Interactions between Allosteric Modulators and 4-DAMP and Other Antagonists at Muscarinic Receptors: Potential Significance of the Distance between the N and Carboxyl C Atoms in the Molecules of Antagonists

Allosteric enhancement of the affinity of muscarinic receptors for their ligands offers a new way to influence cholinergic neurotransmission. The structure of the allosteric binding domain(s) and the features of agonists, antagonists and modulators which determine the occurrence of either positive or negative cooperativity require clarification. We tested interactions between allosteric modulators alcuronium, strychnine and brucine and eight antagonists at muscarinic receptors expressed in CHO cells. In experiments with unlabeled antagonists, all three modulators enhanced the affinity for 4-diphenylacetoxy-N-dimethylpiperidinium (4-DAMP) at the M₂ receptors, and strychnine did so also at the M₄ receptors. Positive interactions were also observed between alcuronium and L-hyoscyamine (M₂) and scopolamine (M₂), between strychnine and butylscopolamine (M₄), L-hyoscyamine (M₂ and M₄) and scopolamine (M₄), and between brucine and scopolamine (M₂). Positive effects of alcuronium, strychnine and brucine on the affinity of the M₂ receptors for 4-DAMP have been confirmed by direct measurements of the binding of [³H]-4-DAMP. A comparison of molecular models of several antagonists which are esters revealed that antagonists in which the distance between the N and the carboxyl C atoms corresponds to five chemical bonds are more likely to display positive cooperativity with alcuronium at the M₂ receptors than the antagonists in which the N-carboxyl C distance corresponds to four chemical bonds.     

Simian immunodeficiency virus (SIV)-specific CTL are present in large numbers in livers of SIV-infected rhesus monkeys

The immunopathogenesis of AIDS-associated hepatitis was explored in the SIV/rhesus monkey model. The livers of SIV-infected monkeys showed a mild hepatitis, with a predominantly CD8+ T lymphocyte infiltration in the periportal fields and sinusoids. These liver-associated CD8+ T cells were comprised of a high percentage of SIV-specific CTL as defined by MHC class I/Gag peptide tetramer binding and Gag peptide epitope-specific lytic activity. There was insufficient viral replication in these livers to account for attracting this large number of functional virus-specific CTL to the liver. There was also no evidence that the predominant population of CTL were functionally end-stage cells trapped in the liver and destined to undergo apoptotic cell death in that organ. Interestingly, we noted that liver tetramer-binding cells showed an increased expression of CD62L, an adhesion molecule usually only rarely expressed on tetramer-binding cells. This observation suggests that the expression of specific adhesion molecules by CTL might facilitate the capture of these cells in the liver. These results demonstrate that functional SIV-specific CD8+ T cells are present in large numbers in the liver of chronically SIV-infected monkeys. Thus, the liver may be a trap for virus-specific cytotoxic T cells.     

MHC class Ib-restricted CTL provide protection against primary and secondary Listeria monocytogenes infection

Infection of B6 mice with the intracellular pathogen Listeria monocytogenes (LM) results in the activation of CD8(+) T cells that respond to Ag presented by both MHC class Ia and class Ib molecules. Enzyme-linked immunospot analysis reveals that these CTL populations expand and contract at different times following a primary sublethal LM infection. Between days 4 and 6 postinfection, class Ib-restricted CTL exhibit a rapid proliferative response that is primarily H2-M3 restricted. The peak response of class Ia-restricted CD8(+) T cells occurs a few days later, after the majority of bacteria have been cleared. Although class Ia-restricted CTL exhibit a vigorous recall response to secondary LM infection, we observe limited expansion of class Ib-restricted memory CTL, even in MHC class Ia-deficient mice (B6.K(b-/-)D(b-/-)). Despite this lack of enhanced expansion in vivo, class Ib-restricted memory CTL retain the ability to proliferate and expand when provided with Ag in vitro. Furthermore, we demonstrate that in vivo depletion of CD8(+) T cells in LM-immune B6.K(b-/-)D(b-/-) mice severely impairs memory protection. Together, these data demonstrate that class Ib-restricted CTL play an important role in clearing a primary LM infection and generate a memory population capable of providing significant protection against subsequent infection.     

Nucleoli undergo structural and molecular modifications during hibernation

The nucleolus is a very dynamic structure able rapidly to adapt its activity to the cellular metabolic state. An interesting physiological model characterized by drastic modifications of cellular metabolism is represented by hibernating animals. In the present study we investigated the hepatocyte nuclei of euthermic and hibernating edible dormice (Glis glis) with the aim of revealing, by means of ultrastructural and immunocytochemical analyses, possible modifications of nucleolar components during hibernation. Our observations demonstrate that, in deep hibernation, nucleoli undergo structural and molecular modifications: (a) they show numerous nucleoplasmic invaginations and clumps of dense fibrillar component extend from the nucleolar surface; (b) they are frequently in contact with coiled bodies and fibro-granular material, two nuclear bodies usually occurring in the nucleoplasm; (c) the dense fibrillar component contains significant amounts of small nuclear ribonucleoproteins, splicing factors usually distributed in the nucleoplasm. Taken together, these results suggest that during hibernation complex relationships are established between the nucleolus and nucleoplasm, probably related to functional activities peculiar to this physiological phase. However, since no evident nucleolar modification was found in early hibernating dormice, it seems likely that the particular structural and molecular arrangement of nucleoli establishes progressively during hibernation, becoming evident only in the deepest phase, and then disappears upon arousal.     

Ascorbate inhibits edema in brain slices

Ascorbate is an essential antioxidant in the CNS, localized predominantly in neuronal cytosol. Slices of mammalian brain rapidly lose ascorbate, however, when incubated in ascorbate-free media; brain slices also take up water and swell. Here we investigated water gain in coronal slices of rat forebrain incubated with and without ascorbate for 1-3 h at 34 degrees C. Slices progressively gained water in ascorbate-free media, with a significant 12% water increase after 3 h at 34 degrees C, compared with the water content of slices after a 1-h recovery period at 24 degrees C, immediately following slice preparation. Inclusion of 400 micro M ascorbate in the medium led to an increase in tissue ascorbate content and prevented water gain at 34 degrees C. By contrast, water gain was not inhibited by isoascorbate or thiourea, which are antioxidants that are not accumulated in brain cells. The oxidant H2O2 enhanced water gain, whereas a cocktail of NMDA and non-NMDA receptor blockers inhibited edema formation to the same extent as ascorbate. These data demonstrate that brain edema, linked to glutamate-receptor activation, can result from intracellular oxidative stress and that this can be prevented by ascorbate.     

Structural and thermodynamic studies of Bergerac-SH3 chimeras

Bergerac-type chimeras of spectrin SH3 were designed by extending a β-hairpin by eight amino acids so that the extension protruded from the domain body like a “nose” being exposed to the solvent. A calorimetric study of several Bergerac-SH3 variants was carried out over a wide range of pH values and protein concentrations and the three-dimensional structure of one of them, SHH, was determined. X-ray studies confirmed that the nose had a well defined β-structure whilst the chimera formed a stable tetramer within the crystal unit because of four tightly packed noses. In the pH range of 4–7 the heat-induced unfolding of some chimeras was complex and concentration dependent, whilst at pH values below 3.5, low protein concentrations of all the chimeras studied, including SHH, seemed to obey a monomolecular two-state unfolding model. The best set of data was obtained for the SHA variant, the unfolding heat effects of which were systematically higher than those of the WT protein (about 16.4 kJ/mol at 323 K), which may be close to the upper limit of the enthalpy gain due to 10 residue β-hairpin folding. At the same time, the chimeras with high nose stability, which, like SHH, have a hydrophobic (IVY) cluster on their surface, showed a lower apparent unfolding heat effect, much closer to that of the WT protein. The possible reasons for this difference are discussed.     

A Delphi Technology Foresight Study: Mapping Social Construction of Scientific Evidence on Metagenomics Tests for Water Safety

Access to clean water is a grand challenge in the 21^st century. Water safety testing for pathogens currently depends on surrogate measures such as fecal indicator bacteria (e.g., E. coli). Metagenomics concerns high-throughput, culture-independent, unbiased shotgun sequencing of DNA from environmental samples that might transform water safety by detecting waterborne pathogens directly instead of their surrogates. Yet emerging innovations such as metagenomics are often fiercely contested. Innovations are subject to shaping/construction not only by technology but also social systems/values in which they are embedded, such as experts’ attitudes towards new scientific evidence. We conducted a classic three-round Delphi survey, comprised of 107 questions. A multidisciplinary expert panel (n = 24) representing the continuum of discovery scientists and policymakers evaluated the emergence of metagenomics tests. To the best of our knowledge, we report here the first Delphi foresight study of experts’ attitudes on (1) the top 10 priority evidentiary criteria for adoption of metagenomics tests for water safety, (2) the specific issues critical to governance of metagenomics innovation trajectory where there is consensus or dissensus among experts, (3) the anticipated time lapse from discovery to practice of metagenomics tests, and (4) the role and timing of public engagement in development of metagenomics tests. The ability of a test to distinguish between harmful and benign waterborne organisms, analytical/clinical sensitivity, and reproducibility were the top three evidentiary criteria for adoption of metagenomics. Experts agree that metagenomic testing will provide novel information but there is dissensus on whether metagenomics will replace the current water safety testing methods or impact the public health end points (e.g., reduction in boil water advisories). Interestingly, experts view the publics relevant in a “downstream capacity” for adoption of metagenomics rather than a co-productionist role at the “upstream” scientific design stage of metagenomics tests. In summary, these findings offer strategic foresight to govern metagenomics innovations symmetrically: by identifying areas where acceleration (e.g., consensus areas) and deceleration/reconsideration (e.g., dissensus areas) of the innovation trajectory might be warranted. Additionally, we show how scientific evidence is subject to potential social construction by experts’ value systems and the need for greater upstream public engagement on metagenomics innovations.