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Microbioma Gastrointestinal , Hipersensibilidad , Síndrome del Colon Irritable , Probióticos , Dieta , Humanos , Níquel , Proyectos PilotoRESUMEN
Essentials Life-threatening maternofetal thrombocytopenias mostly depend on αIIb ß3 antigens. We performed serological, genomic and in vitro studies of two life-threatening thrombocytopenias. Identification of a c.368C>T variation leading to Pro123Leu substitution in GPIX. A rare GPIX variant reported in a genomic database define a new alloantigen. SUMMARY: Background After three miscarriages, a 39-year-old woman gave birth, with a 1-year interval, to two severely thrombocytopenic neonates (4 ×109 L-1 and 33 ×109 L-1 ) with intracranial hemorrhages. Transfusion of platelet concentrates corrected the thrombocytopenia. The outcome was favorable for the first child, but the second one died 10 days after cesarean delivery (31 weeks of gestation + 6 days). Methods Serologic studies were performed with mAb-specific immobilization of platelet antigens and flow cytometry techniques. Human platelet alloantigen (HPA) genotyping was performed with the BioArray HPA BeadChip and PCR-sequence-specific primer techniques. Genomic DNA was studied by direct sequencing of PCR products. The mutant glycoprotein (GP) was expressed in transiently transfected HEK293 cells. Results In MAIPA assay, the maternal serum faintly reacted with GPIbIX from paternal and child 1 platelets, but not with maternal or panel platelets. No maternofetal incompatibility was found in the 22 known HPA systems, tested except for HPA-1b in child 2. A new alloantigen carried by GPIbIX was suspected. Genomic sequencing revealed a paternal GPIX variation (NM_000174.4:c.368C>T). The father and children were heterozygous and incompatible with the mother, who was NM_000174.4:c.368C homozygous. The maternal serum reacted with the GPIX NP_000165.1:p.Leu123 form coexpressed with GPIb in transfected HEK293 cells. The NM_000174.4:c.368T allele (rs202229101) has a minor allele frequency of 0.0002, and was not detected in 120 French subjects (families with fetal and neonatal alloimmune thrombocytopenia [FNAIT]), suggesting that it is rarely implicated in alloimmunization. Conclusion The NP_000165.1:p.Leu123 allele named Cab4b is the first platelet alloantigen described on GPIX. In the absence of other known maternofetal incompatibility, the child 1 case suggests that anti-Cab4b alloantibodies can induce severe thrombocytopenias.
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Antígenos de Plaqueta Humana/genética , Mutación , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Trombocitopenia Neonatal Aloinmune/genética , Antígenos de Plaqueta Humana/sangre , Antígenos de Plaqueta Humana/inmunología , Análisis Mutacional de ADN , Resultado Fatal , Femenino , Predisposición Genética a la Enfermedad , Células HEK293 , Herencia , Humanos , Recién Nacido , Isoanticuerpos/sangre , Linaje , Fenotipo , Complejo GPIb-IX de Glicoproteína Plaquetaria/inmunología , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Transfusión de Plaquetas , Embarazo , Pruebas Serológicas , Trombocitopenia Neonatal Aloinmune/sangre , Trombocitopenia Neonatal Aloinmune/diagnóstico , Trombocitopenia Neonatal Aloinmune/terapia , Transfección , Resultado del TratamientoRESUMEN
The realization of biologically relevant human tissue equivalents as an in vitro model to investigate human diseases, as well as to test the efficacy or toxicity of novel compounds, is emerging as a new challenge in tissue engineering. Currently, the in vitro three-dimensional (3D) dermis model mainly involves the use of cells embedded in exogenous non-human matrices. However, such models feature biological and functional disparities with native dermis, therefore limiting their relevance to the in vivo situation. The purpose of this study was to provide a reliable endogenous human dermal equivalent (HDE) able to recapitulate the extracellular matrix (ECM) remodelling of the native dermis occurring after external damage. To this end, UVA irradiation was used to induce photodamage to both the HDE and to a fibroblast-populated collagen matrix. The photodamage was investigated at the cellular and ECM level and the results showed that, although a cellular response was detected in both systems, no ECM reorganization characteristic of the in vivo photo-aged dermis could be detected in the fibroblast-populated collagen matrix. In contrast in the HDE, the neosynthesized ECM recapitulated the characteristic ageing behaviour of the dermis found in vivo, in terms of collagen and hyaluronic acid synthesis as well as collagen organization remodelling. This study therefore demonstrates the role of the endogenous ECM in recapitulating in vitro the functionality of the human dermis and the proposed HDE as a novel tool for photoprotection trials. Copyright © 2016 John Wiley & Sons, Ltd.
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Dermis , Matriz Extracelular , Fibroblastos , Trastornos por Fotosensibilidad , Ingeniería de Tejidos , Rayos Ultravioleta/efectos adversos , Dermis/metabolismo , Dermis/patología , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Trastornos por Fotosensibilidad/metabolismo , Trastornos por Fotosensibilidad/patologíaRESUMEN
The fabrication of functional tissue units is one of the major challenges in tissue engineering due to their in vitro use in tissue-on-chip systems, as well as in modular tissue engineering for the construction of macrotissue analogs. In this work, we aim to engineer dermal tissue micromodules obtained by culturing human dermal fibroblasts into porous gelatine microscaffold. We proved that such stromal cells coupled with gelatine microscaffolds are able to synthesize and to assemble an endogenous extracellular matrix (ECM) resulting in tissue micromodules, which evolve their biophysical features over the time. In particular, we found a time-dependent variation of oxygen consumption kinetic parameters, of newly formed ECM stiffness and of micromodules self-aggregation properties. As consequence when used as building blocks to fabricate larger tissues, the initial tissue micromodules state strongly affects the ECM organization and maturation in the final macrotissue. Such results highlight the role of the micromodules properties in controlling the formation of three-dimensional macrotissue in vitro, defining an innovative design criterion for selecting tissue-building blocks for modular tissue engineering.
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Matriz Extracelular/fisiología , Fibroblastos/fisiología , Impresión Tridimensional , Piel Artificial , Piel/crecimiento & desarrollo , Andamios del Tejido , Células Cultivadas , Diseño de Equipo , Análisis de Falla de Equipo , Matriz Extracelular/ultraestructura , Fibroblastos/citología , Humanos , Miniaturización , Técnicas de Cultivo de Órganos/instrumentación , Piel/citología , Ingeniería de Tejidos/instrumentación , Ingeniería de Tejidos/métodosRESUMEN
La mastectomía profiláctica contralateral (MPC) en mujeres con diagnóstico de cáncer de mama es un procedimiento quirúrgico que permite obtener un mejor resultado estético y un pronóstico oncológico más favorable, al prevenir el desarrollo contralateral del cáncer. Presentamos nuestra experiencia en 38 casos de mujeres con diagnóstico de cáncer de mama que fueron operadas de mastectomía y reconstrucción inmediata en 2 tiempos mediante expansor tisular/implante. Estas pacientes optaron por una MPC en el segundo tiempo del proceso reconstructivo por motivos psicológicos u oncológicos. Realizamos una técnica ahorradora de piel y del complejo aréola-pezón, que ofrece un resultado natural de la mama una vez reconstruida y, por tanto, un alto nivel de satisfacción de la paciente
Contralateral prophylactic mastectomy (CPM) in women with a diagnosis of breast cancer improves the esthetic outcome and oncological prognosis because it reduces the risk of developing contralateral breast cancer. We report our experience of 38 women with a diagnosis of breast cancer who underwent mastectomy and immediate two-stage tissue expander / implant reconstruction. These patients opted for a CPM in the second stage of their reconstructive process due to psychological or oncological reasons. We performed a skin-sparing, nipple-sparing technique that offers a natural result and consequently a high level of patient satisfaction
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Femenino , Humanos , Mastectomía/métodos , Mamoplastia/instrumentación , Mamoplastia/métodos , Pezones/anomalías , Pezones/citología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/radioterapia , Cirugía Plástica/métodos , Cirugía Plástica/normas , Mastectomía/normas , Mamoplastia/psicología , Mamoplastia/rehabilitación , Pezones/metabolismo , Pezones/patología , Neoplasias de la Mama/rehabilitación , Neoplasias de la Mama/terapia , Cirugía Plástica/psicología , Cirugía PlásticaRESUMEN
Las mujeres que presentan los labios menores hipertróficos o prominentes a menudo presentan trastornos funcionales y psicosociales. Se consideran labios menores hipertróficos aquellos que, en posición y condiciones normales, sobresalen notoriamente de los labios mayores, ocasionando irritación local, dificultades higiénicas, incomodidad mientras se camina o realizan algunas prácticas deportivas e interfiriendo en el desarrollo normal de las relaciones sexuales. Hay diversas técnicas quirúrgicas de labioplastia. Una de las más habituales es la amputación de aquellos segmentos de los labios menores que sobresalen de los labios mayores. Para evitar las complicaciones derivadas de esa técnica, nosotros hemos optado por realizar la labioplastia efectuando una resección con colgajos especulares en forma de «S», con una sutura interdigitada. De esta manera el resultado final es más natural, con bordes suaves y redondeados, lo que reduce en gran medida las complicaciones relacionadas con la contracción cicatricial. A continuación, presentamos nuestra experiencia tras haber aplicado con éxito esta técnica en 7 casos
Women with hypertrophic or enlarged labia minora usually have functional and psychosocial problems. Labial hypertrophy is defined as labia minora that extend beyond the labia majora when the woman is in a normal position. Many affected women have local irritation, interference with sexual intercourse, difficulty with hygiene, and discomfort during walking, sitting, biking or horse-riding. Several surgical techniques can be used to perform labioplasty. One of most common procedures is a simple and straight amputation of the protuberant segments and over sewing of the edge. To prevent the complications associated with this technique, we perform a specular S-shaped resection with interdigitated suturing of the protuberant labium. This technique results in a natural, softer, and more rounded labial edge, with fewer complications. We present our experience in 7 cases with successful outcomes
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Humanos , Femenino , Vulva/anomalías , Anomalías Urogenitales/cirugía , Hipertrofia/cirugía , Procedimientos de Cirugía Plástica/métodosRESUMEN
INTRODUCTION: Irritable bowel syndrome (IBS) is a chronic gastrointestinal (GI) disorder that affects 15-20% of the Western population. BACKGROUND: There are currently few therapeutic options available for the treatment of IBS. The aim of this study is to evaluate the efficacy and the safety of a medical device containing a combination of Simethicone and Bacillus coagulans in the treatment of IBS. PATIENTS AND METHODS: This is a monocentric double-blind, placebo-controlled parallel group clinical trial. Adult subjects suffering from IBS as defined by Rome III criteria were enrolled. Bloating, discomfort, abdominal pain were assessed as primary end point. Subjects received the active treatment or placebo 3 time a day after each meal for 4 weeks of study period. Subjects were submitted to visit at Day 0 (T1), at Days 14 (T2) and 29 (T3). RESULTS: Fifty-two patients were included into the study. Intragroup analysis showed a significant reduction of the bloating, discomfort and pain in Colinox® group (CG) compared to placebo group (PG). Between group analysis confirmed, at T1-T3, significant differences between CG and PG in bloating and discomfort. DISCUSSION: Simethicone is an inert antifoaming able to reduce bloating, abdominal discomfort. Literature offers increasing evidence linking alterations in the gastrointestinal microbiota and IBS and it is well known that probiotics are important to restore the native gut microbiota. The Colinox medical device is specifically targeted against most intrusive symptom of IBS (bloating) and it is also able to counteract the most accredited ethiopathogenetic factor in IBS (alterations of intestinal microbiota). CONCLUSIONS: This is the first randomized double-blind placebo-controlled clinical trial demonstrating the efficacy and safety of a combination of simethicone and Bacillus coagulans in treatment of IBS.
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Antiespumantes/uso terapéutico , Bacillus/crecimiento & desarrollo , Intestinos/efectos de los fármacos , Intestinos/microbiología , Síndrome del Colon Irritable/terapia , Probióticos/uso terapéutico , Simeticona/uso terapéutico , Dolor Abdominal/etiología , Dolor Abdominal/prevención & control , Adulto , Anciano , Antiespumantes/efectos adversos , Bacillus/clasificación , Terapia Combinada , Método Doble Ciego , Femenino , Humanos , Síndrome del Colon Irritable/complicaciones , Síndrome del Colon Irritable/diagnóstico , Síndrome del Colon Irritable/microbiología , Masculino , Persona de Mediana Edad , Probióticos/efectos adversos , Ciudad de Roma , Simeticona/efectos adversos , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND AND OBJECTIVES: In fetal/neonatal thrombocytopenia, maternal alloimmunization is diagnosed by the identification of the maternal alloantibody and the offending paternal antigen inherited by the foetus/neonate. Today, for practical reasons, most laboratories perform platelet genotyping instead of phenotyping. Here, we report the case of a human platelet antigen (HPA)-5 genotype/phenotype discrepancy observed in a mother who delivered a mildly thrombocytopenic newborn. MATERIALS AND METHODS: Platelet antibody detection and platelet phenotyping were performed using the MAIPA assay; platelet genotypes were determined using BeadChip technology (BioArray), PCR-SSP, PCR-RFLP and sequencing. RESULTS: Serological investigations revealed the presence of maternal anti-GPIIbIIIa autoantibodies. No alloantibodies were detected. No feto-maternal platelet incompatibility was observed for HPA-1 to -21. The mother and newborn were genotyped as HPA-5aa using BeadChips, but as HPA-5a (weak b) with PCR-SSP and HPA-5ab with PCR-RFLP. Mother's platelets were phenotyped as HPA-5b(+). GPIa exon 13 sequencing confirmed the HPA-5ab genotype of the mother and newborn, and revealed an NM_002203.3:c.1594A>C mutation near the HPA-5 polymorphism (5' side), leading to an I503L amino acid change. CONCLUSION: Feto-maternal alloimmunization was ruled out: the neonatal thrombocytopenia probably resulted from maternal anti-GPIIbIIIa autoantibodies. This case highlights that platelet typing should be performed using two different methods to avoid false diagnosis.
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Genotipo , Integrina alfa2/genética , Mutación Missense , Polimorfismo de Longitud del Fragmento de Restricción , Adulto , Sustitución de Aminoácidos , Antígenos de Plaqueta Humana/genética , Antígenos de Plaqueta Humana/metabolismo , Femenino , Humanos , Recién Nacido , Isoanticuerpos/sangre , Masculino , Trombocitopenia Neonatal Aloinmune/sangre , Trombocitopenia Neonatal Aloinmune/genéticaRESUMEN
We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca(2+)-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300µg/ml) in combination with acidic lipids at concentrations >10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50µg/ml tubulin, increased PMCA activity >12-fold, whereas tubulin alone at high concentration (≥300µg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca(2+) transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (<50µg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration.
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Calcio/metabolismo , Membrana Celular/enzimología , Lípidos de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Tubulina (Proteína)/metabolismo , Acetilación , Animales , Encéfalo/metabolismo , Química Encefálica/fisiología , Membrana Celular/química , Transporte Iónico/fisiología , Lípidos de la Membrana/química , Proteínas del Tejido Nervioso/química , ATPasas Transportadoras de Calcio de la Membrana Plasmática/química , Ratas , Tubulina (Proteína)/química , Fosfolipasas de Tipo C/químicaAsunto(s)
Implantación de Prótesis/métodos , Testículo/cirugía , Adulto , Humanos , Masculino , Diseño de PrótesisAsunto(s)
Humanos , Masculino , Adulto , Implantación de Prótesis/métodos , Testículo , Orquiectomía/rehabilitaciónRESUMEN
A strong association between type 1 insulin-dependent diabetes mellitus (IDDM1) and coeliac disease (CD) is well documented, but it is known that prevalence values are underestimated. Serum anti-endomysial antibodies (EMA), considered diagnostic for CD because of their high sensitivity and specificity, belong to the IgA class, but the existence of EMA of IgG1 isotype in the presence or absence of IgA deficiency was reported. In order to re-evaluate the occurrence of CD in IDDM1 patients we performed a screening in IDDM1 patients using EMA of both isotypes. Ninety-four adults affected by IDDM1 (unaffected by CD before enrolling) were enrolled and 83 blood donors as controls. All subjects were on a gluten-containing diet. Histology and biopsy culture were performed. EMA IgA and IgG1 in sera and culture supernatants were detected. Serum EMA were positive in 13 of 94 IDDM1 patients (13.8%). Six of 13 presented IgA-EMA, seven of 13 presented IgG1-EMA. No EMA were found in the control population. Total intestinal atrophy was found in all six patients with serum IgA-EMA and in five of seven with serum IgG1-EMA. Diagnosis of CD was confirmed by histology and organ culture in all 13 patients with serum EMA. The prevalence of CD in the patients affected by IDDM1 was 6.4% for IgA-EMA-positive and 7.4% for IgG1-EMA-positive patients. We confirmed the prevalence of CD in the IDDM1 population obtained with IgA-EMA screening only (6.4%). This prevalence value increases dramatically to 13.8% when IgG1-EMA are also used in the screening. We conclude that IgG1-EMA should also be sought whenever an IDDM1 patient undergoes screening for CD.
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Anticuerpos/inmunología , Enfermedad Celíaca/inmunología , Diabetes Mellitus Tipo 1/inmunología , Inmunoglobulina G/inmunología , Adolescente , Adulto , Anciano , Anticuerpos/sangre , Atrofia/inmunología , Enfermedad Celíaca/sangre , Diabetes Mellitus Tipo 1/sangre , Duodeno/inmunología , Femenino , Gliadina/inmunología , Humanos , Inmunoglobulina A/inmunología , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Fibras Musculares Esqueléticas/inmunologíaRESUMEN
Membranes from brain tissue contain tubulin that can be isolated as a hydrophobic compound by partitioning into Triton X-114. The hydrophobic behavior of this tubulin is due to the formation of a complex with the alpha-subunit of Na+,K+-ATPase. In the present work we show that the interaction of tubulin with Na+K+-ATPase inhibits the enzyme activity. We found that the magnitude of the inhibition is correlated with: (1) concentration of the acetylated tubulin isoform present in the tubulin preparation used, and (2) amount of acetylated tubulin isoform isolated as a hydrophobic compound. In addition, some compounds involved in the catalytic action of Na+K+-ATPase were assayed to determine their effects on the inhibitory capability of tubulin on this enzyme. The inhibitory effect of tubulin was only slightly decreased by ATP at relatively low nucleotide concentration (0.06 mM). NaCl (1-160 mM) and KCl (0.2-10 mM) showed no effect whereas inorganic phosphate abolished the inhibitory effect of tubulin in a concentration-dependent manner.
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Encéfalo/enzimología , Membrana Celular/enzimología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Tubulina (Proteína)/metabolismo , Acilación , Adenosina Trifosfato/metabolismo , Animales , Catálisis , Cromatografía en Agarosa , Detergentes/farmacología , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Cinética , Octoxinol/farmacología , Fosfatos/farmacología , Polietilenglicoles/farmacología , Isoformas de Proteínas , Ratas , Cloruro de Sodio/farmacología , Tubulina (Proteína)/químicaRESUMEN
3-Hydroxy-3-methylglutaric aciduria is a rare recessive monogenic disorder that affects ketogenesis and the catabolism of L-leucine. We report the biochemical and molecular characterization of a mutation in the 3-hydroxy-3-methylglutaryl coenzyme A lyase gene in four new probands, three Spanish and one Turkish, affected by 3-hydroxy-3-methylglutaric aciduria, all homozygous for the nonsense mutation Glu37Ter, which was reported by our group in two probands of Portuguese and Moroccan origin (15). In addition to the aberrant mRNAs found in the two previous probands, a novel species of mature HL mRNA was observed in the patients studied here, since a new cDNA, skipped in exons 2 and 3, was obtained from the mRNAs by reverse-transcription PCR (RT-PCR). Thus, three mRNA species were produced in aberrant splicings as a result of this nonsense mutation: (i) one of the expected size that contains the premature stop codon UAA, (ii) another with a deletion of 84 bp corresponding to the whole of exon 2, and (iii) a new species found now, with a deletion of 192 bp corresponding to skipping of the whole of exons 2 and 3, whose translation product led to the loss of seven amino acids in the leader peptide and 57 amino acids in the terminal domain of the mature enzyme. The association of a nonsense mutation with the skipping of the exon that contains it, plus the following exon, is an unusual finding not seen previously in HL deficiencies. The mutation described here shows the highest incidence (> 37%) of total HL deficiencies reported.
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Exones/genética , Meglutol/orina , Mutación , Oxo-Ácido-Liasas/genética , Preescolar , Humanos , Lactante , Masculino , Región MediterráneaRESUMEN
We have previously described that the tubulin isolated from brain membranes as a hydrophobic compound by partitioning into Triton X-114 is a peripheral membrane protein [corrected]. The hydrophobic behavior of this tubulin is due to its interaction with membrane protein(s) and the interaction occurs principally with the acetylated tubulin isotype. In the present work we identified the membrane protein that interacts with tubulin as the Na+,K+-ATPase alpha subunit by amino acid sequencing. Using purified brain Na+,K+-ATPase we were able to isolate part of the total hydrophilic tubulin as a hydrophobic compound which contains a high proportion of the acetylated tubulin isotype.
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Encéfalo/metabolismo , Proteínas de la Membrana/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Tubulina (Proteína)/metabolismo , Acetilación , Secuencia de Aminoácidos , Animales , Datos de Secuencia Molecular , Unión Proteica , Ratas , Análisis de Secuencia , Porcinos , Tubulina (Proteína)/análogos & derivadosRESUMEN
A novel two-base deletion in the 3-hydroxy-3-methylglutaryl coenzyme A lyase (HL) gene was found in a Spanish patient with homozygous 3-hydroxy-3-methylglutaric aciduria. Amplification by RT-PCR of the mRNAs showed that the gene was transcribed into three different mRNAs. One showed the complete deletion of exons 5 and 6 located between nucleotides 348 and 561 of the HL cDNA. The second transcript showed deletion of exon 6 only, and the third contained a two-base deletion CT in exon 6, corresponding to nucleotides 504 and 505 of the HL cDNA. These aberrant mRNAs are predicted to encode three abnormal HMG-CoA lyase proteins; the first (from skipped exons 5 and 6) lacks 71 amino acids, which represents 24% of the mature protein; the second, (from the skipping of exon 6, producing a frameshift) contains only 192 amino acids, the last 26 of which are missense amino acids preceding a stop codon; the third contains only 175 amino acids, the last 7 of which are missense. Northern blot analysis showed that the HL mRNA levels of the patient were 4% of the control. PCR quantitative analysis indicated that the mRNA lacking exons 5 and 6 was the most abundant, representing 88% of the total. The other two mRNAs represented 8% and 4%, respectively. In the genomic DNA only one CT deletion was found at positions +7 and +8 at beginning of exon 6. No mutations were observed in the splice donor, splice acceptor, or pyrimidine-rich sequences of the intronic regions flanking exons 5 and 6. All three aberrant mRNAs resulted only from the deletion of nucleotides CT. We suggest that this deletion may affect the interaction between the small nuclear ribonucleoproteins (snRNPs) and exon 6, and that, as a result, the abnormal splicing of the pre-mRNA produces two different aberrant transcripts.
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Meglutol/orina , Errores Innatos del Metabolismo/enzimología , Oxo-Ácido-Liasas/genética , Eliminación de Secuencia , Adolescente , Secuencia de Aminoácidos , Northern Blotting , Southern Blotting , Femenino , Fibroblastos , Mutación del Sistema de Lectura , Humanos , Errores Innatos del Metabolismo/genética , Datos de Secuencia Molecular , Oxo-Ácido-Liasas/química , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN , Transcripción GenéticaRESUMEN
A novel nonsense mutation associated with the skipping of constitutive exon 2 of the 3-hydroxy-3-methylglutaryl-CoA lyase gene was found in two patients, from Portugal and Morocco, with 3-hydroxy-3-methylglutaric acidemia. By reverse transcriptase PCR and single-strand conformational polymorphism a G-T transversion was located, at nucleotide 109, of the 3-hydroxy-3-methylglutaryl-CoA lyase cDNA, within exon 2. Two mRNAs were produced as a result of this nonsense mutation: one of the expected size that contains the premature stop codon UAA, and the other with a deletion of 84 bp corresponding to the whole of exon 2. This deletion produced the loss of the last seven amino acids of the leader peptide and the first 21 amino acids of the mature protein. The nonsense mutation was found in a purine-rich GGAAG sequence, which is equal to, or similar to, others reported to be exonic splicing enhancers (ESE). We suggest that the nonsense mutation may affect a possible ESE on exon 2, which would hinder the splice site selection and facilitate an aberrant splice with the skipping of this exon. Determination by quantitative PCR shows that the ratio of mRNA with the nonsense mutation to the mRNA with the deletion is approx. 3:1.
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Exones , Mutagénesis , Oxo-Ácido-Liasas/deficiencia , Oxo-Ácido-Liasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Femenino , Humanos , Lactante , Intrones , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
A novel point mutation in the 3-hydroxy-3methyl-glutaryl coenzyme A lyase gene was found in a Turkish patient with homozygous 3-hydroxy-3-methylglutaric acidemia. Amplification by RT-PCR of the mRNA using a six different pairs of oligonucleotides produced no differences in four of the fragments amplified with respect to the control, but generated two fragments of different size. One was representative of a deletion of 126 bp and the other of an insertion of 78 bp. These abnormal mRNAs resulted from a G-->C transversion at the nucleotide +1 of an intron, which changed the invariant GT dinucleotide of the 5' donor splice site. This was associated with the occurrence of an alternative splicing, which led to the skipping of the whole exon of 126 bp, and also with the activation of one cryptic donor splice site in the same intron. These aberrant spliced mRNAs are predicted to encode two abnormal HMG-CoA lyase proteins: the first results in a protein with an internal deletion of 42 amino acids, whose enzyme activity is largely abolished, as the catalytic site was completely removed; the second contains 17 missense amino acids that precede a stop codon. Northern blot analysis showed that the overall content of these aberrantly spliced mRNAs in proband fibroblasts was the same as that found in control fibroblasts. However, hardly any transcript was observed corresponding to the inserted mutated mRNA when it was examined by a specific probe. To quantify the relative proportion of the two mRNAs, a quantitative RT-PCR (the DNA-mimic PCR reaction) was carried out. Results show that the proportion of the inserted mRNAs with respect to the deleted mRNA is only 1.2%. The father, mother, and two brothers of the proband were heterozygous in the G-->C mutation in the +1 nucleotide of the intron considered, while the two alleles of another brother were free of the mutation.
Asunto(s)
Oxo-Ácido-Liasas/genética , Empalme del ARN , ARN Mensajero/metabolismo , Secuencia de Bases , Southern Blotting , Análisis Mutacional de ADN , Exones , Humanos , Recién Nacido , Intrones , Masculino , Datos de Secuencia Molecular , Mutación Puntual , Reacción en Cadena de la Polimerasa , Eliminación de SecuenciaRESUMEN
The present study demonstrates that under conditions of iso or hyperosmolarity, P. aeruginosa utilized carnitine as the carbon, nitrogen or carbon and nitrogen sources. As occurred in the case of choline, the bacteria synthesized cholinesterase (ChE), acid phosphatase (Ac.Pase) and phospholipase C (PLC) under any of these conditions and in the presence of high or low Pi concentrations. Carnitine acted as an osmoprotectant when the cells were grown in the presence of preferred carbon and nitrogen sources and high NaCl concentrations. Under these conditions the three enzyme activities were not produced. The osmotically stressed bacteria grown under any of the above conditions accumulated betaine. Its presence indicated that carnitine may be metabolized by P. aeruginosa to produce betaine which could account for the induction of the three enzyme activities or its action as an osmoprotectant. The phosphatidylcholine encountered in the host cell membranes allows the bacteria to obtain free choline by the coordinated action of PLC and Ac.Pase. Since the consequence of this action may be cell disruption, the increase of free carnitine in the natural environment of the bacteria is also possible. These two compounds, choline and carnitine, acting in conjunction or separately, may increase the production of PLC and Ac.Pase activities by P. aeruginosa and thus enhance the degradative effect upon the host cells.