Role of Histidine Residues in Agonist and Antagonist Binding Sites of A1 Adenosine Receptor

Abstract: The influence of pH on the equilibrium dissociation constant and on kinetic association and dissociation constants was studied for adenosine receptor agonist L-N⁶-[adenine-2,8-³H, ethyl-2-³H]phenylisopropyladenosine ([³H]R-PIA) and antagonist 8-cyclopentyl-1,3-[³H]-dipropylxanthine ([³H]DPCPX). Two ionizable groups, of pK 7.0 and pK 7.4, are involved in the [³H]R-PIA associations with high- and low-affinity states of the receptor, and another group, of pK 6.0, is involved in the association with the low-affinity state. No ionizable group is involved in the dissociation process for the high-affinity state, whereas two ionizable groups, of pK 6.0 and 6.5, are involved in the low-affinity state. For [³H]DPCPX, three ionizable groups (pK 6.0, 7.4, and 8.0) are involved in the association process and only one group, (pK 6.0), is involved in the dissociation step. The apparent pK values obtained agree with histidine residues. We thus studied the effect of diethylpyrocarbonate (DEP), which reacts irreversibly with histidine residues, on agonist and antagonist binding to A₁ adenosine receptors from pig brain cortical membranes. DEP treatment of membrane reduced the affinity (K_D) and the total binding (R) of the agonist and the antagonist. Membrane preincubation with unlabeled ligand (R-PIA or DPCPX) prevented the effect of DEP modification observed when the same ligand, but with label, is added to the same membranes, but did not prevent the DEP modification on different, labeled ligand. The pattern of protective action of R-PIA, DPCPX, adenosine, and guanylylimidodiphosphate in DEP treatment and the displacement curves of radiolabeled agonist and antagonist by both unlabeled ligands indicated that the interaction site for agonist and antagonist binding is the same, although the complete mechanisms for recognition and binding differ.     

Determination of the intrinsic fermentation kinetics of Saccharomyces cerevisiae cells immobilized in Ca-alginate beads and observations on their growth

Continuous fermentation experiments in a well-stirred fermentor with Saccharomyces cerevisiae cells immobilized in Ca-alginate beads of small diameter (approx. 1 mm) have been performed in order to discover their intrinsic fermentation kinetics, and compare them to the fermentation kinetics for free cells, by fitting both sets of results to the same model. The results show similar kinetic parameters for free and immobilized cells. The changes in cell concentration inside the beads and microscopical observations of transverse sections throughout the experiments, allowed discernment of two different scenarios of cell growth inside the beads: low cell density and fully developed growth. Correspondence to: F. Gòdia     

A simple method for N-15 labelling of exocyclic amino groups in synthetic oligodeoxynucleotides

The use of the ammonia deprotection step to introduce ¹⁵N labels at specific exocyclic amino positions of adenine, cytosine, guanine or 2-aminopurine of oligodeoxynucleotides is described.     

Hydroxide and proton migration in aquaporins

Hypothetical hydroxide and proton migration along the linear water chain in Aquaporin GlpF from Escherichia coli are studied by ab initio Car-Parrinello molecular dynamics simulations. It is found that the protein stabilizes a bipolar single file of water. The single file features a contiguous set of water-water hydrogen bonds in which polarization of the water molecules vary with position along the channel axis. Deprotonation of the water chain promotes the reorientation of water molecules while the hydroxide ion rapidly migrates by sequentially accepting protons from the neighboring water molecules. The hydroxide ion is not attracted by a conserved, channel-lining arginine residue, but is immobilized at two centrally located, conserved Asparagine-Proline-Alanine motifs where fourfold coordination stabilizes the ion. Hydroxide transition from the channel vestibules into the channel lumen is strongly influenced by electrostatic coupling to two conserved oppositely aligned macrodipoles. This suggests that the macrodipole's negative poles play a role in preventing hydroxide ions from entering into the channel's inner vestibules. Water protonation within the lumen facilitates water reorientation and subsequent proton expelling occurs. In the periplasmic half-channel, expelling occurs via the Grotthuss mechanism. Protonation within the cytoplasmic half-channel implies wire-breakage at the Asn-Pro-Ala motifs. The proton is here diffusively rejected as (H(5)O(2))(+).     

Adenosine A2A receptor stimulation potentiates nitric oxide release by activated microglia

The absence of adenosine A2A receptors, or its pharmacological inhibition, has neuroprotective effects. Experimental data suggest that glial A2A receptors participate in neurodegeneration induced by A2A receptor stimulation. In this study we have investigated the effects of A2A receptor stimulation on control and activated glial cells. Mouse cortical mixed glial cultures (75% astrocytes, 25% microglia) were treated with the A2A receptor agonist CGS21680 alone or in combination with lipopolysaccharide (LPS). CGS21680 potentiated lipopolysaccharide-induced NO release and NO synthase-II expression in a time- and concentration-dependent manner. CGS21680 potentiation of lipopolysaccharide-induced NO release was suppressed by the A2A receptor antagonist ZM-241385 and did not occur on mixed glial cultures from A2A receptor-deficient mice. In mixed glial cultures treated with LPS + CGS21680, the NO synthase-II inhibitor 1400W abolished NO production, and NO synthase-II immunoreactivity was observed only in microglia. Binding experiments demonstrated the presence of A2A receptors on microglial but not on astroglial cultures. However, the presence of astrocytes was necessary for CGS21680 potentiating effect. In light of the reported neurotoxicity of microglial NO synthase-II and the neuroprotection of A2A receptor inhibition, these data suggest that attenuation of microglial NO production could contribute to the neuroprotection afforded by A2A receptor antagonists.     

Impact of an Educational Program to Reduce Healthcare Resources in Community-Acquired Pneumonia: The EDUCAP Randomized Controlled Trial

Background

Additional healthcare visits and rehospitalizations after discharge are frequent among patients with community-acquired pneumonia (CAP) and have a major impact on healthcare costs. We aimed to determine whether the implementation of an individualized educational program for hospitalized patients with CAP would decrease subsequent healthcare visits and readmissions within 30 days of hospital discharge.

Methods

A multicenter, randomized trial was conducted from January 1, 2011 to October 31, 2014 at three hospitals in Spain. We randomly allocated immunocompetent adults patients hospitalized for CAP to receive either an individualized educational program or conventional information before discharge. The educational program included recommendations regarding fluid intake, adherence to drug therapy and preventive vaccines, knowledge and management of the disease, progressive adaptive physical activity, and counseling for alcohol and smoking cessation. The primary trial endpoint was a composite of the frequency of additional healthcare visits and rehospitalizations within 30 days of hospital discharge. Intention-to-treat analysis was performed.

Results

We assigned 102 patients to receive the individualized educational program and 105 to receive conventional information. The frequency of the composite primary end point was 23.5% following the individualized program and 42.9% following the conventional information (difference, -19.4%; 95% confidence interval, -6.5% to -31.2%; P = 0.003).

Conclusions

The implementation of an individualized educational program for hospitalized patients with CAP was effective in reducing subsequent healthcare visits and rehospitalizations within 30 days of discharge. Such a strategy may help optimize available healthcare resources and identify post-acute care needs in patients with CAP.

Trial Registration

Controlled-Trials.com ISRCTN39531840