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We report on scanning micro-photoluminescence measurements on InAs quantum dots that were patterned using an in vacuo focused ion beam. The focused ion beam produced a square array of holes with an array spacing of 2 μm, upon which multiple layers of InAs and GaAs were deposited. Two-dimensional mapping of the luminescence spatially and spectrally resolves the emission from individual dots. At some positions, multiple emission peaks are observed, which could have originated from (i) different exciton transitions of the same quantum dot, or (ii) different quantum dots. Power dependence measurements reveal that the emission increases linearly with power, and maps of the emission show that the positions of each peak are spatially separated, suggesting that the peaks are ground-state exciton emission from different dots.

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.

We report on scanning microphotoluminescence measurements that spectrally and spatially resolve emission from individual InAs quantum dots that were induced by focused ion beam patterning. Multilayers of quantum dots were spaced 2 μm apart, with a minimum single dot emission line width of 160 μeV, indicating good optical quality for dots patterned using this technique. Mapping 16 array sites, at least 65% were occupied by optically active dots and the spectral inhomogeneity was within 30 meV.

ST-246, a novel compound that inhibits egress of orthopoxvirus from infected cells, is being evaluated as a treatment for pathogenic orthopoxvirus infections in humans. This phase I, double-blind, randomized, placebo-controlled, escalating multiple-dose study was conducted to determine the safety, tolerability, and pharmacokinetics of ST-246 administered as a single daily oral dose of 250, 400, or 800 mg for 21 days to nonfasting healthy human volunteers. ST-246 appeared to be well tolerated, with no serious adverse events (AEs). Headache, for which one subject in the 800-mg group discontinued the study, was the most commonly reported AE in all treatment groups. The multiple-dose pharmacokinetics of ST-246 was well characterized. The day 21 mean elimination half-lives were calculated at 18.8, 19.8, and 20.7 h for each of the 250-, 400-, and 800-mg/day dose groups, respectively. Steady state was reached by day 6 (within 3 to 5 half-lives), saturable absorption was observed at the 800-mg dose level, and the fraction of parent drug excreted in the urine was very low. Based on these results, administration of 400 mg/day ST-246 can be expected to provide plasma concentrations above the efficacious concentration demonstrated in nonhuman primate models in earlier studies.

Cyanovirin-N (CV-N) is a prokaryotic protein under development as a topical anti-HIV microbicide, an urgent and necessary approach to prevent HIV transmission in at-risk populations worldwide. We have expressed recombinant CV-N as inclusion bodies in the cytoplasm of Escherichia coli. A purification scheme has been developed that exploits the physicochemical properties of this protein, in particular its stability in a harsh inclusion body purification scheme. Under the conditions developed, this system yields 140 mg of highly purified CV-N per liter of high-density cell culture, which represents a 14-fold increase over the best recombinant CV-N yield reported to date. This purification scheme results in monomeric CV-N as analyzed by SDS-PAGE, isoelectric focusing, and reverse phase- and size exclusion-HPLC. This recombinantly expressed and refolded CV-N binds to gp120 with nanomolar affinity and retains its potent anti-HIV activities in cell-based assays. The expression and purification system described herein provides a better means for the mass production of CV-N for further microbicide development.

In the fission yeast Schizosaccharomyces pombe , S phase is limited to a single round per cell cycle through cyclin-dependent kinase phosphorylation of critical replication factors, including the Cdc18 replication initiator protein. Because defects in Cdc18 phosphorylation lead to a hyperstable and hyperactive form of Cdc18 that promotes high levels of overreplication in vivo , we wished to identify the components of the Cdc18 proteolysis pathway in fission yeast. In this paper we describe one such component, encoded by the sud1 + gene. sud1 + shares homology with the budding yeast CDC4 gene and is required to prevent spontaneous re-replication in fission yeast. Cells lacking sud1 + accumulate high levels of Cdc18 and the CDK inhibitor Rum1, because they cannot degrade these two key cell cycle regulators. Through genetic analysis we show that hyperaccumulation of Rum1 contributes to re-replication in Δsud1 cells, but is not the cause of the defect in Cdc18 proteolysis. Rather, Sud1 itself is associated with the ubiquitin pathway in fission yeast and binds to Cdc18 in vivo . Most importantly, Sud1-Cdc18 binding requires prior phosphorylation of the Cdc18 polypeptide at CDK consensus sites. These results provide a biochemical mechanism for the phosphorylation-dependent degradation of Cdc18 and other cell cycle regulators, including Rum1. Evolutionary conservation of the Sud1/CDC4 pathway suggests that phosphorylation-coupled proteolysis may be a general feature of nearly all eukaryotic cell cycles.

In all eukaryotes, a conserved group of cyclin-dependent kinases (CDKs) drives the major cell cycle events of DNA replication and mitosis (Forsburg and Nurse 1991; Reed 1992). These CDKs are also involved in quality-control mechanisms (commonly referred to as “checkpoints”), which ensure that, for example, mitosis and cell division occur only after DNA replication has been completed (Enoch and Nurse 1990). Recent work has demonstrated a third major role for CDKs in cell cycle control. Loss of CDK activity in a number of eukaryotic organisms results in ectopic rounds of DNA replication in the absence of mitosis, producing large increases in genome ploidy (Hayles et al. 1994; Dahmann et al. 1995; Sauer et al. 1995). Taken together, these results imply that CDKs actively inhibit the re-firing of replication origins during the G2 and M phases of the cell cycle. These observations can be explained by a two-state model for origin function (Muzi-Falconi et al. 1996b; Nasmyth 1996; Stillman 1996; Jallepalli and Kelly 1997). In this model, a latent pre-initiation state is established at replication origins during the G1 period, when CDK activity is at a minimum. A subsequent increase in CDK activity at the G1 → S transition leads to phosphorylation of critical (but unidentified) initiation factors at origins of replication. This CDK-catalyzed phosphorylation switches origins into an active initiation state that can support DNA replication. Simultaneously, the phosphorylated proteins at fired origins are rendered incapable of re-entering the pre-initiation state. Because formation of the pre-initation state precedes and is required for initiation, CDK phosphorylation at the origin effectively prevents re-initiation until cells have completed mitosis. This CDK-driven “replication switch” can explain why DNA replication occurs precisely once during each cell cycle (Muzi-Falconi et al. 1996b; Nasmyth 1996; Stillman 1996; Jallepalli and Kelly 1997). Validation of this model depends on identifying the major in vivo CDK substrates at eukaryotic replication origins. Despite the large number of replication proteins that are potential or actual CDK substrates in vitro, no data are available on their in vivo regulation by CDK phosphorylation. In this study we have examined the phosphorylation of an important replication initiator, the p65cdc18 protein of fission yeast. p65cdc18 normally accumulates only during the G1 phase of the cell cycle, a consequence of periodic transcription coupled with rapid protein turnover (Nishitani and Nurse 1995; Muzi-Falconi et al. 1996a). p65cdc18 is a member of a family of replication initiator proteins that are evolutionarily conserved from yeast to man (Bell et al. 1995; Gavin et al. 1995; Muzi-Falconi and Kelly 1995; Piatti et al. 1995; Coleman et al. 1996; Rowles et al. 1996; Williams et al. 1997). p65cdc18 interacts physically with components of the fission yeast origin recognition complex (ORC) and the p34cdc2 kinase, the major CDK in fission yeast (Grallert and Nurse 1996; Leatherwood et al. 1996; Brown et al. 1997). Regulation of p65cdc18 abundance and/or activity could be important in the cell cycle control of DNA replication, as constitutive overproduction of cdc18+ induces over-replication in the absence of mitosis (Nishitani and Nurse 1995; Muzi-Falconi et al. 1996a). Our previous genetic studies suggested that p65cdc18 might be an important target for negative regulation by CDKs, as expression of the fission yeast CDK inhibitor p25rum1 (Correa-Bordes and Nurse 1995; Jallepalli and Kelly 1996; Martin-Castellanos et al. 1996) suppressed the cell cycle defect of a temperature-sensitive cdc18 mutant (Jallepalli and Kelly 1996). Our present analysis has revealed that p65cdc18 becomes highly phosphorylated at the G1 → S transition. p65cdc18 phosphorylation depends on the activity of p34cdc2 kinase, the CDK with which it associates. Mutation of the CDK consensus phosphorylation sites within p65cdc18 demonstrates that phosphorylation directly targets p65cdc18 for rapid proteolysis and limits its replication activity in vivo. We propose therefore that phosphorylation of p65cdc18 by p34cdc2 kinase couples CDK activation at the G1 → S boundary to the inactivation and degradation of the p65cdc18 polypeptide. Down-regulation of p65cdc18 by CDK phosphorylation is likely to contribute to the CDK-driven replication switch that limits initiation at chromosomal origins to once per cell cycle (Muzi-Falconi et al. 1996b; Nasmyth 1996; Stillman 1996; Jallepalli and Kelly 1997).

ST-246 is a novel, potent orthopoxvirus egress inhibitor that is being developed to treat pathogenic orthopoxvirus infections of humans. This phase I, double-blind, randomized, placebo-controlled single ascending dose study (first time with humans) was conducted to determine the safety, tolerability, and pharmacokinetics of ST-246 in healthy human volunteers. ST-246 was administered in single oral doses of 500, 1,000, and 2,000 mg to fasting healthy volunteers and 1,000 mg to nonfasting healthy volunteers. ST-246 was generally well tolerated with no serious adverse events, and no subject was withdrawn from the study due to ST-246. The most commonly reported drug-related adverse event was neutropenia, which was found, upon further analysis, not to be treatment related. ST-246 was readily absorbed following oral administration with mean times to maximum concentration from 2 h to 3 h. Absorption was greater in nonfasting volunteers than in fasting volunteers. Administration of ST-246 resulted in exposure levels predicted to be sufficient for inhibiting orthopoxvirus replication compared to exposure levels in nonhuman primates in which ST-246 protected animals from lethal orthopoxvirus infection.