Disease-causing Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator Determine the Functional Responses of Alveolar Macrophages

Alveolar macrophages (AMs) play a major role in host defense against microbial infections in the lung. To perform this function, these cells must ingest and destroy pathogens, generally in phagosomes, as well as secrete a number of products that signal other immune cells to respond. Recently, we demonstrated that murine alveolar macrophages employ the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel as a determinant in lysosomal acidification (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933–944). Lysosomes and phagosomes in murine cftr^−/− AMs failed to acidify, and the cells were deficient in bacterial killing compared with wild type controls. Cystic fibrosis is caused by mutations in CFTR and is characterized by chronic lung infections. The information about relationships between the CFTR genotype and the disease phenotype is scarce both on the organismal and cellular level. The most common disease-causing mutation, ΔF508, is found in 70% of patients with cystic fibrosis. The mutant protein fails to fold properly and is targeted for proteosomal degradation. G551D, the second most common mutation, causes loss of function of the protein at the plasma membrane. In this study, we have investigated the impact of CFTR ΔF508 and G551D on a set of core intracellular functions, including organellar acidification, granule secretion, and microbicidal activity in the AM. Utilizing primary AMs from wild type, cftr^−/−, as well as mutant mice, we show a tight correlation between CFTR genotype and levels of lysosomal acidification, bacterial killing, and agonist-induced secretory responses, all of which would be expected to contribute to a significant impact on microbial clearance in the lung.     

Distinct Structures of Scrapie Prion Protein (PrPSc)-seeded Versus Spontaneous Recombinant Prion Protein Fibrils Revealed by Hydrogen/Deuterium Exchange

The detailed structures of prion disease-associated, partially protease-resistant forms of prion protein (e.g. PrP^Sc) are largely unknown. PrP^Sc appears to propagate itself by autocatalyzing the conformational conversion and oligomerization of normal prion protein (PrP^C). One manifestation of PrP^Sc templating activity is its ability, in protein misfolding cyclic amplification reactions, to seed the conversion of recombinant prion protein (rPrP) into aggregates that more closely resemble PrP^Sc than spontaneously nucleated rPrP amyloids in terms of proteolytic fragmentation and infrared spectra. The absence of posttranslational modifications makes these rPrP aggregates more amenable to detailed structural analyses than bona fide PrP^Sc. Here, we compare the structures of PrP^Sc-seeded and spontaneously nucleated aggregates of hamster rPrP by using H/D exchange coupled with mass spectrometry. In spontaneously formed fibrils, very slow H/D exchange in region ∼163–223 represents a systematically H-bonded cross-β amyloid core structure. PrP^Sc-seeded aggregates have a subpopulation of molecules in which this core region extends N-terminally as far as to residue ∼145, and there is a significant degree of order within residues ∼117–133. The formation of tightly H-bonded structures by these more N-terminal residues may account partially for the generation of longer protease-resistant regions in the PrP^Sc-seeded rPrP aggregates; however, part of the added protease resistance is dependent on the presence of SDS during proteolysis, emphasizing the multifactorial influences on proteolytic fragmentation patterns. These results demonstrate that PrP^Sc has a distinct templating activity that induces ordered, systematically H-bonded structure in regions that are dynamic and poorly defined in spontaneously formed aggregates of rPrP.Transmissible spongiform encephalopathies (TSEs),² or prion diseases, are a group of infectious neurodegenerative disorders that affect many mammalian species and include Creutzfeldt-Jakob disease in humans, scrapie in sheep, chronic wasting disease in cervids, and bovine spongiform encephalopathy (“mad cow” disease) (1–7). All of these diseases appear to be intimately associated with conformational conversion of the normal host-encoded prion protein, termed PrP^C, to a pathological isoform, PrP^Sc (1–5). According to the “protein-only” model, PrP^Sc itself represents the infectious prion agent (1, 8); it is believed to self-propagate by an autocatalytic mechanism involving binding to PrP^C and templating the conversion of the latter protein to the PrP^Sc state (9, 10). Although molecular details of such a mechanism of disease propagation remain largely unknown, the general principle of protein-based infectivity is supported by a wealth of experimental data (1–7).PrP^C is a monomeric glycophosphatidylinositol-linked glycoprotein that is highly protease-sensitive and soluble in nonionic detergents. High resolution NMR data show that the recombinant PrP (rPrP), a nonglycosylated model of PrP^C, consists of a flexible N-terminal region and a folded C-terminal domain encompassing three α-helices and two short β-strands (11–13). Conversely, the PrP^Sc isoform is aggregate in nature, rich in β-sheet structure, insoluble in nonionic detergents, and partially resistant to proteinase K (PK) digestion, with a PK-resistant core encompassing the C-terminal ∼140 residues (1–5, 14, 15). Little specific structural information is available, however, for this isoform beyond low resolution biochemical and spectroscopic characterization. Thus, the structure of PrP^Sc conformer(s) associated with prion infectivity remains one of the best guarded mysteries, hindering efforts to understand the molecular basis of TSE diseases.Many efforts have been made over the years to recapitulate PrP^Sc formation and prion propagation in vitro. Early studies have shown that PrP^C can be converted with remarkable species and strain specificities to a PrP^Sc-like conformation (as judged by PK resistance) simply by incubation with PrP^Sc from prion-infected animals (16, 17). The yields of these original cell-free conversion experiments were low, and no new infectivity could be attributed to the newly converted material (18). An important more recent study showed that both PrP^Sc and TSE infectivity can be amplified indefinitely in crude brain homogenates using successive rounds of sonication and incubation (19), a procedure called protein misfolding cyclic amplification (PMCA) (20). Similar amplification of the TSE infectivity was also accomplished by PMCA employing purified PrP^C as a substrate, although only in the presence of polyanions such as RNA and copurified lipids (21). Unfortunately, the quantities of infectious PrP^Sc generated by PMCA using purified brain-derived PrP^C are very small, precluding most structural studies.In contrast to brain-derived PrP^C, large scale purification can be readily accomplished for bacterially expressed rPrP, a form of PrP lacking glycosylation and the glycophosphatidylinositol anchor. The latter protein can spontaneously polymerize into amyloid fibrils, and much insight has been gained into mechanistic and structural aspects of this reaction (22–28). However, although rPrP fibrils were shown to cause or accelerate a transmissible neurodegenerative disorder in transgenic mice overexpressing a PrP^C variant encompassing residues 89–231, the infectivity titer of these “synthetic prions” was extremely low (29) or absent altogether (4). This low infectivity coincides with much shorter PK-resistant core of rPrP amyloid fibrils compared with brain-derived PrP^Sc (26, 30), raising questions regarding the relationship between these fibrils and the authentic TSE agent. In this context, an important recent development was the finding that the PrP^Sc-seeded PMCA method can be extended to rPrP, yielding protease-resistant recombinant PrP aggregates (rPrP^PMCA or rPrP-res^(Sc)) (31). These aggregates display a PK digestion pattern that is much more closely related to PrP^Sc than that of previously studied spontaneously formed rPrP fibrils, offering a potentially more relevant model for biochemical and biophysical studies. Here, we provide, for the first time, a direct insight into the structure of rPrP^PMCA. H/D exchange data coupled with MS analysis (HXMS) allowed us to identify systematically H-bonded core region(s) of these aggregates, shedding a new light on the mechanisms underlying formation of PK-resistant structures.     

Life-cycles of cestodes of the genus Branchiopodataenia Bondarenko & Kontrimavichus, 2004 (Cestoda: Hymenolepididae) from gulls in Chukotka

Data are presented on the life-cycles and the postembryonal development of four species of Branchiopodataenia Bondarenko & Kontrimavichus, 2004, B. anaticapicirra Bondarenko & Kontrimavichus, 2004, B. arctowskii (Jarecka & Ostas, 1984), B. haldemani (Schiller, 1951) and B. pacifica (Spassky & Jurpalova, 1968), which are specific parasites of gulls. The investigation was carried out in Chukotka (Chaun Bay) between 1971 and 1981 under natural and experimental conditions. Branchiopod crustaceans were exclusive natural and experimental intermediate hosts of all of the species studied; this fact provided additional evidence for the erection of Branchiopodataenia. The metacestodes exhibit a pattern of postembryonal development and a typical larval form, termed a 'cercocyst', which is a modification of a cysticercoid. This modification of the metacestode does not occur in species of Wardium Mayhew, 1925, the genus with which species of Branchiopodataenia had previously been affiliated.     

Biological effects of a 765-kV transmission line: Exposures and thresholds in honeybee colonies

Honeybee colonies exposed under a 765-kV, 60-Hz transmission line at 7 kV/m show the following sequence of effects: 1) increased motor activity with transient increase in hive temperature; 2) abnormal propolization; 3) impaired hive weight gain; 4) queen loss and abnormal production of queen cells; 5) decreased sealed brood; and 6) poor winter survival. When colonies were exposed at 5 different E fields (7, 5.5, 4.1, 1.8, and 0.65–0.85 kV/m) at incremental distances from the line, different thresholds for biologic effects were obtained. Hive net weights showed significant dose-related lags at the following exposures: 7 kV/m, one week; 5.5 kV/m, 2 weeks; and 4.1 kV/m, 11 weeks. The two lowest exposure groups had normal weight after 25 weeks. Abnormal propolization of hive entrances did not occur below 4.1 kV/m. Queen loss occurred in 6 of 7 colonies at 7 kV/m and 1 of 7 at 5.5 kV/m, but not below. Foraging rates were significantly lower only at 7 and 5.5 kV/m. Hive weight impairment and abnormal propolization occur at lower E-field intensity than other effects and limit the “biological effects corridor” of the transmission line to approximately 23 m beyond a ground line projection of each outer phase wire. Intrahive E fields of 15–100 kV/m were measured with a displacement current sensor. Step-potential-induced currents up to 0.5 μA were measured in an electrically equivalent bee model placed on the honeycomb in a hive exposed at 7 kV/m. At 1.8 kV/m body currents were a few nanoamperes, or two orders of magnitude lower, and these colonies showed no effects. E-field versus electric shock mechanisms are discussed.     

A conformational alpha-helix to beta-sheet transition accompanies racemic self-assembly of polylysine: an FT-IR spectroscopic study

The self-assembly of polylysine chains with opposite chiral senses is an intriguing phenomenon, suggesting that subtle hydrational effects may be a driving force of protein aggregation. We have used FT-IR spectroscopy to characterize the alpha-helix-to-beta-sheet conformational transition that accompanies the aggregation of single and mixed enantiomers of polylysine. The preferential racemic self-assembly not only takes place at a lower temperature, but is also less prone to repulsive electrostatic interactions between lysine charged side chains, caused by decreasing pH (pD). While the process is generally irreversible, it yet appears to proceed in a stepwise manner through a sequence of thermodynamically, rather than kinetically controlled events involving gradual destabilization of alpha-helices. Interestingly, although the alpha/beta-transition is in either case (single or mixed enantiomers) an endothermic process, it may also be induced by freezing of water, which leads to markedly more complete (and irreversible) aggregation of the mixed enantiomers. Relevance of these findings has been discussed in the context of protein aggregation studies.     

A catalogue of Lithuanian beetles (Insecta, Coleoptera)

This paper presents the first complete and updated list of all 3597 species of beetles (Insecta: Coleoptera) belonging to 92 familiesfound and published in Lithuania until 2011, with comments also provided on the main systematic and nomenclatural changes since the last monographic treatment in two volumes (Pileckis and Monsevičius 1995, 1997). The introductory section provides a general overview of the main features of the territory of Lithuania, the origins and formation of the beetle fauna and their conservation, the faunistic investigations in Lithuania to date revealing the most important stages of the faunistic research process with reference to the most prominent scientists, an overview of their work, and their contribution to Lithuanian coleopteran faunal research.Species recorded in Lithuania by some authors without reliable evidence and requiring further confirmation with new data are presented in a separate list, consisting of 183 species. For the first time, analysis of errors in works of Lithuanian authors concerning data on coleopteran fauna has been conducted and these errors have been corrected. All available published and Internet sources on beetles found in Lithuania have been considered in the current study. Over 630 literature sources on species composition of beetles, their distribution in Lithuania and neighbouring countries, and taxonomic revisions and changes are reviewed and cited. An alphabetical list of these literature sources is presented. After revision of public beetle collections in Lithuania, the authors propose to remove 43 species from the beetle species list of the country on the grounds, that they have been wrongly identified or published by mistake. For reasons of clarity, 19 previously noted but later excluded species are included in the current checklist with comments. Based on faunal data from neighbouring countries, species expected to occur in Lithuania are matnioned. In total 1390 species are attributed to this category and data on their distribution in neighbouring countries is presented. Completion of this study provides evidence that the Lithuanian coleopteran fauna has yet to be completely investigated and it is estimated that approximately 28 % of beetle species remain undiscovered in Lithuania. More than 85% of beetle species expected for Lithuania have been found in the following families: Cerylonidae, Geotrupidae, Haliplidae, Kateridae, Lycidae, Lucanidae, Mycetophagidae, Scarabaeidae and Silphidae. In families with few species such as Alexiidae, Boridae, Byturidae, Dascilidae, Drilidae, Eucinetidae, Lampyridae, Lymexilidae, Megalopodidae, Nemonychidae, Nosodendridae, Noteridae, Orsodacnidae, Pyrochroidae, Pythidae, Psephenidae, Rhysodidae, Sphaeritidae, Sphaeriusidae, Sphindidae, Stenotrahelidae and Trogidae, all possible species have already been discovered. However in some beetle families such as Aderidae, Bothrideridae, Eucnemidae, Laemoploeidae, Mordellidae, Ptiliidae, Scraptidae and Throscidae less than 50% of all possible species are known. At present the beetle species recorded in Lithuania belong to 92 families, with species from 9 other families such as Agyrtidae, Biphylidae, Deradontidae, Mycteridae, Ochodaeidae, Phleophilidae, Phloeostichidae, Prostomidae, Trachypachidae are expected to be found.A bibliography and a index of subfamily and genus levels are provided. The information published in the monograph will serve to further faunistic and distribution research of beetles and will help to avoid confusion in the identificatation of coleopteran fauna of Lithuania.     

Optimal Superpositioning of Flexible Molecule Ensembles

Analysis of the internal dynamics of a biological molecule requires the successful removal of overall translation and rotation. Particularly for flexible or intrinsically disordered peptides, this is a challenging task due to the absence of a well-defined reference structure that could be used for superpositioning. In this work, we started the analysis with a widely known formulation of an objective for the problem of superimposing a set of multiple molecules as variance minimization over an ensemble. A negative effect of this superpositioning method is the introduction of ambiguous rotations, where different rotation matrices may be applied to structurally similar molecules. We developed two algorithms to resolve the suboptimal rotations. The first approach minimizes the variance together with the distance of a structure to a preceding molecule in the ensemble. The second algorithm seeks for minimal variance together with the distance to the nearest neighbors of each structure. The newly developed methods were applied to molecular-dynamics trajectories and normal-mode ensembles of the Aβ peptide, RS peptide, and lysozyme. These new (to our knowledge) superpositioning methods combine the benefits of variance and distance between nearest-neighbor(s) minimization, providing a solution for the analysis of intrinsic motions of flexible molecules and resolving ambiguous rotations.     

Determination of the volume changes induced by ligand binding to heat shock protein 90 using high-pressure denaturation

The volume changes accompanying ligand binding to proteins are thermodynamically important and could be used in the design of compounds with specific binding properties. Measuring the volumetric properties could yield as much information as the enthalpic properties of binding. Pressure-based methods are significantly more laborious than temperature methods and are underused. Here we present a pressure shift assay (PressureFluor, analogous to the ThermoFluor thermal shift assay) that uses high pressure to denature proteins. The PressureFluor method was used to study the ligand binding thermodynamics of heat shock protein 90 (Hsp90). Ligands stabilize the protein against pressure denaturation, similar to the stabilization against temperature denaturation. The equations that relate the ligand dosing, protein concentration, and binding constant with the volumes and compressibilities of unfolding and binding are presented.