RESUMO
AIMS/HYPOTHESIS: Loss of circadian clocks from all tissues causes defective glucose homeostasis as well as loss of feeding and activity rhythms. Little is known about peripheral tissue clocks, so we tested the hypothesis that an intrinsic circadian clock of the pancreas is important for glucose homeostasis. METHODS: We monitored real-time bioluminescence of pancreas explants from circadian reporter mice and examined clock gene expression in beta cells by immunohistochemistry and in situ hybridisation. We generated mice selectively lacking the essential clock gene Bmal1 (also known as Arntl) in the pancreas and tested mutant mice and littermate controls for glucose and insulin tolerance, insulin production and behaviour. We examined islets isolated from mutants and littermate controls for glucose-stimulated insulin secretion and total insulin content. RESULTS: Pancreas explants exhibited robust circadian rhythms. Clock genes Bmal1 and Per1 were expressed in beta cells. Despite normal activity and feeding behaviour, mutant mice lacking clock function in the pancreas had severe glucose intolerance and defective insulin production; their isolated pancreatic islets had defective glucose-stimulated insulin secretion, but normal total insulin content. CONCLUSIONS/INTERPRETATION: The mouse pancreas has an autonomous clock function and beta cells are very likely to be one of the pancreatic cell types possessing an intrinsic clock. The Bmal1 circadian clock gene is required in the pancreas, probably in beta cells, for normal insulin secretion and glucose homeostasis. Our results provide evidence for a previously unrecognised molecular regulator of pancreatic glucose-sensing and/or insulin secretion.
Assuntos
Ritmo Circadiano/fisiologia , Glucose/metabolismo , Homeostase/fisiologia , Insulina/metabolismo , Pâncreas/metabolismo , Pâncreas/fisiologia , Animais , Teste de Tolerância a Glucose , Imuno-Histoquímica , Hibridização In Situ , Secreção de Insulina , CamundongosRESUMO
Cryptochrome (CRY), a photoreceptor for the circadian clock in Drosophila, binds to the clock component TIM in a light-dependent fashion and blocks its function. In mammals, genetic evidence suggests a role for CRYs within the clock, distinct from hypothetical photoreceptor functions. Mammalian CRY1 and CRY2 are here shown to act as light-independent inhibitors of CLOCK-BMAL1, the activator driving Per1 transcription. CRY1 or CRY2 (or both) showed light-independent interactions with CLOCK and BMAL1, as well as with PER1, PER2, and TIM. Thus, mammalian CRYs act as light-independent components of the circadian clock and probably regulate Per1 transcriptional cycling by contacting both the activator and its feedback inhibitors.
Assuntos
Relógios Biológicos , Ritmo Circadiano , Proteínas de Drosophila , Proteínas do Olho , Flavoproteínas/fisiologia , Regulação da Expressão Gênica , Luz , Proteínas Nucleares/genética , Células Fotorreceptoras de Invertebrados , Células 3T3 , Fatores de Transcrição ARNTL , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Proteínas CLOCK , Proteínas de Ciclo Celular , Células Cultivadas , Criptocromos , Dimerização , Flavoproteínas/metabolismo , Genes Reporter , Sequências Hélice-Alça-Hélice , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , Receptores Acoplados a Proteínas G , Transativadores/antagonistas & inibidores , Transativadores/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Ativação Transcricional , TransfecçãoRESUMO
The mechanism by which mammalian circadian clocks are entrained to light-dark cycles is unknown. The clock that drives behavioral rhythms is located in the suprachiasmatic nucleus (SCN) of the brain, and entrainment is thought to require induction of genes in the SCN by light. A complementary DNA subtraction method based on genomic representational difference analysis was developed to identify such genes without making assumptions about their nature. Four clones corresponded to genes induced specifically in the SCN by light, all of which showed gating of induction by the circadian clock. Among these genes are c-fos and nur77, two of the five early-response genes known to be induced in the SCN by light, and egr-3, a zinc finger transcription factor not previously identified in the SCN. In contrast to known examples, egr-3 induction by light is restricted to the ventral SCN, a structure implicated in entrainment.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Genes fos , Luz , Núcleo Supraquiasmático/fisiologia , Fatores de Transcrição/genética , Animais , Elementos Antissenso (Genética) , Southern Blotting , Ritmo Circadiano , Clonagem Molecular , Cricetinae , DNA Complementar , Proteína 3 de Resposta de Crescimento Precoce , Masculino , Mesocricetus , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares , Receptores de EsteroidesRESUMO
The circadian clock in the suprachiasmatic nucleus (SCN) is thought to drive daily rhythms of behavior by secreting factors that act locally within the hypothalamus. In a systematic screen, we identified transforming growth factor-alpha (TGF-alpha) as a likely SCN inhibitor of locomotion. TGF-alpha is expressed rhythmically in the SCN, and when infused into the third ventricle it reversibly inhibited locomotor activity and disrupted circadian sleep-wake cycles. These actions are mediated by epidermal growth factor (EGF) receptors on neurons in the hypothalamic subparaventricular zone. Mice with a hypomorphic EGF receptor mutation exhibited excessive daytime locomotor activity and failed to suppress activity when exposed to light. These results implicate EGF receptor signaling in the daily control of locomotor activity, and identify a neural circuit in the hypothalamus that likely mediates the regulation of behavior both by the SCN and the retina.
Assuntos
Ritmo Circadiano/fisiologia , Receptores ErbB/metabolismo , Hipotálamo/metabolismo , Atividade Motora , Sono/fisiologia , Núcleo Supraquiasmático/metabolismo , Animais , Relógios Biológicos/efeitos dos fármacos , Relógios Biológicos/fisiologia , Temperatura Corporal/efeitos dos fármacos , Ventrículos Cerebrais/metabolismo , Ritmo Circadiano/efeitos dos fármacos , Cricetinae , Escuridão , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/genética , Feminino , Ligantes , Luz , Masculino , Mesocricetus , Camundongos , Atividade Motora/efeitos dos fármacos , Vias Neurais/fisiologia , Neurônios/metabolismo , Mutação Puntual , Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Transdução de Sinais , Sono/efeitos dos fármacos , Fator de Crescimento Transformador alfa/administração & dosagem , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/metabolismo , Fator de Crescimento Transformador alfa/farmacologiaRESUMO
The period (per) gene likely encodes a component of the Drosophila circadian clock. Circadian oscillations in the abundance of per messenger RNA and per protein (PER) are thought to arise from negative feedback control of per gene transcription by PER. A recently identified second clock locus, timeless (tim), apparently regulates entry of PER into the nucleus. Reported here are the cloning of complementary DNAs derived from the tim gene in a two-hybrid screen for PER-interacting proteins and the demonstration of a physical interaction between the tim protein (TIM) and PER in vitro. A restricted segment of TIM binds directly to a part of the PER dimerization domain PAS. PERL, a mutation that causes a temperature-sensitive lengthening of circadian period and a temperature-sensitive delay in PER nuclear entry, exhibits a temperature-sensitive defect in binding to TIM. These results suggest that the interaction between TIM and PER determines the timing of PER nuclear entry and therefore the duration of part of the circadian cycle.
Assuntos
Ritmo Circadiano/genética , Proteínas de Drosophila , Drosophila melanogaster/metabolismo , Proteínas Nucleares/metabolismo , Proteínas/metabolismo , Animais , Relógios Biológicos/genética , Núcleo Celular/metabolismo , Clonagem Molecular , Citoplasma/metabolismo , DNA Complementar/genética , Drosophila melanogaster/genética , Retroalimentação , Regulação da Expressão Gênica , Genes de Insetos , Mutação , Proteínas Nucleares/genética , Proteínas Circadianas Period , Proteínas/genética , Proteínas/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , TemperaturaRESUMO
Most organisms have circadian clocks consisting of negative feedback loops of gene regulation that facilitate adaptation to cycles of light and darkness. In this study, CRYPTOCHROME (CRY), a protein involved in circadian photoperception in Drosophila, is shown to block the function of PERIOD/TIMELESS (PER/TIM) heterodimeric complexes in a light-dependent fashion. TIM degradation does not occur under these conditions; thus, TIM degradation is uncoupled from abrogation of its function by light. CRY and TIM are part of the same complex and directly interact in yeast in a light-dependent fashion. PER/TIM and CRY influence the subcellular distribution of these protein complexes, which reside primarily in the nucleus after the perception of a light signal. Thus, CRY acts as a circadian photoreceptor by directly interacting with core components of the circadian clock.
Assuntos
Relógios Biológicos , Ritmo Circadiano , Proteínas de Drosophila , Proteínas do Olho , Flavoproteínas/metabolismo , Proteínas de Insetos/metabolismo , Luz , Células Fotorreceptoras de Invertebrados , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Criptocromos , Citoplasma/metabolismo , Escuridão , Dimerização , Drosophila , Flavoproteínas/genética , Proteínas de Fluorescência Verde , Proteínas de Insetos/genética , Proteínas Luminescentes , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , Receptores Acoplados a Proteínas G , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Leveduras/genética , Leveduras/metabolismoRESUMO
The mouse Clock gene encodes a bHLH-PAS protein that regulates circadian rhythms and is related to transcription factors that act as heterodimers. Potential partners of CLOCK were isolated in a two-hybrid screen, and one, BMAL1, was coexpressed with CLOCK and PER1 at known circadian clock sites in brain and retina. CLOCK-BMAL1 heterodimers activated transcription from E-box elements, a type of transcription factor-binding site, found adjacent to the mouse per1 gene and from an identical E-box known to be important for per gene expression in Drosophila. Mutant CLOCK from the dominant-negative Clock allele and BMAL1 formed heterodimers that bound DNA but failed to activate transcription. Thus, CLOCK-BMAL1 heterodimers appear to drive the positive component of per transcriptional oscillations, which are thought to underlie circadian rhythmicity.
Assuntos
Ritmo Circadiano/fisiologia , Proteínas Nucleares/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Fatores de Transcrição ARNTL , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Relógios Biológicos , Proteínas CLOCK , Proteínas de Ciclo Celular , Ritmo Circadiano/genética , Clonagem Molecular , Cricetinae , DNA/metabolismo , Dimerização , Retroalimentação , Expressão Gênica , Sequências Hélice-Alça-Hélice , Masculino , Mesocricetus , Camundongos , Mutação , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , Regiões Promotoras Genéticas , Retina/metabolismo , Núcleo Supraquiasmático/metabolismo , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
The circadian oscillator generates a rhythmic output with a period of about 24 hours. Despite extensive studies in several model systems, the biochemical mode of action has not yet been demonstrated for any of its components. Here, the Drosophila CLOCK protein was shown to induce transcription of the circadian rhythm genes period and timeless. dCLOCK functioned as a heterodimer with a Drosophila homolog of BMAL1. These proteins acted through an E-box sequence in the period promoter. The timeless promoter contains an 18-base pair element encompassing an E-box, which was sufficient to confer dCLOCK responsiveness to a reporter gene. PERIOD and TIMELESS proteins blocked dCLOCK's ability to transactivate their promoters via the E-box. Thus, dCLOCK drives expression of period and timeless, which in turn inhibit dCLOCK's activity and close the circadian loop.
Assuntos
Ritmo Circadiano/fisiologia , Proteínas de Drosophila , Proteínas de Insetos/genética , Proteínas Nucleares/genética , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Fatores de Transcrição ARNTL , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Relógios Biológicos , Proteínas CLOCK , Linhagem Celular , Núcleo Celular/metabolismo , Ritmo Circadiano/genética , Dimerização , Drosophila , Retroalimentação , Expressão Gênica , Sequências Hélice-Alça-Hélice , Proteínas de Insetos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transativadores/genética , Fatores de Transcrição/genética , TransfecçãoRESUMO
The biologically active photoproduct of rhodopsin, metarhodopsin II (M II), exists in a pH-sensitive equilibrium with its precursor, metarhodopsin I (M I). Increasing acidity favors M II, with the midpoint of the pH titration curve at pH 6.4. To test the long-standing proposal that histidine protonation regulates this conformational transition, we characterized mutant rhodopsins in which each of the 6 histidines was replaced by phenylalanine or cysteine. Only mutants substituted at the 3 conserved histidines showed abnormal M I-M II equilibria. Those in which His-211 was replaced by phenylalanine or cysteine formed little or no M II at either extreme of pH, whereas mutants substituted at His-65 or at His-152 showed enhanced sensitivity to protons. The simplest interpretation of these results is that His-211 is the site where protonation strongly stabilizes the M II conformation and that His-65 and His-152 are sites where protonation modestly destabilizes the M II conformation.
Assuntos
Rodopsina/análogos & derivados , Rodopsina/química , Sequência de Aminoácidos , Animais , Bovinos , Análise Mutacional de DNA , Digitonina , Proteínas do Olho/química , Histidina/química , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peptídeos/química , Fotoquímica , Proteínas Recombinantes/química , Opsinas de Bastonetes , Solubilidade , Análise EspectralRESUMO
We report the cloning and mapping of mouse (mTim) and human (hTIM) orthologs of the Drosophila timeless (dtim) gene. The mammalian Tim genes are widely expressed in a variety of tissues; however, unlike Drosophila, mTim mRNA levels do not oscillate in the suprachiasmatic nucleus (SCN) or retina. Importantly, hTIM interacts with the Drosophila PERIOD (dPER) protein as well as the mouse PER1 and PER2 proteins in vitro. In Drosophila (S2) cells, hTIM and dPER interact and translocate into the nucleus. Finally, hTIM and mPER1 specifically inhibit CLOCK-BMAL1-induced transactivation of the mPer1 promoter. Taken together, these results demonstrate that mTim and hTIM are mammalian orthologs of timeless and provide a framework for a basic circadian autoregulatory loop in mammals.
Assuntos
Ritmo Circadiano/genética , Proteínas de Drosophila , Proteínas de Insetos/fisiologia , Proteínas Nucleares/metabolismo , Transativadores/genética , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Fatores de Transcrição ARNTL , Processamento Alternativo/genética , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Relógios Biológicos/genética , Proteínas CLOCK , Proteínas de Ciclo Celular , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 12/genética , Clonagem Molecular , Drosophila , Feminino , Humanos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Nucleares/fisiologia , Proteínas Circadianas Period , Polimorfismo Genético , RNA Mensageiro/biossíntese , Transativadores/antagonistas & inibidores , Fatores de Transcrição/metabolismoRESUMO
A previous study suggested that schizophrenic subjects exhibit an impaired ability to correct their own errors of movement without using exteroceptive signals. However, the performance of schizophrenic subjects was compared to that of only one other psychiatric group (alcoholic subjects), and a relatively small number of subjects was studied. To investigate the specificity of the postulated impairment, 9 schizophrenic, 11 depressed, and 8 normal subjects performed a tracking task designed to prevent the use of exteroceptive cues in correcting errors of movement. The depressed and normal groups did not differ significantly on any performance measure, but the schizophrenic subjects again demonstrated a gross impairment in correcting errors, yet no impairment in initiating correct responses. These findings suggest that the impaired ability to monitor ongoing motor behavior on the basis of internal, self-generated cues may be specific to schizophrenia among major psychiatric disorders.
Assuntos
Atenção , Transtorno Depressivo/psicologia , Desempenho Psicomotor , Psicologia do Esquizofrênico , Adulto , Doença Crônica , Sinais (Psicologia) , Retroalimentação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Escalas de Graduação Psiquiátrica , Esquizofrenia Hebefrênica/psicologia , Esquizofrenia Paranoide/psicologiaRESUMO
Cloned cDNA encoding bovine rhodopsin has been recombined into an expression vector and cotransfected with an antibiotic resistance plasmid into cultured human embryonic kidney cells. The resulting cell lines produce 100-200 micrograms of bovine opsin per liter of saturated tissue culture medium (10(9) cells). Incubation in vitro with 11-cis retinal produces a photolabile pigment the absorbance spectrum of which is indistinguishable from that of bona fide bovine rhodopsin. Expressed rhodopsin accumulates in the plasma membrane as determined by immunoelectron microscopy.
Assuntos
DNA/genética , Pigmentos da Retina/biossíntese , Rodopsina/biossíntese , Animais , Anticorpos Monoclonais , Linhagem Celular , Membrana Celular/análise , Membrana Celular/ultraestrutura , Clonagem Molecular , Expressão Gênica , Humanos , Imuno-Histoquímica , Plasmídeos , Rodopsina/análise , Rodopsina/genética , Espectrofotometria , Suínos , TransfecçãoRESUMO
Circadian clocks are widely distributed in mammalian tissues, but little is known about the physiological functions of clocks outside the suprachiasmatic nucleus of the brain. The retina has an intrinsic circadian clock, but its importance for vision is unknown. Here, we show that mice lacking Bmal1, a gene required for clock function, had abnormal retinal transcriptional responses to light and defective inner retinal electrical responses to light, but normal photoreceptor responses to light and retinas that appeared structurally normal as observed by light and electron microscopy. We generated mice with a retina-specific genetic deletion of Bmal1, and they had defects of retinal visual physiology essentially identical to those of mice lacking Bmal1 in all tissues and lacked a circadian rhythm of inner retinal electrical responses to light. Our findings indicate that the intrinsic circadian clock of the retina regulates retinal visual processing in vivo.
Assuntos
Ritmo Circadiano/fisiologia , Retina/fisiologia , Núcleo Supraquiasmático/fisiologia , Fatores de Transcrição ARNTL , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/deficiência , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Ritmo Circadiano/genética , Primers do DNA/genética , Eletrorretinografia , Expressão Gênica , Luz , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Análise de Sequência com Séries de Oligonucleotídeos , Células Fotorreceptoras de Vertebrados/fisiologia , Células Fotorreceptoras de Vertebrados/ultraestrutura , Retina/efeitos da radiação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Núcleo Supraquiasmático/lesõesRESUMO
We have studied the metarhodopsin I (M I)-metarhodopsin II (M II) equilibria of expressed wild-type and mutant rhodopsins. We studied two classes of mutants with amino acid substitutions in or near the putative transmembrane segments: those in which a charged residue was replaced by a neutral residue (or in one case another charged residue) and those in which a neutral residue likely (or postulated) to be in proximity to the retinylidene Schiff's base was replaced by a charged residue. In the first class, we found mutants that abnormally favored M II (replacements of Asp-83, Glu-134, or Arg-135) as well as one that abnormally favored M I (replacement of Glu-122). In the second class, we found several mutants that abnormally favored M I, the most extreme being those in which glutamate replaced His-211 or Ala-292. These studies suggest that electrostatic forces play a major role in the energetics of the M 1-to-M II transition, and they indicate that electrostatic perturbation in the vicinity of the protonated retinylidene Schiff's base is a plausible mechanism for the change in its pKa that is associated with the M I-M II transition. They further suggest that the highly conserved pair of charged residues homologous to Glu-134 and Arg-135 may play a general role in agonist-dependent conformational changes in G-protein-coupled receptors.
Assuntos
Rodopsina/análogos & derivados , Rodopsina/química , Animais , Bovinos , Concentração de Íons de Hidrogênio , Proteínas de Membrana/química , Mutagênese Sítio-Dirigida , Segmento Externo da Célula Bastonete/química , Bases de Schiff , Análise Espectral , Relação Estrutura-AtividadeRESUMO
The possibility that morphine could be synthesized in animals has long been considered and a pathway in mammalian brain analogous to that in the opium poppy has been proposed. Substances have been detected in mammalian brain that are recognized by antisera raised against morphine. Recently we reported the presence of three such immunoreactive substances in bovine hypothalamus and adrenal, and in rat brain, and the definitive identification of two of them by gas chromatography-mass spectrometry as morphine and codeine. Incorporation of a labelled precursor has demonstrated the biosynthesis of morphine in the opium poppy from tyrosine-derived units (see Fig. 1). Intramolecular coupling of reticuline to form salutaridine is the critical step that generates the morphine skeleton (morphinan) and the stereochemistry of the morphinan series. We now report the conversion in vivo and in vitro of reticuline to salutaridine by rat liver, but this conversion is not detectable in rat brain and bovine adrenal. This is the first direct demonstration of the synthesis of a morphinan in an animal tissue and also supports the hypothesis that morphine and codeine in brain and adrenal are of endogenous origin.
Assuntos
Alcaloides/metabolismo , Benzilisoquinolinas , Isoquinolinas , Fígado/metabolismo , Morfinanos/biossíntese , Morfina/biossíntese , Glândulas Suprarrenais/metabolismo , Animais , Encéfalo/metabolismo , Bovinos , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , RatosRESUMO
Recently, we described the presence of six immunoreactive (ir) morphinans in bovine adrenal and hypothalamus and identified one as morphine [Goldstein, A., Barrett, R. W., James, I. F., Lowney, L. I., Weitz, C. J., Knipmeyer, L. L. & Rapaport, H. (1985) Proc. Natl. Acad. Sci. USA 82, 5203-5207]. We now report that ir morphinans corresponding to the previously reported peak 1 (morphine), peak 4, and peak 5 are consistently present in extracts of bovine hypothalamus and variably present in extracts of bovine adrenal and rat brain. We no longer detect the previously reported peaks 2, 3, or 6, and we have established that they were contamination artifacts. Peak 1 is coeluted with morphine in two distinct reversed-phase HPLC systems, as is peak 4 with codeine. We have purified peak 1 and peak 4 compounds from bovine hypothalamus and determined their identities by gas chromatography/mass spectrometry (GC/MS): peak 1 is confirmed to be morphine and peak 4 is codeine.
Assuntos
Química Encefálica , Codeína/análise , Endorfinas/análise , Morfina/análise , Glândulas Suprarrenais/análise , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Hipotálamo/análise , Radioimunoensaio , RatosRESUMO
Recently, we described three substances in bovine hypothalamus, adrenal, and rat brain recognized by antisera raised against morphine, and we identified one as morphine and another as codeine by GC/MS. We now report the identification of the third immunoreactive (ir) morphinan from bovine brain as 6-acetylmorphine by chemical conversion to morphine, GC/MS, and high-resolution mass measurement. 6-Acetylmorphine has not previously been described as a natural product in plants or animals, but it has long been known as the metabolite in part responsible for the biological properties of heroin. However, we have excluded slaughter-house or laboratory contamination by any morphinan as well as derivation from the morphine in tissues during our procedures. 6-Acetylmorphine is known to be more potent than morphine in vivo chiefly by virtue of its greater penetration into the central nervous system. Should morphinans prove to have physiological functions in animals, the properties of 6-acetylmorphine make it ideal for fulfilling the role of a peripheral-to-central hormone.
Assuntos
Química Encefálica , Derivados da Morfina/análise , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Hipotálamo/análise , Morfina/análise , Morfina/metabolismo , Derivados da Morfina/metabolismo , RatosRESUMO
Tritanopia is an autosomal dominant genetic disorder of human vision characterize by a selective deficiency of blue spectral sensitivity. The defect is manifested within the retina and could be caused by a deficiency in function or numbers (or both) of blue-sensitive cone photoreceptors. We have used PCR, denaturing gradient gel electrophoresis, and DNA sequencing of amplified exons to detect in four of nine unrelated tritanopic subjects two different point mutations in the gene encoding the blue-sensitive opsin, each leading to an amino acid substitution. Segregation analysis within pedigrees and hybridization of oligonucleotides specific for each allele to DNA samples from control subjects support the hypothesis that these mutations cause tritanopia. These results complete the genetic evidence for the trichromatic theory of human color vision.
Assuntos
Defeitos da Visão Cromática/genética , Proteínas do Olho/genética , Pigmentos da Retina/genética , Arginina/genética , Sequência de Bases , Distribuição de Qui-Quadrado , Clonagem Molecular , DNA/análise , Sondas de DNA , Eletroforese em Gel de Campo Pulsado , Genes Dominantes , Glicina/genética , Humanos , Dados de Sequência Molecular , Mutação/genética , Técnicas de Amplificação de Ácido Nucleico , Linhagem , Reação em Cadeia da Polimerase , Opsinas de BastonetesRESUMO
We describe nonpeptide opioids found in extracts of beef hypothalamus and adrenal, which are recognized by antisera raised against morphine. Four have been purified to homogeneity. One is morphine. The structures of the other three have not been determined yet. None of them are derived from morphine or normorphine after extraction from the tissues. It is not known whether the opiates described here are of endogenous or exogenous origin.