The replication of bovine papilloma virus (BPV) DNA in vivo requires two viral-encoded proteins, El and E2, while all other proteins are derived from the host. We described previously the isolation of the El protein and showed that it contains multiple functions required for BPV DNA replication. The BPV transcription factor E2 was shown by others to stimulate BPV DNA replication in vitro. Here, we present results that account for the role of the E2 protein. The El protein bound selectively to the BPV minimal origin of replication. This process required MgCl2 and ATP for maxal effi'ciency. The El protein also catalyzed a BPV origindependent DNA unwinding reaction. In this report, we show that at low levels of El protein, origin binding could be stimulated up to 40-fold by the E2 protein, provided that the DNA contained an E2 binding site. Consistent with this result, the E2 protein stimulated the origin-specific unwinding reaction catalyzed by El, but it had no effect on the nonspecific El-catalyzed helicase activity. In the absence of an E2 binding site, both origin-dependent binding and unwinding reactions with the El protein were unaffected by the E2 protein. These results suggest that E2 participates in the initiation of BPV DNA replication by enhancing El binding to the BPV or4ign via DNA-protein and protein-protein interactions.Bovine papilloma virus (BPV) provides an attractive model system to study the regulation of eukaryotic DNA replication. The viral DNA is maintained in BPV transformed cells as a nuclear plasmid with a constant copy number (1). Recent studies indicate that in vivo BPV DNA replication requires two BPV viral-encoded proteins-the 68-kDa El protein and the 48-kDa E2 protein (2). In addition, the minimal origin sequence that supports BPV DNA replication has been identified (nt 7911-22 of the BPV type 1 genome) (3,4). This sequence contains a binding site for the El protein (El BS) and part of a sequence that acts as a binding site for the E2 protein (E2 BS).The replication of BPV oril DNA in vitro, using extracts of a mouse mammary tumor cell line (FM3A), was recently established in Botchan's laboratory (4). They demonstrated that DNA synthesis required the BPV minimal origin and the El protein, whereas all other required proteins were supplied by uninfected mouse cell extracts. At low concentrations of El protein, the replication reaction was stimulated markedly by the E2 protein (4).We have described the isolation of the El protein and showed that it supported BPV ori+ DNA replication in vitro (5). We also demonstrated that this protein possessed a number of different activities required for BPV DNA replication. These include (i) a DNA helicase activity, for which the protein translocates in the 3' to 5' direction, (ii) a BPV oril DNA binding activity that is stimulated by ATP and MgCl2, and (iii) the capacity to unwind covalently closed circular ori+ DNA leading to highly unwound DNA products (5). Thus, the role of El in the BPV system is analogous to that of large tumor antigen...

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Drug delivery technology is emerging as an interdisciplinary science aimed at improving human health. The controlled delivery of pharmacologically active agents to the specific site of action at the therapeutically optimal rate and dose regimen has been a major goal in designing drug delivery systems. Over the past few decades, there has been considerable interest in developing biodegradable drug carriers as effective drug delivery systems. Polymeric materials from natural sources play an important role in controlled release of drug at a particular site. Polyhydroxyalkanoates, due to their origin from natural sources, are given attention as candidates for drug delivery materials. Biodegradable and biocompatible polyhydroxyalkanoates are linear polyesters produced by microorganisms under unbalanced growth conditions, which have emerged as potential polymers for use as biomedical materials for drug delivery due to their unique physiochemical and mechanical properties. This review summarizes many of the key findings in the applications of polyhydroxyalkanoates and polyhydroxyalkanoate nanoparticles for drug delivery system.

IREs are RNA stem loop sequences found in the 5Ј-UTR 1 of the mRNAs for ferritin and erythroid amino-levulinic acid synthetase and the 3Ј-UTR of transferrin receptor where they function as a binding site for iron regulatory proteins (IRPs) (reviewed in Refs. 1-3). The two known IRPs, IRP1 and IRP2, bind IREs with equal and high affinity and mediate translational repression of transcripts containing ferritin IREs with equal efficacy (4). IRP1 is a bifunctional protein that also functions as cytosolic aconitase, an enzyme that catalyzes the reversible isomerization of citrate to isocitrate via cis-aconitate in the cytosol (5-8). There are two different aconitases in mammalian cells, cytosolic and mitochondrial, which are encoded by two different genes. Both aconitases contain a [4Fe-4S] cluster that is required for enzymatic activity (9), and active site residues are identical between the two proteins (10). Although IRP1 was initially identified as an IRE binding protein, it was shown after comparison of peptide sequences of IRP1 and purified cytosolic aconitase that the two activities were derived from the identical protein (9). IRP1 makes a quantitative transition to the iron-sulfur cluster containing cytosolic aconitase when cells are iron-replete and to the IRE binding form when the iron-sulfur cluster is absent from the protein (6). Whereas the role of mitochondrial aconitase in interconversion of citrate and isocitrate in the citric acid cycle is well understood, the reason that aconitase activity is maintained in the cytosol is not clear. Although the two aconitases share approximately 25% sequence identity, mitochondrial aconitase does not bind IREs (5).Targets of IRP regulation include the transcripts of ferritin, transferrin receptor, and erythroid amino-levulinic acid synthetase. Recently, an IRE has been described in the 5Ј-UTR of the iron protein subunit of succinate dehydrogenase of Drosophila melanogaster (11); the IRE mediates translational regulation by iron of a reporter gene and appears to be responsible for translational regulation of succinate dehydrogenase in Drosophila cell lines (12). A consensus IRE has previously been described in the 5Ј-UTR of porcine aconitase (13,14), but it has been difficult to determine whether the IRE is functional in cells because antibodies that will immunoprecipitate porcine aconitase have not been available.In order to determine whether the IRE in porcine aconitase is functional and physiologically relevant, we have evaluated the transcript by three different approaches. The first is to search data bases for other mammalian mitochondrial aconitase transcripts and to evaluate whether the IRE sequence element is conserved when RACE techniques are used to complete the 5Ј-UTR. The second is to test the efficacy of the 5Ј-UTR in mediating translational regulation in vitro. The third is to measure total levels of mitochondrial aconitase protein in tissues of animals fed on low versus high iron diets to evaluate whether there is a change consistent with translational ...