Hyperosmotic relaxation lysis of chromaffin granules is caused by interactions between the granular membrane and intragranular vesicles

Bovine chromaffin granules undergo irreversible structural changes during osmotic shrinkage in hypertonic sucrose and salt solutions, such that, on reexposure to isoosmotic conditions they do not regain their original morphology, but undergo lysis ('hyperosmotic relaxation lysis'). Irreversible alterations of granules were induced by hypertonic incubations lasting for as little as 1 min. Fluorescence and EPR membrane labelling experiments showed that hypertonicity did not induce membrane loss for instance by inwardly or outwardly directed pinching off of membrane material. The mean sizes of chromaffin granules as a function of increasing and subsequently decreasing osmotic pressure were measured by photon correlation spectroscopy; there was no significant difference in sizes of hyperosmotically pretreated granules as compared with controls. Freeze-fracture electron micrographs showed the formation of 'twins' and 'triplets' under hypertonic conditions. They also revealed intragranular vesicles of 50-200 nm in diameter in both hypertonically and isotonically suspended granules. 'Twin' and 'triplet' granules were formed by the attachment of intragranular vesicles to the granule membranes. We suggest that hyperosmotic relaxation lysis is caused by the fact that this adhesion partly prevents the granule membrane from reexpanding, thus, leading to its rupture.     

Dermal carotenoid measurements via pressure mediated reflection spectroscopy

We describe a reflection‐based method for the quantitative detection of carotenoid antioxidants in living human skin. The skin tissue site of interest is illuminated with broad‐band white light spanning the spectral range from 350–850 nm and the spectral composition of the diffusively reflected light is analyzed in real time. Topical pressure is applied to temporarily squeeze blood out of the illuminated tissue volume. In this way the influence of oxy‐hemoglobin on the reflection spectra is effectively reduced. After a short optical clearing time the carotenoid absorption becomes easily discernable in a 460–500 nm spectral window and its optical density can be calculated with high accuracy. Our empirical methodology provides a non‐invasive rapid determination of skin carotenoid levels, can be used to monitor skin carotenoid concentration changes over time in response to carotenoid containing natural or supplemental diets, and is easily adaptable for applications in clinical and field settings. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Participation of puncta adherentia junctions in the formation of synaptic connections between a dentate fascia graft and the host brain

The fetal dentate fascia of Wistar rats on the 20th day of gestation was heterotopically grafted into the somatosensory neocortex of adult rats. Granule cells of a graft projected their axons (mossy fibers) to the host brain and established synaptic contacts with inappropriate targets. The organization of ectopic mossy fiber synapses was studied by electron microscopy. It was shown that ectopic synapses reproduce the structural determinants of hippocampal giant synapses and induce a subcellular reorganization of postsynaptic neocortex dendrites. Using morphometric analysis, a significant increase was found in the number of discrete puncta adherentia junctions and their total length in ectopic synapses as compared with the control group. The data obtained indicate that puncta adherentia contacts participate in the structural and chemical adaptation of neuronal targets to alien axons growing from transplants.     

A search for Y-chromosomal species-specific markers and their use for hybridization analysis in ground squirrels (Spermophilus: Rodentia, sciuridae)

In four ground squirrel species from the Volga region—yellow (Spermophilus fulvus), russet (S. major), little (S. pygmaeus), and speckled (S. suslicus)—four hybridization variants (major/fulvus, major/pygmaeus, major/suslicus, and pygmaeus/suslicus) have been reliably described. Earlier we have shown that populations of S. major from the Volga region were characterized by wide introgression of mtDNA from S. fulvus and S. pygmaeus, which probably, resulted from ancient hybridization [5]. In this study, the same populations were used to analyze the introgression of the Y chromosome, which (unlike mtDNA) is paternally inherited. Three genes, ZfY, SRY, and SmcY were tested as Y-chromosomal candidate markers. It was demonstrated that Y chromosome of ground squirrels lacked the ZfY gene, while its homologous structure, ZfY(X), was presumably linked to the X chromosome. The SRY region examined was rather conservative. In particular, the sequences determined in S. major and S. fulvus were identical, while three out of four substitutions found in S. pygmaeus were located in the coding region. The SmcY gene was found to be the most suitable marker, providing distinguishing of all of the four ground squirrel species by nine nucleotide substitutions. Introgression at the Y chromosome was observed only in two cases: in one S. major individual (out of 51 phenotypically pure animals) caught in the major/fulvus sympatry zone, and in four (one litter) out of fourteen S. fulvus individuals caught in close vicinity of the sympatry zone of these two species. Among 28 S. pymaeus and 9 S. suslicus individuals, no foreign SmcY genes were detected. Two colonies of the “hybrid swarm” type were examined with eight major/suslicus hybrids analyzed in the first and seventeen major/fulvus hybrids in the second colony. The prevalence of the S. major paternal lineages was observed in both colonies (87.5 and 82.4%, respectively). The data obtained suggest that compared to wide mtDNA introgression, introgression of Y chromosome in the Volga region ground squirrels is statistically significantly less frequent event.     

Lipid and cell membranes in the presence of gadolinium and other ions with high affinity to lipids. 2. A dipole component of the boundary potential on membranes with different surface charge

When Gd3+, a trivalent lanthanide, binds phospholipids with a high affinity, it elicits strong electrostatic effects on the surface of the lipid bilayer. Two experimental methods were applied to monitor the changes in the boundary and surface potentials induced by Gd3+ adsorption on liposomes and planar lipid bilayer membranes (BLM) made from phosphatidylserine (PS), phosphatidylcholine (PC) and their mixtures. The membrane surface charge density was changed by either varying the PS/PC ratio or by changing the degree of PS headgroup ionization in the range of pH between 2.5 and 7.5. The Gouy-Chapman-Stern (GCS) theory combined with the condition of mass balance in the experimental cell was used for quantitative treatment of ion adsorption and related changes in the diffuse part of the electrical double layer (surface potential). Data obtained using microelectrophoresis of liposome suspensions were well described within the framework of the modified GCS theory with constants of 5.10(4) and 10(3) M-1 for Gd3+ association with PS and PC, respectively (Yu. A. Ermakov, A. Z. Averbakh, and S. I. Sukharev, Biol. Membrany 14:434-445 (1997) (in Russian)). The intramembrane field compensation (IFC) technique used to study Gd3+ adsorption on planar lipid bilayers by monitoring the entire boundary potential gave completely different results. An observed drastic difference (approximately 140 mV) between the changes of boundary and surface potential was interpreted as the change in the dipole potential induced by binding of Gd3+. The magnitude of the surface dipole increased with the concentration of PS in PS/PC mixtures and became significant at most negative surface charges (more than 80% of PS in the mixture) and strongly correlated with the degree of PS ionization at different pH. The nature of structural changes at the membrane/water interface induced by Gd(3+)-PS interaction and possible lipid clusterization are discussed in the context of their biological importance.     

Classification of meadows of the South Siberian uplands and mountains

The classification of South Siberian meadows using the Braun-Blanquet approach was carried out on the basis of a geographically wide-ranging data set from the West Siberian Plain and the Altai-Sayanian mountain systems. Two main phytosociological types of meadows were distinguished within the classMolinio-Arrhenatheretea. The natural Siberian forest meadows were included in the orderCarici macrourae-Crepidetalia sibiricae comprising two alliances (Crepidion sibiricae, Aconito barbati-Vicion unijugae) and 7 associations. The anthropogenic types of dry meadows were included in the European-West Siberian orderArrhenatheretalia, comprising one alliance (Festucion pratensis) and 5 associations. DCA ordination revealed clear differences in the floristic composition of the higher units and in the diagnostic importance of the main ecological and phytosociological species groups.     

Preparation and characterization of monoclonal antibodies to human adiponectin for its quantitative measurement in serum

Mouse Monoclonal antibodies against human adiponectin were produced by the routine method and the specificity of antibodies was verified. These monoclonal antibodies (MoAbs) interacted with the monomeric and trimeric forms of recombinant adiponectin according to the results of a Western blot analysis. Human blood serum was fractionated by gel filtration, and the protein of these fractions was stained using labeled MoAbs. It was established that a single high-molecular-weight form (HMW) of endogenous adiponectin was detected by this method. The use of competitive enzymelinked immunoassay on the basis of the obtained MoAbs allowed us to show that the sera of healthy male donors contains lower adiponectin concentrations than that of female donors (8.42 ± 1.59 μg/ml vs. 11.01 ± 2.58 μg/ml, p = 0.01). We also detected statistically significant lower adiponectin levels in the serum of patients with coronary artery disease for both men (6.01 ± 2.73 μg/ml vs. 8.42 ± 1.59 μg/ml, p = 0.015) and women (5.79 ± 2.98 μg/ml vs. 11.01 ± 2.58 μg/ml, p = 0.0003). Therefore, the developed methods for the analysis of the HMW form of adiponectin can be helpful in the diagnostics of the possible implications and assessment of unfavorable prognoses in patients with cardiovascular disorders.     

Plant species richness in continental southern Siberia: effects of pH and climate in the context of the species pool hypothesis

Aim Many high‐latitude floras contain more calcicole than calcifuge vascular plant species. The species pool hypothesis explains this pattern through an historical abundance of high‐pH soils in the Pleistocene and an associated opportunity for the evolutionary accumulation of calcicoles. To obtain insights into the history of calcicole/calcifuge patterns, we studied species richness–pH–climate relationships across a climatic gradient, which included cool and dry landscapes resembling the Pleistocene environments of northern Eurasia. Location Western Sayan Mountains, southern Siberia. Methods Vegetation and environmental variables were sampled at steppe, forest and tundra sites varying in climate and soil pH, which ranged from 3.7 to 8.6. Species richness was related to pH and other variables using linear models and regression trees. Results Species richness is higher in areas with warmer winters and at medium altitudes that are warmer than the mountains and wetter than the lowlands. In treeless vegetation, the species richness–pH relationship is unimodal. In tundra vegetation, which occurs on low‐pH soils, richness increases with pH, but it decreases in steppes, which have high‐pH soils. In forests, where soils are more acidic than in the open landscape, the species richness–pH relationship is monotonic positive. Most species occur on soils with a pH of 6–7. Main conclusions Soil pH in continental southern Siberia is strongly negatively correlated with precipitation, and species richness is determined by the opposite effects of these two variables. Species richness increases with pH until the soil is very dry. In dry soils, pH is high but species richness decreases due to drought stress. Thus, the species richness–pH relationship is unimodal in treeless vegetation. Trees do not grow on the driest soils, which results in a positive species richness–pH relationship in forests. If modern species richness resulted mainly from the species pool effects, it would suggest that historically common habitats had moderate precipitation and slightly acidic to neutral soils.     

Thermodynamic Characterization of ppGpp Binding to EF-G or IF2 and of Initiator tRNA Binding to Free IF2 in the Presence of GDP, GTP, or ppGpp

In addition to their natural substrates GDP and GTP, the bacterial translational GTPases initiation factor (IF) 2 and elongation factor G (EF-G) interact with the alarmone molecule guanosine tetraphosphate (ppGpp), which leads to GTPase inhibition. We have used isothermal titration calorimetry to determine the affinities of ppGpp for IF2 and EF-G at a temperature interval of 5-25 °C. We find that ppGpp has a higher affinity for IF2 than for EF-G (1.7-2.8 μM K_dversus 9.1-13.9 μM K_d at 10-25 °C), suggesting that during stringent response in vivo, IF2 is more responsive to ppGpp than to EF-G. We investigated the effects of ppGpp, GDP, and GTP on IF2 interactions with fMet-tRNA^fMet demonstrating that IF2 binds to initiator tRNA with submicromolar K_d and that affinity is altered by the G nucleotides only slightly. This—in conjunction with earlier reports on IF2 interactions with fMet-tRNA^fMet in the context of the 30S initiation complex, where ppGpp was suggested to strongly inhibit fMet-tRNA^fMet binding and GTP was suggested to strongly promote fMet-tRNA^fMet binding—sheds new light on the mechanisms of the G-nucleotide-regulated fMet-tRNA^fMet selection.