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Four monoclonal antibodies with distinct epitopes were prepared against antithrombin III. None of them is directed against the heparin-binding region nor the active site, yet two mAb namely A36 and B108, interfere with antithrombin III inhibition of thrombin. The epitope of monoclonal antibody A36 is located within amino acid residues 1-393, at a site different from the active site since it recognizes antithrombin III and antithrombin-III-thrombin complexes with the same affinity. A36 partially prevents the intrinsic antithrombin III activity and has no effect on the heparin-enhanced antithrombin III activity when added to the antithrombin-III--heparin complex. If A36 is first reacted with antithrombin III and then heparin is added to the reaction mixture, A36 fixes the conformation of antithrombin III so that heparin binds to antithrombin III, but is not able to induce the conformational change in the antithrombin III molecule required for the enhanced activity. The epitope for monoclonal antibody B108 is located within residues 282-393, close to the active site. It does not recognize antithrombin-III-thrombin complexes by solid-phase radioimmunoassay. Its binding to antithrombin III induces a conformational change that enhances antithrombin III activity in a manner that resembles the heparin effect, but its effect is additive to the heparin effect, since when it was added to a reaction mixture which contained a saturating amount of heparin, inhibition of thrombin was enhanced. The epitope for monoclonal antibody A5 is located within residues 1-393, and its recognition of antithrombin III or antithrombin-III-thrombin is strongly dependent on the integrity of the disulfide bonds. A5 has no effect on antithrombin III activities. The epitope for monoclonal antibody A10 is well defined within a narrow range of 55 amino acid residues, 339-393, on the antithrombin III molecule, close to the active site, yet it has no effect on antithrombin III inhibitory activity. These monoclonal antibodies may be developed for various diagnostic or clinical purposes and offer a powerful tool for studying the conformational changes and structure/activity relationships in the antithrombin III molecule.

Expression of the pS2 gene which is transcriptionally controlled by oestrogens in the breast cancer cell line MCF-7 is oestrogen independent in stomach mucosa. We show here that the level of MCF-7 cell pS2 mRNA can also be increased by the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). We further demonstrate, using transient transfection assays, that the -428 to -332 5' flanking sequence of the pS2 gene contains DNA enhancer elements responsive to oestrogens, TPA, EGF, the c-Ha-ras oncoprotein and the c-jun protein.

A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), strongly stimulates the activity of polyomavirus enhancer in a human erythroleukemia cell line, K562. The target of stimulation was the previously defined A element (from nucleotides 5107 to 5130) of the enhancer. We found that within the A element, two partly overlapping sequence motifs (one from nucleotides 5107 to 5117, the other from nucleotides 5113 to 5121) were independently the targets of TPA stimulation. The former is homologous to the enhancer core sequence of the adenovirus type 5 E1A gene, and the latter shares the consensus AP-1-binding site. In addition, transiently expressed Ha-ras oncogene also stimulated these two subelements in K562 cells, as we reported for NIH 3T3 cells previously.

The relationship between growth signals and transcriptional activator proteins was studied using polyomavirus enhancer as a probe. Transiently expressed Ha-ras gene and a tumor promoting phorbol ester, TPA, strongly stimulated the activity of polyomavirus enhancer in NIH3T3 cells. In both cases, the target of this stimulation was a 24 base pair long A core. At least two nuclear factors, PEBP1 and 2, bind to this core region. The target of stimulation in both cases was the recognition sequence of PEBP1 which is an AP1 consensus sequence. In nuclear extract of NIH3T3 cells stably transformed by Ha-ras gene, however, binding of neither PEBP1 nor PEBP2 was detected. Instead a new factor, PEBP3, emerged to share the binding site with PEBP2. PEBP3 was purified and found to be composed of 2 subunits, alpha and beta. Each of these subunits binds to the same sequence as that of PEBP3. PEBP3 binds to B core, as well as to A core. Preliminary evidence suggests that PEBP2 has an unidentified subunit in addition to alpha and beta. Proper phosphorylation required for PEBP1 for DNA binding and PEBP2 converts to PEBP3 in under-phosphorylation conditions. A repressor, PEBP4, has been identified which partly shares the recognition sequence with PEBP2. This factor is present in F9 embryonal carcinoma cells as well as in those induced to differentiate. On the other hand, neither PEBP1 nor PEBP2 were detected in F9 cells. Both of them became detectable after differentiation. Based on these results, a hypothesis was proposed for developmental regulation and alteration of such regulation in cancer cells.

Dependence of N,N'-dicyclohexylcarbodiimide (DCC)-sensitive H+ secretion of K+ activity was discovered. This dependence took place only in anaerobically grown bacteria, and only at the structural intact DCC-sensitive H+-ATPase complex and K+-ionophore Trk.

245 breast cancer tissues were analysed immunohistochemically, using monoclonal antibody Ki-67, which spezifically reacts with a nuclear antigen of proliferating cells and represents the so-called growth fraction. According to the number of positive cells, three Ki-score categories (I-III) were established. The results were compared with prognostic variables (Histological grading, DNA-cytophotometry), staging parameters, immunocytochemical (ER-ICA, mPRI), and biochemical (DCC) hormone receptor assay. We found a good correlation between Ki-67 and grading (Bloom and Richardson): G 1-tumors had small growth fractions, in contrast to G-III carcinoma, which showed a high percentage of Ki-Score III. Receptor negative cases had a large growth fraction and receptorpositive ones showed an uniform distribution. The comparison between Ki-67 and tumor spread or DNA-Cytophotometry showed no correlation. The majority of 31 recurrent tumors not only had a large number of positive cells, but also a definite dependence was found between Ki-Score and length of the disease-free period. Ki-67 seems to be an important immunohistochemical marker of breast cancer, which contributes to the individual assessment of the disease and can possibly give predictions on tumor development and response to radiation and systemic therapy.

Anemia is an almost invariable feature of chronic renal failure and is particularly severe in children treated by long-term hemodialysis. Recombinant human erythropoietin (rhEPO) offers entirely new aspects in the treatment of renal anemia. This report presents three patients on maintenance hemodialysis aged 10, 10/10 12, and 18 years who were treated with rhEPO. Two suffered from hemochromatosis secondary to multiple transfusions. 100 U/kg rhEPO were administered three times weekly, and venesection after dialysis was performed when a target hematocrit value of 30% was achieved. Hematocrit, reticulocyte-counts and hemoglobin rose within 3 to 6 weeks after initiation of therapy in all patients. Serumferritin levels declined significantly in the two patients with hemochromatosis. No deterioration of the metabolic status (i.e. increase of blood urea nitrogen, serum-creatinine, -phosphate or -potassium) could be detected. Therapy had to be discontinued in one patient who experienced hypertensive ceisis. This patient, however, had suffered from severe hypertension prior to rhEPO therapy. Blood pressure remained stable in the other patients. We conclude that renal anemia can be effectively treated by rhEPO in children. Increase of blood pressure may necessitate discontinuation of therapy especially in primary hypertensive patients. Extensive studies are necessary to eluciate long-term effects of rhEPO in children.

The synergistic effect of antithrombin III (ATIII), activated protein C (APC) and heparin on the tissue thromboplastin (TP)-mediated coagulation cascade was studied. APC prolonged the activated partial thromboplastin time of human plasma with increasing APC concentration but affected the prothrombin time only slightly. Neither ATIII nor heparin prolonged the prothrombin time by itself, while a mixture of APC and heparin strongly inhibited the coagulation. When the effects of APC, heparin and ATIII on the TP-mediated coagulation were examined with a solution consisting of prothrombin, factor V, factor VII, factor X and fibrinogen at physiological concentrations, the coagulation time was prolonged only slightly by the APC-heparin or ATIII-heparin mixture. However, the coagulation time was prolonged markedly by simultaneous addition of APC, ATIII and heparin to the solution. Inhibition of thrombin activity by the ATIII-APC-heparin mixture was weak as compared with that by the ATIII-heparin mixture after a 1-min incubation, but after a 2-min incubation the inhibitory activities were equal. Suppression of thrombin activity by the ATIII-APC-heparin mixture was supposed to be due to the inhibition of the interaction between ATIII and heparin on thrombin by APC because the APC-ATIII-heparin complex was detected by crossed immuno-electrophoresis but not by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis. When the inhibitory effect of APC alone or the APC-heparin mixture on the platelet prothrombin converting activity (PPCA) was examined, heparin accelerated the PPCA inhibition by APC.(ABSTRACT TRUNCATED AT 250 WORDS)