CD45-mediated regulation of extracellular calcium influx in a CD4-transfected human T cell line.

Transfection of a CD4- Jurkat leukemic T cell line with the human wild-type CD4 gene resulted in the constitutive mobilization of calcium by these cells. The altered calcium phenotype was dependent on expression of a completely functional CD4 molecule on the cell surface. Transfectants receiving the vector alone or those in which a mutated CD4 gene lacking a functional Lck binding region failed to generate a constitutive calcium response. In addition, CD4 wild-type transfectants over time lost CD4 expression. These CD4- revertants failed to constitutively mobilize calcium. Treatment of CD4 wild-type transfected cells with either anti-CD45 mAb, EGTA, or PMA rapidly restored the cells to basal levels of intracellular calcium. Analysis of CD45 cross-linking on CD4+ and CD4- normal Jurkat lines demonstrated that CD4 expression was required for CD45-mediated inhibition of TCR induced calcium responses. CD45-mediated inhibition affected the duration of the response rather than its magnitude. These results, taken together with the observations obtained with CD4 transfected Jurkats suggested that influx of extracellular calcium was the predominant reason for the elevated levels of calcium within the cell. In support of this hypothesis, we could find no evidence of phosphorylated phospholipase C gamma 1 or constitutive inositol 1,4,5 trisphosphate generation in the CD4 wild-type transfected cells, yet both were detectable after anti-CD3 mAb-induced activation. Immunokinase assays of Lck and Fyn precipitated from untreated or anti-CD45-treated wild-type transfectants demonstrated that CD45 cross-linking dephosphorylated Lck and reduced its capacity to phosphorylate enolase. In contrast, neither Fyn autophosphorylation nor its activity was affected by CD45 cross-linking.

Characterization of rabbit surfactant-associated proteins.

In the present study, the apolipoproteins associated with a purified surfactant fraction isolated from lung lavage of adult rabbits were characterized. Surfactant purity was assessed by the glycerophospholipid composition and by electron microscopic examination. The purified surfactant was delipidated and the apolipoproteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. By use of this technique at least eight proteins or families of proteins were found to be associated with surfactant. Four of these apolipoproteins were families of proteins of 55-70, 29-36, 26-28 and 22-23 kilodaltons (kDa). All of these apolipoprotein families had acidic isoelectric points (pI less than or equal to 5.6), and were specifically bound to a Con A-Sepharose matrix, indicative that these apolipoprotein families are modified by oligosaccharide side-chains. The finding that neuraminidase treatment degraded the 29-36 kDa family is suggestive that this apolipoprotein family contains sialic acid residues. Three major proteins of 66, 43-45 and 35 kDa and a minor protein of 86 kDa were also observed. These proteins had isoelectric points in the more neutral range (pI 6.0-6.5). The 66 kDa protein (pI 6.4) had the same apparent molecular weight and isoelectric point as the major protein of delipidated rabbit serum and as purified rabbit albumin, which suggests that this protein is albumin. These findings are indicative that the apolipoproteins of surfactant are more numerous and complex than previously reported.