The ability of metals to activate estrogen receptor-alpha (ERalpha) was measured in the human breast cancer cell line, MCF-7. Similar to estradiol, treatment of cells with the divalent metals copper, cobalt, nickel, lead, mercury, tin, and chromium or with the metal anion vanadate stimulated cell proliferation; by d 6, there was a 2- to 5-fold increase in cell number. The metals also decreased the concentration of ERalpha protein and mRNA by 40-60% and induced expression of the estrogen-regulated genes progesterone receptor and pS2 by1.6- to 4-fold. Furthermore, there was a 2- to 4-fold increase in chloramphenicol acetyltransferase activity after treatment with the metals in COS-1 cells transiently cotransfected with the wild-type receptor and an estrogen-responsive chloramphenicol acetyltransferase reporter gene. The ability of the metals to alter gene expression was blocked by an antiestrogen, suggesting that the activity of these compounds is mediated by ERalpha. In binding assays the metals blocked the binding of estradiol to the receptor without altering the apparent binding affinity of the hormone (K(d) = 10(-10) M). Scatchard analysis employing either recombinant ERalpha or extracts from MCF-7 cells demonstrated that (57)Co and (63)Ni bind to ERalpha with equilibrium dissociation constants of 3 and 9.5 x 10(-9) and 2 and 7 x 10(-9) M, respectively. The ability of the metals to activate a chimeric receptor containing the hormone-binding domain of ERalpha suggests that their effects are mediated through the hormone-binding domain. Mutational analysis identified amino acids C381, C447, E523, H524, N532, and D538 as potential interaction sites, suggesting that divalent metals and metal anions activate ERalpha through the formation of a complex within the hormone-binding domain of the receptor.

We present evidence for a complex regulatory interplay between the initiation of DNA replication and deoxyribonucleotide synthesis. In Escherichia coli, the ATP-bound DnaA protein initiates chromosomal replication. Upon loading of the b-clamp subunit (DnaN) of the replicase, DnaA is inactivated as its intrinsic ATPase activity is stimulated by the protein Hda. The b-subunit acts as a matchmaker between Hda and DnaA. Chain elongation of DNA requires a sufficient supply of deoxyribonucleotides (dNTPs), which are produced by ribonucleotide reductase (RNR). We present evidence suggesting that the molecular switch from ATP-DnaA to ADP-DnaA is a critical step coordinating DNA replication with increased deoxyribonucleotide synthesis. Characterization of dnaA and dnaN mutations that result in a constitutively high expression of RNR reveal this mechanism. We propose that the nucleotide bound state of DnaA regulates the transcription of the genes encoding ribonucleotide reductase (nrdAB). Accordingly, the conversion of ATP-DnaA to ADP-DnaA after initiation and loading of the b-subunit DnaN would allow increased nrdAB expression, and consequently, coordinated RNR synthesis and DNA replication during the cell cycle.

SummaryInitiator DnaA and DNA bending proteins, Fis and IHF, comprise prereplication complexes (pre-RC) that unwind the Escherichia coli chromosome's origin of replication, oriC . Loss of either Fis or IHF perturbs synchronous initiation from oriC copies in rapidly growing E. coli . Based on dimethylsulphate (DMS) footprinting of purified proteins, we observed a dynamic interplay among Fis, IHF and DnaA on supercoiled oriC templates. Low levels of Fis inhibited oriC unwinding by blocking both IHF and DnaA binding to low affinity sites. As the concentration of DnaA was increased, Fis repression was relieved and IHF rapidly redistributed DnaA to all unfilled binding sites on oriC . This behaviour in vitro is analogous to observed assembly of pre-RC in synchronized E. coli . We propose that as new DnaA is synthesized in E. coli , opposing activities of Fis and IHF ensure an abrupt transition from a repressed complex with unfilled weak affinity DnaA binding sites to a completely loaded unwound complex, increasing both the precision of DNA replication timing and initiation synchrony.

Initiation of DNA replication from the Escherichia coli chromosomal origin is highly regulated, assuring that replication occurs precisely once per cell cycle. Three mechanisms for regulation of replication initiation have been proposed: titration of free DnaA initiator protein by the datA locus, sequestration of newly replicated origins by SeqA protein and regulatory inactivation of DnaA (RIDA), in which active ATP-DnaA is converted to the inactive ADP-bound form. DNA microarray analyses showed that the level of initiation in rapidly growing cells that lack datA was indistinguishable from that in wild-type cells, and that the absence of SeqA protein caused only a modest increase in initiation, in agreement with flow-cytometry data. In contrast, cells lacking Hda overinitiated replication twofold, implicating RIDA as the predominant mechanism preventing extra initiation events in a cell cycle.

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health (NIH), has developed a Standard Reference Material (SRM) to support technology development in metabolomics research. SRM 1950 Metabolites in Human Plasma is intended to have metabolite concentrations that are representative of those found in adult human plasma. The plasma used in the preparation of SRM 1950 was collected from both male and female donors, and donor ethnicity targets were selected based upon the ethnic makeup of the U.S. population. Metabolomics research is diverse in terms of both instrumentation and scientific goals. This SRM was designed to apply broadly to the field, not toward specific applications. Therefore, concentrations of approximately 100 analytes, including amino acids, fatty acids, trace elements, vitamins, hormones, selenoproteins, clinical markers, and perfluorinated compounds (PFCs), were determined. Value assignment measurements were performed by NIST and the Centers for Disease Control and Prevention (CDC). SRM 1950 is the first reference material developed specifically for metabolomics research.