RESUMEN
OBJECTIVE: To describe the clinical and respiratory characteristics of a cohort of 43 patients with COVID-19 after an evolutive period of 28 days. DESIGN: A prospective, single-center observational study was carried out. SETTING: Intensive care. PATIENTS: Patients admitted due to COVID-19 and respiratory failure. INTERVENTIONS: None. VARIABLES: Automatic recording was made of demographic variables, severity parameters, laboratory data, assisted ventilation (HFO: high-flow oxygen therapy and IMV: invasive mechanical ventilation), oxygenation (PaO2, PaO2/FiO2) and complications. The patients were divided into three groups: survivors (G1), deceased (G2) and patients remaining under admission (G3). The chi-squared test or Fisher exact test (categorical variables) was used, along with the Mann-Whitney U-test or Wilcoxon test for analyzing the differences between medians. Statistical significance was considered for p<0.05. RESULTS: A total of 43 patients were included (G1=28 [65.1%]; G2=10 [23.3%] and G3=5 [11.6%]), with a mean age of 65 years (range: 52-72), 62% males, APACHE II 18 (15-24), SOFA 6 (4-7). Arterial hypertension (30.2%) and obesity (25.6%) were the most frequent comorbidities. High-flow oxygen therapy was used in 62.7% of the patients, with failure in 85%. In turn, 95% of the patients required IMV and 85% received ventilation in prone decubitus. In the general population, initial PaO2/FiO2 improved after 7 days (165 [125-210] vs.194 [153-285]; p=0.02), in the same way as in G1 (164 [125-197] vs. 207 [160-294]; p=0.07), but not in G2 (163 [95-197] vs. 135 [85-177]). No bacterial coinfection was observed. The incidence of IMV-associated pneumonia was high (13 episodes/1000 days of IMV). CONCLUSIONS: Patients with COVID-19 require early IMV, a high frequency of ventilation in prone decubitus, and have a high incidence of failed HFO. The lack of improvement of PaO2/FiO2 at 7 days could be a prognostic marker. .
Asunto(s)
COVID-19/epidemiología , Pandemias , SARS-CoV-2 , Distribución por Edad , Anciano , Antibacterianos/uso terapéutico , Infecciones Bacterianas/complicaciones , Infecciones Bacterianas/tratamiento farmacológico , COVID-19/mortalidad , COVID-19/terapia , Distribución de Chi-Cuadrado , Contraindicaciones de los Procedimientos , Femenino , Mortalidad Hospitalaria , Hospitalización/estadística & datos numéricos , Humanos , Unidades de Cuidados Intensivos , Masculino , Persona de Mediana Edad , Multimorbilidad , Ventilación no Invasiva/efectos adversos , Estudios Prospectivos , Respiración Artificial/métodos , España/epidemiología , Estadísticas no Paramétricas , Centros de Atención Terciaria , Tratamiento Farmacológico de COVID-19RESUMEN
Testicular Leydig cells (LC) regulate the proper development of male individuals, both during fetal life (fetal LC) and puberty (adult LC). In the ovaries of adult women, there are cells that are very similar to Leydig cells, the ovarian hilus cells (OHC), which also produce testosterone. The origin of these cells, in both sexes, remains unknown and is still a matter of debate. We have studied the location, characteristics and relationships of the OHC in 90 patients. The indications for oophorectomy were: metrorrhagia (n=9), prolapse (n=8), endometrial hyperplasia (n=14), cancer (endometrial, myometrial, or cervical) (n=35), uterine leiomyomata (n=14), and various ovarian tumors (cysts and benign tumors, borderline and malignant) (n=10). In addition to the hilus, occasionally the nodules, nests and clusters of OHC were located in the mesovarium, the mesosalpinx, and in the medullar and cortical regions of the ovaries. The morphological (including crystalloids of Reinke) and immunohistochemical (positivity for calretinin and alpha-inhibin) findings were similar to those described for testicular LC. Therefore, OHC can be considered ovarian Leydig cells (OLC). LC are usually found in small numbers in the ovaries, but if one looks for them intentionally, one always finds them. Close relationships were observed between the OLC with nerves and vessels. Moreover, an intraneural location of the OLC was demonstrated in all cases, and these intraneural cells showed similar characteristics to extraneural OLC, suggesting that they derive from endoneural cells which are present in the vegetative nerves of the ovaries.
Asunto(s)
Células Intersticiales del Testículo/patología , Ovario/patología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Inmunohistoquímica , Células Intersticiales del Testículo/metabolismo , Masculino , Persona de Mediana Edad , Neuronas/metabolismo , Neuronas/patología , Enfermedades del Ovario/patología , Enfermedades del Ovario/cirugía , Ovariectomía , Ovario/inervación , Ovario/metabolismoRESUMEN
OBJECTIVE: To describe one case of syringocele in an adult patient. METHODS/RESULTS: We report the case of a 26 year old man who presented frequency, hematuria and fever during one year, mictional cystourethrography showed a syringocele. Treatment consisted in endoscopic surgery, with good results in the follow-up. CONCLUSIONS: The syringocele is a relatively infrequent entity, that is necessary to study in a young patient with voiding symptoms, accompanied or not of haematuria and fever. The diagnosis is based on the cystourethrography, and treatment consisted, usually, in endoscopic surgery.
Asunto(s)
Hernia/diagnóstico por imagen , Médula Espinal/diagnóstico por imagen , Enfermedades Uretrales/diagnóstico por imagen , Adulto , Humanos , Masculino , RadiografíaRESUMEN
Podocalyxin (PODXL) is a mucin protein of the CD34 family expressed in kidney glomerular podocytes, vascular endothelium, progenitor bone marrow and tumor cells. It is assumed that PODXL plays an anti-adherent role in kidney podocytes. CHO cells stably expressing human PODXL (CHO-PODXL) or human tumor cells (Tera-1) inherently expressing PODXL showed increased adherence to platelets. The adherence of cells was inhibited (70%) by blockers of platelet P-selectin, prevented by the soluble ectodomain of human PODXL (PODXL-Delta) or by the arginine-glycine-aspartate (RGDS) peptide and partially impeded by inhibition of integrin alphaVbeta3/alphaVbeta5, suggesting a coordinated action of P-selectin and integrins. Colocalization of platelet P-selectin and PODXL expressed on CHO cells was demonstrated by confocal immunofluorescence. No adherence to platelets was observed when PODXL was expressed in glycomutant CHO cells deficient in sialic acid.
Asunto(s)
Plaquetas/fisiología , Adhesión Celular/fisiología , Sialoglicoproteínas/fisiología , Animales , Secuencia de Bases , Células CHO , Cricetinae , Cricetulus , Cartilla de ADN/genética , ADN Complementario/genética , Glicosilación , Humanos , Integrinas/fisiología , Ratones , Mutación , Selectina-P/fisiología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sialoglicoproteínas/genética , TransfecciónRESUMEN
Paranasal sinuses and nose metastasis are very uncommon. About 50 have been reported. Renal cell carcinoma is the primary neoplasm which most frequently metastasizes in the nasosinusal region, followed by breast and lug. Symptoms are unspecific, but the epistaxis constitutes the most common sign due to the significant vascularizations of the tumor. Prognosis is poor. The survival rate fluctuates between 15-30% at 5 years. Surgery is the elective treatment.
Asunto(s)
Carcinoma de Células Renales/secundario , Neoplasias Renales , Neoplasias del Seno Maxilar/secundario , Antineoplásicos/uso terapéutico , Biopsia , Carcinoma de Células Renales/diagnóstico por imagen , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/radioterapia , Carcinoma de Células Renales/cirugía , Terapia Combinada , Femenino , Humanos , Interferones/uso terapéutico , Neoplasias Renales/cirugía , Seno Maxilar/patología , Neoplasias del Seno Maxilar/diagnóstico por imagen , Neoplasias del Seno Maxilar/tratamiento farmacológico , Neoplasias del Seno Maxilar/patología , Neoplasias del Seno Maxilar/radioterapia , Persona de Mediana Edad , Nefrectomía , Pronóstico , Factores de Tiempo , Tomografía Computarizada por Rayos XRESUMEN
Paranasal sinuses and nose metastasis are very uncommon tumors, about 50 have been reported. Renal cell carcinoma is the primary neoplasm which most frequently metastasizes in the nasosinusal region, followed by breast and lung. Symptoms are unspecific, but the epistaxis constitutes the most common sign due to the significant vascularizations of the tumor. Prognosis is poor. The survival rate fluctuates between 15-30% at 5 years. Surgery is the elective treatment.
Asunto(s)
Carcinoma de Células Renales/secundario , Neoplasias Renales/patología , Neoplasias del Seno Maxilar/diagnóstico por imagen , Neoplasias del Seno Maxilar/secundario , Carcinoma de Células Renales/diagnóstico por imagen , Femenino , Humanos , Persona de Mediana Edad , RadiografíaRESUMEN
Podocalyxin (podxl) is a protein with a peptide bone of approximately 55.5 kDa that undergoes a post-translational glycosylation, yielding a final molecular mass from approximately 145 to approximately 200 kDa. This protein is normally found covering the vascular side of the epithelial glomerular cells, the podocytes, and its presence is essential to maintain a normal renal function. It has also been reported in other cells and tissues although its function has not been yet clarified. The carboxy-terminal intracellular domain of podxl is nearly 100% identical in most species; however, the ectodomain shows considerable variations although the cysteine residues are conserved. Detection of this protein is elusive, most likely due to differences in post-translational modifications. We aimed at producing murine monoclonal antibodies against human podxl. Immunization with Chinese hamster ovarian -hpodxl-green fluorescence protein live cells yielded five different monoclonal antibodies that were characterized by enzyme-linked immunosorbent assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis/western blot, flow cytometry, immunohistochemistry, and immunoprecipitation. The different behavior of these antibodies suggests that some of them may react against epitopes masked by different glycosylated protein moieties.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Sialoglicoproteínas/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Ensayo de Inmunoadsorción Enzimática , Femenino , Eliminación de Gen , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunoquímica , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Sialoglicoproteínas/genéticaRESUMEN
Clear cell renal carcinoma is the most common histological type, representing 70-80% of all renal carcinomas. Metastases are already present in about 25-30% of patients at the time of diagnosis of renal cell carcinoma. Ocular metastasis is extremely rare.
Asunto(s)
Ceguera/etiología , Carcinoma de Células Renales/complicaciones , Carcinoma de Células Renales/secundario , Neoplasias del Ojo/complicaciones , Neoplasias del Ojo/secundario , Neoplasias Renales/patología , Anciano , Humanos , MasculinoRESUMEN
This work reports the study of a patient suffering a bleeding disorder clinically diagnosed as Glanzmann's thrombasthenia (GT). Immunoblotting and flow cytometric analysis showed a low (= 10% of control) platelet content of GPIIb-IIIa, confirming it was indeed a type II GT. The molecular genetic analysis of the proband revealed the presence of a homozygous G188A transition in GPIIb. This mutation alters the consensus sequence of the splice donor site of intron 1 changing arginine 63 for lysine (R63K). No other mutation than [G188A]GPIIb was found in the proband and her parents after complete analysis of GPIIb and GPIIIa coding sequences, and the promoter, 3'-UTR, and intronic flanking regions of GPIIb. The GT phenotype of the proband is the result of a limited availability of GPIIb-mRNA. The etiopathogenic role of the [G188A]GPIIb mutation is supported by the following observations: (i) both parents, who are heterozygous for the [G188A]GPIIb mutation, show a marked decrease in the platelet content of GPIIb-mRNA; (ii) exontrap analysis demonstrated that the G188A mutation leads to a marked reduction in the steady-state level of GPIIb-mRNA. The reduced availability of platelet GPIIb-mRNA associated with the G188A mutation seems to be caused by either inefficient RNA splicing or a preferred utilization of alternative intronic donor sites that generate an in-frame STOP codon with the result of activation of nonsense-mediated mRNA decay, or both.
Asunto(s)
Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria , Mutación Puntual , Sitios de Empalme de ARN/genética , Trombastenia/genética , Empalme Alternativo/genética , Codón sin Sentido , Secuencia Conservada/genética , Salud de la Familia , Femenino , Homocigoto , Humanos , Lactante , Mutación Missense , Estabilidad del ARN/genética , ARN Mensajero/análisisRESUMEN
Bowel is used in urological surgery to replace the bladder, either as a conduit to drain urine to the abdominal wall as a urinary stoma or refashioned to form a substitute bladder. Many factors contribute to stone formation, being urinary stasis, mucus production and bacteriuria the most important. Metabolic changes induced by exposure of segments of the alimentary tract to urine promote struvite, calcium oxalate and calcium phosphate stone formation. Generally, the majority of patients with stones in a urinary diversion can be treated with minimally invasive techniques. Open surgical removal is considered when other modality of treatments cannot be accomplished safely and expeditiously.
Asunto(s)
Complicaciones Posoperatorias , Cálculos Urinarios/etiología , Derivación Urinaria , Reservorios Urinarios Continentes , Trastornos Urinarios/etiología , Carcinoma de Células Transicionales/cirugía , Constricción Patológica , Creatinina/sangre , Cistectomía , Humanos , Obstrucción Intestinal/etiología , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/cirugía , Prostatectomía , Neoplasias de la Vejiga Urinaria/cirugía , Cálculos Urinarios/cirugía , Trastornos Urinarios/cirugíaRESUMEN
Primary retroperitoneal tumours may arise from different structures as neural, mesodermal, urogenital ridge, or embryonic remnant tissues. Lymphangioma is a rare benign tumour of the lymphatic tissue. They result from a developmental failure of the lymphatic system. Although benign, they can compress and infiltrative vital structures. The size of the lesion is more important than its location to the symptomatology development. Intraabdominal and retroperitoneal lymphangioma are the rarest tumour, specially when occurring in adults. The tumour can occur at any age and most are asymptomatic. Preoperative diagnosis is facilitated by ultrasonography and computed tomography. In order to correctly diagnose of these neoplasms it is essential to carry on ultrasound and CT examination. Treatment of choice is always surgical and a complete extirpation should be performed, unless vital structures were are involved.
Asunto(s)
Linfangioma Quístico , Neoplasias Retroperitoneales , Femenino , Humanos , Linfangioma Quístico/diagnóstico , Linfangioma Quístico/diagnóstico por imagen , Linfangioma Quístico/cirugía , Persona de Mediana Edad , Neoplasias Retroperitoneales/diagnóstico , Neoplasias Retroperitoneales/diagnóstico por imagen , Neoplasias Retroperitoneales/cirugía , Tomografía Computarizada por Rayos X , UltrasonografíaRESUMEN
This work reports the molecular cloning and functional characterization of the 5' flanking region of the human mitochondrial malic enzyme (mME) gene. The proximal promoter region has features of housekeeping genes like high G + C-content and absence of TATA or CCAAT boxes. Deletion analysis of the 5' region of the mME showed that maximal transcriptional activity is located within the -205/+86 region. Footprinting analysis showed two protected regions, one comprising potential overlapped AP-1, CREB, and AP-4 sites and a second one encompassing AP-2 and several Sp1 ci-acting elements. Mutation of putative AP-1/AP-4/CREB sites reduced basal promoter activity to less than 50%. Supershift assays demonstrated the specific binding of Sp1 and AP-2 proteins. Moreover, experiments in Drosophila SL2 cells lacking endogenous Sp1 demonstrated that the Sp1 site(s) is essential to maintain a normal basal rate of transcription of this gene. A low-level expression of AP-2 enhanced the activity of a mME promoter construct in HepG2 cells and this effect was prevented by disruption of the putative AP-2 element. In contrast, higher levels of expression of AP-2 induced a DNA-independent inhibitory response. A biphasic regulation of endogenous mME gene is also shown in HepG2 cells transfected with an AP-2 expression plasmid, suggesting that availability of AP-2 protein may control this gene under physiological conditions. A recombinant lambda genomic clone containing a mME pseudogene was also isolated. The high degree of sequence conservation seems to indicate a recent emergency of this human pseudogene.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Malato Deshidrogenasa/química , Malato Deshidrogenasa/genética , Mitocondrias/enzimología , Factor de Transcripción Sp1/fisiología , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Línea Celular , Clonación Molecular , ADN Complementario/metabolismo , Desoxirribonucleasa I/metabolismo , Desoxirribonucleasas/metabolismo , Relación Dosis-Respuesta a Droga , Eliminación de Gen , Biblioteca de Genes , Genes Reporteros , Humanos , Hígado/metabolismo , Datos de Secuencia Molecular , Placenta/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Seudogenes , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factor de Transcripción AP-2 , Transcripción Genética , TransfecciónRESUMEN
This work aimed at investigating the function of the [C674R] mutation in GPIIb that disrupts the intramolecular 674 to 687 disulfide bridge. Individuals heterozygous for this mutation show a platelet GPIIb-IIIa content approximately 30% of normal controls, which is less than expected from one normal functioning allele. Coexpression of normal [674C]GPIIb and mutant [674R]GPIIb with normal GPIIIa produced a [674R]GPIIb concentration-dependent inhibition of surface exposure of GPIIb-IIIa complexes in Chinese hamster ovary (CHO) cells, suggesting that [674R]GPIIb interferes with the association and/or intracellular trafficking of normal subunits. Mutation of either 674C or 687C had similar effects in reducing the surface exposure of GPIIb-IIIa. However, substitution of 674C for A produced a much lesser inhibition than R, suggesting that a positive-charged residue at that position renders a less efficient subunit conformation. The mutant [674R]GPIIb but not normal GPIIb was found associated with the endoplasmic reticulum chaperone BiP in transiently transfected CHO cells. BiP was also found associated with [674R]GPIIb-IIIa heterodimers, but not with normal GPIIIa or normal heterodimers. Overexpression of BiP did not increase the surface exposure of [674R]GPIIb-IIIa complexes, indicating that its availability was not a limiting step. Platelets from the thrombasthenic patient expressing [674R]GPIIb-IIIa were found to bind soluble fibrinogen in response to physiologic agonists or dithiothreitol treatment. Thus, the [674R]GPIIb mutation leads to a retardation of the secretory pathway, most likely related to its binding to the molecular chaperone BiP, with the result of a defective number of functional GPIIb-IIIa receptors in the cell surface.
Asunto(s)
Proteínas de Choque Térmico , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Animales , Células CHO , Proteínas Portadoras/metabolismo , Cricetinae , Chaperón BiP del Retículo Endoplásmico , Humanos , Chaperonas Moleculares/metabolismo , Mutación , Activación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Transducción de Señal , Relación Estructura-Actividad , Trombastenia/sangre , Trombastenia/genéticaRESUMEN
We have recently reported that lymphoblasts from late onset Alzheimer's disease (AD) patients show distinct intracellular pH homeostatic features than those obtained from age-matched healthy donors. Here we report that another distinct feature of AD lymphoblasts is their increased rate of proliferation in serum containing medium, suggesting a different responsiveness of AD cells to serum activators. The increased proliferation of AD cells was accompanied by intracellular alkalinization and was prevented by blockers of the plasma membrane Na+/H+ antiporter (NHE), indicating that the exchanger had to be activated to elicit the cellular responses. The activity of this exchanger can be controlled through several signaling pathways, but only the inhibition of calmodulin activity impeded the serum-induced intracellular alkalinization and enhanced proliferation of AD cells. In contrast, the inhibition of calmodulin did not alter the rate of proliferation of normal cells. Thus, it seems plausible to conclude that the enhanced proliferation of AD cells is the result of a surface receptor-mediated activation of the Ca(2+)-calmodulin signaling pathway. Our observations add further support in favor that AD may be considered a systemic disease which underlying etiopathogenic mechanism may be an altered responsiveness to cell activating agents. Thus, the use of lymphoblastoid cells from AD patients may be a useful model to investigate cell biochemical aspects of this disease.
Asunto(s)
Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/metabolismo , Calmodulina/metabolismo , División Celular/inmunología , Activación de Linfocitos/inmunología , Linfocitos/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Anciano , Enfermedad de Alzheimer/fisiopatología , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Ciclo Celular/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , División Celular/efectos de los fármacos , Línea Celular Transformada/citología , Línea Celular Transformada/inmunología , Línea Celular Transformada/metabolismo , Medios de Cultivo/farmacología , Humanos , Concentración de Iones de Hidrógeno , Líquido Intracelular/metabolismo , Linfocitos/inmunología , Transducción de Señal/inmunología , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Intercambiadores de Sodio-Hidrógeno/inmunologíaRESUMEN
We report the molecular genetic analysis of the Bernard-Soulier syndrome (BSS) phenotype in two related patients showing absence of glycoprotein (GP) Ibalpha and detectable amounts of GPIX on the platelet surface, and a truncated form of GPIbalpha in solubilized platelets and plasma. They both were compound heterozygotes for the GPIbalpha gene: a maternal allele with a T insertion at position 1418 causing a translational frameshift and premature polypeptide termination, and a paternal allele with a T715A substitution chan-ino Cys209 to Ser. Heterozygotes for either one of these mutations were asymptomatic. Transient transfection of cells coexpressing GPIbbeta and GPIX failed to detect surface expression of the GPIbalpha mutants. Cells transfected with [1418insT]GPIbalpha-cDNA showed a truncated protein of the predicted size in both cell lysate and conditioned medium, indicating the inability of the mutant protein to anchor the plasma membrane. In contrast. transfection of [T715A]GPIbalpha-cDNA yield a mutated protein barely detectable in the cell lysate and absent in the medium, indicating that the loss of Cys209 renders GPIbalpha more vulnerable to proteolysis and unable to undergo the normal secretory pathway. Our findings indicate that the additive effects of both mutations are responsible for the BSS phenotype of the patients.
Asunto(s)
Síndrome de Bernard-Soulier/genética , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Adulto , Alelos , Sustitución de Aminoácidos , Animales , Plaquetas/química , Células CHO , Codón sin Sentido , Cricetinae , Cricetulus , Análisis Mutacional de ADN , ADN Complementario/genética , Femenino , Mutación del Sistema de Lectura , Heterocigoto , Humanos , Masculino , Mutagénesis Insercional , Mutación Missense , Fenotipo , Complejo GPIb-IX de Glicoproteína Plaquetaria/química , Mutación Puntual , Reacción en Cadena de la Polimerasa , TransfecciónRESUMEN
We report data on the structural and functional characterization of the 5' flanking region of the human mitochondrial glycerol-3-phosphate dehydrogenase (mtGPDH) gene. We found two regions upstream of 5'-untranslated sequences exhibiting promoter activity in transient transfection assays. Transcription start sites and potential regulatory sites in both promoter regions were defined. The proximal promoter was approximately sevenfold more active than the distal one in most cell lines, but it was only twice as active in a neuroblastoma cell line. These observations seem to indicate that the rate of transcription, as well as the tissue-specific expression of the human mtGPDH gene, is the result of a combinatorial effect of transcription factors on at least two promoters. 3,5,3'-Triiodothyronine failed to alter the transcriptional activity of human mtGPDH promoter(s) constructs in transient transfection assays. Although this finding seems to be in conflict with the reported effect of 3,5,3'-triiodothyronine in rodents, it is consistent with our observation of 3,5, 3'-triiodothyronine stimulation of mtGPDH activity in primary cultures of rat adipocytes, but not human cultured adipocytes, suggesting distinct regulation of this gene in both species.
Asunto(s)
Tejido Adiposo/enzimología , Glicerolfosfato Deshidrogenasa/genética , Mitocondrias/enzimología , Secuencias Reguladoras de Ácidos Nucleicos , Triyodotironina/deficiencia , Secuencia de Bases , Clonación Molecular , ADN/genética , Huella de ADN , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
We report the molecular, genetic and functional analysis of a case of thrombasthenic phenotype. The proband showed absence of platelet glycoprotein (GP)IIb and very low content of GPIIIa, and both his parents showed a marked reduction in the levels of platelet GPIIb-IIIa. Single-stranded conformational polymorphism-polymerase chain reaction (SSCP-PCR) analysis and direct sequencing of PCR-amplified GPIIb exon-12 revealed the presence of a G-->A transition at position 1063 with the expected substitution of glutamate 324 with lysine (K). This mutation did not alter the level of GPIIb mRNA. Co-expression of normal or mutant [324K] GPIIb with normal human GPIIIa in Chinese hamster ovary (CHO) cells failed to show surface exposure of [324K]GPIIb-IIIa complexes. Pulse-chase and immunoprecipitation analysis demonstrated that [324K]GPIIb cDNA was translated into proGPIIb, but neither mutant GPIIb heavy chain (GPIIbH) nor [324K]GPIIb-GPIIIa complexes were detected, suggesting that this mutation is the underlying molecular basis for the thrombasthenic phenotype. Mutation analysis demonstrated that 324E of GPIIb could be replaced by other negatively charged or polar amino acids (AAs) without impairing the surface expression of GPIIb-IIIa. However, substitution of 324E of GPIIb for a positively charged AA other than K prevented the expression of GPIIb-IIIa complexes. These observations suggest that a domain encompassing 324E of GPIIb is essential for heterodimerization with GPIIIa and its substitution for a positively charged residue precludes normal subunit association.
Asunto(s)
Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Mutación Puntual , Trombastenia/genética , Animales , Secuencia de Bases , Plaquetas/metabolismo , Células CHO , Pollos , Preescolar , Cricetinae , Citometría de Flujo , Homocigoto , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Polimorfismo Conformacional Retorcido-Simple , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Trombastenia/sangre , XenopusRESUMEN
This work reports the molecular genetic analysis of two patients who suffer mucocutaneous haemorrhages, prolonged bleeding time and failure of platelets to aggregate, either spontaneously or in response to agonists. The absence of platelet surface glycoprotein (GP)IIb-IIIa complexes confirmed the clinical diagnosis of Glanzmann's thrombasthenia (GT). Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis of exon 2 of GPIIb showed polymorphic bands caused by the homozygous deletion of a cytosine at position 288 relative to the translation start site. causing a shifting of the reading frame and appearance of a premature termination codon. The heterozygous relatives showed a reduced platelet content of GPIIb-IIIa, and a correlation was found between the levels of GPIIb mRNA and surface expression of GPIIb-IIIa complexes. Unlike other mRNAs carrying a nonsense mutation, (288Cdel)GPIIb does not force alternative splicing of GPIIb mRNA. As expected, co-transfection of Chinese hamster ovary (CHO) cells with cDNAs encoding GPIIIa and (288delC)GPIIb failed to enhance the surface exposure of GPIIIa. It is concluded that the (288delC)GPIIb mutation is responsible for the thrombasthenic phenotype of the patients. In addition, it has also been determined that heterodimerization of GPIIb-IIIa requires the integrity of exons 2 and 3 of GPIIb.
Asunto(s)
Eliminación de Gen , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Trombastenia/genética , Adulto , Empalme Alternativo , Plaquetas/metabolismo , Consanguinidad , Exones , Femenino , Citometría de Flujo , Homocigoto , Humanos , Masculino , Linaje , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Polimorfismo Conformacional Retorcido-Simple , Análisis de Secuencia de ARNRESUMEN
The human malic enzyme (hME) promoter contains an inverted palindromic (IP4) 3,5,3'-triiodo-thyronine (T3) response element (T3RE) 15bp downstream from an activating protein-1 (AP-1) site. The purpose of this study was to analyze the functional relationship between both cis-acting elements. The following observations indicate that these two elements operate as a functional unit in controlling the human ME gene:T3 failed to stimulate transcription above the basal levels in cells overexpressing either TRb or TRb/retinoid acid receptor (RXR), indicating that TRbeta acts primarily as a transcriptional repressor in the context of the hME. Moreover, the finding of a repressive effect of TRbeta without DNA binding suggests the existence of both DNA-dependent and independent mechanisms of TRbeta-induced repression of transcription.
Asunto(s)
Malato Deshidrogenasa/genética , Receptores de Hormona Tiroidea/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Elementos de Respuesta/fisiología , Factor de Transcripción AP-1/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Humanos , Malato Deshidrogenasa/metabolismo , Mutación , Regiones Promotoras Genéticas , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Receptores de Hormona Tiroidea/efectos de los fármacos , Receptores de Hormona Tiroidea/genética , Receptores X Retinoide , Factor de Transcripción AP-1/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Triyodotironina/metabolismo , Triyodotironina/farmacologíaRESUMEN
This work was aimed at elucidating the molecular genetic lesion(s) responsible for the thrombasthenic phenotype of a patient whose low platelet content of glycoprotein (GP) IIb-IIIa indicated that it was a case of type II Glanzmann's thrombasthenia (GT). The parents did not admit consanguinity and showed a reduced platelet content of GPIIb-IIIa. Polymerase chain reaction (PCR)-single-stranded conformational polymorphism analysis of genomic DNA showed no mutations in the patient's GPIIIa and two novel mutations in the GPIIb gene: one of them was a heterozygous splice junction mutation, a C-->A transversion, at position +2 of the exon 5-intron 5 boundary [IVS5(+2)C-->A] inherited from the father. The predicted effect of this mutation, insertion of intron 5 (76 bp) into the GPIIb-mRNA, was confirmed by reverse transcription-PCR analysis of platelet mRNA. The almost complete absence of this mutated form of GPIIb-mRNA suggests that it is very unstable. Virtually all of the proband's GPIIb-mRNA was accounted for by the allele inherited from the mother showing a T2113-->C transition that changes Cys674-->Arg674 disrupting the 674-687 intramolecular disulfide bridge. The proband showed a platelet accumulation of proGPIIb and minute amounts of GPIIb and GPIIIa. Moreover, transfection and immunoprecipitation analysis demonstrated that [Arg674]GPIIb is capable of forming a heterodimer complex with GPIIIa, but the rate of subunit maturation and the surface exposure of GPIIb-IIIa are strongly reduced. Thus, the intramolecular 674-687 disulfide bridge in GPIIb is essential for the normal processing of GPIIb-IIIa complexes. The additive effect of these two GPIIb mutations provides the molecular basis for the thrombasthenic phenotype of the proband.