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Mammary carcinoma tissue from 514 primary breast cancer patients were all biochemically and histochemically analyzed for both estrogen receptors and progesterone receptors. The dextran-coated charcoal (DCC) method measured the ER and PR as defined by Scatchard analysis, ligand competition experiments and target organ specificity. The ligands, estradiol-6-carboxymethyloxime-BSA-fluoresceine isothiocyanate and hydroxyprogesteronehemisuccinate-BSA-tetramethylrhodamine isothiocyanate, used for histochemistry, did not bind to either ER or PR and were mainly bound to the membrane fraction of isolated breast cancer cells. Fluorescence was not specifically inhibited by estrogens or progestogens. In addition, "estrogenic" always coincided with "progestogenic" fluorescence. The binding of the fluoresceine compounds to tissue slides depended on the large steroid hormone substitution on the bovine serum albumin molecule. Clinical parameters, known to be related to ER and PR did not correlate with the histochemical results. The observations indicated the impossibility of specific steroid receptor detection by the histochemical method. Therefore, up to the present, evaluation of hormone dependency and prognosis in human breast cancer cannot be based on this approach.

Crystal growth in native collagen gels has been used to determine the role of extracellular matrix macromolecules in biological calcification phenomena. In this system, type I collagen gels containing sodium phosphate and buffered at pH 7.4 are overlayed with a solution containing CaCl2. Crystals form in the collagen gel adjacent to the gel-solution interface. Conditions were determined which permit the growth of crystals of hydroxyapatite [Ca10(PO4)6(OH)2]. At a Ca/P molar ratio of 2:1, the minimum concentrations of calcium and phosphate necessary for precipitation of hydroxyapatite are 10 mM and 5 mM, respectively. Under these conditions, precipitation is initiated at 18-24h, and is maximal between 24h and 6 days. Addition of high concentrations of chondroitin 4-sulphate inhibits the formation of hydroxyapatite in collagen gels; initiation of precipitation is delayed, and the final (equilibrium) amount of precipitation is decreased. Inhibition of hydroxyapatite formation requires concentrations of chondroitin sulphate higher than those required to inhibit calcium pyrophosphate crystal formation.

In an antithrombin-III (AT-III) deficient patient suffering from recurrent episodes of venous and arterial thrombosis requiring major surgery an attempt was made to institute antithrombotic protection by long-term stanozolol treatment supplemented during periods of thrombogenic exposure with subcutaneous heparin and, when needed, infusion of AT-III as plasma or concentrate. Stanozolol raised the plasma levels of AT-III, demonstrating a sparing effect on the AT-III needed. Despite the repeated exposures to major surgery, protection against venous thrombosis was complete, but the arterial disease progressed and led to the demise of the patient.

The influence of Zn+2 on fibrin clot formation was investigated by measuring its effect on the clotting times of fibrinogen exposed to thrombin. It was observed with either human or bovine thrombin that 0.01-0.1 mM Zn+2 induced significant reductions of clotting times in a concentration-dependent manner. The procoagulant effect of Zn+2 occurred in the presence of Ca+2 but was inhibited by metal chelating agents. Higher levels of Zn+2 (greater than 0.2 mM final concentration) were required to accelerate thrombin-induced clot formation in the presence of citrate or oxalate. Similarly with oxalated human plasma, greater than 0.2 mM Zn+2 decreased the clotting time. Cations such as Mg+2 and Mn+2 caused little change in clotting times. As an extension of these findings, we examined the effect of Zn+2 on the inhibition of thrombin by antithrombin-III (AT-III). The presence of as little as 0.006 mM Zn+2 in an incubating mixture of thrombin and AT-III severely reduced the inhibitory activity of AT-III towards thrombin. It was observed that the relative intrinsic fluorescence emission of human thrombin decreased upon exposure to Zn+2 but was unaffected by Mg+2 or Mn+2. It is suggested that Zn+2 can form a complex with thrombin, which results in altered reactivity towards fibrinogen and decreased inhibition by AT-III.