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.     

Observation of Potato Virus X in Tobacco Mosaic Virus (TMV)- Induced Local Lesions and Surrounding Zones in Leaves of Datura stramonium L. in Mixed Infection

In mixed infection by Tobacco Mosaic Virus (TMV) and Potato Virus X (PVX) of leaves of Datura stramonium L., PVX particles were observed in the developing local lesions in both the central part and on the periphery, in addition to TMV. PVX virions were found either separately or together with TMV. Sometimes in local lesions mainly in their periphery, PVX-specific laminar inclusion components were observed and, in certain cases, cylindrical bodies about 120—140 nm in diameter. In 2 mm surrounding zones from the edge of the lesions, TMV particles were not observed. However, in the majority of cells of these zones, PVX intensively accumulated, often forming large masses. In some cases, we observed parts of cells with relatively small amounts of dispersed PVX particles, associated with laminar inclusion bodies. In cell areas with large accumulations of PVX, laminar inclusions were not found.     

The antiviral activity of cyclopentane β,β′-triketones related to secondary metabolites of higher plants

The effect of 20 cyclopentane β,β′-triketones and their sodium salts on the development of infections caused by tobacco mosaic virus (TMV) in leaves of tobacco (Nicotiana tabacum L. cv. Xanthi-nc) has been studied. It has been shown that the strongest antiviral effect is produced by sodium salts of 2-acetyl-4,7-dithio-2,3,4,5,6,7-hexahydro-1H-inden-1,3-dione, 2-acetyl-4-oxa-7-thio-2,3,4,5,6,7-hexahydro-1H-inden-1,3-dione, and 2-acetyl-4,5-didodecylthiocyclopent-4-en-1,3-dione. These compounds at a concentration of 2 mg/mL decrease the number of local TMV-induced necroses formed on inoculated tobacco leaves by 98% and have no toxic effect on leaf tissues.     

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 Study of Accumulation of Two Potato Virus X Strains Differing in Virulence,Hydrolase Activity and Morphological Abnormalities in Datura stramonium L. Leaves

In young systemically infected leaves of Datura stramonium L., a severe strain of Potato virus X (PVX) accumulated to a lower degree than a mild strain. Infected leaves had increased protease and RNase activities in comparison with those of healthy controls. The highest hydrolase activities were found in leaves infected with the severe strain. Negative‐staining electron microscopy of dips from the infected leaves indicated that PVX virions underwent destructive changes, which resulted in the appearance of abnormal (swollen and ‘thin’) particles. Immuno‐electron microscopic assays showed that thin PVX particles, in contrast to those of normal diameter, lost the ability to bind with specific antiserum. The relative number of thin virions in leaves infected with the severe PVX strain was considerably higher than in leaves infected with the mild strain. This shows that a correlation exists between increased protease activity and intracellular destruction of virions. In abnormal virions, the viral RNA appears to be available for RNase attack. Therefore, it seems that high RNase activity together with increased generation of abnormal virions in the leaves infected with the severe strain promote inactivation of the viral RNA with RNase. We suppose that the enhanced hydrolase activities in the leaves infected with severe PVX strain, on the one hand, limit viral accumulation and thus play a defensive role and, on the other hand, cause considerable intracellular pathological changes resulting in severe symptoms.     

Selective resorption in nutritive phagocytes of the sea urchin Anthocidaris crassispina

Phagocytic resorption during spermatogenesis was studied in the sea urchin Anthocidaris crassispina. Nutritive phagocytes in gonad absorbed both waste sperm cells and residual bodies discarded from maturing spermatids, and these materials were subsequently compartmented in heterophagosomes. Based on 180 heterophagosomes examined by transmission electron microscopy, over 99% of heterophagosomes contained either residual bodies or sperm cells only. Simultaneous resorption of sperm cells and residual bodies in a heterophagosome was uncommon, with only approximately 0.56% occurrence, suggesting that heterophagosomes have a selective resorption ability in nutritive phagocytes.     

Growth and ultrastructure of the marine unicellular alga <Emphasis Type="Italic">Dunaliella salina</Emphasis> (Chlorophyta) after chronic selenium intoxication

The effects of selenium (0.01, 0.5, 1, 5 and 10 mg/liter) on the growth and ultrastructure of the microalga Dunaliella salina were investigated following its transfer into clean water. Selenium concentrations of 5 and 10 mg/liter were toxic to D. salina, and reinoculation of microalga into clean water did not prevent it from total mortality. When reinoculated from medium with 0.01 mg Se/liter, the cell population density of D. salina was restored in 14 days. The number of ultrastructural alterations in cells was the same as in the control, while the excretory activity of microalga between days 4 and 10 of this experiment was higher. Cell population growth of D. salina transferred from 0.5 and 1 mg Se/liter was lower than in the control. No ultrastructural defects were observed in microalga reinoculated from medium with a selenium concentration of 0.5 mg/liter and the excretion level corresponded to that at 0.01 mg/liter. Various types of ultrastructural damage were found in microalga from medium with 1 mg Se/liter, which was previously reported to be threshold for D. salina; however, the number of cell injuries decreased with increasing time in clean medium. Excretory activity was decreased at the beginning of experiment; but after 7 days, it was restored to the control level. Though there were no ultrastructural alterations in microalgal cells from medium with 0.5 mg Se/liter, we assume that they had molecular defects that could inhibit the cell population growth. The study of microalgae following their reinoculation from medium containing toxicants into clean medium can be a useful method for evaluating algal survival after toxic exposure.     

Ultrastructure of parenchymal leaf cells of different soybean varieties systemically infected with soybean mosaic virus

A comparative study was made of the ultrastructure of parenchyma leaf cells of different soybean varieties systemically infected with soybean mosaic virus (SMV). It has been shown that virus accumulation and formation of virus-specific cylindrical inclusions (CIs) occur in the infected cells, in addition to intracellular changes showing stimulation of lytic processes, such as activation of smooth endoplasmic reticulum and Golgi apparatus, formation of cytoplasmic vacuoles, cytosegresomes, myelin-like bodies, different disturbances in the structure of cell organelles. Many infected cells demonstrated microbodies with invagination in which cylindrical inclusions were often found showing signs of destruction. It is suggested that such microbodies possess autophagic activity towards CIs. A possible relation of the observed virus-induced ultrastructural cell changes with the degree of SMV affection of investigated varieties is discussed