Redistribution of subpopulations of peritoneal macrophages with Fc gamma receptors under the effect of an immunomodulator

The heterogeneity of resident peritoneal macrophages and peritoneal macrophages was studied in different periods following oral administration of sodium nucleinate according to their ability to bind and phagocytize sheep erythrocytes opsonized by means of specific rabbit IgG. Using a mathematical method developed earlier, it has been possible to demonstrate that resident peritoneal macrophages can be divided into two subpopulations--actively and poorly binding macrophages but, after activation by sodium nucleinate--into three subpopulations. Fractionation of the macrophages according to their ability to adhesion within a temperature gradient has shown that the same peaks are traced in the fractions as in the overall pool of cells, but in different quantitative ratios. It has also been demonstrated that phagocytic activity is reduced in macrophages capable of adhesion to plastic at lower temperatures.     

Effect of periodate treatment and phenol extraction of C. parvum on its capacity to activate macrophages

Summary The chemical modification of the ability of C. parvum to activate macrophages has been studied. It was shown to be possible to decrease this activity in C. parvum by a phenol extraction procedure. Macrophages attracted to the peritoneal cavity by the phenol extract were weakly cytotoxic to tumour cells.Treatment of intact C. parvum with 20 mM periodate produced a rapid decline in the ability of C. parvum to activate macrophages, reaching a basal stimulation after treatment for 8 h. The activity in C. parvum was only slightly affected by incubation with 2 mM periodate for as long as 18 h. The activation of macrophages presented different kinetics when periodate-treated organisms were used as inducers instead of the normal bacteria, although the number of cells attracted to the peritoneal cavity was marginally altered by the periodate treatment.It is concluded that a molecule (or molecules) sensitive to periodate oxidation and partially extractable by phenol is (are) responsible for the activation of macrophages. The possibility that this molecule would be a specific inducer for the subpopulation of macrophages with cytotoxic activity, which has been postulated by other workers, is discussed.     

Repair of the protein structures of ciliated epithelial cells after heat damage

A study has been made of the ability of ciliated pharengial cells of Rana temporaria L. to repair heat injury. The heat injury estimated by cessation of cellular motile activity has been shown to repair, ciliar movements being renewed. The reparation of the ciliar function is due to the restoration of nativity of the intracellular protein structures providing for the ciliar motility. These structures can be repaired only when they are a component of the whole living cell system; they are not capable of spontaneous renativation, however, when isolated as "glycerinized cell models". It is suggested that the reparation of heat injury in these cells is an active process, presumably dependent on the intracellular metabolism.     

Mouse peritoneal cells confer an antiviral state on mouse cell monolayers: role of interferon.

Vesicular stomatitis virus and encephalomyocarditis virus do not multiply in the majority of peritoneal macrophages freshly explanted from 4- to 8-week-old male or female mice. However, when peritoneal macrophages were cultivated in vitro for 3 to 5 days, these cells became permissive for both viruses. The loss of antiviral state in "aged" macrophages paralleled a significant decrease in the intracellular levels of (2'-5')oligo-adenylate synthetase activity. Although biologically active interferon was not detected in the nutrient medium of macrophage cultures, freshly harvested peritoneal cells could confer an antiviral state on monolayer cultures of mouse cells (aged macrophages, embryonic fibroblasts, and L cells) but not on heterologous chicken embryo, rabbit kidney, or human cells infected with vesicular stomatitis virus or encephalomyocarditis virus. The conferred antiviral state required at least 7 h to develop in target cells and was totally inhibited by the presence of antibody to mouse interferon alpha/beta but not to interferon gamma in the cocultures. Heterologous guinea pig and rabbit peritoneal cells could not transfer an antiviral state to target mouse cells. Donor peritoneal cells from mice preinjected with antibody to interferon alpha/beta could not transfer an antiviral state to target mouse cells. This ensemble of results indicating that freshly harvested peritoneal cells transfer interferon (which is responsible for inducing an antiviral state in susceptible mouse target cells) adds further experimental evidence that interferon is spontaneously expressed in normal mice and plays an important role in maintaining some host cells in an antiviral state.     

Effect of oxygen on activation state of complex I and lack of oxaloacetate inhibition of complex II in Langendorff perfused rat heart

Two main entry points for electrons into the mitochondrial respiratory chain are NADH:ubiquinone oxidoreductase (complex I) and succinate:ubiquinone oxidoreductase (complex II). Metabolic regulation of these two respiratory complexes is not understood in detail. It has been suggested that the Krebs cycle metabolic intermediate oxaloacetate (OAA) inhibits complex II in vivo, whereas complex I undergoes a reversible active/de-active transition. In normoxic and anoxic hearts it has been shown that the proportion of complex I in the active and de-active states is different suggesting a possible mode of regulation of the enzyme by oxygen concentration. In the current studies rapid isolation of mitochondrial membranes in a state that preserves the activity of both complex I and complex II has been achieved using Langendorff perfused rat hearts. The findings indicate that the state of activation of complex I is controlled by the oxygen saturation in the perfusate. In addition, these studies show that complex II is fully active in the mitochondrion and not inhibited by OAA regardless of the oxygen concentration.     

Postinspiratory neuronal activities during behavioral control, sleep, and wakefulness.

Cells that discharge in early expiration and inhibit other respiratory cells purportedly cause a separate phase of the respiratory cycle that has been named "postinspiration." Our objective was to study these postinspiratory cells in the intact unanesthetized cat during sleep, wakefulness, and behavioral inhibition of inspiration, but we were unable to find cells with strong and consistent activity confined to early expiration. Instead, we found that various cell types were active in early expiration. They included inspiratory-expiratory phase-spanning cells, retrofacial augmenting expiratory cells with bursts in early expiration, retrofacial decrementing expiratory cells, tonic expiratory cells, and cells with variable activity in the early part of expiration. Just as the cell types active during early expiration were heterogeneous so too were their activities during behavioral inhibition of inspiration and during sleep. These results suggest that the state of early expiration is determined by many different cell types rather than a single class of postinspiratory cells.     

Regulation of cell function in the macrophagal phagocytic system with salmozan

Salmozan has been shown to bring about changes in the phagocytic function of peritoneal macrophages, to produce a labilizing effect on the state of lysosomal membranes and to enhance the intensity of protein synthesis in peritoneal macrophages. The presence of relationship between the functional activity of the cells of the macrophagal phagocytic system, the resistance of the body to infections and the capacity for response to an antigenic stimulus has been revealed. The character of changes observed in this investigation has been found to depend on the interval between the injection of salmozan and the injection of sheep red blood cells or challenge with Listeria. The differences in the functional activity of the cells of the macrophagal phagocytic system, appearing under the influence of salmozan, can be attriduted to the action of this immunomodulating agent on various populations of peritoneal macrophages.     

An indirect and highly sensitive method for the determination of the flow of reducing equivalents in the respiratory chain

Reactions leading to oxido-reduction of TMPD have shown that, in its oxidized form, this compound has among others an extinction maximum at 610 nm; with the exception of cytochrome a, at this wavelength none of the respiratory chain intermediates has the ability to absorb the incident light. This property together with the one of reacting with cytochrome c, has given us the possibility to use TMPD as a "probe" of the reducing equivalents flow in the respiratory chain. Added to mitochondrial suspension, TMPD undergoes redox cycles in relation to the activity of the respiratory chain, modulated by increasing concentrations of succinate or respiratory inhibitors. NEM-induced reversible oxidation on the respiratory intermediates can also be determined by following the TMPD oxidation. The preliminary data obtained are thus consistent with the hypothesis that in appropriate conditions, the TMPD redox state can be used as a probe of the respiratory chain activity.     

Effect of rubomycin, carminomycin and adriamycin on respiration in liver mitochondria in various metabolic states, respiratory control and ADP/O ratio

A comparative study on the effects of antitumour antibiotics of the anthracycline group (rubomycin, carminomycin and adriamycin) on respiration and oxidative phosphorylation in liver mitochondria in various metabolic states has been carried out for the first time. It was shown that the antibiotics under study cause partial inhibition of mitochondrial state 3 respiration, which is eliminated by an uncoupler. Treatment of liver mitochondria with the antibiotics decreases the ADP/O and respiratory control values and stimulates state 4 respiration. The latter is partly inhibited by oligomycin. The uncoupled respiration is decelerated in the presence of the antibiotics. Under these conditions the oxidation of succinate is inhibited by lower concentrations of the antibiotics than that of NAD+-dependent substrates. It was shown that the maximal activity is exerted by the most polar agent carminomycin, while the hydrophobic rubomycin is the least active. The experimental results are discussed in terms of the toxic effect of antitumour antibiotics.     

Dynamics of change in the antibacterial activity of mouse peritoneal cells during the development of experimental influenzal infection

The method of the radiometric determination of the antibacterial activity of peritoneal cells in the process of the development of viral infection has been approved. The genetically determined specific level of the antibacterial activity of these cells in different strains of mice has been established. The development of the lethal form of experimental influenza A in mice has been shown to be accompanied by the mobilization of nonspecific protective factors, including the system of interferons and the antibacterial activity of peritoneal cells. The latter factor is, seemingly, insufficient for ensuring the necessary level of protection in the animals, whose death occurs at a shorter time than that necessary for the development of specific immune response.     

The effect of certain redox dyes on glutathione and on potassium accumulation in normal and mutant yeast

Certain redox dyes have been shown to have an effect on the ability of yeast, to accumulate potassium. It has been postulated that this is due to a generalized breakdown of membrane permeability brought about by the dyes. However, as is described here, a comparative study of normal and respiratory deficient cytoplasmic mutant yeasts in relation both to their ability to accumulate potassium and the glutathione content of the cells, suggests rather that the dyes act primarily on a GSH-like enzyme associated with the cytochrome system. It is concluded that active transport of potassium through the cell membrane involves a coupled GSH-heavy metal catalyst redox system.     

Expression and activity of cGMP-dependent phosphodiesterases is up-regulated by lipopolysaccharide (LPS) in rat peritoneal macrophages

It has been shown that cyclic GMP (cGMP) modulates the inflammatory responses of macrophages, but the underlying molecular mechanisms are still poorly understood. Looking for proteins potentially regulated by cGMP in rat peritoneal macrophages (PMs), in this study we analyzed expression and activity of cGMP-hydrolyzing and cGMP-regulated phosphodiesterases (PDEs). It was found that freshly isolated peritoneal exudate macrophages (PEMs) express enzymes belonging to families PDE1-3, PDE5, PDE10, and PDE11. Analysis of substrate specificity, sensitivity to inhibitors, and subcellular localization showed that PDE2 and PDE3 are the main cGMP-regulated PDE isoforms in PEMs. The profile of PDE expression was altered by maintaining PEMs in culture and treatment with bacterial endotoxin (LPS). After 24 h culture, PDE5 was not present and the levels of PDE2, PDE3, and PDE11 were markedly decreased. However, their expression and activity was recovered after treatment of cultured cells with LPS. A similar pattern of changes was observed for the expression of TNFalpha, but not for guanylyl cyclase A (GC-A). LPS up-regulated PDE expression also in resident peritoneal macrophages (RPMs), although not all PDEs present in PEMs were detected in RPMs. Taken together, our results show that in rat PMs expression of cGMP-dependent PDEs positively correlates with the activation state of cells. Moreover, the fact that most of these PDEs hydrolyze also cAMP indicates that cGMP can play a role of potent regulator of cAMP signaling in macrophages.     

Biochemical and morphological changes in dormant (“Nonculturable”) <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis> cells

Biochemical and morphological changes have been studied during transition of Mycobacterium smegmatis cells into their dormant (“nonculturable”) state. A significant fraction of the population of irreversibly “nonculturable” (NC) cells has a thicker cell wall, condensed cytoplasm, and a decreased number of ribosomes. The lipid contents in the NC cells are lower than in the metabolically active cells, with a relatively decreased amount of phospholipid and neutral lipid. Free mycolic acids, which are abundant in metabolically active cells, were not found in the NC cells. The NC forms are also characterized by decreased respiratory activity on endogenous substrates; however, the respiratory chain enzymes retain their activities in the isolated membranes. Activities of the Krebs cycle and glyoxylate cycle enzymes are markedly decreased. Despite a significant decrease in metabolic activity, NC cells possess membrane potential that seems to provide for reversibility of the NC state of mycobacteria, i.e. their capability of reactivating.     

Effect of Cu++ ions on the morphological, cytological, and physiological state of Candida utilis cells in continuous culture]

Cells of Candida utilis grown in continuous culture under the influence of rather high concentrations of copper ions have been studied biochemically, morphologically, and cytologically. Although under these conditions a new steady state has been adjusted, the population is much more differentiated than under control conditions. With regard to their size, structure, and viability the cells have been arranged into 4 groups. Generally, the "copper cells" in comparison with the "normal cells" are more voluminous (average: 2.5 times; maximum: 4 times) and of altered structure. The appearance of dense particles ("copper containing particles"), globules ("lipid globules"), vacuoles, and the thickening of the cell wall, as well as the disappearance of the mitochondrial cristae have been shown as characteristic indications for damage in the cell substructure caused by the influence of copper ions. Occasionally abnormal cell shapes can be observed. A permanent influence of copper ions in the concentration used was followed also by striking disturbances of the cell metabolism. The cells take up more carbon, phosphorus, and nitrogen, whereas protein synthesis and respiratory activity decrease. Both the synthesis of lipids and polysaccharides and the phosphohydrolase activity increase. A discussion of the experimental data is presented.     

Effect of Low-Intensity Pulsed Electric Fields on the Respiratory Activity and Electrosurface Properties of Bacteria

A change in the respiratory activity (RA) of the resistant to cyanide bacteria Pseudomonas fluorescens under the pulsed electric field treatment has been found experimentally. The bacteria reaction to the field treatment (rectangular impulses with the duration of 1 ms, voltage of 20 V, and frequency of 100 Hz) varied from the RA suppression of active culture to restoration and even stimulation of RA in cells with the decreased respiratory function, caused by the cyanide introduction. The interconnection of energetic membrane processes with the electrosurface properties of biocolloids has been demonstrated: The electrokinetic potential had been varied simultaneously with the respiratory activity change. Theoretical analysis of the experimental data indicates the change of quantity of active respiratory centers in cells under the influence of the pulsed electric field. These changes have reversible character. Depending on the state of bacteria, the processes of inhibition or restoration of the respiratory centers by the pulsed electric field can be observed.     

Influence of phospholipases A2 from snake venoms on survival and neurite outgrowth in pheochromocytoma cell line PC12

To determine whether the ability to induce neurite outgrowth in rat pheochromocytoma cell line PC12 is characteristic of phospholipases of different types, we have studied the influence of phospholipase A(2) (PLA2) from cobra Naja kaouthia venom and two PLA2s from viper Vipera nikolskii venom on PC12 cells. Phospholipases from the viper venom are heterodimers in which only one of the subunits is enzymatically active, while PLA2 from the cobra venom is a monomer. It was found that all three PLA2s induce neurite outgrowth in PC12. The PLA2 from cobra venom exhibits this effect at higher concentrations as compared to the viper enzymes. We have not observed such an activity for isolated subunits of viper PLA2s, since the enzymatically active subunits have very high cytotoxicity, while the other subunits are not active at all. However, co-incubation of active and inactive subunits before addition to the cells leads to a marked decrease in cytotoxicity and to restoration of the neurite-inducing activity. It has also been shown that all enzymatically active PLA2s are cytotoxic, the PLA2 from cobra venom being the least active. Thus, for the first time we have shown that PLA2s from snake venoms can induce neurite outgrowth in PC12 cells.     

The possible role of palmitoyl-CoA in the regulation of the adenine nucleotides transport in mitochondria under different metabolic states

It has been shown that K_M values for ADP when rat liver mitochondria oxidized succinate were strictly dependent on the values of the respiratory control ratios. The K_i values for palmitoyl-CoA inhibition of the ADP-stimulated succinate oxidation and the inhibition of the uncoupler-stimulated ATPase activity were equal to 0.5 μM. Mitochondria from livers of starved rats showed 30% inhibition of the state 3 respiratory rate (compared to the uncoupled respiratory rate) which was abolished by addition of carnitine. It was supposed that this inhibition was due to the influence of acyl-CoAs bound to the inner mitochondrial membrane on the adenienucleotide translocase. Mitochondria from livers of fed rats showed a strong inhibition of succinate oxidation both in state 4 and state 3, although the rate of uncoupled respiration was normal. It was assumed that in this case the changes in mitochondrial behaviour was caused by the decrease in the concentration of ADP and ATP in the matrix space of mitochondria.     

Survival of bacteria in the Artificial Mars unit

The survival of bacteria was studied in the "Artificial Mars" apparatus reproducing a complex of physical extreme factors. Bacteria isolated from microbiocenoses subjected to the action of chemical extreme factors (hydrogen peroxide, catalytically active minerals) were shown to be most resistant among soil heterotrophic bacteria. Cells in the resting state caused by dehydration survived better than vegetative cells. It has been concluded that microorganisms quite different in their physiological requirements and ecological properties can exist in the habitats extreme for life conditions.     

Evaluation of loss of active heparin in the peritoneal cavity during intermittent peritoneal dialysis]

Our studies aimed at determining a loss of active heparin from the peritoneal cavity after its intraperitoneal administration (250 JU/l of dialysis fluid) in 16 patients treated because of the end-stage renal failure with intermittent peritoneal dialysis and at comparing heparin influx clearance with that of glucose. It has been shown that heparin used in this dose loses 60-70% of its activity after 20-minute equilibration of dialysis fluid in the peritoneal cavity. Heparin influx clearance is higher than that of glucose but it depends on utilization of heparin in peritoneal cavity rather than on its penetration to the blood circulation.     

Comparison of transforming growth factor-beta and a macrophage- deactivating polypeptide from tumor cells. Differences in antigenicity and mechanism of action

A factor in medium conditioned by mouse tumor cells was shown previously to suppress the capacity of mouse peritoneal macrophages to undergo a respiratory burst and to kill protozoal pathogens (macrophage deactivation factor, MDF). Recently, pure transforming growth factor-beta (TGF-beta) proved to be a potent macrophage deactivator as well. Two lines of evidence suggest that MDF is not identical with TGF-beta. First, rabbit anti-TGF-beta IgG neutralized the respiratory burst-suppressing activity of TGF-beta without affecting the bioactivity of MDF, even when the latter was treated with acid to activate potentially latent TGF-beta. Second, in contrast to MDF, which decreases the affinity of the NADPH oxidase for NADPH, permeabilized macrophages that had been deactivated with TGF-beta displayed the same Km and Vmax of the oxidase as activated macrophages. As with MDF, TGF-beta had no effect on two other potential control points over the secretion of respiratory burst products, namely, hydrogen peroxide catabolism or glucose uptake. Finally, neither MDF nor TGF-beta affected the extent or affinity of binding of phorbol diesters to macrophages, the activity or cofactor requirements for protein kinase C, or the ability of protein kinase C to translocate quantitatively from cytosol to membrane fractions in response to phorbol diesters. Thus, 1) MDF is not identical with TGF-beta, and 2) in contrast to the activation or deactivation of macrophages by numerous other agents, TGF-beta regulates macrophage respiratory burst capacity at a level other than the apparent affinity of the oxidase for its substrate.