Quantification of mitochondrial morphology in situ

The structural organization of mitochondria reflects their functional status and largely is an index of cell viability. The indirect parameter to assess the functional state of mitochondria in cells is the degree of their fragmentation, i.e., the ratio of long or branched mitochondrial structures to round mitochondria. Such evaluations requires an approach that allows to create an integral pattern of the three-dimensional organization of mitochondrial reticulum using confocal images of mitochondria stained with a fluorescent probe. In the present study, we tested three approaches to analyzing the structural architecture of mitochondria under normal conditions and fission induced by oxidative stress. We revealed that, while the most informative is a three-dimensional reconstruction based on series of confocal images taken along the Z-dimension, with some restrictions it is plausible to use more simple algorithms of analysis, including one that uses unitary twodimensional images. Further improvement of these methods of image analysis will allow more comprehensive study of mitochondrial architecture under normal conditions and different pathological states. It may also provide quantification of a number of mitochondrial parameters determining the morphofunctional state of mitochondria—primarily, their absolute and relative volumes—and give additional information on threedimensional organization of the mitochondrion.     

Optical clearing for improved contrast in second harmonic generation imaging of skeletal muscle

Using second harmonic generation (SHG) imaging microscopy, we have examined the effect of optical clearing with glycerol to achieve greater penetration into specimens of skeletal muscle tissue. We find that treatment with 50% glycerol results in a 2.5-fold increase in achievable SHG imaging depth. Signal processing analyses using fast Fourier transform and continuous wavelet transforms show quantitatively that the periodicity of the sarcomere structure is unaltered by the clearing process and that image quality deep in the tissue is improved with clearing. Comparison of the SHG angular polarization dependence also shows no change in the supramolecular organization of acto-myosin complexes. By contrast, identical treatment of mouse tendon (collagen based) resulted in a strong decrease in SHG response. We suggest that the primary mechanism of optical clearing in muscle with glycerol treatment results from the reduction of cytoplasmic protein concentration and concomitant decrease in the secondary inner filter effect on the SHG signal. The lack of glycerol concentration dependence on the imaging depth indicates that refractive index matching plays only a minor role in the optical clearing of muscle. SHG and optical clearing may provide an ideal mechanism to study physiology in highly scattering skeletal or cardiac muscle tissue with significantly improved depth of penetration and achievable imaging depth.     

The effect of Furolan on the physiological and biochemical characteristics of ripening winter wheat grain

The effects of the preparation Furolan, (2-furyl-2)-1,3-dioxolane, on the degree of mRNA polyadenylation and the pattern of protein synthesis in the ripening grain of several soft winter wheat (Triticum aestivum L.) cultivars were studied. It was demonstrated that Furolan stabilized mRNA in a cultivar-specific manner, thereby accelerating to various degrees the biochemical processes taking place in the ripening grain. Of the wheat cultivars studied, Krasnodarskaya 99 was the most responsive cultivar with respect to a set of changes in nucleic-protein metabolism; the cultivar Deya was next followed by the cultivar Bat’ko. The cultivar Kroshka did not respond to the treatment with Furolan. The cultivar specificity of this preparation allows its practical application to be optimized.     

The symbiotic microflora associated with the tegument of proteocephalidean cestodes and the intestines of their fish hosts

The indigenous symbiotic microflora associated with the tegument of proteocephalidean cestodes and the intestines of their fish hosts has been investigated in morphological and ecological aspects. The indigenous microflora associated with the cestode tegument consists of the nannobacteria population, which was present obligatorily on the surface of tegument, and the "deep microflora". The deep microflora associates with some few species of parasites only. Each individual host-parasite micro-biocenosis includes specific indigenous symbiotic microorganisms, with the differing microfloras of host intestine and parasite. Physiology, biochemistry and/or diet of hosts apparently influence on the symbiotic microflora's structure of parasites. The least bacteria abundance and diversity of their morphotypes were observed in the parasites from baby fishes. The diversity and abundance of bacteria were increased with the fish host ageing and the formation of the definitive structure of its intestine. It is an evidence of the gradual invading of the intestinal parasites (cestodes) tegument by bacterial cells. The invading is realized on the base of the microflora that was present in the food of fish host. The symbiotic microflora has specific morphological features, can regulate the homeostasis of the cestodes and fish hosts and also can maintain equilibrium of alimentary and immune interrelations in the host-parasite system.     

Mitochondria-targeted antioxidant SkQR1 ameliorates gentamycin-induced renal failure and hearing loss

The influence of the mitochondria-targeted antioxidant SkQR1 on gentamycin-induced nephrotoxicity and ototoxicity has been analyzed. SkQR1 reduces the death of kidney epithelium cells and decreases the severity of renal failure caused by gentamycin application and also lowers the animals’ mortality. Treatment with SkQR1 also decreases gentamycininduced hearing loss. Mitochondria-targeted antioxidants, such as SkQR1, are new promising agents for preventing negative consequences of therapy with antibiotics.     

N-terminally glutamate-substituted analogue of gramicidin a as protonophore and selective mitochondrial uncoupler

Limited uncoupling of oxidative phosphorylation could be beneficial for cells by preventing excessive generation of reactive oxygen species. Typical uncouplers are weak organic acids capable of permeating across membranes with a narrow gap between efficacy and toxicity. Aimed at designing a nontoxic uncoupler, the protonatable amino acid residue Glu was substituted for Val at the N-terminus of the pentadecapeptide gramicidin A (gA). The modified peptide [Glu1]gA exhibited high uncoupling activity in isolated mitochondria, in particular, abolishing membrane potential at the inner mitochondrial membrane with the same or even larger efficacy as gA. With mitochondria in cell culture, the depolarizing activity of [Glu1]gA was observed at concentrations by an order of magnitude lower than those of gA. On the contrary, [Glu1]gA was much less potent in forming proton channels in planar lipid bilayers than gA. Remarkably, at uncoupling concentrations, [Glu1]gA did not alter cell morphology and was nontoxic in MTT test, in contrast to gA showing high toxicity. The difference in the behavior of [Glu1]gA and gA in natural and artificial membranes could be ascribed to increased capability of [Glu1]gA to permeate through membranes and/or redistribute between different membranes. Based on the protective role of mild uncoupling, [Glu1]gA and some other proton-conducting gA analogues may be considered as prototypes of prospective therapeutic agents.     

Histone-antihistone interactions in the Escherichia coli-Tetrahymena pyriformis association

Using the Escherichia coli-Tetrahymena pyriformis system, we revealed the involvement of bacterial antihistone activity and protozoan histones in interactions between pro- and eukaryotic microorganisms. Antihistone activity enhanced the viability of E. coli in association with T. pyriformis, according to our data on the dynamics of E. coli cell numbers. The strain with antihistone activity induced incomplete phagocytosis in the infusorians, resulting in cytological changes and ultrastructural alterations that indicated the retention of bacterial cells in phagosomes. Bacteria with antihistone activity located in the T. pyriformis cytoplasm influenced the eukaryotic nucleus. This manifested itself in macronucleus decompactization and a decrease in the average histone content in the population of infusorians. The data obtained suggest that protozoan histone inactivation by bacteria is one of the mechanisms involved in prokaryote persistence in associations with eukaryotic microorganisms.     

Mitochondrial free radical production induced by glucose deprivation in cerebellar granule neurons

Using a fluorescent probe for superoxide, hydroethidine, we have demonstrated that glucose deprivation (GD) activates production of reactive oxygen species (ROS) in cultured cerebellar granule neurons. ROS production was insensitive to the blockade of ionotropic glutamate channels by MK-801 (10 microM) and NBQX (10 microM). Inhibitors of mitochondrial electron transport, i.e. rotenone (complex I), antimycin A (complex III), or sodium azide (complex IV), an inhibitor of mitochondrial ATP synthase--oligomycin, an uncoupler of oxidative phosphorylation--CCCP, a chelator of intracellular Ca2+--BAPTA, an inhibitor of electrogenic mitochondrial Ca2+ transport--ruthenium red, as well as pyruvate significantly decreased neuronal ROS production induced by GD. GD was accompanied by a progressive decrease in the mitochondrial membrane potential and an increase in free cytosolic calcium ions, [Ca2+](i). Pyruvate, BAPTA, and ruthenium red lowered the GD-induced calcium overload, while pyruvate and ruthenium red also prevented mitochondrial membrane potential changes induced by GD. We conclude that GD-induced ROS production in neurons is related to potential-dependent mitochondrial Ca2+ overload. GD-induced mitochondrial Ca2+ overload in neurons in combination with depletion of energy substrates may result in the decrease of the membrane potential in these organelles.     

Morphological and ultrastructural characteristics of symbiotic bacteria colonizing the surface of the helminth Triaenophorus nodulosus and the intestine of pike Esox lucius

The microflora inhabiting the intestinal mucosa of the pike and the tegument surface of the intestinal parasite Triaenophorus nodulosus is investigated. By means of electron microscopy, the ultrastructural features of bacteria and pattern of their interaction with the colonized surfaces are estimated. Specific distribution of microorganisms belonging to different morphotypes and subdivided into subpopulations on the colonized surfaces is found. Predominance of gram-negative bacteria in all studied microbiocenoses and abundance of nanobacteria in the “superficial” subpopulations are observed. The bacteria of “a deep population” associated with the basal parts of microtrichiae and microvilli are described. It is concluded that the helminthes, the fish parasites possess the normal microflora characterized by specific composition, by ultrastructural features of cells, and by patterns of interaction with the helminth surface. The obtained data characterize the mechanisms of symbiotic interaction of prokaryotes and eukaryotes in the system parasite-host-symbiotic microflora.     

Moderate alcohol consumption is associated with better endothelial function: a cross sectional study

Background

Natural heterologous valved conduits with a diameter greater than 22 mm that can be used for right ventricular outflow tract reconstruction in adults are not commercially available. The purpose of this study was to measure by ultrasonography the maximum diameter of the distended jugular veins of horses and cattle, respectively, to identify a population of animals that would be suitable for post-mortem collection of jugular veins at sizes greater than 22 mm.

Methods

The study population included 60 Warmblood horses, 25 Freiberger horses, 20 Brown Swiss cows, and 20 Holstein cows (including 10 Holstein and 10 Red Holstein). The maximum cross-sectional diameter of the distended jugular veins was measured at a location half-way between the mandibular angle and the thoracic inlet. The thoracic circumference (heart girth length) was used as a surrogate of body size. The jugular vein diameters of the different populations were compared by analysis of variance and the association between heart girth length and jugular vein diameter was determined in each of the four study populations by linear regression analysis.

Results

There was considerable individual variation of jugular vein diameters within each of the four study populations. There was no statistically significant relationship between thoracic circumference and jugular vein diameter in any of the populations. The jugular vein diameters of Brown Swiss cows were significantly larger than those of any of the other populations. Warmblood horses had significantly larger jugular vein diameters compared to Freiberger horses.

Conclusion

The results of this study suggest that the production of bovine or equine xenografts with diameters of greater than 22 mm would be feasible. Differences between species and breeds need to be considered. However, prediction of the jugular vein diameter based on breed and heart girth length in an individual animal is inaccurate.