The transport of large preproteins across the Escherichia coli plasma membrane is catalyzed by preprotein translocase, comprised of the peripherally bound SecA subunit and an integrally bound heterotrimeric domain consisting of the SecY, SecE, and SecG subunits. We have now placed the secY, secE, and secG genes under the control of an arabinose-inducible promoter on a multicopy plasmid. Upon induction, all three of the proteins are strongly overexpressed and recovered in the plasma membrane fraction. These membranes show a strong enhancement of 1) translocation ATPase activity, 2) preprotein translocation, 3) capacity for SecA binding, and 4) formation of the membrane-inserted form of SecA. These data establish that SecY, SecE, and SecG constitute the integral membrane domain of preprotein translocase.

The development of vaginal microbicides for the prevention of sexual transmission of HIV is becoming an increasingly important strategy in the battle against the AIDS epidemic. Several first generation microbicide candidates are entering Phase III efficacy trials, and several other candidates are in earlier stages of clinical development. The capacity to make accurate clinical assessments of the safety and efficacy of microbicide formulations is critical. Since microbicide trials will rely on a blinded, randomized, placebo-controlled design, it is important to employ a placebo formulation that does not distort either safety or efficacy assessments. Efficacy of the microbicide would be underestimated if the placebo itself provided a degree of protection. Conversely, a placebo with epithelial toxicity that increased susceptibility would cause an overestimation of microbicide efficacy. To address these issues, a hydroxyethylcellulose (HEC) placebo formulation has been developed and has been adopted for use in clinical evaluations of investigational microbicides as a "universal" placebo. In this report, the chemical and physical properties of this formulation are described, as well as its in vitro and in vivo effects on safety and efficacy. The results show that this "universal" placebo has adequate physical properties, is sufficiently stable as a vaginal gel formulation, and is safe and sufficiently inactive for use in the clinical study of investigational microbicides.

Vaginal delivery of 200 mg or 25 mg dapivirine from intravaginal rings (IVRs) was evaluated over a 7-day period in two phase 1 safety trials (IPM001 and IPM008, respectively) in a total of 25 healthy women 19 to 46 years of age. The IVR was generally safe and well tolerated with similar adverse events observed in the placebo and dapivirine groups. Across both studies, dapivirine concentrations in vaginal fluids measured at the introitus, cervix, and ring area were within the mean range of 0.7-7.1 microg/ml. Mean dapivirine concentrations in vaginal and cervical tissues on day 7 were 0.3-0.7 microg/g in IPM001 and 1.5-3.5 microg/g in IPM008. Mean plasma concentrations of dapivirine were <50 pg/ml. Dapivirine from both IVRs was successfully distributed throughout the lower genital tract at concentrations >1000x the EC(50) against wild-type HIV-1 (LAI) in MT4 cells suggesting that IVR delivery of microbicides is a viable option meriting further study.

Abstract-Recent increased adverse cardiovascular events observed with selective cyclooxygenase-2 inhibition led to the withdrawal of rofecoxib (Vioxx) and valdecoxib (Bextra), but the mechanisms underlying these atherothrombotic events remain unclear. Prostacyclin is the major end product of cyclooxygenase-2 in vascular endothelium. Using a naturally occurring mutation in the prostacyclin receptor, we report for the first time that a deficiency in prostacyclin signaling through its G protein-coupled receptor contributes to atherothrombosis in human patients. We report that a prostacyclin receptor variant (R212C) is defective in adenylyl cyclase activation in both patient blood and in an in vitro COS-1 overexpression system. This promotes increased platelet aggregation, a hallmark of atherothrombosis. Our analysis of patients in 3 separate white cohorts reveals that this dysfunctional receptor is not likely an initiating factor in cardiovascular disease but that it accelerates the course of disease in those patients with the greatest risk factors. R212C was associated with cardiovascular disease only in the high cardiovascular risk cohort (nϭ980), with no association in the low-risk cohort (nϭ2293). In those at highest cardiovascular risk, both disease severity and adverse cardiovascular events were significantly increased with R212C when compared with age-and risk factor-matched normal allele patients. We conclude that for haploinsufficient mutants, such as the R212C, the enhanced atherothrombotic phenotype is likely dependent on the presence of existing atherosclerosis or injury (high risk factors), analogous to what has been observed in the cyclooxygenase-2 inhibition studies or prostacyclin receptor knockout mice studies. [1][2][3][4] and the development of cardiovascular disease in predisposed prostacyclin receptor knockout mice, 5,6 underscores the necessity to better understand the effects of COX-2-derived metabolites on cardiovascular health. Endothelial prostacyclin synthesis requires the COX-2 enzyme 7 and may serve a role in protection from atherothrombosis. 8,9 This cardioprotective role has been supported by recent prostacyclin receptor knockout mice studies showing that the absence of the prostacyclin receptor (IP) (International Union of Pharmacology Receptor classification) leads to intimal hyperplasia, atherosclerosis, and hypercoagulability, 5,6 as well as reperfusion injury, 10 and premenopausal atherogenesis. 11 Despite such accumulating information, controversy remains as to whether prostacyclin deficiency is the etiology of the cardiovascular events observed with COX-2 inhibition, 12 particularly as no human studies have directly implicated defective prostacyclin signaling in the development of cardiovascular disease.The human prostacyclin receptor (hIP) gene (PTGIR) spans approximately 7000 bases along chromosome 19 (locus Original received October 11, 2007; revision received February 12, 2008; accepted February 21, 2008 19q13.3) and is comprised of 3 exons separated by 2 introns, 1 intron...