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Quantitative measurement of steroid receptors including progesterone receptor (PgR) is usually accomplished by the dextran-coated charcoal (DCC) assay. At protein concentrations below about 1 mg/ml, however, serious underestimation of receptor content by DCC occurs, presumably because of adsorption of receptor to the charcoal and possibly to assay tubes, etc. We have therefore developed a modified charcoal-gelatin (MCG) procedure which largely avoids receptor losses even in samples with extremely low protein concentrations. In this MCG procedure, 0.1% gelatin is added to both sample and charcoal suspension, the charcoal content is increased to 1%, and dextran is no longer necessary. Comparison of the MCG procedure with the standard DCC and several other methods at decreasing protein concentrations shows that MCG retains acceptable efficiency for PgR at much lower protein than the others, even as low as 10 micrograms/ml. This MCG procedure will be useful in determining receptors for prognosis in very small human breast cancer biopsies, as shown here, but also for receptor determination in very small tissues such as specific brain regions, and for receptor assay during purification.

In the present study, size exclusion HPLC was used to analyse the properties of Estramustine-binding protein (EMBP) in the cytosol of human benign prostatic hypertrophy (BPH). The typical size exclusion HPLC separation profile of 3H-Estramustine-labelled cytosol of BPH showed four radioactive peaks that corresponded to the V0, 250K, 68K and 45K protein regions. The specific binding protein for Estramustine is contained mainly in the 250K protein region and in part in the V0 region. In the presence of sodium molybdate, the specific Estramustine binding to a 250K protein was increased to a level which was about 2.5 times as much as the value in the absence of sodium molybdate. The specific Estramustine binding to a 250K protein under the condition of no DCC treatment and the addition of sodium molybdate was 600% of the value obtained under the condition of DCC treatment and no addition of sodium molybdate. These results suggested that sodium molybdate stabilized the specific Estramustine-binding activity to a 250K protein and that specific Estramustine binding to a 250K protein seemed to be weaker than the binding of androgen to androgen receptor.

We studied a family with HLA-linked hereditary hemochromatosis in which an informative recombination occurred within the HLA region. The father, an obligate heterozygote for hereditary hemochromatosis, had HLA haplotypes A2,B13 and A11,B27. The mother, also an obligate heterozygote, had HLA haplotypes A29,B44 and A2,B7. Three haplotypes were found among three homozygous affected offspring. Two affected siblings were HLA-identical with haplotypes A2,B13 and A29,B44. The proband had HLA haplotypes A2,B13 and A2,B44, the latter a recombinant haplotype inherited from her mother. Since the maternal hemochromatosis allele was linked to the A29,B44 haplotype, and since the proband has hemochromatosis, the maternal hemochromatosis allele was transmitted to the proband with the B44 antigen. This is the first known example of recombination in an individual with HLA-linked hemochromatosis in whom the hemochromatosis allele appeared to segregate with the HLA-B antigen instead of the -A antigen. The possibility of either a double reciprocal recombination event or a gene conversion event cannot be excluded. Combined with earlier observations of segregation of the hemochromatosis allele with the A locus in HLA recombinants, the findings in this pedigree map the hemochromatosis locus between the HLA-B and HLA-A loci rather than outside the HLA region.

Clonal proliferation of freshly isolated human fetal chondrocytes and adult chondrocytes in response to human insulinlike growth factors I and II (IGF I, IGF II), human biosynthetic insulin, and human growth hormone (GH) was assessed. IGF I (25 ng/ml) stimulated clonal growth of fetal chondrocytes (54 +/- 12 colonies/1,000 inserted cells, mean +/- 1 SD), but IGF II (25 ng/ml) was significantly more effective (106 +/- 12 colonies/1,000 inserted cells, P less than 0.05, unstimulated control: 14 +/- 4 colonies/1,000 inserted cells). In contrast, IGF I (25 ng/ml) was more effective in adult chondrocytes (42 +/- 6 colonies/1,000 inserted cells) than IGF II (25 ng/ml) (21 +/- 6 colonies/1,000 inserted cells; P less than 0.05, unstimulated control: 6 +/- 3 colonies/1,000 inserted cells). GH and human biosynthetic insulin did not affect clonal growth of fetal or adult chondrocytes. The clonal growth pattern of IGF-stimulated fetal and adult chondrocytes was not significantly changed when chondrocytes were first grown in monolayer culture, harvested, and then inserted in the clonal culture system. However, the adult chondrocytes showed a time-dependent decrease of stimulation of clonal growth by IGF I and II. This was not true for fetal chondrocytes. The results are compatible with the concept that IGF II is a more potent stimulant of clonal growth of chondrocytes during fetal life, whereas IGF I is more effective in stimulating clonal growth of chondrocytes during postnatal life.

One hundred seventy orthotopic liver transplants were performed under conventional immunosuppression with azathioprine and steroids with 1- and 5-year survivals of 32.9 per cent and 20.0 per cent, respectively. Since the introduction of cyclosporine-prednisone therapy in March 1980, 313 primary orthotopic liver transplants have been performed. Actuarial survivals at 1 and 5 years have improved to 69.7 per cent and 62.8 per cent, respectively. Biliary atresia is now the most common indication for liver replacement. In adults, primary biliary cirrhosis and sclerosing cholangitis have become more common indications for transplantation, and alcoholic cirrhosis and primary liver malignancy as indications have declined. Early enthusiasm for liver transplantation in patients with hepatic cancer has been tempered by the finding that recurrence is both common and rapid. An increasing number of patients with inborn errors of metabolism originating in the liver are receiving transplants, including patients with Wilson's disease, tyrosinemia, alpha-1-antitrypsin deficiency, glycogen storage disease, familial hypercholesterolemia, and hemochromatosis. Survival in this group of patients has been excellent (74.4 per cent at 1 and 5 years). A hemophiliac who received a transplant for postnecrotic cirrhosis has survived and may have been cured of his hemophilia. About 20 per cent of patients require retransplantation for rejection, technical failure, or primary graft failure. Only four of the patients receiving retransplants under conventional immunosuppression survived beyond 6 months, and all died within 14 months of retransplantation. Sixty-eight patients have received retransplants under cyclosporine-prednisone. Thirty-one patients are surviving, all for at least 1 year. Six of the twelve patients requiring a third transplant are alive 2 to 3 years after the primary operation. An aggressive approach to retransplantation in the patient with a failed graft is justified.

Idiopathic hemochromatosis is an hereditary iron overload. The study of HLA types demonstrated clearly the linkage with HLA system. The preferential correlation established with A3 (72%) but other alleles were linked: B7, B14. HLA alleles were only the markers of hemochromatosis allele (H) and were not implicated in other iron overload. Family studied, defined two linked haplotypes: A3, Cw7, B7, Bw6, BfS, DR2, GLO1 et A3, Cw8, B14, Cw6, BfF, DRw6, GLO2. Demonstration of the recessive mode of inheritance was established by family studies. The affected siblings had the same HLA haplotype that the proband and homozygous or heterozygous expressed state was assessed in siblings. The HLA family types allowed to detect in 147 families 88 potential diseased patients among of them 73% had early blood-drawing.

Insulinlike growth factors (IGF) act qualitatively like insulin on insulin target tissues in vitro. In the circulation in vivo they are bound to specific carrier proteins. In this form or when continuously infused into hypophysectomized (hypox) rats they do not exert acute insulinlike effects on glucose homeostasis. This study definitively shows that intravenous bolus injections of pure IGF I or II act acutely on glucose homeostasis: they lower the blood sugar, enhance the disappearance of U-[14C]glucose from serum and increase its incorporation into diaphragm glycogen in normal and hypox rats in the presence of antiinsulin serum. The same effects were obtained with recombinant human IGF I injected intravenously either with or without antiinsulin serum into normal rats. Free fatty acid levels decreased transiently only in normal animals. Lipid synthesis from glucose in adipose tissue was not stimulated in hypox and barely stimulated in normal rats. The half-life of injected IGF I or II in normal rats (approximately 4 h) is strikingly different from that in hypophysectomized rats (20-30 min) and appears to depend on the growth hormone-induced 150,000-200,000-mol wt IGF carrier protein that is lacking in hypophysectomized rats. 15 min after the bolus serum IGF I and II concentrations were similar to steady state levels during long-term infusion in hypox rats. Free IGF was barely detectable, however, in the infused animals, whereas 40-100% was found free 15 min after the bolus. These observations for the first time confirm the hypothesis that only free IGF, but not the IGF carrier protein complex, is bioavailable to insulin target tissues.

Estrogen (ER) and progesterone receptors (PgR) appear to be a prerequisite to elicit a biologic response by a hormone-target organ. Current methodologies for analysis of these proteins (e.g., dextran-coated charcoal, DCC) in single-label assay (SLA) require relatively large amounts of tissue material, time and laboriousness. Therefore, we have developed for breast cancer tissue an improved dual-label assay (DLA) for simultaneous titration (by DCC) and/or characterization (by sedimentation properties) of ER and PgR on the same sample, using 125I-E2 and 3H-Org 2058 as tracers. The interaction of 125I-E2 with ER and plasma proteins in comparison to 3H-E2 was studied in terms of specificity, time course, affinity binding and sedimentation pattern. 125I-E2 bound the same molecular forms displayed by 3H-E2 (9 and 3S) but with lower titers (about 1.3-fold), irrespective of the technique used, and did not bind to sex hormone-binding globulin. Simultaneous detection of 125I and 3H was achieved by use of a gamma counter plus a beta counter sequentially. ER and PgR titrations with DCC in DLA were in good agreement with those obtained with SLA, in terms of titers and Ka values. An analogous result was obtained with sucrose density gradient (SDG) analysis. Both the DLA methods were highly reproducible (CV less than 8.0%). Between the rotors available for SDG, the vertical one was preferable because of the larger number of samples processed and of less perturbation of sedimenting receptor molecules. Furthermore, a biochemical application of the method is described. In conclusion, the DLA procedure, by simplifying ER and PgR estimation, makes it possible to study, even on small tumor biopsies, the molecular properties of these proteins in relation to the clinical response of the disease.

S-protein, the inhibitor in plasma of the membrane attack complex of complement, appears to have a second function in coagulation. S-protein during clotting enters into a trimolecular complex with thrombin and antithrombin III (ATIII). Functionally, S-protein in the presence of low concentrations of heparin, protects thrombin from inactivation by ATIII. Complex formation between S-protein and thrombin, and between S-protein, thrombin, and ATIII, was demonstrated by agarose gel electrophoresis and by two-dimensional immunoelectrophoresis of purified proteins and in recalcified, clotted plasma. Formation of the trimolecular S-thrombin-ATIII complex was strictly dependent on the presence of thrombin. No association was detectable between S-protein and ATIII or between S-protein and prothrombin. Heparin was not required for the formation of the bimolecular S-protein-thrombin complex or the trimolecular S-protein-ATIII complex. The protective effect of S-protein on inactivation of thrombin by ATIII was demonstrated in functional assays with purified proteins and in plasma only in the presence of low concentrations of heparin. Thus, S-protein may mediate its effect by scavenging heparin required for ATIII activation. It is suggested that the protection of thrombin by S-protein from inactivation by ATIII may be of physiological importance.