Myofibril breakdown during spreading damage. III. Isolated rat muscles

Ultrastructural changes have been studied in fast (EDL) and slow (SOL) isolated muscles of rats during Zenker's degeneration (ZD). The general pattern of ZD is similar in both the muscles and proceeds in the same way as in the fast skeletal muscles of amphibians: vacuolation of the T-system and of the sarcoplasmic reticulum occur prior to necrosis, and then contraction knots are formed. The latter detach from the rest of the fiber, and myofibrils degrade into clots of electron dense material. Differences in the development of ZD in this muscle are as follows: vacuolation is more strongly pronounced in EDL, contraction and supercontraction of sarcomeres being more characteristic of SOL. In this ZD region of both the muscles, additional membranes are found in some intracrystal spaces of mitochondria, this phenomenon occuring more frequently in SOL. Selective lysis of the Z-line during ZD has been detected only in single EDL Fibres.     

Effect of the ion composition of the calcium-free medium and cardiomyocyte damage during "calcium paradox" in rats]

The findings reveal that the degree of myocardial damage in the "calcium paradox" does not depend on the contracture strength, that the contracture attenuation due to a decreased concentration in the Ca-free medium is not equal to the cardiocytes protection as compared with other means. Mg2+ and the studied means of myocardial protection seem to play a major role in assessment of the "calcium paradox" development and explain the difference in results of the hyposodium medium effects in the "calcium paradox".     

Electric breakdown during the pulsed current spreading in the sand

Processes of spreading of the pulsed current from spherical electrodes and an electric breakdown in the quartz sand are studied experimentally. When the current density on the electrode exceeds the critical value, a nonlinear reduction occurs in the grounding resistance as a result of sparking in the soil. The critical electric field strengths for ionization and breakdown are determined. The ionization-overheating instability is shown to develop on the electrode, which leads to the current contraction and formation of plasma channels.     

Visualization of freezing damage. II. Structural alterations during warming

There is a growing amount of indirect evidence which suggests that the loss in viability of rapidly cooled cells is due to recrystallization of intracellular ice. This possibility was tested by an evaluation of the formation of morphological artifacts in rapidly cooled cells to determine whether this process can account for the loss in viability. Samples of the common yeast Saccharomyces cerevisiae were frozen at 1.8 or 1500 °C/min, and the structure of the frozen cells was examined by the use of freeze-fracturing techniques. Other cells cooled at the same rate were warmed to temperatures ranging from ?20 ° to ?50 °C and then rapidly cooled to ?196 °C, a procedure that should cause small ice crystals to coalesce by the process of migratory recrystallization. Cells cooled at 1500 °C/min and then warmed to temperatures above ?40 °C formed large intracellular ice crystals within 30 min, and appreciable recrystallization occurred at temperatures as low as ?45 °C. Cells cooled at 1.8 °C/min and warmed to temperatures as high as ?20 °C underwent little structural alteration. These results demonstrate that intracellular ice can cause morphological artifacts. The correlation between the temperature at which rapid recrystallization begins and the temperature at which the cells are inactivated indicates that recrystallization is responsible for the death of rapidly cooled cells.     

Disruption of myofibrils during spreading degeneration. I. Increased Ca2+ concentration]

The breakdown of sarcomeres of frog's twitch skeletal muscles during Zenker's (spreading) degeneration has been studied. The speed of propagation of the destruction process was accelerated by increasing CaCl2 concentration in Ringer's solution up to 8mM. An hour after local injury, the fibres were fixed just before separation of the next retraction clot or at the stage of granular destruction (Fig. 1). The dominating features of the ultrastructure of a fibre at the necrotic boundary are the coagulation of small bundles of supercontracted myofibrils and breakdown of uncontracted sarcomeres into separate A- and I-bands and then into small bundles of A- and I-protofibrils (Fig. 2,3). The same breakdown of sarcomers is observed in several small regions at a distance of about 100 micron from the necrotic boundary (Fig. 5). Besides this, fusion of a few myofibrils followed by the disappearence of M- and Z-bands occurs in the same region of the fibre (Fig. 4, 6). The diameter of the majority of myofibrils decreases towards the necrotic boundary due to longitudinal splitting and loss of peripheral protofibrils, presumably, as a result of lysis (Fig. 7).     

Bacterial breakdown of benomyl. II. Mixed cultures

Experiments with mixed bacterial cultures grown in liquid media which contained the benzimidazole fungicide benomyl, with or without Na-lactate, as source of carbon provided circumstantial evidence for cleavage of the benzimidazole heterocyclic ring. Yet, neither 2-aminobenzimidazole (2-AB) nor benzimidazole, as sole source of carbon, supported any bacterial growth. Total ¹⁴C-balance analysis experiments conclusively showed production of ¹⁴CO₂ from [2-¹⁴C] methyl benzimidazol-2-yl carbamate (MBC), and thus cleavage of the benzimidazole nucleus; bioassays, however, showed that the actual rate of benomyl and MBC breakdown was only small, the parent compound benomyl being still recovered in substantial quantities after up to 80 days of incubation. Therefore, cleavage of the benzimidazole ring is probably a matter of cometabolism, n-butylamine which originates from the butylcarbamoyl side chain serving as the proper source of carbon.Besides radiolabelled 2-AB and CO₂, an unknown metabolite was isolated which showed characteristics of a 2-AB-nucleotide. Probably, 2-AB was incorporated into bacterial DNA, which upon lysis of the bacterial cells gave rise to the nucleotide in question. Therefore, 2-AB might exert its inhibitory action by interfering with the normal functioning of DNA.     

Substance P like-immunoreactivity release from enterochromaffin cells of rat caecum mucosa. Inhibition by serotonin and calcium-free medium

Location, endogenous contents, and release of Substance P like-immunoreactivity were investigated in the rat caecum, using Immunohistochemistry and RadioImmunoAssay. Our immunohistochemical results indicate that Substance P was present both in the neuromuscular and mucosal compartments of this intestinal structure. However, detection of the peptide in the enterochromaffin cells of the mucosa remained very difficult. That may be explained by the very low endogenous contents of Substance P detected in the mucosa, using RIA. As we have already described a serotonin release from rat caecum mucosa, we show, now, that Substance P like-immunoreactivity may be released from the same structure. This release was stable, calcium-dependent, inhibited by serotonin, and not influenced by the chemical depolarization. Our data demonstrate an active release of Substance P like-immunoreactivity from intestinal mucosa, in the rat caecum. It seems that the endogenous pool of Substance P like-immunoreactivity is involved as a functional pool. The mechanisms responsible of this release seem to be different than that observed for the serotonin release. Substance P like-immunoreactivity may be released in precise physiological conditions, or even, in pathological conditions.     

Characterization of alpha1-adrenoceptor subtypes mediating noradrenaline-induced contraction of rat epididymal vas deferens in calcium-free medium.

The alpha1-adrenoceptor subtype mediating noradrenaline (NA)-induced contractions of rat epididymal vas deferens in Ca2+-free/EGTA (1 mM) medium was studied using competitive antagonists. The effects of chloroethylclonidine (CEC) was investigated in Ca2+-free and normal Krebs' medium and RT-PCR was used to identify alpha1-adrenoceptor specific mRNA in epididymal vas deferens. In Ca2+-free medium, NA evoked sustained contractions but was less potent (pD2, 5.9) than in normal Krebs' medium (pD2, 7.3). The contractions in Ca2+-free medium were inhibited by prazosin (pA2, 9.3), 5-methylurapidil (pA2, 8.4), spiperone (pA2, 7.6) and BMY 7378 (pK(B), 6.8) consistent with activation of alpha1A-subtype. Repeated pretreatment with CEC (100 microM) reduced the potency of NA and maximum contractions in normal and Ca2+-free media. CEC-sensitivity in normal Krebs' medium was enhanced by prior treatment with phenoxybenzamine. mRNA for alpha1a- and alpha1d- but not alpha1b-adrenoceptors were detected in epididymal vas deferens. These results suggest that NA contracts the tissue in Ca2+-free medium by the stimulation of alpha1A-adrenoceptors. Two factors affecting CEC-sensitivity of NA-induced contractions in this tissue are discussed.     

Phospholipase activation during monocyte adherence and spreading.

Phospholipase activation is an important element in cellular signal transduction. In our study we investigated the role and regulation of phospholipase activation during human monocyte adherence and spreading. In human monocytes, phospholipase inhibition (with bromophenacyl bromide (BPB) or manoalide) impaired cell adherence and spreading. In contrast, neither cyclooxygenase/lipoxygenase inhibition nor platelet activating factor receptor blockade affected these responses. The impaired adherence and spreading induced by phospholipase inhibition with BPB could be partially reversed by the addition of nM levels of arachidonate (20:4(n - 6)). Dihomogammalinolenic acid (20:3(n - 6)) could substitute for arachidonate, but other polyunsaturated fatty acids were ineffective in this regard. The phospholipase inhibitor, BPB was selective in its effects on cellular phospholipase activities. BPB inhibited adherence/spreading-related and PMA-stimulated phospholipase activities, but not Ca2+ ionophore-stimulated phospholipase activity. To further probe for the role of Ca2+ in monocyte adherence and spreading, monocytes were loaded with MAPTAM (bis-(2-amino-5-methylphenoxy)-ethane-N,N,N',N', tetraacetic acid tetraacetoxymethyl ester), an EGTA analog. In contrast to phospholipase inhibition, intracellular Ca2+ chelation with MAPTAM did not affect monocyte adherence but did inhibit monocyte spreading. MAPTAM partially inhibited adherence/spreading-stimulated phospholipase activity, but did not inhibit PMA-stimulated phospholipase activity. These data suggest that human monocyte adherence and spreading may sequentially activate Ca(2+)-independent and then Ca(2+)-dependent phospholipases to release arachidonate. The activation of phospholipase and the release of arachidonate appear to be integral parts of the adhesion process.     

Cortical afterdischarges during Leao's cortical spreading depression.

Cortical spreading depression (CSD) has been employed in unanesthetized curarized rats, in order to analyse the role of the cerebral cortex in the generation of epileptic self-sustained parozysms produced by direct cortical electrical stimulation. CSD was preferred because it is reversible and may be repeated several times in the same animal. CSD evoked in the hemisphere contralateral to the stimulated cortex decreased the duration of the afterdischarge by 40% and modified its form and amplitude both at the cortical and reticular levels. The possible role of cortical and subcortical structures in the development of after-discharges is discussed.     

Differential localization of unconventional myosin I and nonmuscle myosin II during B cell spreading

Cross-linking of CD44 in vitro promotes chemokinesis and actin-based dendrite formation in T and B cells. However, the mechanisms by which the adhesion molecule CD44 induces cytoskeleton activation in lymphocytes are still poorly understood. In this study, we have investigated whether myosin isoforms are involved in CD44-dependent dendrite formation in activated B cells. Pharmacological inhibition of myosin with 2,3-butanedione monoxime strongly affected spreading and dendrite formation, suggesting that these cellular motors may participate in these phenomena. Furthermore, immunofluorescence analysis showed differences in subcellular localization of class I and class II myosin during B cell spreading. In response to CD44 cross-linking, myosin-1c was polarized to lamellipodia, where F-actin was high. In contrast, the distribution of cytosplasmic nonmuscle class II myosin was not altered. Expressions of myosin-1c and II were also demonstrated in B cells by Western blot. Although the inhibition of PLCgamma, PI3K and MEK-1 activation affected the spreading and dendrite formation in activated B cells, only PLCgamma and MEK-1 inhibition correlated with absence of myosin-1c polarization. Additionally, myosin-1c polarization was observed upon cross-linking of other surface molecules, suggesting a common mechanism for B cell spreading. This work shows that class I and class II myosin are expressed in B cells, are differentially distributed, and may participate in the morphological changes of these cells.     

Cholesterol attenuates and prevents bilayer damage and breakdown in lipoperoxidized model membranes. A spin labeling EPR study

The stabilizing effect of cholesterol on oxidized membranes has been studied in planar phospholipid bilayers and multilamellar 1-palmitoyl-2-linoleoyl-phosphatidylcholine vesicles also containing either 1-palmitoyl-2-glutaroyl-phosphatidylcholine or 1-palmitoyl-2-(13-hydroxy-9,11-octadecanedienoyl)-phosphatidylcholine oxidized phosphatidylcholine in variable ratio. Lipid peroxidation-dependent membrane alterations in the absence and in the presence of cholesterol were analyzed using Electron Paramagnetic Resonance spectroscopy of the model membranes spin labelled with either cholestane spin label (3-DC) or phosphatidylcholine spin label (5-DSPC). Cholesterol, added to lipid mixtures up to 40% final molar ratio, decreased the inner bilayer disorder as compared to cholesterol-free membranes and strongly reduced bilayer alterations brought about by the two oxidized phosphatidylcholine species. Furthermore, Sepharose 4B gel-chromatography and cryo electron microscopy of aqueous suspensions of the lipid mixtures clearly showed that cholesterol is able to counteract the micelle forming tendency of pure 1-palmitoyl-2-glutaroyl-phosphatidylcholine and to sustain multilamellar vesicles formation. It is concluded that membrane cholesterol may exert a beneficial and protective role against bilayer damage caused by oxidized phospholipids formation following reactive oxygen species attack to biomembranes.     

Macrophage spreading in vitro : II. Manganese and other metals as inducers or as co-factors for induced spreading

The cation requirement for the spreading of macrophages on glass and in simple media was examined. Several divalents allowed spreading induced by glass-bound immune-complexes. Their rank was MnCo > Ni, Zn > Cd > Mg > Fe in bicarbonate-buffered saline (SBK). Concentrations of Mn of 3μM allowed for extensive spreading in SBK while 10 μM were required for full spreading in Tris- or imidazole-buffered saline. Similar concentrations of Mn permitted spreading induced by dithiothreitol. Ca²⁺, Al³⁺ or Th⁴⁺, among other metals, were ineffective in immune-complex triggered spreading. Spreading in the presence of Mg was inhibited by EDTA but not by EGTA, giving further support for lack of a Ca requirement. At higher concentrations, Mn and several other metals induced macrophage spreading in the absence of other inducers. Features of the spreading promoted by 1 mM Mn were similar to those previously found for other inducers [23], and cells induced to spread by Mn were fully capable of phagocytosis. The specificity and the low concentrations needed for co-factor activity are incompatible with a charge reduction role of divalent cations and are consistent with a metal activated enzymatic reaction step. The similarity between macrophage spreading and cell to substrate adhesion in regard to Mg requirements and other features, suggests that cell ‘adhesion’, as usually measured, may be dependent on the extent of cell spreading.     

Impulse photoconductivity of solutions of chlorophyll and its analogs. II. Effect of medium polarity on the yield of ion-radicals during the photoxidation of chlorophyll a ]

The effect of the dielectric constant epsilon of the solvent on the yield delta n of ion-radicals during photooxidation of chlorophy-l a with n-benzoquinone was studied by the method of impulse photoconductivity. It was shown that in the studied range of dielectric constant valve (epsilon congruent to 5-25) delta n monotonously increased with an increase of epsilon the relationship delta n (epsilon) being of clearly pronounced S-like character with a bend in the region of epsilon congruent to 10. A half-quantitative explanation of the data obtained is presented.     

Mutational analysis of flagellum-independent surface spreading of Serratia marcescens 274 on a low-agar medium.

In a previous study (J. O'Rear, L. Alberti, and R. M. Harshey, J. Bacteriol. 174:6125-6137, 1992) we reported the isolation of several transposon mutants of Serratia marcescens 274 that were defective either in swarming alone or in both swimming and swarming motility. All the nonflagellate (Fla-) mutants, while defective in both types of motility, were able to spread rapidly on the surface of low-agar (0.35%) media. We show here that some of the swarming-defective mutants are defective in the production of serrawettin W1, an extracellular cyclic lipopeptide produced by S. marcescens 274. When combined with a Fla defect, the serrawettin (Swt) mutants are deficient in spreading on low-agar media. The spreading deficiency can be overcome by serrawettin supplied extracellularly. Introduction of Fla defects into chemotaxis mutants does not affect this mode of surface translocation. These results suggest that spreading may be a passive form of translocation. We also report that swarming defects in all mutants showing a Dps phenotype (able to swarm within the inoculated area but unable to move outward) in the earlier study can be overcome by changing the commercial source of agar.