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Samples of human breast cancer tissue were analyzed for estrogen receptor content (ER) with a hydroxylapatite method (HAP) as well as a dextran-coated charcoal method (DCC). It was found that both methods revealed artificially low ER concentrations in cytosols with low protein concentration. It was also found that the DCC assay underestimated the ER activity in the tumors compared to the HAP assay. No differences in terms of low affinity binding nor Kd-values of high affinity estrogen binding were observed. A significant higher background radioactivity was obtained in the HAP assay, and in some tumors one assay would classify them as ER-negative and another as ER-positive.

Normal mouse spleen cells are not capable of mounting a primary cytotoxic T lymphocyte (Tc) response to non-H-2 alloantigens in vitro, although a good secondary H-2-restricted response is observable after in vivo immunization of the responder animals. Suppressor cells are generated in such a primary responses provided a Mls incompatibility exists between the responder and stimulator. These suppressors are not antigen specific, are Thy-1+, Lyt-1+, 2-, I-J-, and are highly radiosensitive. The suppressor cell precursors in normal spleen express the same phenotype. These suppressor cells are probably implicated in the lack of a primary Tc response in a primary mixed lymphocyte reaction across non-H-2 incompatibilities that include an Mls difference.

To investigate whether recurrent mutation has contributed to the high frequency of the beta E-globin gene in Southeast Asia, we used the haplotypes at three polymorphic restriction sites within and to the 3' side of the beta-globin gene to predict the framework of 23 beta E-globin genes. These haplotypes suggested that beta E-globin genes are present in two different beta-globin gene frameworks. DNA sequence determination of one gene representing each framework demonstrated that the same mutation (GAG leads to AAG at codon 26) was present in both frameworks. Moreover, the frameworks differed at three nucleotide positions known to be polymorphic in Mediterraneans. These polymorphic sites are located 70 nucleotides to the 5' side of the beta E mutation and 382 and 1032 nucleotides to the 3' side of it. The existence of the beta E mutation in these two beta-globin gene frameworks can be explained by (i) recurrent mutation giving rise to beta E-globin, (ii) a double crossing-over event, or (iii) two single crossing-over events. Mathematical analysis suggests that the first alternative, recurrent mutation of G leads to A at the first nucleotide of codon 26, is most likely.

No abstract available.

Factor VII clotting activity increases about five-fold when blood is clotted in glass. Prior studies suggested that this results from activation induced by activated factor IX (IXa). However, in purified systems containing phospholipid and calcium, activated factor X (Xa) is known to activate factor VII rapidly. Therefore, we studied activation of factor VII by IXa and X, in systems using purified human factors. Concentrations of IXa and Xa were calculated from total activated protein concentrations rather than from active site concentrations. In the presence of phospolipid and calcium, both IXa and Xa activated factor VII 25-fold; however, Xa was roughly 800 times more efficient than IXa. Without added phospholipid, activation of factor VII by both Xa and IXa was markedly slowed, and Xa was roughly 20 times more efficient than IXa. When both phospholipid and calcium were omitted, activation of factor VII by either enzyme was negligible. Adding normal prothrombin, but not decarboxylated prothrombin, substantially slowed activation of factor VII by both Xa and IXa. Adding thrombin-activated factor VIII and antithrombin-III did not change rates of factor VII activation by either enzyme. These results from purified systems do not provide an explanation for the prior data from plasma systems.

Standard unfractionated heparin is known to have two actions on blood clotting. Unfractionated heparin enhances the rates at which antithrombin III inactivates activated clotting factors, and inhibits the activation of both Factor X and prothrombin by disrupting the calcium and phospholipid dependent assembly of the Factor X and prothrombin activator complexes. This latter inhibitory action of heparin occurs independently of antithrombin III. A heparin fraction with low affinity to antithrombin III was prepared from standard heparin by affinity chromatography on antithrombin-III-Sepharose and its properties compared with unfractionated heparin. The low affinity heparin fraction and the unfractionated heparin had equivalent inhibitory effects on prothrombin activation in antithrombin III depleted plasma. In normal plasma, the low affinity fraction inhibited the activation of prothrombin. Unlike the unfractionated heparin, however, the fraction of heparin with low affinity to antithrombin III did not enhance the inactivation of either Factor Xa or thrombin. This antithrombin III independent inhibition of the activation of prothrombin was also evident when activated platelets were used as the source of the procoagulant phospholipids. The antithrombin III independent effect of heparin is unlikely to be important therapeutically, however, if this property of heparin is shared by other naturally occurring glycosaminoglycans, it could be important in maintaining the fluidity of blood under physiological conditions.