Chronic Exposure of NG108-15 Cells to Amyloid β Peptide (Aβ1–42) Abolishes Calcium Influx via N-Type Calcium Channels

We investigated whether amyloid--peptide (A_1–42) has an effect on the elevations of the intracellular concentration of Ca²⁺ ions ([Ca²⁺]_i) induced by depolarizations of NG108-15 cells and on related Ca²⁺ channels. A_1–42 (10-1000 nM) had no immediate effect on depolarization-induced [Ca²⁺]_i elevations. [Ca²⁺]_i increases were slightly diminished in cells grown in the presence of 100 or 1000 nM A_1–42. Nifedipine (1 M) reduced these elevations equally in cells grown in the absence or presence of A_1–42. In contrast, the ability of -conotoxin GVIA to diminish the depolarization-induced [Ca²⁺]_i responses became lost in cells grown in the presence of 100 nM A_1–42. This indicates that the influx of calcium through the N-type Ca²⁺ channels was compromised by the chronic exposure of cells to a submicromolar concentration of A_1–42, presumably because of impairement of their function or diminished expression. This may be important in the pathogeny of Alzheimer's dementia in view of the pivotal role of N-type Ca²⁺ channels in neurotransmitter release.     

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.     

Reflection and absorption of millimeter waves by thin absorbing films

Reflection, transmission, and absorption of mm-waves by thin absorbing films were determined at two therapeutic frequencies: 42. 25 and 53.57 GHz. Thin filter strips saturated with distilled water or an alcohol-water solution were used as absorbing samples of different thicknesses. The dependence of the power reflection coefficient R(d) on film thickness (d) was not monotonic. R(d) passed through a pronounced maximum before reaching its steady-state level [R(infinity)]. Similarly, absorption, A(d), passed two maximums with one minimum between them, before reaching its steady-state level [A(infinity)]. At 42.25 GHz, A(d) was compared with absorption in a semi-infinite water medium at a depth d. When d < 0.3 mm, absorption by the film increased: at d = 0.1 mm the absorption ratio for the thin layer sample and the semi-infinite medium was 3.2, while at d = 0.05 mm it increased up to 5.8. Calculations based on Fresnel equations for flat thin layers adequately described the dependence of the reflection, transmission, and absorption on d and allowed the determination of the refractive index (n), dielectric constant (epsilon), and penetration depth (delta) of the absorbing medium for various frequencies. For water samples, epsilon was found to be 12.4-19.3j, delta = 0.49 mm at 42.25 GHz, and epsilon = 9.0-19.5j, delta = 0.36 mm at 53.57 GHz. The calculated power density distribution within the film was strongly dependent on d. The measurements and calculations have shown that the reflection and absorption of mm-waves by thin absorbing layers can significantly differ from the reflection and absorption in similar semi-infinite media. The difference in reflection, absorption, and power density distribution in films, as compared to semi-infinite media, are caused by multiple internal reflections from the film boundaries. That is why, when using thin phantoms and thin biological samples, the specifics of the interaction of mm-waves with thin films should be taken into account.     

Dynamics of milk ejection reflex during continuous rhythmic stimulation of areola-nipple complex of the mammary gland

In continuous 10-minute simultaneous action of the vacuum and compression stimuli at a 1 cycle/second frequency, the rate of the milk ejection from the breast was periodically changing in lactating women. The milk flow first peaks latency was 0.5-3.0 minutes, the number of peaks being 2-7 per minute with the inter-peak interval 2-4 minutes during a 10-minute milk ejection. The women revealed an individual pattern of the milk flow peaks. The findings suggest that a central gate mechanism intervenes to set the periodicity of the milk ejection.     

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.     

Single amino acid substitutions in the Ca2+-binding site of recoverin.II.The unusual behavior of the protein upon the binding of calcium ions

The structural properties of myristoylated forms of recombinant recoverin of the wild type and of its mutants with damaged second and/or third Ca(2+)-binding sites were studied by fluorimetry and circular dichroism. The interaction of wild-type recoverin with calcium ions was shown to induce unusual structural rearrangements in its molecule. In particular, protein binding with Ca2+ ions results in an increase in the mobility of the environment of Trp residues, in higher hydrophobicity, and in elevated thermal stability (its thermal transition shifts by 15 degrees C to higher temperatures) but has almost no effect on its secondary structure. Similar structural changes induced by Ca2+ are also characteristic of the -EF2 mutant of recoverin whose second Ca(2+)-binding site is modified and cannot bind calcium ions. The structural properties of the -EF3 and -EF2,3 mutants (whose third or simultaneously second and third Ca(2+)-binding sites, respectively, are modified and damaged) are practically indifferent to calcium ions.     

Ligand-insensitive State of Cardiac ATP-sensitive K+ Channels : Basis for Channel Opening

The mechanism by which ATP-sensitive K⁺ (K_ATP) channels open in the presence of inhibitory concentrations of ATP remains unknown. Herein, using a four-state kinetic model, we found that the nucleotide diphosphate UDP directed cardiac K_ATP channels to operate within intraburst transitions. These transitions are not targeted by ATP, nor the structurally unrelated sulfonylurea glyburide, which inhibit channel opening by acting on interburst transitions. Therefore, the channel remained insensitive to ATP and glyburide in the presence of UDP. “Rundown” of channel activity decreased the efficacy with which UDP could direct and maintain the channel to operate within intraburst transitions. Under this condition, the channel was sensitive to inhibition by ATP and glyburide despite the presence of UDP. This behavior of the K_ATP channel could be accounted for by an allosteric model of ligand-channel interaction. Thus, the response of cardiac K_ATP channels towards inhibitory ligands is determined by the relative lifetime the channel spends in a ligand-sensitive versus -insensitive state. Interconversion between these two conformational states represents a novel basis for K_ATP channel opening in the presence of inhibitory concentrations of ATP in a cardiac cell.     

Evidence for Direct Physical Association between a K+ Channel (Kir6.2) and an ATP-Binding Cassette Protein (SUR1) Which Affects Cellular Distribution and Kinetic Behavior of an ATP-Sensitive K+ Channel

Structurally unique among ion channels, ATP-sensitive K⁺ (K_ATP) channels are essential in coupling cellular metabolism with membrane excitability, and their activity can be reconstituted by coexpression of an inwardly rectifying K⁺ channel, Kir6.2, with an ATP-binding cassette protein, SUR1. To determine if constitutive channel subunits form a physical complex, we developed antibodies to specifically label and immunoprecipitate Kir6.2. From a mixture of Kir6.2 and SUR1 in vitro-translated proteins, and from COS cells transfected with both channel subunits, the Kir6.2-specific antibody coimmunoprecipitated 38- and 140-kDa proteins corresponding to Kir6.2 and SUR1, respectively. Since previous reports suggest that the carboxy-truncated Kir6.2 can form a channel independent of SUR, we deleted 114 nucleotides from the carboxy terminus of the Kir6.2 open reading frame (Kir6.2ΔC37). Kir6.2ΔC37 still coimmunoprecipitated with SUR1, suggesting that the distal carboxy terminus of Kir6.2 is unnecessary for subunit association. Confocal microscopic images of COS cells transfected with Kir6.2 or Kir6.2ΔC37 and labeled with fluorescent antibodies revealed unique honeycomb patterns unlike the diffuse immunostaining observed when cells were cotransfected with Kir6.2-SUR1 or Kir6.2ΔC37-SUR1. Membrane patches excised from COS cells cotransfected with Kir6.2-SUR1 or Kir6.2ΔC37-SUR1 exhibited single-channel activity characteristic of pancreatic K_ATP channels. Kir6.2ΔC37 alone formed functional channels with single-channel conductance and intraburst kinetic properties similar to those of Kir6.2-SUR1 or Kir6.2ΔC37-SUR1 but with reduced burst duration. This study provides direct evidence that an inwardly rectifying K⁺ channel and an ATP-binding cassette protein physically associate, which affects the cellular distribution and kinetic behavior of a K_ATP channel.     

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.