RESUMEN
BACKGROUND: The formation of novel N-substituted-1,2,3,4-tetrahydro[1,3]-dioxolo-[6,7]-5H-[1]benzopyrano [3,4-c]pyridines were observed unexpectedly during the acid-mediated ketal removal of ethylenedioxy ketal protected 4-piperidones. The literature revealed that benzopyranopyridine derivatives are of scientific interest and some exhibit interesting biological activities. Diastereomeric resolution was utilized to isolate optically pure chiral molecules. RESULTS: The acid catalyzed deprotection of N-substituted-4,4-ethylenedioxy-3- [(1,3-benzodioxol-5-yloxy)methyl]piperidines, prepared by condensation of the corresponding phenols and mesylate derivatives, unexpectedly resulted in cyclodehydration leading to new benzopyrano derivatives, N-substituted-1,2,3,4-tetrahydro[1,3]-dioxolo-[6,7]-5H-[1]benzopyrano [3,4-c]pyridines. The process involves the deprotection of the carbonyl protecting group, and then the cyclization reaction occurs followed by dehydration to give the final product.These N-substituted-1,2,3,4-tetrahydro[1,3]-dioxolo-[6,7]-5H-[1]benzopyrano [3,4-c] pyridines were dealkylated giving the corresponding N-unsubstituted derivatives. The cis-1,3,4,4a,5,10b-hexahydro-[6,7]-2H-[1]benzopyrano [3,4-c]pyridine derivative was also obtained from the N-benzylated-1,2,3,4-tetrahydro[1,3]-dioxolo-[6,7]-5H-[1]benzopyrano [3,4-c]pyridine via catalytic hydrogenation. The resolution of the enantiomers was carried out using D-(-)-mandelic acid as chiral reagent. The absolute configuration of the S,S-mandelate salt derivative was determined by X-ray crystallographic analysis. CONCLUSION: The approach led to the construction of N-substituted-1,2,3,4-tetrahydro[1,3]-dioxolo-[6,7]-5H-[1]benzopyrano [3,4-c] pyridines ring systems involving the one-pot deprotection, cyclization and dehydration of N-substituted-4,4-ethylenedioxy-3- [(1,3-benzodioxol-5-yloxy)methyl]piperidines. The hydrogenation of the N-benzylated benzopyrano [3,4-c]pyridine derivative followed by resolution led to the formation of a new compound.
RESUMEN
To examine the role of the beta-myosin heavy chain (betaMyHC) distal muscle CAT (MCAT) element in muscle fiber type-specific expression and mechanical overload (MOV) responsiveness, we conducted transgenic and in vitro experiments. In adult transgenic mice, mutation of the distal MCAT element led to significant reductions in chloramphenicol acetyltransferase (CAT) specific activity measured in control soleus and plantaris muscles when compared with wild type transgene beta293WT but did not abolish MOV-induced CAT specific activity. Electrophoretic mobility shift assay revealed the formation of a specific low migrating nuclear protein complex (LMC) at the betaMyHC MCAT element that was highly enriched only when using either MOV plantaris or control soleus nuclear extract. Scanning mutagenesis of the betaMyHC distal MCAT element revealed that only the nucleotides comprising the core MCAT element were essential for LMC formation. The proteins within the LMC when using either MOV plantaris or control soleus nuclear extracts were antigenically related to nominal transcription enhancer factor 1 (NTEF-1), poly(ADP-ribose) polymerase (PARP), and Max. Only in vitro translated TEF-1 protein bound to the distal MCAT element, suggesting that this multiprotein complex is tethered to the DNA via TEF-1. Protein-protein interaction assays revealed interactions between nominal TEF-1, PARP, and Max. Our studies show that for transgene beta293 the distal MCAT element is not required for MOV responsiveness but suggest that a multiprotein complex likely comprised of nominal TEF-1, PARP, and Max forms at this element to contribute to basal slow fiber expression.
Asunto(s)
Regulación de la Expresión Génica , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/fisiología , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Proteínas Nucleares , Animales , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Sitios de Unión , Fenómenos Biomecánicos , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Sustancias Macromoleculares , Ratones , Ratones Transgénicos , Complejos Multiproteicos , Mutagénesis Sitio-Dirigida , Sondas de Oligonucleótidos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Proteínas Recombinantes/metabolismo , Factores de Transcripción de Dominio TEA , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The definition of the term "environment" has broadened in the past 40 years to include knowledge generated from sequencing genes. Studies on animal responses to the environment have expanded to include selective lifestyle behaviors. Environmental lifestyle components interact with susceptibility genes to pass a threshold of biological significance such that a disease requires clinical treatment. Examples of environmental-gene interactions producing cystic fibrosis and asthma are described. The contributing role of physical inactivity to the epidemic of type 2 diabetes is presented with some of its underlying effectors. A lack of contractile activity by skeletal muscle is associated with less GLUT4 protein in the sarcolemma and a lower glucose uptake into the muscle. The pathways by which contractile activity signals an increase in glucose uptake differs from insulin signaling, but is remarkably similar to how hypoxia stimulates muscle to increase its glucose uptake.
Asunto(s)
Hipoxia/etiología , Proteínas Musculares , Altitud , Animales , Asma/genética , Fibrosis Quística/genética , Fibrosis Quística/microbiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Diabetes Mellitus Tipo 2/genética , Ambiente , Ejercicio Físico , Transportador de Glucosa de Tipo 4 , Humanos , Hipoxia/genética , Hipoxia/metabolismo , Estilo de Vida , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas de Transporte de Monosacáridos/fisiología , Músculo Esquelético/metabolismoRESUMEN
In adult mouse skeletal muscle, beta-myosin heavy chain (betaMyHC) gene expression is primarily restricted to slow-type I fibers but can be induced in fast-type II fibers by mechanical overload (MOV). Our previous transgenic analyses have delimited an 89-base pair (bp) MOV-responsive region (-293 to -205), and shown that mutation of the MCAT and C-rich elements within this region did not abolish betaMyHC transgene induction by MOV. In this study we describe an A/T-rich element (betaA/T-rich; -269 5'-GGAGATATTTTT-3' -258) located within this 89-bp region that, only under MOV conditions, revealed enriched binding as characterized by electrophoretic mobility shift assays and dimethyl sulfate and diethyl pyrocarbonate interference footprinting. Direct, competition, and supershift electrophoretic mobility shift assays revealed highly enriched specific binding activity at the betaA/T-rich element that was antigenically distinct from GATA-4, MEF2A-D, SRF, and Oct-1, nuclear proteins that were previously shown to bind A/T-rich elements. In vitro translated GATA-4, MEF2C, SRF, and Oct-1 bound to consensus GATA, MEF2, SRE, and Oct-1 elements, respectively, but not to the betaA/T-rich element. Two-dimensional UV cross-linking of the bromodeoxyuridine-substituted betaA/T-rich element with mechanically overloaded plantaris (MOV-P) nuclear extract detected two proteins (44 and 48 kDa). Our results indicate that the betaA/T-rich element may function in vivo as a betaMyHC MOV-inducible element during hypertrophy of adult skeletal muscle by binding two distinct proteins identified only in MOV-P nuclear extract.
Asunto(s)
Secuencia Rica en At , Núcleo Celular/metabolismo , Regulación de la Expresión Génica , Músculo Esquelético/fisiología , Cadenas Pesadas de Miosina/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Sitios de Unión , Cricetinae , Humanos , Ratones , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Biosíntesis de Proteínas , Conejos , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Porcinos , Transcripción Genética , Troponina T/genética , Soporte de PesoRESUMEN
Our previous transgenic analyses revealed that a 600-base pair beta-myosin heavy chain (betaMyHC) promoter conferred mechanical overload (MOV) and non-weight-bearing (NWB) responsiveness to a chloramphenicol acetyltransferase reporter gene. Whether the same DNA regulatory element(s) direct betaMyHC expression following MOV or NWB activity in vivo remains unknown. We now show that a 293-base pair betaMyHC promoter fused to chloramphenicol acetyltransferase (beta293) responds to MOV, but not NWB activity, indicating a segregation of these two diverse elements. Inclusion of the betaMyHC negative regulatory element (-332 to -300; betaNRE) within transgene beta350 repressed expression in all transgenic lines. Electrophoretic mobility shift assays showed highly enriched binding activity only in NWB soleus nuclear extracts that was specific to the distal region of the betaNRE sense strand (dbetaNRE-S; -332 to -311). Supershift electrophoretic mobility shift assay revealed that the binding at the distal region of the betaNRE sense strand was antigenically distinct from cellular nucleic acid-binding protein and Y-box-binding factor 1, two proteins shown to bind this element. Two-dimensional UV cross-linking and shift Southwestern blotting analyses detected two proteins (50 and 52 kDa) that bind to this element. These in vivo results demonstrate that segregated betaMyHC promoter elements transcriptionally regulate betaMyHC transgene expression in response to two diverse modes of neuromuscular activity.