RESUMO
The maize Vp1 gene and abi3 gene of Arabidopsis are believed to be orthologs based on similarities of the mutant phenotypes and amino acid sequence conservation. Here we show that expression of VP1 driven by the 35S promoter can partially complement abi3-6, a deletion mutant allele of abi3. The visible phenotype of seed produced from VP1 expression in the abi3 mutant background is nearly indistinguishable from wild type. VP1 fully restores abscisic acid (ABA) sensitivity of abi3 during seed germination and suppresses the early flowering phenotype of abi3. The temporal regulation of C1-beta-glucuronidase (GUS) and chlorophyll a/b binding protein (cab3)-GUS reporter genes in developing seeds of 35S-VP1 lines were similar to wild type. On the other hand, two qualitative differences are observed between the 35S-VP1 line and wild type. The levels of CRC and C1-GUS expression are markedly lower in the seeds of 35S-VP1 lines than in wild type suggesting incomplete complementation of gene activation functions. Similar to ectopic expression of ABI3 (Parcy et al., 1994), ectopic expression of VP1 in vegetative tissue enhances ABA inhibition of root growth. In addition, 35S-VP1 confers strong ABA inducible expression of the normally seed-specific cruciferin C (CRC) gene in leaves. In contrast, ectopic ABA induction of C1-GUS is restricted to a localized region of the root elongation zone. The ABA-dependent C1-GUS expression expanded to a broader area in the root tissues treated with exogenous application of auxin. Interestingly, auxin-induced lateral root formation is completely suppressed by ABA in 35S-VP1 plants but not in wild type. These results indicate VP1 mediates a novel interaction between ABA and auxin signaling that results in developmental arrest and altered patterns of gene expression.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis , Genes de Plantas , Ácidos Indolacéticos/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Transativadores/genética , Ácido Abscísico/farmacologia , Arabidopsis/genética , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Germinação/efeitos dos fármacos , Sementes , Transdução de Sinais , Supressão Genética , Fatores de Transcrição , Ativação Transcricional , Transgenes , Zea mays/genéticaRESUMO
Abscisic acid (ABA) is a key regulator of seed dormancy and plant responses to environmental challenges. ABA is synthesized via an oxidative cleavage of 9-cis epoxy-carotenoids, the first committed and key regulatory step in the ABA biosynthetic pathway. Vp14 of maize encodes an epoxy-carotenoid dioxygenase that is soluble when expressed in E. coli. An important goal has been to determine how the soluble VP14 protein is targeted to epoxy-carotenoid substrates that are located in the thylakoid and envelope membranes of chloroplasts and other plastids. Using an in vitro chloroplast import assay, we have shown that VP14 is imported into chloroplasts with cleavage of a short stroma-targeting domain. The mature VP14 exists in two forms, one which is soluble in stroma and the other bound to thylakoid membranes. Analysis of a series of truncated VP14 mutants mapped the membrane targeting signal to the 160 amino acid N-terminal sequence. A putative amphipathic alpha-helix within this region is essential, but not sufficient, for the membrane targeting. Either deletion of or insertion of helix breaking residues into this region abolished the membrane binding, whereas a chimeric protein carrying just the amphipathic region fused with bacterial glutathione S-transferase failed to associate with the thylakoid membrane. The membrane-bound VP14 was partially resistant to chaotropic washes such as 0.1 M Na2CO3 (pH 11.5) and 6 M urea. Unlabelled recombinant VP14 inhibited the tight binding of imported VP14, suggesting that VP14 is associated with specific components of the thylakoid membrane.
Assuntos
Cloroplastos/metabolismo , Membranas Intracelulares/metabolismo , Oxigenases/metabolismo , Tilacoides/metabolismo , Sequência de Aminoácidos , Ligação Competitiva , Compartimento Celular , Dioxigenases , Dados de Sequência Molecular , Oxigenases/genética , Proteínas de Plantas , Ligação Proteica , Sinais Direcionadores de Proteínas , Estrutura Secundária de Proteína , Transporte Proteico , Proteínas Recombinantes/metabolismoRESUMO
In the present studies we have examined the effects of a new calcium channel blocker, LY393615 ((N-Butyl-[5,5-bis-(4-fluorophenyl)tetrahydrofuran-2-yl]methylamine hydrochloride, NCC1048) in a model of hypoxia-hypoglycaemia in vitro and in a gerbil model of global and in two rat models of focal cerebral ischaemia in vivo. Results indicated that LY393615 protected against hypoxia-hypoglycaemic insults in brain slices and also provided significant protection against ischaemia-induced hippocampal damage in gerbil global cerebral ischaemia when dosed at 10, 12.5 (P<0.05) or 15 mg/kg i.p. (P<0.01) 30 min before and 2 h 30 min after occlusion. The compound penetrated the brain well after a 15 mg/kg i.p. dose and had a half-life of 2.5 h. In further studies LY393615 was protective 1 h post-occlusion when administered at 15 mg/kg i.p. followed by 2 doses of 5 mg/kg i.p. 2 and 3 h later. LY393615 dosed at 15 mg/kg i.p. followed by 2 further doses of 5 mg/kg i.p. (2 and 3 h later) also produced a significant reduction in the infarct volume following Endothelin-1 (Et-1) middle cerebral artery occlusion in the rat when administration was initiated immediately (P<0.01) or 1 h (P<0.05) after occlusion. The compound was also evaluated in the intraluminal monofilament model of focal ischaemia. The animals had the middle cerebral artery occluded for 2 h, and 15 min after reperfusion LY393615 was administered at 15 mg/kg i.p. followed by 2 mg/kg/h i.v. infusion for 6 h. There was no reduction in infarct volume using this dosing protocol. In conclusion, in the present studies we have reported that a novel calcium channel blocker, LY393615, with good bioavailability protects against neuronal damage caused by hypoxia-hypoglycaemia in vitro and both global and focal cerebral ischaemia in vivo. The compound is neuroprotective when administered post-occlusion and may therefore be a useful anti-ischaemic agent.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Butilaminas/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Furanos/farmacologia , Fármacos Neuroprotetores/farmacologia , Bloqueadores dos Canais de Sódio , Animais , Isquemia Encefálica/patologia , Butilaminas/química , Bloqueadores dos Canais de Cálcio/química , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Furanos/química , Gerbillinae , Hipocampo/irrigação sanguínea , Hipocampo/patologia , Técnicas In Vitro , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/patologia , Masculino , Fármacos Neuroprotetores/química , Células de Purkinje/efeitos dos fármacos , Células de Purkinje/patologia , Ratos , Ratos WistarRESUMO
In the present studies, we have evaluated the effects of N-[4-(2-¿[(3-Chlorophenyl)methyl]amino¿ethyl)phenyl]-2-thiophenecarbo ximidamide dihydrochloride (ARL 17477) on recombinant human neuronal NOS (nNOS) and endothelial NOS (eNOS). We then carried out pharmacokinetic studies and measured cortical nitric oxide synthase (NOS) inhibition to determine that the compound crossed the blood brain barrier. Finally, the compound was evaluated in a model of global ischaemia in the gerbil and two models of transient focal ischaemia in the rat. The IC(50) values for ARL 17477 on human recombinant human nNOS and eNOS were 1 and 17 microM, respectively. ARL 17477 (50 mg/kg i.p.) produced a significant reduction in the ischaemia-induced hippocampal damage following global ischaemia when administered immediately post-occlusion, but failed to protect when administration was delayed until 30 min post-occlusion. In the endothelin-1 model of focal ischaemia, ARL 17477 (1 mg/kg i.v.) significantly attenuated the infarct volume when administered at either 0, 1 or 2 h post-endothelin-1 (P<0.05). In the intraluminal suture model, ARL 17477 at both 1 and 3 mg/kg i.v. failed to reduce the infarct volume measured at 1, 3 or 7 days post-occlusion. These results demonstrate that ARL 17477 protects against global ischaemia in gerbils and provides some reduction in infarct volume following transient middle cerebral artery occlusion in rats, indicating that nNOS inhibition may be a useful treatment of ischaemic conditions.
Assuntos
Amidinas/farmacocinética , Isquemia Encefálica/tratamento farmacológico , Inibidores Enzimáticos/farmacocinética , Fármacos Neuroprotetores/farmacocinética , Óxido Nítrico Sintase/antagonistas & inibidores , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Isquemia Encefálica/enzimologia , Isquemia Encefálica/fisiopatologia , Linhagem Celular , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Interações Medicamentosas/fisiologia , Endotelina-1/farmacologia , Gerbillinae , Humanos , Masculino , Óxido Nítrico Sintase/efeitos dos fármacos , Óxido Nítrico Sintase/metabolismo , Ratos , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/fisiopatologia , Fatores de TempoRESUMO
The VP1 transcription factor functions as both a repressor and an activator of gene expression in the developing aleurone. Vp1 activation of the anthocyanin pathway exhibits strict cell autonomy in aleurone. In contrast, Vp1-mediated repression of hydrolase genes in aleurone cells during seed development is determined by a combination of cell autonomous and cell non-autonomous signals. To analyze signaling between the embryo and aleurone during seed development, a T-B3La chromosome translocation was used to create seed that has non-concordant embryo and endosperm genotypes. We show that de-repression of an Amy-GUS reporter gene in developing vp1 mutant aleurone cells strongly depends on the presence of a viviparous embryo. Genetic ablation of the developing embryo in vp1 mutant and Vp1 seeds through the introduction of an early embryo mutation caused a similar enhancement of Amy-GUS expression in the aleurone, suggesting that the quiescent embryo present in normal seed is a critical source of inhibitory signals. Analysis of an ABA deficient vp1 vp5 double mutant indicates that ABA synthesized in the embyro interacts additively with Vp1 to prevent precocious induction of alpha-amylase genes in the aleurone of the developing seed. A lack of ABA synthesis, however, does not account for the strongly synergistic interaction between a viviparous vp1 embryo and mutant aleurone suggesting that a quiescent embyro is a source of other inhibitory signals.
Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/fisiologia , Sementes/genética , Transdução de Sinais , Transativadores/fisiologia , Zea mays/genética , alfa-Amilases/genética , Ácido Abscísico/genética , Ácido Abscísico/fisiologia , Genótipo , Germinação , Giberelinas/genética , Modelos Biológicos , Mosaicismo , Mutação , Fenótipo , Reguladores de Crescimento de Plantas/genética , Reguladores de Crescimento de Plantas/fisiologia , Proteínas de Plantas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Sementes/embriologia , Sementes/enzimologia , Sementes/fisiologia , Transativadores/genética , Translocação Genética/genética , Zea mays/citologia , Zea mays/embriologia , Zea mays/enzimologiaRESUMO
The notabilis (not) mutant of tomato has a wilty phenotype due to a deficiency in the levels of the plant hormone abscisic acid (ABA). The mutant appears to have a defect in a key control step in ABA biosynthesis--the oxidative cleavage of a 9-cis xanthophyll precursor to form the C15 intermediate, xanthoxin. A maize mutant, viviparous 14 (vp14) was recently obtained by transposon mutagenesis. This maize genetic lesion also affects the oxidative cleavage step in ABA synthesis. Degenerate primers for PCR, based on the VP14 predicted amino acid sequence, have been used to provide probes for screening a wilt-related tomato cDNA library. A full-length cDNA clone was identified which is specific to the not gene locus. The ORFs of the tomato cDNA and maize Vp14 are very similar, apart from parts of their N-terminal sequences. The not mutation has been characterized at the DNA level. A specific A/T base pair deletion of the coding sequence has resulted in a frameshift mutation, indicating that not is a null mutant. This observation is discussed in connection with the relatively mild phenotype exhibited by not mutant homozygotes.
Assuntos
Ácido Abscísico/genética , Mutação , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Zea mays/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA , DNA Complementar , Dioxigenases , Dados de Sequência Molecular , Oxigenases/química , Oxigenases/genética , Homologia de Sequência de AminoácidosRESUMO
The present work describes the neuroprotective effects of the free radical spin trap, MDL 101,002, in models of permanent and transient focal ischemia. Permanent focal ischemia was carried out by occlusion of the distal segment of the middle cerebral artery (MCA) and CCA's in Spontaneously Hypertensive (SH) and Wistar rats. Transient focal ischemia was undertaken by occluding the origin of the MCA for 180 min by the intraluminar monofilament method in Wistar rats. With permanent distal MCA occlusion in SH rats, 100 mg/kg i.v. at 30 min post-ischemia resulted in a significant 40% reduction in infarct volume. Similarly, a 75 mg/kg bolus + 45 mg/kg-h dose of MDL 101,002 given i.v. at 5 min post-ischemia resulted in a 90% or 60% decrease in infarct volume in the mixed permanent/transient distal MCA model with Wistar rats using 120 or 180 min of CCA occlusion, respectively. When full reperfusion was established, after 180 min of occlusion in the proximal MCA model, a dose of 40 mg/kg + infusion and 75 mg/kg + infusion resulted in a significant 50% and 70% decrease in ischemic damage, respectively. MDL 101,002 is clearly an effective neuroprotective agent in all models examined. This work would suggest that this novel cyclic nitrone spin trap affords effective neuroprotection and is useful for the treatment of ischemic stroke.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Isoquinolinas/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Óxidos de Nitrogênio/uso terapêutico , Animais , Temperatura Corporal/efeitos dos fármacos , Sequestradores de Radicais Livres , Isoquinolinas/farmacologia , Fármacos Neuroprotetores/farmacologia , Óxidos de Nitrogênio/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Marcadores de SpinRESUMO
Propentofylline (HWA 285, 3-methyl-1-(5-oxo-hexyl)-7-propylxanthine) is an adenosine uptake and phosphodiesterase inhibitor that has been shown to be neuroprotective in both global and permanent focal ischemia animal models. However, to date, the efficacy of propentofylline has never been examined in an animal model of temporary focal ischemia or the 'therapeutic window' systematically examined in a focal ischemia model. The present experiments were designed to investigate these. Temporary (3 h) middle cerebral artery occlusion was accomplished by the monofilament method. Infarct volumes were determined at 24 h from 2,3,5-triphenyltetrazolieum chloride (TTC) stained coronal slices. Animals were dosed with vehicle or propentofylline at 3 mg/kg bolus and/or a 6 mg/kg per h infusion (24 h infusion) at 30 min, 1 h or 3 h post ischemia onset. Physiological monitoring on a subset of animals indicated no changes in mean arterial pressure, blood gases, blood pH, and glucose levels with either ischemia or drug treatment. Propentofylline treatment resulted in a statistically significant decrease in infarct volume when an infusion dose of 6 mg/kg per h was initiated at 30 min or when a bolus of 3 mg/kg plus an infusion dose was initiated at 1 h but not 3 h post ischemia. Therefore, propentofylline is neuroprotective in a model of temporary focal ischemia. This suggests that combination therapy with propentofylline might lead to clinical improvement beyond that which would occur with thrombolytics alone. The apparent short window of opportunity for effective dosing is consistent with the proposed mechanism of action for propentofylline.
Assuntos
Ataque Isquêmico Transitório/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Xantinas/uso terapêutico , Animais , Gasometria , Glicemia/metabolismo , Encéfalo/patologia , Infarto Cerebral/tratamento farmacológico , Infarto Cerebral/patologia , Ataque Isquêmico Transitório/patologia , Masculino , Ratos , Ratos Wistar , Fatores de TempoRESUMO
BACKGROUND AND PURPOSE: Stroke patients often experience a significant temporal delay between the onset of ischemia and the time to initiation of therapy. Thus, there is a need for neuroprotectants with a long therapeutic window of opportunity. The efficacy of a potent, central nervous system-penetrating calpain inhibitor (MDL 28,170) was evaluated in a temporary model of focal cerebral ischemia to determine the window of opportunity for intracellular protease inhibition. METHODS: An ex vivo brain protease inhibition assay established pharmacodynamic dosing parameters for MDL 28,170. Middle cerebral artery (MCA) occlusion was accomplished by advancing a monofilament through the internal carotid artery to the origin of the MCA. Postmortem infarct volumes were determined by quantitative image analysis of triphenyltetrazolium-stained brain sections. RESULTS: Maximal inhibition of brain protease activity was observed 30 minutes after injection of MDL 28,170 with an estimated pharmacodynamic half-life of 2 hours. MDL 28,170 caused a dose-dependent reduction in infarct volume when administered 30 minutes after MCA occlusion. A window of opportunity study was conducted to determine the maximal delay between the onset of ischemia and the initiation of efficacious therapy. MDL 28,170 reduced infarct volume when therapy was delayed for 0.5, 3, 4, and 6 hours after the initiation of ischemia. The protective effect of MDL 28,170 was lost after an 8-hour delay. CONCLUSIONS: These data indicate that the therapeutic window of opportunity for calpain inhibition is at least 6 hours in a reversible focal cerebral ischemia model. This protection is observed despite the lethal hypoxic and excitotoxic challenge, suggesting that calpain activation may be an obligatory, downstream event in the ischemic cell death cascade.
Assuntos
Calpaína/antagonistas & inibidores , Inibidores de Cisteína Proteinase/uso terapêutico , Dipeptídeos/uso terapêutico , Ataque Isquêmico Transitório/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Animais , Encéfalo/enzimologia , Artéria Carótida Interna , Morte Celular , Doenças Arteriais Cerebrais/complicações , Infarto Cerebral/tratamento farmacológico , Infarto Cerebral/enzimologia , Infarto Cerebral/etiologia , Infarto Cerebral/patologia , Corantes , Inibidores de Cisteína Proteinase/administração & dosagem , Inibidores de Cisteína Proteinase/farmacocinética , Dipeptídeos/administração & dosagem , Dipeptídeos/farmacocinética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ativação Enzimática , Meia-Vida , Hipóxia/etiologia , Processamento de Imagem Assistida por Computador , Ataque Isquêmico Transitório/enzimologia , Ataque Isquêmico Transitório/etiologia , Ataque Isquêmico Transitório/patologia , Masculino , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/farmacocinética , Neurotoxinas/efeitos adversos , Ratos , Ratos Wistar , Sais de Tetrazólio , Fatores de TempoRESUMO
Abscisic acid (ABA), an apocarotenoid synthesized from cleavage of carotenoids, regulates seed maturation and stress responses in plants. The viviparous seed mutants of maize identify genes involved in synthesis and perception of ABA. Two alleles of a new mutant, viviparous14 (vp14), were identified by transposon mutagenesis. Mutant embryos had normal sensitivity to ABA, and detached leaves of mutant seedlings showed markedly higher rates of water loss than those of wild type. The ABA content of developing mutant embryos was 70% lower than that of wild type, indicating a defect in ABA biosynthesis. vp14 embryos were not deficient in epoxy-carotenoids, and extracts of vp14 embryos efficiently converted the carotenoid cleavage product, xanthoxin, to ABA, suggesting a lesion in the cleavage reaction. vp14 was cloned by transposon tagging. The VP14 protein sequence is similar to bacterial lignostilbene dioxygenases (LSD). LSD catalyzes a double-bond cleavage reaction that is closely analogous to the carotenoid cleavage reaction of ABA biosynthesis. Southern blots indicated a family of four to six related genes in maize. The Vp14 mRNA is expressed in embryos and roots and is strongly induced in leaves by water stress. A family of Vp14-related genes evidently controls the first committed step of ABA biosynthesis. These genes are likely to play a key role in the developmental and environmental control of ABA synthesis in plants.
Assuntos
Ácido Abscísico/biossíntese , Dioxigenases , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Zea mays/genética , Sequência de Aminoácidos , Carotenoides , Regulação da Expressão Gênica no Desenvolvimento , Biblioteca Gênica , Dados de Sequência Molecular , Mutação , Oxigenases/genética , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Sementes/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Sesquiterpenos/metabolismoRESUMO
The plant growth regulator abscisic acid (ABA) is formed by the oxidative cleavage of an epoxy-carotenoid. The synthesis of other apocarotenoids, such as vitamin A in animals, may occur by a similar mechanism. In ABA biosynthesis, oxidative cleavage is the first committed reaction and is believed to be the key regulatory step. A new ABA-deficient mutant of maize has been identified and the corresponding gene, Vp14, has been cloned. The recombinant VP14 protein catalyzes the cleavage of 9-cis-epoxy-carotenoids to form C25 apo-aldehydes and xanthoxin, a precursor of ABA in higher plants.
Assuntos
Carotenoides/metabolismo , Dioxigenases , Proteínas de Plantas/metabolismo , Xantofilas , Zea mays/metabolismo , beta Caroteno/análogos & derivados , Ácido Abscísico/biossíntese , Cromatografia Líquida de Alta Pressão , Mutação , Oxirredução , Oxigenases/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Zea mays/genética , beta Caroteno/metabolismoRESUMO
The biochemical activities that underlie the genetically defined activator and repressor functions of the VIVIPAROUS1 (VP1) protein have resisted in vitro analysis. Here, we show that a glutathione S-transferase (GST) fusion protein, including only the highly conserved B3 domain of VP1, has a highly cooperative, sequence-specific DNA binding activity. GST fusion proteins that include larger regions of the VP1 protein have very low activity, indicating that removal of the flanking protein sequences is necessary to elicit DNA binding in vitro. DNA competition and DNase I footprinting analyses show that B3 binds specifically to the Sph element involved in VP1 activation of the C1 gene, whereas binding to the G-box-type VP1-responsive element is of low affinity and is nonspecific. Footprint analysis of the C1 promoter revealed that sequences flanking the core TCCATGCAT motif of Sph also contribute to the recognition of the Sph element in its native context. The salient features of the in vitro GST-B3 DNA interaction are in good agreement with the protein and DNA sequence requirements defined by the functional analyses of VP1 and VP1-responsive elements in maize cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Zea mays/metabolismo , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Sequência Conservada , Sondas de DNA , Proteínas de Ligação a DNA/biossíntese , Desoxirribonuclease I , Glutationa Transferase/biossíntese , Dados de Sequência Molecular , Proteínas de Plantas , Proteínas Recombinantes de Fusão/biossíntese , Transativadores , Fatores de Transcrição/biossínteseRESUMO
MDL 74,721 (R)-2-(N1,N1-dipropylamino)-8-methylaminosulfonylmethyl-1,2,3,4-te trahydronaphthalene, a sulfonamidotetralin, has been found to exhibit a 10,000-fold greater potency in neurogenic versus vascular models of migraine. Sumatriptan, a relatively pure 5-HT1D/5-HT1B receptor agonist, also showed higher potency versus neurogenic inflammation. However, for sumatriptan the potency difference (100-fold) in the two pathophysiological models was less pronounced than seen for MDL 74,721. The affinity profile of MDL 74,721 at 5-HT1 receptor subtypes may in part explain its ability to differentiate these two physiological responses. MDL 74,721 demonstrated nanomolar affinity for 5-HT1A (12.7 +/- 0.3 nM) and 5-HT1D (41.3 +/- 10.9 nM) but considerably lower affinity for 5-HT1B receptors (> 1000 nM). Serotonin-like activity was seen in in vitro functional assays including inhibition of forskolin-stimulated cAMP accumulation in human 5-HT1D receptor-transfected fibroblasts or eliciting vasoconstriction in isolated human pial arteries. The intrinsic activity (relative to 5 - HT[E(Amax)]) and affinity (pD2) for the human cerebrovascular 5-HT receptors were: 5-HT (100%, 7.51 +/- 0.09), sumatriptan (94%, 6.85 +/- 0.1) and MDL 74,721 (66%, 5.70 +/- 0.23). In anaesthetised cats, treatment with MDL 74,721 resulted in a dose-related reduction in the percentage of carotid flow going through the arteriovenous anastomoses to the lungs, with an ED50 of 0.3 mg/kg i.v., the same as sumatriptan. However, in the guinea-pig neurogenic model, MDL 74,721 inhibited plasma protein extravasation with an ED50 of 0.023 microg/kg compared to 2.5 microg/kg for sumatriptan. MDL 74,721 was also effective in this model (in rats) after oral administration. In conclusion, MDL 74,721 demonstrates a preclinical profile consistent with anti-migraine efficacy. Its marked preference for inhibiting neurogenic inflammation makes this compound a useful tool for assessing the relative contribution of this pathophysiological mechanism to the human disease state.
Assuntos
Artérias Cerebrais/efeitos dos fármacos , Transtornos de Enxaqueca/fisiopatologia , Receptores de Serotonina/efeitos dos fármacos , Agonistas do Receptor de Serotonina/farmacologia , Sulfonamidas/farmacologia , Sumatriptana/farmacologia , Tetra-Hidronaftalenos/farmacologia , Vasoconstritores/farmacologia , Animais , Ligação Competitiva , Gatos , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Cobaias , Hemodinâmica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Masculino , Transtornos de Enxaqueca/tratamento farmacológico , Núcleos da Rafe/irrigação sanguínea , Núcleos da Rafe/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptor 5-HT1D de Serotonina , Receptores de Serotonina/metabolismo , Receptores 5-HT1 de Serotonina , Nervo Trigêmeo/irrigação sanguíneaRESUMO
A series of vp1 alleles distinguish at least two classes of maturation-related genes that are regulated by the VP1 factor and abscisic acid (ABA). The intermediate vp1-c821708 and vp1-McW alleles have quiescent (non-viviparous) anthocyanin-deficient phenotypes while maintaining significant levels of maturation-specific gene expression in the developing embryo. However, expression of the C1 regulatory gene of the anthocyanin pathway is not detected in these mutants. Reduced steady-state levels of structurally altered VP1 proteins are detected in quiescent mutant embryos. The VP1-McW protein sequence lacks the highly conserved region encoded by exons 3-5 of the Vp1 gene. A sensitive RT-PCR assay was used to rule out significant amounts of intact transcripts in the vp1-McW mutant that could account for the quiescent phenotype. In transient expression assays, the VP1-McW protein and other mutants with a truncated B3 domain of VP1 retained a strong capacity to synergistically enhance ABA-regulation of the Em-GUS reporter gene; whereas transactivation of both Em-GUS and C1-sh-GUS genes in the absence of hormone was strongly inhibited. These results indicate that the largest conserved region in VP1 homologs (B3) is critical for gene activation at low or insignificant ABA dosages; whereas the N-terminal domain provides a key interface with ABA signaling pathways in the developing seed.
Assuntos
Ácido Abscísico/fisiologia , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Sementes/fisiologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Sequência Conservada , Éxons , Genes de Plantas , Íntrons , Dados de Sequência Molecular , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Transativadores , Transcrição Gênica , Ativação Transcricional , Zea maysRESUMO
The maize crinkly4 (cr4) mutation affects leaf epidermis differentiation such that cell size and morphology are altered, and surface functions are compromised, allowing graft-like fusions between organs. In the seed, loss of cr4 inhibits aleurone formation in a pattern that reflects the normal progression of differentiation over the developing endosperm surface. The cr4 gene was isolated by transposon tagging and found to encode a putative receptor kinase. The extracellular domain contains a cysteine-rich region similar to the ligand binding domain in mammalian tumor necrosis factor receptors (TNFRs) and seven copies of a previously unknown 39-amino acid repeat. The results suggest a role for cr4 in a differentiation signal.
Assuntos
Proteínas de Plantas , Proteínas Quinases/fisiologia , Zea mays/citologia , Sequência de Aminoácidos , Diferenciação Celular , Clonagem Molecular , Elementos de DNA Transponíveis , Genes de Plantas , Dados de Sequência Molecular , Mutagênese , Fenótipo , Folhas de Planta/citologia , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/química , Receptores do Fator de Necrose Tumoral/química , Sementes/citologia , Zea mays/química , Zea mays/genética , Zea mays/crescimento & desenvolvimentoRESUMO
Invertase and sucrose synthase catalyze the two known paths for the first step in carbon use by sucrose-importing plant cells. The hypothesis that sugar-modulated expression of these genes could provide a means of import adjustment was initially suggested based on data from sucrose synthases alone; however, this hypothesis remained largely conjectural without critical evidence for invertases. Toward this end, a family of maize invertases was cloned and characterized. Here, we show that invertases are indeed sugar modulated and, surprisingly, like the sucrose synthase genes, fall into two classes with contrasting sugar responses. In both families, one class of genes is upregulated by increasing carbohydrate supply (Sucrose synthase1 [Sus1] and Invertase2 [Ivr2]), whereas a second class in the same family is repressed by sugars and upregulated by depletion of this resource (Shrunken1 [Sh1] and Invertase1 [Ivr1]). The two classes also display differential expression during development, with sugar-enhanced genes (Sus1 and Ivr2) expressed in many importing organs and sugar-repressed, starvation-tolerant genes (Sh1 and Ivr1) upregulated primarily during reproductive development. Both the Ivr1 and Ivr2 invertase mRNAs are abundant in root tips, very young kernels, silk, anthers, and pollen, where a close relationship is evident between changes in message abundance and soluble invertase activity. During development, patterns of expression shift as assimilate partitioning changes from elongating silks to newly fertilized kernels. Together, the data support a model for integrating expression of genes differentially responsive to carbohydrate availability (i.e., feast and famine conditions) with developmental signals. The demonstration that similar regulatory patterns occur in both paths of sucrose metabolism indicates a potential to influence profoundly the adjustment of carbon resource allocation.
RESUMO
The C1 regulatory gene of the maize anthocyanin pathway is regulated by a combination of developmental and environmental signals that include the Viviparous1 (Vp1) gene, abscisic acid (ABA), and light. Using protoplast electroporation and particle bombardment assays, we have defined c/s-acting elements that are necessary and sufficient for the activation of C1 by ABA, VP1, and light, respectively. The sequence from positions -142 to -132 (CGTCCATGCAT) is essential for VP1 activation, whereas a larger overlapping element from -147 to -132 (CGTGTCGTCCATGCAT) is necessary and sufficient for activation by ABA. A separate light (blue and red)-responsive c/s element is located between positions -116 and -59. Light interacts synergistically with the ABA and VP1 responses in transient expression assays, suggesting that combinatorial interaction between modules plays a role in integrating these signals in the developing seed.
Assuntos
Ácido Abscísico/farmacologia , Proteínas de Ligação a DNA , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Genes Reguladores , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Zea mays/genética , Antocianinas/metabolismo , Sequência de Bases , Caulimovirus/genética , Escuridão , Eletroporação , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/efeitos dos fármacos , Genes Reguladores/efeitos dos fármacos , Íntrons , Luz , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Proteínas de Plantas/genética , Protoplastos/efeitos dos fármacos , Protoplastos/metabolismo , TATA Box , Transativadores , Fatores de Transcrição/genética , Zea mays/metabolismoRESUMO
In preclinical studies, [R-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl)ethyl]-4- piperidinemethanol] [formula: see text] (MDL 100,907), a putative atypical antipsychotic, was characterized in vitro as a potent and selective ligand for the serotonin2A (5-HT2A) receptor and was evaluated in vitro and in vivo as a potent 5-HT2A receptor antagonist. Furthermore, MDL 100,907's potential CNS safety profile and selectivity as a potential antipsychotic agent were evaluated and compared with benchmark compounds. MDL 100,907 demonstrated low nanomolar or subnanomolar binding in vitro at the 5-HT2A receptor and showed a > 100-fold separation from all other receptors measured. MDL 100,907 had subnanomolar potency as a 5-HT2A antagonist in vitro in reversing 5-HT-stimulated inositol phosphate accumulation in NIH 3T3 cells transfected with the rat 5-HT2A receptor. In vivo, MDL 100,907 potently inhibited 5-methoxy-N, N-dimethyltryptamine-induced head twitches in mice or 5-hydroxytryptophan-induced head twitches in rats. In vivo functional tests in mice revealed a > 500-fold separation between doses that produced 5-HT2A antagonism and doses that produced alpha 1-adrenergic or striatal D2 antagonism. Using inhibition of D-amphetamine-stimulated locomotion in mice as a measure of potential antipsychotic efficacy, MDL 100,907 showed a superior CNS safety index relative to the reference compounds, haloperidol, clozapine, risperidone, ritanserin, and amperozide, in each of five tests for side effect potential, including measures of ataxia, general depressant effects, alpha 1-adrenergic antagonism, striatal D2 receptor antagonism, and muscle relaxation. MDL 100,907 did not antagonize apomorphine-induced stereotypes in rats, suggesting that it potentially lacks extrapyramidal side effect liability. MDL 100,907 showed selectivity as a potential antipsychotic in that it lacked consistent activity in selected rodent models of anticonvulsant, antidepressant, analgesic, or anxiolytic activity. In summary, these preclinical data indicate that MDL 100,907 is a potent and selective ligand at the 5-HT2A receptor. MDL 100,907's potent 5-HT2A antagonist activity might account for its activity in preclinical models of antipsychotic potential. Ongoing clinical evaluation with MDL 100,907 will test the hypothesis that 5-HT2A receptor antagonism is sufficient for antipsychotic activity in humans.
Assuntos
Antipsicóticos/farmacologia , Encéfalo/efeitos dos fármacos , Fluorbenzenos/farmacologia , Piperidinas/farmacologia , Antagonistas da Serotonina/farmacologia , Animais , Ansiolíticos/farmacologia , Fluorbenzenos/toxicidade , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Piperidinas/toxicidade , RatosRESUMO
Assignment of particular transcription factors to specific roles in promoter elements can be problematic, especially in systems such as the G-box, where multiple factors of overlapping specificity exist. In the Arabidopsis alcohol dehydrogenase (Adh) promoter, the G-box regulates expression in response to cold and dehydration, presumably through the action of abscisic acid (ABA), and is bound by a nuclear protein complex in vivo during expression in cell cultures. In this report, we test the conventional wisdom of biochemical approaches used to identify DNA binding proteins and assess their specific interactions by using the G-box and a nearby half G-box element of the Arabidopsis Adh promoter as a model system. Typical in vitro assays demonstrated specific interaction of G-box factor 3 (GBF3) with both the G-box and the half G-box element. Dimethyl sulfate footprint analysis confirmed that the in vitro binding signature of GBF3 essentially matches the footprint signature detected in vivo at the G-box. Because RNA gel blot data indicated that GBF3 is itself induced by ABA, we might have concluded that GBF3 is indeed the GBF responsible in cell cultures for binding to the Adh G-box and is therefore responsible for ABA-regulated expression of Adh. Potential limitations of this conclusion are exposed by the fact that other GBFs bind the G-box with the same signature as GBF3, and subtle differences between in vivo and in vitro footprint signatures indicate that factors other than or in addition to GBF3 interact with the half G-box element.