Partly folded states of members of the lysozyme/lactalbumin superfamily: a comparative study by circular dichroism spectroscopy and limited proteolysis

The partly folded states of protein members of the lysozyme (LYS)/alpha-lactalbumin (LA) superfamily have been analyzed by circular dichroism (CD) measurements and limited proteolysis experiments. Hen, horse, dog, and pigeon LYSs and bovine LA were used in the present study. These are related proteins of 123- to 129-amino-acid residues with similar three-dimensional structures but low similarity in amino acid sequences. Moreover, notable differences among them reside in their calcium-binding properties and capability to adopt partly folded states or molten globules in acid solution (A-state) or on depletion of calcium at neutral pH (apo-state). Far- and near-UV CD measurements revealed that although the structures of hen and dog LYS are rather stable in acid at pH 2.0 or at neutral pH in the absence of calcium, conformational transitions to various extents occur with all other LYS/LA proteins herewith investigated. The most significant perturbation of tertiary structure in acid was observed with bovine LA and LYS from horse milk and pigeon egg-white. Pepsin and proteinase K were used as proteolytic probes, because these proteases show broad substrate specificity, and therefore, their sites of proteolysis are dictated not by the specific amino acid sequence of the protein substrate but by its overall structure and dynamics. Although hen LYS at pH 2.0 was fully resistant to proteolysis by pepsin, the other members of the LYS/LA superfamily were cleaved at different rates at few sites of the polypeptide chain and thus producing rather large protein fragments. The apo-form of bovine LA, horse LYS, and pigeon LYS were attacked by proteinase K at pH 8.3, whereas dog and hen LYSs were resistant to proteolysis when reacted under identical experimental conditions. Briefly, it has been found that the proteolysis data correlate well with the extent of conformational transitions inferred from CD spectra and with existing structural informations regarding the proteins herewith investigated, mainly derived from NMR and hydrogen exchange measurements. The sites of initial proteolytic cleavages in the LYS variants occur at the level of the beta-subdomain (approximately chain region 34-57), in analogy to those observed with bovine LA. Proteolysis data are in agreement with the current view that the molten globule of the LYS/LA proteins is characterized by a structured alpha-domain and a largely disrupted beta-subdomain. Our results underscore the utility of the limited proteolysis approach for analyzing structure and dynamics of proteins, even if adopting an ensemble of dynamic states as in the molten globule.     

Clinical perspectives on the association between respiratory syncytial virus and reactive airway disease

Asthma is a leading cause of morbidity and mortality among children worldwide, as is respiratory syncytial virus (RSV). This report reviews controlled retrospective and prospective studies conducted to investigate whether there is an association between RSV bronchiolitis in infancy and subsequent development of reactive airway disease or allergic sensitization. Findings indicate that such a link to bronchial obstructive symptoms does exist and is strongest for children who experienced severe RSV illness that requires hospitalization. However, it is not yet clear what roles genetic predisposition and environmental or other risk factors may play in the interaction between RSV bronchiolitis and reactive airway disease or allergic sensitization. Randomized, prospective studies utilizing an intervention against RSV, such as a passive immunoprophylactic agent, may determine whether preventing RSV bronchiolitis reduces the incidence of asthma.     

Management Effects on Bryophytes in Estonian Forests

Bryophyte species richness and composition of six different forest types and three different management rates was analysed in 36 Estonian forests. Analyses were done separately for six different bryophyte species groups: total species number, hepatic, hemerophobic, common, non-epigeic and epigeic species. The differences in species richness between unmanaged and moderately managed forests were significant for all species groups, while the differences between moderately and intensively managed forests were significant only for total species number, and numbers of common and non-epigeic species. Our results showed also that in comparison of unmanaged and moderately managed forests, management effect was significant for species richness of all species groups except non-epigeic species, while in comparison of moderately and intensely managed forests, management rate remained significant only for total species richness and richness of common species. Also the species composition was affected by forest management. Only the compositions of unmanaged forest types were found to be significantly different between each other. In conclusion, even moderate management eliminates a significant number of bryophyte species, and the species composition of different forest types become more similar.     

Headgroup-specific exposure of phospholipids in ABCA1-expressing cells

ABCA1 has been established to be required for the efflux of cholesterol and phospholipids to apolipoproteins such as apoA-I. At present, it is unclear whether ABCA1-mediated lipid exposure is specific with regard to lipid headgroups and whether it requires calcium activation and the presence of a lipid acceptor. In the present work, we found exofacial exposure of endogenous phosphatidylserine in the absence of apoA-I to be enhanced in ABCA1-GFP expressing MDCKII and HeLa cells compared with control cells. By using C6-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-labeled phospholipid analogues, we observed elevated redistribution of phosphatidylserine and phosphatidylethanolamine but not of phosphatidylcholine analogues from the cytoplasmic to the exoplasmic leaflet of the plasma membrane of ABCA1-GFP expressing cells. Whereas glyburide affected neither the level of exofacial endogenous PS nor the outward movement of the amino phospholipid analogues, the latter was sensitive to intracellular Ca2+ in ABCA1-GFP expressing cells, further enhancing outward analogue redistribution with respect to control cells. Both receptor-mediated endocytosis and fluidphase endocytosis were reduced in MDCKII cells expressing ABCA1-GFP. Glyburide raised the level of receptor-mediated endocytosis in the ABCA1-GFP expressing cell to the level of control cells in the absence of glyburide. In control cells, however, fluid-phase endocytosis but not receptor-mediated endocytosis was significantly reduced upon glyburide treatment.     

A circular dichroic study of Cu(II) binding to bovine alpha-lactalbumin.

The visible and ultraviolet circular dichroic spectra resulting from the interaction of bovine alpha-lactalbumin with successive Cu(II) ions have been recorded under a variety of conditions. Analysis of the observed change-transfer and d-d band transitions can be made in terms of two kinds of binding sites: at a histidyl group and at the N-terminal amino group, respectively. At basic pH the amide nitrogens of the peptide backbone progressively take part in the coordination. The occupation of the high affinity calcium binding site by Ca(II) and Mn(II) does not influence the Cu(II) binding process, suggesting that there is no direct interaction between this site and the Cu(II) binding sites.     

Influence of polymer addition on biogenic sulfuric acid attack of concrete

A simple and reproducible microbiological simulation procedure in combination with a chemical procedure was used to test concrete for its potential resistance towards biogenic sulfuric acid. Concerning fundamental aspects of the corrosion reaction, it was shown that particularly the penetration of H₂S inside the concrete crevices accelerated the corrosion process. The influence of different polymer types and silica fume additions on the resistance of the concrete samples was determined. The addition of the styrene acrylic ester polymer resulted in an increased resistance while the addition of the acrylic polymer or silica fume caused less resistant concrete. For the vinylcopolymer and the styrene butadiene polymer, no significant effect was observed on the resistance of the concrete samples. The results of the two different test methods confirmed the difference between corrosion due to purely chemical sulfuric acid and corrosion due to microbiologically produced sulfuric acid.     

A Comparative Study of Early Afterdepolarization-Mediated Fibrillation in Two Mathematical Models for Human Ventricular Cells

Early afterdepolarizations (EADs), which are abnormal oscillations of the membrane potential at the plateau phase of an action potential, are implicated in the development of cardiac arrhythmias like Torsade de Pointes. We carry out extensive numerical simulations of the TP06 and ORd mathematical models for human ventricular cells with EADs. We investigate the different regimes in both these models, namely, the parameter regimes where they exhibit (1) a normal action potential (AP) with no EADs, (2) an AP with EADs, and (3) an AP with EADs that does not go back to the resting potential. We also study the dependence of EADs on the rate of at which we pace a cell, with the specific goal of elucidating EADs that are induced by slow or fast rate pacing. In our simulations in two- and three-dimensional domains, in the presence of EADs, we find the following wave types: (A) waves driven by the fast sodium current and the L-type calcium current (Na-Ca-mediated waves); (B) waves driven only by the L-type calcium current (Ca-mediated waves); (C) phase waves, which are pseudo-travelling waves. Furthermore, we compare the wave patterns of the various wave-types (Na-Ca-mediated, Ca-mediated, and phase waves) in both these models. We find that the two models produce qualitatively similar results in terms of exhibiting Na-Ca-mediated wave patterns that are more chaotic than those for the Ca-mediated and phase waves. However, there are quantitative differences in the wave patterns of each wave type. The Na-Ca-mediated waves in the ORd model show short-lived spirals but the TP06 model does not. The TP06 model supports more Ca-mediated spirals than those in the ORd model, and the TP06 model exhibits more phase-wave patterns than does the ORd model.     

From Affective Experience to Motivated Action: Tracking Reward-Seeking and Punishment-Avoidant Behaviour in Real-Life

Many of the decisions and actions in everyday life result from implicit learning processes. Important to psychopathology are, for example, implicit reward-seeking and punishment-avoidant learning processes. It is known that when specific actions get associated with a rewarding experience, such as positive emotions, that this will increase the likelihood that an organism will engage in similar actions in the future. Similarly, when actions get associated with punishing experiences, such as negative emotions, this may reduce the likelihood that the organism will engage in similar actions in the future. This study examines whether we can observe these implicit processes prospectively in the flow of daily life. If such processes take place then we expect that current behaviour can be predicted by how similar behaviour was experienced (in terms of positive and negative affect) at previous measurement moments. This was examined in a sample of 621 female individuals that had participated in an Experience Sampling data collection. Measures of affect and behaviour were collected at 10 semi-random moments of the day for 5 consecutive days. It was examined whether affective experience that was paired with certain behaviours (physical activity and social context) at previous measurements modified the likelihood to show similar behaviours at next measurement moments. Analyses were performed both at the level of observations (a time scale with units of ± 90 min) and at day level (a time scale with units of 24 h). As expected, we found that affect indeed moderated the extent to which previous behaviour predicted similar behaviour later in time, at both beep- and day-level. This study showed that it is feasible to track reward-seeking and punishment-avoidant behaviour prospectively in humans in the flow of daily life. This opens up a new toolbox to examine processes determining goal-oriented behaviour in relation to psychopathology in humans.     

The Leber Congenital Amaurosis Protein AIPL1 and EB Proteins Co-Localize at the Photoreceptor Cilium

Purpose

The aim of this study was to investigate the interaction and co-localization of novel interacting proteins with the Leber congenital amaurosis (LCA) associated protein aryl hydrocarbon receptor interacting protein-like 1 (AIPL1).

Methods

The CytoTrapXR yeast two-hybrid system was used to screen a bovine retinal cDNA library. A novel interaction between AIPL1 and members of the family of EB proteins was confirmed by directed yeast two-hybrid analysis and co-immunoprecipitation assays. The localization of AIPL1 and the EB proteins in cultured cells and in retinal cryosections was examined by immunofluorescence microscopy and cryo-immunogold electron microscopy.

Results

Yeast two-hybrid (Y2H) analysis identified the interaction between AIPL1 and the EB proteins, EB1 and EB3. EB1 and EB3 were specifically co-immunoprecipitated with AIPL1 from SK-N-SH neuroblastoma cells. In directed 1:1 Y2H analysis, the interaction of EB1 with AIPL1 harbouring the LCA-causing mutations A197P, C239R and W278X was severely compromised. Immunofluorescent confocal microscopy revealed that AIPL1 did not co-localize with endogenous EB1 at the tips of microtubules, endogenous EB1 at the microtubule organising centre following disruption of the microtubule network, or with endogenous β-tubulin. Moreover, AIPL1 did not localize to primary cilia in ARPE-19 cells, whereas EB1 co-localized with the centrosomal marker pericentrin at the base of primary cilia. However, both AIPL1 and the EB proteins, EB1 and EB3, co-localized with centrin-3 in the connecting cilium of photoreceptor cells. Cryo-immunogold electron microscopy confirmed the co-localization of AIPL1 and EB1 in the connecting cilia in human retinal photoreceptors.

Conclusions

AIPL1 and the EB proteins, EB1 and EB3, localize at the connecting cilia of retinal photoreceptor cells, but do not co-localize in the cellular microtubule network or in primary cilia in non-retinal cells. These findings suggest that AIPL1 function in these cells is not related to the role of EB proteins in microtubule dynamics or primary ciliogenesis, but that their association may be related to a specific role in the specialized cilia apparatus of retinal photoreceptors.