The main finding of this work is that providing a relatively low cell concentration is used in IonWorks HT, the potency information generated correlates well with that determined using conventional electrophysiology. The effect on potency of increasing cell concentration may relate to a reduced free concentration of test compound owing to partitioning into cell membranes. In summary, the IonWorks HT hERG assay can generate pIC50 values based on a direct assessment of channel function in a timeframe short enough to influence chemical design.

Non-differentiating bacteria adapt to starvation induced growth arrest by a complex turn-on/turn-off pattern of protein synthesis. This response shows distinct similarities with those of spore formation in differentiating organisms. A substantial amount of information on the non-growth biology of non-differentiating bacteria can be derived from studies on Vibrio strains. One important result is that carbon rather than nitrogen or phosphorus starvation leads to the development of a starvation and stress resistant cell in these organisms. Hence, we have attempted to characterize the carbon starvation stimulon. By the use of two-dimensional gel electrophoresis of pulse-labelled cells and transposon mutagenesis, using reporter gene constructs, the identity and function of some members of the carbon starvation stimulon have been elucidated. Moreover, regulatory genes of the starvation response have been identified with these techniques. Current studies primarily address the identity and function of these genes. The role of transcript modification and stability for both long term persistence during starvation as well as the efficient recovery of cells which occurs upon nutrient addition is also addressed. It is suggested that an understanding of the functionality of the translational machinery is essential for the understanding of these adaptive pathways. This contribution also discusses the diversity of the differentiation-like response to starvation in different bacteria and whether a general starvation induced programme exists.

Three gonococcal genes have been identified which encode proteins with substantial similarities to known components of the type IV pilus biogenesis pathway in Pseudomonas aeruginosa. Two of the genes were identified based on their hybridization with a DNA probe derived from the pilB gene of P. aeruginosa under conditions of reduced stringency. The product of the gonococcal pilF gene is most closely related to the pilus assembly protein PilB of P. aeruginosa while the product of the gonococcal pilT gene is most similar to the PilT protein of P. aeruginosa which is involved in pilus-associated twitching motility and colony morphology. The products of both of these genes display canonical nucleoside triphosphate binding sites and are predicted to be to cytoplasmically localized based on their overall hydrophilicity. The gonococcal pilD gene, identified by virtue of its linkage to the pilF gene, is homologous to a family of prepilin leader peptidase genes. When expressed in Escherichia coli, the gonococcal PilD protein functions to process gonococcal prepilin in a manner consistent with its being gonococcal prepilin peptidase. These results suggest that Neisseria gonorrhoeae is capable of expressing many of the essential elements of a highly conserved protein translocation system and that these gene products are probably involved in pilus biogenesis.

In order to evaluate the role of the stringent response in starvation adaptations of the marine Vtbrio sp. strain S14, we have cloned the relA gene and generated relaxed mutants of this organism. The concentrations of biologically available nutrients are generally low in natural bacterial habitats (34, 35), and adaptations to restrictions in nutrient and energy availability may contribute significantly to the fitness of bacteria in such environments. Marine vibrios are adequate models for studies of starvation responses, and their adaptations include the development of general stress resistance and the ability to survive extended periods of complete carbon and energy starvation. Alterations in cell surface components, adhesive properties, nutrient scavenging, and uptake systems are commonly observed (reviewed in references 21, 22, and 33). The marine isolate Vibrio sp. strain S14 has served as a model organism in many of these studies (38). It responds to multiplenutrient starvation by a developmental process comprising the temporally ordered induction of more than 66 starvationinduced (Sti) proteins (39). The identities of the Sti proteins are, with some exceptions (17), unknown, but it is assumed that phenotypic adaptations during the starvation response can be ascribed to the induction of specific Sti proteins. That is the case with Escherichia coli, in which several of the phenotypes that are expressed during stationary phase are dependent on specific members of the as or RpoS regulon (14).The transition of growing Vbrio sp. strain S14 cells to multiple-nutrient starvation includes the induction of the stringent response (39), a regulatory network with pleiotropic effects on cellular physiology, primarily studied as it occurs in E. coli (3). Evidence for a role of the stringent response in the starvation adaptation of Vibrio sp. strain S14 was obtained

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The recovery of Vibrio sp. S14 cells from energy-and nutrient-starvation was monitored after the addition of glucose minimal medium. Upshift experiments were done throughout a starvation period of 200 h to determine whether cells were more responsive to nutrient addition at the onset of starvation, or if the previously described programme of starvation-induced cellular reorganization had to be completed before cells could become committed to recovery following nutritional upshifts. The kinetics of macromolecular synthesis (RNA, protein and DNA), the rate of respiration and changes in median cell volume in response to nutritional upshifts at different times of starvation were examined. The relative rates of RNA and protein synthesis increased immediately upon addition of glucose minimal medium; the increase in protein synthesis was not dependent on a parallel increase in RNA synthesis, indicating that the starved cells may have an excess of protein synthesizing machinery, including stable RNA and functional ribosom?. The subsequent increase in the rate of DNA replication was initiated approximately 60min before the first apparent cell division at approximately one doubling of the theoretical minimal cell volume ( Vu). Two-dimensional gel electrophoresis was used to demonstrate the fate of starvationspecific proteins during the upshift, and also the synthesis of recovery-induced proteins that were not found in starving cells. Most starvation-inducible proteins were repressed immediately at the onset of the nutritional upshift, while 11 of the 21 recovery-induced proteins identified were expressed exclusively during the maturation phase and were subsequently repressed at the onset of regrowth. The possible role of such maturation-specific proteins in the rapid formation of a reproductive cell committed to growth and division is discussed.

Positive cooperativity (auto-and heteroactivation) of drug oxidation, a potential cause of drug interactions, is well established in vitro for cytochrome P450 (P450) 3A4 but to a much lesser extent for other drug-metabolizing P450 isoforms. Using a high throughput fluorescent-based CYP2C9 effector assay, we identified Ͼ30 heteroactivators from a set of 1504 structurally diverse compounds. Several potent heteroactivators of CYP2C9-mediated 7-methoxy-4-trifluoromethyl-coumarin metabolism are marketed drugs or endogenous compounds (amiodarone, niclosamide, liothyronine, meclofenemate, zafirlukast, estropipate, and dichlorphenamide, yielding 150% control reaction velocity at 0.04, 0.09, 0.5, 1, 1.2, 1.5, and 2.5 M, respectively). Some heteroactivators are also known CYP2C9 substrates or inhibitors, suggesting potential multiple binding sites and substrate-dependent effects. v 150% , the concentration of effector giving 150% of control reaction velocity, was used as pharmacophore modeling parameter based on enzyme kinetic assumptions. The generated pharmacophore (training set: n ϭ 36, v 150% 0.04 -150 M) contains one hydrogen bond acceptor, one aromatic ring, and two hydrophobes. v 150% values for 94% of the training set heteroactivators were predicted within 1 log unit for the residual (r [log observed v 150% ] versus [log predicted v 150% ] ϭ 0.71; r 2 0.50). The model also correctly identifies close to 70% of potent inhibitors (IC 50 Ͻ 1 M) as high-affinity CYP2C9 binders, suggesting that heteroactivators and inhibitors share some common structural CYP2C9 binding features, supporting the previously suggested hypothesis that CYP2C9 heteroactivators can bind within the active site.

In-silico models were generated to predict the extent of inhibition of cytochrome P450 isoenzymes using a set of relatively interpretable descriptors in conjunction with partial least squares (PLS) and regression trees (RT). The former was chosen due to the conservative nature of the resultant models built and the latter to more effectively account for any non-linearity between dependent and independent variables. All models are statistically significant and agree with the known SAR and they could be used as a guide to P450 liability through a classification based on the continuous pIC50 prediction given by the model. A compound is classified as having either a high or low P450 liability if the predicted pIC(50) is at least one root mean square error (RMSE) from the high/low pIC(50) cut-off of 5. If predicted within an RMSE of the cut-off we cannot be confident a compound will be experimentally low or high so an indeterminate classification is given. Hybrid models using bulk descriptors and fragmental descriptors do significantly better in modeling CYP450 inhibition, than bulk property QSAR descriptors alone.