Effect of intracellular Ca2+ concentration on endothelin-1 secretion

AbstractThe role of intracellular free Ca2+ concentration ([Ca2+]i in cellular regulation of endothelin-(ET-1) secretion was investigated in cultured porcine aortic endothelial cells of first passage. Intracellular Ca2+ concentration were adjusted between 50 nM and 1 μM using EGTA and thapsigargin, respectively. ET-1 secretion was maximal at [Ca2+]i of 190–470 nM, and reduced at low (50 and 110 nM) and high (470 nM) [Ca2+]i. The Ca2+ ionophores A23187 and ionomycin (each 1 μM), both of which raise [Ca2+]i above 1 μM, also potently inhibited ET-1 secretion under basal and stimulated conditions. The A23187-induced reduction in ET-1 secretion was not affected by NG-nitro-l-arginine (0.1 mM). Our results provide evidence that basal ET-1 secretion is regulated by Ca2+ and that Ca2+ ionophores reduce ET-1 secretion due to the inhibitory effect of high [Ca2+]i

Lifeworld-oriented education with older participants in precarious living situations. Experiences from the participatory project "Caring-Living-Labs Graz: Gut leben im Alter" (Living well in old age)

Menschen, die in prekären Lebenslagen altern und zudem eine Migrationsbiografie haben, sind in organisierten Bildungsveranstaltungen selten anzutreffen. Das Forschungs und Bildungsprojekt „Caring Living Labs Graz” (2022 2024) versucht, sozial benachteiligte Personen bzw. Menschen mit Migrationsgeschichte im dritten und vierten Lebensalter aktiv in die Weiterentwicklung sogenannter „Caring Communities” einzubeziehen. Das sind partizipativ gestaltete Communities im Lebensumfeld der Bürger*innen, die ihre Anliegen berücksichtigen, ein solidarisches Miteinander stärken und Gesundheitskompetenz (Health Literacy) fördern. Zunächst wurden die Bedarfe, Sorgeerfahrungen und Lebenssituationen älterer Menschen mit Migrationsbiografie oder in prekären Lebensverhältnissen in Graz qualitativ erhoben. Parallel dazu starteten erste reflexiv angelegte Aktivitäten zum Empowerment der genannten Gruppen, etwa „Philosophische Erzähl Cafés”, Workshops und konkrete Gestaltungsmaßnahmen in einer Gemeindebau Siedlung. Es folgte das erste Caring Living Lab vor Ort in Form einer partizipativen fünfteiligen Workshopreihe. Zukunftsforen dienen dazu, das gesammelte Wissen strukturell in die kommunale Politik zu bringen. Theoretisch verortet wird das Projekt im Lebensweltkonzept (Alfred Schütz) sowie in der Ethik der kommunalen Sorge, die auf die alltäglichen Lebens und Beziehungszusammenhänge von Menschen fokussiert. (DIPF/Orig.)People who age in precarious living situations and/or have a migration biography are seldom encountered in organized educational programmes. The research and educational project CaringLivingLabs Graz (20222024) attempts to actively include socioeconomically disadvantaged people in the third and fourth stages of life in the development of so-called “caring communities”. These are participatory communities in the citizens’ social environment that take into account their concerns, strengthen solidarity as they live and work together and promote health literacy. First, a qualitative survey is conducted of the needs, experiences and living situations of older people in Graz with a migration biography or in precarious circumstances. Parallel to this, the first activities involving reflection are started to empower this group, for example philosophical Erzählcafés (storytelling cafés), workshops and specific design measures on a social housing estate. The first Living Lab took place on site in the form of a fivepart series of participatory workshops. Future forums serve to bring collective knowledge into the structures of community politics. The theoretical basis for the project is provided by the concept of the lifeworld (Alfred Schütz) and the ethics of communal care, which is focused on the everyday life and relationships of people. (DIPF/Orig.

Nonparametric Bayesian inference for meta-stable conformational dynamics

Analyses of structural dynamics of biomolecules hold great promise to deepen the understanding of and ability to construct complex molecular systems. To this end, both experimental and computational means are available, such as fluorescence quenching experiments or molecular dynamics simulations, respectively. We argue that while seemingly disparate, both fields of study have to deal with the same type of data about the same underlying phenomenon of conformational switching. Two central challenges typically arise in both contexts: (i) the amount of obtained data is large, and (ii) it is often unknown how many distinct molecular states underlie these data. In this study, we build on the established idea of Markov state modeling and propose a generative, Bayesian nonparametric hidden Markov state model that addresses these challenges. Utilizing hierarchical Dirichlet processes, we treat different meta-stable molecule conformations as distinct Markov states, the number of which we then do not have to set a priori. In contrast to existing approaches to both experimental as well as simulation data that are based on the same idea, we leverage a mean-field variational inference approach, enabling scalable inference on large amounts of data. Furthermore, we specify the model also for the important case of angular data, which however proves to be computationally intractable. Addressing this issue, we propose a computationally tractable approximation to the angular model. We demonstrate the method on synthetic ground truth data and apply it to known benchmark problems as well as electrophysiological experimental data from a conformation-switching ion channel to highlight its practical utility

Genetic Diversity of Potassium Ion Channel Proteins Encoded by Chloroviruses That Infect Chlorella heliozoae

Chloroviruses are large, plaque-forming, dsDNA viruses that infect chlorella-like green algae that live in a symbiotic relationship with protists. Chloroviruses have genomes from 290 to 370 kb, and they encode as many as 400 proteins. One interesting feature of chloroviruses is that they encode a potassium ion (K+) channel protein named Kcv. The Kcv protein encoded by SAG chlorovirus ATCV-1 is one of the smallest known functional K+ channel proteins consisting of 82 amino acids. The KcvATCV-1 protein has similarities to the family of two transmembrane domain K+ channel proteins; it consists of two transmembrane -helixes with a pore region in the middle, making it an ideal model for studying K+ channels. To assess their genetic diversity, kcv genes were sequenced from 103 geographically distinct SAG chlorovirus isolates. Of the 103 kcv genes, there were 42 unique DNA sequences that translated into 26 new Kcv channels. The new predicted Kcv proteins differed from KcvATCV-1 by 1 to 55 amino acids. The most conserved region of the Kcv protein was the filter, the turret and the pore helix were fairly well conserved, and the outer and the inner transmembrane domains of the protein were the most variable. Two of the new predicted channels were shown to be functional K+ channels

Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers

Recent studies have shown that membrane proteins can be efficiently synthesized in vitro before spontaneously inserting into soluble nanoscale lipid bilayers called nanodiscs (NDs). In this paper, we present experimental details that allow a combination of in vitro translation of ion channels into commercially available NDs followed by their direct reconstitution from these nanobilayers into standard bilayer setups for electrophysiological characterization. We present data showing that two model K+ channels, Kcv and KcsA, as well as a recently discovered dual-topology F-channel, Fluc, can be reliably reconstituted from different types of NDs into bilayers without contamination from the in vitro translation cocktail. The functional properties of Kcv and KcsA were characterized electrophysiologically and exhibited sensitivity to the lipid composition of the target DPhPC bilayer, suggesting that the channel proteins were fully exposed to the target membrane and were no longer surrounded by the lipid/protein scaffold. The single-channel properties of the three tested channels are compatible with studies from recordings of the same proteins in other expression systems. Altogether, the data show that synthesis of ion channels into NDs and their subsequent reconstitution into conventional bilayers provide a fast and reliable method for functional analysis of ion channels

Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers

Recent studies have shown that membrane proteins can be efficiently synthesized in vitro before spontaneously inserting into soluble nanoscale lipid bilayers called nanodiscs (NDs). In this paper, we present experimental details that allow a combination of in vitro translation of ion channels into commercially available NDs followed by their direct reconstitution from these nanobilayers into standard bilayer setups for electrophysiological characterization. We present data showing that two model K+ channels, Kcv and KcsA, as well as a recently discovered dual-topology F− channel, Fluc, can be reliably reconstituted from different types of NDs into bilayers without contamination from the in vitro translation cocktail. The functional properties of Kcv and KcsA were characterized electrophysiologically and exhibited sensitivity to the lipid composition of the target DPhPC bilayer, suggesting that the channel proteins were fully exposed to the target membrane and were no longer surrounded by the lipid/protein scaffold. The single-channel properties of the three tested channels are compatible with studies from recordings of the same proteins in other expression systems. Altogether, the data show that synthesis of ion channels into NDs and their subsequent reconstitution into conventional bilayers provide a fast and reliable method for functional analysis of ion channels