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.     

Comparative analysis of the localization and mobility of retrotransposons in sibling species Drosophila simulans and Drosophila melanogaster]

The distribution of four retrotransposon families (MDG1, MDG3, MDG4 and copia) on polytene chromosomes of different (from 9 to 15) Drosophila simulans strains is studied. The mean number of MDG1 and copia euchromatic hybridization sites (3 sites for each element) is drastically decreased in D. simulans in comparison with D. melanogaster (24 and 18 sites respectively). The mean number of MDG3 sites of hybridization is 5 in D. simulans against 12 in D. melanogaster. As for MDG4 both species have on the average about 2-3 euchromatic sites. The majority of MDG1 and copia and about a half of MDG3 euchromatic copies are localized in restricted number of sites (hot spots) on D. simulans polytene chromosomes. In D. melanogaster these elements are scattered along the chromosomes though there are some hot spots too. It appears that euchromatic copies of MDG1 and copia are considerably less mobile in D. simulans in contrast to D. melanogaster. Some common hot spots of retrotransposon localization in D. simulans and D. melanogaster were earlier described as intercalary heterochromatin regions in D. melanogaster. The level of interstrain variability of MDG4 hybridization sites is comparable in both species. Comparative blot-analysis of adult and larval salivary gland DNA shows that MDG1 and copia are situated mainly in euchromatic regions of D. melanogaster chromosomes. In D. simulans genome they are located mainly in heterochromatic regions underreplicated in salivary gland polytene chromosomes. There are interspecies differences in the distribution of retrotransposons in beta-heterochromatic chromosome regions.     

Cellular energy functions of a subline of mouse fibroblasts resistent to the action of ethidium bromide]

The respiration of subline Leb-25 cells, resistant to ethidium bromide (EB, 25 g/ml), is 2.5 times slower than the respiration of parental L cells of mouse fibroblasts. The EB resistant cells have a normal level of ATP. Disturbances of mitochondrial functions can be observed such as a defect of the succinate dehydrogenase complex and the uncoupling of respiration and oxidative phosphorylation in mitochondria of Leb-25 cells.     

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.     

Comparative characterization of laboratory-made selective nutrient media for Salmonella accumulation

In this work the results of the trial of three selective nutrient media for Salmonella accumulation, viz. Leifson's selenite broth produced by the Research and Manufacturing Amalgamation (RMA) "Nutrient Media", tetrarathionate broth (Müller's medium), laboratory-prepared, and newly developed MA-broth produced by the RMA "Nutrient Media", are presented. The results of 6- and 24-hour incubation in these selective media was evaluated. As found in this study, the laboratory-prepared medium was most effective for the accumulation of salmonellae (S. paratyphi, S. typhi, S. gallinarum). With respect to the possible concomitant microflora (Escherichia coli and shigellae), the inhibiting properties of MA-broth were superior to those of Leifson's medium, but inferior to those of Müller's medium.