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PubMed2005

Investigation of the relationship between protein-protein interaction and catalytic activity of a heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) by protein microarray.

Ec DOS, a heme-regulated phosphodiesterase from Escherichia coli, is composed of an N-terminal heme-bound PAS domain and a C-terminal phosphodiesterase domain. The heme redox state in the PAS domain regulates Ec DOS phosphodiesterase activity. Interestingly, the isolated heme-bound PAS fragment enhances phosphodiesterase activity of full-length Ec DOS. The enhancement is also regulated by the heme redox state of the isolated PAS domain. In the present study, we used a newly developed protein microarray system to examine the relationship between catalytic activity and the interaction of full-length Ec DOS and the isolated PAS fragment. Adenosine 3',5'-cyclic monophosphate (cAMP), a substrate of the Ec DOS phosphodiesterase, was found to be indispensable for the interaction between Ec DOS and the PAS fragment, and two phosphodiesterase inhibitors, 3-isobutyl-methyl-xanthine and etazolate hydrochloride, hindered the interaction. In addition, an enzyme with a mutation in the putative cAMP-binding sites (H590 and H594) was unable to interact with Ec DOS and lacked enzymatic activity. These results strongly suggest a close relationship between Ec DOS phosphodiesterase activity and interaction with the isolated PAS fragment. Therefore, this study provides insights into the mechanism of how the isolated PAS domain activates Ec DOS, which has important implications for the general role of the isolated PAS domain in cells. Moreover, we found that multiple microscale analyses using the protein microarray system had several advantages over conventional affinity column methods, including the quantity of protein needed, the sensitivity, the variability of immobilized protein, and the time required for the experiment.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2002

Characterization of a direct oxygen sensor heme protein from Escherichia coli. Effects of the heme redox states and mutations at the heme-binding site on catalysis and structure.

A protein containing a heme-binding PAS (PAS is from the protein names in which imperfect repeat sequences were first recognized: PER, ARNT, and SIM) domain from Escherichia coli has been implied a direct oxygen sensor (Ec DOS) enzyme. In the present study, we isolated cDNA for the Ec DOS full-length protein, expressed it in E. coli, and examined its structure-function relationships for the first time. Ec DOS was found to be tetrameric and was obtained as a 6-coordinate low spin ferric heme complex. Its alpha-helix content was calculated as 53% by CD spectroscopy. The redox potential of the heme was found to be +67 mV versus SHE. Mutation of His-77 of the isolated PAS domain abolished heme binding, whereas mutation of His-83 did not, suggesting that His-77 is one of the heme axial ligands. Ferrous, but not ferric, Ec DOS had phosphodiesterase (PDE) activity of nearly 0.15 min(-1) with cAMP, which was optimal at pH 8.5 in the presence of Mg(2+) and was strongly inhibited by CO, NO, and etazolate, a selective cAMP PDE inhibitor. Absorption spectral changes indicated tight CO and NO bindings to the ferrous heme. Therefore, the present study unequivocally indicates for the first time that Ec DOS exhibits PDE activity with cAMP and that this is regulated by the heme redox state.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1981

Interaction of putative anxiolytic agents with central adenosine receptors.

The benzodiazepine anxiolytics flurazepam and diazepam and CL 218872, zopiclone and two beta-carboline ethyl carboxyl esters, compounds which are potent displacers of specific [3H]diazepam binding from rat brain membranes, have little or no activity in displacing [3H]2-chloroadenosine ([3H]2-CADO) from central A1-adenosine receptors. Conversely, the purine agonists, 1-N6-phenylisopropyladenosine, N6-cyclohexyladenosine, 2-chloroadenosine, and the adenosine antagonist 8-phenyltheophylline have no significant effect on [3H]diazepam binding. Etazolate (SQ 20009) and Avermectin B1a which enhance [3H]diazepam binding in vitro were also without significant effect on [3H]2-CADO binding. The lack of correlation of the activities of the compounds examined in the two binding assays is discussed in relation to the hypothesis that purine-like compounds may be involved in the molecular mechanisms related to anxiolytic action at the receptor level.

Animals↗
PubMed2005

Global expression profiling of theophylline response genes in macrophages: evidence of airway anti-inflammatory regulation.

BACKGROUND: Theophylline has been used widely as a bronchodilator for the treatment of bronchial asthma and has been suggested to modulate immune response. While the importance of macrophages in asthma has been reappraised and emphasized, their significance has not been well investigated. We conducted a genome-wide profiling of the gene expressions of macrophages in response to theophylline. METHODS: Microarray technology was used to profile the gene expression patterns of macrophages modulated by theophylline. Northern blot and real-time quantitative RT-PCR were also used to validate the microarray data, while Western blot and ELISA were used to measure the levels of IL-13 and LTC4. RESULTS: We identified dozens of genes in macrophages that were dose-dependently down- or up-regulated by theophylline. These included genes related to inflammation, cytokines, signaling transduction, cell adhesion and motility, cell cycle regulators, and metabolism. We observed that IL-13, a central mediator of airway inflammation, was dramatically suppressed by theophylline. Real-time quantitative RT-PCR and ELISA analyses also confirmed these results, without respect to PMA-treated THP-1 cells or isolated human alveolar macrophages. Theophylline, rolipram, etazolate, db-cAMP and forskolin suppressed both IL-13 mRNA expression (~25%, 2.73%, 8.12%, 5.28%, and 18.41%, respectively) and protein secretion (<10% production) in macrophages. These agents also effectively suppressed LTC4 expression. CONCLUSION: Our results suggest that the suppression of IL-13 by theophylline may be through cAMP mediation and may decrease LTC4 production. This study supports the role of theophylline as a signal regulator of inflammation, and that down regulation of IL-13 by theophylline may have beneficial effects in inflammatory airway diseases.

Anti-Inflammatory Agents↗
PubMed1997

Differentiation of African trypanosomes is controlled by a density sensing mechanism which signals cell cycle arrest via the cAMP pathway.

Differentiation of African trypanosomes from replicating slender bloodstream forms to nondividing stumpy forms limits the parasite population size, allowing survival of the mammalian host and establishment of a stable host-parasite relationship. Using a novel in vitro culture system we have shown that slender to stumpy differentiation is induced by parasite density alone and thus is independent of host cues. Here we investigate the density sensing mechanism and show that trypanosomes release a soluble activity of low relative molecular mass, termed stumpy induction factor (SIF), which accumulates in conditioned medium. SIF activity triggers cell cycle arrest in G1/G0 phase and induces differentiation with high efficiency and rapid kinetics. Membrane-permeable derivates of cAMP or the phosphodiesterase inhibitor etazolate perfectly mimic SIF activity. Furthermore, SIF activity elicits an immediate two- to threefold elevation of intracellular cAMP content upon addition to slender forms. We conclude that SIF and hence density sensing operate through the cAMP signalling pathway. Temporal correlation of markers indicates that cell cycle arrest invariably precedes differentiation. Thus, our results indicate that the cell cycle regulation of bloodstream forms is under dominant control of cAMP signalling. Irreversible commitment to the quiescent state is elicited by a cAMP agonist within a period shorter than one complete cell cycle.

Animals↗
PubMed1980

[Experimental changes of GABA receptor binding in the central nervous system (author's transl)].

The effects of etazolate and cartazolate, two pyrazolopyridine derivatives with anxiolytic actions, on the receptor binding of gamma-aminobutyric acid (GABA) was studied in vitro using membrane fractions prepared from rat brain. Both compounds increased the specific GABA receptor binding. This effect was markedly enhanced in the presence of several anions, predominantly halide ions. Pyrazolopyridines appear to modulate the binding properties of GABA receptors by acting on a site closely related to the chloride ion channel of the membrane. These results indicate that activation of the GABA system is a possible mechanism of action of pyrazolopyridines. In the second part of the study binding experiments were used to provide evidence for the presynaptic localisation of GABA receptors on primary afferent terminals in the dorsal horn of the rat spinal cord. Capsaicin-induced degeneration of primary afferent fibres significantly reduced the number of GABA receptors in the dorsal spinal cord, whereas the number of benzodiazepine and glycine receptors remained unchanged. The results support the role of GABA as transmitter involved in presynaptic inhibition in the spinal cord.

Animals↗
PubMedDate not supplied

[Sulfanilamide binding and distribution in the liver cells of rats].

Experiments on white noninbred rats were made to study the binding by isolated organoids and distribution in liver cells of etazole, norsulfazole, sulfamonomethoxine and sulfalene. The latter drug was found to be absorbed by organoids to the greatest degree. Drug complexes with organoids were discovered to be capable of dissociating with a partial release of unbound drug. Intracellular distribution of sulfanilamides is marked by their accumulation primarily by the nuclei and to a lesser degree by the mitochondria and microsomes.

Animals↗
PubMed1993

Bicuculline-produced regional differences in the modulation of 35S-TBPS binding by GABA, pentobarbital and diazepam in mouse cerebellum and cortex.

The modulation of 35S-t-butylbicyclophosphorothionate (35S-TBPS) binding by in vitro addition of gamma-aminobutyric acid (GABA), diazepam, pentobarbital, etomidate and etazolate was studied in the presence of KCl (100 mM) in the cerebellum and in the cortex of C57Bl mice. In the cortex, all of the depressant drugs caused a biphasic effect (stimulation followed by inhibition) on 35S-TBPS binding, whereas in the cerebellum only inhibition was observed. Saturation analysis revealed that the enhancement of 35S-TBPS binding was due to an increase in the affinity of 35S-TBPS for its binding sites. The introduction of a GABAA receptor antagonist, bicuculline methiodide (10 microM), into assay media altered the effects of the depressants, except for those of diazepam, on 35S-TBPS binding in a complex manner. Our results indicate that bicuculline, in addition to its GABAA receptor blocking properties, also influenced the binding of 35S-TBPS through some other, as yet unknown, mechanism(s). Thus, bicuculline not only produced a rightward shift of the dose-response curves of the central depressant drugs in the cortex, but also increased the maximal stimulation of 35S-TBPS binding. Furthermore, in the cerebellum, the previously observed drug-induced inhibition of 35S-TBPS binding was replaced by stimulation followed by inhibition in the presence of bicuculline. Finally, it was found that the in vitro addition of bicuculline had no effect on the diazepam-induced stimulation of 35S-TBPS binding. It is suggested that the regional differences in the modulation of 35S-TBPS binding in various brain structures may be due to endogenous differences in the molecular composition of GABAA receptors in various brain areas.

Animals↗
PubMed1981

Cyclic AMP, 5-HT, and the modulation of transmitter release at the crayfish neuromuscular junction.

In this study it was found that several agents which elevate cAMP levels in cells also increase dramatically the quantity of transmitter released from crayfish excitatory nerve terminals in response to a stimulus. With respect to time course and magnitude, the increase produced by one of these agents, the cyclic nucleotide phosphodiesterase inhibitor Squibb 20,009 (SQ 20,009), is unlike any reported for such a drug at a synapse. Additionally, SQ 20,009 potentiated the facilitation of transmitter release produced by serotonin (5-HT) at this synapse. These results establish a possible role for cAMP in the control and modulation of transmitter release at the crayfish neuromuscular junction (NMJ). They further suggest that 5-HT functions here by activation of a presynaptically located adenylate cyclase.

Animals↗
PubMed1982

Inhibition of a high affinity cyclic AMP phosphodiesterase and relaxation of canine tracheal smooth muscle.

The cyclic nucleotide phosphodiesterase (PDE) activity of canine tracheal smooth muscle (CTS,) was examined. Column chromatography of soluble CTSM-PDE revealed five peaks of activity. One of these peaks (V) was examined further in this study and showed a high affinity for adenosine 3',5'-cyclic monophosphate (Km = 0.63 microM). Seven pharmacological PDE inhibitors were tested for their abilities to inhibit the peak V enzyme and also for their abilities to cause mechanical relaxation of CTSM strips in isolated tissue baths. A strong correlation (P greater than 0.001) between peak V PDE inhibition (-log Ki) and airway muscle relaxation (-log ED50) was found.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1980

In vitro modulation by SQ 20009 and SQ 65396 of GABA receptor binding in rat CNS membranes.

SQ 20009 and SQ 65396, two new pyrazolopyridines with anxiolytic properties in animals, reversibly increased Na+-independent 3H-GABA binding to rat cerabral cortex membranes in vitro. This modulation was partially chloride dependent and sensitive to Triton-X 100. Kinetic analysis of 3H-muscimol binding revealed an increase of the apparent number of binding sites. The results are compatible with a functional association between Na+-independent GABA binding sites and the GABA/benzodiazepine receptor complex.

Animals↗