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By the conventional steroid-binding assay method for receptor, 3% of 1,095 primary breast cancers (or 10.6% of 263 premenopausal tumors) were classified as negative for estrogen receptor (ER), but positive for progesterone receptor (PR). The true ER status in this rare group of tumors was further investigated by the enzyme-immunoassay (EIA) or immunocytochemical (ICA) staining method using monoclonal antibodies H222 and D547. Immunoreactive ER was present in nine ER-/PR+ tumors studied, whereas it was not detectable in nine age-matched ER-/PR- tumors. Immunoreactive ER was also present in 24 ER+ breast cancers studied, and was particularly higher in tumors that were PR+. Measurement of immunoreactive ER by monoclonal antibody method provides certain advantages over the conventional dextran-coated charcoal (DCC) method, especially in ER-/PR+ tumors.

The authors analyze estrogen receptors in 501 cases of breast cancer and the therapeutic response achieved with treatment of patients who relapsed. The method utilized to determine the presence of estrogen receptor was cytosol protein-counting and Dextran coated charcoal (DCC) saturation analysis. Patients were treated with chemotherapy when the primary tumor was estrogen-receptor negative. If receptor was present, anti-estrogen drug (Tamoxifen) was added. Patients previously treated with the anti-estrogen presented a better response to anti-estrogen therapy and a lower recurrence rate. Recurrences were more frequent in estrogen receptor negative cases and better responses to treatment were found in estrogen receptor positive ones.

The sex pheromone inhibitor iPD1, which is excreted by Streptococcus faecalis donor strains harboring bacteriocin plasmid pPD1 and which inhibits sex pheromone cPD1, was isolated, and its structure was determined. Its molecular weight is 828, and its amino acid sequence is H-Ala-Leu-Ile-Leu-Thr-Leu-Val-Ser-OH.

The antithrombin III (ATIII) isoform pattern of a number of serum and plasma samples was analysed by isoelectric focusing and immuno-blotting. A novel ATIII isoform pattern which was observed in 4/80 children with acute lymphatic leukaemia (ALL) and in 1/4 children with Ewing's sarcoma, has been shown by family studies to be due to a mutant form of ATIII (AT Dublin) in the heterozygous state. The coagulation properties of AT Dublin heterozygotes were normal. In addition the immunological and activity levels of their ATIII were normal. The effects of thrombin and heparin on the mutant ATIII were similar to controls. Neuraminidase treatment reduced the ATIII isoforms to one in controls and two in the mutant. Two-dimensional gel analysis showed the mutant ATIII to have an identical molecular size distribution to the normal form. This mutant is, thus, most likely due to an amino acid substitution giving a more basic molecule that is clinically silent (at the coagulation level). It may be of interest that the frequency of AT Dublin in the ALL group is significantly higher than in the control group (3/430) studied (P less than 0.001).

To test the hypothesis that steroid hormone receptor expression is particularly pronounced in breast tumours when compared with non-neoplastic tissue, mastectomy samples were divided into 16 sectors. Multifocal tumours, of varying receptor phenotype were found in 4 patients and in addition different regions of large tumours also showed varying receptor contents. Remaining samples were found to consist of normal tissue, with fat, connective tissue and some benign breast disease. In the 9 patients with oestrogen receptor positive tumours (ER), ER content was invariably much greater in the tumours than in the remainder of the breast. Progesterone receptor (PR) content was not closely related to ER, and was lowest in the poorly differentiated tumours. This relation to differentiation was not seen in ER. The data support the view that ER concentration in ER positive tumours may reflect the transformed nature of neoplastic tissue.

To determine the interaction of platelet factor 4 (PF4) and protamine sulfate in the neutralization of heparin in plasma in vitro studies were carried out using a tritium-labeled heparin and a PF4 tagged with 14C. Plasmas treated with various combinations of PF4, protamine and heparin were chromatographed on Sephadex G200 and the fractions were tested for both radioactivity and antithrombin activity. PF4 was comparable to protamine in its ability to neutralize heparin, but the complexes formed with heparin were different. In contrast to protamine, when heparinized plasma was treated with an excess of PF4, no large PF4-heparin complexes were formed and none of the PF4-heparin complexes which did form were able to activate antithrombin III (ATIII). Also, incubation of PF4-neutralized, heparinized plasma at 37 degrees C did not result in liberation of heparin and prolongation of the thrombin clotting time as was found with protamine-neutralized plasma. The action of protamine and PF4 is complimentary. When half the neutralizing dose of each was added together to heparinized plasma, no immediate antithrombin activity remained. When a neutralizing dose of protamine was added to PF4-neutralized, heparinized plasma, the protamine displaced the PF4 from its complexes with heparin. The large protamine-heparin complexes which formed also contained PF4 but could not activate fresh ATIII as has been demonstrated with protamine-heparin complexes without PF4. On incubation of the protamine-PF4-neutralized, heparinized plasmas for 5 hours at 37 degrees C, the large complexes were broken down but no active heparin appeared. The results of these experiments may have some bearing on the amount of protamine needed for the neutralization of heparin following extracorporeal bypass procedures, when large amounts of PF4 may have been released from activated or disrupted platelets.

As part of our continuing search for new agents which might be useful for the treatment of sickle-cell anemia, we have synthesized two cyclic tetrapeptide homologs, cyclo(-Val-Glu[-Thr-Pro-]-OH) (1a) and cyclo(-Phe-Glu[-Thr-Pro-]-OH (1b), and a tetrapeptide lactone homolog cyclo(H-Thr-Pro-Val-Glu-OH) (2). The intent was that these peptides would mimic a tetrapeptide region around the mutation site of HbS and thus be able to bind at the acceptor site of HbS and thereby inhibit polymerization. The synthesis of the linear peptides was accomplished in solution using both the polymeric reagent (PHBT) and DCC/HOBT methods; cyclization was accomplished by an improved method. 13C-n.m.r. studies were performed which allowed us to assign the conformation about the Thr-Pro bond in 1a and 2 as trans. The cyclic peptides were tested for their ability to increase the solubility of HbS under deoxygenating conditions, but only 1a had any antigelling activity, albeit low.

A methodology, originally built up in our laboratory, for the simultaneous determination of estrogen (ER) and progesterone (PR) receptors on small samples of tumor tissues, has been adapted to fine needle aspirates (FNA) of breast tumors. The method is based on a simultaneous incubation of aliquots of high-salt (0.4 M KCl) cytosols with tritiated estrogen and progestin tags. The mixture of receptor-bound ligands is isolated by dextran-coated charcoal and extracted by ethanol. The extracted ligands are separated quantitatively by HPLC and counted by liquid scintillation. FNA provides sufficient cellular material for ER and/or PR assay by single point (5 nM) dextran-coated charcoal (DCC) assay. FNA samples, being contaminated by blood, [3H] R 2858 and [3H] O 2058 are appropriate tags for ER and PR respectively, giving lowest non-specific binding. Sixty-five simultaneous estrogen and progestin receptor determinations on FNA at time of diagnosis were compared to the individual assays performed on the surgical sample obtained from the same patients a few weeks after diagnosis. The correlation between the 2 sets of determinations is excellent in 60 cases with sufficient FNA cellularity. This correlation validates the reliability of estrogen and progestin receptor determination on FNA in the majority of clinical cases.