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1.
FASEB J ; 35(7): e21727, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34117802

RESUMEN

We previously discovered the implication of membrane-type 5-matrix metalloproteinase (MT5-MMP) in Alzheimer's disease (AD) pathogenesis. Here, we shed new light on pathogenic mechanisms by which MT5-MMP controls the processing of amyloid precursor protein (APP) and the fate of amyloid beta peptide (Aß) as well as its precursor C99, and C83. We found in human embryonic kidney cells (HEK) carrying the APP Swedish familial mutation (HEKswe) that deleting the C-terminal non-catalytic domains of MT5-MMP hampered its ability to process APP and release the soluble 95 kDa form (sAPP95). Catalytically inactive MT5-MMP variants increased the levels of Aß and promoted APP/C99 sorting in the endolysosomal system, likely through interactions of the proteinase C-terminal portion with C99. Most interestingly, the deletion of the C-terminal domain of MT5-MMP caused a strong degradation of C99 by the proteasome and prevented Aß accumulation. These discoveries reveal new control of MT5-MMP over APP by proteolytic and non-proteolytic mechanisms driven by the C-terminal domains of the proteinase. The targeting of these non-catalytic domains of MT5-MMP could, therefore, provide new insights into the therapeutic regulation of APP-related pathology in AD.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Metaloproteinasas de la Matriz Asociadas a la Membrana/metabolismo , Fragmentos de Péptidos/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Línea Celular , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteolisis
2.
Mol Psychiatry ; 26(5): 1606-1618, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-32327736

RESUMEN

Autism spectrum disorders (ASD) are complex neurodevelopmental disorders with a very large number of risk loci detected in the genome. However, at best, each of them explains rare cases, the majority being idiopathic. Genomic data on ASD derive mostly from post-mortem brain analyses or cell lines derived from blood or patient-specific induced pluripotent stem cells (iPSCS). Therefore, the transcriptional and regulatory architecture of the nervous system, particularly during early developmental periods, remains highly incomplete. To access the critical disturbances that may have occurred during pregnancy or early childhood, we recently isolated stem cells from the nasal cavity of anesthetized patients diagnosed for ASD and compared them to stem cells from gender-matched control individuals without neuropsychiatric disorders. This allowed us to discover MOCOS, a non-mutated molybdenum cofactor sulfurase-coding gene that was under-expressed in the stem cells of most ASD patients of our cohort, disturbing redox homeostasis and synaptogenesis. We now report that a divergent transcription upstream of MOCOS generates an antisense long noncoding RNA, to which we coined the name COSMOC. Surprisingly, COSMOC is strongly under-expressed in all ASD patients of our cohort with the exception of a patient affected by Asperger syndrome. Knockdown studies indicate that loss of COSMOC reduces MOCOS expression, destabilizes lipid and energy metabolisms of stem cells, but also affects neuronal maturation and splicing of synaptic genes. Impaired expression of the COSMOC/MOCOS bidirectional unit might shed new lights on the origins of ASD that could be of importance for future translational studies.


Asunto(s)
Síndrome de Asperger , Trastorno del Espectro Autista , Células Madre Pluripotentes Inducidas , Trastornos del Neurodesarrollo , Sulfurtransferasas/genética , Trastorno del Espectro Autista/genética , Humanos , Sistema Nervioso
3.
Methods ; 191: 59-67, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-32599056

RESUMEN

The widespread availability of recombineered vectors and gene targeted embryonic stem cells from large-scale repositories facilitates the generation of mouse models for functional genetic studies. Southern blotting validates the structure of these targeted alleles produced by homologous recombination, as well as indicating any additional integrations of the vector into the genome. Traditionally this technique employs radioactively-labelled probes; however, there are many laboratories that are restricted in their use of radioactivity. Here, we present a widely applicable protocol for Southern blot analysis using cold probes and alternative procedures employing radioactive probes. Furthermore, the probes are designed to recognise standardised regions of gene-targeting cassettes and so represent universally applicable reagents for assessing allelic integrity.


Asunto(s)
Radiactividad , Alelos , Animales , Southern Blotting , Marcación de Gen , Vectores Genéticos , Recombinación Homóloga , Ratones
4.
Clin Endocrinol (Oxf) ; 94(2): 277-289, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33098107

RESUMEN

CONTEXT: The international GENHYPOPIT network collects phenotypical data and screens genetic causes of non-acquired hypopituitarism. AIMS: To describe main phenotype patterns and their evolution through life. DESIGN: Patients were screened according to their phenotype for coding sequence variations in 8 genes: HESX1, LHX3, LHX4, PROP1, POU1F1, TBX19, OTX2 and PROKR2. RESULTS: Among 1213 patients (1143 index cases), the age of diagnosis of hypopituitarism was congenital (24%), in childhood (28%), at puberty (32%), in adulthood (7.2%) or not available (8.8%). Noteworthy, pituitary hormonal deficiencies kept on evolving during adulthood in 49 of patients. Growth Hormone deficiency (GHD) affected 85.8% of patients and was often the first diagnosed deficiency. AdrenoCorticoTropic Hormone deficiency rarely preceded GHD, but usually followed it by over 10 years. Pituitary Magnetic Resonance Imaging (MRI) abnormalities were common (79.7%), with 39.4% pituitary stalk interruption syndrome (PSIS). The most frequently associated extrapituitary malformations were ophthalmological abnormalities (16.1%). Prevalence of identified mutations was 7.3% of index cases (84/1143) and 29.5% in familial cases (n = 146). Genetic analysis in 449 patients without extrapituitary phenotype revealed 36 PROP1, 2 POU1F1 and 17 TBX19 mutations. CONCLUSION: This large international cohort highlights atypical phenotypic presentation of constitutional hypopituitarism, such as post pubertal presentation or adult progression of hormonal deficiencies. These results justify long-term follow-up, and the need for systematic evaluation of associated abnormalities. Genetic defects were rarely identified, mainly PROP1 mutations in pure endocrine phenotypes.


Asunto(s)
Hipopituitarismo , Adulto , Estudios de Cohortes , Proteínas de Homeodominio/genética , Humanos , Hipopituitarismo/genética , Imagen por Resonancia Magnética , Mutación , Factores de Transcripción/genética
5.
Int Orthop ; 42(9): 2263-2272, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29744650

RESUMEN

PURPOSE: Degenerative disc disease involves sequential events that lead to the loss of cells, a decrease in disc matrix production, disc dehydration, and alteration of its biomechanical properties. The aim of this study was to determine whether cryoinjury of the nucleus pulposus performed through endplate perforation contributes to disc degeneration and to compare this technique with standard methods. METHOD: Under general anesthesia, the lumbar discs of six pigs were exposed and randomly submitted to needle puncture of the annulus fibrosus (NeP), isolated endplate injury (EP), or cryoinjury using a 2.5-J Thompson cryoprobe applied through a single endplate perforation (EP+cryo). The remaining discs served as controls. Animals were sacrificed at two months and the harvested lumbar spines were submitted to CT scan and MRI investigations. Histologic analysis was performed to assess the degree of disc degeneration. RESULTS: CT scan showed that decrease in average disc height was more important after cryoinjury (49.3%) than after endplate perforation (16.9%) (P < 0.0001) or needle puncture (19.4%) (P < 0.0001). On MRI, the dehydration ratio was significantly more important after EP+cryo (60%) than after NP (40%) or EP (30%) (P < 0.0001). After cryoinjury, the histologic score developed for this study was significantly higher than after needle puncture or endplate perforation (P < 0.0001). CONCLUSIONS: Imaging and histological analysis showed that disc cryoinjury applied through endplate perforation was superior to the classical NeP and EP models to induce experimental disc degeneration. This model appears suitable for testing safety and efficacy of novel treatments of intervertebral disc degeneration.


Asunto(s)
Criocirugía/métodos , Degeneración del Disco Intervertebral/etiología , Disco Intervertebral/lesiones , Animales , Criocirugía/veterinaria , Modelos Animales de Enfermedad , Femenino , Disco Intervertebral/patología , Degeneración del Disco Intervertebral/veterinaria , Vértebras Lumbares/patología , Imagen por Resonancia Magnética , Agujas , Distribución Aleatoria , Porcinos , Tomografía Computarizada por Rayos X
6.
Nat Methods ; 10(2): 125-7, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23263690

RESUMEN

We established a conditional site-specific recombination system based on dimerizable Cre recombinase-mediated recombination in the apicomplexan parasite Toxoplasma gondii. Using a new single-vector strategy that allows ligand-dependent, efficient removal of a gene of interest, we generated three knockouts of apicomplexan genes considered essential for host-cell invasion. Our findings uncovered the existence of an alternative invasion pathway in apicomplexan parasites.


Asunto(s)
Genoma de Protozoos , Toxoplasma/genética , Técnicas de Inactivación de Genes/métodos , Interacciones Huésped-Parásitos/genética , Integrasas
7.
Radiat Res ; 201(5): 429-439, 2024 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-38253061

RESUMEN

The current geopolitical context has brought the radiological nuclear risk to the forefront of concerns. High-dose localized radiation exposure leads to the development of a musculocutaneous radiation syndrome affecting the skin and subcutaneous muscles. Despite the implementation of a gold standard treatment based on an invasive surgical procedure coupled with autologous cell therapy, a muscular defect frequently persists. Targeting the modulation of the Hedgehog (Hh) signaling pathway appears to be a promising therapeutic approach. Activation of this pathway enhances cell survival and promotes proliferation after irradiation, while inhibition by Cyclopamine facilitates differentiation. In this study, we compared the effects of three antagonists of Hh, Cyclopamine (CA), Vismodegib (VDG) and Sonidegib (SDG) on differentiation. A stable cell line of murine myoblasts, C2C12, was exposed to X-ray radiation (5 Gy) and treated with CA, VDG or SDG. Analysis of proliferation, survival (apoptosis), morphology, myogenesis genes expression and proteins production were performed. According to the results, VDG does not have a significant impact on C2C12 cells. SDG increases the expression/production of differentiation markers to a similar extent as CA, while morphologically, SDG proves to be more effective than CA. To conclude, SDG can be used in the same way as CA but already has a marketing authorization with an indication against basal cell cancers, facilitating their use in vivo. This proof of concept demonstrates that SDG represents a promising alternative to CA to promotes differentiation of murine myoblasts. Future studies on isolated and cultured satellite cells and in vivo will test this proof of concept.


Asunto(s)
Proteínas Hedgehog , Músculo Esquelético , Regeneración , Transducción de Señal , Animales , Ratones , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/antagonistas & inhibidores , Músculo Esquelético/efectos de la radiación , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/citología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Línea Celular , Regeneración/efectos de los fármacos , Regeneración/efectos de la radiación , Piridinas/farmacología , Alcaloides de Veratrum/farmacología , Anilidas/farmacología , Compuestos de Bifenilo/farmacología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Desarrollo de Músculos/efectos de los fármacos , Desarrollo de Músculos/efectos de la radiación
8.
Science ; 385(6711): eado2032, 2024 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-39172837

RESUMEN

Clathrin-mediated endocytosis has characteristic features in neuronal dendrites and presynapses, but how membrane proteins are internalized along the axon shaft remains unclear. We focused on clathrin-coated structures and endocytosis along the axon initial segment (AIS) and their relationship to the periodic actin-spectrin scaffold that lines the axonal plasma membrane. A combination of super-resolution microscopy and platinum-replica electron microscopy on cultured neurons revealed that AIS clathrin-coated pits form within "clearings", circular areas devoid of actin-spectrin mesh. Actin-spectrin scaffold disorganization increased clathrin-coated pit formation. Cargo uptake and live-cell imaging showed that AIS clathrin-coated pits are particularly stable. Neuronal plasticity-inducing stimulation triggered internalization of the clathrin-coated pits through polymerization of branched actin around them. Thus, spectrin and actin regulate clathrin-coated pit formation and scission to control endocytosis at the AIS.


Asunto(s)
Actinas , Axones , Clatrina , Endocitosis , Espectrina , Animales , Humanos , Ratas , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Axones/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Clatrina/metabolismo , Vesículas Cubiertas por Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Células HEK293 , Plasticidad Neuronal , Neuronas/metabolismo , Espectrina/metabolismo
9.
J Neuroendocrinol ; 35(6): e13287, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-37322808

RESUMEN

Deficient anterior pituitary with common variable immune deficiency (DAVID) syndrome is a rare condition characterized by adrenocorticotropic hormone (ACTH) deficiency and primary hypogammaglobulinemia. It is due to heterozygous mutations of the nuclear factor kappa-B subunit 2 (NFKB2) gene. Only a few isolated cases have been reported since its first description by our team. Through the international multicenter GENHYPOPIT network, we identified a new case of DAVID syndrome. We then conducted an extensive review of the DAVID syndrome cases published from 2012 to 2022. A 7-year-old boy was diagnosed with symptomatic hypoglycemia revealing ACTH deficiency. Laboratory tests showed asymptomatic hypogammaglobulinemia. He harbored a heterozygous point mutation in NFKB2 gene (c.2600C > T, p.Ala867Val). His management included hydrocortisone replacement treatment, and he also received subcutaneous immunoglobulins during the Covid-19 pandemic. We analyzed 28 cases of DAVID syndrome with ACTH deficiency. ACTH deficiency was the only hormone deficiency in 79% of patients, but some patients harbored growth hormone (GH) and thyroid stimulating hormone (TSH) deficiencies. The first presenting symptoms were sinus/pulmonary infections (82%, mean age of 3 years) and alopecia (mean age of 4.7 years). ACTH deficiency was the third presenting condition (mean age at diagnosis of 8.6 years). All patients had hypogammaglobulinemia (decreased IgA and IgM levels), and 57% of patients had at least one autoimmune manifestation. Heterozygous mutations at the 3'end of the NFKB2 gene, coding for the C-terminal domain of the protein, were identified in all cases. Better knowledge of DAVID syndrome will help clinicians make an early diagnosis to avoid life-threatening complications.


Asunto(s)
Inmunodeficiencia Variable Común , Hormonas Adenohipofisarias , Adulto , Niño , Femenino , Humanos , Masculino , Hormona Adrenocorticotrópica/deficiencia , Agammaglobulinemia/complicaciones , Autoinmunidad , Inmunodeficiencia Variable Común/complicaciones , Inmunodeficiencia Variable Común/genética , Inmunodeficiencia Variable Común/inmunología , Inmunodeficiencia Variable Común/fisiopatología , Heterocigoto , Hormona de Crecimiento Humana/deficiencia , Infecciones/complicaciones , Madres , Mutación , Fenotipo , Hormonas Adenohipofisarias/deficiencia , Síndrome , Tirotropina/deficiencia
10.
J Cell Biol ; 222(10)2023 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-37578754

RESUMEN

The architecture of the actin cytoskeleton that concentrates at presynapses remains poorly known, hindering our understanding of its roles in synaptic physiology. In this work, we measure and visualize presynaptic actin by diffraction-limited and super-resolution microscopy, thanks to a validated model of bead-induced presynapses in cultured neurons. We identify a major population of actin-enriched presynapses that concentrates more presynaptic components and shows higher synaptic vesicle cycling than their non-enriched counterparts. Pharmacological perturbations point to an optimal actin amount and the presence of distinct actin structures within presynapses. We directly visualize these nanostructures using Single Molecule Localization Microscopy (SMLM), defining three distinct types: an actin mesh at the active zone, actin rails between the active zone and deeper reserve pools, and actin corrals around the whole presynaptic compartment. Finally, CRISPR-tagging of endogenous actin allows us to validate our results in natural synapses between cultured neurons, confirming the role of actin enrichment and the presence of three types of presynaptic actin nanostructures.


Asunto(s)
Actinas , Nanoestructuras , Neuronas , Sinapsis , Neuronas/fisiología , Sinapsis/fisiología , Vesículas Sinápticas , Citoesqueleto , Células Cultivadas
11.
J Cell Biochem ; 113(6): 2047-56, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22274864

RESUMEN

Mesenchymal stem cells (MSC) can differentiate into osteoblasts upon activation of Wnt signaling. Identifying targets of Wnt signaling in MSC may help promote MSC osteoblast differentiation for bone regeneration. In this study, using microarray analysis we found that Wnt3a upregulates neuregulin 1 (NRG-1) during Wnt3a-induced osteoblast differentiation in primary human MSC and murine C3H10T1/2 mesenchymal cells. Western blot and qPCR analyses confirmed that NRG-1 is upregulated by Wnt3a, and that this effect was counterbalanced by decreased expression of the NRG-1 receptor ErbB3. Consistently, exogenous NRG-1 had no effect on alkaline phosphatase (ALP) activity, an early marker of osteoblast differentiation. In contrast, small interfering RNA-mediated silencing of endogenous NRG-1 increased basal and Wnt3a-induced ALP activity in MSC. We showed that short hairpin (sh) ErbB3 and Wnt3a additively increased ß-catenin transcriptional activity and ALP activity in MSC. These effects were abrogated by DKK1, indicating that cross-talk between Wnt3a and ErbB3 control MSC osteoblast differentiation via Wnt/ß-catenin signaling. Furthermore, ErbB3 silencing decreased Src expression. Pharmacological inhibition of Src signaling promoted ErbB3- and Wnt-induced ALP activity, suggestive of a role of Src signaling in the modulation of osteoblast differentiation by ErbB3 and Wnt3a. The results indicate that downregulation of ErbB3 induced by Wnt3a contributes to Wnt3a-induced early osteoblast differentiation of MSCs through increased canonical Wnt/ß-catenin signaling and decreased Src signaling.


Asunto(s)
Células Madre Mesenquimatosas/citología , Neurregulina-1/biosíntesis , Osteoblastos/citología , Osteoblastos/metabolismo , Osteogénesis , Receptor ErbB-3/biosíntesis , Proteína Wnt3A/metabolismo , Fosfatasa Alcalina/biosíntesis , Androstadienos/farmacología , Animales , Apoptosis , Diferenciación Celular , Línea Celular , Proliferación Celular , Regulación hacia Abajo , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C3H , Neurregulina-1/genética , Neurregulina-1/metabolismo , Osteogénesis/genética , Interferencia de ARN , ARN Interferente Pequeño , Transcripción Genética , Vía de Señalización Wnt , Proteína Wnt3A/genética , Wortmanina , beta Catenina/biosíntesis , beta Catenina/genética , Familia-src Quinasas/metabolismo
12.
Cell Rep ; 40(7): 111200, 2022 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-35977506

RESUMEN

Apolipoprotein E4 (APOEε4) is the major allelic risk factor for late-onset sporadic Alzheimer's disease (sAD). Inflammation is increasingly considered as critical in sAD initiation and progression. Identifying brain molecular mechanisms that could bridge these two risk factors remain unelucidated. Leveraging induced pluripotent stem cell (iPSC)-based strategies, we demonstrate that APOE controls inflammation in human astrocytes by regulating Transgelin 3 (TAGLN3) expression and, ultimately, nuclear factor κB (NF-κB) activation. We uncover that APOE4 specifically downregulates TAGLN3, involving histone deacetylases activity, which results in low-grade chronic inflammation and hyperactivated inflammatory responses. We show that APOE4 exerts a dominant negative effect to prime astrocytes toward a pro-inflammatory state that is pharmacologically reversible by TAGLN3 supplementation. We further confirm that TAGLN3 is downregulated in the brain of patients with sAD. Our findings highlight the APOE-TAGLN3-NF-κB axis regulating neuroinflammation in human astrocytes and reveal TAGLN3 as a molecular target to modulate neuroinflammation, as well as a potential biomarker for AD.


Asunto(s)
Enfermedad de Alzheimer , Apolipoproteína E4 , Apolipoproteínas E/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Enfermedad de Alzheimer/metabolismo , Apolipoproteína E3/metabolismo , Apolipoproteína E4/metabolismo , Apolipoproteínas E/genética , Astrocitos/metabolismo , Humanos , Inflamación/metabolismo , FN-kappa B/metabolismo
13.
Med Sci (Paris) ; 25(3): 267-72, 2009 Mar.
Artículo en Francés | MEDLINE | ID: mdl-19361390

RESUMEN

Despite constant progress in radiotherapy techniques such as tumour imaging and cartography, techniques of radiation delivery or fractionation schedules, damage to normal gastro-intestinal tissues is inevitably associated with radiation therapy of pelvic tumours. Acute radiation enteritis concerns 80% of patients. It is related to stem cell loss, default in epithelial regenerating capacity and inflammation-induced mucosal dystrophy and ulceration. Chronic injury may develop in 5 to 10% of patients and is characterized by intestinal wall fibrosis resulting from an exaggerated scarring process, chronic inflammation and tissue necrosis. Research in mechanistic processes of normal tissue damage paved the way for new therapeutic approaches to emerge. These new targets include mucosal regeneration, reduction of vascular activation, inflammation and thrombosis, and fight against mesenchymal cells sustained activation. Effective strategies are multiple on preclinical models, but numerous efforts have to be made to achieve the complicated goal of protection of normal tissues from the side effects of radiation therapy.


Asunto(s)
Enfermedades Gastrointestinales/etiología , Neoplasias/radioterapia , Neoplasias Pélvicas/radioterapia , Traumatismos por Radiación/epidemiología , Radioterapia/efectos adversos , Animales , Enteritis/epidemiología , Humanos , Ratas
14.
Eur J Hum Genet ; 27(2): 216-225, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30262920

RESUMEN

LHX3 is an LIM domain transcription factor involved in the early steps of pituitary ontogenesis. We report here functional studies of three allelic variants, including the first heterozygous variant of LHX3 NM_178138.5(LHX3):c.587T>C (p.(Leu196Pro)) that may be responsible for a milder phenotype of hypopituitarism. Our functional studies showed that NM_178138.5(LHX3):c.587T>C (p.(Leu196Pro)) was not able to activate target promoters in vitro, as it did not bind DNA, and likely affected LHX3 function via a mechanism of haplo-insufficiency. Our study demonstrates the possibility that patients with a heterozygous variant of LHX3 may have pituitary deficiencies, with a milder phenotype than patients with homozygous variants. It is thus of vital to propose an optimal follow-up of such patients, who, until now, were considered as not being at risk of presenting pituitary deficiency. The second variant NM_178138.5(LHX3):c.622C>G (p.(Arg208Gly)), present in a homozygous state, displayed decreased transactivating ability without loss of binding capacity in vitro, concordant with in silico analysis; it should thus be considered to affect LHX3 function. In contrast, the NM_178138.5(LHX3):c.929G>C (p.(Arg310Pro)) variant, in a heterozygous state, also predicted as deleterious in silico, proved functionally active in vitro, and should thus still be classified as a variant of unknown significance. Our study emphasizes the need for functional studies due to the limits of software-based predictions of new variants, and the possible association of a pituitary phenotype to heterozygous LHX3 variants.


Asunto(s)
Hipopituitarismo/genética , Proteínas con Homeodominio LIM/genética , Mutación Missense , Fenotipo , Factores de Transcripción/genética , Adulto , Animales , Células COS , Preescolar , Chlorocebus aethiops , Femenino , Pruebas Genéticas/métodos , Pruebas Genéticas/normas , Células HEK293 , Heterocigoto , Humanos , Hipopituitarismo/patología , Proteínas con Homeodominio LIM/química , Proteínas con Homeodominio LIM/metabolismo , Masculino , Unión Proteica , Conformación Proteica , Factores de Transcripción/química , Factores de Transcripción/metabolismo
15.
Genesis ; 46(4): 193-9, 2008 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-18395834

RESUMEN

We examined the use of ERT2-iCre-ERT2 (Cre2ERT2), a tamoxifen-regulated form of Cre that has been described to have a background activity lower than that of other tamoxifen-regulated Cre constructs, for establishing performant conditional deleter mouse lines. Cre2ERT2 was inserted by homologous recombination into the Rosa26 locus. These mice were mated with R26R Cre-reporter mice. No recombination could be observed in the progenies in the absence of tamoxifen treatment. Tamoxifen treatment at E13-14 led to a high level, albeit variable, recombination in most of the tissues examined: liver, heart, kidney, brain, lung etc. Treatment of adult animals also induced recombination in these tissues, although at a lower level. Northern blot and qPCR studies suggested that these differences are not linked to significant variations of the level of expression of Cre2ERT2. Thus, Cre2ERT2 appears to be a good alternative to existing modulatable Cre systems, displaying a lack of background activity and a high-level inducibility in vivo.


Asunto(s)
Técnicas de Transferencia de Gen , Integrasas/genética , Animales , Células Cultivadas , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas/genética , ARN no Traducido , Recombinación Genética/genética
16.
Health Phys ; 115(1): 37-48, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29787429

RESUMEN

The cutaneous radiation syndrome is the clinical consequence of local high-dose irradiation. It is characterized by extensive inflammation, necrosis, and poor revascularization of the skin, resulting in muscle inflammation and fibrosis. Based on these physiopathological processes, subcutaneous injections of adipose-tissue-derived stem/stromal cells have shown favorable effects on skin-wound healing in a minipig model of cutaneous radiation syndrome, in which muscle fibrosis persisted. Since fibrosis is mainly due to the inflammatory processes that often affect underlying tissues as well, the beneficial effects of intramuscular injections of adipose-tissue-derived stem/stromal cells on tissue recovery were evaluated. The polarization of the inflammatory response of irradiated muscle in a minipig model of cutaneous radiation syndrome was determined after acute local irradiation with 50 Gy gamma rays in a preliminary study (six minipigs). Analysis of the main inflammatory cytokines of the inflammatory response M1 (IL-1-beta and IL-6) and M2 (IL-10 and TGF-beta) by western blotting and in situ hybridization, as well as analysis of CD80/CD206 M1/M2 macrophage-specific markers by immunohistochemistry on minipig muscle samples, was performed 76 d after irradiation. The treatment of irradiated muscles with autologous adipose-tissue-derived stem/stromal cells led to an increase in IL-10 and TGF-beta, being associated with an increase in CD68+/CD206+ cells in this area. This highlights a polarization of M2 in the inflammatory response and indicates that adipose-tissue-derived stem/stromal cells may direct the irradiated tissues' inflammatory response towards a proregenerative outcome.


Asunto(s)
Tejido Adiposo/citología , Inflamación/inmunología , Rayos Infrarrojos/efectos adversos , Músculo Esquelético/efectos de la radiación , Traumatismos por Radiación/inmunología , Enfermedades de la Piel/inmunología , Trasplante de Células Madre/métodos , Células Madre/citología , Animales , Células Cultivadas , Femenino , Inflamación/etiología , Inflamación/terapia , Músculo Esquelético/inmunología , Músculo Esquelético/lesiones , Traumatismos por Radiación/etiología , Traumatismos por Radiación/terapia , Enfermedades de la Piel/etiología , Enfermedades de la Piel/terapia , Porcinos , Porcinos Enanos , Cicatrización de Heridas/inmunología
17.
Ann Endocrinol (Paris) ; 78(2): 77-79, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28483366

RESUMEN

Through the multicentric international GENHYPOPIT network, 10 transcription factor genes involved in pituitary development have been screened in more than 1200 patients with constitutional hypopituitarism over the past two decades. The present report summarizes the main lessons learned from this phenotype-based genetic screening: (1) genetically determined hypopituitarism does not necessarily present during childhood; (2) constitutional hypopituitarism may be characterized by a pure endocrine phenotype or by various combinations of endocrine deficits and visceral malformations; (3) syndromic hypopituitarism may also be observed in patients with POU1F1 or PROP1 mutations; (4) in cases of idiopathic hypopituitarism, extensive genetic screening identifies gene alterations in a minority of patients; (5) functional studies are imperfect in determining the involvement of an allelic variant in a specific pituitary phenotype.


Asunto(s)
Hormona de Crecimiento Humana/deficiencia , Hipopituitarismo/etiología , Adulto , Niño , Proteínas de Homeodominio/genética , Humanos , Hipopituitarismo/genética , Hipopituitarismo/patología , Hipófisis/patología , Factor de Transcripción Pit-1/genética
18.
Horm Res Paediatr ; 88(3-4): 274-280, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28898885

RESUMEN

BACKGROUND/AIM: Congenital hypothyroidism (CH) is a heterogeneous entity. Neonatal screening programs based on thyrotropin (TSH) determination allow primary CH diagnosis but miss central CH (CCH). CCH causes morbidity, alerts to other pituitary deficiencies, and is more prevalent than previously thought. We aimed at developing a pilot neonatal screening program for CCH detection. PATIENTS AND METHODS: A prospective 2-year pilot neonatal screening study based on simultaneous dried blood specimen TSH and thyroxine (T4) measurements was implemented in term newborns aged 2-7 days. Those with T4 ≤4.5 µg/dL (-2.3 SDS) and TSH <10 mIU/L were recalled (suspicious of CCH) and underwent clinical and biochemical assessment performed by expert pediatric endocrinologists. RESULTS: A total of 67,719 newborns were screened. Primary CH was confirmed in 24 (1: 2,821). Forty-four newborns with potential CCH were recalled (recall rate 0.07%) at a mean age of 12.6 ± 4.8 days. In this group, permanent CCH was confirmed in 3 (1: 22,573), starting L-T4 treatment at a mean age of 12.3 ± 6.6 days; 14 boys showed T4-binding globulin deficiency (1: 4,837); 24 had transient hypothyroxinemia (21 non-thyroidal illness and 3 healthy); and 3 died before the confirmation stage. According to initial free T4 measurements, CCH patients had moderate hypothyroidism. CONCLUSIONS: Adding T4 to TSH measurements enabled the identification of CCH as a prevalent condition and contributed to improving the care of newborns with congenital hypopituitarism and recognizing other thyroidal disorders.


Asunto(s)
Hipotiroidismo Congénito/diagnóstico , Tamizaje Neonatal/métodos , Hipotiroidismo Congénito/sangre , Femenino , Humanos , Recién Nacido , Masculino , Proyectos Piloto , Estudios Prospectivos , Pruebas de Función de la Tiroides , Tirotropina/sangre , Tiroxina/sangre , Triyodotironina/sangre
19.
FEBS J ; 273(8): 1772-81, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16623712

RESUMEN

Somatic angiotensin-converting enzyme (ACE) contains two homologous domains, each bearing a functional active site. Studies on the selectivity of these ACE domains towards either substrates or inhibitors have mostly relied on the use of mutants or isolated domains of ACE. To determine directly the selectivity properties of each ACE domain, working with wild-type enzyme, we developed an approach based on the combined use of N-domain-selective and C-domain-selective ACE inhibitors and fluorogenic substrates. With this approach, marked differences in substrate selectivity were revealed between rat, mouse and human somatic ACE. In particular, the fluorogenic substrate Mca-Ala-Ser-Asp-Lys-DpaOH was shown to be a strict N-domain-selective substrate of mouse ACE, whereas with rat ACE it displayed marked C-domain selectivity. Similar differences in selectivity between these ACE species were also observed with a new fluorogenic substrate of ACE, Mca-Arg-Pro-Pro-Gly-Phe-Ser-Pro-DpaOH. In support of these results, changes in amino-acid composition in the binding site of these three ACE species were pinpointed. Together these data demonstrate that the substrate selectivity of the N-domain and C-domain depends on the ACE species. These results raise concerns about the interpretation of functional studies performed in animals using N-domain and C-domain substrate selectivity data derived only from human ACE.


Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Oligopéptidos/farmacología , Peptidil-Dipeptidasa A/química , Ácidos Fosfínicos/farmacología , Animales , Sitios de Unión , Combinación de Medicamentos , Colorantes Fluorescentes , Humanos , Ratones , Ratones Endogámicos C57BL , Peptidil-Dipeptidasa A/metabolismo , Ratas , Ratas Endogámicas Lew , Relación Estructura-Actividad , Especificidad por Sustrato
20.
Nucleic Acids Res ; 31(21): e131, 2003 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-14576331

RESUMEN

Cre recombinase is extensively used to engineer the genome of experimental animals. However, its usefulness is still limited by the lack of an efficient temporal control over its activity. To overcome this, we have developed DiCre, a regulatable fragment complementation system for Cre. The enzyme was split into two moieties that were fused to FKBP12 (FK506-binding protein) and FRB (binding domain of the FKBP12-rapamycin-associated protein), respectively. These can be efficiently heterodimerized by rapamycin. Several variants, based on splitting Cre at different sites and using different linker peptides, were tested in an indicator cell line. The fusion proteins, taken separately, had no recombinase activity. Stable transformants, co-expressing complementing fragments based on splitting Cre between Asn59 and Asn60, displayed low background activity affecting 0.05-0.4% of the cells. Rapamycin induced a rapid recombination, reaching 100% by 48-72 h, with an EC50 of 0.02 nM. Thus, ligand-induced dimerization can efficiently regulate Cre, and should be useful to achieve a tight temporal control of its activity, such as in the case of the creation of conditional knock-out animals.


Asunto(s)
Integrasas/química , Integrasas/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Dimerización , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Prueba de Complementación Genética , Integrasas/genética , Ligandos , Modelos Moleculares , Fragmentos de Péptidos/genética , Estructura Cuaternaria de Proteína/efectos de los fármacos , Ratas , Eliminación de Secuencia/genética , Sirolimus/farmacología , Transfección , Proteínas Virales/genética
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