RESUMEN
Light-induced oxidation of the reaction center dimer and periplasmic cytochromes was detected by fast kinetic difference absorption changes in intact cells of wild type and cytochrome mutants (cycA, cytC4 and pufC) of Rubrivivax gelatinosus and Rhodobacter sphaeroides. Constant illumination from a laser diode or trains of saturating flashes enabled the kinetic separation of acceptor and donor redox processes, and the electron contribution from the cyt bc1 complex via periplasmic cytochromes. Under continuous excitation, concentrations of oxidized cytochromes increased in three phases where light intensity, electron transfer rate and the number of reduced cytochromes were the rate liming steps, respectively. By choosing suitable flash timing, gradual steps of cytochrome oxidation in whole cells were observed; each successive flash resulted in a smaller, damped oxidation. We attribute this damping to lowered availability of reduced cytochromes resulting from both exchange (unbinding/binding) of the cytochromes and electron transfer at the reaction center interface since a similar effect is observed upon deletion of genes encoding periplasmic cytochromes. In addition, we present a simple model to calculate the damping effect; application of this method may contribute to understanding the function of the diverse range of c-type cytochromes in the electron transport chains of anaerobic phototrophic bacteria.
Asunto(s)
Proteínas del Complejo del Centro de Reacción Fotosintética , Rhodobacter sphaeroides , Grupo Citocromo c/genética , Citocromos/metabolismo , Transporte de Electrón , Cinética , Oxidación-Reducción , Proteínas del Complejo del Centro de Reacción Fotosintética/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Rhodobacter sphaeroides/metabolismoRESUMEN
Antenna systems serve to absorb light and to transmit excitation energy to the reaction center (RC) in photosynthetic organisms. As the emitted (bacterio)chlorophyll fluorescence competes with the photochemical utilization of the excitation, the measured fluorescence yield is informed by the migration of the excitation in the antenna. In this work, the fluorescence yield concomitant with the oxidized dimer (P+) of the RC were measured during light excitation (induction) and relaxation (in the dark) for whole cells of photosynthetic bacterium Rhodobacter sphaeroides lacking cytochrome c2 as natural electron donor to P+ (mutant cycA). The relationship between the fluorescence yield and P+ (fraction of closed RC) showed deviations from the standard Joliot-Lavergne-Trissl model: (1) the hyperbola is not symmetric and (2) exhibits hysteresis. These phenomena originate from the difference between the delays of fluorescence relative to P+ kinetics during induction and relaxation, and in structural terms from the non-random distribution of the closed RCs during induction. The experimental findings are supported by Monte Carlo simulations and by results from statistical physics based on random walk approximations of the excitation in the antenna. The applied mathematical treatment demonstrates the generalization of the standard theory and sets the stage for a more adequate description of the long-debated kinetics of fluorescence and of the delicate control and balance between efficient light harvest and photoprotection in photosynthetic organisms.
Asunto(s)
Fluorescencia , Luz , Fotosíntesis , Proteínas del Complejo del Centro de Reacción Fotosintética/química , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Rhodobacter sphaeroides/metabolismo , Transporte de Electrón , Cinética , Procesos Fotoquímicos , TermodinámicaRESUMEN
Ras homolog enriched in brain (RHEB1) is a member within the superfamily of GTP-binding proteins encoded by the RAS oncogenes. RHEB1 is located at the crossroad of several important pathways including the insulin-signaling pathways and thus plays an important role in different physiological processes. To understand better the physiological relevance of RHEB1 protein, the expression pattern of RHEB1 was analyzed in both embryonic (at E3.5-E16.5) and adult (1-month old) mice. RHEB1 immunostaining and X-gal staining were used for wild-type and Rheb1 gene trap mutant mice, respectively. These independent methods revealed similar RHEB1 expression patterns during both embryonic and postnatal developments. Ubiquitous uniform RHEB1/ß-gal and/or RHEB1 expression was seen in preimplantation embryos at E3.5 and postimplantation embryos up to E12.5. Between stages E13.5 and E16.5, RHEB1 expression levels became complex: In particular, strong expression was identified in neural tissues, including the neuroepithelial layer of the mesencephalon, telencephalon, and neural tube of CNS and dorsal root ganglia. In addition, strong expression was seen in certain peripheral tissues including heart, intestine, muscle, and urinary bladder. Postnatal mice have broad spatial RHEB1 expression in different regions of the cerebral cortex, subcortical regions (including hippocampus), olfactory bulb, medulla oblongata, and cerebellum (particularly in Purkinje cells). Significant RHEB1 expression was also viewed in internal organs including the heart, intestine, urinary bladder, and muscle. Moreover, adult animals have complex tissue- and organ-specific RHEB1 expression patterns with different intensities observed throughout postnatal development. Its expression level is in general comparable in CNS and other organs of mouse. Thus, the expression pattern of RHEB1 suggests that it likely plays a ubiquitous role in the development of the early embryo with more tissue-specific roles in later development.
Asunto(s)
Embrión de Mamíferos/metabolismo , Proteínas de Unión al GTP Monoméricas/análisis , Proteínas de Unión al GTP Monoméricas/metabolismo , Neuronas/metabolismo , Animales , Animales Recién Nacidos , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión al GTP Monoméricas/genética , Especificidad de ÓrganosRESUMEN
BACKGROUND: Neuronal polarization is an essential step of morphogenesis and connectivity in the developing brain. The serine/threonine kinase LKB1 is a key regulator of cell polarity, metabolism, tumorigenesis, and is required for axon formation. It is allosterically regulated by two related and evolutionarily conserved pseudokinases, STe20-Related ADapters (STRADs) α and ß. The roles of STRADα and STRADß in the developing nervous system are not fully defined, nor is it known whether they serve distinct functions. RESULTS: We find that STRADα is highly spliced and appears to be the primal STRAD paralog. We report that each STRAD is sufficient for axogenesis and promoting cell survival in the developing cortex. We also reveal a reciprocal protein-stabilizing relationship in vivo between LKB1 and STRADα, whereby STRADα specifically maintains LKB1 protein levels via cytoplasmic compartmentalization. CONCLUSIONS: We demonstrate a novel role for STRADß in axogenesis and also show for the first time in vivo that STRADα, but not STRADß, is responsible for LKB1 protein stability.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Axones/fisiología , Corteza Cerebral/metabolismo , Neuronas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Animales , Axones/metabolismo , Axones/ultraestructura , Corteza Cerebral/citología , Ratones , Neuronas/citologíaRESUMEN
Arachidonic acid (AA) is generated in the anterior pituitary gland upon stimulation by the ACTH secretagogue, CRH. Using the patch clamp technique, we examined the action of AA on the excitability of single pituitary corticotropes obtained from a transgenic mouse strain that expresses the enhanced green fluorescent protein driven by the proopiomelanocortin promoter. CRH evoked depolarization, but AA caused hyperpolarization. Under voltage clamp condition, AA caused a rapid inhibition of the delayed rectifier K(+) current and then increased a background K(+) current. Inhibition of AA metabolism did not prevent the activation of the K(+) current by AA, suggesting a direct action of AA. The sensitivity of the AA-activated K(+) current to fluoxetine, chlorpromazine, extracellular acidification, diphenylbutylpiperidine antipsychotics, and the membrane permeable cAMP analog [8-(4-chlorophenylthio)-cAMP] suggest that the current is mediated via TWIK-related K(+) channel (TREK)-1 channels. Activation of the CRH receptors that are coupled to the adenylate cyclase pathway suppressed the activation of TREK-1 current by AA and reversed the AA-mediated hyperpolarization. Intracellular acidification (pH 7.0) increased the basal amplitude of TREK-1 current and resulted in hyperpolarizaton. CRH suppressed the basal TREK-1 current in cells with intracellular acidification and caused depolarization. Our finding indicates that TREK-1 channels are important in setting the resting potential in corticotropes. The opposing actions of CRH and AA on the excitability of corticotropes raise the possibility that AA may act as a negative feedback regulator to reduce the stimulatory action of CRH and thus prevent excessive ACTH release during chronic stress.
Asunto(s)
Ácido Araquidónico/farmacología , Corticotrofos/fisiología , Hormona Liberadora de Corticotropina/farmacología , Canales de Potasio de Dominio Poro en Tándem/fisiología , Animales , Células Cultivadas , Clorpromazina/farmacología , Corticotrofos/citología , Corticotrofos/metabolismo , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Fluoxetina/farmacología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Concentración de Iones de Hidrógeno , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Transgénicos , Fármacos Neuroprotectores/farmacología , Técnicas de Placa-Clamp , Tionucleótidos/farmacologíaRESUMEN
Phytochromes are red/far-red light photoreceptors found in plants, cyanobacteria and heterotrophic bacteria. Biochemical analyses have established that the genes bphO and bphP (PA4116 and PA4117) of Pseudomonas aeruginosa encode both phytochrome components: BphO, a heme oxygenase that produces the linear tetrapyrrole chromophore biliverdin IXalpha, and BphP, the apo-phytochrome. Reverse transcription-PCR established that both genes form a bicistronic operon. Expression of the bphOP operon was induced in the stationary phase, indicating an involvement of the P. aeruginosa quorum-sensing system and/or the stationary-phase alternative sigma factor RpoS. Bioinformatic analyses of the promoter region revealed a potential binding site for the quorum sensing regulators LasR and/or RhlR. While a direct involvement of the quorum-sensing system could be ruled out, the dependence of bphOP expression on RpoS was clearly demonstrated. Chromosomal knock-out mutants showed identical growth behavior as a wild type under various conditions but increased levels of pyocyanin were detected in the DeltabphO strain. Additionally, this strain showed decreased heat tolerance in the stationary phase, indicating a potential protective role of the BphO reaction product biliverdin. Therefore, BphO might have an additional function besides providing the chromophore for BphP and both proteins are likely to fulfill a task in the stationary phase.
Asunto(s)
Fitocromo/metabolismo , Pseudomonas aeruginosa/metabolismo , Regulón/fisiología , Factor sigma/fisiología , Biliverdina/metabolismo , Regulación Bacteriana de la Expresión Génica , Hemo/metabolismo , Hemo Oxigenasa (Desciclizante)/fisiología , Operón , Fitocromo/química , Fitocromo/genética , Pseudomonas aeruginosa/genética , Percepción de Quorum/genéticaRESUMEN
We provide in vivo genetic and in vitro biochemical evidence that RegA directly regulates bacteriochlorophyll and carotenoid biosynthesis in Rhodobacter capsulatus. beta-Galactosidase expression assays with a RegA-disrupted strain containing reporter plasmids for Mg-protoporphyrin IX monomethyl ester oxidative cyclase (bchE), Mg-protoporphyrin IX chelatase (bchD), and phytoene dehydrogenase (crtI) demonstrate RegA is responsible for fourfold anaerobic induction of bchE, threefold induction of bchD, and twofold induction of crtI. Promoter mapping studies, coupled with DNase I protection assays, map the region of RegA binding to three sites in the bchE promoter region. Similar studies at the crtA and crtI promoters indicate that RegA binds to a single region equidistant from these divergent promoters. These results demonstrate that RegA is directly responsible for anaerobic induction of bacteriochlorophyll biosynthesis genes bchE, bchD, bchJ, bchI, bchG, and bchP and carotenoid biosynthesis genes crtI, crtB, and crtA.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bacterioclorofilas/biosíntesis , Carotenoides/biosíntesis , Rhodobacter capsulatus/metabolismo , Transactivadores/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Huella de ADN , Desoxirribonucleasa I , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Rhodobacter capsulatus/genética , Transactivadores/genéticaRESUMEN
Proopiomelanocortin (POMC) is synthesized predominantly in pituitary corticotrophs, melanotrophs, and arcuate hypothalamic neurons. Corticotroph-derived ACTH mediates basal and stress-induced glucocorticoid secretion, but it is uncertain whether POMC peptides produced in the brain also regulate the hypothalamic-pituitary-adrenal axis. To address this question, we generated neuron-specific POMC-deficient mice by transgenic (Tg) replacement of pituitary POMC in a global Pomc(-/-) background. Selective restoration of pituitary POMC prevented the adrenal insufficiency and neonatal mortality characteristic of Pomc(-/-) mice. However, adult Pomc(-/-)Tg/+ mice expressing the pituitary-specific transgene exhibited adrenal cortical hypertrophy, elevated basal plasma corticosterone, elevated basal but attenuated stress-induced ACTH secretion, and inappropriately elevated CRH expression in the hypothalamic paraventricular nucleus. In addition, Pomc(-/-)Tg/+, Pomc(+/-)Tg/+, and Pomc(+/-) mice, which all displayed varying degrees of elevated CRH, frequently developed melanotroph adenomas after 1 yr of age, whereas Pomc(-/-) mice, with maximal CRH expression and glucocorticoid disinhibition, developed corticotroph and melanotroph adenomas. These results indicate that neuronal POMC peptides are necessary to regulate CRH within physiological limits and that a chronic reduction or absence of hypothalamic POMC leads to trophic stimulation of pituitary cells directly or indirectly through elevated CRH levels.
Asunto(s)
Encéfalo/metabolismo , Sistema Hipotálamo-Hipofisario/fisiología , Sistema Hipófiso-Suprarrenal/fisiología , Proopiomelanocortina/fisiología , Adenoma/etiología , Adenoma/patología , Corteza Suprarrenal/patología , Insuficiencia Suprarrenal/etiología , Insuficiencia Suprarrenal/mortalidad , Hormona Adrenocorticotrópica/metabolismo , Animales , Corticosterona/sangre , Hormona Liberadora de Corticotropina/metabolismo , Femenino , Hipertrofia , Sistema Hipotálamo-Hipofisario/fisiopatología , Hipotálamo/metabolismo , Masculino , Melanotrofos/patología , Ratones , Ratones Noqueados , Ratones Transgénicos , Núcleo Hipotalámico Paraventricular/metabolismo , Hipófisis/metabolismo , Neoplasias Hipofisarias/etiología , Neoplasias Hipofisarias/patología , Sistema Hipófiso-Suprarrenal/fisiopatología , Proopiomelanocortina/deficiencia , Proopiomelanocortina/genética , Estrés Fisiológico/metabolismo , Estrés Fisiológico/fisiopatologíaRESUMEN
We demonstrate that the expression of hem genes in Rhodobacter capsulatus is transcriptionally repressed in response to the exogenous addition of heme. A high-copy suppressor screen for regulators of hem gene expression resulted in the identification of an LysR-type transcriptional regulator, called HbrL, that regulates hem promoters in response to the availability of heme. HbrL is shown to activate the expression of hemA and hemZ in the absence of exogenous hemin and repress hemB expression in the presence of exogenous hemin. Heterologously expressed HbrL apoprotein binds heme b and is purified with bound heme b when expressed in the presence of 5-aminolevulinic acid. Electrophoretic gel shift analysis demonstrated that HbrL binds the promoter region of hemA, hemB, and hemZ as well as its own promoter and that the presence of heme increases the binding affinity of HbrL to hemB.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Rhodobacter capsulatus/genética , Transcripción Genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Consenso , Hemo/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Rhodobacter capsulatus/metabolismo , Tetrapirroles/biosíntesisRESUMEN
Null mutations of the proopiomelanocortin gene (Pomc) cause obesity in humans and rodents, but the contributions of central versus pituitary POMC deficiency are not fully established. To elucidate these roles, we introduced a POMC transgene (Tg) that selectively restored peripheral melanocortin and corticosterone secretion in Pomc mice. Rather than improving energy balance, the genetic replacement of pituitary POMC in PomcTg mice aggravated their metabolic syndrome with increased caloric intake and feed efficiency, reduced oxygen consumption, increased subcutaneous, visceral, and hepatic fat, and severe insulin resistance. Pair-feeding of PomcTg mice to the daily intake of lean controls normalized their rate of weight gain but did not abolish obesity, indicating that hyperphagia is a major but not sole determinant of the phenotype. Replacement of corticosterone in the drinking water of Pomc mice recapitulated the hyperphagia, excess weight gain and fat accumulation, and hyperleptinemia characteristic of genetically rescued PomcTg mice. These data demonstrate that CNS POMC peptides play a critical role in energy homeostasis that is not substituted by peripheral POMC. Restoration of pituitary POMC expression to create a de facto neuronal POMC deficiency exacerbated the development of obesity, largely via glucocorticoid modulation of appetite, metabolism, and energy partitioning.
Asunto(s)
Glucocorticoides/metabolismo , Resistencia a la Insulina , Neuronas/metabolismo , Obesidad/metabolismo , Proopiomelanocortina/metabolismo , Animales , Peso Corporal , Ingestión de Alimentos , Metabolismo Energético , Femenino , Glucosa/metabolismo , Homeostasis , Humanos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Neuronas/citología , Oxígeno/metabolismo , Fenotipo , Proopiomelanocortina/genéticaRESUMEN
The nucleus tractus solitarius (NTS) receives dense terminations from cranial visceral afferents, including those from the gastrointestinal (GI) system. Although the NTS integrates peripheral satiety signals and relays this signal to central feeding centers, little is known about which NTS neurons are involved or what mechanisms are responsible. Proopiomelanocortin (POMC) neurons are good candidates for GI integration, because disruption of the POMC gene leads to severe obesity and hyperphagia. Here, we used POMC-enhanced green fluorescent protein (EGFP) transgenic mice to identify NTS POMC neurons. Intraperitoneal administration of cholecystokinin (CCK) induced c-fos gene expression in NTS POMC-EGFP neurons, suggesting that they are activated by afferents stimulated by the satiety hormone. We tested the synaptic relationship of these neurons to visceral afferents and their modulation by CCK and opioids using patch recordings in horizontal brain slices. Electrical activation of the solitary tract (ST) evoked EPSCs in NTS POMC-EGFP neurons. The invariant latencies, low failure rates, and substantial paired-pulse depression of the ST-evoked EPSCs indicate that NTS POMC-EGFP neurons are second-order neurons directly contacted by afferent terminals. The EPSCs were blocked by the glutamate antagonist 2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline. CCK increased the amplitude of the ST-stimulated EPSCs and the frequency of miniature EPSCs, effects attenuated by the CCK1 receptor antagonist lorglumide. In contrast, the orexigenic opioid agonists [D-Ala(2), N-Me-Phe(4), Gly-ol(5)]-enkephalin and met-enkephalin inhibited both ST-stimulated EPSCs and the frequency of miniature EPSCs. These findings identify a potential satiety pathway in which visceral afferents directly activate NTS POMC-EGFP neurons with excitatory inputs that are appropriately modulated by appetite regulators.
Asunto(s)
Colecistoquinina/farmacología , Narcóticos/farmacología , Neuronas/efectos de los fármacos , Proopiomelanocortina/metabolismo , Núcleo Solitario/citología , Aferentes Viscerales/fisiología , Animales , Recuento de Células/métodos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Estimulación Eléctrica/métodos , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Encefalina Metionina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Potenciales Postsinápticos Excitadores/efectos de la radiación , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Antagonistas de Hormonas/farmacología , Inmunohistoquímica/métodos , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Técnicas de Placa-Clamp/métodos , Proopiomelanocortina/genética , Proglumida/análogos & derivados , Proglumida/farmacología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Quinoxalinas/farmacología , Factores de TiempoRESUMEN
The proopiomelanocortin (POMC) gene is expressed in the pituitary and arcuate neurons of the hypothalamus. POMC arcuate neurons play a central role in the control of energy homeostasis, and rare loss-of-function mutations in POMC cause obesity. Moreover, POMC is the prime candidate gene within a highly significant quantitative trait locus on chromosome 2 associated with obesity traits in several human populations. Here, we identify two phylogenetically conserved neuronal POMC enhancers designated nPE1 (600 bp) and nPE2 (150 bp) located approximately 10 to 12 kb upstream of mammalian POMC transcriptional units. We show that mouse or human genomic regions containing these enhancers are able to direct reporter gene expression to POMC hypothalamic neurons, but not the pituitary of transgenic mice. Conversely, deletion of nPE1 and nPE2 in the context of the entire transcriptional unit of POMC abolishes transgene expression in the hypothalamus without affecting pituitary expression. Our results indicate that the nPEs are necessary and sufficient for hypothalamic POMC expression and that POMC expression in the brain and pituitary is controlled by independent sets of enhancers. Our study advances the understanding of the molecular nature of hypothalamic POMC neurons and will be useful to determine whether polymorphisms in POMC regulatory regions play a role in the predisposition to obesity.
Asunto(s)
Núcleo Arqueado del Hipotálamo/metabolismo , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica , Filogenia , Proopiomelanocortina/genética , Animales , Núcleo Arqueado del Hipotálamo/citología , Secuencia de Bases , Secuencia Conservada , Análisis Mutacional de ADN , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Humanos , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Neuronas/química , Neuronas/metabolismo , Obesidad/genética , Adenohipófisis/citología , Adenohipófisis/metabolismo , Polimorfismo Genético , Proopiomelanocortina/análisis , Proopiomelanocortina/metabolismo , Eliminación de SecuenciaRESUMEN
The original strain of proopiomelanocortin (POMC)-deficient mice (Pomc-/-) was generated by homologous recombination in 129X1/SvJ (A(w)/A(w))-derived embryonic stem cells using a targeting construct that deleted exon 3, encoding all the known functional POMC-derived peptides including alpha MSH, from the Pomc gene. Although these Pomc-/- mice exhibited adrenal hypoplasia and obesity similar to the syndrome of POMC deficiency in children, their agouti coat color was only subtly altered. To further investigate the mechanism of hair pigmentation in the absence of POMC peptides, we studied wild-type (Pomc+/+), heterozygous (Pomc+/-), and homozygous (Pomc-/-) mice on a nonagouti (a/a) 129;B6 hybrid genetic background. All three genotypes had similar black fur pigmentation with yellow hairs behind the ears, around the nipples, and in the perianal area characteristic of inbred C57BL/6 mice. Histologic and electron paramagnetic resonance spectrometry examination demonstrated that hair follicles in back skin of Pomc-/- mice developed with normal structure and eumelanin pigmentation; corresponding molecular analyses, however, excluded local production of alpha MSH and ACTH because neither Pomc nor putative Pomc pseudogene mRNAs were detected in the skin. Thus, 129;B6 Pomc null mutant mice produce abundant eumelanin hair pigmentation despite their congenital absence of melanocortin ligands. These results suggest that either the mouse melanocortin receptor 1 has sufficient basal activity to trigger and sustain eumelanogenesis in vivo or that redundant nonmelanocortin pathway(s) compensate for the melanocortin deficiency. Whereas the latter implies feedback control of melanogenesis, it is also possible that the two mechanisms operate jointly in hair follicles.
Asunto(s)
Cabello/metabolismo , Melaninas/metabolismo , Pigmentación , Proopiomelanocortina/fisiología , Glándulas Suprarrenales/patología , Alelos , Animales , Cruzamientos Genéticos , ADN Complementario/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Exones , Eliminación de Gen , Color del Cabello , Heterocigoto , Homocigoto , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Mutación , Fenotipo , Reacción en Cadena de la Polimerasa , Proopiomelanocortina/genética , alfa-MSH/metabolismoRESUMEN
Biosynthetic pathways for heme and chlorophyll share common intermediates from 5-aminolevulinic acid through protoporphyrin IX. To obtain a better understanding of how photosynthetic organisms coordinate heme and chlorophyll biosynthesis, we have undertaken detailed analysis of the expression pattern of numerous heme biosynthesis genes in the purple photosynthetic bacterium Rhodobacter capsulatus. beta-Galactosidase reporter assays demonstrated that expression of hemA, hemB, hemC, hemE and hemZ genes is elevated under conditions that give rise to elevated bacteriochlorophyll synthesis. Heme gene expression is shown to be affected by mutations in previously identified transcriptional regulators RegA, FnrL, CrtJ, and AerR, which also control expression of genes involved in bacteriochlorophyll and carotenoid synthesis, and synthesis of the apoprotein subunits of the photosynthetic and electron transport apparatus. High-resolution primer extension analysis of hem mRNA reveals the presence of numerous putative RegA, FnrL and CrtJ binding sites in several hem promoter regions.
Asunto(s)
Proteínas Bacterianas/fisiología , Regulación Bacteriana de la Expresión Génica , Hemo/genética , Proteínas del Complejo del Centro de Reacción Fotosintética/fisiología , Rhodobacter capsulatus/genética , Secuencia de Bases , ADN Bacteriano , Oxidación-Reducción , Regiones Promotoras Genéticas , Rhodobacter capsulatus/crecimiento & desarrolloRESUMEN
Neurogenesis in the dentate gyrus continues into adulthood, yet little is known about the function of newly born neurons or how they integrate into an existing network of mature neurons. We made transgenic mice that selectively and transiently express enhanced green fluorescent protein (EGFP) in newly born granule cells of the dentate gyrus under the transcriptional control of proopiomelanocortin (POMC) genomic sequences. Analysis of transgenic pedigrees with truncation or deletion mutations indicated that EGFP expression in the dentate gyrus required cryptic POMC promoter regions dispensable for arcuate hypothalamic or pituitary expression. Unlike arcuate neurons, dentate granule cells did not express the endogenous POMC gene. EGFP-positive neurons had immature properties, including short spineless dendrites and small action potentials. Colocalization with bromodeoxyuridine indicated that EGFP-labeled granule cells were approximately 2 weeks postmitotic. EGFP-labeled cells expressed markers for immature granule cells but not the glial marker GFAP. The number of EGFP-labeled neurons declined with age and increased with exercise, paralleling neurogenesis. Our results indicate that POMC-EGFP marks immature granule cells and that adult-generated granule cells integrate quite slowly into the hippocampal circuitry.
Asunto(s)
Giro Dentado/metabolismo , Neuronas/metabolismo , Transgenes/fisiología , Potenciales de Acción/fisiología , Envejecimiento/metabolismo , Animales , Biomarcadores/análisis , Bromodesoxiuridina , Recuento de Células , División Celular/fisiología , Movimiento Celular , Giro Dentado/citología , Genes Reporteros , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Molécula L1 de Adhesión de Célula Nerviosa/biosíntesis , Esfuerzo Físico/fisiología , Proopiomelanocortina/genética , Regiones Promotoras Genéticas/fisiología , Ácidos Siálicos/biosíntesisRESUMEN
C75, a recently derived compound that potently suppresses feeding and induces weight loss, has been proposed to act mainly by inhibiting fatty acid synthase (FAS) in central neurons that control feeding. For example, normal, fasting- associated, hypothalamic increases in neuropeptide Y (NPY)/Agouti-related protein (AGRP) expression and decreases in proopiomelanocortin (POMC)/cocaine and amphetamine regulated transcript (CART) expression were reported to be blocked by C75. Using loose-patch extracellular recording in acute slices, we tested the effect of C75 on anorexigenic POMC neurons and orexigenic NPY neurons of the hypothalamic arcuate nucleus, which were identified by promoter-driven GFP expression, as well as on feeding-unrelated cerebellar Purkinje neurons. We expected C75 to activate POMC neurons, inhibit NPY neurons, and have no effect on Purkinje neurons. Instead, C75 activated all cell types, suggesting that it lacks target specificity. This activation was probably not caused by FAS inhibition, because the classical FAS inhibitor, cerulenin, did not have this effect when tested on POMC and NPY neurons. Nonspecific neuronal activation and resulting neurological effects might contribute to the decreased feeding reported to follow centrally administered C75. Injection, i.p., of C75 induced severe loosening or liquefaction of stools, weight loss, and decreased food intake in both wild-type and melanocortin-4 receptor knockout mice. In contrast, cerulenin failed to loosen stools, even at a molar dose over 9-fold greater than C75, and had a much smaller effect on body weight. FAS inhibitory activity, by itself, seems to be insufficient to reproduce all of the effects of i.p.-injected C75.
Asunto(s)
4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacología , Núcleo Arqueado del Hipotálamo/citología , Ácido Graso Sintasas/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Células de Purkinje/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Animales , Ingestión de Alimentos/efectos de los fármacos , Ingestión de Alimentos/fisiología , Heces , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/fisiología , Neuropéptido Y/fisiología , Técnicas de Placa-Clamp , Proopiomelanocortina/fisiología , Células de Purkinje/fisiología , Pérdida de Peso/efectos de los fármacos , Pérdida de Peso/fisiologíaRESUMEN
It is now established that the hypothalamus is essential in coordinating endocrine, autonomic, and behavioral responses to changes in energy availability. However, the interaction of key peptides, neuropeptides, and neurotransmitters systems within the hypothalamus has yet to be delineated. Recently, we investigated the mechanisms through which central serotonergic (5-hydroxytryptamine, 5-HT) systems recruit leptin-responsive hypothalamic pathways, such as the melanocortin systems, to affect energy balance. Through a combination of functional neuroanatomy, feeding, and electrophysiology studies in rodents, we found that 5-HT drugs require functional melanocortin pathways to exert their effects on food intake. Specifically, we observed that anorectic 5-HT drugs activate pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (Arc). We provide evidence that the serotonin 2C receptor (5-HT(2C)R) is expressed on POMC neurons and contributes to this effect. Finally, we found that 5-HT drug-induced hypophagia is attenuated by pharmacological or genetic blockade of downstream melanocortin 3 and 4 receptors. We review candidate brain regions expressing melanocortin 3 and 4 receptors that play a role in energy balance. A model is presented in which activation of the melanocortin system is downstream of 5-HT and is necessary to produce the complete anorectic effect of 5-HT drugs. The data reviewed in this paper incorporate the central 5-HT system to the growing list of metabolic signals that converge on melanocortin neurons in the hypothalamus.
Asunto(s)
Metabolismo Energético/fisiología , Homeostasis , Proopiomelanocortina/metabolismo , Serotonina/metabolismo , Animales , Fenfluramina/metabolismo , Humanos , Hipotálamo/metabolismo , Neuronas/metabolismo , Serotoninérgicos/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Mice deficient in proopiomelanocortin peptides (Pomc(-/-)) generated on a 129 (A(w)/ A(w)) genetic background were back-crossed onto the C57BL/6 (a/a) genetic background. These mice exhibited most of the phenotypic characteristics previously reported on the 129 genetic background (Yaswen et al. 1999. Nat. Med. 5: 1066-1070). Adult mice became obese, their adrenals were atrophied, and they had undetectable plasma corticosterone in basal and stressed states. The partial perinatal lethality previously reported was also present on the C57BL/6 background. In addition, we found that both male and female homozygote (-/-) adults were fertile, but when homozygous males were intercrossed with homozygous females, all the pups died in the perinatal period. Attempts to rescue the perinatal lethality of pups from homozygous breeder pairs by supplementing the mother's drinking water with glucocorticoids were unsuccessful. Furthermore, failure to stimulate adrenal development and corticosterone production/release with daily exogenous adreno-corticotropin-stimulating hormone (ACTH) injections indicates an adrenal dependence on POMC peptides for normal development and function. While the original Pomc(-/-) mice, bred on a mixed white-bellied agouti (A(w)/ A(w)) 129 genetic background, had patchy alternations in their coat color, they clearly were not a uniform yellow like the lethal yellow (A(y)/a) mice. Our Pomc(-/-) mice bred onto the C57BL/6 (a/a) genetic background had a black coat color indistinguishable from that of the wild-type C57BL/6 mice, further suggesting that the POMC peptide melanocyte-stimulating hormone (alpha-MSH) is not essential for the production of eumelanin (black/brown) pigmentation.
Asunto(s)
Glándulas Suprarrenales/fisiopatología , Melanocitos/metabolismo , Obesidad/metabolismo , Péptidos/metabolismo , Pigmentación/fisiología , Proopiomelanocortina/metabolismo , Glándulas Suprarrenales/anatomía & histología , Animales , Núcleo Arqueado del Hipotálamo/citología , Núcleo Arqueado del Hipotálamo/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/genética , Tamaño de los Órganos , Péptidos/genética , Fenotipo , Pigmentación/genética , Hipófisis/citología , Hipófisis/metabolismo , Proopiomelanocortina/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Hypothalamic proopiomelanocortin (POMC) neurons are critical for controlling homeostatic functions in the mammal. We used a transgenic mouse model in which the POMC neurons were labeled with enhanced green fluorescent protein to perform visualized, whole-cell patch recordings from prepubertal female hypothalamic slices. The mouse POMC-enhanced green fluorescent protein neurons expressed the same endogenous conductances (a transient outward K(+) current and a hyperpolarization-activated, cation current) that have been described for guinea pig POMC neurons. In addition, the selective micro -opioid receptor agonist DAMGO induced an outward current (maximum of 12.8 +/- 1.2 pA), which reversed at K(+) equilibrium potential (E(K+)), in the majority (85%) of POMC neurons with an EC(50) of 102 nM. This response was blocked by the opioid receptor antagonist naloxone with an inhibition constant of 3.1 nM. In addition, the gamma-aminobutyric acid(B) receptor agonist baclofen (40 micro M) caused an outward current (21.6 +/- 4.0 pA) that reversed at E(K+) in these same neurons. The ATP-sensitive potassium channel opener diazoxide also induced an outward K(+) current (maximum of 18.7 +/- 2.2 pA) in the majority (92%) of POMC neurons with an EC(50) of 61 micro M. The response to diazoxide was blocked by the sulfonylurea tolbutamide, indicating that the POMC neurons express both Kir6.2 and sulfonylurea receptor 1 channel subunits, which was verified using single cell RT-PCR. This pharmacological and molecular profile suggested that POMC neurons might be sensitive to metabolic inhibition, and indeed, we found that their firing rate varied with changes in glucose concentrations. Therefore, it appears that POMC neurons may function as an integrator of metabolic cues and synaptic input for controlling homeostasis in the mammal.
Asunto(s)
Glucosa/farmacología , Hipotálamo/citología , Neuronas/fisiología , Canales de Potasio de Rectificación Interna/genética , Proopiomelanocortina/genética , Transportadoras de Casetes de Unión a ATP , Adenosina Trifosfato/metabolismo , Analgésicos Opioides/farmacología , Animales , Antihipertensivos/farmacología , Baclofeno/farmacología , Diazóxido/farmacología , Conductividad Eléctrica , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Femenino , Agonistas del GABA/farmacología , Agonistas de Receptores GABA-B , Proteínas Fluorescentes Verdes , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Canales de Potasio/genética , Canales de Potasio de Rectificación Interna/agonistas , Canales de Potasio de Rectificación Interna/metabolismo , Proopiomelanocortina/metabolismo , ARN Mensajero/análisis , Receptores de Droga , Receptores de GABA-B/fisiología , Receptores Opioides mu/agonistas , Receptores Opioides mu/fisiología , Receptores de SulfonilureasRESUMEN
D-fenfluramine (d-FEN) was once widely prescribed and was among the most effective weight loss drugs, but was withdrawn from clinical use because of reports of cardiac complications in a subset of patients. Discerning the neurobiology underlying the anorexic action of d-FEN may facilitate the development of new drugs to prevent and treat obesity. Through a combination of functional neuroanatomy, feeding, and electrophysiology studies in rodents, we show that d-FEN-induced anorexia requires activation of central nervous system melanocortin pathways. These results provide a mechanistic explanation of d-FEN's anorexic actions and indicate that drugs targeting these downstream melanocortin pathways may prove to be effective and more selective anti-obesity treatments.