RESUMEN

INTRODUCTION: Regulatory and competitive pressure to reduce the QT interval prolongation risk of potential new drugs has led to focus on methods to test for inhibition of the human ether-a-go-go-related gene (hERG)-encoded K+ channel, the primary molecular target underlying this safety issue. Here we describe the validation of a method that combines medium-throughput with direct assessment of channel function. METHODS: The electrophysiological and pharmacological properties of hERG were compared using two methods: conventional, low-throughput electrophysiology and planar-array-based, medium-throughput electrophysiology (IonWorks HT). A pharmacological comparison was also made between IonWorks HT and an indirect assay (Rb+ efflux). RESULTS: Basic electrophysiological properties of hERG were similar whether recorded conventionally (HEK cells) or using IonWorks HT (CHO cells): for example, tail current V1/2 -12.1+/-5.0 mV (32) for conventional and -9.5+/-6.0 mV (46) for IonWorks HT (mean+/-S.D. (n)). A key finding was that as the number of cells per well was increased in IonWorks HT, the potency reported for a given compound decreased. Using the lowest possible cell concentration (250,000 cells/ml) and 89 compounds spanning a broad potency range, the pIC50 values from IonWorks HT (CHO-hERG) were found to correlate well with those obtained using conventional methodology (HEK-hERG)(r=0.90; p<0.001). Further validation using CHO-hERG cells with both methods confirmed the correlation (r=0.94; p<0.001). In contrast, a comparison of IonWorks HT and Rb+ efflux data with 649 compounds using CHO-hERG cells showed that the indirect assay consistently reported compounds as being, on average, 6-fold less potent, though the differences varied depending on chemical series. DISCUSSION: 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.

Asunto(s)

RESUMEN

The gastrointestinal pathogen Helicobacter pylori requires supplementation with either fetal calf serum (FCS), bovine serum albumin (BSA), or (2,6-dimethyl)-beta-cyclodextrin (CD) for growth in a complex or defined medium. Because the availability of medium in which all components were chemically defined would facilitate metabolic studies of H. pylori, growth of the type strain, ATCC 43504, was compared in a defined medium with different growth additives. The dependency of H. pylori growth on FCS or BSA in a defined medium could partially be replaced by dependency on CD and cholesterol when the last two components were both added to the defined medium. Growth and cell yield were not affected by the addition of glucose, but the culture viability (numbers of CFU per milliliter was extended. Because therapeutic antifoams are used to relieve gastrointestinal symptoms we studied whether the unique susceptibility of H. pylori to the emulsifier polyoxyethylene-20-stearylether (Brij 78) was growth dependent or medium specific. The bactericidal activity exerted in buffer at pH 5 was independent of the preculture medium, and a 5-h exposure of the bacteria to 1.28 to 2.56 microg of Brij 78 per ml reduced the numbers of viable bacteria by >5 log10. The MICs (0.16 to 0.32 microg/ml) were lower than the corresponding minimal bactericidal concentrations in different growth media and were affected by FCS or BSA. In conclusion, CD plus cholesterol promotes the growth of H. pylori in a serum-free defined medium in which glucose enhances cell viability. The antibacterial activity exerted by Brij 78 is neither growth dependent nor medium specific.

Asunto(s)

RESUMEN

The previously described pLOFKm transposon delivery plasmid (J.Bacteriol. (1990) 172, 6557-6567) was engineered such that a promotorless lacZ gene was cloned within the transposon cassette, generating the vector pLBT. Using pLBT, stable insertion mutations were generated at high frequencies in Vibrio sp. S141 and Pseudomonas sp. S91, and the interrupted genes could be monitored for their pattern of regulation. Genetic screens isolated mutants defective in a variety of activities. We describe the construction and use of pLBT as a tool for reporter gene mutant analysis in bacteria other than well-characterized laboratory strains.

Asunto(s)

RESUMEN

In order to evaluate the role of the stringent response in starvation adaptations of the marine Vibrio sp. strain S14, we have cloned the relA gene and generated relaxed mutants of this organism. The Vibrio relA gene was selected from a chromosomal DNA library by complementation of an Escherichia coli delta relA strain. The nucleotide sequence contains a 743-codon open reading frame that encodes a polypeptide that is identical in length and highly homologous to the E. coli RelA protein. The amino acid sequences are 64% identical, and they share some completely conserved regions. A delta relA::kan allele was generated by replacing 53% of the open reading frame with a kanamycin resistance gene. The Vibrio relA mutants displayed a relaxed control of RNA synthesis and failed to accumulate ppGpp during amino acid limitation. During carbon and energy starvation, a relA-dependent burst of ppGpp synthesis concomitant with carbon source depletion and growth arrest was observed. Also, in the absence of the relA gene, there was an accumulation of ppGpp during carbon starvation, but this was slower and smaller than that which occurred in the stringent strains, and it was preceded by a marked decrease in the [ATP]/[ADP] ratio. In both the wild-type and the relaxed strains, carbon source depletion caused an immediate decrease in the size of the GTP pool and a block of net RNA accumulation. The relA mutation did not affect long-term survival or the development of resistance against heat, ethanol, and oxidative stress during carbon starvation of Vibrio sp. strain S14.

Asunto(s)

RESUMEN

The decay rate of the potential to synthesize proteins after complete inhibition of transcription by rifampicin was analyzed to determine the functional mRNA stability of exponentially growing and glucose-starved Escherichia coli B and K12 cells. We found the following: (i) The half-life of the mRNA pool increased 2.2-fold during a period of 2 h of starvation (from 1.8 min in exponentially growing cells to 4.0 min for cells starved for 2 h); (ii) the effect on transcript stability appeared to be global since transcripts of genes that were induced, repressed or unaltered in their expression during starvation exhibited more or less the same increased stability; (iii) the rate of peptide chain elongation, as measured by the synthesis time for beta-galactosidase, decreased 1.9-fold during the starvation period studied and may, at least in part, account for the global stabilization of transcripts in starved cells.

Asunto(s)

RESUMEN

Proline iminopeptidase (Pip) is a hydrolase elaborated by virtually all strains of Neisseria gonorrhoeae that selectively removes N-terminal proline residues from peptides. Escherichia coli clones expressing the gonococcal gene coding for Pip were identified in a genomic cosmid library using a synthetic colorimetric substrate. Nucleotide sequence determination and analyses of polypeptides detected by coupled in vitro transcription/translation reactions revealed that Pip is a 311-amino-acid polypeptide with a M(r) of 35 kDa and a pI of 5.4. Southern hybridization showed that the pip gene is present in a single copy on the chromosome of N. gonorrhoeae strain MS11 which maps immediately upstream of the previously identified opaA locus. The transcriptional start site of pip in E. coli, determined by primer extension analysis, was characteristic of an NtrA or sigma-54-dependent promotor. Complementation of an E. coli mutant deficient in both proline biosynthesis and dipeptide uptake confirmed that Pip is capable of releasing biologically active proline from peptides. Pip expression was found to be non-essential for in vitro growth of N. gonorrhoeae, based on the viability of a Pip- gonococcal mutant.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

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.

Asunto(s)

RESUMEN

The uptake kinetics of D-glucose were examined in the marine Vibrio sp. S14 during a period of 168 h of complete energy and nutrient starvation. Two glucose transport systems were distinguished in Vibrio sp. S14: a low affinity system (Km = 4.6 +/- 0.9 microM) at the onset of starvation, and a high affinity system (Km = 0.55 +/- 0.15 microM) after 168 h of starvation. Both systems had a narrow substrate specificity, and both were osmotic shock-sensitive.

Asunto(s)

RESUMEN

Exoprotease activity during 120 h of total energy and nutrient starvation was examined in two marine bacteria, Vibrio sp. strain S14 and Pseudomonas sp. strain S9. The activity was determined by spectrophotometric measurement of the rate of release of soluble color from an insoluble azure dye derivative of hide powder (hide powder azure). Starved cells of both strains (5 h for S14, and 4 or 24 h for S9) showed greater extracellular proteolytic activity than at the onset of starvation. The exoprotease activity of cells starved for longer periods of time then decreased, but was found to be present at significant levels throughout the starvation period studied (120 h). The accumulation of exoprotease activity in the bulk phase during starvation indicated that both strains constitutively excreted extracellular proteases. As deduced from experiments with chloramphenicol, de novo protein synthesis during starvation was required for the production and/or release of the exoproteases into the surrounding environment. The degradation of hide powder azure allowed an immediate increase in respiration rate, also by long-term-starved cells. This suggests that metabolic systems are primed to respond to the availability of substrates, allowing the cells to recover rapidly. The regulation of exoprotease activity was also studied and found to be different in the two strains. Casamino Acids repressed exoprotease activity in Pseudomonas sp. strain S9, whereas a mechanism similar to catabolite repression was found for Vibrio sp. strain S14 in that glucose repressed activity and cyclic AMP reversed this effect. The exoproteases appeared to be metalloproteinases because the addition of EDTA to cell-free starvation supernatants from both strains significantly inhibited the activity of the proteases.

RESUMEN

Changes in membrane and periplasmic protein profiles induced by starvation conditions in the marine Vibrio sp. S14 were examined by one-dimensional gel electrophoresis. Analysis by densitometry resolved at least six periplasmic proteins, nine outer membrane proteins, and four cytoplasmic membrane proteins induced at various times during 120 h of nutrient and energy starvation. Eight of these were also synthesized by heat- and/or ethanol-shocked cells. Pulse-labelling indicated that the starvation-induced proteins were not products of degradation, and that their synthesis was differently modulated during starvation. The most pronounced changes occurred during the initial hours of nutrient and energy deprivation. The correlation between the initial changes in protein composition and utilization of the intracellular energy reserve poly-beta-hydroxybutyrate is discussed. The rate of proteolysis during the initial hours of starvation was approximately 16 times greater than that during exponential growth.

Asunto(s)

RESUMEN

The little compound 1a has been prepared and was found to be a strong, orally active agonist with narcotic--antagonist properties. 1a was prepared by two independent routes: (1) nitration of volazocine and subsequent reduction and (b) a sequence involving dissolving metal reduction of cyclazocine methyl ether, followed by oximination and Semmler--Wolff rearrangement. Several analogues were prepared and tested.

Asunto(s)

RESUMEN

May's benzomorphan synthesis leads not only to the alpha or cis isomer and the beta or trans isomer but also to a position isomer hereinafter called the gamma isomer. The structure and synthesis of this isomer are described. Biological activities of the alpha and gamma isomers are compared.

Asunto(s)

Asunto(s)

RESUMEN

In a benzomorphan bearing an antagonist side chain, introduction on the methano bridge of a hydroxyl oriented away from nitrogen has little effect on antagonist activity whereas a hydroxyl oriented toward nitrogen enhances this activity. Hydroxylation tends to decrease analgesic activity.

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)

Asunto(s)