Cytotoxicity of a novel lipid-like bacterial product

A highly cytotoxic, lipid-like compound was isolated from a Serratia marcescens strain currently under identification. We have named the compound DCX for its direct cytotoxic activity on various cell types in culture. DCX was purified by preparative thin layer chromatography from chloroform: methanol = 4:1 extracts of whole bacteria, and is chromatographically homogeneous. The effect of DCX on cells is dose, time, and temperature dependent. DCX is particularly toxic to the mastocytoma cell line P815 (TD50 = 75 pg/ml). Three other malignant or transformed murine cell lines were sensitive to the cytotoxic action of DCX. The effect of DCX was also tested on normal cells (human gingival fibroblasts), which showed greater resistance to DCX than the other cells tested.     

Heparin inhibits the inositol 1,4,5-trisphosphate-dependent, but not the independent, calcium release induced by guanine nucleotide in vascular smooth muscle

The effects of heparin on the release of intracellular Ca2+, assessed by tension development in saponin-permeabilized rabbit main pulmonary artery, were determined. Heparin inhibited (IC50 = 5 micrograms/ml) inositol 1,4,5-trisphosphate (InsP3)-induced, but not caffeine-induced, Ca2+ release. The initial (InsP3-dependent) component of GTP gamma S-induced Ca2+-release was also inhibited by heparin, but the InsP3-independent component was resistant to both heparin and procaine. These results support the existence of a G protein activated mechanism of Ca2+ release that is not mediated by InsP3 or by Ca2+-induced Ca2+ release.     

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.     

Bridging structures spanning the junctioning gap at the triad of skeletal muscle

The membrane systems of skeletal muscle were examined after tannic acid fixation. A new structure consisting of bridges spanning the junctional gap is described, and a model is proposed in which the cytoplasmic but not the luminal membrane leaflets of the transverse tubule and of the junctional sarcoplasmic reticulum (SR) are continuous. The globular particles (presumably the Ca-binding proteins) within the terminal cisternae were arranged in longitudinal rows and appeared adherent to the junctional membrane. The junctional gap was present in negatively stained, frozen thin sections of fixed muscles. Negatively staining material occured within the junctional gap. The cytoplasmic leaflets of the longitudinal, intermediate, and terminal cisterna regions of the SR exhibited a thick coat of densely staining material compatible with the presence of the Ca-ATPase. Similar bridges were also observed at the surface membrane-SR close coupling sites of vascular smooth muscle.     

Vascular smooth muscle mitochondria: Magnesium content and transport

Endogenous magnesium content and magnesium transport of isolated bovine vascular smooth muscle mitochondria were studied. Mitochondria isolated from atherosclerotic bovine arteries contained two to three times as much magnesium (178 nmol/mg of mitochondrial protein) as those isolated from normal arteries (67 nmol/mg of mitochondrial protein). Electron-opaque granules were visible in the unstained unfixed mitochondria and could be shown with electron probe analysis to consist of magnesium, calcium, and phosphorus. At concentrations of external Mg²⁺ from 0 to 6 mm, the vascular smooth muscle mitochondria exhibited respiratory substrate-supported release of Mg²⁺ as studied with metallochromic indicator, eriochrome blue, using dual-wavelength spectrophotometry. The maximal velocity of energized release (3 nmol of Mg²⁺/s/mg of mitochondrial protein) was observed at 4 mm external Mg²⁺ and the half-maximal transport occurred at 0.5 mm.     

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.     

Smooth muscle myosin: regulation and properties

The relationship of the biochemical states to the mechanical events in contraction of smooth muscle cross-bridges is reviewed. These studies use direct measurements of the kinetics of Pi and ADP release. The rate of release of Pi from thiophosphorylated cycling cross-bridges held isometric was biphasic with turnovers of 1.8 s-1 and 0.3 s-1, reflecting properties and forces directly acting on cross-bridges through mechanisms such as positive strain and inhibition by high-affinity MgADP binding. Fluorescent transients reporting release of an ADP analogue 3'-deac-edaADP were significantly faster in phasic than in tonic smooth muscles. Thiophosphorylation of myosin regulatory light chains (RLCs) increased and positive strain decreased the release rate around twofold. The rates of ADP release from rigor cross-bridges and the steady-state Pi release from cycling isometric cross-bridges are similar, indicating that the ADP-release step or an isomerization preceding it may limit the ATPase rate. Thus ADP release in phasic and tonic smooth muscles is a regulated step with strain- and dephosphorylation-dependence. High affinity of cross-bridges for ADP and slow ADP release prolong the fraction of the duty cycle occupied by strongly bound AM.ADP state(s) and contribute to the high economy of force that is characteristic of smooth muscle. RLC thiophosphorylation led to structural changes in smooth muscle cross-bridges consistent with our findings that thiophosphorylation and strain modulate product release.     

Cryo-atomic force microscopy of unphosphorylated and thiophosphorylated single smooth muscle myosin molecules

The purpose of this study was to determine whether steric blockage of one head by the second head of native two-headed myosin was responsible for the inactivity of nonphosphorylated two-headed myosin compared with the high activity of single-headed myosin, as suggested on the basis of electron microscopy of two-dimensional crystals of heavy meromyosin (Wendt, T., Taylor, D., Messier, T., Trybus, K. M., and Taylor, K. A. (1999) J. Cell Biol. 147, 1385-1390; and Wendt, T., Taylor, D., Trybus, K. M., and Taylor, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4361-4366). Our earlier cryo-atomic force microscopy (cryo-AFM) (Zhang, Y., Shao, Z., Somlyo, A. P., and Somlyo, A. V. (1997) Biophys. J. 72, 1308-1318) indicates that thiophosphorylation of the regulatory light chain increases the separation of the two heads of a single myosin molecule, but the thermodynamic probability of steric hindrance by strong binding between the two heads was not determined. We now report this probability determined by cryo-AFM of single whole myosin molecules shown to have normal low ATPase activity (0.007 s-1). We found that the thermodynamic probability of the relative head positions of nonphosphorylated myosin was approximately equal between separated heads as compared with closely apposed heads (energy difference of 0.24 kT (where k is a Boltzman constant and T is the absolute temperature)), and thiophosphorylation increased the number of molecules having separated heads (energy advantage of -1.2 kT (where k is a Boltzman constant and I is the absolute temperature)). Our results do not support the suggestion that strong binding of one head to the other stabilizes the blocked conformation against thermal fluctuations resulting in steric blockage that can account for the low activity of nonphosphorylated two-headed myosin.