Urban community reaction to health facilities in residential areas: lessons from the placement of methadone facilities in New York City.

Neighborhood characteristics associated with negative reaction to community-based methadone maintenance treatment programs (MMTPs) were studied using the MMTPs in Brooklyn, Bronx, Manhattan, and Queens (71 census tracts). A measure of community opposition to the establishment of the clinics was developed (CRMC). Census and survey data were combined for data analysis. Results are presented following a fourfold conceptual framework focused on the community's social ecology, patient characteristics, clinic administration, and the physical ecology of the clinic location. Eight specific characteristics found within these four conceptual areas accounted for 49% of the variation in CRMC.

Steroid receptor-nuclear matrix interactions. The role of DNA.

The interaction of sex steroid hormone receptors with the nuclear matrix (NM) of target and non-target tissue was investigated using a simple in vitro binding assay. Steroid receptors can recognize acceptor sites on the NM of target cells; androgen receptor binds with the highest apparent affinity to rat prostate NM; similarly estrogen receptor binds with the highest apparent affinity to uterine NM. Furthermore, the steroid receptor-NM interaction depends upon the hormonal status of the animal. The binding of androgen receptor to rat prostate NM was drastically reduced upon hormone withdrawal (castration) and fully recovered upon hormonal stimulation. When NM were prepared by an alternate method (DNase I digestion prior to high salt extraction) known to digest "active" chromatin, no preferential receptor binding to target tissue NM was observed. Although the NM fraction contains less than 1% of the total nuclear DNA, the matrix-associated DNA sequences seem to be, at least in part, responsible for specific receptor recognition. DNA extracted from the prostate NM was shown to be a potent competitor for androgen receptor binding as measured by DNA-cellulose competition experiments. Moreover, this DNA recognition also depends upon the hormonal status of the animal. These studies are consistent with the notion that hormonal manipulation induces changes in the NM-associated DNA sequences of steroid hormone target tissue.

Assessment of estrogen receptor--histone interactions.

Several different in vitro binding assays show that the estrogen receptor from rabbit uterus interacts selectively with purified histones from calf thymus. The estrogen receptor binds strongly to histones H2B and H2A, moderately to histones H3 and H4, and poorly to histone H1. In the presence of histones H2B or H2A, the position at which the estrogen receptor focuses in an isoelectric gradient is shifted to a more basic zone. Kinetic experiments show that, if histone H2B is bound to a DNA, the estrogen receptor dissociates more slowly from that DNA. The portion of the estrogen receptor molecule required for binding to histone H2B is relatively stable to tryptic digestion; in contrast, the portion of the receptor molecule responsible for DNA binding is promptly lost during limited tryptic digestion. These in vitro findings suggest that the mechanism by which the estrogen receptor selectively alters gene expression may involve specific contacts with histone molecules.

RNA inhibits estrogen receptor binding to DNA.

Cytosol from MTW9 rat mammary tumor contains a high molecular weight inhibitor of estrogen receptor binding to DNA. RNase treatment of crude preparations destroys inhibitory activity, whereas DNase or trypsin treatment are without affect, suggesting that some RNA molecule might be involved; in addition, RNA extracted from tumor cytosol was an effective inhibitor of estrogen receptor binding to DNA. A number of synthetic and natural RNA species were analyzed for inhibitory activity; a definite specificity was observed, with poly(G) > poly(U) > poly(A) or poly(C).

Effect of photooxidation on the estrogen receptor from rat mammary tumor.

Photooxidation of estradiol receptor form MTW9 rat mammary tumor cytosol leads to a time-dependent loss of DNA-binding activity; this effect is pH-dependent. Binding of receptor to DNA does not protect the DNA-binding site from photooxidation. The estrogen binding site of the receptor is also susceptible to photooxidation, although the rate of loss of estradiol binding capacity is considerably less than that of DNA-binding capacity; this process is independent of pH. Binding of receptor to estradiol substantially protects the estradiol binding site from photooxidation. Sucrose gradient analysis of photooxidized receptor preparations showed no qualitative difference compared with nonirradiated controls. Similarly, the isoelectric focusing pattern of photooxidized receptor showed two peaks of radioactivity at pH approximately 5.6 and 6.3, as did nonirradiated receptor.

Estrogen receptor has enhanced affinity for bromodeoxyuridine-substituted DNA.

The estrogen receptor (E2R) from rabbit uterus has an enhanced affinity for BrdUrd-substituted DNA compared to unsubstituted DNA. Increasing levels of BrdUrd substitution (from 0 to 100%) in a DNA sample are associated with increasingly tighter binding of E2R to that DNA as measured by equilibrium competition experiments and by rates of dissociation (or receptor transfer experiments). Although the rates of dissociation of E2R-DNA complexes vary greatly, depending on the BrdUrd-substitution level in the DNA, no differences were detected between the rates of association of E2R with unsubstituted and fully BrdUrd-substituted DNA. The E2R-DNA complex dissociates more rapidly in 150 mM KCl than in 50 mM KCl; but, at both ionic strengths, BrdUrd substitution in the DNA confers enhanced stability on the complex. The demonstration that a specific mammalian regulatory protein has an enhanced affinity for BrdUrd-substituted DNA further strengthens the possibility that BrdUrd modulates gene expression through an altered binding of regulatory proteins.

Photochemical attachment of cyclic AMP binding protein(s) to the nuclear genome.

The nature of the cyclic AMP-receptor-nucleus interactions was examined by a novel combination of two photoreactions. A photosensitive derivative of cyclic AMP, N6-butyryl cyclic AMP, was covalently attached to its cytoplasmic receptod by photo-affinity labelling and this receptor complex was photo-crosslinked by the DNA in the rat liver nuclei. The photolytic reactions seemed to be specific since stable links were formed only when substantial noncovalent binding occured.

Covalent attachment of diethylstilbestrol to glutamate dehydrogenase: implications for allosteric regulation.

An affinity labeling reagent for the estrogenic-binding site of bovine liver L-glutamate dehydrogenase (EC 1.4.1.3) was prepared by conversion of diethylstilbestrol to its alkylating analogue, bromoacetyldiethylstilbestrol. Under standard assay conditions, the analogue acted as a reversible allosteric ligand with regulatory activity much like that of diethylstilbestrol. However, incubation of the enzyme with the alkylating agent in the presence of DPNH resulted in a permanent decrease in glutamate (X form) and an increase in alanine (Y form) activities, and in covalent attachment of diethylstilbestrol in the ratio of 1 mol per subunit (of particle weight 52,000). The brominated analogue behaved as an affinity label that mimicked the allosteric effects of diethylstilbestrol. Diethylstilbestrol protection of the enzyme against alkylation by bromoacetylated sterol suggested competition for the same binding site, while ADP protection indicated a shift of protein equilibrium into the X form. The diethylstilbestrol-enzyme compound was desensitized (relative to the native enzyme) to allosteric reagents such as ADP and GTP. The results were consistent with conformational freezing of the modified protein molecule into the Y form.