High content imaging-based assay to classify estrogen receptor-α ligands based on defined mechanistic outcomes.

Estrogen receptor-α (ER) is an important target both for therapeutic compounds and endocrine disrupting chemicals (EDCs); however, the mechanisms involved in chemical modulation of regulating ER transcriptional activity are inadequately understood. Here, we report the development of a high content analysis-based assay to describe ER activity that uniquely exploits a microscopically visible multi-copy integration of an ER-regulated promoter. Through automated single-cell analyses, we simultaneously quantified promoter occupancy, recruitment of transcriptional cofactors and large-scale chromatin changes in response to a panel of ER ligands and EDCs. Image-derived multi-parametric data was used to classify a panel of ligand responses at high resolution. We propose this system as a novel technology providing new mechanistic insights into EDC activities in a manner useful for both basic mechanistic studies and drug testing.

Femoral sulcus angle measurements.

We sought to determine whether the femoral sulcus angle measurement is consistent on different radiographic projections of the knee. Seventy-four cadaveric knees were radiographed using Merchant, Laurin, and Hughston projections. The measurements of the sulcus angle (mean, 138.6 degrees; SD, 6.9 degrees; and range, 120 degrees to 155 degrees) obtained with the Merchant technique were similar (P = 0.0007) to those obtained with the Hughston (mean, 137.4 degrees; SD, 7.6 degrees; range, 122 degrees to 155 degrees) and Laurin (mean, 141.6 degrees; SD, 6.9 degrees; and range, 125 degrees to 162 degrees) projections. Therefore, we submit that either the Hughston or Laurin view can be used instead of the Merchant view to reliably measure the femoral sulcus angle.

Mathematics instruction for secondary students with learning disabilities.

Secondary students with learning disabilities generally make inadequate progress in mathematics. Their achievement is often limited by a variety of factors, including prior low achievement, low expectations for success, and inadequate instruction. This article will discuss techniques that have been demonstrated to be effective with secondary students who have learning disabilities in mathematics.

Highly dispersive photonic band-gap prism.

We propose the concept of a photonic band-gap (PBG) prism based on two-dimensional PBG structures and realize it in the millimeter-wave spectral regime. We recognize the highly nonlinear dispersion of PBG materials near Brillouin zone edges and utilize the dispersion to achieve strong prism action. Such a PBG prism is very compact if operated in the optical regime, ~20 mm in size for lambda ~ 700 nm, and can serve as a dispersive element for building ultracompact miniature spectrometers.

Comparing outcomes in a statewide program for adolescent mothers with outcomes in a national sample.

One-year outcomes for 1,004 participants in a statewide program for pregnant and parenting teenagers were compared with outcomes for 790 adolescent mothers included in the National Longitudinal Survey of Youth. The program group was substantially younger and included more black women and fewer married teenagers than the national sample. Controlling for these and other baseline differences revealed that program participants were significantly more likely than the national sample to be enrolled in school, to be employed and to have avoided a subsequent pregnancy 12 months later. Several baseline variables, including age at first birth, ethnicity, education and living arrangements, were also predictive of one-year outcomes.

The links between types of maltreatment and demographic characteristics of children.

This paper examines the relative impact of demographic characteristics of the child, family structure, and economic variables on types of child abuse and neglect. The current analysis is based on data from the second National Incidence Study of Child Abuse and Neglect (NIS-2), which collected information from both CPS and non-CPS agencies (e.g., schools, hospitals) in a national sample of 29 counties (Westat, 1988). The NIS-2 offers a unique opportunity to examine abuse and neglect issues with a large, national data set. This paper looks at a series of exploratory logistic regression models to distinguish between four different types of maltreatment: (a) physical abuse, (b) sexual abuse, (c) emotional maltreatment, and (d) physical neglect. Our findings show that physical neglect, in comparison with the other types of abuse, is the most predictable and distinguishable. It is most clearly related to economic factors such as low income and Aid to Families with Dependent Children (AFDC) status, regardless of race. Additionally, both sexual abuse and physical neglect occur at younger ages than previously shown. The policy implications for these findings are discussed.

Structure-activity analysis of microsomal antigen/thyroid peroxidase.

The interaction between thyroid microsomal autoantibodies and thyroid microsomal antigen/thyroid peroxidase (TPO) has been studied using both intact antigen preparations and their water-soluble trypsin fragments. In an analysis of sera from 30 patients with Graves' or Hashimoto's diseases, microsomal antibodies showed similar reactivity towards trypsin fragments (with TPO activity) and intact detergent (sodium deoxycholate, DOC)-solubilized human microsomal antigen preparations (r = 0.96). This raised the possibility that both the peroxidase-active site and the major autoantigenic site(s) of microsomal antigen were present on the same trypsin fragments. Studies with porcine TPO showed that only a few sera contained microsomal antibodies which cross-reacted strongly with the porcine preparations. Further analysis was carried out by immunoprecipitation of 125I-labelled microsomal antigen followed by SDS-PAGE and autoradiography. These studies suggest that intact human microsomal antigen (a single-chain protein with Mr = 110,000) contains an intrachain loop of amino acids formed by a disulphide bridge. Trypsin treatment cleaves the antigen close to its transmembrane section and releases a water-soluble fragment (Mr = 100,000), containing the intact disulphide-linked loop of amino acids. Further trypsin action causes cleavage of the peptide bonds within the loop in some preparations. Consequently, three major water-soluble trypsin fragments (Mr = 100,000, 73,000 and 68,000) are formed all of which contain an intact disulphide bridge and have microsomal antibody binding activities. The integrity of the disulphide bridge in intact antigen/TPO preparations and their trypsin fragments is essential for autoantibody binding activity.

Assessment of the shape and molecular size of TSH-TSH receptor complexes.

Photoaffinity labelling and analysis under denaturing conditions (SDS-PAGE) have shown that the porcine TSH receptor contains an A subunit (Mr = 47,000) which forms the binding site for TSH and a B subunit (Mr = 25,000) linked to the A subunit by a disulphide bridge. In order to assess the size and shape of the receptor under non-denaturing conditions we have solubilized photoaffinity-labelled porcine TSH receptors using the small micelle-sized detergent sodium deoxycholate and analysed the preparations by sucrose density gradient centrifugation and gel filtration. Under these conditions, the cross-linked TSH-TSH receptor complex showed an S20,w of 6.4 S and a frictional ratio f/f0 of 1.8. These values were consistent with those which might be expected from an elongated protein complex with a molecular weight of about 100,000 (the value obtained by SDS-PAGE). Analysis of another thyroid membrane protein, human thyroid microsomal antigen (Mr = 110,000 by SDS-PAGE) under the same conditions gave an S20,w of 6.0 S and f/f0 = 1.3, suggesting that this protein has a compact structure. The TSH receptor A subunit cross-linked to TSH (Mr = 70,000 by SDS-PAGE) gave an S20,w of 4.6 S and f/f0 = 1.8 and these values could be compared with those obtained for the A subunit alone (S20,w = 3.6 S; f/f0 = 1.4; Mr by SDS-PAGE = 47,000) and TSH alone (S20,w = 2.6 S; f/f0 = 1.6; Mr = 28,000.(ABSTRACT TRUNCATED AT 250 WORDS)

The interaction between the TSH receptor and Graves' sera with TSH agonist or antagonist properties.

The A subunit of the TSH receptor was prepared by reduction of human thyroid membranes with dithiothreitol, and partially purified by gel filtration. The ability of Graves' sera to inhibit TSH binding to the TSH receptor and to stimulate cyclic AMP release from isolated thyroid cells was abolished by incubation with crude and partially purified preparations of the A subunit.

An investigation of the properties of ornithine aminotransferase after inactivation by the 'suicide' inhibitor aminohexynoate and use of the compound as a probe of intracellullar protein turnover.

Ornithine aminotransferase is shown to bind 1 mol of amino[14C]hexynoate per mol of coenzyme in the 'suicide' inactivation process. At the same time the coenzyme pyridoxal phosphate becomes irreversibly bound to the enzyme protein. Apart from the inactivation, the labelled enzyme is indistinguishable from native ornithine aminotransferase by several separation techniques. Because the rate of degradation of the labelled enzyme is the same as that of the normal enzyme it is concluded that loss of coenzyme does not initiate turnover. Free aminohexynoate is rapidly eliminated from the liver, and 70% of the compound is excreted unchanged in 7.5 h. Inactivated ornithine aminotransferase accounts for 11% of the total labelled liver protein and significant amounts of label are found in aspartate aminotransferase which is also extensively inactivated. The rate of return of enzyme activity is determined and found to be more rapid than expected for a process in which the enzyme is synthesized at a constant rate and degraded in a single, first-order process.

1-pps 2-microsec pulse high-energy frequency-doubled Nd:YAG/ glass laser for studying combustion processes.

A frequency-doubled Nd:YAG/glass laser system has been constructed for use at the Combustion Research Facility at Sandia National Laboratories, Livermore. This laser system will be used to make single-shot spontaneous Raman spectroscopy measurements of ongoing combustion processes. The output of the system is 5 J of 1.064-microm radiation and 0.8 J of 532-nm radiation in a 2-microsec pulse. The repetition rate of the system is 1 pps. A microprocessor-based diagnostic and control system for the operation is described.

5-J 1.8-microsec pulse 10-pps dye laser for combustion applications.

A 5-J/pulse 1.8-microsec pulse width 10-Hz flashlamp-pumped tunable dye laser system has been built for performing spontaneous Raman scattering experiments in combusting media at the Combustion Research Facility, Sandia National Laboratories, Livermore. Details of construction, including a microprocessor-based diagnostic and control system, are described, and performance of the system in terms of various operating parameters is noted.

Enzyme-induced inactivation of transminases by acetylenic and vinyl analogues of 4-aminobutyrate.

The reactions of two analogues of 4-aminobutyrate, namely 4-aminohex-5-ynoate and 4-aminohex-5-enoate, with three transaminases were studied. Three pure enzymes were used, aminobutyrate transaminase (EC 2.6.1.19), ornithine transaminase (EC 2.6.1.13) and aspartate transaminase (EC 2.6.1.1), and the course of the reactions was studied by observing changes in the absorption spectrum of the bound coenzyme and by observing loss of activity. All of the enzymes were inactivated by either inhibitor, but amino-hexenoate showed a marked specificity for aminobutyrate transaminase. Aminohexynoate was most potent towards ornithine transaminase, and with this enzyme transamination of the inhibitor is an important factor in protecting the enzyme. Most of the reactions could be analysed as first order, with the observed rate constant showing a hyperbolic dependence on inhibitor concentration.

Pulsed acousto-optical modulator driver.

A gated rf driver for an acousto-optic modulator used to actively mode-lock a laser is described. The modulator is driven in a pulsed mode, so that much higher rf power can be applied to obtain high modulation indices.

Control of temporal and spectral jitter in single mode pulsed ND:YAG oscillators.

We report reliable operation of a repetitively pulsed (3 pps) passively Q-switched Nd:YAG oscillator on a single longitudinal mode with a high degree of temporal and spectral stability. The total output is 3.5 mJ in a 7-ns bandwidth limited pulse with diffraction limited divergence of 0.33 mrad.