Effects of phenothiazine and dibenzazepine derivatives on the muscarinic cholinergic system]

Interactions of some dialkylaminoalkyl (DAL) and dialkylaminoacyl (DAC) derivatives of phenothiazine and dibenzazepine with muscarinic cholinergic receptors (MR) of rabbit striatum and heart and rat brain were investigated. DAC derivatives were more active at brain and heart MR in some cases. The most active preparation was G-512, DA-analogue of chlorpromazine. Some cardiotropic properties of antianginal preparation nonachlazine may be connected with its central antimuscarinic activity.     

Differences in the efficiency of their interaction with membrane receptors of the dialkylaminoalkyl and dialkylaminoacyl derivatives of phenothiazine

New dialkylaminoacyl phenothiazine derivatives (DAC) were compared with their dialkylaminoalkyl analogues (neuroleptics chlorpromazine, trifluoperazine and fluphenazine) as well as with anti-arrhythmia drugs ethmozine and ethacizine for their receptor-blocking potencies. It was established that DAC are significantly less potent with dopamine alpha 1-adrenergic and H1-histamine receptors of calf and rabbit brain, which can explain the absence of neuroleptic effect of DAC drugs. DAC affinities to muscarinic and alpha-adrenergic receptors of both types are very similar to those of ethmozine and ethacizine. New DAC substance G-512 (chlorpromazine analogue) demonstrated high affinity to M1-muscarinic receptors of rabbit brain cortex (Ki = 4.2 nM) and to M2-muscarinic receptors of the rabbit heart (Ki = 48 nM).     

Identification of the ureidoglycolate hydrolase gene in the DAL gene cluster of Saccharomyces cerevisiae.

This report describes the isolation of the genes encoding allantoicase (DAL2) and ureidoglycolate hydrolase (DAL3), which are components of the large DAL gene cluster on the right arm of chromosome IX of Saccharomyces cerevisiae. During this work a new gene (DAL7) was identified and found to be regulated in the manner expected for an allantoin pathway gene. Its expression was (i) induced by allophanate, (ii) sensitive to nitrogen catabolite repression, and (iii) responsive to mutation of the DAL80 and DAL81 loci, which have previously been shown to regulate the allantoin degradation system. Hybridization probes generated from these cloned genes were used to analyze expression of the allantoin pathway genes in wild-type and mutant cells grown under a variety of physiological conditions. When comparison was possible, the patterns of mRNA and enzyme levels observed in various strains and physiological conditions were very similar, suggesting that the system is predominantly regulated at the level of gene expression. Although all of the genes seem to be controlled by a common mechanism, their detailed patterns of expression were, at the same time, highly individual and diverse.     

Circadian phase-dependent antinociceptive reaction in mice determined by the hot-plate test and the tail-flick test after intravenous injection of dalargin-loaded nanoparticles

Peptides normally do not cross the blood-brain barrier (BBB). Previously, it has been shown that the hexapeptide enkephalin analogue dalargin with polysorbate-80-coated nanoparticles (DAL/NP) can be transported across the BBB and is able to exhibit an antinociceptive effect in mice. In the present study, the circadian time and dose dependencies of the antinociceptive effect of different dalargin preparations were investigated. The active preparation (DAL/NP, 5 mg/kg, 10 mg/kg), as well as a dalargin solution in phosphate buffered saline (DAL/SOL, 10 mg/kg) were injected intravenously to groups of 10-12 inbred DBA/2 mice at 12 different circadian times; mice were synchronized to a light-dark (LD) 12:12 regimen. The antinociceptive effect was determined 15 minutes postinjection by the hot-plate test. Experiments with DAL/NP were repeated using the tail-flick test system at two selected times (08:00 and 20:00) to test for dose dependency (2.5, 5, 7.5, 10 mg/kg). Hot-plate latencies were rhythmic under baseline and after DAL/SOL, with acrophases in the dark phase; DAL/SOL did not influence latency time. In contrast, DAL/NP significantly increased reaction time dose dependency; the maximal possible effect was rhythmic with the 10 mg/kg preparation, with a peak effect in the early light phase. Results were confirmed by the tail-flick test. The experiments demonstrate that an enkephalin analogue coated with nanoparticles can easily cross the BBB and is able to display a dose- and time-dependent antinociceptive effect.     

The DAL82 protein of Saccharomyces cerevisiae binds to the DAL upstream induction sequence (UIS).

Expression of the DAL2, DAL4, DAL7, DUR1,2, and DUR3 genes in S. cerevisiae is induced by allophanate, the last intermediate in the allantoin catabolic pathway. Analysis of the DAL7 promoter identified a dodecanucleotide, the DAL7 UIS, which was required for inducer-responsiveness. Operation of the DAL7 UIS required functional DAL81 and DAL82 gene products. Since the DAL81 product was not an allantoin pathway-specific regulatory factor, the DAL82 product was considered as the more likely candidate to be the DAL UIS binding protein. Using an E. coli expression system, we showed that DAL82 protein specifically bound to wild type but not mutant DAL UIS sequences. DNA fragments containing DAL UIS elements derived from various DAL gene promoters bound DAL82 protein with different affinities which correlate with the degree of inducer-responsiveness the genes displayed.     

Location of the Genes That Control Induction of the Allantoin-Degrading Enzymes in SACCHAROMYCES CEREVISIAE

In an effort to understand the regulation of allantoin degradation in Saccharomyces cerevisiae, we isolated two classes of mutants, each defective in the induction process associated with production of the pathway enzymes. Mutation at one locus (DAL80) results in constitutive expression of the genes involved in allantoin catabolism. Mutation at the second locus (DAL-81) results in the loss of ability to induce these enzymes. This report describes genetic data indicating that the DAL80 and DAL81 loci are situated approximately 13 cM from the centromere on the right arm of chromosome XI and 9 cM proximal to the DAL1 locus on chromosome IX, respectively.     

Opiate Binding Sites in Bovine Adrenal Medulla

Abstract

Previous studies using a variety of opiate ligands have suggested the existence of several subclasses of opiate receptors in crude membrane fractions of rat brain, and a similar diversity in bovine adrenal medulla. To examine the receptor profile of bovine adrenal medulla in detail we have studied the binding of classical ligands for mu (μ), delta (δ) and kappa (k) opiate receptors. [³H]naloxone ([³H]NAL), [³H] morphine ([³H]MOR), [³H]D-Ala²-D-Leu⁵-enkephalin ([³H]DAL) and [³H]ethyl-ketocyclazocine ([³H]EKCZ) were used as tracers; unlabeled competitors were NAL, MOR, DAL and ketocyclazocine (KCZ). In adrenal medulla [³H]NAL was specifically bound with a hierarchy of displacement NAL > MOR > KCZ ? DAL. No specific binding of [³H]DAL or [³H]EKCZ was found; for [³H]MOR very low levels of binding were seen, with no displacement by NAL or DAL, inconsistent displacement by KCZ and substantial displacement by MOR with an ED₅₀ of 1.5 nM. In parallel studies rat brain membranes bound each labeled ligand with affinity and specificity consistent with previously published reports. Identical results were obtained in membranes from both tissues prepared with a preincubation step including 100 mM Na⁺, suggesting that the results were not influenced by occupation of binding sites by endogenous ligands. We interpret these data as supporting the existence of opiate receptors of the μ subtype in bovine adrenal medulla. We find, however, no evidence of δ or k sites in this tissue.     

Regulatory circuit for responses of nitrogen catabolic gene expression to the GLN3 and DAL80 proteins and nitrogen catabolite repression in Saccharomyces cerevisiae.

We demonstrate that expression of the UGA1, CAN1, GAP1, PUT1, PUT2, PUT4, and DAL4 genes is sensitive to nitrogen catabolite repression. The expression of all these genes, with the exception of UGA1 and PUT2, also required a functional GLN3 protein. In addition, GLN3 protein was required for expression of the DAL1, DAL2, DAL7, GDH1, and GDH2 genes. The UGA1, CAN1, GAP1, and DAL4 genes markedly increased their expression when the DAL80 locus, encoding a negative regulatory element, was disrupted. Expression of the GDH1, PUT1, PUT2, and PUT4 genes also responded to DAL80 disruption, but much more modestly. Expression of GLN1 and GDH2 exhibited parallel responses to the provision of asparagine and glutamine as nitrogen sources but did not follow the regulatory responses noted above for the nitrogen catabolic genes such as DAL5. Steady-state mRNA levels of both genes did not significantly decrease when glutamine was provided as nitrogen source but were lowered by the provision of asparagine. They also did not respond to disruption of DAL80.     

Doubling the size of the glucocorticoid receptor ligand binding pocket by deacylcortivazol

A common feature of nuclear receptor ligand binding domains (LBD) is a helical sandwich fold that nests a ligand binding pocket within the bottom half of the domain. Here we report that the ligand pocket of glucocorticoid receptor (GR) can be continuously extended into the top half of the LBD by binding to deacylcortivazol (DAC), an extremely potent glucocorticoid. It has been puzzling for decades why DAC, which contains a phenylpyrazole replacement at the conserved 3-ketone of steroid hormones that are normally required for activation of their cognate receptors, is a potent GR activator. The crystal structure of the GR LBD bound to DAC and the fourth LXXLL motif of steroid receptor coactivator 1 reveals that the GR ligand binding pocket is expanded to a size of 1,070 ų, effectively doubling the size of the GR dexamethasone-binding pocket of 540 ų and yet leaving the structure of the coactivator binding site intact. DAC occupies only ~50% of the space of the pocket but makes intricate interactions with the receptor around the phenylpyrazole group that accounts for the high-affinity binding of DAC. The dramatic expansion of the DAC-binding pocket thus highlights the conformational adaptability of GR to ligand binding. The new structure also allows docking of various nonsteroidal ligands that cannot be fitted into the previous structures, thus providing a new rational template for drug discovery of steroidal and nonsteroidal glucocorticoids that can be specifically designed to reach the unoccupied space of the expanded pocket.     

Arabidopsis DAL1 and DAL2, two RING finger proteins homologous to Drosophila DIAP1, are involved in regulation of programmed cell death

Programmed cell death (PCD) is a precise, genetically controlled cellular process with important roles in plant growth, development, and response to biotic and abiotic stress. However, the genetic mechanisms that control PCD in plants are unclear. Two Arabidopsis genes, DAL1 and DAL2 (for Drosophila DIAP1 like 1 and 2), encoding RING finger proteins with homology to DIAP1 were identified, and a series of experiments were performed to elucidate their roles in the regulation of PCD and disease resistance. Expression of DAL1 and DAL2 genes was induced in Arabidopsis plants after inoculation with virulent and avirulent strains of Pseudomonas syrinage pv. tomato (Pst) DC3000 or after infiltration with fumonisin B1 (FB1). Plants with mutations in the DAL1 and DAL2 genes displayed more severe disease after inoculation with an avirulent strain of Pst DC3000, but they showed similar disease severity as the wild-type plant after inoculation with a virulent strain of Pst DC3000. Significant accumulations of reactive oxygen species (ROS) and increased cell death were observed in the dal1 and dal2 mutant plants after inoculation with the avirulent strain of Pst DC3000. The dal mutant plants underwent extensive PCD upon infiltration of FB1 and displayed higher levels of ROS accumulation, callose deposition, and autofluorescence than the wild-type plants. Our data suggest that DAL1 and DAL2 may act as negative regulators of PCD in Arabidopsis.