RESUMO
19-Hydroxyeicosatetraenoic acid (19-HETE, 1), a metabolically and chemically labile cytochrome P450 eicosanoid, has diverse biological activities including antagonism of the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE, 2). A SAR study was conducted to develop robust analogs of 1 with improved in vitro and in vivo efficacy. Analogs were screened in vitro for inhibition of 20-HETE-induced sensitization of rat renal preglomerular microvessels toward phenylephrine and demonstrated to normalize the blood pressure of male Cyp4a14(-/-) mice that display androgen-driven, 20-HETE-dependent hypertension.
Assuntos
Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Família 4 do Citocromo P450/fisiologia , Hipertensão/tratamento farmacológico , Glomérulos Renais/efeitos dos fármacos , Microvasos/efeitos dos fármacos , Animais , Ácidos Hidroxieicosatetraenoicos/toxicidade , Hipertensão/induzido quimicamente , Hipertensão/patologia , Glomérulos Renais/irrigação sanguínea , Masculino , Camundongos , Camundongos Knockout , Fenilefrina/toxicidade , Vasoconstritores/toxicidadeRESUMO
A facile, one-pot reaction cascade condenses 1,1,1-trichloroalkanes with alpha,beta-unsaturated ketones to unexpectedly furnish moderate to good yields of (E)-2-alkylidenecyclobutanols.
Assuntos
Ciclobutanos/química , Ciclobutanos/síntese química , Cetonas/química , EstereoisomerismoRESUMO
We have characterized the positional specificity of the mammalian and yeast VIP/diphosphoinositol pentakisphosphate kinase (PPIP5K) family of inositol phosphate kinases. We deployed a microscale metal dye detection protocol coupled to a high performance liquid chromatography system that was calibrated with synthetic and biologically synthesized standards of inositol pyrophosphates. In addition, we have directly analyzed the structures of biological inositol pyrophosphates using two-dimensional 1H-1H and 1H-31P nuclear magnetic resonance spectroscopy. Using these tools, we have determined that the mammalian and yeast VIP/PPIP5K family phosphorylates the 1/3-position of the inositol ring in vitro and in vivo. For example, the VIP/PPIP5K enzymes convert inositol hexakisphosphate to 1/3-diphosphoinositol pentakisphosphate. The latter compound has not previously been identified in any organism. We have also unequivocally determined that 1/3,5-(PP)2-IP4 is the isomeric structure of the bis-diphosphoinositol tetrakisphosphate that is synthesized by yeasts and mammals, through a collaboration between the inositol hexakisphosphate kinase and VIP/PPIP5K enzymes. These data uncover phylogenetic variability within the crown taxa in the structures of inositol pyrophosphates. For example, in the Dictyostelids, the major bis-diphosphoinositol tetrakisphosphate is 5,6-(PP)2-IP4 ( Laussmann, T., Eujen, R., Weisshuhn, C. M., Thiel, U., Falck, J. R., and Vogel, G. (1996) Biochem. J. 315, 715-725 ). Our study brings us closer to the goal of understanding the structure/function relationships that control specificity in the synthesis and biological actions of inositol pyrophosphates.
Assuntos
Fosfatos de Inositol/química , Fosfotransferases (Aceptor do Grupo Fosfato)/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Animais , Dictyosteliida/enzimologia , Humanos , Fosfatos de Inositol/metabolismo , Camundongos , Estrutura Molecular , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Filogenia , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Especificidade por Substrato/fisiologia , Células Swiss 3T3RESUMO
Inositol pyrophosphates are recognized components of cellular processes that regulate vesicle trafficking, telomere length, and apoptosis. We observed that pancreatic beta cells maintain high basal concentrations of the pyrophosphate diphosphoinositol pentakisphosphate (InsP7 or IP7). Inositol hexakisphosphate kinases (IP6Ks) that can generate IP7 were overexpressed. This overexpression stimulated exocytosis of insulin-containing granules from the readily releasable pool. Exogenously applied IP7 dose-dependently enhanced exocytosis at physiological concentrations. We determined that IP6K1 and IP6K2 were present in beta cells. RNA silencing of IP6K1, but not IP6K2, inhibited exocytosis, which suggests that IP6K1 is the critical endogenous kinase. Maintenance of high concentrations of IP7 in the pancreatic beta cell may enhance the immediate exocytotic capacity and consequently allow rapid adjustment of insulin secretion in response to increased demand.
Assuntos
Exocitose , Fosfatos de Inositol/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Vesículas Secretórias/metabolismo , Animais , Linhagem Celular , Cricetinae , Capacitância Elétrica , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Camundongos , Técnicas de Patch-Clamp , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Ácido Fítico/metabolismo , Interferência de RNA , Ratos , TransfecçãoRESUMO
Fe(0) was investigated as a cost-effective, environmentally friendly alternative to Cr(II) for the olefination of carbonyls by activated polyhalides. In many instances, Fe(0) was equivalent or superior to Cr(II). Notably, Fe(0), but not Cr(II), proved compatible with a wide range of functionality, inter alia, unprotected phenol, aryl nitro, carboxylic acid, and alkyl nitrile. A surprising reversal of stereoselectivity for aldehydes versus ketones was observed using both metals. The resultant alpha-halo-alpha,beta-unsaturated or alpha,beta-unsaturated carboxylic acids, esters, and nitriles are common structural elements in numerous compounds of interest as well as key intermediates in the preparation of other functionality.
Assuntos
Alcenos/síntese químicaRESUMO
Treatment of cyclic tert-trihalomethylcarbinols with CrCl(2) in THF/HMPA in the presence of aryl or aliphatic aldehydes initiates a cascade sequence of one carbon ring expansion-olefination affording conjugated exocyclic ketones. Acyclic tert-trihalomethylcarbinols undergo a comparable cascade of one carbon homologation-olefination.
Assuntos
Aldeídos/química , Metanol/química , CiclizaçãoRESUMO
The O-linked N-acetylglucosamine (O-GlcNAc) modification of serine/threonine residues is an abundant posttranslational modification present in cytosolic and nuclear proteins. The functions and subproteome of O-GlcNAc modification remain largely undefined. Here we report the application of the tagging-via-substrate (TAS) approach for global identification of O-GlcNAc-modified proteins. The TAS method utilizes an O-GlcNAc azide analogue for metabolic labeling of O-GlcNAc-modified proteins, which can be chemoselectively conjugated for detection and enrichment of the proteins for proteomics studies. Our study led to the identification of 199 putative O-GlcNAc-modified proteins from HeLa cells, among which 23 were confirmed using reciprocal immunoprecipitation. Functional classification shows that proteins with diverse functions are modified by O-GlcNAc, implying that O-GlcNAc might be involved in the regulation of multiple cellular pathways.
Assuntos
Acetilglucosamina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Proteômica/métodos , Acetilglucosamina/química , Cromatografia Líquida de Alta Pressão , Células HeLa , Humanos , N-Acetilglucosaminiltransferases/metabolismo , Proteínas/química , Espectrometria de Massas em TandemRESUMO
The four stereoisomers of the endothelial-derived vasorelaxant 11,12,15(S)-trihydroxyeicosatrienoic acid [1, 11,12,15(S)-THETA] were prepared by a triply convergent, asymmetric route that exploited the stereospecific, copper mediated cross-coupling of alpha,beta-dialkoxystannanes with organic electrophiles and the utility of dialkylthionocarbamates as orthogonal alcohol protective groups. Only 11(R),12(S),15(S)-THETA was comparable to natural material by HPLC, GC/MS, and in vitro bioassay.