The oncogenic property of the adenovirus (Ad) transforming E1A protein is linked to its capacity to induce cellular DNA synthesis which occurs as a result of its interaction with several host proteins, including pRb and p300/CBP. While the proteins that contribute to the forced induction of cellular DNA synthesis have been intensively studied, the nature of the cellular DNA replication that is induced by E1A in quiescent cells is not well understood. Here we show that E1A expression in quiescent cells leads to massive cellular DNA rereplication in late S phase. Using a single-molecule DNA fiber assay, we studied the cellular DNA replication dynamics in E1A-expressing cells. Our studies show that the DNA replication pattern is dramatically altered in E1A-expressing cells, with increased replicon length, fork velocity, and interorigin distance. The interorigin distance increased by about 3-fold, suggesting that fewer DNA replication origins are used in E1A-expressing cells. These aberrant replication events led to replication stress, as evidenced by the activation of the DNA damage response. In earlier studies, we showed that E1A induces c-Myc as a result of E1A binding to p300. Using an antisense c-Myc to block c-Myc expression, our results indicate that induction of c-Myc in E1A-expressing cells contributes to the induction of host DNA replication. Together, our results suggest that the E1A oncogene-induced cellular DNA replication stress is due to dramatically altered cellular replication events and that E1A-induced c-Myc may contribute to these events. The adenovirus (Ad) transforming E1A protein [a 243-aminoacid E1A protein, also referred to as small E1A protein [1,2]) has the capacity to induce S phase in quiescent cells, and in the presence of activated ras or virus-encoded E1B19K or 55K proteins, E1A can transform rodent cells in culture (1, 2). The S-phase induction and cell transformation activities of the small E1A protein are genetically linked and are dependent on the N-terminal region of E1A binding to cellular protein complexes, including TRRAP/p400/GCN5, histone acetyltransferase p300/CBP, and the Rb family tumor suppressor proteins (1-4). E1A-Rb interactions result in the release of the progrowth E2F family transcription factors from the Rb-histone deacetylase (HDAC) repressor complexes and the induction of the S phase (1, 5). However, studies have shown that in order for E1A to induce S phase efficiently, it must bind to p300/CBP and Rb family proteins simultaneously, suggesting that E1A must also alter the functions of p300/CBP (3, 6).Although a large number of studies have focused on the cellular proteins that contribute to the forced induction of host DNA synthesis in E1A-expressing cells, the nature of the cellular DNA that replicates in these cells is not well understood. Previous studies have shown that the E1A-expressing cells fail to undergo proper mitosis and that such cells accumulate in the S and G 2 /M phases (7-10). Mammalian cells contain a large number of DNA replication origins, a...

JMI-thrombin is used as topical hemostatic agent. While earlier clinically available JMI were reported to produce immunologic responses upon repeated exposure, the improved JMI, Recothrom?, and Evithrom? are claimed to be less immunogenic. Recothrom, despite its reduced immunogenic nature, upon repeated administration may result in the generation of antibodies (Abs) and that may cross react with bovine and human thrombin. Therefore, groups of rabbits were challenged repeatedly with Recothrom, Evithrom, and JMI over a 9-month period. Pre-immune blood and antiserum were collected from each rabbit on different time point. To determine their relative cross reactivity, JMI, Recothrom, and Evithrom were evaluated by western blotting using the rabbit IgG fractions. The results suggest that anti-Recothrom Abs cross-react with Evithrom and JMI in a time dependent fashion. Anti-JMI Abs did not cross-react with Recothrom, and Evithrom. Also, anti-Evithrom did not show any cross-reactivity with Recothrom and JMI at any time.

4321 Recombinant versions of hirudin such as lepirudin (Refludan®) and desirudin (Iprivask®) are currently used as parenteral anticoagulants for various indications. The recombinant hirudin preparations differ from the natural hirudin in lacking a sulfate group on tyrosine at the 63rd position and a single amino acid substitution. It is hypothesized that these minor differences in the natural and recombinant versions of hirudins contribute to a differential immunogenic behavior. The purpose of this investigation was to compare the relative immunoreactivity of the three forms of hirudin to a sheep antin–hirudin antibody. Antibodies (anti-n-hirudin) were generated in sheep treated with natural hirudin isolated from the medicinal leech (Hirudo medicinalis ). SDS-PAGE immunoblot and Western transfer were conducted using the native hirudin and two recombinant hirudins against the sheep anti-n-hirudin IgG antibodies. Cross-reactivity was quantified by measuring total optical density of the bands using a UVP densitometric scanning system and chemiluminescence detection, by comparing the protein-antibody complex band density from the western blot showed a comparable behavior of the Desirudin and Lepirudin in contrast to the normal or native hirudin which exhibited a much higher band density (2-fold increase). Moreover, the native hirudin exhibited specific bands representing mono, di- and tetra-meric forms, whereas the two recombinant forms exhibited primarily monomeric and dimeric forms. Interestingly several other preclinical versions of recombinant hirudins exhibited fifferent immunoblotting patterns. Although, the structural differences and the molecular weight represent relatively minor variations in the natural and recombinant hirudins, these studies strongly suggest a differential behavior of natural and recombinant hirudins in terms of their cross-reactivity with anti-n-hirudin antibodies. Disclosures: No relevant conflicts of interest to declare.