RESUMEN
Steroid 5 alpha-reductase catalyzes the reduction of testosterone (T) into the very potent androgen dihydrotestosterone (DHT). The different tissue expression patterns of the two isoforms of 5 alpha-reductase, namely type-1 and type-2 5 alpha-reductase (5 alpha-R1 and 5 alpha-R2, respectively), have prompted studies directed towards the synthesis of selective 5 alpha-R1 or 5 alpha-R2 inhibitors. In this present work, we have performed a structure/activity study on the inhibitory potential of indole carboxylic acids against hair follicle 5 alpha-reductase activity. We have demonstrated that this class of molecules were potent inhibitors of either 5 alpha-R1 or 5 alpha-R2 or both depending on (i) substituents in positions 4, 5 or 6 and (ii) the presence of a free carboxylic group. We have also found that only 5 alpha-R1 or 5 alpha-R1/R2 inhibitors were able to inhibit 5 alpha-reductase activity in plucked hairs from female volunteers or in freshly isolated female hair follicles, selective 5 alpha-R2 inhibitors being inactive.
Asunto(s)
3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/metabolismo , Folículo Piloso/enzimología , Isoenzimas/metabolismo , 3-Oxo-5-alfa-Esteroide 4-Deshidrogenasa/química , Inhibidores de 5-alfa-Reductasa , Animales , Células COS , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacología , Disección , Inhibidores Enzimáticos/farmacología , Femenino , Haplorrinos , Humanos , Indoles/química , Indoles/farmacología , Relación Estructura-Actividad , Testosterona/metabolismoRESUMEN
TTD is a rare human genetic disease caused by mutations in XPB and XPD, two subunits of the transcription/repair factor TFIIH, and whose outstanding clinical characteristic is a lack of most human UHS proteins resulting in sulfur-deficient brittle hair. In an attempt to understand this transcription defect, we report here the genomic cloning of two highly related UHS keratin genes specifically expressed in follicular and epidermal cells. In addition to a high degree of nucleotide homology (87%), both genes also have a similar 90-nt promoter sequence. In-vivo and in-vitro studies allowed us to specify the position of the start sites, the TATA-boxes and some regulatory regions. Results indicate that both genes present common features in the regulation of their transcription and suggest that control of their expression might be affected by mutations in TFIIH subunits.
Asunto(s)
Queratinas/genética , Regiones Promotoras Genéticas/genética , Factores de Transcripción TFII , Secuencia de Aminoácidos , Clonación Molecular , Reparación del ADN/genética , Regulación de la Expresión Génica/genética , Enfermedades del Cabello/genética , Humanos , Queratinocitos , Datos de Secuencia Molecular , Mapeo Restrictivo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Factor de Transcripción TFIIH , Factores de Transcripción/genética , Transcripción Genética/genéticaAsunto(s)
Articulación del Tobillo , Artritis Infecciosa/microbiología , Infecciones por Fusobacterium/microbiología , Fusobacterium necrophorum , Adulto , Amputación Quirúrgica , Artritis Infecciosa/cirugía , Infecciones por Fusobacterium/cirugía , Humanos , Masculino , Necrosis , Faringitis/complicacionesRESUMEN
The diversity of isoforms of retinoic acid (RA) receptors (RARs) and of DNA sequences of retinoic acid-responsive elements (RAREs) suggests the existence of selectivities in the RAR/RARE recognition or in the subsequent gene modulation. Such selectivities might be particularly important for RAREs involved in positive feedback, eg. the RAR beta RARE. In the present work we found that in several epithelial cell lines, reporter constructs containing the RAR beta RARE linked to the HSV-tk promoter were transactivated in the presence of RA by endogenous RARs and co-transfected RAR alpha 1 and RAR beta 2 isoforms, but not by RAR gamam 1. On the contrary, this latter isoform behaved towards the RAR beta RARE as an inhibitor of the transactivation produced by endogenous RARs and by cotransfected RAR alpha 1 and RAR beta 2. RAR gamma 1 also behaved as an antagonist of the transactivation produced by cotransfected RXR alpha. The natural RAR beta gene promoter or RAR beta RARE tk constructs were not activated by the endogenous receptors of normal human keratinocytes (NHK), which are known to contain predominantly RAR gamma 1. It was, however, possible to activate to a certain extent RAR beta RARE-reporter constructs in NHK by co-transfecting RAR alpha 1, RAR beta 2 or RXR alpha. The antagonist behavior of RAR gamma 1 towards the RAR beta RARE may explain why in certain cell types such as keratinocytes, RAR beta is neither expressed nor induced by RA.
Asunto(s)
Proteínas Portadoras/genética , Tretinoina/metabolismo , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Regulación de la Expresión Génica , Células HeLa , Humanos , Queratinocitos/metabolismo , Regiones Promotoras Genéticas , Receptores de Ácido Retinoico , Activación Transcripcional , TransfecciónRESUMEN
We have studied the consequences of decreasing the donor site-branch site distance on splicing factor-splice site interactions by analyzing alternative splicing of adenovirus E1A pre-mRNAs in vitro. We show that the proximal 13S donor site has a cis-inhibiting effect on the 9S and 12S mRNA reactions when it is brought too close to the common branch site, suggesting that the factor interactions in the common 3' part of the intron are impaired by the U1 small nuclear ribonucleoprotein particle (snRNP) binding to the displaced 13S donor site. Further analysis of the interactions was carried out by studying complex assembly and the accessibility to micrococcal nuclease digestion of 5'-truncated E1A substrates containing only splice sites for the 13S mRNA reaction. A deletion which brings the donor site- branch site distance to 49 nucleotides, which is just below the minimal functional distance, results in a complete block of the U4-U5-U6 snRNP binding, whereas a deletion 15 nucleotides larger results in a severe inhibition of the formation of the U2 snRNP-containing complexes. Sequence accessibility analyses performed by using the last mini-intron-containing transcript demonstrate that the interactions of U2 snRNP with the branch site are strongly impaired whereas the initial bindings of U1 snRNP to the donor site and of specific factors to the 3' splice site are not significantly modified. Our results strongly suggest that the interaction of U1 snRNP with the donor site of a mini-intron is stable enough in vitro to affect the succession of events leading to U2 snRNP binding with the branch site.
Asunto(s)
Intrones , Proteínas Oncogénicas Virales/genética , Empalme del ARN , ARN Mensajero/genética , Ribonucleoproteínas/fisiología , Proteínas Precoces de Adenovirus , Adenovirus Humanos/genética , Clonación Molecular , Análisis Mutacional de ADN , Sustancias Macromoleculares , Nucleasa Microcócica/farmacología , Precursores de Ácido Nucleico/metabolismo , ARN Viral/genética , Ribonucleoproteínas Nucleares Pequeñas , Relación Estructura-ActividadRESUMEN
The nuclear non-polyadenylated RNA from HeLa cells infected with adenovirus-2 was examined for the presence of molecules containing the first intervening sequence (IVS1) of the major late premessenger RNA. Four molecules with the approximate size of free IVS1 in sucrose gradients (1021 nucleotides) were separated by polyacrylamide gel electrophoresis and characterized by complementary methods: S1 nuclease mapping, susceptibility to debranching enzyme, RNAase-H-directed cleavage. The results indicate that the most abundant RNA form is the excised lariat IVS1. We also find linear IVS1 and a randomly nicked lariat, the latter probably being made during RNA isolation. The fourth RNA is a leader 1-IVS1 molecule. No truncated IVS1 which might indicate that IVS1 is excised by several cycle of cleavage-ligation was detected. A study of the distribution of the four RNAs in hnRNP shows that they are part of RNPs of about 70 S. However, each RNP has distinct sedimentation characteristics and sensitivity to salt dissociation. Together, the results suggest that the excised lariat IVS1 is released from the large late premRNP under the form of a 70 S RNP, where it is linearized.
Asunto(s)
Adenovirus Humanos/genética , Intrones , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Ribonucleoproteínas/metabolismo , Centrifugación por Gradiente de Densidad , Electroforesis en Gel de Poliacrilamida , Endonucleasas/metabolismo , Endorribonucleasas/metabolismo , Células HeLa , Humanos , Hibridación de Ácido Nucleico , ARN Mensajero/genética , ARN Viral/genética , Ribonucleasa H , Endonucleasas Específicas del ADN y ARN con un Solo Filamento , Transcripción GenéticaRESUMEN
The ejection fraction as determined by gated blood pool studies was compared with the ejection fraction as evaluated by the following conventional methods: 1) biplane cineangiography employing the Simpson rule. Correlation was r = 0.805 (n = 25); 2) the same patients using the area-length method (r = 0.88, n = 25); 3) monoplane cineangiography using the method of Greene et al. (5) (r = 0.86, n = 35). In 20 patients the maximal contraction velocity as evaluated by radionuclide ventriculography was compared with the mean systolic ejection rate. The observed correlation was weak (r = 0.62, n = 20); however, it should be noted that the radionuclide method determines the maximum contraction velocity whereas cinceangiography measures an average value over the whole systole.