Sara E. Bryan

Angiotensin II receptors in chromatin fragments generated by micrococcal nuclease.

Rat liver nuclei were digested with micrococcal nuclease following incubation with 125I-angiotensin II (AII) or with 125I-AII and excess unlabeled hormone. Chromatin enriched in 125I was solubilized after 3 min and was applied to a BIO-GEL A-5 M column. Labeled hormone was 40-60% displaceable by unlabeled hormone, in nucleoprotein eluting with a V/Vo near 1.9, indicating that these solubilized chromatin fragments contained specific receptors for AII. Furthermore, a discrete AII binding nucleoprotein particle was resolved on DNP gel electrophoresis. Additionally, binding to specific AII nuclear receptors appeared to bring about changes in chromatin structure consistent with the induction of transcriptional activity.

Effect of cadmium on RNA-polymerase and protein synthesis in rat liver

By studying the intracellular movement of cadmium we have shown that the metal enters rat liver nuclei and attains a maximum concentration (3.0 X 1 OM3 pmol Cd/mg DNA) after one hour in rats received 20 pmol of cadmium/kg [ 1 ] and have correlated the uptake and disappearance of cadmium from the nucleus with the appearance of a cytoplasmic cadmium binding protein (CdBP). Other investigators [2,3] have shown that RNA polymerase (a nuclear bound enzyme) is either completely or severely inhibited by adding cadmium to nuclei at concentrations which are 30-800 times greater than those found in vivo. Weser and Htibner [2] have reported that cadmium does not inhibit the RNA polymerase activity of rat liver nuclei 10 hours after animals were injected with 10 pmol of Cd/kg. In an attempt to reconcile these in vivo and in vitro data, we have studied the effect of cadmium on RNA polymerase activity prior to and after 10 hours of exposure. In this report we show that cadmium inhibits RNA polymerase activity in vivo. The metal also inhibits, perhaps independently, protein synthesis in the liver. Our data suggest that RNA polymerase activity is dependent on nuclear cadmium levels up to seven hours after intraperitoneal cadmium injection, while at eleven hours RNA polymerase activity is no longer a function of nuclear cadmium levels under our given experimental conditions.

Nuclear 115cadmium: Uptake and disappearance correlated with cadmium-binding protein synthesis

Abstract The intracellular distribution of 115cadmium was determined following a pulsed exposure to the metal. The uptake and disappearance of label from rat liver nuclei was correlated with the appearance of a cytoplasmic Cd-binding protein. By coupling in vivo - in vitro experiments it was shown that unspecifically bound cadmium is free to enter the nucleus while specifically bound cadmium remains in the cytoplasm.

Nuclear-hormone mediated changes in chromatin solubility

Rat liver nuclei were incubated with either thyroid hormone or angiotensin (AII) at varying concentrations or with buffer (control) prior to digestion with micrococcal nuclease. Concentrations of hormones greater than 10(-10)M were effective in increasing the solubilization of chromatin with physiological levels (10(-9)M) of AII showing an approximate 2.4 fold increase over control. Nuclei were also isolated from animals treated in-vivo with either AII or buffer (control) and chromatin solubility was increased in the AII treated nuclei even prior to the addition of exogenous nuclease, presumably from the action of endogenous nucleases. The data suggest that hormone-induced increases in solubility are a reflection of structural changes in chromatin which enhance the accessibility of DNA to endonuclease attack.

Partitioning of zinc and copper in fetal liver subfractions: Appearance of metallothionein-like proteins during development☆

Abstract The appearance and subsequent accumulation of cytosolic metallothionein (MT)-like proteins in developing fetal rat liver begin as early as 14 days postfertilization (P.F.). Fetal MT-like proteins increase steadily; while in contrast, maternal MT-like protein levels remain fairly constant throughout pregnancy. The accumulation of these proteins seems to be related to the differential partitioning of zinc and copper in several cellular subfractions: zinc accumulates in the fetal hepatic cytosol, while copper specifically localizes in the particulate fraction. We report an approximate 10-fold increase in the percentage of cytosolic zinc bound to MT-like proteins between 15 and 17 days (P.F.). Furthermore, from Day 17 (P.F.) until term, the percentage zinc associated with MT-like proteins remains relatively constant, with a slight increase from 21 to 23 days (P.F.). We are suggesting that this initial partitioning of zinc within the MT fraction may be one of the biochemical signals that elicits the shift from proliferation to differentiation during hepatic development in the rat.

Intranuclear Localization of Mercury in vivo

Purified nuclei isolated from mice challenged with nonlethal levels of mercury chloride (10-3M) in drinking water for 4 to 7 weeks (experimental) and from animals given deionized water (control) were fractionated and the subsequent fractions were analyzed for mercury by flameless atomic absorption. Control (active) euchromatin contained 1.75 � 0.53 micrograms of mercury per milligram of DNA. There was a 12- to 15-fold enrichment of mercury in the euchromatin fraction of challenged animals. Mercury was not detected in control (inactive) heterochromatin, and only trace levels (parts per billion) appeared in experimental heterochromatin. It seems likely that mercury can be incorporated into chromatin as a metal-protein complex, but the possibility of protein-mercury-DNA or mercury-DNA complexes within euchromatin cannot be excluded.

Functional Intracellular Renin-Angiotensin Systems May Exist in Multiple Tissues

The renin-angiotensin system is customarily thought of as a homeostatic feedback loop involved in the regulation of intravascular volume and pressure. However, over the last three decades, renin has been identified in a wide variety of nonrenal tissues and indeed our laboratory has confirmed synthesis of renin in at least one extrarenal site, namely, the arterial smooth muscle cell (1). Although an important neuroregulatory role for angiotensin II is likely and a paracrine role for renin in peripheral tissues is possible, a local cellular or intracellular role cannot be excluded. Other workers have provided evidence suggestive of intracellular angiotensin II receptors (2) and our laboratory has detected the presence of high-affinity specific angiotensin II receptors on isolated hepatic nuclei (3). Additionally, we have provided evidence suggesting a direct effect of angiotensin II on RNA polymerase II activity (4) as well as on conformational changes in chromatin of the sort associated with enhanced trans...

Cadmium-115 bound to nuclear and cytoplasmic proteins

Abstract The distribution of cadmium-115 in DNA, histone, and nonhistone proteins, and with two cytoplasmic subfractions of cadmium-biding protein was measured in rat liver after a single cadmium exposure (20 μmol of CdCl 2 /kg). The nuclear cadmium (per milligram of protein) was twice as concentrated in nonhistone proteins as in the histone proteins. The cytoplasmic cadmium was unevenly distributed with two cadmium-binding protein subfractions, however, the relative amounts of these proteins, with respect to each other, remained constant throughout the period of the study (188-hr maximum). These data provide further evidence for the specificity of the in vivo interactions of cadmium with cellular components.

Localization and uptake of copper into chromatin

Analysis of total liver, liver nuclei, and nuclear fractions obtained from mice given either deionized water (control) or deionized water containing cupric chloride (experimental) revealed the presence of copper in all samples tested including control nuclei and control heterochromatin and euchromatin. Copper content in control euchromatin appear to remain constant at approximately 0.3 μg/mg DNA, while the concentration in control heterochromatin seem to be subject to wider variations. An elevation in total liver copper followed prolonged treatment with metal (in vivo) and was accompanied by an increase in total nuclear copper. A preferential uptake of copper by heterochromatin was suggested by the majority of metal binding to this fraction; there was no significant metal accumulation in euchromatin. Dialysis equilibrium experiments were carried out at 25°C, pH 6.0 to determine in vitro binding of the two nuclear fractions. Under physiologic ionic strength (154 mm) heterochromatin binds considerable copper, whereas euchromatin fails to bind appreciable amounts. This biological specificity is lost under conditions of low ionic strength (<2 mm NaCl).

Effects of ionic strength on endogenous nuclease activity in chelated and nonchelated chromatin

Calf thymus chromatin, isolated using a standard (low ionic strength, but nonchelating) isolation protocol, dialyzed against either Tris-PMSF or Tris-EDTA, was reconstituted in a high salt compacting buffer (COM) or a low salt dispersing buffer (DIS) prior to digestion with endogenous nucleases. A greater level of enzyme activity occurred when chromatin was in a condensed state (COM buffer) and not chelated prior to digestion. In contrast, chromatin chelated by dialysis against Tris-EDTA prior to digestion showed higher levels of enzyme activity in the dispersed state (DIS buffer). Nonchelated undigested chromatin contained 0.280 +/- 0.16 ug copper/mg DNA and and 0.305 +/+- 0.09 ug zinc/mg DNA. Chelation removed about 78% of copper per mg DNA and approximately 65% of zinc per mg DNA. In COM buffer after a 20 min digestion, the solubilized fraction was enriched in copper showing about 20 X more metal per mg DNA than nonchelated chromatin. Approximately the same amount of zinc was found in both chelated and nonchelated chromatin while there was less zinc in chelated chromatin solubilized in DIS buffer. Thus, chelation has important effects on the digestibility of chromatin and on the type of ionic environment that provides the most favorable conditions for endogenous nuclease activity.