RESUMO
Depending on endoplasmic reticulum (ER) stress levels, the ER transmembrane multidomain protein IRE1α promotes either adaptation or apoptosis. Unfolded ER proteins cause IRE1α lumenal domain homo-oligomerization, inducing trans autophosphorylation that further drives homo-oligomerization of its cytosolic kinase/endoribonuclease (RNase) domains to activate mRNA splicing of adaptive XBP1 transcription factor. However, under high/chronic ER stress, IRE1α surpasses an oligomerization threshold that expands RNase substrate repertoire to many ER-localized mRNAs, leading to apoptosis. To modulate these effects, we developed ATP-competitive IRE1α Kinase-Inhibiting RNase Attenuators-KIRAs-that allosterically inhibit IRE1α's RNase by breaking oligomers. One optimized KIRA, KIRA6, inhibits IRE1α in vivo and promotes cell survival under ER stress. Intravitreally, KIRA6 preserves photoreceptor functional viability in rat models of ER stress-induced retinal degeneration. Systemically, KIRA6 preserves pancreatic ß cells, increases insulin, and reduces hyperglycemia in Akita diabetic mice. Thus, IRE1α powerfully controls cell fate but can itself be controlled with small molecules to reduce cell degeneration.
Assuntos
Estresse do Retículo Endoplasmático , Endorribonucleases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Regulação Alostérica , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Endorribonucleases/química , Endorribonucleases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Retina/metabolismo , Ribonucleases/antagonistas & inibidoresRESUMO
MASEI is the main validated ultrasound score for the evaluation of enthesis. The lack of studies facing the agreement to achieve for the interpretation of the MAdrid Sonographic Enthesis Index (MASEI) among researchers from different centers in multicenter studies is of concern. The aim of this multicenter was to evaluate the interobserver reliability of MASEI. An experienced ultrasonographer-rheumatologist performed ultrasound scans of the areas included in MASEI index in three patients with Ankylosing Spondylitis and Psoriatic Arthritis. Videos were captured. The videos were then evaluated by 24 rheumatologists of the ultrasound working group of the Catalan Society of Rheumatology (EcoCAT). A face-to-face training meeting was held. Ten days after the workshop, the study participants evaluated the videos. A reliability assessment was performed. The ICC for the MASEI scores after the workshop was of 0.97 (95% CI 89-99). Reliability did not vary statistically with examiner experience. Globally, no problems of reliability by structures were seen, and all the ICCs were above 0.90 and improved slightly after the educational program. However, the correlation observed between examiners at plantar aponeursis and triceps tendon was weak. The small variability observed in the results of the index validation in our study, suggests that the MASEI index is reproducible by different observers when those are well trained and show awesome results of the enthesis when examined by ultrasound.
Assuntos
Sistema Musculoesquelético/diagnóstico por imagem , Espondiloartropatias/diagnóstico por imagem , Ultrassonografia/métodos , Adulto , Idoso , Feminino , Humanos , Masculino , Variações Dependentes do Observador , Reprodutibilidade dos Testes , Reumatologia/educação , Reumatologia/métodos , Índice de Gravidade de DoençaRESUMO
Gi-GPCRs, G protein-coupled receptors that signal via Gα proteins of the i/o class (Gαi/o), acutely regulate cellular behaviors widely in mammalian tissues, but their impact on the development and growth of these tissues is less clear. For example, Gi-GPCRs acutely regulate insulin release from pancreatic ß cells, and variants in genes encoding several Gi-GPCRs--including the α-2a adrenergic receptor, ADRA2A--increase the risk of type 2 diabetes mellitus. However, type 2 diabetes also is associated with reduced total ß-cell mass, and the role of Gi-GPCRs in establishing ß-cell mass is unknown. Therefore, we asked whether Gi-GPCR signaling regulates ß-cell mass. Here we show that Gi-GPCRs limit the proliferation of the insulin-producing pancreatic ß cells and especially their expansion during the critical perinatal period. Increased Gi-GPCR activity in perinatal ß cells decreased ß-cell proliferation, reduced adult ß-cell mass, and impaired glucose homeostasis. In contrast, Gi-GPCR inhibition enhanced perinatal ß-cell proliferation, increased adult ß-cell mass, and improved glucose homeostasis. Transcriptome analysis detected the expression of multiple Gi-GPCRs in developing and adult ß cells, and gene-deletion experiments identified ADRA2A as a key Gi-GPCR regulator of ß-cell replication. These studies link Gi-GPCR signaling to ß-cell mass and diabetes risk and identify it as a potential target for therapies to protect and increase ß-cell mass in patients with diabetes.
Assuntos
Proliferação de Células , Diabetes Mellitus Tipo 2/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Células Secretoras de Insulina/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Transdução de Sinais , Animais , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Glucose/genética , Glucose/metabolismo , Células Secretoras de Insulina/patologia , Camundongos , Camundongos Transgênicos , Receptores Adrenérgicos alfa 2/genéticaRESUMO
To gain further insight on the estrogen-dependent transcriptional regulation of the uteroglobin (UG) gene, we cloned the 5'-flanking region of the UG gene from the phylogenetically ancient volcano rabbit (Romerolagus diazi; Rd). The cloned region spans 812 base pairs (bp; -812/-1) and contains a noncanonical TATA box (TACA). The translation start site is 48 bp downstream from the putative transcription initiation site (AGA), and is preceded by a consensus Kozak box. Comparison of the Rd-UG gene with that previously isolated from rabbits (Oryctolagus cuniculus) showed 93% in sequence identity as well as a number of conserved cis-acting elements, including the estrogen-response element (ERE; -265/-251), which differs from the consensus by two nucleotides. In MCF-7 cells, 17ß-estradiol (E(2)) induced transcription of a luciferase reporter driven by the Rd-UG promoter in a similar manner as in an equivalent rabbit UG reporter; the Rd-UG promoter was 30% more responsive to E(2) than the rabbit promoter. Mutagenesis studies on the Rd-ERE confirmed this cis-element as a target of E(2) as two luciferase mutant reporters of the Rd-promoter, one with the rabbit and the other with the consensus ERE, were more responsive to the hormone than the wild-type reporter. Gel shift and super-shift assays showed that estrogen receptor-α indeed binds to the imperfect palindromic sequence of the Rd-ERE.
Assuntos
Estrogênios/fisiologia , Lagomorpha/genética , Regiões Promotoras Genéticas , Elementos de Resposta , Uteroglobina/genética , Animais , Sequência de Bases , Linhagem Celular Tumoral , Clonagem Molecular , Ensaio de Desvio de Mobilidade Eletroforética , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Regulação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Iniciação Traducional da Cadeia Peptídica , Coelhos/genética , Sequências Reguladoras de Ácido Nucleico , Alinhamento de Sequência , TATA Box/genética , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.
Assuntos
Glândulas Mamárias Animais/crescimento & desenvolvimento , Mosaicismo , Organoides/crescimento & desenvolvimento , Tripsina/metabolismo , Animais , Diferenciação Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio/crescimento & desenvolvimento , Matriz Extracelular/metabolismo , Feminino , Camundongos , Técnicas de Cultura de Tecidos , Fixação de TecidosRESUMO
Growth cone response to the bifunctional guidance cue netrin-1 is regulated by the activity of intracellular signaling intermediates such as protein kinase C-alpha (PKCalpha) and adenylyl cyclase. Among the diverse cellular events these enzymes regulate is receptor trafficking. Netrin-1, itself, may govern the activity of these signaling intermediates, thereby regulating axonal responses to itself. Alternatively, other ligands, such as activators of G protein-coupled receptors, may regulate responses to netrin-1 by governing these signaling intermediates. Here, we investigate the mechanisms controlling activation of PKCalpha and the subsequent downstream regulation of cell surface UNC5A receptors. We report that activation of adenosine receptors by adenosine analogs, or activation of the putative netrin-1 receptor, the G protein-coupled receptor adenosine A2b receptor (A2bR) results in PKCalpha-dependent removal of UNC5A from the cell surface. This decrease in cell surface UNC5A reduces the number of growth cones that collapse in response to netrin-1 and converts repulsion to attraction. We show these A2bR-mediated alterations in axonal response are not because of netrin-1 because netrin-1 neither binds A2bR, as assayed by protein overlay, nor stimulates PKCalpha-dependent UNC5A surface loss. Our results demonstrate that netrin-1-independent A2bR signaling governs the responsiveness of a neuron to netrin-1 by regulating the levels of cell surface UNC5A receptor.
Assuntos
Axônios/metabolismo , Membrana Celular/metabolismo , Fatores de Crescimento Neural/fisiologia , Receptor A2B de Adenosina/metabolismo , Proteínas Supressoras de Tumor/fisiologia , Agonistas do Receptor A2 de Adenosina , Animais , Axônios/efeitos dos fármacos , Células COS , Células Cultivadas , Galinhas , Chlorocebus aethiops , Receptores de Netrina , Netrina-1 , Ligação Proteica/fisiologia , Ratos , Receptor A2B de Adenosina/fisiologia , Receptores de Superfície Celular/agonistas , Receptores de Superfície Celular/metabolismoRESUMO
It is well established that DNA damage induces checkpoint-mediated interphase arrest in higher eukaryotes, but recent studies demonstrate that DNA damage delays entry into anaphase as well. Damaged DNA in syncytial and gastrulating Drosophila embryos delays the metaphase/anaphase transition . In human cultured cells, DNA damage also induces a delay in mitosis . However, the mechanism by which DNA damage delays the anaphase onset is controversial. Some studies implicate a DNA damage checkpoint , whereas other studies invoke a spindle checkpoint . To resolve this issue, we compared the effects of random DNA breaks induced by X-irradiation to site-specific I-CreI endonuclease-induced chromosome breaks on cell-cycle progression in wild-type and checkpoint-defective Drosophila neuroblasts. We found that both the BubR1 spindle checkpoint pathway and the Grp/Chk1 DNA damage checkpoint pathway are involved in delaying the metaphase/anaphase transition after extensive X-irradiation-induced DNA damage, whereas Grp/Chk1, but not BubR1, is required to delay anaphase onset in the presence of I-CreI-induced double-strand breaks. On the basis of these results, we propose that DNA damage in nonkinetochore regions produces a Grp/Chk1 DNA-damage-checkpoint-mediated delay in the metaphase/anaphase transition.
Assuntos
Anáfase/fisiologia , Dano ao DNA/fisiologia , Drosophila/crescimento & desenvolvimento , Metáfase/fisiologia , Proteínas Quinases/fisiologia , Animais , Encéfalo/fisiologia , Proteínas de Ciclo Celular , Quinase 1 do Ponto de Checagem , Análise Mutacional de DNA , Enzimas de Restrição do DNA , Drosophila/genética , Proteínas de Drosophila , Larva/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Raios XRESUMO
Breast tumor progression is accompanied by changes in the surrounding extracellular matrix (ECM) that increase stiffness of the microenvironment. Mammary epithelial cells engage regulatory pathways that permit dynamic responses to mechanical cues from the ECM. Here, we identify a SLIT2/ROBO1 signaling circuit as a key regulatory mechanism by which cells sense and respond to ECM stiffness to preserve tensional homeostasis. We observed that Robo1 ablation in the developing mammary gland compromised actin stress fiber assembly and inhibited cell contractility to perturb tissue morphogenesis, whereas SLIT2 treatment stimulated Rac and increased focal adhesion kinase activity to enhance cell tension by maintaining cell shape and matrix adhesion. Further investigation revealed that a stiff ECM increased Robo1 levels by down-regulating miR-203. Consistently, patients whose tumor expressed a low miR-203/high Robo1 expression pattern exhibited a better overall survival prognosis. These studies show that cells subjected to stiffened environments up-regulate Robo1 as a protective mechanism that maintains cell shape and facilitates ECM adherence.
Assuntos
Adesão Celular/genética , Forma Celular/genética , Matriz Extracelular/genética , Quinase 1 de Adesão Focal/genética , MicroRNAs/genética , Proteínas do Tecido Nervoso/genética , Receptores Imunológicos/genética , Proteínas rac de Ligação ao GTP/genética , Animais , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Forma Celular/fisiologia , Microambiente Celular/genética , Microambiente Celular/fisiologia , Regulação para Baixo/genética , Células Epiteliais/fisiologia , Matriz Extracelular/fisiologia , Homeostase/genética , Homeostase/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Glândulas Mamárias Humanas/fisiologia , Camundongos , Morfogênese/genética , Morfogênese/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas RoundaboutRESUMO
OBJECTIVE: Ostomy patients face a number of problems that impact negatively on their personal welfare. The aim of this research is determine the nature and intensity of the relationship between the level of self-concept adaptive mode and the consistent use of coping strategies of older adults with a stoma. METHODOLOGY: Quantitative, correlational and transversal. VIVEROS 03 and CAPS surveys were applied in 3 hospitals in the City of Durango, México. The study included 90 older adults with an intestinal elimination stoma with complications. Kendall's Tau-b coefficient was the non-parametric test used to measure this association. RESULTS: Most older adults analyzed (61.3 < % < 79.9) are not completely adapted to the condition of living with an intestinal stoma. There is also a moderate positive correlation (0,569) between the level of adaptation of the older adults with a stoma and the conscious use of coping strategies. CONCLUSIONS: The presence of an intestinal stoma represents a physical and psychological health problem that is reflected in the level of adaptation of the self-image. Elderly people with a stoma use only a small part of defense mechanisms as part of coping process. This limits their ability to face the adversities related to their condition, potentially causing major health complications.
Assuntos
Adaptação Psicológica , Mecanismos de Defesa , Enterostomia/psicologia , Autoimagem , Idoso , Estudos Transversais , Enterostomia/efeitos adversos , Feminino , Humanos , MasculinoRESUMO
The mammary gland develops through several distinct stages. The first transpires in the embryo as the ectoderm forms a mammary line that resolves into placodes. Regulated by epithelialmesenchymal interactions, the placodes descend into the underlying mesenchyme and produce the rudimentary ductal structure of the gland present at birth. Subsequent stages of developmentpubertal growth, pregnancy, lactation, and involutionoccur postnatally under the regulation of hormones. Puberty initiates branching morphogenesis, which requires growth hormone (GH) and estrogen, as well as insulin-like growth factor 1 (IGF1), to create a ductal tree that fills the fat pad. Upon pregnancy, the combined actions of progesterone and prolactin generate alveoli, which secrete milk during lactation. Lack of demand for milk at weaning initiates the process of involution whereby the gland is remodeled back to its prepregnancy state. These processes require numerous signaling pathways that have distinct regulatory functions at different stages of gland development. Signaling pathways also regulate a specialized subpopulation of mammary stem cells that fuel the dramatic changes in the gland occurring with each pregnancy. Our knowledge of mammary gland development and mammary stem cell biology has significantly contributed to our understanding of breast cancer and has advanced the discovery of therapies to treat this disease.
Assuntos
Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Morfogênese , Animais , Diferenciação Celular , Linhagem da Célula , Feminino , Hormônios/metabolismo , Humanos , Mamíferos/embriologia , Mamíferos/crescimento & desenvolvimento , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Humanas/citologia , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
In the field of breast biology, there is a growing appreciation for the "gatekeeping function" of basal cells during development and disease processes yet mechanisms regulating the generation of these cells are poorly understood. Here, we report that the proliferation of basal cells is controlled by SLIT/ROBO1 signaling and that production of these cells regulates outgrowth of mammary branches. We identify the negative regulator TGF-ß1 upstream of Robo1 and show that it induces Robo1 expression specifically in the basal layer, functioning together with SLIT2 to restrict branch formation. Loss of SLIT/ROBO1 signaling in this layer alone results in precocious branching due to a surplus of basal cells. SLIT2 limits basal cell proliferation by inhibiting canonical WNT signaling, increasing the cytoplasmic and membrane pools of ß-catenin at the expense of its nuclear pool. Together, our studies provide mechanistic insight into how specification of basal cell number influences branching morphogenesis.
Assuntos
Proliferação de Células , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Glândulas Mamárias Animais/metabolismo , Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Receptores Imunológicos/fisiologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Proteína Axina , Western Blotting , Adesão Celular , Movimento Celular , Proteínas do Citoesqueleto/fisiologia , Feminino , Fatores de Transcrição Forkhead/fisiologia , Glândulas Mamárias Animais/citologia , Camundongos , Camundongos Knockout , Camundongos Nus , Morfogênese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta1/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Proteínas RoundaboutRESUMO
To gain further insights on the genetic divergence and the species-specific characteristics of the follicle-stimulating hormone receptor (FSHR), we cloned 946 bp of the 5'-flanking region of the FSHR gene from the volcano mouse (Neotomodon alstoni alstoni), and compared its features with those from other mammalian species. The sequence of neotomodon FSHR (nFSHR) gene from the translation initiation site to -946 is 74, 71, 64, and 59% homologous to rat, mouse (129/J), human, and sheep, respectively. The nFSHR 5'-flanking region exhibits new interesting putative cis-regulatory elements including those for the SRY transcription factor, which had not been previously related to the FSHR gene. The transcriptional regulation properties of nFSHR gene were studied in mouse Sertoli (MSC-1) and non-Sertoli (H441) cell lines, and compared with those obtained with similar 129/J constructs. All constructs tested were more active in H441 than in MSC-1 cells. The low transcription levels detected in MSC-1 cells probably reflect the recruitment of Sertoli cells-specific nuclear factors that repress transcription of the FSHR gene. In H441 cells, 129/J constructs were more active than their neotomodon counterparts, indicating important species-specific differences in their transcription pattern. Functional analysis of a series of progressive 5'-deletion mutants identified regions involved in positive and negative transcriptional regulation as well as the strongest minimal promoter spanning 260 bp upstream the translation initiation site. The identification of inhibitory nuclear transcription factors, which are apparently expressed in MSC-1 cells, may contribute to a better understanding of the transcriptional regulation of the FSHR gene.
Assuntos
Arvicolinae/genética , Arvicolinae/metabolismo , Regiões Promotoras Genéticas , Receptores do FSH/química , Receptores do FSH/genética , Região 5'-Flanqueadora/genética , Sequência Rica em At , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Genes Reporter , Genes sry , Masculino , Camundongos , Camundongos da Linhagem 129 , Dados de Sequência Molecular , Receptores do FSH/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Células de Sertoli/metabolismoRESUMO
The genes encoding Slits and their Robo receptors are silenced in many types of cancer, including breast, suggesting a role for this signaling pathway in suppressing tumorigenesis. The molecular mechanism underlying these tumor-suppressive effects has not been delineated. Here, we show that loss of Slits, or their Robo1 receptor, in murine mammary gland or human breast carcinoma cells results in coordinate up-regulation of the Sdf1 and Cxcr4 signaling axis, specifically within mammary epithelium. This is accompanied by hyperplastic changes in cells and desmoplastic alterations in the surrounding stroma. A similar inverse correlation between Slit and Cxcr4 expression is identified in human breast tumor tissues. Furthermore, we show in a xenograft model that Slit overexpression down-regulates CXCR4 and dominantly suppresses tumor growth. These studies classify Slits as negative regulators of Sdf1 and Cxcr4 and identify a molecular signature in hyperplastic breast lesions that signifies inappropriate up-regulation of key prometastatic genes.
Assuntos
Neoplasias da Mama/patologia , Carcinoma/patologia , Quimiocina CXCL12/genética , Inativação Gênica/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Receptores CXCR4/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma/genética , Carcinoma/metabolismo , Proliferação de Células , Quimiocina CXCL12/metabolismo , Regulação para Baixo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Glândulas Mamárias Humanas/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Camundongos Nus , Proteínas do Tecido Nervoso/genética , Receptores CXCR4/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/fisiologia , Transplante Heterólogo , Células Tumorais Cultivadas , Proteínas RoundaboutRESUMO
Analysis of the transcriptional regulation of the Clara cell secretory protein (CCSP) gene has resulted in the characterization of several trans-acting factors that regulate the activity of this gene. However, little is known about negative regulatory elements involved in CCSP gene transcription. Using transient transfections of luciferase reporter constructs driven by various fragments of the Neotomodon CCSP (nCCSP) promoter, we identified an inhibitory region that contains an inverted CCAAT box located -225 to -221 bp upstream of the transcriptional start site. Sequence analysis in a broad region of the nCCSP promoter (-744/+33) identified another potentially important CCAAT motif (-459/-455). Gel shift and supershift assays indicated that the transcription factor NF-Y binds to both CCAAT boxes. Mutation of the CCAAT motif prevented the in vitro binding of NF-Y and led to a significant increase of CCSP promoter activity in both pulmonary (H441) and non-pulmonary (HeLa and MCF-7) cells, suggesting that NF-Y is involved in a negative transcriptional regulation that may potentially contribute to the highly cell-specific expression of the anti-inflammatory CCSP gene.
Assuntos
Fator de Ligação a CCAAT/genética , Fator de Ligação a CCAAT/metabolismo , Regulação da Expressão Gênica/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Uteroglobina/metabolismo , Animais , Camundongos , Regiões Promotoras Genéticas/genética , Uteroglobina/genéticaRESUMO
To better understand the phylogenetic divergence and the species-specific characteristics of the Clara cell secretory protein (CCSP), we cloned the cDNA encoding the neotomodon CCSP (nCCSP) and analyzed its tissue-specific expression. The full-length cDNA is 451bp long and predicts an amino acid sequence of 93 residues. Northern blot analysis from different neotomodon tissues demonstrated that the mRNA of CCSP appears to be solely expressed in the lung. To study the transcriptional regulation of the CCSP gene, we cloned the 5'-flanking region of the nCCSP gene and compared its features with those previously reported for the hamster gene. The neotomodon and hamster genes share 89% sequence homology in their promoter region as well as a number of conserved cis-acting elements. However, in H441 cells the expression of a reporter gene driven by the nCCSP promoter was about 4-fold greater than its hamster counterpart. Functional analysis of progressive 5'-deletion mutants identified a region involved in the higher transcriptional activity of the neotomodon promoter.
Assuntos
Região 5'-Flanqueadora/genética , DNA Complementar/química , DNA Complementar/genética , Análise de Sequência de Proteína/métodos , Homologia de Sequência de Aminoácidos , Uteroglobina/química , Uteroglobina/genética , Sequência de Aminoácidos , Animais , Cricetinae , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Filogenia , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA/métodos , Especificidade da EspécieRESUMO
El potencial eléctrico a través de la membrana (gama) se cuantificó mediante la acumulación del catión radiactivo trifenilmetilfosfonio (TPMP+) en la membrana espermática. Los espermatozoides previamente lavados se incubaron en presencia de TPMP+ en un medio denominado K+ - baja o K+ - elevada hasta que se logra el estado de equilibrio (20 min a 37 grados centigrados)., El valor obtenido por diferencia se inserta en la ecuación de Nernst y se obtiene el valor de -69 + - 2 mV. La presencia de cationes como el Zn ++ y Mg++ en el medio de incubación inducen una hiperpolarización de un 10 y 8.6% respectivamente. La adición de reactivos específicos como el p-cloromercuribenzoato-ácido sulofónico y etilendiaminotetra-acetato sódico, disminuye el gama en un 35 y 58% respectivamente. Los agentes que actúan sobre los componentes de la superficie de espermatozoide como el ditiotreitol y progesterona inducen una hiperpolarización y despolarización de la membrana en un 16 y 40%, respectivamente. La presencia de propanolol y L-alfa-lisofosfatidilcolina, los cuales afectan los gradientes iónicos presentes a través de la membrana inducen una despolarización en un 43 y 92%, respectivamente. Finalmente, la adición de tetraffenilboron (TPB -) en el medio de incubación aumentó el valor del gama 75%. La importancia de estos estudios es que mediante la utilización de agentes hiperpolarizantes y despolarizantes se obtienen cambios en el gama desde -80 + - 2.5 mV hasta -6 + - 0.6 mV es decir, cambios hasta de 74 +- 1.5 mV a través de la membrana espermática.(au)