ZAP-70 Tyrosine Kinase Is Required for LFA-1–dependent T Cell Migration

The ZAP-70 tyrosine kinase is essential for T cell activation by the T cell receptor. We show that ZAP-70 is also required for migration of T cells that is dependent on the integrin LFA-1. Invasion of TAM2D2 T cell hybridoma cells into fibroblast monolayers, which is LFA-1–dependent, was blocked by overexpression of dominant-negative ZAP-70 and by piceatannol but not by herbimycin A. The Syk inhibitor piceatannol blocks the Syk homologue ZAP-70, which is expressed by TAM2D2 cells, with the same dose dependence as the inhibition of invasion. Dominant-negative ZAP-70 completely inhibited the extensive metastasis formation of TAM2D2 cells to multiple organs upon i.v. injection into mice. Migration of TAM2D2 cells through filters coated with the LFA-1 ligand ICAM-1, induced by 1 ng/ml of the chemokine SDF-1, was blocked by anti–LFA-1 mAb and also abrogated by dominant-negative ZAP-70 and piceatannol. In contrast, migration induced by 100 ng/ml SDF-1 was independent of both LFA-1 and ZAP-70. LFA-1 cross-linking induced tyrosine phosphorylation, which was blocked by dominant-negative ZAP-70 and piceatannol. We conclude that LFA-1 engagement triggers ZAP-70 activity that is essential for LFA-1–dependent migration.     

Effects of intermittent and continuous pheromone stimulation on the flight behaviour of the oriental fruit moth, Grapholita molesta

ABSTRACT. When male oriental fruit moths, Grapholita molesta (Busck) (Tortricidae), casting in clean air entered an airstream permeated with pheromone their flight tracks changed immediately on initial contact with pheromone, but after a few seconds returned to casting as if in clean air. The degree of change in the flight track was directly related to the concentration of pheromone. Although little net uptunnel movement occurred in response to the continuous stimulation provided by a uniformly permeated airstream, when an intermittent stimulus provided by a point-source plume was superimposed onto the permeated airstream moths were able to 'lock on' and zigzag uptunnel in the plume. The percentage of moths doing so corresponded to the difference between the peak concentration within the plume and the background concentration of pheromone permeating the airstream. Moths also locked onto, and flew upwind along the pheromone-clean-air boundary formed along a pheromone-permeated side corridor. Because a similar response was observed along a horizontal edge between a pheromone-permeated floor corridor and clean air, we conclude that the intermittent stimulation at the edge perpetuated the narrow zigzagging response to pheromone.     

The role of phosphoinositide-3-kinase in the control of shape and directional movement of the Physarum polycephalum plasmodium

The effects of wortmannin and LY294002, specific inhibitors of phosphoinositide-3-kinase, on the shape, locomotive behavior, and glucose chemotaxis were studied using the Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the self-oscillatory mode of locomotive behavior. Both inhibitors were shown to cause a reduction in the plasmodium frontal edge and a decrease in the efficiency of mass transfer during migration. They also suppressed the chemotaxis towards glucose and eliminated the characteristic changes in self-oscillatory behavior normally observed in response to the treatment of the whole plasmodium with glucose. The manifestation of these effects depended on the inhibitor concentration, treatment duration, and size of plasmodium. The involvement of phosphoinositide-3-kinase in formation of the frontal edge and control of P. polycephalum plasmodium chemotaxis suggests that the correlation of polar shape and directional movement of amoeboid cells with the distribution of phosphoinositides in the plasma membrane has a universal nature.     

Effect of nifedipine administration on the functional capacity of neutrophils

Administration of nifedipine to mice over a period of six months caused a significant (p<0.05) decrease in neutrophilic functions viz superoxide generation, coupled to NADPH oxidase activity as well as NADPH production by HMP shunt. Properties like chemotaxis and phagocytosis showed a similar decrease. From this study, it is seen that nifedipine causes neutrophil functional abrogation which is therefore an apparent concern for the prolonged usage of the drug. However, relevance of the mouse model to clinical situation needs further investigation.     

Membrane topology of the methyl-accepting chemotaxis protein DcrA fromDesulfovibrio vulgaris Hildenborough

Alkaline phosphatase fusions were used to study the membrane topology of DcrA, a protein of 668 amino acids fromDesulfovibrio vulgaris Hildenborough, which has two potentially membrane-spanning hydrophobic sequences at residues 11 to 29 and 188 to 207. A fusion at amino acid residue 170 in the proposed periplasmic domain exhibited high alkaline phosphatase activity, while low activity was observed for a fusion at amino acid residue 284 in the proposed cytoplasmic domain. The data support a topological model for DcrA similar to that of the methyl-accepting chemotaxis proteins of the enteric bacteria.     

A Method for Assessing the Migratory Response of Naegleria fowleri Utilizing [3H]Uridine-Labeled Amoebae

ABSTRACT. A new procedure is described to assay the migratory response of Naegleria fowleri (ATCC 30894) amoebae to potential chemoattractants. The method utilizes a blind-well Boyden chemotaxis chamber, two micropore filters of different construction, and amoebae-labeled with [³H]uridine. The technique was standardized by determining the influence of incubation time, filter construction, filter pore size and geometry, amoebae to filter pore ratio, and chemoattractant concentration. Radiolabeled amoebae were placed in Boyden chambers that contained the combination of an upper polycarbonate filter with distinct pores with a diameter of 8 μm and a lower filter of nitrocellulose with a 150-μm depth to separate the wells. A ratio of two amoebae to one filter pore and a 2-h incubation period were chosen to obtain optimal migration conditions. Nerve cell extract was used as the chemoattractant. The migratory responses of both highly pathogenic and weakly pathogenic strains of N. fowleri to nerve cell extract were compared using either the radiolabel procedure or the conventional single filter, leading-front method. Using either method, a highly pathogenic cloned strain of N. fowleri amoebae moved in a directiona manner (chemotactically)in vitro to B103 rat neuroblastoma cell extract. In contrast, a weakly pathogenic strain of amoebae responded in a nondirectional manner (chemokinetically) to nerve cell extract. While both the leading-front assay and the radiolabel assay give accurate results, the measurement of radiolabeled cells allows one to test a greater number of attractants in one assay and the procedure eliminates observer bias.