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Insect cells of an established line, Drosophila Kc cells, take up and metabolize juvenile hormone (JH). The cytoplasm of these cells contains a protein that binds JH with specificity, saturability, and high affinity (K(d) = 1.56 x 10(-8) M). The kinetics for the specific binding and dissociation of JH I were independently measured, and the rate constants were found to be k(a) = 1.3 x 10(6) M(-1) min(-1), k(d) = 1.3 x 10(-2) min(-1), respectively. All three juvenile hormones bind to the protein with comparable affinities; the corresponding acid or diol metabolites of JH I are not bound. About 2500 hormone-binding protein molecules are present per cell. The protein has a molecular weight of 80,000 as estimated by gel permeation chromatography and by sucrose gradient sedimentation. The properties of this protein suggest that it functions as a cytoplasmic receptor for juvenile hormone.

Glucocorticoid receptors (RG) and mammary tumor virus (MM-TV) DNA sequences were extracted by micrococcal nuclease digestion from the nuclei of C3H mouse mammary tumor cells in order to specify their relative distribution in chromatin. RG was labelled and translocated into the nuclei by incubating cells with 3H Dexamethasone (3H Dex). The purified nuclei were then treated at 2 degrees C with micrococcal nuclease. Three chromatin fractions were successively obtained: an isotonic extract (ne3H1), ahypotonic extract (ne2) and the residual pellet (P). The Dex-RG complexes were measured by the hydroxyapatite technique. The MMTV DNA sequences were titrated by molecular hybridization with an excess of MMTV radioactive cDNA probe. Up to 75% of the nuclear 3H Dex and the MMTV radioactive cDNA probe. Up to 75% of the nuclear 3H Dex and MMTV DNA sequences were extracted in a concentration dependent manner while only 10-15% of nucleic acids became soluble in 10% perchloric acid. The extracted 3H Dex-RG complex was found to be partly bound to soluble chromatin and partly free. The free complex displayed similar sedimentation constants (4S, 7S) and DNA binding ability to the cytosol receptor. The 3H Dex-RG complexes were 2 to 8 fold more concentrated in ne1, which is known to be enriched in active chromatin, than in ne2. Conversely, the concentration of MMTV DNA sequences per microgram DNA was the same in the three nuclear fractions. These results suggest that the Dex-RG complexes are concentrated in an active fraction of chromatin. We propose that, among the 20-30 copies of MMTV genes per haploid genome, only a small proportion are transcribed or regulated.

A homogeneous substrate-labeled fluorescent immunoassay has been developed to measure amikacin levels in human serum. Amikacin is covalently labeled with the fluorogenic enzyme substrate beta-galactosyl-umbelliferone. This beta-galactosyl-umbelliferone-amikacin conjugate is nonfluorescent under assay conditions until it is hydrolyzed by beta-galactosidase to yield a fluorescent product. When antiserum to amikacin binds the substrate-labeled drug, the antibody complex formation inhibits hydrolysis of the fluorogenic substrate. Reaction mixtures containing a constant level of substrate-labeled amikacin and a limiting amount of antiserum enable labeled and unlabeled amikacin to compete for the antibody-binding sites. Unbound substrate-labeled drug is hydrolyzed by the enzyme to release a fluorescent product that is proportional to the unlabeled amikacin concentration. The amikacin levels found in clinical serum samples with this method were comparable (r = 0.987) to those obtained by radioimmunoassay. The fluorescent immunoassay is rapid and simple to perform and requires only 2 microliters of serum.

A method for determining antithrombin-III heparin cofactor activity that requires no defibrinogenation of plasma, can be used with heparinized samples, and is specific for antithrombin-III heparin cofactor activity was developed. The procedure requires minimal pipetting, uses small amounts of sample over a single short incubation period, and is relatively inexpensive. The antithrombin-III assay appears to be superior to other commercially available procedures when employed in the diagnosis of deficiencies and in the identification of decreased levels of antithrombin III associated with liver disease and thrombotic disorders.

Histochemical analyses estrogen (ER) and progesterone (PgR) receptors in breast cancer were statistically correlated with results of dextran-coated charcoal (DDC) and sucrose gradient assays. Correlated for ER was 91% of 363 cases, and for PgR 88% of 255 specimens. Breast cancer ER/PgR positivity by histochemistry correlated with a favorable clinical response to endocrine therapies in 72% of 25 cases, while ER/PgR negativity correlated with a lack of response in 96% of 22 cases with Stage IV disease. Nuclear ER/PgR correlated with a poor response to therapy in 8 of 12 patients. An in vitro technique to detect nuclear translocation of ER revealed two groups of ER positive cases, with 11 of 17 exhibiting translocation and 6 not displaying translocation. In prostatic carcinoma, 72% of 65 men were positive for ER and/or androgen receptor. Comparison of specimens obtained without and with electrocautery revealed a preponderance of nuclear binding in the latter, suggesting heat-induced nuclear translocation of receptor. coumestrol, a naturally fluorescent, entirely unaltered estrogen was also used for histochemical detection of ER. Results correlated with ER by DCC in 87% of 61 breast cancers. Coumestrol was additionally used to visually observe receptor and nuclear translocation of ER in intact whole cells in culture.

A case-control study of fibrinolytic activity and antithrombin-III (AT-III) was conducted comparing 16 women within the childbearing ages who had a recent history of idiopathic thromboembolism to 29 age- and race-matched female controls without a history of thromboembolism. All subjects had stopped using oral contraceptives at least 12 months prior to study. No difference in AT-III was evident between cases and controls. The mean fibrinolytic response to venous occlusion in cases was significantly lower; however, baseline fibrinolysis was essentially identical. These data demonstrated a bimodal distribution of fibrinolytic response among controls. By dichotomizing fibrinolytic response between the two modes in the controls, we estimated that the rate of low fibrinolytic response was 6 times as frequent in cases as controls. Thus, low fibrinolytic response may signal a natural predisposition to thromboembolism which could be triggered by use of synthetic estrogens.