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1.
BMC Med Genomics ; 17(1): 213, 2024 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-39148116

RESUMEN

BACKGROUND: Myopathy, lactic acidosis and inherited sideroblastic anemia (MLASA) are a group of rare intriguing disorders with wider pathophysiological implications. One of the causes of MLASA is the mutation in PUS1 gene that encodes for pseudouridine synthase. This PUS1 mutation results in MLASA in which anemia and myopathy predominate. Severe pulmonary arterial hypertension has not been previously reported in patients with PUS1 gene mutation. CASE REPORT: A 17 year old girl with congenital sideroblastic anemia presented with worsening of breathlessness. Severe pulmonary artery hypertension was documented on investigations. A homozygous variant in exon 3 of gene PUS1,( chromosome 12:g.131932301 C > T c.430 C > T) was found on sanger sequencing. CONCLUSION: We document severe pulmonary arterial hypertension in a patient of congenital sideroblastic anemia from PUS1 gene. We hypothesis that cross talk with TGFb pathways might occur in PUS1 mutation, and that might cause severe PAH. This observation might have therapeutic implications.


Asunto(s)
Anemia Sideroblástica , Hidroliasas , Mutación , Humanos , Anemia Sideroblástica/genética , Anemia Sideroblástica/complicaciones , Femenino , Adolescente , Hidroliasas/genética , Hidroliasas/deficiencia , Hipertensión Arterial Pulmonar/genética
2.
Nature ; 629(8010): 184-192, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38600378

RESUMEN

Glucocorticoids represent the mainstay of therapy for a broad spectrum of immune-mediated inflammatory diseases. However, the molecular mechanisms underlying their anti-inflammatory mode of action have remained incompletely understood1. Here we show that the anti-inflammatory properties of glucocorticoids involve reprogramming of the mitochondrial metabolism of macrophages, resulting in increased and sustained production of the anti-inflammatory metabolite itaconate and consequent inhibition of the inflammatory response. The glucocorticoid receptor interacts with parts of the pyruvate dehydrogenase complex whereby glucocorticoids provoke an increase in activity and enable an accelerated and paradoxical flux of the tricarboxylic acid (TCA) cycle in otherwise pro-inflammatory macrophages. This glucocorticoid-mediated rewiring of mitochondrial metabolism potentiates TCA-cycle-dependent production of itaconate throughout the inflammatory response, thereby interfering with the production of pro-inflammatory cytokines. By contrast, artificial blocking of the TCA cycle or genetic deficiency in aconitate decarboxylase 1, the rate-limiting enzyme of itaconate synthesis, interferes with the anti-inflammatory effects of glucocorticoids and, accordingly, abrogates their beneficial effects during a diverse range of preclinical models of immune-mediated inflammatory diseases. Our findings provide important insights into the anti-inflammatory properties of glucocorticoids and have substantial implications for the design of new classes of anti-inflammatory drugs.


Asunto(s)
Antiinflamatorios , Glucocorticoides , Inflamación , Macrófagos , Mitocondrias , Succinatos , Animales , Femenino , Humanos , Masculino , Ratones , Antiinflamatorios/farmacología , Carboxiliasas/metabolismo , Carboxiliasas/antagonistas & inhibidores , Ciclo del Ácido Cítrico/efectos de los fármacos , Ciclo del Ácido Cítrico/genética , Citocinas/inmunología , Citocinas/metabolismo , Glucocorticoides/farmacología , Glucocorticoides/metabolismo , Hidroliasas/deficiencia , Hidroliasas/genética , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Complejo Piruvato Deshidrogenasa/metabolismo , Receptores de Glucocorticoides/metabolismo , Succinatos/metabolismo , Activación Enzimática/efectos de los fármacos
3.
Am J Med Genet A ; 194(5): e63519, 2024 05.
Artículo en Inglés | MEDLINE | ID: mdl-38214124

RESUMEN

Metabolic pathways are known to generate byproducts-some of which have no clear metabolic function and some of which are toxic. Nicotinamide adenine dinucleotide phosphate hydrate (NAD(P)HX) is a toxic metabolite that is produced by stressors such as a fever, infection, or physical stress. Nicotinamide adenine dinucleotide phosphate hydrate dehydratase (NAXD) and nicotinamide adenine dinucleotide phosphate hydrate epimerase (NAXE) are part of the nicotinamide repair system that function to break down this toxic metabolite. Deficiency of NAXD and NAXE interrupts the critical intracellular repair of NAD(P)HX and allows for its accumulation. Clinically, deficiency of NAXE manifests as progressive, early onset encephalopathy with brain edema and/or leukoencephalopathy (PEBEL) 1, while deficiency of NAXD manifests as PEBEL2. In this report, we describe a case of probable PEBEL2 in a patient with a variant of unknown significance (c.362C>T, p.121L) in the NAXD gene who presented after routine immunizations with significant skin findings and in the absence of fevers.


Asunto(s)
Encefalopatías , Inmunización , Humanos , Inmunización/efectos adversos , Leucoencefalopatías/etiología , Racemasas y Epimerasas/deficiencia , Racemasas y Epimerasas/genética , Hidroliasas/deficiencia , Hidroliasas/genética , Encefalopatías/etiología
4.
Cancer Sci ; 112(11): 4799-4811, 2021 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-34449934

RESUMEN

Epstein-Barr virus (EBV)-associated gastric cancer belongs to 1 of the 4 subtypes of gastric cancer and accounts for 10% of total gastric cancers. However, most cases of gastric cancer have a history of Helicobacter pylori infection. Therefore, we investigated the possibility that H. pylori infection promotes the development of EBV-associated gastric cancer. H. pylori was exposed to principal EBV receptor, CD21, negative gastric epithelial cells, and then infected with EBV recombinant expressing enhanced green fluorescent protein. Changes in EBV infectivity due to prior H. pylori exposure were analyzed using flow cytometry. The treatment of gastric epithelial cells with H. pylori increased the efficiency of EBV infection. An increase was also observed when CagA-deficient, VacA-deficient, and FlaA-deficient H. pylori strains were used, but not when cag pathogenicity island-deficient H. pylori was used. The treatment of epithelial cells with H. pylori induced the expression of accessory EBV receptors, EphA2 and NMHC-IIA, and increased the efficiency of EBV infection depending on their expression levels. When gastric epithelial cells were treated with EPHA2 or NMHC-IIA siRNA, EBV infection via H. pylori attachment was decreased. The adhesion of H. pylori induced the expression of accessory EBV receptors in gastric epithelial cells and increased the efficiency of EBV infection.


Asunto(s)
Infecciones por Virus de Epstein-Barr/etiología , Infecciones por Helicobacter/complicaciones , Helicobacter pylori/fisiología , Herpesvirus Humano 4 , Neoplasias Gástricas/virología , Antígenos Bacterianos/metabolismo , Sitios de Ligazón Microbiológica/fisiología , Adhesión Bacteriana/fisiología , Proteínas Bacterianas/metabolismo , Línea Celular Tumoral , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Proteínas Fluorescentes Verdes/metabolismo , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/genética , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 4/patogenicidad , Humanos , Hidroliasas/deficiencia , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Oxidorreductasas/deficiencia , ARN Interferente Pequeño/farmacología , Receptor EphA2/genética , Receptor EphA2/metabolismo , Receptores de Complemento 3d/metabolismo , Neoplasias Gástricas/microbiología
5.
Cell Rep ; 34(10): 108756, 2021 03 09.
Artículo en Inglés | MEDLINE | ID: mdl-33691097

RESUMEN

Itaconate is a unique regulatory metabolite that is induced upon Toll-like receptor (TLR) stimulation in myeloid cells. Here, we demonstrate major inflammatory tolerance and cell death phenotypes associated with itaconate production in activated macrophages. We show that endogenous itaconate is a key regulator of the signal 2 of NLR family pyrin domain containing 3 (NLRP3) inflammasome activation after long lipopolysaccharide (LPS) priming, which establishes tolerance to late NLRP3 inflammasome activation. We show that itaconate acts synergistically with inducible nitric oxide synthase (iNOS) and that the ability of various TLR ligands to establish NLRP3 inflammasome tolerance depends on the pattern of co-expression of IRG1 and iNOS. Mechanistically, itaconate accumulation upon prolonged inflammatory stimulation prevents full caspase-1 activation and processing of gasdermin D, which we demonstrate to be post-translationally modified by endogenous itaconate. Altogether, our data demonstrate that metabolic rewiring in inflammatory macrophages establishes tolerance to NLRP3 inflammasome activation that, if uncontrolled, can result in pyroptotic cell death and tissue damage.


Asunto(s)
Inflamasomas/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Succinatos/farmacología , Adenosina Trifosfato/farmacología , Animales , Caspasa 1/metabolismo , Hidroliasas/deficiencia , Hidroliasas/genética , Hidroliasas/metabolismo , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Proteínas de Unión a Fosfato/genética , Proteínas de Unión a Fosfato/metabolismo , Poli I-C/farmacología , Piroptosis/efectos de los fármacos , Sepsis/inducido químicamente , Sepsis/metabolismo , Sepsis/patología , Transducción de Señal/efectos de los fármacos , Receptores Toll-Like/química , Receptores Toll-Like/metabolismo
6.
Brain ; 142(1): 50-58, 2019 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-30576410

RESUMEN

Physical stress, including high temperatures, may damage the central metabolic nicotinamide nucleotide cofactors [NAD(P)H], generating toxic derivatives [NAD(P)HX]. The highly conserved enzyme NAD(P)HX dehydratase (NAXD) is essential for intracellular repair of NAD(P)HX. Here we present a series of infants and children who suffered episodes of febrile illness-induced neurodegeneration or cardiac failure and early death. Whole-exome or whole-genome sequencing identified recessive NAXD variants in each case. Variants were predicted to be potentially deleterious through in silico analysis. Reverse-transcription PCR confirmed altered splicing in one case. Subject fibroblasts showed highly elevated concentrations of the damaged cofactors S-NADHX, R-NADHX and cyclic NADHX. NADHX accumulation was abrogated by lentiviral transduction of subject cells with wild-type NAXD. Subject fibroblasts and muscle biopsies showed impaired mitochondrial function, higher sensitivity to metabolic stress in media containing galactose and azide, but not glucose, and decreased mitochondrial reactive oxygen species production. Recombinant NAXD protein harbouring two missense variants leading to the amino acid changes p.(Gly63Ser) and p.(Arg608Cys) were thermolabile and showed a decrease in Vmax and increase in KM for the ATP-dependent NADHX dehydratase activity. This is the first study to identify pathogenic variants in NAXD and to link deficient NADHX repair with mitochondrial dysfunction. The results show that NAXD deficiency can be classified as a metabolite repair disorder in which accumulation of damaged metabolites likely triggers devastating effects in tissues such as the brain and the heart, eventually leading to early childhood death.


Asunto(s)
Hidroliasas/deficiencia , Enfermedades Neurodegenerativas/genética , Preescolar , Simulación por Computador , Femenino , Fiebre/complicaciones , Fiebre/metabolismo , Fibroblastos/metabolismo , Vectores Genéticos , Humanos , Hidroliasas/genética , Lactante , Cinética , Lentivirus , Masculino , Mitocondrias/metabolismo , Mutación , NAD/análogos & derivados , NAD/metabolismo , Enfermedades Neurodegenerativas/complicaciones , Enfermedades Neurodegenerativas/metabolismo , Cultivo Primario de Células , Secuenciación Completa del Genoma
7.
J Clin Invest ; 128(9): 3794-3805, 2018 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-29920191

RESUMEN

Control of cellular metabolism is critical for efficient cell function, although little is known about the interplay between cell subset-specific metabolites in situ, especially in the tumor setting. Here, we determined how a macrophage-specific (Mϕ-specific) metabolite, itaconic acid, can regulate tumor progression in the peritoneum. We show that peritoneal tumors (B16 melanoma or ID8 ovarian carcinoma) elicited a fatty acid oxidation-mediated increase in oxidative phosphorylation (OXPHOS) and glycolysis in peritoneal tissue-resident macrophages (pResMϕ). Unbiased metabolomics identified itaconic acid, the product of immune-responsive gene 1-mediated (Irg1-mediated) catabolism of mitochondrial cis-aconitate, among the most highly upregulated metabolites in pResMϕ of tumor-bearing mice. Administration of lentivirally encoded Irg1 shRNA significantly reduced peritoneal tumors. This resulted in reductions in OXPHOS and OXPHOS-driven production of ROS in pResMϕ and ROS-mediated MAPK activation in tumor cells. Our findings demonstrate that tumors profoundly alter pResMϕ metabolism, leading to the production of itaconic acid, which potentiates tumor growth. Monocytes isolated from ovarian carcinoma patients' ascites fluid expressed significantly elevated levels of IRG1. Therefore, IRG1 in pResMϕ represents a potential therapeutic target for peritoneal tumors.


Asunto(s)
Macrófagos Peritoneales/metabolismo , Neoplasias Peritoneales/metabolismo , Succinatos/metabolismo , Animales , Carboxiliasas , Línea Celular Tumoral , Progresión de la Enfermedad , Ácidos Grasos/metabolismo , Femenino , Glucólisis , Humanos , Hidroliasas/deficiencia , Hidroliasas/genética , Hidroliasas/metabolismo , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/metabolismo , Neoplasias Ováricas/metabolismo , Fosforilación Oxidativa , Neoplasias Peritoneales/genética , Neoplasias Peritoneales/patología , Proteínas/metabolismo , ARN Interferente Pequeño/genética , Especies Reactivas de Oxígeno/metabolismo , Carga Tumoral
8.
Sci Rep ; 6: 26202, 2016 05 20.
Artículo en Inglés | MEDLINE | ID: mdl-27197761

RESUMEN

Mitochondrial myopathy with lactic acidosis and sideroblastic anemia (MLASA) is an oxidative phosphorylation disorder, with primary clinical manifestations of myopathic exercise intolerance and a macrocytic sideroblastic anemia. One cause of MLASA is recessive mutations in PUS1, which encodes pseudouridine (Ψ) synthase 1 (Pus1p). Here we describe a mouse model of MLASA due to mutations in PUS1. As expected, certain Ψ modifications were missing in cytoplasmic and mitochondrial tRNAs from Pus1(-/-) animals. Pus1(-/-) mice were born at the expected Mendelian frequency and were non-dysmorphic. At 14 weeks the mutants displayed reduced exercise capacity. Examination of tibialis anterior (TA) muscle morphology and histochemistry demonstrated an increase in the cross sectional area and proportion of myosin heavy chain (MHC) IIB and low succinate dehydrogenase (SDH) expressing myofibers, without a change in the size of MHC IIA positive or high SDH myofibers. Cytochrome c oxidase activity was significantly reduced in extracts from red gastrocnemius muscle from Pus1(-/-) mice. Transmission electron microscopy on red gastrocnemius muscle demonstrated that Pus1(-/-) mice also had lower intermyofibrillar mitochondrial density and smaller mitochondria. Collectively, these results suggest that alterations in muscle metabolism related to mitochondrial content and oxidative capacity may account for the reduced exercise capacity in Pus1(-/-) mice.


Asunto(s)
Hidroliasas/deficiencia , Síndrome MELAS/patología , Músculos/patología , Músculos/fisiología , Animales , Modelos Animales de Enfermedad , Histocitoquímica , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión
9.
J Biosci Bioeng ; 118(4): 448-54, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24794851

RESUMEN

Production of pharmaceutical glycoproteins, such as therapeutic antibodies and cytokines, in plants has many advantages in safety and reduced costs. However, plant-made glycoproteins have N-glycans with plant-specific sugar residues (core ß-1,2-xylose and α-1,3-fucose) and a Lewis a (Le(a)) epitope, Galß(1-3)[Fucα(1-4)]GlcNAc. Because it is likely that these sugar residues and glycan structures are immunogenic, many attempts have been made to delete them. Previously, we reported the simultaneous deletion of the plant-specific core α-1,3-fucose and α-1,4-fucose residues in Le(a) epitopes by repressing the GDP-D-mannose 4,6-dehydratase (GMD) gene, which is associated with GDP-L-fucose biosynthesis, in Nicotiana benthamiana plants (rGMD plants, renamed to ΔGMD plants) (Matsuo and Matsumura, Plant Biotechnol. J., 9, 264-281, 2011). In the present study, we generated a core ß-1,2-xylose residue-repressed transgenic N. benthamiana plant by co-suppression of ß-1,2-xylosyltransferase (ΔXylT plant). By crossing ΔGMD and ΔXylT plants, we successfully generated plants in which plant-specific sugar residues were repressed (ΔGMDΔXylT plants). The proportion of N-glycans with deleted plant-specific sugar residues found in total soluble protein from ΔGMDΔXylT plants increased by 82.41%. Recombinant mouse granulocyte/macrophage-colony stimulating factor (mGM-CSF) and human monoclonal immunoglobulin G (hIgG) harboring N-glycans with deleted plant-specific sugar residues were successfully produced in ΔGMDΔXylT plants. Simultaneous repression of the GMD and XylT genes in N. benthamiana is thus very useful for deleting plant-specific sugar residues.


Asunto(s)
Regulación de la Expresión Génica de las Plantas , Hidroliasas/deficiencia , Nicotiana/genética , Pentosiltransferasa/deficiencia , Proteínas de Plantas/genética , Animales , Secuencia de Carbohidratos , Fucosa/metabolismo , Glicosilación , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Humanos , Hidroliasas/genética , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/química , Inmunoglobulina G/genética , Manosa/metabolismo , Ratones , Datos de Secuencia Molecular , Pentosiltransferasa/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Polisacáridos/química , Polisacáridos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Nicotiana/metabolismo , Xilosa/metabolismo
10.
Clin Vaccine Immunol ; 20(4): 572-81, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23408524

RESUMEN

Johne's disease (JD) is prevalent worldwide and has a significant impact on the global agricultural economy. In the present study, we evaluated the protective efficacy of a leuD (Δleud) mutant and gained insight into differential immune responses after challenge with virulent M. avium subsp. paratuberculosis in a caprine colonization model. The immune response and protective efficacy were compared with those of the killed vaccine Mycopar. In vitro stimulation of peripheral blood mononuclear cells with johnin purified protein derivative showed that Mycopar and ΔleuD generated similar levels of gamma interferon (IFN-γ) but significantly higher levels than unvaccinated and challenged phosphate-buffered saline controls. However, only with ΔleuD was the IFN-γ response maintained. Flow cytometric analysis showed that the increase in IFN-γ correlated with proliferation and activation (increased expression of CD25) of CD4, CD8, and γδT cells, but this response was significantly higher in ΔleuD-vaccinated animals at some time points after challenge. Both Mycopar and ΔleuD vaccines upregulated Th1/proinflammatory and Th17 cytokines and downregulated Th2/anti-inflammatory and regulatory cytokines at similar levels at almost all time points. However, significantly higher levels of IFN-γ (at weeks 26 and 30), interleukin-2 (IL-2; week 18), IL-1b (weeks 14 and 22), IL-17 (weeks 18 and 22), and IL-23 (week 18) and a significantly lower level of IL-10 (weeks 14 and 18) and transforming growth factor ß (week 18) were detected in the ΔleuD-vaccinated group. Most importantly, ΔleuD elicited an immune response that significantly limited colonization of tissues compared to Mycopar upon challenge with wild-type M. avium subsp. paratuberculosis. In conclusion, the ΔleuD mutant is a promising vaccine candidate for development of a live attenuated vaccine for JD in ruminants.


Asunto(s)
Vacunas Bacterianas/inmunología , Hidroliasas/deficiencia , Mycobacterium avium subsp. paratuberculosis/genética , Mycobacterium avium subsp. paratuberculosis/inmunología , Paratuberculosis/prevención & control , Animales , Proteínas Bacterianas , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Cabras , Leucocitos Mononucleares/inmunología , Masculino , Paratuberculosis/inmunología , Subgrupos de Linfocitos T/inmunología , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología
11.
PLoS One ; 8(1): e53688, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23308274

RESUMEN

Molecular basis of D-bifunctional protein (D-BP) deficiency was studied with wild type and five disease-causing variants of 3R-hydroxyacyl-CoA dehydrogenase fragment of the human MFE-2 (multifunctional enzyme type 2) protein. Complementation analysis in vivo in yeast and in vitro enzyme kinetic and stability determinants as well as in silico stability and structural fluctuation calculations were correlated with clinical data of known patients. Despite variations not affecting the catalytic residues, enzyme kinetic performance (K(m), V(max) and k(cat)) of the recombinant protein variants were compromised to a varying extent and this can be judged as the direct molecular cause for D-BP deficiency. Protein stability plays an additional role in producing non-functionality of MFE-2 in case structural variations affect cofactor or substrate binding sites. Structure-function considerations of the variant proteins matched well with the available data of the patients.


Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/química , 17-Hidroxiesteroide Deshidrogenasas/deficiencia , Disgenesia Gonadal 46 XX/genética , Pérdida Auditiva Sensorineural/genética , Hidroliasas/química , Hidroliasas/deficiencia , Mutación , Peroxisomas/genética , 17-Hidroxiesteroide Deshidrogenasas/genética , Dominio Catalítico , Niño , Preescolar , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli/genética , Ácidos Grasos/metabolismo , Femenino , Prueba de Complementación Genética , Disgenesia Gonadal 46 XX/enzimología , Pérdida Auditiva Sensorineural/enzimología , Humanos , Hidroliasas/genética , Cinética , Metabolismo de los Lípidos , Masculino , Modelos Moleculares , Oxidación-Reducción , Proteína-2 Multifuncional Peroxisomal , Peroxisomas/enzimología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Relación Estructura-Actividad , Especificidad por Sustrato
12.
Ann Hematol ; 92(1): 1-9, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22983749

RESUMEN

Sideroblastic anemia is characterized by anemia with the emergence of ring sideroblasts in the bone marrow. There are two forms of sideroblastic anemia, i.e., congenital sideroblastic anemia (CSA) and acquired sideroblastic anemia. In order to clarify the pathophysiology of sideroblastic anemia, a nationwide survey consisting of clinical and molecular genetic analysis was performed in Japan. As of January 31, 2012, data of 137 cases of sideroblastic anemia, including 72 cases of myelodysplastic syndrome (MDS)-refractory cytopenia with multilineage dysplasia (RCMD), 47 cases of MDS-refractory anemia with ring sideroblasts (RARS), and 18 cases of CSA, have been collected. Hemoglobin and MCV level in CSA are significantly lower than those of MDS, whereas serum iron level in CSA is significantly higher than those of MDS. Of 14 CSA for which DNA was available for genetic analysis, 10 cases were diagnosed as X-linked sideroblastic anemia due to ALAS2 gene mutation. The mutation of SF3B1 gene, which was frequently mutated in MDS-RS, was not detected in CSA patients. Together with the difference of clinical data, it is suggested that genetic background, which is responsible for the development of CSA, is different from that of MDS-RS.


Asunto(s)
Anemia Sideroblástica/congénito , 5-Aminolevulinato Sintetasa/deficiencia , 5-Aminolevulinato Sintetasa/genética , 5-Aminolevulinato Sintetasa/metabolismo , Transportadoras de Casetes de Unión a ATP/deficiencia , Transportadoras de Casetes de Unión a ATP/genética , Adolescente , Adulto , Edad de Inicio , Anciano , Anemia Sideroblástica/sangre , Anemia Sideroblástica/clasificación , Anemia Sideroblástica/epidemiología , Anemia Sideroblástica/genética , Niño , Preescolar , Aberraciones Cromosómicas , Femenino , Frecuencia de los Genes , Genes Ligados a X , Enfermedades Genéticas Ligadas al Cromosoma X/sangre , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Glutarredoxinas/deficiencia , Glutarredoxinas/genética , Encuestas Epidemiológicas , Humanos , Hidroliasas/deficiencia , Hidroliasas/genética , Lactante , Recién Nacido , Japón/epidemiología , Masculino , Proteínas de Transporte de Membrana/deficiencia , Proteínas de Transporte de Membrana/genética , Persona de Mediana Edad , Proteínas de Transporte de Membrana Mitocondrial/deficiencia , Proteínas de Transporte de Membrana Mitocondrial/genética , Síndromes Mielodisplásicos/sangre , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/epidemiología , Síndromes Mielodisplásicos/genética , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Factores de Empalme de ARN , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/deficiencia , Ribonucleoproteína Nuclear Pequeña U2/genética , Resultado del Tratamiento , Vitamina B 6/uso terapéutico , Adulto Joven
13.
Orphanet J Rare Dis ; 7: 90, 2012 Nov 22.
Artículo en Inglés | MEDLINE | ID: mdl-23181892

RESUMEN

BACKGROUND: D-bifunctional protein (DBP) deficiency is typically apparent within the first month of life with most infants demonstrating hypotonia, psychomotor delay and seizures. Few children survive beyond two years of age. Among patients with prolonged survival all demonstrate severe gross motor delay, absent language development, and severe hearing and visual impairment. DBP contains three catalytically active domains; an N-terminal dehydrogenase, a central hydratase and a C-terminal sterol carrier protein-2-like domain. Three subtypes of the disease are identified based upon the domain affected; DBP type I results from a combined deficiency of dehydrogenase and hydratase activity; DBP type II from isolated hydratase deficiency and DBP type III from isolated dehydrogenase deficiency. Here we report two brothers (16½ and 14 years old) with DBP deficiency characterized by normal early childhood followed by sensorineural hearing loss, progressive cerebellar and sensory ataxia and subclinical retinitis pigmentosa. METHODS AND RESULTS: Biochemical analysis revealed normal levels of plasma VLCFA, phytanic acid and pristanic acid, and normal bile acids in urine; based on these results no diagnosis was made. Exome analysis was performed using the Agilent SureSelect 50Mb All Exon Kit and the Illumina HiSeq 2000 next-generation-sequencing (NGS) platform. Compound heterozygous mutations were identified by exome sequencing and confirmed by Sanger sequencing within the dehydrogenase domain (c.101C>T; p.Ala34Val) and hydratase domain (c.1547T>C; p.Ile516Thr) of the 17ß-hydroxysteroid dehydrogenase type 4 gene (HSD17B4). These mutations have been previously reported in patients with severe-forms of DBP deficiency, however each mutation was reported in combination with another mutation affecting the same domain. Subsequent studies in fibroblasts revealed normal VLCFA levels, normal C26:0 but reduced pristanic acid beta-oxidation activity. Both DBP hydratase and dehydrogenase activity were markedly decreased but detectable. CONCLUSIONS: We propose that the DBP phenotype seen in this family represents a distinct and novel subtype of DBP deficiency, which we have termed type IV based on the presence of a missense mutation in each of the domains of DBP resulting in markedly reduced but detectable hydratase and dehydrogenase activity of DBP. Given that the biochemical testing in plasma was normal in these patients, this is likely an underdiagnosed form of DBP deficiency.


Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/deficiencia , 17-Hidroxiesteroide Deshidrogenasas/genética , Hidroliasas/deficiencia , Hidroliasas/genética , Ataxia Cerebelosa/sangre , Ataxia Cerebelosa/genética , Ataxia Cerebelosa/orina , Ácidos Grasos/sangre , Ácidos Grasos/orina , Pérdida Auditiva Sensorineural/sangre , Pérdida Auditiva Sensorineural/genética , Pérdida Auditiva Sensorineural/orina , Heterocigoto , Mutación , Proteína-2 Multifuncional Peroxisomal , Ácido Fitánico/sangre , Polineuropatías/sangre , Polineuropatías/genética , Polineuropatías/orina , Retinitis Pigmentosa/sangre , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/orina
14.
Methods Mol Biol ; 908: 229-50, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22843403

RESUMEN

Single and multiple T-DNA knockouts of genes encoding arogenate dehydratases (ADTs) in Arabidopsis were obtained in homozygous form. These were analyzed for potential differences in lignin contents and compositions, as well as for distinct phenotypes over growth and development. Of these different lines, distinct reductions in lignin contents were obtained, with those having different G:S ratios depending upon the combination of ADT genes being knocked out. Results from pyrolysis GC/MS analyses indicated that differential carbon flux occurred into the vascular bundles (vb) and interfascicular fibers (if). These results provide additional new insight into factors controlling lignin heterogeneity and configuration.


Asunto(s)
Arabidopsis/química , Hidroliasas/deficiencia , Captura por Microdisección con Láser/métodos , Lignina/química , Conformación Molecular , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , ADN Bacteriano , Cromatografía de Gases y Espectrometría de Masas , Técnicas de Inactivación de Genes/métodos , Hidroliasas/genética , Lignina/genética , Análisis de Secuencia de Proteína , Espectrofotometría Ultravioleta
15.
Phytochemistry ; 82: 22-37, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22818526

RESUMEN

Phe is formed from arogenate in planta through the action of arogenate dehydratase (ADT), and there are six ADT isoenzymes in the "model" vascular plant species Arabidopsis thaliana. This raised the possibility that specific ADTs may be differentially regulated so as to control Phe biosynthesis for protein synthesis vs its much more massive deployment for phenylpropanoid metabolism. In our previous reverse genetics study using 25 single/multiple ADT knockout (KO) lines, a subset of these knockouts was differentially reduced in their lignin contents. In the current investigation, it was hypothesized that Phe pool sizes might correlate well with reduction in lignin contents in the affected KO lines. The free amino acid contents of these KO lines were thus comprehensively analyzed in stem, leaf and root tissues, over a growth/developmental time course from 3 to 8 weeks until senescence. The data obtained were then compared to, and contrasted with, the differential extent of lignin deposition occurring in the various lines. Relative changes in pool sizes were also analyzed by performing a pairwise confirmatory factor analysis for Phe:Tyr, Phe:Trp and Tyr:Trp, following determination of the deviation from the mean for Phe, Tyr and Trp in each plant line. It was found that the Phe pool sizes measured were differentially reduced only in lignin-deficient lines, and in tissues and at time points where lignin biosynthesis was constitutively highly active (in wild type lines) under the growth conditions employed. In contrast, this trend was not evident across all ADT KO lines, possibly due to maintenance of Phe pools by non-targeted isoenzymes, or by feedback mechanisms known to be in place.


Asunto(s)
Arabidopsis/genética , Arabidopsis/metabolismo , Carbono/metabolismo , Hidroliasas/genética , Fenilalanina/metabolismo , Arabidopsis/enzimología , Hidroliasas/deficiencia , Hidroliasas/metabolismo , Nitrógeno/metabolismo , Fenilalanina/biosíntesis
16.
J Inherit Metab Dis ; 35(6): 963-73, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22729819

RESUMEN

OBJECTIVES: The present study summarizes clinical and biochemical findings, current treatment strategies and follow-up in patients with tetrahydrobiopterin (BH(4)) deficiencies. METHODS: We analyzed the clinical, biochemical and treatment data of 626 patients with BH(4) deficiencies [355 with 6-pyruvoyl-tetrahydropterin synthase (PTPS), 217 with dihydropteridine reductase (DHPR), 31 with autosomal recessive GTP cyclohydrolase I (GTPCH), and 23 with pterin-4a-carbinolamine dehydratase (PCD) deficiencies] from the BIODEF Database. Patients with autosomal dominant GTPCH and SR deficiencies will not be discussed in detail. RESULTS: Up to 57 % of neonates with BH(4) deficiencies are already clinically symptomatic. During infancy and childhood, the predominant symptoms are muscular hypotonia, mental retardation and age-dependent movement disorders, including dystonia. The laboratory diagnosis of BH(4) deficiency is based on a positive newborn screening (NBS) for phenylketonuria (PKU), characteristic profiles of urinary or dried blood spot pterins (biopterin, neopterin, and primapterin), and the measurement of DHPR activity in blood. Some patients with autosomal recessive GTPCH deficiency and all with sepiapterin reductase deficiency may be diagnosed late due to normal blood phenylalanine in NBS. L-dopa, 5-hydroxytryptophan, and BH(4) are supplemented in PTPS and GTPCH-deficient patients, whereas L-dopa, 5-hydroxytryptophan, folinic acid and diet are used in DHPR-deficient patients. Medication doses vary widely among patients, and our understanding of the effects of dopamine agonists and monoamine catabolism inhibitors are limited. CONCLUSIONS: BH(4) deficiencies are a group of treatable pediatric neurotransmitter disorders that are characterized by motor dysfunction, mental retardation, impaired muscle tone, movement disorders and epileptic seizures. Although the outcomes of BH(4) deficiencies are highly variable, early diagnosis and treatment result in improved outcomes.


Asunto(s)
Biopterinas/análogos & derivados , Fenilcetonurias/metabolismo , Biopterinas/deficiencia , Recolección de Datos , Bases de Datos Factuales , Dihidropteridina Reductasa/genética , Femenino , Humanos , Hidroliasas/deficiencia , Hidroliasas/genética , Lactante , Recién Nacido , Internacionalidad , Masculino , Fenilcetonurias/diagnóstico , Fenilcetonurias/genética , Fenilcetonurias/terapia , Liasas de Fósforo-Oxígeno/deficiencia , Liasas de Fósforo-Oxígeno/genética
18.
J Biol Chem ; 287(14): 11446-59, 2012 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-22311980

RESUMEN

How carbon flux differentially occurs in vascular plants following photosynthesis for protein formation, phenylpropanoid metabolism (i.e. lignins), and other metabolic processes is not well understood. Our previous discovery/deduction that a six-membered arogenate dehydratase (ADT1-6) gene family encodes the final step in Phe biosynthesis in Arabidopsis thaliana raised the fascinating question whether individual ADT isoenzymes (or combinations thereof) differentially modulated carbon flux to lignins, proteins, etc. If so, unlike all other lignin pathway manipulations that target cell wall/cytosolic processes, this would be the first example of a plastid (chloroplast)-associated metabolic process influencing cell wall formation. Homozygous T-DNA insertion lines were thus obtained for five of the six ADTs and used to generate double, triple, and quadruple knockouts (KOs) in different combinations. The various mutants so obtained gave phenotypes with profound but distinct reductions in lignin amounts, encompassing a range spanning from near wild type levels to reductions of up to ∼68%. In the various KOs, there were also marked changes in guaiacyl:syringyl ratios ranging from ∼3:1 to 1:1, respectively; these changes were attributed to differential carbon flux into vascular bundles versus that into fiber cells. Laser microscope dissection/pyrolysis GC/MS, histochemical staining/lignin analyses, and pADT::GUS localization indicated that ADT5 preferentially affects carbon flux into the vascular bundles, whereas the adt3456 knock-out additionally greatly reduced carbon flux into fiber cells. This plastid-localized metabolic step can thus profoundly differentially affect carbon flux into lignins in distinct anatomical regions and provides incisive new insight into different factors affecting guaiacyl:syringyl ratios and lignin primary structure.


Asunto(s)
Carbono/metabolismo , Hidroliasas/metabolismo , Lignina/metabolismo , Acetatos/metabolismo , Arabidopsis/citología , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/metabolismo , Cloroplastos/enzimología , Cloroplastos/metabolismo , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Glucuronidasa/genética , Hidroliasas/deficiencia , Hidroliasas/genética , Isoenzimas/deficiencia , Isoenzimas/genética , Isoenzimas/metabolismo , Fenotipo , Transporte de Proteínas
19.
Mol Cell ; 44(4): 660-6, 2011 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-22099312

RESUMEN

How pseudouridylation (Ψ), the most common and evolutionarily conserved modification of rRNA, regulates ribosome activity is poorly understood. Medically, Ψ is important because the rRNA Ψ synthase, DKC1, is mutated in X-linked dyskeratosis congenita (X-DC) and Hoyeraal-Hreidarsson (HH) syndrome. Here, we characterize ribosomes isolated from a yeast strain in which Cbf5p, the yeast homolog of DKC1, is catalytically impaired through a D95A mutation (cbf5-D95A). Ribosomes from cbf5-D95A cells display decreased affinities for tRNA binding to the A and P sites as well as the cricket paralysis virus internal ribosome entry site (IRES), which interacts with both the P and the E sites of the ribosome. This biochemical impairment in ribosome activity manifests as decreased translational fidelity and IRES-dependent translational initiation, which are also evident in mouse and human cells deficient for DKC1 activity. These findings uncover specific roles for Ψ modification in ribosome-ligand interactions that are conserved in yeast, mouse, and humans.


Asunto(s)
Proteínas de Ciclo Celular/deficiencia , Disqueratosis Congénita/genética , Retardo del Crecimiento Fetal/genética , Hidroliasas/deficiencia , Hidroliasas/metabolismo , Discapacidad Intelectual/genética , Microcefalia/genética , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Nucleares/deficiencia , ARN Ribosómico/metabolismo , ARN de Transferencia/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/deficiencia , Saccharomyces cerevisiae/genética , Animales , Sitios de Unión , Proteínas de Ciclo Celular/genética , Disqueratosis Congénita/enzimología , Retardo del Crecimiento Fetal/enzimología , Genes Reporteros , Humanos , Hidroliasas/genética , Discapacidad Intelectual/enzimología , Luciferasas/análisis , Ratones , Microcefalia/enzimología , Proteínas Asociadas a Microtúbulos/genética , Mutación , Proteínas Nucleares/genética , Plásmidos , Biosíntesis de Proteínas , ARN Ribosómico/química , ARN Ribosómico/genética , ARN de Transferencia/química , ARN de Transferencia/genética , Ribonucleoproteínas Nucleares Pequeñas/genética , Ribosomas/química , Ribosomas/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Transducción Genética
20.
J Biol Chem ; 286(50): 43123-33, 2011 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-22027835

RESUMEN

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis through binding to TRAIL receptors, death receptor 4 (DR4), and DR5. TRAIL has potential therapeutic value against cancer because of its selective cytotoxic effects on several transformed cell types. Fucosylation of proteins and lipids on the cell surface is a very important posttranslational modification that is involved in many cellular events. Recently, we found that a deficiency in GDP-mannose-4,6-dehydratase (GMDS) rendered colon cancer cells resistant to TRAIL-induced apoptosis, resulting in tumor development and metastasis by escape from tumor immune surveillance. GMDS is an indispensable regulator of cellular fucosylation. In this study, we investigated the molecular mechanism of inhibition of TRAIL signaling by GMDS deficiency. DR4, but not DR5, was found to be fucosylated; however, GMDS deficiency inhibited both DR4- and DR5-mediated apoptosis despite the absence of fucosylation on DR5. In addition, GMDS deficiency also inhibited CD95-mediated apoptosis but not the intrinsic apoptosis pathway induced by anti-cancer drugs. Binding of TRAIL and CD95 ligand to their cognate receptors primarily leads to formation of a complex comprising the receptor, FADD, and caspase-8, referred to as the death-inducing signaling complex (DISC). GMDS deficiency did not affect formation of the primary DISC or recruitment to and activation of caspase-8 on the DISC. However, formation of secondary FADD-dependent complex II, comprising caspase-8 and cFLIP, was significantly inhibited by GMDS deficiency. These results indicate that GMDS regulates the formation of secondary complex II from the primary DISC independent of direct fucosylation of death receptors.


Asunto(s)
Neoplasias del Colon/metabolismo , Hidroliasas/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Receptor fas/metabolismo , Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Caspasa 8/metabolismo , Neoplasias del Colon/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Citometría de Flujo , Células HCT116 , Humanos , Hidroliasas/deficiencia , Hidroliasas/genética , Inmunoprecipitación , Unión Proteica , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Receptor fas/antagonistas & inhibidores
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