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Recently we reported the isolation and partial biochemical characterization of a novel polypeptide, h3, from the human brain and liver. Thin-layer isoelectric focusing showed that the polypeptide was ubiquitously distributed throughout the human brain. Immunophosphatase transfer electrophoresis showed that this protein was localized in several mammalian species and different tissues. In addition, h3 or h3-like protein was demonstrated in subsets of tissues from one avian species. Protein h3 was present in epithelial and muscular tissue, as well as in nervous tissue; however, for all species investigated, it was most abundant in CNS and muscle.

The aim of this study was to evaluate the results obtained from a new enzyme immunoassay (Abbott-ER-EIA) for the determination of estrogen receptor levels in tumor cytosols in comparison with the currently used DCC method. One hundred and fifteen consecutive primary breast cancer specimens were examined; 66 of the women were postmenopausal and 49 were premenopausal. A good correlation (r = 0.88, p less than 0.001 and a slope of 1.3) was found between ER-EIA and the steroid binding assay (DCC). When these data were analyzed according to menopausal status, no differences were observed for the slopes and correlation coefficients in pre' and postmenopausal groups. The ER-EIA appears to produce results comparable to those obtained with the conventional DCC method for the determination of ER in breast tumor cytosols.

The coordinate expression of the nuclear p53 protein, cytoplasmic intermediate filament vimentin (VIM) and membrane epidermal growth factor receptor (EGF-R) was significantly associated with oestrogen receptor immunocytochemical nuclear stain (ER-ICA) negative breast carcinomas. Twenty-three (51.1%), 26 (57.8%) and 27 (60%) of 45 ER-ICA -ve cancers were respectively p53 +ve, VIM +ve and EGF-R +ve; whereas of 151 ER-ICA +ve tumours 8 (5.3%) were p53 +ve (P less than 0.0001), 23 (15.2%) VIM +ve (P less than 0.001) and 40 (26.5%) EGF-R +ve P less than 0.001). Thirty-six of 45 (80%) ER-ICA -ve carcinomas were positive for at least one of the markers versus 55/151 (36.4%) ER-ICA +ve cases (chi 2 = 28.92, P less than 0.001). A prevalence of high grade carcinomas was found among p53 +ve, VIM +ve cases; the latter subset of tumours also had a larger mean diameter. These results suggest that ER -ve breast carcinoma cells display a coordinate expression of cell cycle-related proteins and marked changes of both the cytoskeleton and the membrane receptor repertoire.

Antithrombin Rouen-II, a new inherited variant of antithrombin-III, was found in two members of a family with no definite history of thrombosis. The subjects had normal antigenic concentrations of antithrombin and normal progressive inhibitory activity. However, the variant had defective heparin and heparan sulfate cofactor activities, and was not activated by a synthetic pentasaccharide representing the minimum heparin sequence. The abnormal antithrombin was isolated using heparin-Sepharose chromatography, and on electrophoresis at pH 8.6 migrated more anodally than normal. Two-dimensional peptide mapping of tryptic and Staphylococcus aureus V8 protease digests was performed and the abnormal peptide was located by tryptophan staining. Amino acid sequence studies demonstrated a substitution of arginine at residue 47 by a serine. Evidence strongly suggests that arginine 47 is a prime heparin binding site in antithrombin and that it forms part of a proposed positively charged linear site (to which heparin binds) that stretches across the surface of the molecule from the A to the D helix.

Hereditary deficiency of antithrombin-III (AT-III), the major heparin cofactor in human plasma, is a well-established cause of recurrent venous thrombosis. Cross-reactivity of heparin cofactor II (HC II) in assays of AT-III may, in some cases, interfere with the ability to diagnose hereditary deficiency of AT-III. For that reason, we have evaluated the interference by HC II in the new DuPont aca antithrombin assay. Response of the assay to purified AT-III and HC II was compared. Inhibition of the bovine thrombin in the assay was sixfold less per molecule of HC II than of AT-III. This level of selectivity should be adequate to prevent misdiagnosis of patients. Analysis of patient samples showed close correlation (r = 0.91) of values from the automated aca assay with those of a manual assay (Coatest antithrombin, Helena Laboratories).

The abnormal Antithrombin-III (AT-III) Toyama has no ability to interact with heparin in the mast cells or heparin-like glycosaminoglycans on the surface of vascular endothelial cells, so that it has low heparin cofactor activity. In the present study, we have measured the binding capacity of 125I-labeled AT-III to cultured porcine aortic endothelial cells and estimated the inactivation of thrombin by AT-III in the presence of endothelial cells. The abnormal AT-III Toyama had low binding capacity to endothelial cells and had lower heparin cofactor antithrombin activity in the presence of endothelial cells than that of normal AT-III. Our results suggested that heparin-like glycosaminoglycans on endothelial cells play as important a role as heparin does in the regulation of antithrombin activity.