Ultrastructure of resting cells of some non-spore-forming bacteria

Using electron microscopy (ultrathin sections and freeze-fractures), we investigated the ultrastructure of the resting cells formed in the cultures of Micrococcus luteus, Arthrobacter globiformis, and Pseudomonas aurantiaca under conditions of prolonged incubation (up to 9 months). These resting cells included cyst-like forms that were characterized by complex cell structure and the following ultrastructural properties: (i) a thickened or multiprofiled cell wall (CW), typically made up of a layer of the preexisting CW and one to three de novo synthesized murein layers; (ii) a thick, structurally differentiated capsule; (iii) presence of large intramembrane particles (d = 180-270 A), occurring both on the PF and EF sides of the membrane fractures of M. luteus and A. globiformis; (iv) a peculiar structure of the cytoplasm, which was either fine-grained or lumpy (coarse-grained) in different parts of the cell population; and (v) a condensed nucleoid. Intense formation of cyst-like cells occurred in aged (2- to 9-month-old) bacterial cultures grown on diluted complex media or on nitrogen-, carbon-, and phosphorus-limited synthetic media, as well as in suspensions of cells incubated in media with sodium silicate. The general morphological properties, ultrastructural organization, and physiological features of cyst-like cells formed during the developmental cycle suggest that constitutive dormancy is characteristic of non-spore-forming bacteria.     

Effect of the cationic detergent CTAB on the involvement of ADP/ATP antiporter and aspartate/glutamate antiporter in fatty acid-induced uncoupling of liver mitochondria

The influence of the positively charged amphiphilic compound cetyltrimethyl ammonium bromide (CTAB) on palmitate- and laurate-induced uncoupling and on carboxyatractylate and glutamate recoupling effects in liver mitochondria have been studied. CTAB (40 M) in the presence of 3 mM MgCl₂ had little (if any) effect on the palmitic acid-stimulated respiration of mitochondria; the glutamate recoupling effect increased, and the carboxyatractylate recoupling effect decreased to the same degree with the combined effect (about 80%) remaining unchanged. Thus, CTAB decreases the ADP/ATP antiporter involvement and increases to the same extent the aspartate/glutamate antiporter involvement in the fatty acid-induced uncoupling. The carboxyatractylate and glutamate recoupling effects were less pH dependent in the presence of CTAB than in its absence. These data could be interpreted with the assumption that fatty acid anions are more accessible to the ADP/ATP antiporter and their neutral forms are more accessible to the aspartate/glutamate antiporter, and that CTAB changes the relative anion carrier involvement in the fatty acid-induced uncoupling as it forms neutral complexes with fatty acid anions.     

Cations SkQ1 and MitoQ accumulated in mitochondria delay opening of ascorbate/FeSO4-induced nonspecific pore in the inner mitochondrial membrane

It is known that an addition of FeSO4 in the presence of ascorbic acid to cells or mitochondria can injure energy coupling and some other functions in mitochondria. The present study demonstrates that decrease in ascorbate concentration from 4 to 0.2 mM in the presence of the same low concentrations of FeSO4 accelerates the nonspecific pore opening, while cyclosporin A prevents and under some conditions reverses the pore opening. Hydrophobic cations SkQ1 and MitoQ (structural analogs of plastoquinone and coenzyme Q(10), respectively) delay pore opening, SkQ1 being more efficient. It is known that an increase in matrix ADP concentration delays pore opening, while an addition of carboxyatractylate to mitochondria accelerates the beginning of pore opening. Preliminary addition of SkQ1 into a mitochondrial suspension increased the effect of ADP and decreased the effect of carboxyatractylate. These results suggest that under the conditions used SkQ1 protects mitochondria from oxidative damage as an antioxidant when added at extremely low concentrations.     

Metabolic state of liver mitochondria and lymphocytes after T-activin administration under condition of hypothermia

After 10 days of swimming (10 min per day, water temperature +20 degrees C) the oxygen consumption in rat liver mitochondria increased via the external pathway of NADH oxidation from 3.6 +/- 0.3 to 4.4 +/- 0,2 nmoles O2 x min-1 x mg-1 protein; when the rats were simultaneously injected with an endogenous immunomodulator T-activin (5 micrograms/100 g body weight) daily, this rate increased up to 6.5 +/- 0.6 nmoles O2 x min-1 x mg-1 protein. In the control group, the uncoupled respiration rate is also higher, while the ascorbate+ +TMPD oxidation rate is lower than in the cold- and cold + T-activin-treated groups. The metabolic states of lymphocyte mitochondria did not differ in the three experimental groups. The respiration rates and delta psi m (monitored by diS-C3-(5) fluorescence) of lymphocyte mitochondria in these three groups were also identical.     

Effect of palmitate on energy coupling in lymphocyte mitochondria

In the presence of 0.1 micrograms/ml of oligomycin, DNP (40-60 microM) increases lymphocyte respiration 10-fold and more. Palmitate taken at the same concentration stimulates the respiration of isolated mitochondria (1-2 mg prot/ml) in the presence of 1 mg/ml of BSA and the respiration of lymphocytes (10(8) cells/ml). When BSA and EGTA are absent in mitochondria isolation media, the mitochondrial respiration does not increase after DNP or ADP addition. Lymphocyte preparations are mostly distinguished by mitochondrial morphology in the presence of the uncoupler; they differ less by changes in dis-C3-(5) fluorescence after addition of 5-10 microM DNP and only insignificantly by the stimulation of respiration by DNP and palmitate. These results may be explained by the increase in the uncoupler-induced permeability of mitochondria for K+ and by partial transformation of delta psi m into delta pH in some cells, which may increase the cell resistance to damaging influences.     

Participation of the ATP/ADP antiporter and fatty acids in oxidative phosphorylation uncoupling in squirrel liver mitochondria during winter hibernation and awakening]

The parameters of energy coupling of mitochondria isolated from the livers of hibernating and awakening gophers were studied. The ATP/ADP-antiporter inhibitor carboxyatractylate slowed down the respiration rate, increased delta psi and decreased the ionic conductivity of the inner mitochondrial membrane as measured by the rate of the delta psi decline after addition of cyanide (in the presence of oligomycin and EGTA). A similar effect was produced by BSA, carboxyatractylate being fairly ineffective in the presence of BSA. In hibernating gophers the maximal rate of the uncoupled respiration and the ionic conductivity of the inner mitochondrial membrane were markedly decreased as compared with awakening gophers. The data obtained suggest that in awakening animals fatty acids induce the uncoupling of oxidative phosphorylation by the ATP/ADP-antiporter, this process being simultaneous with the activation of the respiratory chain.     

Changes in the Fine Structure of Microbial Cells Induced by Chaotropic Salts

The electron microscopic examination of thin sections of cells of the yeasts Saccharomyces cerevisiae and Pichia pastoris and the gram-positive bacteria Micrococcus luteus and Bacillus subtilis showed that cell treatment with the chaotropic salts guanidine hydrochloride (6 M) and guanidine thiocyanate (4 M) at 37°C for 3–5 h or at 100°C for 5–6 min induced degradative processes, which affected almost all cellular structures. The cell wall, however, retained its ultrastructure, integrity, and rigidity, due to which the morphology of cells treated with the chaotropic salts did not change. High-molecular-weight DNA was localized in a new cell compartment, the ectoplasm (a peripheral hydrophilic zone). The chaotropic salts destroyed the outer and inner membranes and partially degraded the outer and inner protein coats of Bacillus subtilis spores, leaving their cortex (the murein layer) unchanged. The spore core became accessible to stains and showed the presence of regions with high and low electron densities. The conditions of cell treatment with the chaotropic salts were chosen to provide for efficient in situ PCR analysis of the 16S and 18S rRNA genes with the use of oligonucleotide primers.     

Inhibitors of the ATP/ADP antiporter suppress stimulation of mitochondrial respiration and H+ permeability by palmitate and anionic detergents

The action of ATP/ADP-antiporter inhibitors upon the uncoupling effect of palmitate, detergents and 'classical' uncouplers has been studied. The uncoupling effect was estimated by stimulation of succinate oxidation and of H+ permeability of rat liver mitochondria in the presence of oligomycin. It is shown that carboxyatractylate (CAtr) and pyridoxal 5-phosphate (PLP) suppress the uncoupling induced by palmitate and the anionic detergents SDS and cholate, but do not affect that induced by the cationic detergents CTAB, by the non-ionic detergent Triton X-100, as well as by the 'classical' uncouplers FCCP and DNP. The results are discussed in terms of a concept assuming that the ATP/ADP-antiporter facilitates the electrophoretic export of hydrophobic anions from mitochondria.     

The effects of cold stress on respiration of diaphragm muscle

The mechanism of the stimulating effect of short-term cold exposure of animal on tissue respiration has been studied. Oxygen consumption by a piece of the rat diaphragm muscle, fixed in a frame and put into a polarographic cell, was measured with an oxygen electrode. It is found that 13 min exposure of the rat to an air temperature of +2°C induces (1) an increase in respiration rate in the diaphragm tissue, (2) a decrease in stimulation of respiration by dinitrophenol (DNP) and (3) a decrease in the sensitivity of respiration to amytal. The maximal respiration rate observed in the presence of 40 M DNP is unaffected. Acclimation for two weeks of rats at +2°C does not influence the measured parameters. Effects similar to those produced by short-term cold exposure can be obtained by treatment of the cold acclimated animals with norepinephrine. In non-acclimated rats, norepinephrine is ineffective. The effects of cold exposure (or norepinephrine) can be reproduced on the diaphragm from a non-treated animal by the addition of 0.2 mM oleate or 10 mM pyruvate to the incubation medium. All effects of cold exposure or norepinephrinein vivo, or oleate and pyruvatein vitro are inhibited by oubain or replacement of Na⁺ by Li⁺ in the incubation mixture. The role of fatty acids and Na,K ATP-ase in the thermoregulatory responses of the tissue respiration is discussed.