DnaA occupies only the three highest-affinity binding sites in E. coli oriC throughout most of the cell cycle. Immediately prior to initiation of chromosome replication, DnaA interacts with additional recognition sites, resulting in localized DNA-strand separation. These two DnaA-oriC complexes formed during the cell cycle are functionally and temporally analogous to yeast ORC and pre-RC. After initiation, SeqA binds to hemimethylated oriC, sequestering oriC while levels of active DnaA are reduced, preventing reinitiation. In this paper, we investigate how resetting of oriC to the ORC-like complex is coordinated with SeqA-mediated sequestration. We report that oriC resets to ORC during sequestration. This was possible because SeqA blocked DnaA binding to hemimethylated oriC only at low-affinity recognition sites associated with GATC but did not interfere with occupation of higher-affinity sites. Thus, during the sequestration period, SeqA repressed pre-RC assembly while ensuring resetting of E. coli ORC.

Purpose: Study distribution, pharmacokinetics, and safety of intraperitoneal (IP) 212 Pb-TCMC-trastuzumab in patients with HER-2-expressing malignancy. Experimental Design: IP 212 Pb-TCMC-trastuzumab was delivered, after 4 mg/kg intravenous (IV) trastuzumab, to 3 patients with HER-2-expressing cancer who had failed standard therapies. Patients were monitored for toxicity and pharmacokinetics/dosimetry parameters. Results: Imaging studies after 0.2 mCi/m 2 (7.4 MBq/m 2 ) show little redistribution out of the peritoneal cavity and no significant uptake in major organs. Peak blood level of the radiolabeled antibody, determined by decay corrected counts, was < 23% injected dose at 63 hours; maximum blood radioactivity concentration was 6.3nCi/mL at 18 hours. Cumulative urinary excretion was £ 6% in 2.3 half-lives. The maximum external exposure rate immediately post-infusion at skin contact over the abdomen averaged 7.67 mR/h and dropped to 0.67 mR/h by 24 hours. The exposure rates at the other positions monitored (axilla, chest, and femur) decreased as a function of distance from the abdomen. The data points correlate closely with 212 Pb physical decay (T 1/2 = 10.6 hours). Follow-up >6 months showed no evidence of agent-related toxicity. Conclusions: Pharmacokinetics and imaging after 0.2 mCi/m 2 IP 212 Pb-TCMC-trastuzumab in patients with HER-2-expressing malignancy showed minimal distribution outside the peritoneal cavity, £ 6% urinary excretion, and good tolerance.

Our purpose was to study the safety, distribution, pharmacokinetics, immunogenicity and tumor response of intraperitoneal (IP) 212Pb-TCMC-trastuzumab (TCMC is S-2-(4-isothiocyantobenzl)-1, 4, 7, 10-tetraaza-1, 4, 7, 10=tetra (2-carbamoylmethl) cyclododecane) in patients with HER-2 expressing malignancy. Methods In a standard 3+3 Phase 1 design for dose escalation, 212Pb-TCMC-trastuzumab was delivered IP less than 4 hours after giving 4mg/kg IV trastuzumab to patients with peritoneal carcinomatosis who had failed standard therapies. Results Five dosage levels (7.4, 9.6, 12.6, 16.3, 21.1 MBq/m2) showed minimal toxicity at >1 year for the first group and >4 months for others. The lack of substantial toxicity was consistent with the dosimetry assessments (mean equivalent dose to marrow = 0.18 mSv/MBq). Radiation dosimetry assessment was performed using pharmacokinetics data obtained in the initial cohort (n=3). Limited redistribution of radioactivity out of the peritoneal cavity to circulating blood, which cleared via urinary excretion and no specific uptake in major organs was observed in 24 hours. Maximum serum concentration of the radiolabeled antibody was 22.9% at 24h (decay corrected to injection time) and 500 Bq/mL (decay corrected to collection time). Non-decay corrected cumulative urinary excretion was ≤6% in 24h (2.3 half lives). Dose rate measurements performed at 1m from the patient registered less than 5μSv/hr (using portable detectors) in the latest cohort, significantly less than what is normally observed using nuclear medicine imaging agents. Anti-drug antibody assays performed on serum from the first 4 cohorts were all negative. Conclusions Five dose levels of IP 212Pb-TCMC-trastuzumab treatment of patients with peritoneal carcinomatosis showed little agent related toxicity, consistent with the dosimetry calculations.

Eukaryotic initiator proteins form origin recognition complexes (ORCs) that bind to replication origins during most of the cell cycle and direct assembly of prereplication complexes (pre-RCs) before the onset of S phase. In the eubacterium Escherichia coli, there is a temporally similar nucleoprotein complex comprising the initiator protein DnaA bound to three high-affinity recognition sites in the unique origin of replication, oriC. At the time of initiation, this high-affinity DnaA-oriC complex (the bacterial ORC) accumulates additional DnaA that interacts with lower-affinity sites in oriC, forming a pre-RC. In this paper, we investigate the functional role of the bacterial ORC and examine whether it mediates low-affinity DnaAoriC interactions during pre-RC assembly. We report that E. coli ORC is essential for DnaA occupation of low-affinity sites. The assistance given by ORC is directed primarily to proximal weak sites and requires oligomerization-proficient DnaA. We propose that in bacteria, DnaA oligomers of limited length and stability emerge from single highaffinity sites and extend toward weak sites to facilitate their loading as a key stage of prokaryotic pre-RC assembly.R egulating chromosome duplication requires precisely timed formation of nucleoprotein complexes that comprise initiator proteins bound to replication origins and that direct assembly of new replisomes (1-6). Among the best-studied examples of such nucleoprotein complexes are the origin recognition complexes (ORCs) bound to origins in budding yeast (7,8), and the complexes formed by DnaA binding to the unique origin of chromosomal replication, oriC, in Escherichia coli (6, 9). Yeast ORC subunits share structural motifs with DnaA as well as archeal Orc1 (9, 10), and all are members of the AAAϩ family of ATPases (11). This structural conservation among initiator proteins suggests the intriguing possibility that mechanisms used by all cell types to initiate DNA synthesis could be fundamentally similar (12).Examination of the binding patterns of initiator proteins to origins during the cell cycle (5,13,14) has revealed that in addition to structural similarities, there are temporal similarities in nucleoprotein complex formation at eukaryotic and prokaryotic replication origins. Yeast ORCs bind to replication origins throughout the cell cycle and recruit additional initiator proteins needed to form the prereplicative complexes (pre-RCs) that load helicase and unwind origin DNA before entry into S phase (7,8,14,15). In E. coli, a temporally similar nucleoprotein complex is formed by DnaA binding to three high-affinity (K d Ͻ 200 nM), 9-bp recognition sites (R1, R2, and R4) within oriC (Fig. 1); like yeast ORC, this binding persists throughout the majority of the cell cycle (13,16,17), except at the time of initiation, when additional initiator DnaA binds to lower-affinity (K d Ͼ 200 nM) sites in oriC (13, 18). The additional DnaA causes localized strand separation within an AT-rich, 13-mer repeat region that is adjacent to the left side of t...