Interaction of phenylisothiocyanate with human erythrocyte band 3 protein. II. Topology of phenylisothiocyanate binding sites and influence of p-sulfophenylisothiocyanate on phenylisothiocyanate modification

The two structurally related probes, the apolar phenylisothiocyanate and the polar, water-soluble p-sulfophenylisothiocyanate, were analysed for their topological interaction with human erythrocyte band 3 protein. Upon thermolytic and peptic digestion of labeled erythrocyte ghosts, the membrane-integrated segments of band 3 protein, the 17,000 and 10,000 dalton peptides, were isolated. At 2 mM initial label concentration, 90% of the hydrophobic probe phenylisothiocyanate was recovered in the 10,000 dalton peptide, the remaining amount of label being associated with the 17,000 dalton fragment. Pretreatment of the membranes with 5 mM p-sulfophenylisothiocyanate followed by labeling with 2 mM phenylisothiocyanate results in a consistent reduction in binding of phenylisothiocyanate by 1 mol/mol isolated band 3 protein. p-Sulfophenylisothiocyanate reportedly binds to the 17,000 dalton fragment (Drickamer, K. (1977), J. Biol. Chem. 252, 6909-6917). The interaction of the polar probe with the membrane protein affects binding of phenylisothiocyanate to the 10,000 dalton peptide by the equivalent of 1 mol/mol isolated peptide. The topological interrelation of the membrane-integrated segments is concluded.     

Interaction of aluminium and gallium with human lymphocytes: the role of transferrin

Aluminium-transferrin (Al-Tf) and gallium-transferrin caused a dose-dependent decrease in proliferation of human peripheral blood lymphocytes cultured for 3 days with phytohaemagglutinin (PHA). Addition of apotransferrin reduced the inhibitory effect. Al added as AlCl3 or aluminium citrate had no effect, and there was no significant difference in the response of cells from renal failure patients with or without high serum Al levels or controls. Lymphocytes cultured in the presence of Al-Tf showed a dose-dependent uptake of Al, whereas uptake from aluminium citrate was low and not dose-dependent. Uptake from AlCl3 was very high but probably involved a nonspecific uptake mechanism. Levels of Al in freshly isolated lymphocytes were approximately 1.6 ng/10(6) cells, there being no difference between cells from patients and controls. It is concluded that Al, when bound to transferrin, may have a detrimental effect on lymphocyte function and might contribute to the decreased immune responsiveness of renal failure patients on haemodialysis. However, lymphocyte Al levels are probably not useful as a marker of Al overload in such patients.     

Molecular screening of partners of cystic fibrosis heterozygotes.

We have screened 100 partners of known or suspected CF heterozygotes for ten CF mutations which account for 88% of the CF mutations seen by us on CF chromosomes. We have identified six CF heterozygotes amongst the 100 low-risk people screened. As two of the six people at high-risk of being CF carriers were subsequently shown not to be CF carriers this gave rise to four couples with a one in four risk of CF in a pregnancy and so far to two PND's. The risk of CF in the remaining 94 couples was reduced to less than one in 800. We have concentrated on the screening of partners for the commoner CF mutations rather than haplotyping them for the CF linked markers XV-2c/TaqI and KM19/PstI which are in linkage disequilibrium with CF. For individuals shown not to carry these ten mutations, a five fold difference in risk of being a CF carrier remains between those who have the XV-2c/KM19 genotypes associated with the highest risk(BB), as compared to those with the lowest risk(CC). Nevertheless we feel that effort is better expended in mutation screening rather than haplotyping.     

IL-2 induction of IL-1 beta mRNA expression in monocytes. Regulation by agents that block second messenger pathways

We have previously shown that in mixed cultures of PBL incubation with human rIL-2 induces the rapid expression of IL-1 alpha and IL-1 beta mRNA. Because studies have demonstrated that IL-2R can be expressed on the surface of human peripheral blood monocytes, we chose to investigate whether IL-1 beta mRNA could be directly induced in purified human monocytes by treatment with Il-2 and, if so, to analyze the second messenger pathways by which it may be controlled. Human monocytes do not spontaneously express IL-1 beta mRNA, but can express the gene as soon as 1 h after treatment with IL-2. The level of IL-1 beta mRNA induced by IL-2 at 5 h in human monocytes was about one-fourth that induced by LPS. LPS induction of IL-1 beta mRNA in human monocytes can be blocked by either an inhibitor of protein kinase C (PKc) 1-(5-isoquinolinesulfonyl)-2-methylpiperazine or an inhibitor of calcium/calmodulin (CaM) kinase N-(6-aminohexyl) 5-chloro-1-naphthalenesulfonamide, suggesting that both PKc and CaM kinase are involved in transducing signals initiated by LPS. In contrast, IL-2 induction of IL-1 beta mRNA expression is blocked only by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, suggesting that PKc, and not CaM kinase, is activated by IL-2. These data suggest that overlapping but distinct second messenger pathways are involved in the transduction of signals initiated by IL-2 and LPS.