotin uptake. These findings suggest that the effect of S. typhimurium infection is indirect and is likely mediated by proinflammatory cytokines, the levels of which were markedly induced in the intestine of S. typhimurium-infected mice. Consistent with this hypothesis, exposure of NCM460 cells to the proinflammatory cytokines TNF-α and IFN-γ led to a significant inhibition of biotin uptake, sodium-dependent multivitamin transporter expression, and activity of the SLC5A6 promoter. The latter effects appear to be mediated, at least in part, via the NF-κB signaling pathway. These results demonstrate that S. typhimurium infection inhibits intestinal biotin uptake, and that the inhibition is mediated via the action of proinflammatory cytokines.
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Biotina/metabolismo , Mucosa Intestinal/metabolismo , Salmonelosis Animal/metabolismo , Salmonella typhimurium/patogenicidad , Simportadores/metabolismo , Animales , Transporte Biológico , Células CACO-2 , Modelos Animales de Enfermedad , Interacciones Huésped-Patógeno , Humanos , Mediadores de Inflamación/metabolismo , Interferón gamma/metabolismo , Intestinos/inmunología , Intestinos/microbiología , Ratones , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , Salmonelosis Animal/genética , Salmonelosis Animal/inmunología , Salmonelosis Animal/microbiología , Salmonella typhimurium/inmunología , Transducción de Señal , Simportadores/genética , Transfección , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The human malaria parasite Plasmodium falciparum is absolutely dependent on the acquisition of host pantothenate for its development within human erythrocytes. Although the biochemical properties of this transport have been characterized, the molecular identity of the parasite-encoded pantothenate transporter remains unknown. Here we report the identification and functional characterization of the first protozoan pantothenate transporter, PfPAT, from P. falciparum. We show using cell biological, biochemical, and genetic analyses that this transporter is localized to the parasite plasma membrane and plays an essential role in parasite intraerythrocytic development. We have targeted PfPAT to the yeast plasma membrane and showed that the transporter complements the growth defect of the yeast fen2Δ pantothenate transporter-deficient mutant and mediates the entry of the fungicide drug, fenpropimorph. Our studies in P. falciparum revealed that fenpropimorph inhibits the intraerythrocytic development of both chloroquine- and pyrimethamine-resistant P. falciparum strains with potency equal or better than that of currently available pantothenate analogs. The essential function of PfPAT and its ability to deliver both pantothenate and fenpropimorph makes it an attractive target for the development and delivery of new classes of antimalarial drugs.
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Membrana Celular/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Simportadores/metabolismo , Secuencia de Aminoácidos , Animales , Antimaláricos/farmacología , Cloroquina/farmacología , Resistencia a Medicamentos/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Eritrocitos/parasitología , Eritrocitos/ultraestructura , Prueba de Complementación Genética , Células HEK293 , Interacciones Huésped-Parásitos/efectos de los fármacos , Humanos , Malaria Falciparum/parasitología , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Morfolinas/metabolismo , Morfolinas/farmacología , Mutación , Ácido Pantoténico/metabolismo , Ácido Pantoténico/farmacología , Filogenia , Plasmodium falciparum/genética , Plasmodium falciparum/fisiología , Proteínas Protozoarias/genética , Pirimetamina/farmacología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Simportadores/clasificación , Simportadores/genéticaRESUMEN
Biotin is essential for the normal function of pancreatic beta cells. These cells obtain biotin from their surroundings via transport across their cell membrane. Little is known about the uptake mechanism involved, how it is regulated, and how it is affected by internal and external factors. We addressed these issues using the mouse-derived pancreatic beta-TC-6 cells and freshly isolated mouse and human primary pancreatic beta cells as models. The results showed biotin uptake by pancreatic beta-TC-6 cells occurs via a Na(+)-dependent, carrier-mediated process, that is sensitive to desthiobiotin, as well as to pantothenic acid and lipoate; the process is also saturable as a function of concentration (apparent Km = 22.24 ± 5.5 µM). These cells express the sodium-dependent multivitamin transporter (SMVT), whose knockdown (with doxycycline-inducible shRNA) led to a sever inhibition in biotin uptake. Similarly, uptake of biotin by mouse and human primary pancreatic islets is Na(+)-dependent and carrier-mediated, and both cell types express SMVT. Biotin uptake by pancreatic beta-TC-6 cells is also adaptively regulated (via transcriptional mechanism) by extracellular substrate level. Chronic treatment of pancreatic beta-TC-6 cells with bacterial lipopolysaccharides (LPS) leads to inhibition in biotin uptake. This inhibition is mediated via a Toll-Like receptor 4-mediated process and involves a decrease in membrane expression of SMVT. These findings show, for the first time, that pancreatic beta cells/islets take up biotin via a specific and regulated carrier-mediated process, and that the process is sensitive to the effect of LPS.
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Biotina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Islotes Pancreáticos/efectos de los fármacos , Lipopolisacáridos/farmacología , Simportadores/efectos de los fármacos , Animales , Transporte Biológico , Biotina/análogos & derivados , Biotina/farmacología , Línea Celular , Humanos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Cinética , Ratones , Ácido Pantoténico/farmacología , Interferencia de ARN , Simportadores/genética , Simportadores/metabolismo , Ácido Tióctico/farmacología , Receptor Toll-Like 4/metabolismo , TransfecciónRESUMEN
The characterization of negative ion resonances poses a fundamental challenge to density functional methods due to the unbound nature of resonances. To overcome this challenge, we propose one-particle nonlocal exchange-correlation (xc) potentials combining the exact-exchange (EXX) and the random phase approximation (RPA) correlation potentials. The negative ion resonances are identified by perturbing the real Hermitian nonlocal xc potentials using complex absorbing local potentials. Our studies show that the nonlocal EXX+RPA potential significantly enhances the description of positions and widths of negative ion resonance states compared to potentials that exclude dynamic polarization in RPA or include only EXX. The use of low-scaling algorithms simplifies the computation of the RPA potential, thereby providing a practical solution for resonance-state characterization within the density functional framework. A theoretical framework and the underlying assumptions required for combining real Hermitian nonlocal xc potentials with complex local potentials are discussed.
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Barrett's esophagus (BE) is a known precursor to esophageal adenocarcinoma (EAC). Guidelines recommend BE screening in populations with multiple risk factors, for which non-endoscopic esophageal cell collection with biomarker testing is considered as an acceptable alternative to esophagogastroduodenoscopy (EGD). The aim of this study was to evaluate analytical performance characteristics of EsoGuard® (EG), a DNA methylation biomarker assay, as a laboratory-developed test (LDT) in esophageal samples collected with the swallowable EsoCheck® (EC) device. EG is a next-generation sequencing (NGS) assay that evaluates methylated vimentin (VIM) and cyclin A1 (CCNA1), clinically validated biomarkers for the detection of BE and EAC. The studies were conducted according to standards of College of American Pathology (CAP), Clinical Laboratory Improvement Amendments (CLIA), and New York (NY) state requirements for the analytical validation of molecular assays. Comparison to Sanger sequencing showed that EG was 100% accurate at all 31 CpG sites evaluated by the assay. The analytical sensitivity, specificity, and accuracy of the assay were 89%, 100%, and 96%, respectively. Intra- and inter-assay precision was 100%. The limit of detection (LOD) was 1 in 400 methylated cells, and the reference range was 84%. In summary, EsoGuard demonstrates high analytical accuracy, repeatability, and reproducibility in samples collected using the EsoCheck device.
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Infections with enteric pathogens like enterotoxigenic Escherichia coli (ETEC) is a major health issue worldwide and while diarrhea is the major problem, prolonged, severe, and dual infections with multiple pathogens may also compromise the nutritional status of the infected individuals. There is almost nothing currently known about the effect of ETEC infection on intestinal absorptions of water-soluble vitamins including thiamin. We examined the effect of ETEC infection on intestinal uptake of the thiamin using as a model the human-derived intestinal epithelial Caco-2 cells. The results showed that infecting confluent Caco-2 monolayers with live ETEC (but not with boiled/killed ETEC or nonpathogenic E. coli) or treatment with bacterial culture supernatant led to a significant inhibition in thiamin uptake. This inhibition appears to be caused by a heat-labile and -secreted ETEC component and is mediated via activation of the epithelial adenylate cyclase system. The inhibition in thiamin uptake by ETEC was associated with a significant reduction in expression of human thiamin transporter-1 and -2 (hTHTR1 and hTHTR2) at the protein and mRNA levels as well as in the activity of the SLC19A2 and SLC19A3 promoters. Dual infection of Caco-2 cells with ETEC and EPEC (enteropathogenic E. coli) led to compounded inhibition in intestinal thiamin uptake. These results show for the first time that infection of human intestinal epithelial cells with ETEC causes a significant inhibition in intestinal thiamin uptake. This inhibition is mediated by a secreted heat-labile toxin and is associated with a decrease in the expression of intestinal thiamin transporters.
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Escherichia coli Enterotoxigénica/fisiología , Infecciones por Escherichia coli/metabolismo , Absorción Intestinal , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Tiamina/metabolismo , Células CACO-2 , Infecciones por Escherichia coli/patología , Humanos , Absorción Intestinal/fisiología , Mucosa Intestinal/patología , Tiamina/antagonistas & inhibidoresRESUMEN
Vitamin B2 (riboflavin, RF) is essential for normal human health. Mammals obtain RF from exogenous sources via intestinal absorption and prevent its urinary loss by reabsorption in the kidneys. Both of these absorptive events are carrier-mediated and involve specific RF transporters (RFVTs). Chronic alcohol consumption in humans is associated with a high prevalence of RF deficiency and suboptimal levels, but little is known about the effect of chronic alcohol exposure on physiological and molecular parameters of the intestinal and renal RF transport events. We addressed these issues using rats chronically fed an alcohol liquid diet and pair-fed controls as a model. The results showed that chronic alcohol feeding significantly inhibits carrier-mediated RF transport across the intestinal brush-border and basolateral membrane domains of the polarized enterocytes. This inhibition was associated with a parallel reduction in the expression of the rat RFVT-1 and -3 at the protein, mRNA, and heterogeneous nuclear RNA (hnRNA) levels. Chronic alcohol feeding also caused a significant inhibition in RF uptake in the colon. Similarly, a significant inhibition in carrier-mediated RF transport across the renal brush-border and basolateral membrane domains was observed, which again was associated with a significant reduction in the level of expression of RFVT-1 and -3 at the protein, mRNA, and hnRNA levels. These findings demonstrate that chronic alcohol exposure impairs both intestinal absorption and renal reabsorption processes of RF and that these effects are, at least in part, mediated via transcriptional mechanism(s) involving the slc52a1 and slc52a3 genes.
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Consumo de Bebidas Alcohólicas/metabolismo , Etanol/farmacología , Absorción Intestinal/efectos de los fármacos , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Riboflavina/metabolismo , Consumo de Bebidas Alcohólicas/fisiopatología , Animales , Transporte Biológico/efectos de los fármacos , Colon/efectos de los fármacos , Colon/metabolismo , Colon/patología , Dieta , Enterocitos/efectos de los fármacos , Enterocitos/metabolismo , Enterocitos/patología , Regulación de la Expresión Génica , Humanos , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Intestino Delgado/patología , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/patología , Masculino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Microvellosidades/efectos de los fármacos , Microvellosidades/metabolismo , Microvellosidades/patología , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transcripción GenéticaRESUMEN
The sodium-dependent multivitamin transporter (SMVT) plays an important role in biotin uptake in the intestine and other cell types. While significant knowledge has been gained with regard to regulation and cell biology of the SMVT system, there is little known about its structure-function relationships. Here we examined the role of each of the ten conserved (among species) cysteine residues in the function of the human SMVT (hSMVT) using site-directed mutagenesis. Our results showed a significant impairment in biotin uptake only in cells transfected with hSMVT mutated at Cys(294), but not at the other conserved cysteine residues; the impairment in biotin uptake caused by mutating Cys(294) was not related to the polar status of substituting amino acid. The inhibition in hSMVT function upon mutating Cys(294) was mediated via a significant reduction in the V(max), but not the apparent K(m), of the biotin uptake process, suggesting a decrease in the number (and/or activity) of hSMVT but not affinity. Biotinylation assay confirmed this suggestion by showing a marked reduction in the level of expression of the mutated protein at the cell membrane, without affecting total cellular level of induced hSMVT. These results show an important role for Cys(294) in the function and cell biology of hSMVT.
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Membrana Celular/metabolismo , Cisteína/metabolismo , Células Epiteliales/metabolismo , Retina/metabolismo , Simportadores/metabolismo , Transporte Biológico , Biotina/metabolismo , Biotinilación , Membrana Celular/genética , Cisteína/genética , Cartilla de ADN/química , Cartilla de ADN/genética , Células Epiteliales/citología , Humanos , Cinética , Mutagénesis Sitio-Dirigida , Mutación , Reacción en Cadena de la Polimerasa , Retina/citología , Relación Estructura-Actividad , Simportadores/química , Simportadores/genética , TransfecciónRESUMEN
The Slc5a6 gene expresses a plasma membrane protein involved in the transport of the water-soluble vitamin biotin; the transporter is commonly referred to as the sodium-dependent multivitamin transporter (SMVT) because it also transports pantothenic acid and lipoic acid. The relative contribution of the SMVT system toward carrier-mediated biotin uptake in the native intestine in vivo has not been established. We used a Cre/lox technology to generate an intestine-specific (conditional) SMVT knockout (KO) mouse model to address this issue. The KO mice exhibited absence of expression of SMVT in the intestine compared with sex-matched littermates as well as the expected normal SMVT expression in other tissues. About two-thirds of the KO mice died prematurely between the age of 6 and 10 wk. Growth retardation, decreased bone density, decreased bone length, and decreased biotin status were observed in the KO mice. Microscopic analysis showed histological abnormalities in the small bowel (shortened villi, dysplasia) and cecum (chronic active inflammation, dysplasia) of the KO mice. In vivo (and in vitro) transport studies showed complete inhibition in carrier-mediated biotin uptake in the intestine of the KO mice compared with their control littermates. These studies provide the first in vivo confirmation in native intestine that SMVT is solely responsible for intestinal biotin uptake. These studies also provide evidence for a casual association between SMVT function and normal intestinal health.
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Biotina/metabolismo , Absorción Intestinal/fisiología , Mucosa Intestinal/metabolismo , Simportadores/genética , Animales , Western Blotting , Células Madre Embrionarias/trasplante , Intestinos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ácido Pantoténico/metabolismo , Fenotipo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Intestinal epithelial cells undergo differentiation as they move from the crypt to the villi, a process that is associated with up- and downregulation in expression of a variety of genes, including those involved in nutrient absorption. Whether the intestinal uptake process of vitamin B(2) [riboflavin (RF)] also undergoes differentiation-dependent regulation and the mechanism through which this occurs are not known. We used human-derived intestinal epithelial Caco-2 cells and native rat intestine as models to address these issues. Caco-2 cells showed a significantly higher carrier-mediated RF uptake in post- than preconfluent cells. This upregulation was associated with a significantly higher level of protein and mRNA expression of the RF transporters hRFVT-1 and hRFVT-3 in the post- than preconfluent cells; it was also accompanied with a significantly higher rate of transcription of the respective genes (SLC52A1 and SLC52A3), as indicated by the higher level of expression of heterogeneous nuclear RNA and higher promoter activity in post- than preconfluent cells. Studies with native rat intestine also showed a significantly higher RF uptake by epithelial cells of the villus tip than epithelial cells of the crypt; this again was accompanied by a significantly higher level of expression of the rat RFVT-1 and RFVT-3 at the protein, mRNA, and heterogeneous nuclear RNA levels. These findings show, for the first time, that the intestinal RF uptake process undergoes differentiation-dependent upregulation and suggest that this is mediated (at least in part) via transcriptional mechanisms.
Asunto(s)
Absorción Intestinal/fisiología , Mucosa Intestinal/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Riboflavina/farmacocinética , Animales , Células CACO-2 , Diferenciación Celular/fisiología , Regulación de la Expresión Génica/fisiología , Humanos , Mucosa Intestinal/citología , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana/genética , Regiones Promotoras Genéticas/fisiología , ARN Mensajero/metabolismo , Ratas , Receptores Acoplados a Proteínas G/genética , Transcripción Genética/fisiología , Complejo Vitamínico B/farmacocinéticaRESUMEN
BACKGROUND: Ascorbic acid (AA) is required for normal human health and development. Human intestine expresses two sodium-dependent vitamin C transporters (hSVCT-1 and -2) that mediate cellular AA transport, with hSVCT1 targeting to the apical membrane of polarized epithelia. Studies have shown a role for the Rab8a in the apical membrane targeting of transporters in intestinal cells. AIMS: The purpose of this study was to determine whether Rab8a impacts the function and/or targeting of hSVCT1, and intestinal AA uptake. METHODS: We used human intestinal cells and cells from a Rab8a knockout mouse. (14)C-AA uptake was performed to determine functionality. PCR and western blotting were performed to determine RNA and protein expression, respectively. Confocal imaging was performed to determine co-localization. RESULTS: We show that hSVCT1 co-localized with Rab8a in intestinal cells. Knockdown of Rab8a lead to a significant inhibition in AA uptake and cell surface biotinylation studies revealed a lower cell surface expression of hSVCT1 in Rab8a siRNA-treated cells. Similarly, in the small intestine of a Rab8a knockout mouse, AA uptake was significantly inhibited. This effect again resulted from a decreased expression level of mSVCT1 protein, even though mRNA expression of SVCT1 was similar in intestinal cells from Rab8a knockout and wild-type litter-mates. The latter data are suggestive of enhanced lysosomal degradation of hSVCT1 protein in Rab8a-deficient cells; indeed, confocal imaging of Rab8a siRNA-treated intestinal cells revealed a strong overlap between hSVCT1-YFP and LAMP1-RFP. CONCLUSIONS: These findings show a role for Rab8a in the physiological function of hSVCT1 in intestinal epithelia.
Asunto(s)
Ácido Ascórbico/metabolismo , Mucosa Intestinal/metabolismo , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Aminoácidos/metabolismo , Animales , Western Blotting , Células CACO-2 , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Humanos , Ratones , Ratones Noqueados , Transporte de Proteínas , ARN Interferente Pequeño , Reacción en Cadena en Tiempo Real de la Polimerasa , Transportadores de Sodio Acoplados a la Vitamina C/genética , Proteínas de Unión al GTP rab/genéticaRESUMEN
The sodium-dependent multivitamin transporter (SMVT) is a major biotin transporter in a variety of tissues including the small intestine. The human SMVT (hSMVT) polypeptide is predicted to have four N-glycosylation sites and two putative PKC phosphorylation sites but their role in the function and regulation of the protein is not known and was examined in this investigation. Our results showed that the hSMVT protein is glycosylated and that this glycosylation is important for its function. Studies utilizing site-directed mutagenesis revealed that the N-glycosylation sites at positions Asn(138) and Asn(489) are important for the function of hSMVT and that mutating these sites significantly reduces the V(max) of the biotin uptake process. Mutating the putative PKC phosphorylation site Thr(286) of hSMVT led to a significant decrease in the PMA-induced inhibition in biotin uptake. The latter effect was not mediated via changes in the level of expression of the hSMVT protein and mRNA or in its level of expression at the cell membrane. These findings demonstrate that the hSMVT protein is glycosylated, and that glycosylation is important for its function. Furthermore, the study shows a role for the putative PKC-phosphorylation site Thr(286) of hSMVT in the PKC-mediated regulation of biotin uptake.