Studies of RyR function in situ

The ryanodine receptors (RyRs) are intracellular Ca2+ release channels of the sarcoplasmic reticulum (SR) involved in many cellular responses, including muscle excitation-contraction coupling. Multiple biochemical and biophysical methods are available to study RyR functions. However, most of them are somewhat limited because they can only be used to examine channels which are purified from the SR and no longer in their natural environment. In this review we discuss optical methods for studying RyR functions in situ. We describe several techniques for the investigation of local (microscopic) intracellular Ca2+ signals (a.k.a Ca2+ sparks) by means of confocal microscopy and flash photolysis of caged compounds. We discuss how these studies can and will continue to contribute to our understanding of RyR function in physiological and pathological conditions.     

Biogeochemistry of organic carbon, CO2, CH4, and trace elements in thermokarst water bodies in discontinuous permafrost zones of Western Siberia

Active processes of permafrost thaw in Western Siberia increase the number of soil subsidencies, thermokarst lakes and thaw ponds. In continuous permafrost zones, this process promotes soil carbon mobilisation to water reservoirs, as well as organic matter (OM) biodegradation, which produces a permanent flux of carbon dioxide (CO₂) to the atmosphere. At the same time, the biogeochemical evolution of aquatic ecosystems situated in the transition zone between continuous permafrost and permafrost-free terrain remains poorly known. In order to better understand the biogeochemical processes that occur in thaw ponds and lakes located in discontinuous permafrost zones, we studied ~30 small (1–100,000 m²) shallow (<1 m depth) lakes and ponds formed as a result of permafrost subsidence and thaw of the palsa bog located in the transition zone between the tundra and forest-tundra (central part of Western Siberia). There is a significant increase in dissolved CO₂ and methane (CH₄) concentration with decreasing water body surface area, with the largest supersaturation with respect to atmospheric CO₂ and CH₄ in small (<100 m²) permafrost depressions filled with thaw water. Dissolved organic carbon (DOC), conductivity, and metal concentrations also progressively increase from large lakes to thaw ponds and depressions. As such, small water bodies with surface areas of 1–100 m² that are not accounted for in the existing lake and pond databases may significantly contribute to CO₂ and CH₄ fluxes to the atmosphere, as well as to the stocks of dissolved trace elements and organic carbon. In situ lake water incubation experiments yielded negligible primary productivity but significant oxygen consumption linked to the mineralisation rate of dissolved OM by heterotrophic bacterioplankton, which produce a net CO₂ flux to the atmosphere of 5 ± 2.5 mol C m² year^?1. The most significant result of this study, which has long-term consequences on our prediction of aquatic ecosystem development in the course of permafrost degradation is CO₂, CH₄, and DOC concentrations increase with decreasing lake age and size. As a consequence, upon future permafrost thaw, the increase in the number of small water bodies, accompanied by the drainage of large thermokarst lakes to the hydrological network, will likely favour (i) the increase of DOC and colloidal metal stocks in surface aquatic systems, and (ii) the enhancement of CO₂ and CH₄ fluxes from the water surface to the atmosphere. According to a conservative estimation that considers that the total area occupied by water bodies in Western Siberia will not change, this increase in stocks and fluxes could be as high as a factor of ten.     

Pathways of abnormal stress-induced Ca influx into dystrophic mdx cardiomyocytes

In Duchenne muscular dystrophy, deficiency of the cytoskeletal protein dystrophin leads to well-described defects in skeletal muscle, but also to dilated cardiomyopathy, accounting for about 20% of the mortality. Mechanisms leading to cardiomyocyte cell death and cardiomyopathy are not well understood. One hypothesis suggests that the lack of dystrophin leads to membrane instability during mechanical stress and to activation of Ca²⁺ entry pathways. Using cardiomyocytes isolated from dystrophic mdx mice we dissected the contribution of various putative Ca²⁺ influx pathways with pharmacological tools. Cytosolic Ca²⁺ and Na⁺ signals as well as uptake of membrane impermeant compounds were monitored with fluorescent indicators using confocal microscopy and photometry. Membrane stress was applied as moderate osmotic challenges while membrane current was quantified using the whole-cell patch-clamp technique. Our findings suggest a major contribution of two primary Ca²⁺ influx pathways, stretch-activated membrane channels and short-lived microruptures. Furthermore, we found evidence for a secondary Ca²⁺ influx pathway, the Na⁺–Ca²⁺ exchange (NCX), which in cardiac muscle has a large transport capacity. After stress it contributes to Ca²⁺ entry in exchange for Na⁺ which had previously entered via primary stress-induced pathways, representing a previously not recognized mechanism contributing to subsequent cellular damage. This complexity needs to be considered when targeting abnormal Ca²⁺ influx as a treatment option for dystrophy.     

Potassium and sodium balance in U937 cells during apoptosis with and without cell shrinkage.

Staurosporine (STS) and etoposide (Eto) induced apoptosis of the human histiocytic lymphoma cells U937 were studied to determine the role of monovalent ions in apoptotic cell shrinkage. Cell shrinkage, defined as cell dehydration, was assayed by measurement of buoyant density of cells in continuous Percoll gradient. The K+ and Na+ content in cells of different density fractions was estimated by flame emission analysis. Apoptosis was evaluated by confocal microscopy and flow cytometry of acridine orange stained cells, by flow DNA cytometry and by effector caspase activity. Apoptosis of U937 cells induced by 1 muM STS for 4 h was found to be paralleled by an increase in buoyant density indicating cell shrinkage. An increase in density was accompanied by a decrease in K+ content (from 1.1 to 0.78 mmol/g protein), which exceeded the increase in Na+ content (from 0.30 to 0.34 mmol/g) and resulted in a significant decrease of the total K+ and Na+ content (from 1.4 to 1.1 mmol/g). In contrast to STS, 50 microM Eto for 4 h or 0.8-8 microM Eto for 18-24 h induced apoptosis without triggering cell shrinkage. During apoptosis of U937 cells induced by Eto the intracellular K(+)/Na+ ratio decreased like in the cells treated with STS, but the total K+ and Na+ content remained virtually the same due to a decrease in K+ content being nearly the same as an increase in Na+ content. Apoptotic cell dehydration correlated with the shift of the total cellular K+ and Na+ content. There was no statistically significant decrease in K+ concentration per cell water during apoptosis induced by either Eto (by 13.5%) or STS (by 8%), whereas increase in Na+ concentration per cell water was statistically significant (by 27% and 47%, respectively). The data show that apoptosis can occur without cell shrinkage-dehydration, that apoptosis with shrinkage is mostly due to a decrease in cellular K+ content, and that this decrease is not accompanied by a significant decrease of K+ concentration in cell water.     

The Effect of Acute Hiperthermia on Levels of Hsp70 and Lipid Peroxidation in Laboratory-Cultured Pulmonate Mollusk <Emphasis Type="Italic">Stagnicola corvus</Emphasis>

This study evaluates dynamic changes in levels of Hsp70s and products of lipid peroxidation due to hyperthermia in the laboratory-raised freshwater pulmonate mollusk Stagnicola corvus (Gmelin, 1791). It is concluded that acute hyperthermia in S. corvus leads to the activation of nonspecific mechanisms of stress adaptation, which is manifested in an elevated Hsp70s level in the beginning of exposure, followed by a reduction in protein concentration, and the further preservation of the Hsp70 at a level exceeding the constitutive forms. At the same time, the level of LPO products tends to decrease, which indicates strengthening of the antioxidant defense processes in response to stress.     

Variation of Otolith Structure in Amur Sleeper Perccottus glenii (Odontobutidae) Populations from Central Russia

Journal of Ichthyology - Development of annual increments on the otoliths (sagittae) of Amur sleeper Perccottus glenii, as well as the sagitta shape and bilateral asymmetry, is analyzed in the...     

Structural determinants of odorant recognition by the human olfactory receptors OR1A1 and OR1A2

An interaction of odorants with olfactory receptors is thought to be the initial step in odorant detection. However, ligands have been reported for only 6 out of 380 human olfactory receptors, with their structural determinants of odorant recognition just beginning to emerge. Guided by the notion that amino acid positions that interact with specific odorants would be conserved in orthologs, but variable in paralogs, and based on the prediction of a set of 22 of such amino acid positions, we have combined site-directed mutagenesis, rhodopsin-based homology modelling, and functional expression in HeLa/Olf cells of receptors OR1A1 and OR1A2. We found that (i) their odorant profiles are centred around citronellic terpenoid structures, (ii) two evolutionary conserved amino acid residues in transmembrane domain 3 are necessary for the responsiveness of OR1A1 and the mouse ortholog Olfr43 to (S)-(-)-citronellol, (iii) changes at these two positions are sufficient to account for the differential (S)-(-)-citronellol responsiveness of the paralogs OR1A1 and OR1A2, and (iv) the interaction sites for (S)-(-)-citronellal and (S)-(-)-citronellol differ in both human receptors. Our results show that the orientation of odorants within a homology modelling-derived binding pocket of olfactory receptor orthologs is defined by evolutionary conserved amino acid positions.     

Effect of cycloheximide and actinomycin D on the phospholipid and neutral lipid content of Cunninghamella japonica

Changes in the composition of neutral lipids and phospholipids were studied during the growth of Cunninghamella japonica under the action of cycloheximide and actinomycin D. The data were used to discuss how the synthesis of enzymes catalysing the formation of individual lipid classes was regulated and whether it would be possible to control the composition of phospholipids and neutral lipids using inhibitors of RNA and protein synthesis. The results are also indicative of a certain correlation between growth phases of the fungus and changes in certain characteristics of membrane lipids.