RESUMEN
The outbreak of SARS-CoV-2 pandemic highlighted the worldwide lack of surgical masks and personal protective equipment, which represent the main defense available against respiratory diseases as COVID-19. At the time, masks shortage was dramatic in Italy, the first European country seriously hit by the pandemic: aiming to address the emergency and to support the Italian industrial reconversion to the production of surgical masks, a multidisciplinary team of the University of Bologna organized a laboratory to test surgical masks according to European regulations. The group, driven by the expertise of chemical engineers, microbiologists, and occupational physicians, set-up the test lines to perform all the functional tests required. The laboratory started its activity on late March 2020, and as of the end of December of the same year 435 surgical mask prototypes were tested, with only 42 masks compliant to the European standard. From the analysis of the materials used, as well as of the production methods, it was found that a compliant surgical mask is most likely composed of three layers, a central meltblown filtration layer and two external spunbond comfort layers. An increase in the material thickness (grammage), or in the number of layers, does not improve the filtration efficiency, but leads to poor breathability, indicating that filtration depends not only on pure size exclusion, but other mechanisms are taking place (driven by electrostatic charge). The study critically reviewed the European standard procedures, identifying the weak aspects; among the others, the control of aerosol droplet size during the bacterial filtration test results to be crucial, since it can change the classification of a mask when its performance lies near to the limiting values of 95 or 98%.
RESUMEN
Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome characterized by fetal macrosomia, macroglossia, and abdominal wall defects. BWS patients are at risk to develop Wilms tumor, neuroblastoma, hepatoblastoma, and adrenal tumors. A young woman with BWS features, but with inconclusive genetic evidence for the disease, came to clinical observation for signs of virilization at the age of 16 years. An adrenocortical tumor was diagnosed and surgically resected. The tumor underwent 2 local relapses that were also surgically treated. The patient was also operated to remove a breast fibroadenoma. SNP arrays were used to analyze chromosome abnormalities in normal and tumor samples from the patient and her parents. The patient presented genome-wide mosaic paternal uniparental disomy (patUPD) both in the adrenocortical and the breast tumors, with different degrees of loss of heterozygosity (LOH). The more recent relapses of the adrenocortical tumor showed a loss of part of chromosome 17p that was absent in the first tumor. Analysis of a skin biopsy sample also showed mosaic patUPD with partial LOH, while no LOH was detected in leukocyte DNA. This case shows that virilizing adrenocortical tumors may be a clinical feature of patients with BWS. The SNP array technology is useful to diagnose genome-wide patUPD mosaicism in BWS patients with an inconclusive molecular diagnosis and underlines the tumorigenic potential of the absence of the maternal genome combined with an excess of the paternal genome.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/genética , Síndrome de Beckwith-Wiedemann/genética , Disomía Uniparental , Virilismo/genética , Adolescente , Femenino , Hirsutismo/genética , Humanos , Polimorfismo de Nucleótido Simple , Adulto JovenRESUMEN
A relevant gender difference exists in adrenal physiology and propensity to disease. In mice, a remarkable sexual dimorphism is present in several components of the hypothalamic-pituitary-adrenal axis, with females displaying higher adrenal weight, plasma ACTH, corticosterone, and aldosterone levels than males. The molecular bases of this sexual dimorphism are little known. We have compared global gene expression profiles in males vs. female mouse adrenal glands and also studied the effect that testosterone treatment and castration have on adrenal gene expression in female vs. male mice, respectively. Our study evidenced a set of 71 genes that are coordinately modulated according to sex and hormonal treatments and represent the core sexually dimorphic expression program in the mouse adrenal gland. Moreover, we show that some genes involved in steroid metabolism have a remarkable sexual dimorphic expression and identify new potential markers for the adrenal X-zone, a transitory cellular layer in the inner adrenal cortex, which spontaneously regresses at puberty in males and during the first pregnancy in females and has an uncertain physiological role. Finally, sexually dimorphic expression of the transcriptional regulators Nr5a1 and Nr0b1 may explain at least in part the differences in adrenal steroidogenesis between sexes.
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Glándulas Suprarrenales/metabolismo , Regulación de la Expresión Génica , Genoma/genética , Caracteres Sexuales , Glándulas Suprarrenales/efectos de los fármacos , Animales , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Testosterona/farmacologíaAsunto(s)
Neoplasias de la Corteza Suprarrenal/fisiopatología , Corteza Suprarrenal/fisiología , Factor Esteroidogénico 1/fisiología , Corteza Suprarrenal/crecimiento & desarrollo , Neoplasias de la Corteza Suprarrenal/veterinaria , Animales , Hurones , Regulación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Factor Esteroidogénico 1/genéticaRESUMEN
The molecular hallmark of the Ewing's family of tumors is the presence of balanced chromosomal translocations, leading to the formation of chimerical transcription factors (that is, EWS/FLI1) that play a pivotal role in the pathogenesis of Ewing's tumors by deregulating gene expression. We have recently demonstrated that DAX1 (NR0B1), an orphan nuclear receptor that was not previously implicated in cancer, is induced by the EWS/FLI1 oncoprotein and is highly expressed in Ewing's tumors, suggesting that DAX1 is a biologically relevant target of EWS/FLI1-mediated oncogenesis. In this study we demonstrate that DAX1 is a direct transcriptional target of the EWS/FLI1 oncoprotein through its binding to a GGAA-rich region in the DAX1 promoter and show that DAX1 is a key player of EWS/FLI1-mediated oncogenesis. DAX1 silencing using an inducible model of RNA interference induces growth arrest in the A673 Ewing's cell line and severely impairs its capability to grow in semisolid medium and form tumors in immunodeficient mice. Gene expression profile analysis demonstrated that about 10% of the genes regulated by EWS/FLI1 in Ewing's cells are DAX1 targets, confirming the importance of DAX1 in Ewing's oncogenesis. Functional genomic analysis, validated by quantitative RT-PCR, showed that genes implicated in cell-cycle progression, such as CDK2, CDC6, MCM10 or SKP2 were similarly regulated by EWS/FLI1 and DAX1. These findings indicate that DAX1 is important in the pathogenesis of the Ewing's family of tumors, identify new functions for DAX1 as a cell-cycle progression regulator and open the possibility to new therapeutic approaches based on DAX1 function interference.
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Neoplasias Óseas/genética , Ciclo Celular/genética , Proteínas de Unión al ADN/fisiología , Receptores de Ácido Retinoico/fisiología , Proteínas Represoras/fisiología , Sarcoma de Ewing/genética , Animales , Secuencia de Bases , Neoplasias Óseas/patología , Ciclo Celular/fisiología , Proliferación Celular/efectos de los fármacos , Análisis por Conglomerados , Receptor Nuclear Huérfano DAX-1 , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen/fisiología , Humanos , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Fusión Oncogénica/fisiología , Proteína Proto-Oncogénica c-fli-1 , ARN Interferente Pequeño/farmacología , Proteína EWS de Unión a ARN , Receptores de Ácido Retinoico/antagonistas & inhibidores , Receptores de Ácido Retinoico/genética , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Sarcoma de Ewing/patología , Factores de Transcripción/fisiología , Células Tumorales CultivadasRESUMEN
The MC2-Receptor (melanocortin 2 receptor, MC2-R) is a Gs-protein coupled receptor that is upregulated by its own ligand ACTH and by forskolin. The mechanisms regulating MC2-R expression are still unclear. We therefore investigated the role of the stimulatory transcription factors CREB and CREM and the inhibitory factor ICER for regulation of human MC2-R expression. We cotransfected mouse adrenocortical Y1 cells with luciferase reporter gene vectors containing full length and deleted human MC2-R promoter constructs with expression plasmids for CREB, CREBS133A, CREMtau, CREMtauS117A, or ICER. Direct protein-DNA interaction was investigated by EMSA. Wild type CREB did not significantly affect promoter activity due to high endogenous CREB activity. However, CREBS133A decreased forskolin stimulated MC2-R promoter activity by 48+/-5% (mean+/-SEM) while unstimulated values remained unchanged. CREMtau moderately increased basal and forskolin stimulated luciferase activity in a dose-dependent manner (maximum effect 252+/-24% and 186+/-13% VS. control vector, respectively). While this effect required the full length promoter, cAMP stimulation was retained in shorter constructs. ICER reduced basal luciferase activity in Y1 cells by 17+/-28%, but completely abolished forskolin stimulation. Although 5'-deletion constructs mapped the minimum promoter region required for ICER effect to the shortest -64/+40 construct, direct protein DNA interaction in this promoter region could not be identified by EMSA. Moreover, mutation of the SF-1 binding sites, which retained ICER dependent inhibition, excluded SF-1 to be required for this effect. We conclude from these data that transcription factors of the CREB/CREM/ATF family have a moderate effect on human MC2-R promoter activity, but seem to play a minor role in transmitting stimulation of the cAMP pathway to increased MC2-R expression.
Asunto(s)
Corteza Suprarrenal/metabolismo , Modulador del Elemento de Respuesta al AMP Cíclico/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Receptor de Melanocortina Tipo 2/genética , Animales , Línea Celular , Colforsina/farmacología , AMP Cíclico/farmacología , Regulación de la Expresión Génica , Humanos , Ratones , Regiones Promotoras Genéticas/efectos de los fármacos , TransfecciónRESUMEN
The cAMP-responsive element modulator (CREM) gene plays a pivotal role in the mouse spermatogenesis, but its role in the human infertility has not been fully established. We performed a mutation screening in 13 Slovenian men with round spermatid arrest and in six controls. Eleven genetic changes have been identified in the human CREM gene, three novel single-nucleotide polymorphisms [within the promoters P1, P3 and intervening sequence 1 (IVS1)], one insertion (IVS2) and one non-sense mutation (exon gamma). Some infertile patients seem to accumulate potentially harmful genetic changes. We identified a patient with no CREM immunoreactive protein that was homozygous for the nucleotide changes in all promoters, IVS 1, 2, 6, and was heterozygous for the mutation in exon gamma. Interestingly, insertion in IVS2 (IVS2-58_55insT) results in a four-fold decrease in binding of nuclear proteins. Computer predictions suggested the presence of a potential novel CREM promoter, however, random amplification of cDNA ends from the human testis cDNA library was not successful in confirming a novel transcription start site of the CREM gene. Screening of a larger number of patients and controls is required to elucidate whether the observed combinations of genetic changes in the CREM gene can explain some forms of male infertility.
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Modulador del Elemento de Respuesta al AMP Cíclico/genética , AMP Cíclico/fisiología , Infertilidad Masculina/genética , Elementos de Respuesta/genética , Adulto , Secuencia de Bases , Cromosomas Artificiales Bacterianos , Modulador del Elemento de Respuesta al AMP Cíclico/metabolismo , Marcadores Genéticos , Haplotipos , Humanos , Masculino , Datos de Secuencia Molecular , Mutación , Alineación de Secuencia , Análisis de Secuencia de ADN , Testículo/metabolismoRESUMEN
This study was aimed at evaluating serum osteoprotegerin (OPG) concentrations in a cohort of patients with hyperthyroidism before and after methimazole (MMI) treatment. One hundred fourteen hyperthyroid patients [93 with Graves disease (GD) and 21 with toxic nodular goitre (TNG)] and 68 matched for sex and age healthy subjects were evaluated for serum free-thyroxine (FT4), free-triiodiothyronine (FT3), thyrotropin (TSH), TSH receptor antibodies (TRAb), bone alkaline phosphatase (BALP), C-telopeptides of type-1 collagen (CrossLaps), OPG levels, and bone mineral density (BMD). In hyperthyroid patients, the biochemical evaluations were performed before and after 6 and 12 months of MMI treatment, whereas BMD was measured at baseline and after 12 months of treatment. Hyperthyroidism was more severe in GD than TNG patients. Serum OPG levels were found to be significantly higher in hyperthyroid patients than in the healthy subjects (4.3 pmol/l, range: 1.6-12.0, vs. 2.2 pmol/l, range: 1.4-6.0; P < 0.001), the values being higher in GD patients than TNG. A significant correlation between serum OPG levels and age was found in the healthy subjects (r: 0.48; P < 0.001) but not in hyperthyroid patients (r: -0.03; P = 0.8). In the healthy subjects, serum OPG levels were also positively correlated with both serum FT4 (r: 0.23; P = 0.03) and FT3 (r: 0.24; P = 0.04) levels. In hyperthyroid patients, however, serum OPG was still correlated with FT3 levels (r: 0.38; P < 0.001), whereas the correlation with serum FT4 was lost (r: 0.19; P = 0.06). In hyperthyroid patients, but not in the healthy subjects, serum OPG levels were correlated positively with CrossLaps (r: 0.20; P = 0.03) and negatively with BALP (r: -0.24; P = 0.01) and BMD (r: -0.33; P = 0.01). After 6 months of MMI treatment, serum OPG concentrations decreased significantly in TNG patients (from 3.5 pmol/l, range: 1.6-8.0, to 2.3 pmol/l, range: 1.0-4.3; P < 0.001), whereas a not significant change in OPG levels occurred in GD patients (from 4.8 pmol/l, range: 1.8-12.0, to 4.2 pmol/l, range: 1.0-14.0; P = 0.7). At Month 12 of treatment, serum OPG concentrations were significantly lower than those measured at baseline in both TNG (2.5 pmol/l, range: 1.0-3.1, vs. 3.5 pmol/l, range: 1.6-8.0; P < 0.001) and GD (2.1 pmol/l, range: 1.0-8.6, vs. 4.8 pmol/l, range: 1.8-12.0; P < 0.001). At this time, no significant differences in serum OPG, CrossLaps, and BALP values were found between patients and control subjects. At the end of follow-up, BMD was higher than those measured at baseline but still significantly lower than those measured in the control subjects. This study shows that hyperthyroid patients have serum OPG concentrations significantly higher in comparison with euthyroid subjects, in relation to thyroid hormone excess and high bone turnover. Medical treatment of hyperthyroidism normalizes serum OPG levels in temporal relationship with the normalization of bone metabolism markers, even in presence of persistent abnormal bone structure as determined by ultrasonography.
Asunto(s)
Glicoproteínas/sangre , Hipertiroidismo/sangre , Hipertiroidismo/tratamiento farmacológico , Metimazol/uso terapéutico , Receptores Citoplasmáticos y Nucleares/sangre , Adulto , Anciano , Distribución de Chi-Cuadrado , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Osteoprotegerina , Receptores del Factor de Necrosis Tumoral , Estadísticas no Paramétricas , Hormonas Tiroideas/sangreRESUMEN
Transcriptional induction by cAMP is mediated through the interaction of the cAMP response-element binding protein (CREB) with a cAMP response element (CRE) in the promoter of target genes. The steroidogenic acute regulatory (StAR) protein gene is regulated by cAMP-mediated signaling in steroidogenic cells even though its promoter lacks a consensus CRE. Previously, we have identified three highly conserved 5'-CRE half-sites within the -96/-67 bp region of the mouse StAR gene, and a member of the CREB family (CREB/CRE modulator (CREM)) was shown to be involved in its expression and regulation. Here we show that CREB and CREMtau (but not CREMalpha and CREMbeta) have qualitatively similar effects on StAR promoter activity in response to (Bu)(2)cAMP. Studies on the effects of the functional integrity of the CRE half-sites on CREB-dependent (Bu)(2)cAMP-mediated StAR gene transcription demonstrated the greater importance of the CRE2 site in comparison with the CRE1 and CRE3 sites. The CRE2 sequence was also found to bind specifically to recombinant CREB protein and nuclear extract from MA-10 mouse Leydig tumor cells. The cAMP and CREB/CREM responsive region (-151/-1 bp) of the mouse StAR promoter also contains three recognition motifs for steroidogenic factor 1 (SF-1). Electrophoretic mobility shift assays and reporter gene analyses demonstrated the involvement of different SF-1 elements in StAR gene expression with the order of importance being SF-1/3>SF-1/1>SF-1/2. Specific mutations that eliminated the binding sites of CRE and SF-1 elements, either alone or in combination, resulted in an attenuation of StAR promoter activity, indicating that CREB and SF-1 can regulate StAR gene transcription in a cooperative fashion. In addition, mammalian two-hybrid assays revealed a high affinity protein-protein interaction between CREB/CREMtau and SF-1 which appeared to be dependent upon CREB protein phosphorylation. These findings further demonstrate CREB's role in StAR gene transcription and also provide evidence that the combined action of CREB/CREMtau and SF-1 results in enhanced activation of the StAR promoter.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Proteínas de Unión al ADN/fisiología , Fosfoproteínas/genética , Factores de Transcripción/fisiología , Transcripción Genética/fisiología , Animales , Secuencia de Bases , Cartilla de ADN , Ensayo de Cambio de Movilidad Electroforética , Factores de Transcripción Fushi Tarazu , Proteínas de Homeodominio , Ratones , Receptores Citoplasmáticos y Nucleares , Factor Esteroidogénico 1 , Células Tumorales CultivadasRESUMEN
Here we describe the uncommon case of a Caucasian male with secondary hyperparathyroidism due to 8 parathyroid glands discovered in the course of a surgical exploration. The patient (age 49 yr) with a 21-yr history of chronic renal failure came to our observation in June 1999 complaining of depression, muscle weakness, bone and joint pain, movement hindrance. The biochemical evaluation evidenced low-normal serum calcium, high phosphorus and PTH levels. The symptoms and the biochemical findings were suggestive for secondary hyperparathyroidism. The neck US revealed an increase of thyroid gland volume with diffuse hyperechogenity; two nodules of 20 and 25 mm as maximum diameter were found in the thyroid parenchyma, while 4 hypoechogenous nodules (maximum diameter ranging from 13.0 to 30.0 mm) with clean borders and anechogenous areas inside were evidenced in the rear side of the thyroid lobes. The parathyroid scan with 99mTc and 201 Tl demonstrated increased uptake bilaterally in the inferior side of the neck. The patient underwent a total parathyroidectomy with near total thyroidectomy in November 1999. Histological examination of surgical specimen evidenced 6 hyperplastic parathyroid glands in back side of the 2 lobes (3 on the right and 3 on the left), and the examination of the thyroid gland showed 2 hyperplastic parathyroids (5 mm and 15 mm maximum diameter) into the 2 nodules previously evidenced by US. The physiopathological and clinical and therapeutic implications of this observation are discussed.
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Hiperparatiroidismo Secundario/etiología , Hiperparatiroidismo Secundario/cirugía , Glándulas Paratiroides/anomalías , Adulto , Humanos , Masculino , Glándulas Paratiroides/diagnóstico por imagen , Glándulas Paratiroides/patología , Glándulas Paratiroides/cirugía , Paratiroidectomía , Glándula Tiroides/diagnóstico por imagen , Glándula Tiroides/patología , Glándula Tiroides/cirugía , Tiroidectomía , UltrasonografíaRESUMEN
A key regulatory point in fine tuning of steroidogenesis is the synthesis of steroidogenic acute regulatory protein, which transfers cholesterol into mitochondria. Heat shock and toxic insults reduce steroidogenic acute regulatory protein, severely compromising steroid synthesis. As the molecular mechanisms for this reduction remain elusive, we tested the hypothesis that heat shock directly interferes with transcription of the steroidogenic acute regulatory protein gene. We show that, in mouse MA-10 Leydig tumor cells, heat shock caused drastic declines in (Bu)(2)cAMP-induced progesterone accumulation and steroidogenic acute regulatory protein transcript abundance. A proximal steroidogenic acute regulatory protein promoter fragment (-85 to +39) is sufficient to direct both cAMP inducibility and heat shock inhibition. Nuclear extracts from MA-10 cells displayed binding to this proximal promoter fragment as a low mobility complex in gel shift experiments. This complex disappeared in nuclear extracts taken at 5 and 10 min after initiation of heat shock and reappeared in extracts taken at 2 and 8 h. Similar low- mobility complexes formed on oligonucleotides representing the overlapping subfragments of the minimal steroidogenic acute regulatory protein promoter fragment sensitive to the heat shock effect. Extracts from heat-shocked MA-10 cells displayed reduced complex formation to each of the subfragments. We conclude that heat shock reduces progesterone synthesis, steroidogenic acute regulatory protein mRNA abundance, and steroidogenic acute regulatory protein promoter activity and disrupts binding of nuclear proteins to the proximal region of the steroidogenic acute regulatory protein promoter. Together these observations provide strong evidence for a mechanism of transcriptional inhibition in the down-regulation of steroidogenic acute regulatory protein expression by heat shock.
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Calor , Fosfoproteínas/genética , Esteroides/biosíntesis , Transcripción Genética , Animales , Northern Blotting , Western Blotting , Bucladesina/farmacología , Colesterol/metabolismo , ADN/metabolismo , Tumor de Células de Leydig/metabolismo , Ratones , Mitocondrias/metabolismo , Proteínas Nucleares/metabolismo , Progesterona/biosíntesis , Regiones Promotoras Genéticas , ARN Mensajero/análisis , Transfección , Células Tumorales CultivadasRESUMEN
The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) that is responsible for detoxifying isocaproaldehyde generated by steroidogenesis. In adrenocortical cell cultures, hormonal regulation of MVDP gene occurs through the cAMP pathway. We show that in adrenals, the pituitary hormone ACTH regulates MVDP gene expression in a coordinate fashion with steroidogenic genes. Cell transfection and DNA-binding studies were used to investigate the molecular mechanisms underlying MVDP gene regulation in Y1 adrenocortical cells. Progressive deletions of upstream regulatory regions identified a -121/+41 fragment that was sufficient for basal and cAMP-mediated transcriptional activities. Gel shift assays showed that CTF1/nuclear factor 1 (NF1), CCAAT enhancer binding protein-ss (C/EBPss), and selective promoter factor 1 (Sp1) factors bound to cis-acting elements at positions -76, -61, and -52, respectively. We report that the cell-specific steroidogenic factor-1 (SF-1) interacts specifically with a novel regulatory element located in the downstream half-site of the proximal androgen response element (AREp) at position -102. Functional analysis of SF-1 and NF1 sites in the -121/+41 promoter showed that mutation of one of them decreases both constitutive and forskolin-stimulated promoter activity without affecting the fold induction (forskolin stimulated/basal). Individual mutations of C/EBP and Sp1 sites resulted in a loss of more than 50% of the cAMP-dependent induction. When both sites were mutated simultaneously, cAMP responsiveness was nearly abolished. Thus, in adrenocortical cells, both SF-1 and NF1 are required for high expression of the MVDP promoter while Sp1 and C/EBPss functionally interact in an additive manner to mediate cAMP-dependent regulation. Furthermore, we report that MVDP gene regulation is impaired in stably transfected Y1 clones expressing DAX-1. Taken together, our findings suggest that detoxifying enzymes of the aldose reductase family may constitute new potential targets for regulators of adrenal and gonadal differentiation and function, e.g. SF-1 and DAX-1.
Asunto(s)
Corteza Suprarrenal/enzimología , Aldehído Reductasa/genética , Proteínas Potenciadoras de Unión a CCAAT/fisiología , Proteínas de Unión al ADN/fisiología , Proteínas Represoras , Factor de Transcripción Sp1/fisiología , Factores de Transcripción/fisiología , Aldo-Ceto Reductasas , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT/química , Proteínas Potenciadoras de Unión a CCAAT/genética , Línea Celular , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/farmacología , Receptor Nuclear Huérfano DAX-1 , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/farmacología , Factores de Transcripción Fushi Tarazu , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio , Masculino , Ratones , Mutagénesis Sitio-Dirigida , Factores de Transcripción NFI , ARN Mensajero/análisis , Receptores Citoplasmáticos y Nucleares , Receptores de Ácido Retinoico , Secuencias Reguladoras de Ácidos Nucleicos , Factor de Transcripción Sp1/química , Factor de Transcripción Sp1/genética , Factor Esteroidogénico 1 , Relación Estructura-Actividad , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/farmacologíaRESUMEN
Currently, replacement recombinant GH (rGH) therapy in GH-deficient (GHD) adults is performed in daily injections. This modality of treatment is not complied with by the totality of GHD patients, who are supposed to receive life-long replacement. The aim of our study was to compare daily vs. thrice weekly (TIW) rGH injection effects on lipid profile, body composition, bone metabolism, and bone density in 34 GHD patients (13 women and 21 men; median age, 39 yr; range, 30-55 yr) randomly assigned to different therapeutic regimens. Group A included 18 patients receiving daily rGH injections, and group B included 16 patients receiving TIW injections of rGH. The starting dose of rGH was 10 microg/kg x day in both groups. Subsequently, the dose was adjusted to maintain serum insulin-like growth factor I (IGF-I) concentrations in the normal age-adjusted range. IGF-I levels were assessed before and after 1, 3, 6, and 12 months of rGH treatment, and lipid profile, body composition, bone metabolism, and bone density were evaluated before and after 6 and 12 months of treatment. Thirty-four healthy subjects served as controls. In the basal condition, lipid profile, body composition, bone metabolism, and bone density were significantly different in patients compared to controls. Conversely, patients included in groups A and B had similar serum IGF-I levels, lipid profile, body composition, bone metabolism, and bone density. After 3 months of rGH treatment, IGF-I levels were normalized in 15 of 18 patients (83.3%) in group A and in 7 of 16 patients (43.7%) in group B (chi2 = 4.21; P = 0.04). At this time point, serum IGF-I levels in patients in group A (202+/-57.5 microg/L) were significantly higher than those in patients in group B (155+/-45.1 microg/L; P = 0.001). After 6 months of therapy, serum IGF-I levels were normalized in all patients and were similar in both groups (223+/-35.2 vs. 212+/-41.4 microg/L, A vs. B, respectively). IGF-I levels remained normal until the 12-month follow-up. After 6 months of rGH replacement, total cholesterol, low density lipoprotein cholesterol, triglycerides, bioelectrical impedance, and body fat mass were significantly reduced, whereas high density lipoprotein cholesterol levels and lean body mass were significantly increased in both groups of patients, without any difference between them. No further change in lipid profile and body composition was observed after 12 months of treatment. Serum bone GLA protein and procollagen III levels were significantly increased after 6 months, and a downward trend was observed after 12 months of rGH replacement. However, a slight, but significant, increase in bone mineral density was observed in both groups only after 12 months (P = 0.0001). All patients in group B had good compliance to the TIW treatment, whereas 5 patients in group A had poor compliance to the treatment (chi2 = 3.2; P = 0.07). In conclusion, our randomized, prospective, and controlled study confirmed that rGH therapy with TIW injection regimen is effective in normalizing IGF-I levels and improving lipid profile, body composition, bone metabolism, and bone density. It also demonstrated that this efficacy is comparable to that observed in patients treated with daily rhGH therapy, with few side-effects and good compliance.
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Hormona del Crecimiento/administración & dosificación , Hormona del Crecimiento/uso terapéutico , Hormona de Crecimiento Humana/deficiencia , Adulto , Envejecimiento/metabolismo , Composición Corporal/fisiología , Densidad Ósea , Huesos/metabolismo , Femenino , Prueba de Tolerancia a la Glucosa , Hormona del Crecimiento/efectos adversos , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Lípidos/sangre , Masculino , Persona de Mediana Edad , Estudios ProspectivosRESUMEN
An increasing body of evidence suggests that an endogenous mammalian bufadienolide (BD) may be involved in the regulation of Na(+),K(+)-ATPase activity and the pathogenesis of arterial hypertension. We developed a purification scheme for marinobufagenin (MBG), an amphibian cardiotonic BD, and applied it to purify and characterize material in human plasma, culture medium conditioned by Y-1 adrenocortical cells, and rat adrenal tissue. MBG immunoreactivity purified from plasma and measured by ELISA showed important similarities (chromatography and antibody cross-reactivity) to material secreted into cell culture medium by Y-1 cells. This observation indicates that circulating mammalian BD may have an adrenocortical origin. Release of mammalian BD from adrenocortical cells grown in the absence of exogenous cholesterol was reduced by treatment of cultures with mevastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. Supplementation of the serum and cholesterol-free cell culture medium with the LDL fraction of human plasma increased the production of MBG material in the presence of mevastatin, supporting its origin from cholesterol. We used Y-1 cell lines transfected with genes shown to inhibit steroidogenesis through cholesterol side-chain cleavage (Y-1/DAX and Y-1/RIAB) to investigate the dependence of MBG biosynthesis on side-chain cleavage. Our results indicate that the mammalian BD is synthesized in the adrenal cortex from cholesterol and shares important similarities with the amphibian BD MBG, that its biosynthesis is independent of transfer of cholesterol to the side-chain cleavage enzyme complex mediated by steroidogenic acute regulatory protein, and that neither cAMP nor protein kinase A appears to be a critical component of the pathway controlling its biosynthesis.
Asunto(s)
Corteza Suprarrenal/metabolismo , Bufanólidos/metabolismo , Colesterol/metabolismo , Animales , Femenino , Humanos , Ratones , Preeclampsia/metabolismo , Embarazo , Ratas , Células Tumorales CultivadasRESUMEN
The DAX-1 (NR0B1) gene encodes an unusual member of the nuclear hormone receptor superfamily which acts as a transcriptional repressor. Mutations in the human DAX-1 gene cause X-linked adrenal hypoplasia congenita (AHC) associated with hypogonadotropic hypogonadism (HHG). We have studied the intracellular localization of the DAX-1 protein in human adrenal cortex and mouse Leydig tumor cells and found it to be both nuclear and cytoplasmic. A significant proportion of DAX-1 is associated with polyribosomes and is found complexed with polyadenylated RNA. DAX-1 directly binds to RNA, two domains within the protein being responsible for cooperative binding activity and specificity. Mutations in DAX-1 found in AHC-HHG patients significantly impair RNA binding. These findings reveal that DAX-1 plays multiple regulatory roles at the transcriptional and posttranscriptional levels.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Polirribosomas/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Proteínas Represoras , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Corteza Suprarrenal/metabolismo , Hiperplasia Suprarrenal Congénita/genética , Animales , Sitios de Unión , Línea Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Receptor Nuclear Huérfano DAX-1 , Humanos , Hipogonadismo/genética , Células Intersticiales del Testículo/metabolismo , Células Intersticiales del Testículo/patología , Masculino , Ratones , Mutación , Poli A/metabolismo , Biosíntesis de Proteínas , Proteínas de Unión al ARN/genética , Secuencias Repetitivas de Aminoácido , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/ultraestructura , Temperatura , Neoplasias Testiculares/metabolismo , Neoplasias Testiculares/patologíaRESUMEN
DAX-1 is an unusual member of the nuclear hormone receptor superfamily whose expression is mainly, but not uniquely, restricted to steroidogenic tissues. We have recently shown that DAX-1 can block the first and rate-limiting step in steroid biosynthesis by repressing StAR (steroidogenic acute regulatory protein) expression. Here we show that DAX-1 blocks steroid production at multiple levels in the Y-1 mouse adrenocortical tumor cell line. Expression of DAX-1 in Y-1 cells significantly impairs both basal and cAMP-stimulated steroid production, without affecting the functionality of the cAMP-responsive PKA pathway. Experiments using an hydroxylated cholesterol derivative show that biochemical steps in steroidogenesis subsequent to cholesterol delivery to mitochondria are also impaired in Y-1 cells expressing DAX-1. This is explained by the repression of P450scc and 3beta-HSD expression, in addition to StAR. DAX-1 expression in Y-1 cells results in the inhibition of the activity of the StAR, P450scc and 3beta-HSD promoters. An inappropriate steroidogenic block in the male fetus might have an important role in the pathogenesis of sex reversal syndromes caused by a duplication of the genomic region of the X chromosome containing the DAX-1 gene.
Asunto(s)
Proteínas Represoras , Esteroides/biosíntesis , 3-Hidroxiesteroide Deshidrogenasas/biosíntesis , 3-Hidroxiesteroide Deshidrogenasas/genética , Animales , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/biosíntesis , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , AMP Cíclico/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Receptor Nuclear Huérfano DAX-1 , Proteínas de Unión al ADN/fisiología , Factores de Transcripción Fushi Tarazu , Proteínas de Homeodominio , Masculino , Ratones , Fosfoproteínas/biosíntesis , Fosfoproteínas/genética , Regiones Promotoras Genéticas , Receptores Citoplasmáticos y Nucleares , Receptores de Ácido Retinoico/fisiología , Factor Esteroidogénico 1 , Factores de Transcripción/fisiología , Células Tumorales CultivadasRESUMEN
The DAX-1 gene encodes an orphan nuclear hormone receptor essential for normal fetal development of the adrenal cortex. Recently, DAX-1 has been shown to act as a transcriptional repressor of steroidogenic acute regulatory protein gene expression (StAR), suppressing steroidogenesis. We, therefore, investigated the expression of DAX-1 in a variety of adrenocortical tumors and compared the results with StAR mRNA expression. We found low or absent DAX-1 expression in aldosterone-producing adenomas (n = 11: 35 +/- 11%; normal adrenals: 100 +/- 17%) and in aldosterone-producing adrenocortical carcinomas (n = 2: 24 and 36%). Cortisol-producing adenomas showed intermediate DAX-1 expression (n = 8; 92 +/- 16), as did 3 non-aldosterone-producing carcinomas (72, 132 and 132%). High DAX-1 expression was present in nonfunctional adenomas (n = 3; 160 +/- 17%). In contrast to DAX-1, StAR mRNA expression did not show significant variations between groups. We did not detect the expected negative correlation between DAX-1 and StAR in adrenocortical tumors. These data suggest that high DAX-1 expression in adrenocortical tumors is associated with a non-functional phenotype whereas low DAX-1 expression favors mineralocorticoid secretion. These effects on steroidogenesis are mediated by mechanisms other than repression of StAR gene expression. Our results indicate that DAX-1 may be one of the factors influencing the steroid biosynthesis of adrenocortical neoplasms.
Asunto(s)
Corticoesteroides/biosíntesis , Neoplasias de la Corteza Suprarrenal/genética , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica/fisiología , Receptores de Ácido Retinoico/genética , Proteínas Represoras , Esteroides/biosíntesis , Factores de Transcripción/genética , Adolescente , Adenoma Corticosuprarrenal/genética , Carcinoma Corticosuprarrenal/genética , Adulto , Anciano , Análisis de Varianza , Estudios de Casos y Controles , Niño , Preescolar , Receptor Nuclear Huérfano DAX-1 , Femenino , Humanos , Lactante , Masculino , Persona de Mediana EdadRESUMEN
Mutations in the DAX-1 gene are responsible for congenital X-linked adrenal hypoplasia, a disease that is associated with hypogonadotropic hypogonadism. DAX-1 expression is tissue-specific and is finely regulated throughout development, suggesting that it has a role in both adrenal and gonadal function. DAX-1 is an unusual member of the nuclear-receptor superfamily of transcription factors which contains no canonical zinc-finger or any other known DNA-binding motif. Binding sites for DAX-1 are found in the promoters of the dax-1 and StAR (for steroidogenic acute regulatory protein) genes. Here we show that DAX-1 binds DNA and acts as a powerful transcriptional repressor of StAR gene expression, leading to a drastic decrease in steroid production. We provide in vitro and in vivo evidence that DAX-1 binds to DNA hairpin structures. Our results establish DAX-1 as the first member of the nuclear receptor superfamily with novel DNA-binding features and reveal that it has regulatory properties critical to the understanding of its physiological functions.