Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-μm diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.     

Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

Interaction of intracellular signalling and metabolic pathways at inhibition of mitochondrial aconitase by fluoroacetate

Mitochondrial aconitase has been shown to be inactivated under the effects of many compounds and critical states. Fluoroacetate (FA) is the best-known aconitase-inhibiting toxic agent. The biochemistry of the toxic action of FA has been rather well studied; however, no effective therapy has been developed over the past six decades. To search for new approaches to the development of possible antidotes, experiments were carried out in vitro with rat liver mitochondria, Ehrlich ascite tumor (EAT) cells, and cardiomyocytes exposed to FA or fluorocitrate (FC). FA produced its effects at much higher concentrations as compared with FC; in experiments with mitochondria these effects depended on respiratory substrates: with pyruvate, FA induced a slow oxidation and/or a leak of pyridine nucleotides and inhibition of respiration. Oxidation of pyridine nucleotides (PN) was prevented by the incubation of mitochondria with cyclosporin A. Studies of the PN level and dynamics of Ca²⁺ in EAT cells during activation by ATP also revealed the PN leak from mitochondria, which led to a shift in the balance of mitochondrial and cytosolic NAD(P)H under action of FA. Moreover, an increase of cytosolic Ca²⁺ was revealed in the cells exposed to FA, which could be explained by the activation of plasma membrane calcium channels. This mechanism could affect the amplitude and rate of calcium waves in cardiomyocytes under the effects of FA. We emphasize the reciprocal relationship between intracellular PN dynamics and calcium balance and discuss possible pathways of metabolic modulation in the context of development of effective therapy of poisoning with FA and other aconitase inhibitors.     

Destabilization of the cytosolic calcium level and the death of cardiomyocytes in the presence of derivatives of long-chain fatty acids

By means of fluorescent microscopy, long-chain fatty acid derivatives, myristoylcarnitine and palmitoylcarnitine, were shown to exert the most toxic effect on rat ventricular cardiomyocytes. The addition of 20–50 μM acylcarnitines increased calcium concentration in cytoplasm ([Ca²⁺]_i) and caused cell death after a lag-period of 4–8 min. This effect was independent of extracellular calcium level and Ca²⁺ inhibitors of L-type channels. Free myristic and palmitic acids at concentrations of 300–500 μM had little effect on [Ca²⁺]_i within 30 min. We suggest that the toxic effect is due to the activation of calcium channels of sarcoplasmic reticulum by acylcarnitines and/or arising acyl-CoA. Mitochondria play a role of calcium-buffer system under these conditions. The calcium capacity of the buffer determines the duration of the lag-period. Phosphate increases the calcium capacity of mitochondria and the lag-period. In the presence of rotenone and oligomycin, the elevation of [Ca²⁺]_i after the addition of acylcarnitines occurs without the lag-period. The exhaustion of the mitochondrial calcium-buffer capacity or significant depolarization of mitochondria leads to a rapid release of calcium from mitochondria and cell death. Thus, the activation of reticular calcium channels is the main reason of the toxicity of myristoylcarnitine and palmitoylcarnitine.     

L1 (LINE-1) retrotransposable elements provide a "fossil" record of the phylogenetic history of murid rodents

The single most difficult problem in phylogenetic analysis is deciding whether a shared taxonomic character is due to common ancestry or one that appeared independently due to convergence, parallelism, or reversion to an ancestral state. Mammalian L1 retrotransposons undergo periodic amplifications in which multiple copies of the elements are interspersed in the genome. Because these elements apparently are transmitted only by inheritance and are retained in the genome, a shared L1 amplification event can only be an inherited ancestral character. We propose that L1 amplification events can be an excellent tool for analyzing mammalian evolution and demonstrate here how we addressed several refractory problems in rodent systematics using L1 DNA as a taxonomic character.      

A method for the detection and characterization of GABA(A) receptors by calcium-sensitive fluorescent probes

A method for the detection and characterization of GABA(A) receptors of neurons has been developed, which is based on the measurement of the activity of potential-dependent calcium channels using the fluorescence of the two-wavelength calcium-sensitive probe Fura-2. The method makes it possible to detect the ligands of GABA(A) receptors and determine the constants of activation and inhibition as well as the type of inhibition. The object of investigation was a young (two- to four-day-old) rat hippocampal cell culture in which GABA induces the depolarization and a transient increase in Ca2+ concentration in the cytosol of neurons due to the activation of potential-dependent calcium channels. It was shown that a short-time application of GABA induces a decrease in the amplitude of calcium responses to subsequent addition of the depolarizing agents GABA or KCl. However, at low amplitudes of calcium responses to the addition of GABA, this reducing effect on the subsequent addition of KCl was insignificant. It was found that the amplitudes of calcium responses to KCl and GABA are linearly dependent on the angular coefficient b = 3.41. This enabled one to develop a method of normalizing calcium signals, which makes it possible to compare experiments performed on different days and different cultures. By using this normalization technique, the values of EC50 = 2.21 +/- 0.14 ?M and the Hill coefficient = 1.9 +/- 0.2 were estimated. The blocker of potential-dependent calcium channels nifedipine suppressed simultaneously the amplitudes of calcium responses to the addition of KCl and GABA. In this case, the linear relationship between the amplitudes of calcium responses to the addition of KCl and GABA was retained. To verify the validity of the method, the constant of inhibition of a calcium signal and the type of inhibition for known noncompetitive and competitive antagonists of GABA(A) receptors were determined.     

DNA methylation-associated colonic mucosal immune and defense responses in treatment-na?ve pediatric ulcerative colitis

Inflammatory bowel diseases (IBD) are emerging globally, indicating that environmental factors may be important in their pathogenesis. Colonic mucosal epigenetic changes, such as DNA methylation, can occur in response to the environment and have been implicated in IBD pathology. However, mucosal DNA methylation has not been examined in treatment-naïve patients. We studied DNA methylation in untreated, left sided colonic biopsy specimens using the Infinium HumanMethylation450 BeadChip array. We analyzed 22 control (C) patients, 15 untreated Crohn’s disease (CD) patients, and 9 untreated ulcerative colitis (UC) patients from two cohorts. Samples obtained at the time of clinical remission from two of the treatment-naïve UC patients were also included into the analysis. UC-specific gene expression was interrogated in a subset of adjacent samples (5 C and 5 UC) using the Affymetrix GeneChip PrimeView Human Gene Expression Arrays. Only treatment-naïve UC separated from control. One-hundred-and-twenty genes with significant expression change in UC (> 2-fold, P < 0.05) were associated with differentially methylated regions (DMRs). Epigenetically associated gene expression changes (including gene expression changes in the IFITM1, ITGB2, S100A9, SLPI, SAA1, and STAT3 genes) were linked to colonic mucosal immune and defense responses. These findings underscore the relationship between epigenetic changes and inflammation in pediatric treatment-naïve UC and may have potential etiologic, diagnostic, and therapeutic relevance for IBD.     

“Arginine paradox” in cardiomyocytes of Sprague Dawley and spontaneously hypertensive rats: α<Subscript>2</Subscript>-adrenoreceptor-mediated regulation of L-type Ca<Superscript>2+</Superscript> currents by <Emphasis Type="Italic">L</Emphasis>-arginine

The “arginine paradox” in cardiomyocytes isolated from the left ventricle of Spraque Dawlay (SD) and spontaneously hypertensive rats (SHR) was studied. With 1 mM L-arginine in the bath, the addition of 5 mM L-arginine to incubation medium increased NO production and inhibited amplitude of L-type Ca²⁺ currents in SD cardiomyocytes. A variety of compounds, including the antagonist of α₂-adrenoceptors yohimbine and inhibitors of PI3 kinase (wortmanine), NO synthase (7NI), and cGMP-dependent protein kinase (KT5823), dramatically weakened the inhibitory effects of 5 mM L-arginine on Ca²⁺ currents. The agonist of α₂-adrenoceptors guanabenz acetate increased NO production and inhibited Ca²⁺ currents, while wortmanine, 7NI, and KT5823 antagonized guanabenz. In SHR cardiomyocytes, the “arginine paradox” was not observed: 5 mM L-arginine affected neither NO production nor Ca²⁺ currents. Consistently, guanabenz acetate did not alter NO production and inhibited Ca²⁺ currents to a much smaller extent in SHR cardiomyocytes as compared to SD cardiomyocytes. Taken together, the data of the inhibitory analysis suggest that millimolar L-arginine serves as an agonist of α₂-adrenoceptors, which are coupled to PI3K-Akt pathway as well as downstream NO-cGMP pathway to control activity of L-type Ca²⁺ channels, thus providing new insights into the “arginine paradox” in cardiomyocytes.     

Flora Europaea Notulae Systematicae ad Floram Europaeam spectantes No. 20 Corrigenda et Addenda

Following the publication of the last of the series of Flora Europaea Notulae, No. 20 in the Botanical Journal of the Linnean Society , 76: 297–384 (1978), a number of additions or alterations have been drawn to our attention. These are published in continuation.