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We investigated the G1 and S phases of synchronization using sodium n-butyrate, MTX and excess thymidine. The flow cytometry system was employed for cell cycle analysis while the receptor assay was adopted dextran coated charcoal (DCC) and the wash method. The results were as follows: S phase synchronization by MTX was 138% (control 100%) and by excess thymidine in the block and release method 210%. G1 phase synchronization by sodium n-butyrate was 140%. The progesterone receptor level, by E2 priming increased to 1.45 fmol/ug DNA being more than five times that of the control PR. The estrogen receptor level increased to 18.29 fmol/ug DNA in the G1 phase synchronization, seven times that of the control ER. From this study, the functional increase of the steroid receptor was most significant in the G1 phase.

A cDNA library was prepared in lambda gt11 bacteriophage from poly(A)+ RNA isolated from primary cultures of endothelial cells from human umbilical vein. Approximately 2.5 million independent recombinants were screened and 2 of those were found to synthesize a fusion protein with beta-galactosidase that reacted with rabbit antibody against human von Willebrand factor. Comparison of the amino acid sequence translated from the cDNA insert of the two clones with the amino acid sequence determined by Edman degradation of the protein established that both phage isolates code for von Willebrand factor. The first clone (lambda HvWF1) contained an insert of 404 nucleotides that corresponded to amino acid residues 1-110 in the mature protein circulating in blood, in addition to a portion (24 amino acids) of a prepro leader sequence. The second cDNA clone (lambda HvWF3) contained an insert of 4.9 kilobases that coded for the carboxyl-terminal 1525 amino acids of von Willebrand factor, a stop codon of TGA, 134 nucleotides of 3' noncoding sequence, and a poly(A) tail of 150 nucleotides. The two clones together code for greater than 80% of the molecule circulating in blood. The same carboxyl-terminal lysine residue was identified in the mature protein as well as in the cDNA, indicating that all of the proteolytic processing that occurs during the biosynthesis and assembly of von Willebrand factor is associated with the amino-terminal portion of the precursor protein. The amino acid sequence of von Willebrand factor indicates the presence of two different internal gene duplications and one triplication. These repetitive amino acid sequences account for about one-half of the amino acids present in the mature protein. The tetrapeptide sequence of Arg-Gly-Asp-Ser, which mediates the cell attachment and platelet binding activity of fibronectin, was also identified in the carboxyl-terminal portion of von Willebrand factor.

The competitive binding assay described will specifically and accurately measure concentrations of administered heparin in biological fluids with a sensitivity of 60 ng ml-1. Neither endogenous glycosaminoglycans, nor plasma proteins such as ATIII and PF4 interfere in the assay. Semi-synthetic highly sulphated heparinoids and LMW heparin can also be measured. Using this assay heparin clearance followed simple first-order kinetics over the dose range 100-5,000 units, but the half-life was strongly dose-dependent. There was good correlation with heparin activity measurements by APTT and anti-Xa clotting assays. Plasma concentrations were measurable for at least 5 h following subcutaneous injection of 10,000 units of heparin. Excretion in the urine could be followed after all but the lowest intravenous dose. This assay, used in conjunction with measurements of heparin anticoagulant activity, will be valuable in the elucidation of mechanisms of action of heparin and the heparinoids, and in the assessment and management of problems related to heparin therapy.

It is very important to detect the intracellular distribution and dynamics of estrogen receptor (ER) in order to make the mechanisms of estrogen action clear. In a recent report, monoclonal antibodies localized ER only in the nucleus of target cells under both the presence and absence of estrogen. We purified cow and human uterine ER and induced specific polyclonal anti-ER antibodies. In this report, the specificity of immunological reactivity of anti-ER antibodies was examined, and the distribution of ER in the target tissues was detected by the indirect immunoperoxidase method. The specific staining was observed in the nucleus and cytoplasm of the endo- and myometrium of cow, rat and human uterus. In the nontarget organs, such as kidney and muscle, the specific staining was not detected. The cytoplasm of MCF-7 cells and immature rat uterine tissues were mainly stained without estrogen; however, the nuclear staining increased obviously after the estrogen priming. The cytoplasmic and/or nuclear staining was positive in 63% of 21 human breast cancer cases. This immunohistochemical assay (IHC) method of ER measurement was compared with the DCC assay method, a biochemical assay based on hormone binding ability. The concomitant positivity or negativity in DCC and IHC were observed as 61.9% (13 cases) or 19.0% (4 cases), respectively. Therefore, these two methods showed the same assay results in 81.0% of human breast cancer tissues.

Heparin cofactor II (HCII) is a glycoprotein in human plasma which inactivates thrombin rapidly in the presence of heparin or dermatan sulfate. We have developed a functional assay for HCII in which inhibition of thrombin by plasma is determined in the presence of dermatan sulfate. The assay is specific for HCII by the following criteria: (a) under the conditions of the assay, 125I-thrombin forms complexes in plasma which comigrate with the thrombin-HCII complex during sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); (b) activity detected by the assay is decreased in plasma absorbed with monospecific antibodies against HCII; and (c) purified antithrombin III (ATIII) is unreactive in the assay system. Addition of Polybrene to the assay permits determination of HCII activity in samples containing less than or equal to 12 U/mL of heparin. The range of HCII concentrations in normal individuals is 1.2 +/- 0.4 mumol/L (mean +/- 2 SD, n = 34). HCII activity was determined in 54 consecutive patients undergoing evaluation for the possibility of disseminated intravascular coagulation (DIC). Ten of the 11 patients with documented DIC had decreased HCII activity as compared with only 7 of the 43 patients without DIC (chi 2 = 19.3, P less than .0001). The concentrations of HCII and ATIII varied in parallel in most of the patients tested. A significant correlation between decreased HCII activity and decreased serum albumin concentration was also observed in these patients and in eight additional patients with hepatic failure in the absence of DIC. We conclude that HCII activity is decreased in many patients with DIC and hepatic failure.

When manual assays of antithrombin-III with use of different synthetic chromogenic peptide substrates are modified for a centrifugal analyzer, the downward deflection of the reference curve at the zero point raises a serious problem, because the position of the zero value is included in the calculation of the reference curve. Complete linearity was obtained by addition of polyethylene glycol 6000 to the thrombin solution and Tween 80 to the substrate solution in final concentrations of 1 g/L and 100 mg/L, respectively. The additives increased the concentration of active thrombin in the solutions by preventing its adsorption and aggregation, and the solubility of the substrates in aqueous media is increased. The precision of the optimized method, tested in routine assays during three months, yielded an estimated day-to-day CV of 3.4%.

Microheterogeneity of antithrombin III (ATIII) was examined in pooled normal human plasma and serum by IEF/PA and immunoblotting techniques. A pattern consisting of 8 bands varying in isoelectric point from 4.7 to 5.2 was observed in citrated plasma following IEF in a pH range of 4-6. A similar pattern was observed in all 149 healthy adults examined. The reactivity of ATIII with heparin or, thrombin was investigated using this technique. Characteristic shifts of the banding pattern to the anode or, cathode respectively were observed.