Boundary lipids of the nicotinic acetylcholine receptor: Spontaneous partitioning via coarse-grained molecular dynamics simulation

Reconstituted nicotinic acetylcholine receptors (nAChRs) exhibit significant gain-of-function upon addition of cholesterol to reconstitution mixtures, and cholesterol affects the organization of nAChRs within domain-forming membranes, but whether nAChR partitions to cholesterol-rich liquid-ordered (“raft” or l_o) domains or cholesterol-poor liquid-disordered (l_do) domains is unknown. We use coarse-grained molecular dynamics simulations to observe spontaneous interactions of cholesterol, saturated lipids, and polyunsaturated (PUFA) lipids with nAChRs. In binary Dipalmitoylphosphatidylcholine:Cholesterol (DPPC:CHOL) mixtures, both CHOL and DPPC acyl chains were observed spontaneously entering deep “non-annular” cavities in the nAChR TMD, particularly at the subunit interface and the β subunit center, facilitated by the low amino acid density in the cryo-EM structure of nAChR in a native membrane. Cholesterol was highly enriched in the annulus around the TMD, but this effect extended over (at most) 5–10 Å. In domain-forming ternary mixtures containing PUFAs, the presence of a single receptor did not significantly affect the likelihood of domain formation. nAChR partitioned to any cholesterol-poor l_do domain that was present, regardless of whether the l_do or l_o domain lipids had PC or PE headgroups. Enrichment of PUFAs among boundary lipids was positively correlated with their propensity for demixing from cholesterol-rich phases. Long n-3 chains (tested here with Docosahexaenoic Acid, DHA) were highly enriched in annular and non-annular embedded sites, partially displacing cholesterol and completely displacing DPPC, and occupying sites even deeper within the bundle. Shorter n-6 chains were far less effective at displacing cholesterol from non-annular sites.     

Early Indicators of Fatal Leptospirosis during the 2010 Epidemic in Puerto Rico

Background

Leptospirosis is a potentially fatal bacterial zoonosis that is endemic throughout the tropics and may be misdiagnosed as dengue. Delayed hospital admission of leptospirosis patients is associated with increased mortality.

Methodology/Principal Findings

During a concurrent dengue/leptospirosis epidemic in Puerto Rico in 2010, suspected dengue patients that tested dengue-negative were tested for leptospirosis. Fatal and non-fatal hospitalized leptospirosis patients were matched 1:1–3 by age. Records from all medical visits were evaluated for factors associated with fatal outcome. Among 175 leptospirosis patients identified (4.7 per 100,000 residents), 26 (15%) were fatal. Most patients were older males and had illness onset during the rainy season. Fatal case patients first sought medical care earlier than non-fatal control patients (2.5 vs. 5 days post-illness onset [DPO], p < 0.01), but less frequently first sought care at a hospital (52.4% vs. 92.2%, p < 0.01). Although fatal cases were more often diagnosed with leptospirosis at first medical visit (43.9% vs. 9.6%, p = 0.01), they were admitted to the hospital no earlier than non-fatal controls (4.5 vs. 6 DPO, p = 0.31). Cases less often developed fever (p = 0.03), but more often developed jaundice, edema, leg pain, hemoptysis, and had a seizure (p ≤ 0.03). Multivariable analysis of laboratory values from first medical visit associated with fatal outcome included increased white blood cell (WBC) count with increased creatinine (p = 0.001), and decreased bicarbonate with either increased WBC count, increased creatinine, or decreased platelet count (p < 0.001).

Conclusions/Significance

Patients with fatal leptospirosis sought care earlier, but were not admitted for care any earlier than non-fatal patients. Combinations of routine laboratory values predictive of fatal outcome should be considered in admission decision-making for patients with suspected leptospirosis.     

Effect of inhibition of abscisic Acid accumulation on the spatial distribution of elongation in the primary root and mesocotyl of maize at low water potentials

Previous work showed that accumulation of endogenous abscisic acid (ABA) acts both to maintain primary root growth and inhibit shoot growth in maize seedlings at low water potentials (ψ_w) (IN Saab, RE Sharp, J Pritchard, GS Voetberg [1990] Plant Physiol 93: 1329-1336). In this study, we have characterized the growth responses of the primary root and mesocotyl of maize (Zea mays L. cv FR27 × FRMo 17) to manipulation of ABA levels at low ψ_w with a high degree of spatial resolution to provide the basis for studies of the mechanism(s) of ABA action. In seedlings growing at low ψ_w and treated with fluridone to inhibit carotenoid (and ABA) biosynthesis, ABA levels were decreased in all locations of the root and mesocotyl growing zones compared with untreated seedlings growing at the same ψ_w. In the root, low ψ_w (−1.6 megapascals) caused a shortening of the growing zone, as reported previously. The fluridone treatment was associated with severe inhibition of root elongation rate, which resulted from further shortening of the growing zone. In the mesocotyl, low ψ_w (−0.3 megapascal) also resulted in a shortened growing zone. In contrast with the primary root, however, fluridone treatment prevented most of the inhibition of elongation and the shortening of the growing zone. Final cell length measurements indicated that the responses of both root and mesocotyl elongation to ABA manipulation at low ψ_w involve large effects on cell expansion. Measurements of the relative changes in root and shoot water contents and dry weights after transplanting to a ψ_w of −0.3 megapascal showed that the maintenance of shoot elongation in fluridone-treated seedlings was not attributable to increased water or seed-reserve availability resulting from inhibition of root growth. The results suggest a developmental gradient in tissue responsiveness to endogenous ABA in both the root and mesocotyl growing zones. In the root, the capacity for ABA to protect cell expansion at low ψ_w appears to decrease with increasing distance from the apex. In the mesocotyl, in contrast, the accumulation of ABA at low ψ_w appears to become increasingly inhibitory to expansion as cells are displaced away from the meristematic region.     

Neuroendocrine control of reduced persistence of egg-laying in domestic hens: evidence for the development of photorefractoriness.

Egg-laying in hens exposed for more than 11 months to photostimulatory daylengths was intermittent and associated with a reduction in numbers of yellow-yolky ovarian follicles. Old laying hens (105 weeks) had lower concentrations of luteinizing hormone (LH) in the pituitary gland and plasma and reduced pituitary gland responsiveness to chicken LH-releasing-hormones (LHRH-I and II) in vivo when compared with young laying hens (28 weeks). Four weeks after transfer from 14 to 8 h light/day, egg production almost stopped in old, but not in young hens, although plasma LH concentrations decreased in all birds. After transfer from 14 to 20 h light/day, plasma LH increased in young, but not in old, hens, without a change in the rate of egg production. Reproductive function was enhanced in old hens returned to long days after induction of a moult and ovarian regression by reducing daylength and dietary restriction. Moulted hens had a greater rate of egg production, higher concentrations of plasma LH and a greater pituitary-gland responsiveness to LHRH-II in vivo than unmoulted control hens. After transfer from 14 to 8 h light/day, egg-laying decreased more rapidly in unmoulted than in moulted hens; transfer to 17 h light/day increased egg production in moulted, but not in unmoulted, birds. Induction of ovarian regression in old hens by dietary restriction alone also enhanced reproductive function after the dietary restriction was relaxed. Egg-laying was more persistent in hens brought into lay for a second year by transferring them from 3 to 11 h light/day than in hens transferred from 3 to 20 h light/day. Egg production was stimulated in hens maintained on 3 or 11 h light/day for 42 weeks, after transfer to 20 h light/day. Egg production ceased in hens maintained on 20 h light/day for 46 weeks, after transfer to 3 h light/day. These observations are consistent with the view that poor persistence of laying in hens less than 2 years old and exposed continuously to long days is caused, in part, by a reduction in hypothalamic-gonadotroph function. This reduction in neuroendocrine function may be due, in part, to the development of relative photorefractoriness.     

Monospecific antibodies against a synthetic peptide predicted from the alpha-3 nicotinic receptor cDNA inhibit binding of [3H]nicotine to rat brain nicotinic cholinergic receptor

Polyclonal antibodies were raised against a synthetic decapeptide (designated S3) predicted from a segment of the alpha-3 subunit cDNA (amino acid residues 130-139) encoding the rat brain nicotinic cholinergic receptor. This segment was selected because it may be proximate to the nicotine/acetylcholine-binding site of the receptor (1). By radioligand binding assays and sucrose density gradient centrifugation, these monospecific antibodies were shown to inhibit the binding of [3H]nicotine to both the large molecular weight rat brain receptor (240 kDa) and to an SDS-disaggregated nicotine-binding subunit species (80 kDa), in a dose-dependent manner. The neutralizing effect of the anti-S3 antibodies supports the view that this region of the protein is closely related to the agonist binding site.     

MALE‐BIASED FITNESS EFFECTS OF SPONTANEOUS MUTATIONS IN DROSOPHILA MELANOGASTER

In populations with males and females, sexual selection may often represent a major component of overall selection. Sexual selection could act to eliminate deleterious alleles in concert with other forms of selection, thereby improving the fitness of sexual populations. Alternatively, the divergent reproductive strategies of the sexes could promote the maintenance of sexually antagonistic variation, causing sexual populations to be less fit. The net impact of sexual selection on fitness is not well understood, due in part to limited data on the sex‐specific effects of spontaneous mutations on total fitness. Using a set of mutation accumulation lines of Drosophila melanogaster, we found that mutations were deleterious in both sexes and had larger effects on fitness in males than in females. This pattern is expected to reduce the mutation load of sexual females and promote the maintenance of sexual reproduction.     

Studies on Shell Formation : VIII. Electron Microscopy of Crystal Growth of the Nacreous Layer of the Oyster Crassostrea virginica

Electron microscope observations have been made by means of the replica method on growth processes of calcite crystals of the nacreous layer of the shell of the oyster, Crassostrea virginica. Layer formation is initiated by the secretion of a conchiolin matrix and the deposition of rounded crystal seeds on or in this material. In some areas crystal seeds are elongate and within a given area show a similar orientation, probably due to slower deposition. The seeds appear to increase in size by dendritic growth, and smaller seeds become incorporated into larger ones which come into contact to form a single layer. With further growth, crystals overlap, forming a step-like arrangement. The direction of growth is frequently different in neighboring regions. Crystal seeds deposited on crystal surfaces are usually elongate and oriented. Well developed crystals have a tabular idiomorphic form and are parallel in their growth. Rounded and irregular crystals were also observed. The crystals show reticular structure with units of the order of 100 A and striations corresponding with the rhombohedral axes of the crystals. The role of the mantle is discussed in relation to the growth patterns of crystals and shell structure.     

Evidence that plasma membrane electrical potential is required for vesicular stomatitis virus infection of MDCK cells: A study using fluorescence measurements through polycarbonate supports

Summary We used fluorescence microscopy of Madin-Darby Canine Kidney (MDCK) cells grown on polycarbonate filters to study a possible link between plasma membrane electrical potential (_pm) and infectivity of vesicular stomatitis virus (VSV). Complete substitution of K⁺ for extracellular Na⁺blocks VSV infection of MDCK cells as well as baby hamster kidney (BHK) cells. When we independently perfused the apical and basal-lateral surfaces of high resistance monolayers, high K⁺ inhibited VSV infection of MDCK cells only when applied to the basal-lateral side; high K⁺ applied apically had no effect on VSV infection. This morphological specificity correlates with a large decrease in _pm of MDCK cells when high K⁺ buffer is perfused across the basal-lateral surface. Depolarization of the plasma membrane by 130 mm basal K⁺ causes a sustained increase of cytosol pH in MDCK cells from 7.3 to 7.5 as reported by the fluorescent dye BCECF. Depolarization also causes a transient increase of cytosol Ca²⁺ from 70 to 300 nm as reported by the dye Fura-2. Neither increase could explain the block of VSV infectivity by plasma membrane depolarization. One alternative hypothesis is that _pm facilitates membrane translocation of viral macromolecules as previously described for colicins, mitochondrial import proteins, and proteins secreted by Escherichia coli.We thank Kenneth Spring for many helpful discussions concerning fluorescence digitized imaging systems, James Russell for his collaboration in the design of our imaging system, Herbert Chase for suggestions on dye loading into MDCK cells, and Manfred Schubert and George Harmison for providing expertise on VSV.     

ThehapC gene ofAspergillus nidulans is involved in the expression of CCAAT-containing promoters

The 5 regulatory region of theamdS gene ofAspergillus nidulans, which encodes an acetamidase required for growth on acetamide as a carbon and nitrogen source, contains a CCAAT sequence which is required for setting the basal level ofamdS expression. Mobility shift studies have identified a factor inA. nidulans nuclear extracts which binds to this CCAAT sequence. InSaccharomyces cerevisiae theHAP3 gene encodes one component of a multisubunit complex that binds CCAAT sequences. A search of the EMBL and SwissProt databases has revealed anA. nidulans sequence with significant homology to theHAP3 gene adjacent to the previously cloned regulatory geneamdR. Sequencing of the remainder of this region has confirmed the presence of a gene, designatedhapC, with extensive homology toHAP3. The predicted amino acid sequence of HapC shows extensive identity to HAP3 in the central conserved domain, but shows little conservation in the flanking sequences. A haploid carrying ahapC deletion has been created and is viable, but grows poorly on all media tested. This null mutant grows especially slowly on acetamide as a sole carbon and nitrogen source, indicating thathapC plays a role inamdS expression. In agreement with this notion, it has been shown that thehapC deletion results in reduced levels of expression of anamdS::lacZ reporter gene and this effect is particularly evident under conditions of carbon limitation. Nuclear extracts prepared from thehapC deletion mutant show no CCAAT binding activity to theamdS orgatA promoters, indicating thathapC may encode a component of the complex binding at this sequence.