Ultrastructural features of chromatin nu bodies.

Spread chromatin fibers and isolated chromatin fragments prepared from chicken erythrocyte nuclei were stained with dilute aqueous uranyl acetate. High-resolution electron micrographs reveal two new morphological features exhibited by many of the chromatin nu bodies: (a) lateral association of the nu body with the connecting strand, and (b) a centrally stained spot approximately 15 A wide, possibly corresponding to a hole or crevice within the nu body.

Chromatin fragments resembling v bodies.

Fragments of chromatin resembling the spheroid chromatin units (v bodies) have been isolated from formaldehyde-fixed and sonicated chicken erythrocyte nuclei. Ultracentrifugal analyses demonstrated that monomer v bodies have a molecular weight of about 300,000 per particle, exhibit a protein to DNA ratio (by weight) of 1.22:1, and contain a DNA fragment with a molecular weight of approximately 140,000 per v body.

Progesterone, glucocorticoid and estradiol receptors in MCF-7 cells bind to chromatin.

MCF-7 cells contain progesterone, estradiol and glucocorticoid receptors. Following addition of these hormones to the growth medium of the cells, hormone-receptor complexes were found to sediment with chromatin fragments produced by trace digestion with micrococcal nuclease. The binding in all cases could be competed by excess unlabeled hormone. In each case the fragments with which the hormone-receptor complexes were associated tended to be smaller than the bulk chromatin fragments, indicating a greater sensitivity of those chromatin regions to the nuclease. The mononucleosomes released by more extensive digestion with micrococcal nuclease contained different amounts of each of the three hormone-receptor complexes. Progesterone could usually be detected on mononucleosomes only after very brief sedimentation analyses, whereas glucocorticoid- and estradiol-labeled mononucleosomes were stable during long centrifugations. Comparison of glucocorticoid- and estradiol-labeled mononucleosomes indicated that their sedimentation rates differed from one another and from bulk nucleosomes. Estradiol nucleosomes from MCF-7 cells and rat uterus (Senior and Frankel, 1978) sediment significantly faster than bulk nucleosomes, while glucocorticoid nucleosomes from MCF-7 cells and rat hepatoma cells sediment with, or even fractionally slower than, bulk nucleosomes.

The utility of the TDx test in the assessment of fetal lung maturity.

Accurate assessment of fetal lung maturity is essential in the management of high-risk obstetric patients. New rapid techniques have been developed to supplement time-consuming chromatographic methods. We compared one of these newer methods, the TDx-FLM, to the standard tests for fetal pulmonary maturity. There was an excellent correlation between the TDx and the lecithin-sphingomyelin ratio (r = 0.78). Although a TDx value of 70 or greater is considered mature, we found a value of 50 or greater predictive of fetal lung maturity in 100% of cases, and have chosen to redefine a mature value as 50 or greater in our institution. This value has greatly enhanced the clinical applicability of the test, allowing use of a large number of specimens from the previously poorly understood and often disregarded borderline category.

The estrogen-receptor complex is bound at unusual chromatin regions.

The estrogen receptor of MCF-7 cells labeled with high specific activity estradiol was used to mark the chromatin binding sites for this regulatory molecule. Many of these sites are especially sensitive to nuclease, and produce on digestion a series of uniquely sedimenting products. Several of these have been examined in some detail in this paper. These include a form of receptor that sediments in trace digests at 9S but in more extensive digests at 7S, fast mononucleosomes of about 12.5S, and a species at 15S. Two components of digests, fast mononucleosomes and dinucleosomes were isolated and subjected to further digestion. Much of the hormone on these isolated particles was found to be sensitive to additional hydrolysis, although some was nuclease resistant. It appears that a major fraction of the hormone receptor complexes bound to MCF-7 cell chromatin occurs at nucleosome-free regions which can be detected as transient hydrolysis intermediates.

Effect of formaldehyde on the circular dichroism of chicken erythrocyte chromatin.

Formaldehyde (HCHO) fixation of chicken erythrocyte chromatin produces a marked decrease in its positive circular dichroism (CD), above 260 nm, and the appearance of s small negative ellipticity around 295 nm. The ultraviolet spectrum of chromatin is unaffected, nor does HCHO produce any changes in the uv or CD spectra of chicken erythrocyte DNA. The extent of the circular dichroism transition from the native chromatin to the suppressed spectrum is dependent on the concentration of HCHO and salt concentration. The kinetics of the reactions are complex, implicating at least two reactive species. Studies of the reaction of HCHO with chromatin in ethylene glycol and CD measurements of aqueous chromatin solution with added glutaraldehyde preclude simple dehydration and general cross-linking effects as causes of the CD changes observed. The results are interpreted as indicating a conformational change of the DNA in chromatin caused by histone-DNA or histone-histone cross-linking.

Potent inhibition of mammalian progesterone synthesis by 2 alpha-cyanoprogesterone.

2 alpha-Cyanoprogesterone potently inhibits the conversion of [3H]pregnenolone into progesterone catalysed by bovine corpora lutea, bovine adrenal cortex and human term placenta microsomes (microsomal fractions), yielding IC50 (concentration causing 50% inhibition) values of 66 nM, 120 nM and 700 nM respectively. By contrast, it is an exceedingly poor inhibitor of the isomerization of pregn-5-ene-3,20-dione, yielding IC50 values between 50 and 70 microM. On this basis, 2 alpha-cyanoprogesterone would appear to be an extraordinarily selective inhibitor of the 3 beta-hydroxysteroid dehydrogenase. Dixon plots indicate that it is a very-tight-binding competitive inhibitor of the corpus-luteum enzyme, yielding a Ki of 15 nM. In the bovine adrenal cortex and human placenta the steroid is less potent and inhibits the dehydrogenase non-competitively with Ki values of 150 nM and 1.0 microM respectively. Thus 2 alpha-cyanoprogesterone inhibits the corpus-luteum dehydrogenase with substantial selectivity. Because of its high affinity for the ovarian enzyme, the presence of low-micromolar concentrations of 2 alpha-cyanoprogesterone can promote a complete cessation of progesterone synthesis in corpora-lutea microsomes for several hours. Since this effect is observed in the presence of saturating concentrations of pregnenolone (50 microM), it is predicted that this inhibitor may be even more potent in vivo. 2 alpha-Cyanoprogesterone displays very low affinity for the human progesterone receptor, yielding a Kd of 600 nM as against a Kd of 1.6 nM for progesterone. It is suggested that 2 alpha-cyanoprogesterone may be a selective inhibitor of ovarian progesterone synthesis and may act as an effective anti-gestational agent in vivo.

Assessment of proton nuclear magnetic resonance spectroscopy for detection of malignancy.

Water-suppressed proton nuclear magnetic resonance spectra were generated (by using 360 and 500 MHz systems) from human plasma and serum samples taken from 35 apparently healthy individuals, 52 patients with overt malignancies, and 37 patients with hypertriglyceridemia (triglycerides greater than 200 mg/dL or 2.26 mmol/L). The line widths from the lipoprotein-lipid methylene and methyl resonances at approximately 1.3 and 0.9 ppm were averaged by the method of Fossel et al. (N Engl J Med 1986;315:1369-76), but, contrary to their findings, we were unable to distinguish normal individuals from those with malignant tumors (e.g., mean +/- SD line width at 360 MHz: normal group = 32.9 +/- 3.6 Hz, malignant group = 28.3 +/- 4.9 Hz). The average line-width measurements (y), however, varied with the triglyceride content (x, mg/dL) of the plasma or serum as follows (logarithmic transformation of the data determined at 360 MHz and regression analysis): y = 110 (x-0.27). Data from both nonmalignant and malignant specimens fit this equation, the coefficient of correlation being -0.91. These findings suggest that considerable caution should be used in interpreting water-suppressed proton NMR spectra for cancer detection.

Estrogen and progesterone receptors in epithelial ovarian malignancies.

Cytoplasmic estrogen receptor levels (ER) were measured in ovarian epithelial malignancies from 44 patients using a dextran-coated charcoal assay with analysis of the data by Scatchard plots. In 31 cases, progesterone receptor levels (PR) were also determined. In untreated patients, 68.8% of malignancies were ER-positive (greater than or equal to 10 fmole receptor/mg protein) and 35.0% were PR-positive (greater than or equal to 50 fmole/mg protein). Receptor levels in ovarian epithelial malignancies were not dependent on patient age, menopausal status, tumor stage, or histologic grade. However, ovarian endometrioid carcinomas contained significantly more PR than other histologic variants (P less than 0.02). Excluding endometrioid carcinoma, tissues from patients treated with chemotherapy contained less ER and PR than tissues from untreated patients. ER-positive tumors from three patients assayed before cyclophosphamide or combination chemotherapy became ER-negative after therapy. In addition, tamoxifen increased cytoplasmic PR (124 to 333 fmol/mg) and decreased cytoplasmic ER (65 to 4 fmol/mg) in a single patient with endometrioid carcinoma.

Kinetic investigation of unfolding and partial refolding of a crab satellite (dA-dT)n.

Crab (dA-dT)n was isolated from the testes of Cancer borealis by a procedure involving separation of DNA and segregation of the satellite fraction by Hg2+ binding/Cs2SO4 density gradient ultracentrifugation. The titration of crab (dA-dT)n samples at 10 degrees indicated a sharp absorbance change at pH 11.98 in agreement with the pHm value observed for synthetic poly(dA-dT) under identical conditions. The reversal of the titration, however, resulted only in about 50% recovery of the original absorbance (at 260 nm) in marked contrast to the complete reversibility of the synthetic material. pH-jump experiments were carried out for the purpose of characterizing the rates and mechanisms of conformational transitions brought about by changes in the solution environment. It was found that the disintegration of the putative native structure of crab (dA-dT)n starts with a very fast reaction (occurring within the 6-msec deadtime of the instrument and comprising 65% of the total absorbance change) and it is completed via a slower first-order reaction (k = 66 sec minus 1). It is postulated that the first process is due to the rapid untwisting of end regions and, perhaps, some short hairpin-like helical branches present on the macromolecules. The second reaction is believed to be the end-to-end type unwinding of the double-helical backbone of crab (dA-dT)n. In the presence of low concentration (3 mug/ml) of Hg2+ ions the overall rate of disintegration process decreased drastically. pH jumps from pH values above pHm to values below were used to study the rates of absorbance changes corresponding to the refolding of the strands of denatured crab (DA-dT)n. A concentration independent process consisting of two phases was observed. The first phase was a gradual nonexponential process spanning the first second of the reaction, and the other, a very slow first-order process characterized by the rate constant value of 0.053 sec minus 1. It is proposed that the first part of the process (involving about 24% of nucleotide residues) is an intramolecular formation of helical hairpins (frequently interrupted by mismatching bases) and the second part is a manifestation of some association of the extant unpaired bases during the folding of the branched structure. Refolded crab (dA-dT)n samples when subjected again to pH greater than pHm in the stopped-flow apparatus displayed not the disintegration pattern of the native crab (dA-dT)n but rather that of synthetic poly(dA-dT. The marked facility of crab (dA-dT)n macromolecules for rapid conformational transitions induced by slight changes in the solution environment might be relevant to the biological function of this DNA.

Delineation of false-positive HIV antibody response in patients with renal failure and history of multiple transfusions.

Four patients with a history of multiple blood transfusions who awaited renal transplantation were tested for human immunodeficiency virus (HIV) infection and found to be positive on enzyme immunoassay (EIA) and negative on Western blot. None of these patients had any clinical evidence of HIV infection. Absorption of these patients' sera with B-lymphoblastoid cell lines (B-LCL) positive for the serologic specificities DR3, DR4 (Dw4, Dw10, Dw14), and DR5 resulted in EIAs that were negative for HIV. Treatment of the B-LCL with an anti-DR monoclonal antibody (L243) interfered with the absorption of the serum sample by B-LCL. This indicates that the initial false-positive EIA results may be due to HLA antibodies. Furthermore, it was shown that these HLA antibodies are not limited in specificity to the HLA type of the host cell used in the preparation of the EIA reagents, but can consist of other DR specificities.