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Heparin covalently-linked to polyvinyl alcohol (PVA) is a biomaterial which is of potential value as a non-thrombogenic coating. 125I-labelled thrombin adsorbed to heparin-PVA beads was not dislodged by phosphate-buffered saline, pH 7.4, although radioactivity was progressively displaced from the adsorbent by fibrinogen-free human plasma. Analysis by gel filtration and affinity chromatography showed that the released radioactivity was distributed between (thrombin-antithrombin-III) complex (approx. 70%) and, probably, (thrombin-alpha-2-macroglobulin) complex (approx. 30%). Less efficient thrombin displacement was obtained by either bovine serum albumin (5% w/v) or antithrombin-III-free human plasma: in the latter case, the dislodged enzyme was presumably associated with alpha-2-macroglobulin. Purified alpha-2-macroglobulin did not displace thrombin from heparin-PVA. The quantity of thrombin displaced by an alpha-2-macroglobulin-free plasma fraction compared well with fibrinogen-free plasma: The eluted enzyme was largely associated with antithrombin-III. Purified antithrombin-III did not displace thrombin from heparin-PVA despite causing greater than 70% inactivation of the bound enzyme. Subsequent treatment with fibrinogen-free plasma dislodged (thrombin-antithrombin-III) at a similar rate to that of bound thrombin. We conclude that plasma contains a component(s) which displaces (thrombin-antithrombin-III) complex from immobilised heparin: presumably this leaves the heparin sites free for further use in enzyme inactivation.

Sex steroid receptor proteins were studied in human normal liver and hepatocellular carcinoma (HCC). Oestrogen receptor (ER) was detected in nucleosol and cytosol of 4 normal adult and 5 malignant liver specimens and in the cytosol of 6 foetal liver samples. Levels were 27.6-500 fmol mg-1 soluble protein in normal adults (Kd 1.48 X 10(-8) -1.12 X 10(-10) mol 1(-1) ), 45-290 fmol mg-1 in malignant liver tissue (Kd 3.26 X 10(-9) -3.64 X 10(-10) mol 1(-1] and a mean of 93 fmol mg-1 in foetal tissue (Kd 1.55 X 10(-9) mol 1(-1]. Androgen receptors (AR) were found only in cytosol and nucleosol of HCC (23-370 fmol mg-1) and in cytosol from foetal liver (29 fmol mg-1) with Kd from 2.90 X 10(-9) to 3.734 X 10(-10) mol 1(-1]. AR was distinguished from sex hormone binding globulin, which was also present in all cytosol samples, by the former's ability to selectively bind to methyltrienolone and the latter's absence from nucleosol. These findings provide further support for suggestions that oestrogen-related hepatic functions in man may be mediated by receptors and raise the possibility that hepatocellular carcinoma may be androgen dependent.

The Ia antigens, encoded within the I region of the major histocompatibility complex, are a group of cell surface glycoproteins that are involved in the control of immune responsiveness. We isolated and determined the sequence of a 1,045-base-pair cDNA clone for one of the murine immune response genes, Ek beta. Comparison of the predicted amino acid sequence of the product of Ek beta with that of Ed beta shows that most of the amino acid differences are clustered in short stretches of peptide sequence in the first external protein domain. This clustering of allelic variation suggests that observed haplotype-specific immune responsiveness to certain antigens may be controlled, at least in part, by differences in configuration defined by these regions of allelic variability in the NH2-terminal domain of the E beta chain.

The marked propensity of insulin to self-associate into large aggregates causes significant mechanical problems in insulin delivery devices and may also stimulate production of a tissue-amyloid A precursor in some patients. Although conventionally prepared sulfated insulin (SI) resists aggregation, clinical application has been limited by major insulin bioactivity losses that occur during synthesis. To eliminate this problem, insulin sulfation was carried out in the organic solvent dimethylformamide in the presence of condensing agents such as N,N'-dicyclohexyl carbodiimide (DCC) and a sulfate donor. With this new procedure, the degree of sulfation could be controlled over an eightfold range by varying the amount of condensing agent. The bioactivity of these new SI derivatives varied between 78% and 87% of unmodified insulin. Insulin aggregation, induced by passage through a syringe and needle, did not occur with derivatives having two or more sulfate moieties per insulin molecule. Diffusion velocity studies using "non-aggregated" insulin solutions demonstrated that aggregates were present in crystalline zinc and sodium porcine insulin. In contrast, SI having more than 0.5 mole sulfate per mole of insulin dialyzed as it were predominantly in the monomeric form. Results from the studies described in this report now provide the means for selectively designing and preparing specific high-potency, non-aggregating insulins, which may be necessary for optimal use of current and future insulin delivery devices.

By using the intra-I-region recombinant mouse strain, B10.BASR1 (H-2as4), the immune response (Ir) genes for LDH-B and MOPC-173 were genetically and serologically separated, as assayed by T cell proliferation. Previous work demonstrated that the H-2s and H-2b strains respond to LDH-B and MOPC-173, whereas the H-2a and H-2k strains failed to respond due to haplotype-specific suppression of I-Ak-activated T helper cells by I-Ek-activated T suppressor cells. In the experiments reported here, B10.BASR1 mice, which lack I-Ek expression, mounted a significant T cell proliferative response to MOPC-173 but not to LDH-B. Separation of the Ia determinants used in restricting these two antigen responses was further confirmed when pretreatment of B10.S(9R) (A alpha sA beta sE beta sJk) macrophages with A.TL anti-B10.HTT (anti-A beta sE beta sJs) serum absorbed with B10.BASR1 spleen cells blocked the LDH-B response but not the MOPC-173 response. Unabsorbed serum blocked both antigen responses. The primary immunogenic determinant recognized by LDH-B or MOPC-173 immune T cells was not present on both antigens, as MOPC-173-primed T cells and LDH-B-primed T cells responded only to the priming antigen. Lastly, by using the A beta mutant strain, B6CH-2bm12, it was shown that the Ir gene and Ia determinants affected by this mutation had no effect on the LDH-B and MOPC-173 proliferative responses. These results suggest the possibility of an intragenic recombinatorial event in either the A alpha or A beta chain resulting in the separation of these two immune response gene functions.

Five patients who presented with arthritis as the sole manifestation of hereditary hemochromatosis and 51 family members were studied. Studies included clinical evaluation for the presence of arthritis and hemochromatosis, roentgenography of hands, knees, and pelvis, serum iron and serum ferritin measurements, complete HLA typing for 50 of the A and B loci, and, when indicated, liver biopsy. Arthritis occurred in 45 percent of persons with hemochromatosis. Although typical involvement of second and third metacarpophalangeal joints was observed in all five patients and some family members, two with typical arthritis did not have characteristic radiographic changes, two had constitutional symptoms without arthropathy, and one had unilateral hand changes. A specific HLA haplotype (A2/B17 in Family 1 and A29/B15 in Family 2) correlated with hereditary hemochromatosis but not with the arthropathy. Phlebotomy alleviated the early constitutional symptoms but did not help advanced arthritis. Anti-inflammatory drugs, intraarticular injections of glucocorticoids, and resection osteotomies of metacarpal heads were other treatment modalities.

Effects of Kampo (Chinese) drugs on hypothalamo-pituitary-gonadal system were investigated in immature male rats of Holtzman strain. Hachimijiogan(H), its components and Ninjin(N) were administered orally to the animals for 28 days, and the weight of the various organs, pituitary content of FSH, LH and PRL, and serum concentration of FSH, LH, PRL and testosterone were measured. H caused a significant increase in the weight of seminal vesicles while H, Sanyaku(S) and N caused a significant increase in the weight of the prostate, respectively. The pituitary content of FSH was significantly increased by H and S, respectively, while the testosterone concentration was significantly decreased by Jio, S and N, respectively. No increase in the weight of the prostate or seminal vesicles followed the administration of H, S and N to orchiectomied immature rats. This result clearly shows that these three drugs have no androgenic activity. After the administration of H to immature male rats, the character of dihydrotestosterone(DHT) receptor was studied by the DCC method. The number of binding sites of DHT receptor was significantly increased by 32% (p less than 0.001) by H. These results demonstrate that H has a stimulatory effect on androgen target organs through the increase in androgen receptor in these organs.

In a previous paper (Otsuka, H. & Yamakawa, T. (1981) J. Biochem. 90, 247-254), we reported the separation of acidic glycolipids by droplet counter-current chromatography using 500 columns with a commercially available DCC apparatus and described the precise conditions of the separation. In this paper, separation of neutral glycolipids from rabbit and human erythrocytes by DCC is described. For efficient separation of neutral glycolipids, addition of a definite amount of benzene was required. The solvent system of chloroform: benzene: methanol: water = 50: 50: 70: 20 gave clear separation of these glycolipids and further modification of this solvent system to chloroform: benzene: methanol: water = 50: 25: 65: 30 gave satisfactory results for the separation of Globoside I from human erythrocytes due to the difference between the amide linked fatty acids. Also under the same conditions, the clear separation of two peaks with blood group A activity was demonstrated by a hemagglutination inhibition test.

Nucleotide sequence analysis revealed that a DNA length polymorphism 5' to the human antithrombin III gene is due to the presence of 32bp or 108bp nonhomologous nucleotide sequences (variable segments) 345bp upstream from the translation initiation codon. Sequences at the 3' borders of both variable segments can form intrastrand inverted repeat structures with sequences further downstream. An inverted repeat is also found immediately 5' to the site where the variable segments are located. Thus, cruciform structures may form flanking the variable segments of both alleles of this DNA length polymorphism. DNA secondary structure may be detected with single strand specific nucleases. S1 nuclease sensitive sites were mapped in recombinant plasmids containing the cloned alleles of the ATIII length polymorphism. The site most sensitive to S1 is located upstream from the variable segments in an AT-rich segment flanked by 6bp direct repeats. A region of lesser nuclease sensitivity was also observed in the AT-rich loops formed between the inverted repeats 5' to the variable segments.