Evolution of mechanisms of Ca<Superscript>2+</Superscript>-signaling: Role of calcium ions in signal transduction in prokaryotes

The review considers current concepts of mechanisms of Ca²⁺-signaling in cells of prokaryotes. Problems of structural-functional organization of the Ca²⁺-messenger system, mechanisms of membrane transport, and Ca²⁺ homeostasis are discussed. A special attention is paid to analysis of role of calcium ions and Ca²⁺-receptor proteins in regulation of intracellular processes in bacteria.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 1, 2005, pp. 12–17.Original Russian Text Copyright © 2005 by Shemarova, Nesterov.     

T-channels and Na<Superscript>+</Superscript>,Ca<Superscript>2+</Superscript>-exchangers as components of the Ca<Superscript>2+</Superscript>-system of regulation of activity of the heart myocardium of the frog <Emphasis Type="Italic">Rana temporaria</Emphasis>

Earlier we have shown that regulation of rhythm and strength of the frog heart contractions, mediated by transmitters of the autonomic nervous system, is of the Ca²⁺-dependent character. In the present work, we studied chronoand inotropic effect of verapamil—an inhibitor of Ca²⁺-channels of the L-type, of nickel chloride-an inhibitor of Ca²⁺—channels of the T-type and of Na⁺,Ca²⁺exchangers as well as of adrenaline and acetylcholine (ACh) after nickel chloride. It has been found that the intracardially administered NiCh₂ at a dose of 0.01 μg/kg produced a sharp fall of amplitude of action potential (AP) and an almost twofold deceleration of heart rate (HR). The intracardiac administration of NiCh₂ (0.01 μg/kg) on the background of action of verapamil (6 mg/kg, i/m) led as soon as after 3 min to even more prominent HR deceleration and to further fall of the AP amplitude by more than 50% as compared with norm. An intracardiac administration of adrenaline (0.5 mg/kg) partly restored the cardiac activity. However, preservation of the myocardium electrical activity in such animals was brief and its duration did not exceed several minutes. Administration of Ni²⁺ on the background of acetylcholine (3.6 mg/kg) led to almost complete cessation of cardiac activity. As soon as 3 min after injection of this agent the HR decreased to 2 contractions/min. On electrograms (EG), the 10-fold fall of the AP amplitude was recorded. To elucidate role of extraand intracellular Ca²⁺ in regulation of strength of heart contractions, isometric contraction of myocardium preparations was studied in response to action of NiCl₂ (10–200 μM), verapamil (70 μM), adrenaline (5 μM), and acetylcholine (0.2 μM) after NiCl₂. It has been found that Ni²⁺ causes a dose-dependent increase of the muscle contraction amplitude. Minimal change of the contraction amplitude (on average, by 14.9% as compared with control) was recorded at a Ni²⁺ concentration of 100 μM. An increase of Ni²⁺ in the sample to 200 μM increased the cardiac contraction strength, on average, by 41%. The negative inotropic action of verapamil was essentially reduced by 100 μM Ni²⁺. Adrenaline added to the sample after Ni²⁺ produced stimulating effect on the cardiac muscle, with an almost twofold rise of the contraction amplitude. ACh (0.2 μM) decreased the cardiac contraction amplitude, on average, by 56.3%, whereas Ni²⁺ (200 μM) administered after ACh not only restored, but also stimulated partly the myocardial work. Within several parts of percent there was an increase of such isometric contraction parameters as amplitude of the effort developed by muscle, maximal rate, maximal acceleration, time of semirise and semifall. The obtained experimental results indicate that the functional activity of the frog pacemaker and contractile cardiomyocytes is regulated by Ca²⁺-dependent mechanisms. Structure of these mechanisms includes the potential-controlled Land T-channels of the plasma membrane as well as Na⁺,Ca²-exchangers characteristic exclusively of contractile cardiomyocytes. The existence of these differences seems to be due to the cardiomyocyte morphological peculiarities that appeared in evolution at the stage of the functional cell specialization.     

Antibody fingerprints in lyme disease deciphered with high density peptide arrays

Lyme disease is the most common tick‐borne infectious disease in Europe and North America. Previous studies discovered the immunogenic role of a surface‐exposed lipoprotein (VlsE) of Borreliella burgdorferi. We employed high density peptide arrays to investigate the antibody response to the VlsE protein in VlsE‐positive patients by mapping the protein as overlapping peptides and subsequent in‐depth epitope substitution analyses. These investigations led to the identification of antibody fingerprints represented by a number of key residues that are indispensable for the binding of the respective antibody. This approach allows us to compare the antibody specificities of different patients to the resolution of single amino acids. Our study revealed that the sera of VlsE‐positive patients recognize different epitopes on the protein. Remarkably, in those cases where the same epitope is targeted, the antibody fingerprint is almost identical. Furthermore, we could correlate two fingerprints with human autoantigens and an Epstein‐Barr virus epitope; yet, the link to autoimmune disorders seems unlikely and must be investigated in further studies. The other three fingerprints are much more specific for B. burgdorferi. Since antibody fingerprints of longer sequences have proven to be highly disease specific, our findings suggest that the fingerprints could function as diagnostic markers that can reduce false positive test results.     

The immunofluorescent detection of phosphorylated receptors of the epidermal growth factor during their internalization in A-431 cells]

Phosphorylated receptors of the epidermal growth factor (EGF) were localized in the human epidermoid carcinoma cells using immunofluorescent staining with antibody to phosphotyrosine. The application of EGF at 4 degrees C was seen to induce a characteristic fluorescence of the cell margins, whereas no cell staining occurs in the absence of EGF. After a 1 hour incubation of cells at 37 degrees C, within which the internalized EGF receptor complexes are accumulated in the juxtanuclear compartment near the para-Golgi region, the staining with antiphosphotyrosine antibody reveals the receptors in this region. It is concluded that the internalized EGF-receptor complexes may remain in the phosphorylated state.     

Structural characteristics of the mediastinal pleura and its blood vessels

By means of impregnation of the microcirculatory bed vessels after V. V. Kupriyanov and transmissive electron microscopy, topological picture of hemomicrovessels has been estimated in the glomerular constructions of the dog mediastinal pleura. The composition of cell elements and peculiarities in distribution of the latter in the zone of the milk spots are examined. Their structural characteristics are manifested as presence of mesothelium of a decomplexated type accumulation of macrophagal and lymphocyto-plasma cells, glomerular organization of the capillary network and postcapillary venules, two types of endothelial lining of the metabolic microvessels. The role of the milk spots in ensuring immune protection, in migration of macrophages and in filtration of the pleural liquid is discussed.     

Mersalyl prevents the Tl+-induced permeability transition pore opening in the inner membrane of Ca2+-loaded rat liver mitochondria

It was earlier shown that the calcium load of rat liver mitochondria in medium containing TlNO₃ and KNO₃ resulted in the Tl⁺-induced mitochondrial permeability transition pore (MPTP) opening in the inner membrane. This opening was accompanied by an increase in swelling and membrane potential dissipation and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration. This respiratory decrease was markedly leveled by mersalyl (MSL), the phosphate symporter (PiC) inhibitor which poorly stimulated the calcium-induced swelling, but further increased the potential dissipation. All of these effects of Ca²⁺ and MSL were visibly reduced in the presence of the MPTP inhibitors (ADP, N-ethylmaleimide, and cyclosporine A). High MSL concentrations attenuated the ability of ADP to inhibit the MPTP. Our data suggest that the PiC can participate in the Tl⁺-induced MPTP opening in the inner membrane of Ca²⁺-loaded rat liver mitochondria.     

Biochemical conditionality of differentiation of halophytes by the type of regulation of salt metabolism in Prieltonye

The elemental composition and the content of pigments, proteins, lipids, free amino acids, and antioxidants of five wild halophyte species in Prieltonye were investigated. Plants differed in systematic location (Chenopodiaceae, Plumbaginaceae, Asteraceae), the type of regulation of salt metabolism (eu-, cryno, and glycohalophytes), life form (annual grasses, shrubs), and the water regime (mesoxerophytes, xeromesophytes). A decrease in the ion content of K, Na, Ca among Suaeda linifolia > Salicornia perennans > Halocnemum strobilaceum > Limonium gmelinii > Artemisia santonica was noted. The reversed pattern was observed for the content of C. The increase in the total content of C in glyco-, cryno-, and euhalophytes was accompanied by an increased content of total and membrane lipids, proteins, and pigments. Halophytes varied considerably in terms of components of the antioxidant system—the content of endogenous proline, soluble protein, and lipid peroxidation and the level of total SOD activity. Cluster analysis revealed that the differentiation of the studied halophyte species by the type of regulation of salt metabolism was mostly determined by biochemical parameters.     

Anisakiasis in the Caspian seal

On the basis of 548 helminthological dissections the main foci of Anisakis infection of Caspian seal and the character of pathomorphological process in the stomach infected with the nematode Anisakis schupakovi have been established.     

Effect of dansylcadaverine on the epidermal growth factor-stimulated phosphorylation of uridine

The increase of uridine phosphorylation during the first hour after epidermal growth factor (EGF) stimulation (1.25 nM) of Swiss 3T3 cells is completely blocked by 100 microM dansylcadaverine (DC). Lack of the effect of DC on uridine transport, uridine kinase activity in cell homogenate, intracellular ATP concentration and plasma membrane permeability for phosphorylated uridine derivatives makes it possible to propose the inhibition by DC (100 microM) of the activated state of uridine kinase. The rapidity of the inhibition of EGF effect and the lack of influence of DC (in tested concentration) upon the clustering of EGF-receptor complexes, rate of their internalization (Sorkin, 1985; Nikol'ski? et al., 1987) and pH value of intracellular compartments (Sorkin et al., 1985; Teslenko et al., 1986) may suggest an association of DC inhibitory action with blocking of some steps of the receptor mediated endocytosis. Accumulation of DC in cell membranes, rather than in intracellular compartments with acidic pH, is a necessary factor for its blocking effect. Possibilities of DC action through the influence on calmodulin-dependent proteins or EGF-induced cell protein phosphorylation are discussed.