[Investigation of the impact of cowpox virus BTB/kelch gene deletion on some characteristics of infection in vitro].

The biological properties of cowpox virus (CPXV) mutants with target deletion of 4 of the 6 BTB/kelch genes (D11L, C18L, G3L, and A56R) were examined in CV-1 cell cultures. There were changes in mutant temperature sensitivity and a reduction in a viral cytopathic effect. The mutant-infected culture yielded a smaller number of cells with actin-related long cellular protrusions (63 of 300 cells) as compared with wild CPXV (127 of 300). The length of the protrusions was 20-60 and 40-120 microm, respectively). Confocal microscopy revealed the formation of large globed structures containing both actin and CPXV antigens in the cells infected with quadruple mutants. These globed structures were recognized as incomplete protrusions. The findings show that the formation of long protrusions in the cells infected with wild type CPXV represents a type of specific viral potency related to the activity of BTB/kelch genes whose deletion results in cellular insufficiency to form full-fledged protrusions.

Suppression of endotoxin-induced inflammation by taxol.

The pathogenesis of acute lung injury includes transendothelial diapedesis of leukocytes into lung tissues and disruption of endothelial/epithelial barriers leading to protein-rich oedema. In vitro studies show that the microtubule network plays a role in the regulation of endothelial permeability as well as in neutrophil locomotion. It was hypothesised that the microtubule-stabilising agent, taxol, might attenuate inflammation and vascular leak associated with acute lung injury in vivo. The effect of intravenously delivered taxol was assessed using a model of murine lung injury induced by intratracheal lipopolysaccharide (LPS) administration. Parameters of lung injury and inflammation were assessed 18 h after treatment. Intravenously delivered taxol significantly reduced inflammatory histological changes in lung parenchyma and parameters of LPS-induced inflammation: infiltration of proteins and inflammatory cells into bronchoalveolar lavage fluid, lung myeloperoxidase activity, and extravasation of Evans blue-labelled albumin into lung tissue. Taxol alone (in the absence of LPS) had no appreciable effect on these parameters. In addition to lung proteins, intravenous taxol reduced accumulation of leukocytes in ascitic fluid in a model of LPS-induced peritonitis. Taken together, the present data demonstrate that microtubule stabilisation with taxol systemically attenuates lipopolysaccharide-induced inflammation and vascular leak.

Volume-dependent regulation of ion carriers in human and rat erythrocytes: role of cytoskeleton and protein phosphorylation.

This study examines the effect of heat-induced cytoskeleton transitions and phosphoprotein phosphatase inhibitors on the activity of shrinkage-induced Na+, K+, 2Cl- cotransport and Na+/H+ exchange in rat erythrocytes and swelling-induced K+, Cl- cotransport in human and rat blood cells. Preincubation of human and rat erythrocytes at 49 degrees C drastically activated K+, Cl- cotransport and completely (rat) or partly (human) abolished its volume-dependent regulation. The same procedure did not affect basal activity of Na+, K+, 2Cl- cotransport but completely abolished its activation by shrinkage thus suggesting the involvement of a thermosensitive element of cytoskeleton network in the volume-dependent regulation of cotransporters. Both the shrinkage- and electrochemical proton gradient-induced Na+/H+ exchange was inhibited by the heat treatment to the same extent (50-70%), thus indicating the different signaling pathways involved in the activation of Na+, K+, 2Cl- cotransport and Na+/H+ exchange by cell shrinkage. This suggestion is in accordance with data on the different kinetics of volume-dependent activation and inactivation of these carriers as well as on their sensitivity to medium osmolality. Both swelling- and heat-induced increments of K+, Cl- cotransport activity were diminished by inhibitors of phosphoprotein phosphatases (okadaic acid and calyculin). In rat erythrocytes these compounds potentiate shrinkage-induced Na+/H+ exchange. On the contrary, neither basal nor shrinkage-induced Na+, K+, 2Cl- cotransport was affected by these compounds. Our results indicate a key role of cytoskeleton network in volume-dependent activation of K+, Cl- and Na+, K+, 2Cl- cotransport and the involvement of protein phosphorylation-dephosphorylation cycle in regulation of the activity of K+, Cl- cotransport and Na+/H+ exchange.

[Na+/H+- and Na+/Na+-countertransport in human, rabbit, and rat erythrocytes: evidence for the existence of two independent ion-transporting systems]. / Na+/H+- i Na+/Na+-protivotransport v éritrotsitakh cheloveka, krolika,i krysy: dokaztel'stva sushestvovaniia dvukh nezavisimykh ion-transportiruiushchikh sistem.

The activity and regulatory features of the Na+/H(+)- and Na+/Na(+)-exchange were studied in human, rabbit and rat red blood cells. No basal activity of the Na+/H(+)-exchange (the amyloride-inhibited component of the 22Na+ influx) in erythrocytes of these species was observed. The rate of 22Na+ influx increased rapidly when the experiments were carried out on acid-loaded cells in an alkaline (pH0 = 8.0) incubation medium (delta mu H(+)-induced Na+/H(+)-exchange). The ratio of delta mu H(+)-induced Na+/H(+)-exchange activities in human, rabbit and rat red blood cells was 1.0 : 1.1 : 2.3, respectively, whereas that of the Na+/Na(+)-exchange activities (the phloretin-inhibited component of the 22Na+ influx) in erythrocytes of these species was 1.0 : 4.6 : 0.2. The osmotic shrinkage of rat and rabbit erythrocytes led to the stimulation of the Na+/H(+)- (but not Na+/Na+) exchange. Amyloride (1 mM) inhibited the shrinkage-induced 22Na+ entry as well as the delta mu H(+)-induced 22Na+ entry--by 95 and 10-20%, respectively. Heat treatment (10 min, 49-51 degrees C), disturbing the membrane cytoskeleton suppressed both the shrinkage-induced activation and the delta mu H(+)-induced activation of the Na+/H(+)-exchange. The data obtained indicate that the both transport systems are mediated by two distinct transport carriers. It may be suggested that the delta mu H(+)-induced Na+/H(+)-exchange, on the one hand, and the shrinkage-induced Na+/H(+)-exchange, on the other, are mediated by two different Na+/H(+)-exchanger subtypes.

Kinetics and peculiarities of thermal inactivation of volume-induced Na+/H+ exchange, Na+,K+,2Cl- cotransport and K+,Cl- cotransport in rat erythrocytes.

The kinetics of the volume-dependent activation of Na+/H+ exchange, Na+,K+,2Cl(-)-cotransport and K+,Cl(-)-cotransport in rat erythrocytes was studied. The significant increase in the rate of Na+/H+ exchange is observed within 15 min after hypertonic shrinkage while the maximum transport rate is reached by 20 min. A delay of about 5 min was found in activation of Na+,K+,2Cl(-)-cotransport, the maximum transport rate being reached 10 min after shrinkage. Activation of K+,Cl(-)-cotransport by hypotonic swelling was registered within 10 min after cell swelling, with a simultaneous achievement of the constant transport rate. Preincubation of cells at 49 degrees C has no effect on the basal Na+/H+ exchange and Na+,K+,2Cl(-)-cotransport but suppresses the activation of these systems by osmotic shrinkage. On the contrary, the rate of K+,Cl(-)-cotransport in isosmotic medium is raised 10-fold after preincubation at 49 degrees C. The thermal treatment at 49 degrees C blocks the activation of K+,Cl(-)-cotransport by swelling. On the basis of the data on thermal denaturation of spectrin at the same temperature it was suggested that the cytoskeleton of erythrocyte membrane is involved in volume regulation of the ion-transporting systems and that the molecular mechanisms which underlie the activation of Na+/H+ exchange, Na+,K+,2Cl(-)-cotransport and K+,Cl(-)-cotransport are essentially different.

[The role of GTP-binding proteins in regulating the activation of Na+/H+-exchange and Na+, K+, 2Cl- cotransport: effect of fluoride ion]. / Rol' GTP-sviazyvaiushchikh belkov v reguliatsii aktivatsii Na+/H+-obmena i Na+, K+, 2Cl- kotransporta: deistvie ftorid-iona.

The effects of NaF and AlCl3 on basal and stimulated by hyperosmotic shock activities of Na+/H(+)-exchange and Na+, K+, 2Cl(-)-cotransport in rat smooth muscle and red blood cells have been studied. Preincubation of cells with 10 mM NaF leads to inhibition of basal and hyperosmotic shock-activated Na+, K+, 2Cl(-)-cotransport in smooth muscle cells--by 45% and 190%, respectively. Sodium fluoride causes 80% activation of basal Na+, K+, 2Cl(-)-cotransport in red blood cells with no effect on the transport induced by hyperosmotic shock. The effect of NaF on Na+, K+, 2Cl(-)-cotransport in red blood cells does not depend on Al3+. Basal Na+/H(+)-exchange is stimulated by NaF by 780% in smooth muscle cells and is insensitive to the fluoride in red blood cells. Under hyperosmotic conditions NaF activates Na+/H(+)-exchange by 100% in smooth muscle cells and by 130% in red blood cells; in the latter case its effect is potentiated by Al3+. The data obtained suggest that GTP-binding proteins are involved in the activation of Na+/H(+)-exchange under hyperosmotic conditions.

Evidence for the involvement of G proteins in the modulation of 22Na and 45Ca fluxes in myogenic L6 and aortic smooth muscle cells.

Preincubation of rat skeletal muscle derived L6 myoblasts with 10 mM NaF increased, the activity of Na+/H+ exchanger, and the uptake of 45Ca by 2 and 5 folds, respectively. In cultured vascular smooth muscle cells (CVSMC) of rat aorta, NaF increased the activity of Na+,K(+)-pump, Na+/H+ exchanger, passive permeability for Na+, and 45Ca uptake by 1.6, 9, 2, and 9 folds, respectively. Both, in CVSMC and L6 cells, the effect of NaF on the Na+/H+ exchanger and 45Ca uptake were significantly augmented by 20 microM AlCl3. No effect of AlCl3 on the NaF dependent changes in ion flux was seen in rat erythrocytes. The results suggest that in L6 and CVSMC cells, the Na+/H+ exchanger and Ca++ uptake pathway(s) are activated by GTP-binding proteins.