RESUMEN
HYDROXYPROLINE O-ARABINOSYLTRANSFERASEs (HPATs) initiate a post-translational protein modification (Hyp-Ara) found abundantly on cell wall structural proteins. In Arabidopsis thaliana, HPAT1 and HPAT3 are redundantly required for full pollen fertility. In addition to the lack of Hyp-Ara in hpat1/3 pollen tubes (PTs), we also found broadly disrupted cell wall polymer distributions, particularly the conversion of the tip cell wall to a more shaft-like state. Mutant PTs were slow growing and prone to rupture and morphological irregularities. In a forward mutagenesis screen for suppressors of the hpat1/3 low seed-set phenotype, we identified a missense mutation in exo70a2, a predicted member of the vesicle-tethering exocyst complex. The suppressed pollen had increased fertility, fewer morphological defects and partially rescued cell wall organization. A transcriptional null allele of exo70a2 also suppressed the hpat1/3 fertility phenotype, as did mutants of core exocyst complex member sec15a, indicating that reduced exocyst function bypassed the PT requirement for Hyp-Ara. In a wild-type background, exo70a2 reduced male transmission efficiency, lowered pollen germination frequency and slowed PT elongation. EXO70A2 also localized to the PT tip plasma membrane, consistent with a role in exocyst-mediated secretion. To monitor the trafficking of Hyp-Ara modified proteins, we generated an HPAT-targeted fluorescent secretion reporter. Reporter secretion was partially dependent on EXO70A2 and was significantly increased in hpat1/3 PTs compared with the wild type, but was reduced in the suppressed exo70a2 hpat1/3 tubes.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/enzimología , Pared Celular/metabolismo , Pentosiltransferasa/metabolismo , Tubo Polínico/crecimiento & desarrollo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Hidroxiprolina/metabolismo , Mutación , Pentosiltransferasa/genética , Pentosiltransferasa/fisiología , Tubo Polínico/metabolismoRESUMEN
BACKGROUND/OBJECTIVES: The cellular and extracellular matrix (ECM) interactions that regulate adipose tissue homeostasis are incompletely understood. Proteoglycans (PGs) and their sulfated glycosaminoglycans (GAGs) provide spatial and temporal signals for ECM organization and interactions with resident cells by impacting growth factor and cytokine activity. Therefore, PGs and their GAGs could be significant to adipose tissue homeostasis. The purpose of this study was to determine the role of ECM sulfated GAGs in adipose tissue homeostasis. METHODS: Adipose tissue and metabolic homeostasis in mice deficient in xylosyltransferase 2 (Xylt2-/-) were examined by histologic analyses, gene expression analyses, whole body fat composition measurements, and glucose tolerance test. Adipose tissue inflammation and adipocyte precursors were characterized by flow cytometry and in vitro culture of mesenchymal stem cells. RESULTS: Xylt2-/- mice have low body weight due to overall reductions in abdominal fat deposition. Histologically, the adipocytes are reduced in size and number in both gonadal and mesenteric fat depots of Xylt2-/- mice. In addition, these mice are glucose intolerant, insulin resistant, and have increased serum triglycerides as compared to Xylt2 + / + control mice. Furthermore, the adipose tissue niche has increased inflammatory cells and enrichment of proinflammatory factors IL6 and IL1ß, and these mice also have a loss of adipose tissue vascular endothelial cells. Lastly, xylosyltransferease-2 (XylT2) deficient mesenchymal stem cells from gonadal adipose tissue and bone marrow exhibit impaired adipogenic differentiation in vitro. CONCLUSIONS: Decreased GAGs due to the loss of the key GAG assembly enzyme XylT2 causes reduced steady state adipose tissue stores leading to a unique lipodystrophic model. Accumulation of an adipocytic precursor pool of cells is discovered indicating an interruption in differentiation. Therefore, adipose tissue GAGs are important in the homeostasis of adipose tissue by mediating control of adipose precursor development, tissue inflammation, and vascular development.
Asunto(s)
Tejido Adiposo , Lipodistrofia/metabolismo , Pentosiltransferasa , Tejido Adiposo/química , Tejido Adiposo/metabolismo , Animales , Glucemia/metabolismo , Peso Corporal/fisiología , Citocinas/metabolismo , Matriz Extracelular/química , Femenino , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Lipodistrofia/genética , Masculino , Ratones , Ratones Noqueados , Pentosiltransferasa/deficiencia , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo , Pentosiltransferasa/fisiología , UDP Xilosa Proteína XilosiltransferasaRESUMEN
Glucoside xylosyltransferase2 (GXYLT2), a member of the human α-1,3-D-xylosyltransferases, functions to modify the first xylose to the O-Glucose residue on epidermal growth factor (EGF) repeats of Notch receptors. It is well-established that the Notch signaling pathway plays a critical role in proper development and homeostasis. However, the regulatory role of EGF xylosylation in Notch signaling and different cell activities in human cells remains unknown. In this study, we showed that knockdown of GXYLT2 suppressed human cell proliferation and induced G1/S phase cell cycle arrest. GXYLT2 downregulation also inhibited cell migration and invasion, whereas the overexpression of GXYLT2 had the opposite effects. Additionally, GXYLT2 activated Notch signaling and promoted the phosphorylation of MAPKs but not PI3K and Akt. Taken together, our findings indicated that GXYLT2 plays an important role in cell activities via regulation of the Notch signaling.
Asunto(s)
Neoplasias de la Mama/genética , Movimiento Celular/genética , Proliferación Celular/genética , Glicosiltransferasas/genética , Pentosiltransferasa/fisiología , Neoplasias de la Mama/patología , Factor de Crecimiento Epidérmico/genética , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Glucosa/genética , Humanos , Pentosiltransferasa/genética , Receptores Notch/genética , Xilosa/genéticaRESUMEN
BACKGROUND Salivary pleomorphic adenoma is one of the most common salivary gland tumors. It has a relatively high tendency to recur and a high risk of malignant transformation. The present study aimed to study the effect of XT-I gene silencing on the implanting growth of salivary pleomorphic adenoma. MATERIAL AND METHODS Primary cultures of SPA cells and fibroblasts from the same patient were assessed. The adenovirus vector Ad-shRNA-XT-I was constructed and transfected into SPA cells. The expression of XT-I gene and XT-I protein was detected by real-time PCR and Western blot. The contents of proteoglycans were detected. The SPA cells transfected with Ad-shRNA-XT-I (group SPA-XT-I) and Ad-shRNA-HK (group SPA-HK), as well as without transfection (group SPA), were implanted into ADM scaffold with fibroblasts and then transferred into 18 BALB/C-nu nude mice for 3 months. RESULTS Primary cultures showed SPA cells were positive for human CK and S-100 protein and the fibroblasts were positive for human vimentin. The expressions of XT-I gene and protein were decreased by 51% and 51.31%, respectively. The content of proteoglycans was reduced by 48.45%. The results of the implanting growth in vitro and in vivo of nude mice indicated that no tumors grew in the SPA-XT-I group, whereas SPA grew in groups SPA-HK and SPA positive for human a-SMA, S-100 protein, and calponin. CONCLUSIONS XT-I gene silencing effectively inhibited the implanting growth of SPA.
Asunto(s)
Adenoma Pleomórfico/genética , Pentosiltransferasa/genética , Pentosiltransferasa/fisiología , Adenoma Pleomórfico/metabolismo , Adulto , Animales , Fibroblastos/metabolismo , Silenciador del Gen/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica/genética , Recurrencia Local de Neoplasia/genética , Cultivo Primario de Células , Interferencia de ARN , ARN Interferente Pequeño/genética , Neoplasias de las Glándulas Salivales/genética , Neoplasias de las Glándulas Salivales/metabolismo , UDP Xilosa Proteína XilosiltransferasaRESUMEN
Uracil phosphoribosyltransferase (UPRT) is a pyrimidine salvage pathway enzyme that catalyzes the conversion of uracil to uridine monophosphate (UMP). The enzyme is highly conserved from prokaryotes to humans and yet phylogenetic evidence suggests that UPRT homologues from higher-eukaryotes, including Drosophila, are incapable of binding uracil. Purified human UPRT also do not show any enzymatic activity in vitro, making microbial UPRT an attractive candidate for anti-microbial drug development, suicide-gene therapy, and cell-specific mRNA labeling techniques. Nevertheless, the enzymatic site of UPRT remains conserved across the animal kingdom indicating an in vivo role for the enzyme. We find that the Drosophila UPRT homologue, krishah (kri), codes for an enzyme that is required for larval growth, pre-pupal/pupal viability and long-term adult lifespan. Our findings suggest that UPRT from all higher eukaryotes is likely enzymatically active in vivo and challenges the previous notion that the enzyme is non-essential in higher eukaryotes and cautions against targeting the enzyme for therapeutic purposes. Our findings also suggest that expression of the endogenous UPRT gene will likely cause background incorporation when using microbial UPRT as a cell-specific mRNA labeling reagent in higher eukaryotes.
Asunto(s)
Envejecimiento/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Longevidad/fisiología , Pentosiltransferasa/fisiología , Animales , Regulación del Desarrollo de la Expresión Génica/fisiología , Larva/crecimiento & desarrollo , Larva/metabolismoRESUMEN
All cells of an adult plant are ultimately derived from divisions that occur in small groups of cells distributed throughout the plant, termed meristems. A new study shows that carbohydrate post-translational modification of a peptide signal influences meristem and, as a consequence, fruit size in tomato.
Asunto(s)
Meristema/enzimología , Pentosiltransferasa/fisiología , Proteínas de Plantas/fisiología , Solanum lycopersicum/enzimologíaRESUMEN
Shoot meristems of plants are composed of stem cells that are continuously replenished through a classical feedback circuit involving the homeobox WUSCHEL (WUS) gene and the CLAVATA (CLV) gene signaling pathway. In CLV signaling, the CLV1 receptor complex is bound by CLV3, a secreted peptide modified with sugars. However, the pathway responsible for modifying CLV3 and its relevance for CLV signaling are unknown. Here we show that tomato inflorescence branching mutants with extra flower and fruit organs due to enlarged meristems are defective in arabinosyltransferase genes. The most extreme mutant is disrupted in a hydroxyproline O-arabinosyltransferase and can be rescued with arabinosylated CLV3. Weaker mutants are defective in arabinosyltransferases that extend arabinose chains, indicating that CLV3 must be fully arabinosylated to maintain meristem size. Finally, we show that a mutation in CLV3 increased fruit size during domestication. Our findings uncover a new layer of complexity in the control of plant stem cell proliferation.
Asunto(s)
Meristema/enzimología , Pentosiltransferasa/fisiología , Proteínas de Plantas/fisiología , Solanum lycopersicum/enzimología , Secuencia de Bases , Flores/enzimología , Flores/genética , Flores/crecimiento & desarrollo , Frutas/enzimología , Frutas/genética , Frutas/crecimiento & desarrollo , Glicosilación , Solanum lycopersicum/genética , Solanum lycopersicum/crecimiento & desarrollo , Meristema/genética , Meristema/crecimiento & desarrollo , Datos de Secuencia Molecular , Mutación , Procesamiento Proteico-PostraduccionalRESUMEN
In addition to its role as a redox coenzyme, NAD is a substrate of various enzymes that split the molecule to either catalyze covalent modifications of target proteins or convert NAD into biologically active metabolites. The coenzyme bioavailability may be significantly affected by these reactions, with ensuing major impact on energy metabolism, cell survival, and aging. Moreover, through the activity of the NAD-dependent deacetylating sirtuins, NAD behaves as a beacon molecule that reports the cell metabolic state, and accordingly modulates transcriptional responses and metabolic adaptations. In this view, NAD biosynthesis emerges as a highly regulated process: it enables cells to preserve NAD homeostasis in response to significant NAD-consuming events and it can be modulated by various stimuli to induce, via NAD level changes, suitable NAD-mediated metabolic responses. Here we review the current knowledge on the regulation of mammalian NAD biosynthesis, with focus on the relevant rate-limiting enzymes. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.
Asunto(s)
Señales (Psicología) , NAD/biosíntesis , Animales , Humanos , Nicotinamida Fosforribosiltransferasa/fisiología , Pentosiltransferasa/fisiología , Sirtuinas/fisiologíaRESUMEN
The redox coenzyme NAD(+) is also a rate-limiting co-substrate for several enzymes that consume the molecule, thus rendering its continuous re-synthesis indispensable. NAD(+) biosynthesis has emerged as a therapeutic target due to the relevance of NAD(+) -consuming reactions in complex intracellular signaling networks whose alteration leads to many neurologic and metabolic disorders. Distinct metabolic routes, starting from various precursors, are known to support NAD(+) biosynthesis with tissue/cell-specific efficiencies, probably reflecting differential expression of the corresponding rate-limiting enzymes, i.e. nicotinamide phosphoribosyltransferase, quinolinate phosphoribosyltransferase, nicotinate phosphoribosyltransferase and nicotinamide riboside kinase. Understanding the contribution of these enzymes to NAD(+) levels depending on the tissue/cell type and metabolic status is necessary for the rational design of therapeutic strategies aimed at modulating NAD(+) availability. Here we report a simple, fast and sensitive coupled fluorometric assay that enables simultaneous determination of the four activities in whole-cell extracts and biological fluids. Its application to extracts from various mouse tissues, human cell lines and plasma yielded for the first time an overall picture of the tissue/cell-specific distribution of the activities of the various enzymes. The screening enabled us to gather novel findings, including (a) the presence of quinolinate phosphoribosyltransferase and nicotinamide riboside kinase in all examined tissues/cell lines, indicating that quinolinate and nicotinamide riboside are relevant NAD(+) precursors, and (b) the unexpected occurrence of nicotinate phosphoribosyltransferase in human plasma.
Asunto(s)
NAD/biosíntesis , Animales , Vías Biosintéticas , Línea Celular , Sistema Libre de Células , Pruebas de Enzimas , Fluorometría , Humanos , Hígado/enzimología , Ratones , Ratones Endogámicos C57BL , NAD/química , Mononucleótido de Nicotinamida/análogos & derivados , Mononucleótido de Nicotinamida/química , Nicotinamida-Nucleótido Adenililtransferasa/química , Nicotinamida-Nucleótido Adenililtransferasa/fisiología , Especificidad de Órganos , Pentosiltransferasa/química , Pentosiltransferasa/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiologíaRESUMEN
The assumption that mutagens have linear dose-responses recently has been challenged. In particular, ethyl methanesulfonate (EMS), a DNA-reactive mutagen and carcinogen, exhibited sublinear or thresholded dose-responses for LacZ mutation in transgenic Muta™Mouse and for micronucleus (MN) frequency in CD1 mice (Gocke E and Müller L [2009]: Mutat Res 678:101-107). In order to explore variables in establishing genotoxicity dose-responses, we characterized the genotoxicity of EMS using gene mutation assays anticipated to have lower spontaneous mutant frequencies (MFs) than Muta™Mouse. Male gpt-delta transgenic mice were treated daily for 28 days with 5 to 100 mg/kg EMS, and measurements were made on: (i) gpt MFs in liver, lung, bone marrow, kidney, small intestine, and spleen; and (ii) Pig-a MFs in peripheral blood reticulocytes (RETs) and total red blood cells. MN induction also was measured in peripheral blood RETs. These data were used to calculate Points of Departure (PoDs) for the dose responses, i.e., no-observed-genotoxic-effect-levels (NOGELs), lower confidence limits of threshold effect levels (Td-LCIs), and lower confidence limits of 10% benchmark response rates (BMDL10 s). Similar PoDs were calculated from the published EMS dose-responses for LacZ mutation and CD1 MN induction. Vehicle control gpt and Pig-a MFs were 13-40-fold lower than published vehicle control LacZ MFs. In general, the EMS genotoxicity dose-responses in gpt-delta mice had lower PoDs than those calculated from the Muta™Mouse and CD1 mouse data. Our results indicate that the magnitude and possibly the shape of mutagenicity dose responses differ between in vivo models, with lower PoDs generally detected by gene mutation assays with lower backgrounds.
Asunto(s)
Daño del ADN/efectos de los fármacos , Proteínas de Escherichia coli/fisiología , Metanosulfonato de Etilo/toxicidad , Mutágenos/toxicidad , Tasa de Mutación , Mutación/genética , Pentosiltransferasa/fisiología , Animales , Daño del ADN/genética , Relación Dosis-Respuesta a Droga , Hipoxantina Fosforribosiltransferasa/genética , Operón Lac/genética , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Pruebas de Micronúcleos , Reticulocitos/efectos de los fármacos , Bazo/efectos de los fármacosRESUMEN
The long bones of the vertebrate body are built by the initial formation of a cartilage template that is later replaced by mineralized bone. The proliferation and maturation of the skeletal precursor cells (chondrocytes) within the cartilage template and their replacement by bone is a highly coordinated process which, if misregulated, can lead to a number of defects including dwarfism and other skeletal deformities. This is exemplified by the fact that abnormal bone development is one of the most common types of human birth defects. Yet, many of the factors that initiate and regulate chondrocyte maturation are not known. We identified a recessive dwarf mouse mutant (pug) from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. pug mutant skeletal elements are patterned normally during development, but display a ~20% length reduction compared to wild-type embryos. We show that the pug mutation does not lead to changes in chondrocyte proliferation but instead promotes premature maturation and early ossification, which ultimately leads to disproportionate dwarfism. Using sequence capture and high-throughput sequencing, we identified a missense mutation in the Xylosyltransferase 1 (Xylt1) gene in pug mutants. Xylosyltransferases catalyze the initial step in glycosaminoglycan (GAG) chain addition to proteoglycan core proteins, and these modifications are essential for normal proteoglycan function. We show that the pug mutation disrupts Xylt1 activity and subcellular localization, leading to a reduction in GAG chains in pug mutants. The pug mutant serves as a novel model for mammalian dwarfism and identifies a key role for proteoglycan modification in the initiation of chondrocyte maturation.
Asunto(s)
Huesos/embriología , Condrocitos/metabolismo , Osteogénesis/genética , Pentosiltransferasa/fisiología , Animales , Secuencia de Bases , Huesos/metabolismo , Diferenciación Celular/genética , Proliferación Celular , Enanismo/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Hedgehog/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Pentosiltransferasa/genética , Análisis de Secuencia de ADN , Transducción de Señal/genética , UDP Xilosa Proteína XilosiltransferasaRESUMEN
The recently developed Pig-a mutation assay is based on flow cytometric enumeration of glycosylphosphatidylinositol (GPI) anchor-deficient red blood cells caused by a forward mutation in the Pig-a gene. Because the assay can be conducted in nontransgenic animals and the mutations accumulate with repeat dosing, we believe that the Pig-a assay could be integrated into repeat-dose toxicology studies and provides an alternative to transgenic rodent (TGR) mutation assays. The capacity and characteristics of the Pig-a assay relative to TGR mutation assays, however, are unclear. Here, using transgenic gpt delta mice, we compared the in vivo genotoxicity of single oral doses of N-ethyl-N-nitrosourea (ENU, 40 mg/kg), benzo[a]pyrene (BP, 100 and 200 mg/kg), and 4-nitroquinoline-1-oxide (4NQO, 50 mg/kg) in the Pig-a (peripheral blood) and gpt (bone marrow and liver) gene mutation assays. Pig-a assays were conducted at 2, 4, and 7 weeks after the treatment, while gpt assays were conducted on tissues collected at the 7-week terminal sacrifice. ENU increased both Pig-a and gpt mutant frequencies (MFs) at all sampling times, and BP increased MFs in both assays but the Pig-a MFs peaked at 2 weeks and then decreased. Although 4NQO increased gpt MFs in the liver, only weak, nonsignificant increases (two- or threefold above control) were detected in the bone marrow in both the Pig-a and the gpt assay. These findings suggest that further studies are needed to elucidate the kinetics of the Pig-a mutation assay in order to use it as an alternative to the TGR mutation assay.
Asunto(s)
4-Nitroquinolina-1-Óxido/toxicidad , Benzo(a)pireno/toxicidad , Daño del ADN/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Proteínas de Escherichia coli/fisiología , Etilnitrosourea/toxicidad , Mutágenos/toxicidad , Pentosiltransferasa/fisiología , Animales , Bioensayo , Citometría de Flujo , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Pruebas de Mutagenicidad , Mutación/genéticaRESUMEN
Xyloglucan (XyG) is the dominant hemicellulose present in the primary cell walls of dicotyledonous plants. Unlike Arabidopsis (Arabidopsis thaliana) XyG, which contains galactosyl and fucosyl substituents, tomato (Solanum lycopersicum) XyG contains arabinofuranosyl residues. To investigate the biological function of these differing substituents, we used a functional complementation approach. Candidate glycosyltransferases were identified from tomato by using comparative genomics with known XyG galactosyltransferase genes from Arabidopsis. These candidate genes were expressed in an Arabidopsis mutant lacking XyG galactosylation, and two of them resulted in the production of arabinosylated XyG, a structure not previously found in this plant species. These genes may therefore encode XyG arabinofuranosyltransferases. Moreover, the addition of arabinofuranosyl residues to the XyG of this Arabidopsis mutant rescued a growth and cell wall biomechanics phenotype, demonstrating that the function of XyG in plant growth, development, and mechanics has considerable flexibility in terms of the specific residues in the side chains. These experiments also highlight the potential of reengineering the sugar substituents on plant wall polysaccharides without compromising growth or viability.
Asunto(s)
Glucanos/química , Pentosiltransferasa/genética , Proteínas de Plantas/genética , Solanum lycopersicum/enzimología , Xilanos/química , Arabidopsis/genética , Pared Celular/metabolismo , Pentosiltransferasa/metabolismo , Pentosiltransferasa/fisiología , Filogenia , Proteínas de Plantas/metabolismo , Proteínas de Plantas/fisiología , Plantas Modificadas Genéticamente/metabolismoRESUMEN
The relation of ethambutol resistance to embB mutations remains unclear, and there are no reports on ethambutol resistance from the caribbean. We examined the sequence of embB in 57 distinct Multi-Drug Resistant (MDR) and non-MDR strains of Mycobacterium tuberculosis, mostly from Cuba and the Dominican Republic. embB306 codon mutations were found exclusively in MDR-TB, but in both ethambutol sensitive and resistant strains. Valine substitutions predominated in ethambutol resistant strains, while isoleucine replacements were more common in sensitive strains. Three ethambutol resistant MDR strains without embB306 substitutions had replacements in embB406 or embB497, but these were also found in ethambutol sensitive MDR strains. The results confirm previous findings that amino acid substitutions in EmbB306, EmbB406 and EmbB497 are found only in MDR-TB strains but in both phenotypically resistant and sensitive strains. One ethambutol resistant non-MDR strain did not have any embB mutation suggesting that other undefined mutations can also confer ethambutol resistance.
Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Microbiana/genética , Etambutol/farmacología , Mycobacterium tuberculosis/genética , Pentosiltransferasa/genética , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Sustitución de Aminoácidos , Codón/genética , Cuba/epidemiología , Análisis Mutacional de ADN , ADN Bacteriano/genética , República Dominicana/epidemiología , Relación Dosis-Respuesta a Droga , Farmacorresistencia Bacteriana Múltiple/genética , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Pentosiltransferasa/fisiología , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Tuberculosis Resistente a Múltiples Medicamentos/epidemiologíaRESUMEN
The relation of ethambutol resistance to embB mutations remains unclear, and there are no reports on ethambutol resistance from the caribbean. We examined the sequence of embB in 57 distinct Multi-Drug Resistant (MDR) and non-MDR strains of Mycobacterium tuberculosis, mostly from Cuba and the Dominican Republic. embB306 codon mutations were found exclusively in MDR-TB, but in both ethambutol sensitive and resistant strains. Valine substitutions predominated in ethambutol resistant strains, while isoleucine replacements were more common in sensitive strains. Three ethambutol resistant MDR strains without embB306 substitutions had replacements in embB406 or embB497, but these were also found in ethambutol sensitive MDR strains. The results confirm previous findings that amino acid substitutions in EmbB306, EmbB406 and EmbB497 are found only in MDR-TB strains but in both phenotypically resistant and sensitive strains. One ethambutol resistant non-MDR strain did not have any embB mutation suggesting that other undefined mutations can also confer ethambutol resistance.
Asunto(s)
Antituberculosos/farmacología , Etambutol/farmacología , Mycobacterium tuberculosis/genética , Pentosiltransferasa/genética , Farmacorresistencia Microbiana/genética , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Análisis Mutacional de ADN , Cuba/epidemiología , Codón/genética , ADN Bacteriano/genética , Farmacorresistencia Bacteriana Múltiple/genética , Humanos , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Pentosiltransferasa/fisiología , Relación Dosis-Respuesta a Droga , Reproducibilidad de los Resultados , República Dominicana/epidemiología , Sensibilidad y Especificidad , Sustitución de Aminoácidos , Pruebas de Sensibilidad Microbiana , Tuberculosis Resistente a Múltiples Medicamentos/epidemiologíaRESUMEN
The relation of ethambutol resistance to embB mutations remains unclear, and there are no reports on ethambutol resistance from the caribbean. We examined the sequence of embB in 57 distinct Multi-Drug Resistant (MDR) and non-MDR strains of Mycobacterium tuberculosis, mostly from Cuba and the Dominican Republic. embB306 codon mutations were found exclusively in MDR-TB, but in both ethambutol sensitive and resistant strains. Valine substitutions predominated in ethambutol resistant strains, while isoleucine replacements were more common in sensitive strains. Three ethambutol resistant MDR strains without embB306 substitutions had replacements in embB406 or embB497, but these were also found in ethambutol sensitive MDR strains. The results confirm previous findings that amino acid substitutions in EmbB306, EmbB406 and EmbB497 are found only in MDR-TB strains but in both phenotypically resistant and sensitive strains. One ethambutol resistant non-MDR strain did not have any embB mutation suggesting that other undefined mutations can also confer ethambutol resistance.
Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Microbiana/genética , Etambutol/farmacología , Mycobacterium tuberculosis/genética , Pentosiltransferasa/genética , Tuberculosis Resistente a Múltiples Medicamentos/microbiología , Sustitución de Aminoácidos , Codón/genética , Cuba/epidemiología , Análisis Mutacional de ADN , ADN Bacteriano/genética , República Dominicana/epidemiología , Relación Dosis-Respuesta a Droga , Farmacorresistencia Bacteriana Múltiple/genética , Humanos , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Pentosiltransferasa/fisiología , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Tuberculosis Resistente a Múltiples Medicamentos/epidemiologíaRESUMEN
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne disease in animals and MAP involvement in human Crohn disease has been recently emphasized. Evidence from M. tuberculosis studies suggests mycobacterial proteins activate dendritic cells (DCs) via Toll-like receptor (TLR) 4, eventually determining the fate of immune responses. Here, we investigated whether MAP CobT contributes to the development of T cell immunity through the activation of DCs. MAP CobT recognizes TLR4, and induces DC maturation and activation via the MyD88 and TRIF signaling cascades, which are followed by MAP kinases and NF-κB. We further found that MAP CobT-treated DCs activated naive T cells, effectively polarized CD4(+) and CD8(+) T cells to secrete IFN-γ and IL-2, but not IL-4 and IL-10, and induced T cell proliferation. These data indicate that MAP CobT contributes to T helper (Th) 1 polarization of the immune response. MAP CobT-treated DCs specifically induced the expansion of CD4(+)/CD8(+)CD44(high)CD62L(low) memory T cells in the mesenteric lymph node of MAP-infected mice in a TLR4-dependent manner. Our results indicate that MAP CobT is a novel DC maturation-inducing antigen that drives Th1 polarized-naive/memory T cell expansion in a TLR4-dependent cascade, suggesting that MAP CobT potentially links innate and adaptive immunity against MAP.
Asunto(s)
Complejos Multienzimáticos/genética , Mycobacterium avium subsp. paratuberculosis/metabolismo , Nucleotidiltransferasas/genética , Pentosiltransferasa/genética , Animales , Proteínas Bacterianas/metabolismo , Células Dendríticas/citología , Femenino , Sistema Inmunológico , Memoria Inmunológica , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Complejos Multienzimáticos/fisiología , Nucleotidiltransferasas/fisiología , Pentosiltransferasa/fisiología , Proteínas Recombinantes/química , Linfocitos T/inmunología , Células TH1/citología , Receptor Toll-Like 4/metabolismoRESUMEN
Loss of glycosaminoglycan (GAG) chains of proteoglycans (PGs) is an early event of osteoarthritis (OA) resulting in cartilage degradation that has been previously demonstrated in both huma and experimental OA models. However, the mechanism of GAG loss and the role of xylosyltransferase-I (XT-I) that initiates GAG biosynthesis onto PG molecules in the pathogenic process of human OA are unknown. In this study, we have characterized XT-I expression and activity together with GAG synthesis in human OA cartilage obtained from different regions of the same joint, defined as "normal", "late-stage" or adjacent to "late-stage". The results showed that GAG synthesis and content increased in cartilage from areas flanking OA lesions compared to cartilage from macroscopically "normal" unaffected regions, while decreased in "late-stage" OA cartilage lesions. This increase in anabolic state was associated with a marked upregulation of XT-I expression and activity in cartilage "next to lesion" while a decrease in the "late-stage" OA cartilage. Importantly, XT-I inhibition by shRNA or forced-expression with a pCMV-XT-I construct correlated with the modulation of GAG anabolism in human cartilage explants. The observation that XT-I gene expression was down-regulated by IL-1ß and up-regulated by TGF-ß1 indicates that these cytokines may play a role in regulating GAG content in human OA. Noteworthy, expression of IL-1ß receptor (IL-1R1) was down-regulated whereas that of TGF-ß1 was up-regulated in early OA cartilage. Theses observations may account for upregulation of XT-I and sustained GAG synthesis prior to the development of cartilage lesions during the pathogenic process of OA.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Glicosaminoglicanos/metabolismo , Osteoartritis/metabolismo , Pentosiltransferasa/fisiología , Receptores Tipo I de Interleucina-1/metabolismo , Anciano , Cartílago Articular/metabolismo , Progresión de la Enfermedad , Fémur/patología , Perfilación de la Expresión Génica , Humanos , Interleucina-1beta/metabolismo , Persona de Mediana Edad , Modelos Biológicos , Factor de Crecimiento Transformador beta1/metabolismo , UDP Xilosa Proteína XilosiltransferasaAsunto(s)
Epistasis Genética , Redes Reguladoras de Genes/fisiología , Modelos Biológicos , Biología de Sistemas , Ciclo del Ácido Cítrico/genética , Fumarato Hidratasa/genética , Fumarato Hidratasa/metabolismo , Fumarato Hidratasa/fisiología , NAD/biosíntesis , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo , Pentosiltransferasa/fisiologíaRESUMEN
Psoriasis patients are frequently associated with metabolic syndromes. Such associations are possibly mediated by adipokines. We investigated the in vitro effects of visfatin (an adipokine) on chemokine expression in human keratinocytes. Normal human keratinocytes were incubated with visfatin, and their chemokine production was analyzed by ELISA and RT-PCR. Visfatin enhanced TNF-α-induced CXC chemokine ligand (CXCL) 8, CXCL10, and CC chemokine ligand (CCL) 20 secretion and mRNA expression in keratinocytes, although visfatin alone was ineffective. A small interfering RNA against nuclear factor-κB (NF-κB) p65 suppressed the visfatin-induced production of CXCL8, CXCL10, and CCL20 whereas a small interfering RNA against signal transducer and activator of transcription (STAT) 3 suppressed CXCL8 induction. This indicates the involvement of NF-κB in CXCL8, CXCL10, and CCL20 induction by visfatin and the involvement of STAT3 in CXCL8 induction. Visfatin alone increased the transcriptional activity and tyrosine phosphorylation of STAT3, which was suppressed by Janus kinase (JAK) 2 inhibitor. Visfatin enhanced basal and TNF-α-induced NF-κB activity and inhibitory κB (IκB) α phosphorylation, which was suppressed by IκB kinase inhibitor. Visfatin induced the tyrosine and serine phosphorylation of JAK2 and IκB kinase α/ß, respectively. Intraperitoneal injection of visfatin elevated mRNA and protein levels of CXCL1, CXCL10, and CCL20 in murine skin. These results suggest that visfatin enhances CXCL8, CXCL10, and CCL20 production in human keratinocytes and homologous chemokine production in murine skin. Visfatin may induce the infiltration of type 1 or type 17 helper T cells or neutrophils to the skin via chemokine induction and thus link metabolic syndromes to psoriasis.