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Estrogen receptors (ER) were evaluated in 634 breast cancer patients by the dextran-coated charcoal method (DCC). In 206, ER and progesterone receptors (PR) were also tested by cytochemistry (Lee method), and in 124 ER were tested by immunofluorescence (Pertschuk method). The median follow-up is 3.7 years. Comparisons have been made between receptor content and: anatomical and clinical features, disease-free survival (DFS), and survival. The following conclusions can be drawn: there is no correlation between ER determinations by DCC and by immunofluorescence or cytochemical methods; there is no evidence of association between ER and PR determined by Lee's method and anatomical and clinical features; a highly significant positive association was found between ER rich specimens and age, post-menopausal status, lobular and tubular histologic types; there is no association between ER values and TNM stage, WHO grading, pathologic prognostic factors of primary tumor and of lymph nodes; and the DFS was not affected by ER status, except for tumors with more than 50 fmol/mg protein.

Estrogen receptor determinations were performed on 241 cytosols from 160 breast cancer tumors, using both dextran coated charcoal method (D.C.C.) and monoclonal antibodies (Abbott's ER-EIA kit) in order to compare the two methods, and to evaluate the clinical usefulness of this new immunological simplified assay. Intra- and inter-assay reproducibility were studied during a six month period. Intra-assay of both methods were lower than 5%. Inter-assay variation coefficients of ER-EIA studied on 35 standard curves varied from 12.5% (standard: 0) to 6.7% (standard: 250 fmoles/ml). ER determination of 80 human breast cancers were performed both by EIA and Scatchard analysis. The regression curve obtained was (EIA) = 1.04 (Scatchard) + 21 fmoles/mg of proteins (r = 0.963). With 153 breast cancer cytosols whose volume was too small for multipoint Scatchard analysis, EIA results were compared to results obtained by a "near saturating" concentration of tritiated ligand (5 nM). The regression curve obtained was (EIA) = 1.34 (5nM) + 5 fmoles/mg proteins (r = 0.978). These results can be compared with those obtained between Scatchard and 5 nM: (Scatchard) = 1.29 (5nM) - 9 fmoles/mg proteins (r = 0.985). Reproducibility was studied on clinical specimens assayed at two different periods during the clinical evaluation. Regression curves obtained were (2nd assay) = 1.05 (1st assay) - 5.5 fmoles/mg proteins for EIA, and (2nd assay) = 0.96 (1st assay) + 9 fmoles/mg proteins for DCC method. EIA assay presented an high stability for protein concentrations very low, up to 0.2 mg/ml. Finally, a very good correlation was obtained between ER-EIA and DCC method, and ER-EIA seemed specially well fitted to small tumors.

Human testosterone-estradiol-binding globulin (hTeBG) has been purified to apparent homogeneity by several laboratories using procedures which, in most instances, were labor intensive. In this report, hTeBG was purified from pregnancy serum by a newly developed two step procedure involving sequential affinity chromatography and ion-exchange high performance liquid chromatography (ion-exchange HPLC). The purity of the final product was confirmed by silver stained SDS-polyacrylamide gel and reverse phase HPLC monitored at 206 nm. hTeBG purified by ion-exchange-HPLC maintained binding activity by Dextran coated charcoal (DCC) assay and size heterogeneity on SDS-polyacrylamide gels which were indistinguishable from those of the proteins purified by conventional chromatography. Removal of the carbohydrate moiety from the molecule by both enzymatic and chemical treatment reduced the apparent molecular size and eliminated lectin binding of hTeBG subunits. Deglycosylation did not, however, abolish or alter the distribution of the protomeric forms of this subunit. We conclude that hTeBG is a dimer whose monomer exhibits two protomeric forms which is not a result of carbohydrate heterogeneity. In addition, disialylated and deglycosylated hTeBG exhibited antigenic determinants identical to the native protein.

Biotin has been converted to 2-(biotinylamido)ethanol and condensed to phosphorylated oligonucleotides in a solid phase synthesis. The 5'-biotinylated oligonucleotides were enzymatically coupled to other DNA fragments by T4 DNA ligase or T4 RNA ligase. The hybridization properties of such biotin-labeled oligonucleotide probes were studied.

Maximum likelihood analysis of linkage between antithrombin III (ATIII) DNA polymorphisms and ATIII deficiency in a large Utah kindred suggests that thrombotic disease in this family is caused by a dysfunctional ATIII gene. ATIII-deficient family members were identified on the basis of: (1) reduced anticoagulant activity and (2) the presence of an electrophoretically abnormal inhibitor molecule in their plasmas. Affected individuals have two copies of the ATIII structural gene, and both alleles appear normal at the resolution of whole genome Southern blotting. However, family studies revealed statistically significant cosegregation of ATIII-deficiency trait and a particular ATIII DNA polymorphism haplotype (lod = 3.35; theta = 0.0); this result is consistent with the presence of a dysfunctional ATIII gene on a chromosome of the +, S haplotype.

The anti-tumour activities of 1,2-diphenylethane oestrogens (hexoestrol and orthohexoestrol) and anti-oestrogens (metahexoestrol, tetramethylHES, and metatetramethylHES) were studied on the human MCF-7 and MDA-MB-231 breast cancer cell lines. On the E2R-positive MCF-7 cell line, all test compounds exhibited a dose-dependent inhibition of cell proliferation, but no correlation between anti-proliferative activity and binding affinity for the E2R was found. Tested on the E2R-negative MDA-MB-231 cell line, metahexoestrol also showed dose-dependent inhibitory effects, but higher concentrations were necessary than on the MCF-7 cell line. From this it is concluded that the anti-proliferative effect is specific and at least partially mediated via the E2R. Combination of metahexoestrol (10(-6)M) with E2 (10(-9) to 10(-7)M) gave no rescue effect. It is therefore suggested that this compound might be useful for therapy in the presence of high oestrogen levels, i.e. in pre-menopausal patients. The test compounds (10(-8) to 10(-6)M) could rescue the inhibitory effect of tamoxifen (10(-6)M) in a dose-dependent manner, except in the cases of metahexoestrol (10(-6)M) and tetramethylHES (10(-6)M). The latter compound exhibited a strongly additive effect at this concentration.

Estrogen (ER) and progesterone (PR) receptors in 20 primary carcinomas of the ovary were studied by the customary DCC method. One of the receptors examined was found in 12 patients (60%) and both of them in 5 patients (25%). Serous types of tumour revealed a higher concentration of receptors than mucinous ones. The study of this kind is considered useful for the selection of patients in whom the adjuvant hormonal therapy could be applied.

This report describes the molecular species composition of phosphatidylcholines (PC) transferred from human erythrocytes to acceptor vesicles composed of cholesterol and single PC species in the presence of PC-specific transfer protein from bovine liver. The compositions of the PC isolated from the vesicles were determined by capillary GLC as the diacylglycerol trimethylsilyl ethers. The cellular PC species appearing in the acceptor vesicles were enriched in unsaturated species and showed a low content of dipalmitoyl PC compared to untreated erythrocytes. This trend was independent of the composition of the PC used to construct the acceptor vesicles and it was possible to determine that the relative rates of efflux of the palmitoyl-containing phosphatidylcholines decreased in the order: palmitoyl-linoleoyl greater than palmitoyl-oleoyl greater than dipalmitoyl and in the stearoyl series, stearoyl-linoleoyl greater than stearoyl-oleoyl. No clear trend was distinguished for the influence of chain-length on the efflux, thus preventing an unambiguous assignment of the order of removal of all species from the cell membrane. Results derived for arachidonoyl-containing species were compromised by evidence for oxidation occurring during incubations at 37 degrees C. To confirm that acyl selectivity was also possible during transfer in the absence of the transfer protein, the efflux of 14C-labeled soya PC and [14C]dipalmitoyl PC from prelabeled erythrocytes was measured using plasma as the acceptor. As predicted by the chromatographic analyses, 14C-labeled soya PC effused up to 10-times faster than [14C]dipalmitoyl PC from the red cell membrane. Thus, the more rapid transfer of unsaturated PC cannot be explained entirely as a specificity of the transfer protein and is consistent with the hypothesis that intermolecular interactions involving PC molecules within the erythrocyte membrane, become weaker with increasing unsaturation. The results suggest a potential role of PC-specific transfer protein as a probe of the nature of PC interactions within biological membranes.

Methotrexate (MTX) was first conjugated to antibovine serum albumin IgG (antiBSA) or its F(ab)2 fragment to define conditions for retention of drug and antibody activity. With identical drug: protein molar ratios, incorporation in the F(ab)2 fragment was lower than in intact antiBSA, an observation consistent with analysis of the number of lysine residues (22 in F(ab)2 compared to 40 in antiBSA). In either case, up to approximately 10 mol MTX could be incorporated per mol protein, with recovery of 70% of the protein. At an incorporation ratio of 6 mol MTX per mol protein, MTX-antiBSA retained 100% of antibody activity and MTX-F(ab)2-antiBSA retained 75%. MTX-antiBSA and MTX-F(ab)2antiBSA were equally potent in vitro inhibitors of dihydrofolate reductase. Conjugates prepared from antiEL4 IgG (AELG) and from F(ab)2AELG significantly increased survival in EL4 lymphoma-bearing mice compared with mice receiving equal amounts (5 mg MTX/kg) of free MTX, MTX linked to the F(ab)2 fragment of normal rabbit IgG, or a simple mixture of MTX and F(ab)2AELG. MTX-AELG at this dose level produced longer survival than MTX-F(ab)2AELG (0.005 less than P less than 0.01).

Breast cancer specimens from 184 patients were analyzed for estrogen binding by two different histochemical techniques using conjugates of estradiol, bovine serum albumin, and fluorescein. In one conjugate estradiol was bound at position 6, in the other at position 17. Results were in agreement in 64% (p less than .001), but obvious differences in ligand distribution were noted. Results were also correlated with estrogen receptor (ER) analysis by dextran-coated charcoal assay (DCC) and were in accord in 65% and 67% of specimens respectively (p less than .001). In 114 cases, the tissue samples were also studied with the estrogen receptor immunocytochemical assay (ERICA) of Greene and his colleagues, which employs monoclonal antibodies to ER protein. Results were in accord with DCC in 86% (p less than .001). The pattern of staining with ERICA differed from that of either histochemical method. In 43 cases assay results were correlated with clinical endocrine response. Overall, the best statistical prognostic parameters were obtained with ERICA. Analysis of combined assay results revealed that patients with assays positive by all techniques were the most likely to respond to hormonal treatment (p less than .001), whereas if one or more assays were negative the chances for a good response were significantly less favorable. These data suggest that DCC and ERICA are both a measure of the same estrogen binding site (type I) while the histochemical methods apparently identify two other separate and distinct sites (putative type II sites). A degree of positive interaction may exist between these multiple estrogen binding sites.

The response of components of the coagulation and fibrinolytic systems has been examined in patients undergoing minor and major elective surgery and receiving or not receiving subcutaneously administered prophylactic low molecular weight (LMW) heparin. Blood samples were withdrawn pre-operation (PO), post-anaesthesia (PA), during operation (DO) and 24 hours post-operation. Release of plasminogen activator occurred post anaesthesia at a time when factor VIII components were unchanged or decreased. Induction of anaesthesia decreased plasminogen (p less than 0.005) fibrinogen (p less than 0.02) ATIII (p less than 0.002) and fast a2-antiplasmin (p less than 0.005). During operation plasminogen activator release reached a peak in association with a moderate increase in factor VIII components. Heparin treatment produced a prolongation of APTT at DO (p less than 0.05) and at 24hr (p less than 0.002) stages, this prolongation being apparently unrelated to its concentration but did not prevent the rise of factor VIII components observed at 24 hr (p less than 0.02). Prekallikrein (p less than 0.05) and antithrombin III levels (p less than 0.05) were significantly higher whereas fast a2-antiplasmin (p less than 0.002) was significantly lower at 24 hours in patients undergoing major operation and treated with heparin. Protein C levels fell significantly at 24 hours in the untreated patient (p less than 0.014) and in the heparin treated group the fall was greater than the control group (p less than 0.007). At 24 hours other haemostatic and fibrinolytic components were unaffected by heparin treatment.

During the last three years we have analyzed, in 94 patients with chronic arterial occlusive disease, in order to assess the hemorheological and hemocoagulative balance, the following parameters: prothrombin time, partial thromboplastin time, antithrombin III, antiplasmin, fibrinogen, factor VIII, erythrocyte sedimentation rate, platelet aggregation, erythrocyte filterability and hematocrit values. The main findings were: in 56 patients the hematocrit value was higher than 44%; Katz index was above normal values in 61.7% of the cases; ATIII showed a trend to lower values; platelet aggregation was increased; no significant variations in comparison to normal values were found in FVIII, antiplasmin levels, prothrombin time and partial thromboplastin time. Erythrocyte filterability values were decreased at a level as lower as more advanced was the clinical stage of the disease. These findings show, in agreement with those of other authors, the presence of hyperviscosity and hypercoagulative state in patients with chronic arterial occlusive disease.

Abnormal factor IX variant proteins were isolated from the plasmas of three unrelated severe hemophilia-B families that had been previously shown to contain functionally impaired molecules immunologically similar to normal factor IX. The families studied were: (1) a patient with markedly prolonged ox brain prothrombin time, designated factor IX Bm Lake Elsinore (IXBmLE); (b) three patients (brothers) with moderately prolonged ox brain prothrombin time, designated factor IX Long Beach (IXLB); and (c) a patient with normal ox brain prothrombin time designated factor IX Los Angeles (IXLA). Each variant molecule comigrates with normal factor IX (IXN) both in the sodium dodecyl sulfate and in the nondenaturing alkaline gel electrophoresis. All three variant proteins are indistinguishable from IXN in their amino acid compositions, isoelectric points, carbohydrate distributions and number of gamma-carboxyglutamic acid residues. Each variant protein undergoes a similar pattern of cleavage by factor XIa/Ca2+ and by factor VIIa/Ca2+/tissue factor, and is activated at a rate similar to that observed for IXN. All of the three variant proteins also react with an anti-IXN monoclonal antibody that interferes with the binding of activated IXN(IXaN) to thrombin-treated factor VIIIC. However, in contrast to IXaN, the cleaved IXBmLE has negligible activity (approximately 0.2%), and cleaved forms of IXLA and IXLB have significantly reduced activity (approximately 5-6%) in binding to antithrombin-III/heparin, and in activating factor VII (plus Ca2+ and phospholipid) or factor X (plus Ca2+ and phospholipid) +/- factor VIII. These data, taken together, strongly indicate that the defect in these three variant proteins resides near or within the latent catalytic site. This results in virtually a complete loss of catalytic activity of the cleaved IXBmLE molecule and approximately 95% loss of catalytic activity of the cleaved IXLA and IXLB molecules.

45 malignant melanomas, 10 benign nevi and 17 basal cell carcinomas were investigated for estradiol (ER) and progesterone (PR) receptor activity by a single saturating-dose method (5 nM for ER and 10 nM for PR) using DCC. ER were found respectively in 7%, 10% and 24% of the studied samples and PR in 18%, 40% and 29% of the same samples. The low rates of ER+ and PR+ samples were discussed according to the data of the literature, more specially to the weak results obtained with anti-estrogenic therapy and to the possibility of tyrosinase to mimic the estrogen binding detected in melanomas.

The human antithrombin III (ATIII) structural gene was mapped by in situ hybridization and quantitative analysis of ATIII gene dosage in DNA isolated from carriers of chromosome 1 deletions. These studies indicate that the ATIII structural gene maps to human chromosome q23-q25 and so is likely identical to AT3.

Ultrasonically guided fine needle aspiration biopsy is known to be of great value in the diagnosis of malignant liver disease, with an overall accuracy rate of 73-94 p. 100. However, investigators have essentially reported cases of liver metastases. In this report, we examined the diagnostic value of this method in the specific case of tumors associated with cirrhosis. Twenty-seven patients with cirrhosis (20 alcoholic, 4 post-hepatitis, 3 hemochromatosis) with ultrasonically suspected hepatic malignancy were studied. They all presented severe blood clotting disturbances and/or ascites. At the end of the study, all patients had proven malignancy (by post mortem biopsy in 14 cases and/or serum AFP greater than 500 microgram/l in 17 cases). There were 25 primary and 2 metastatic tumors. Twenty-nine fine needle aspiration biopsies were performed under ultrasonic guidance. material suitable for cytologic evaluation was obtained in 25 patients. In 14 cases, a diagnosis of malignant involvement of the liver was firmly established by cytological examination; it was suggested in 4 other cases. Tumor typing was possible in 12 primary and 2 metastatic tumors, in agreement with the proven diagnosis. The present study shows that fine needle aspiration biopsy under ultrasound guidance is a safe and accurate diagnostic procedure in malignant liver disease associated with cirrhosis.

3 individuals from 2 unrelated families with recurrent thromboses and quantitative deficiencies of antithrombin III (AT-III) were treated with danazol, 600 mg daily for 4 months. Significant increases of AT-III (p less than 0.025) measured as heparin cofactor activity were noted in 1 female and 1 male patient. Failure to augment levels in the other male patient may have been due to poor absorption of the drug following small bowel resection for mesenteric infarction. Side effects of estrogen deficiency necessitated dosage reduction in the female patient. The 2 males experienced no adverse side effects except for prolongation of the prothrombin time in 1 who was receiving oral anticoagulants. We conclude that danazol causes a significant increase in some individuals with familial AT-III deficiency. Additional studies are necessary to determine whether this form of therapy may prove to be a suitable alternative to long-term anticoagulation and to assess the long-term clinical benefits in individuals with recurrent thrombosis.

It was shown that increased antiheparin activity of the blood plasma of women in labour is evoked by increased level of low density lipoproteids (LDL). This activity does not depend on changes neither of the level of other compounds neutralizing heparin (fibrinogen, acid alpha 1-glycoproteid, globulins, basic proteins) nor conditioning its action (antithrombin-III). The intensity of antiheparin action of LDL increases in the presence of calcium ions.

TLC, DCC and HPLC were used to study the ginsenoside composition of the main root, lateral root, rhizome, leaves and seeds of Panax ginseng cultivated in Jilin, China. Each of these methods has advantages of its own and the ensemble reveal the special features of Jilin ginseng. Total saponin content of various plant parts in Jilin ginseng showed a mid-range value as compared to those in ginsengs reported in literature. Fresh as well as sun-dried specimens from the same batch possessed a high percentage of Rg1 in the main root and this might account for the traditional preference of this plant part despite its lowest percentage of saponin in the whole plant. Large amounts of polar saponins were also observed in roots and rhizome of fresh Jilin ginseng, the nature and significance of which remained to be investigated.

The effect of Placental Protein 5 (PP5) on thrombin-induced coagulation of diluted fibrinogen is described. In contrast to previous reports which failed to demonstrate antithrombin-like activity for PP5 on synthetic substrates, we show in this study that PP5 inhibits thrombin activity in a dose-dependent manner. PP5 acts thus in a very similar way to antithrombin III (ATIII). Although PP5 binds to heparin, it does not show any heparin-cofactor activity. Whereas catalytic amounts of heparin accelerate greatly the inhibitory effect of ATIII, no accelerating effect of heparin on PP5 could be observed under the same conditions. In spite of its anti-thrombin activity, PP5 cannot be considered as a pregnancy-analogue of ATIII, as it lacks the heparin-cofactor activity.

Lyophilized calf uterine cytosol standards for estradiol receptor (ER) were prepared separately in two institutes. The ER binding capacity was determined by dextran-coated charcoaL (DCC)-technique using a multi-point Scatchard analysis. The intra- and inter-assay coefficient of variation determined on the Hungarian preparation were 13.10% and 14.70%. The dissociation constant (Kd) values of all the samples ranged between 1.2 X 10(-9) M and 9.35 X 10(-10) M. Standards stored at--70 degrees C and at 0-4 degrees C proved to be stable for several months. At ambient temperature, a progressive decline of the ER activity was observed during a 1 year storage period. The use of the lyophilized calf uterine cytosol standards stored at 0-4 degrees C is recommended for both intra- and inter-laboratory quality control study in multicentric clinical trials of breast cancer.

The presence of ER and PgR in human cervical tissues was investigated. The material consisted of receptor measurements in tissue sample obtained from normal and abnormal colposcopical pictures (leukoplakia, mosaic, punctation, atypical transformation), in 38 outpatients. The receptor assays were performed by a dextran-coated-charcoal (DCC) technique.