In situ assay for identifying inhibitors of bacterial transglycosylase

An in situ transglycosylase assay has been developed using endogenously synthesized lipid II. The assay involves the preferential synthesis and accumulation of lipid II in a reaction mixture containing the cell wall membrane material isolated from Escherichia coli, exogenously supplied UDP-MurNAc-pentapeptide, and radiolabeled UDP-GlcNAc. In the presence of Triton X-100, the radiolabeled product formed is almost exclusively lipid II, while the subsequent formation of peptidoglycan is inhibited. Removal of the detergent resulted in the synthesis of peptidoglycan (25% incorporation of radiolabeled material) from the accumulated lipid II. This reaction was inhibited by moenomycin, a known transglycosylase inhibitor. In addition, tunicamycin, which affects an earlier step of the pathway by inhibiting MraY, had no effect on the formation of peptidoglycan in this assay, as expected. Similarly, ampicillin and bacitracin did not inhibit the formation of peptidoglycan under the conditions established.     

Identification of arylamine N -acetyltransferase inhibitors as an approach towards novel anti-tuberculars

New anti-tubercular drugs and drug targets are urgently needed to reduce the time for treatment and also to identify agents that will be effective against Mycobacterium tuberculosis persisting intracellularly. Mycobacteria have a unique cell wall. Deletion of the gene for arylamine N-acetyltransferase (NAT) decreases mycobacterial cell wall lipids, particularly the distinctive mycolates, and also increases antibiotic susceptibility and killing within macrophage of Mycobacterium bovis BCG. The nat gene and its associated gene cluster are almost identical in sequence in M. bovis BCG and M. tuberculosis. The gene cluster is essential for intracellular survival of mycobacteria. We have therefore used pure NAT protein for high-throughput screening to identify several classes of small molecules that inhibit NAT activity. Here, we characterize one class of such molecules— triazoles—in relation to its effects on the target enzyme and on both M. bovis BCG and M. tuberculosis. The most potent triazole mimics the effects of deletion of the nat gene on growth, lipid disruption and intracellular survival. We also present the structure-activity relationship between NAT inhibition and effects on mycobacterial growth, and use ligand-protein analysis to give further insight into the structure-activity relationships. We conclude that screening a chemical library with NAT protein yields compounds that have high potential as anti-tubercular agents and that the inhibitors will allow further exploration of the biochemical pathway in which NAT is involved.     

QTc dispersion as a novel marker in identifying patients requiring an epicardial approach for ablation of scar mediated ventricular tachycardia

IntroductionEpicardial exit sites of ventricular tachycardia (VT) are frequently encountered during VT ablation requiring an epicardial ablation approach for successful elimination of VT. We sought to assess the utility of repolarization markers in identifying individuals requiring an epicardial ablation approach in addition to an endocardial approach.Methods32 patients who underwent successful ablation for scar mediated VT were included in the study. Fourteen patients who required a combined endocardial and epicardial VT ablation were defined as epicardial VT group (Epi) whereas 18 patients who were successfully ablated from the endocardium alone constituted the endocardial VT group (Endo). Repolarization markers during sinus rhythm were compared between the two groups.ResultsA higher QTc max and QTc dispersion were seen in the Epi group compared to Endo group (479 ± 34 vs 449 ± 20, p = 0.008 and 63 ± 13 vs 38 ± 8, p = 0.001, respectively). Ts-p and Ts-p/Tp-e were higher in the Epi group (166 ± 23 vs 143 ± 23, p = 0.008 and 1.55 ± 0.26 vs 1.3 ± 0.21, p < 0.005). On multivariate regression, QTc dispersion was an independent predictor of the need for an epicardial approach to ablation. A QTc dispersion more than 51.5 msec identified individuals requiring a combined epicardial and endocardial approach to ablation with a sensitivity of 92.9% and a specificity of 100%.ConclusionsPatients requiring an epicardial ablation have a higher QTc dispersion. A value greater than 51.5 msec reliably differentiates between the two groups with high sensitivity and specificity.     

In situ random primer extension of metaphase chromosomes

We have developed a technique of random primer extension of fixed chromosomes that is applicable to both mouse and man. Human chromosomes are not homogeneously labeled with this technique; those regions corresponding to R-bands appear to be more sensitive than those identified as G-bands, whereas centromeric regions are not labeled. These results not only corroborate specific structural differences between distinct regions of mammalian genomes but also open up the possibility of assays with specific primers to test whether primer extension is useful for the identification of genes and families of sequences on chromosomes.     

Two novel aminooligosaccharides isolated from the culture of Streptomyces coelicoflavus ZG0656 as potent inhibitors of alpha-amylase

Two novel aminooligosaccharides were separated from the culture filtrate of Streptomyces coelicoflavus ZG0656. Their chemical structures were determined by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and 2D nuclear magnetic resonance (NMR) spectroscopy. Because of their acarviosine core structures, the names acarviostatins II23 and II13 were given to the novel compounds. The two acarviostatins were both mixed noncompetitive inhibitors of porcine pancreatic alpha-amylase (PPA), with inhibition constants (K(i)) of 0.009 microM (acarviostatin II23) and 0.010 microM (acarviostatin II13). Therefore, acarviostatin II23 and acarviostatin II13 are, respectively, 231 and 208 times more potent than acarbose.     

A novel approach for identifying the heme-binding proteins from mouse tissues

Heme is a key cofactor in aerobic life, both in eukaryotes and prokaryotes. Because of the high reactivity of ferrous protoporphyrin IX, the reactions of heme in cells are often carried out through heme-protein complexes. Traditionally studies of heme-binding proteins have been approached on a case by case basis, thus there is a limited global view of the distribution of heme-binding proteins in different cells or tissues. The procedure described here is aimed at profiling heme-binding proteins in mouse tissues sequentially by 1) purification of heme-binding proteins by heme-agarose, an affinity chromatographic resin; 2) isolation of heme-binding proteins by SDS-PAGE or two-dimensional electrophoresis; 3) identification of heme-binding proteins by mass spectrometry. In five mouse tissues, over 600 protein spots were visualized on 2DE gel stained by Commassie blue and 154 proteins were identified by MALDI-TOF, in which most proteins belong to heme related. This methodology makes it possible to globally c     

A novel approach to identifying beta-secretase inhibitors: bis-statine peptide mimetics discovered using structure and spot synthesis

Beta-secretase 1 (BACE1) is an aspartic protease believed to play a critical role in Alzheimer's disease. Inhibitors of this enzyme have been designed by incorporating the non-cleavable hydroxyethylene and statine isosteres into peptides corresponding to BACE1 substrate sequences. We sought to develop new methods to quickly characterize and optimize inhibitors based on the statine core. Minimal sequence requirements for binding were first established using both crystallography and peptide spot synthesis. These shortened peptide inhibitors were then optimized by using spot synthesis to perform iterative cycles of substitution and deletion. The present study resulted in the identification of novel "bis-statine" inhibitors shown by crystallography to have a unique binding mode. Our results demonstrate the application of peptide spot synthesis as an effective method for enhancing peptidomimetic drug discovery.     

Pharmacophore modeling study based on known Spleen tyrosine kinase inhibitors together with virtual screening for identifying novel inhibitors

In this investigation, chemical features based 3D pharmacophore models were developed based on the known inhibitors of Spleen tyrosine kinase (Syk) with the aid of hiphop and hyporefine modules within catalyst. The best quantitative pharmacophore model, Hypo1, was used as a 3D structural query for retrieving potential inhibitors from chemical databases including Specs, NCI, MayBridge, and Chinese Nature Product Database (CNPD). The hit compounds were subsequently subjected to filtering by Lipinski’s rule of five and docking studies to refine the retrieved hits. Finally 30 compounds were selected from the top ranked hit compounds and conducted an in vitro kinase inhibitory assay. Six compounds showed a good inhibitory potency against Syk, which have been selected for further investigation.     

Dihydroquinolines as novel n-NOS inhibitors

Dihydroquinolines have been synthesized and have been shown to be potent n-NOS inhibitors. Selectivity versus e-NOS was increased to approximately 100-fold through appropriate substitution at the benzene ring.     

In situ assembly of enzyme inhibitors using extended tethering

Cysteine aspartyl protease-3 (caspase-3) is a mediator of apoptosis and a therapeutic target for a wide range of diseases. Using a dynamic combinatorial technology, 'extended tethering', we identified unique nonpeptidic inhibitors for this enzyme. Extended tethering allowed the identification of ligands that bind to discrete regions of caspase-3 and also helped direct the assembly of these ligands into small-molecule inhibitors. We first designed a small-molecule 'extender' that irreversibly alkylates the cysteine residue of caspase-3 and also contains a thiol group. The modified protein was then screened against a library of disulfide-containing small-molecule fragments. Mass-spectrometry was used to identify ligands that bind noncovalently to the protein and that also form a disulfide linkage with the extender. Linking the selected fragments with binding elements from the extenders generates reversible, tight-binding molecules that are druglike and distinct from known inhibitors. One molecule derived from this approach inhibited apoptosis in cells.     

CpG_MI: a novel approach for identifying functional CpG islands in mammalian genomes

CpG islands (CGIs) are CpG-rich regions compared to CpG-depleted bulk DNA of mammalian genomes and are generally regarded as the epigenetic regulatory regions in association with unmethylation, promoter activity and histone modifications. Accurate identification of CpG islands with epigenetic regulatory function in bulk genomes is of wide interest. Here, the common features of functional CGIs are identified using an average mutual information method to differentiate functional CGIs from the remaining CGIs. A new approach (CpG mutual information, CpG_MI) was further explored to identify functional CGIs based on the cumulative mutual information of physical distances between two neighboring CpGs. Compared to current approaches, CpG_MI achieved the highest prediction accuracy. This approach also identified new functional CGIs overlapping with gene promoter regions which were missed by other algorithms. Nearly all CGIs identified by CpG_MI overlapped with histone modification marks. CpG_MI could also be used to identify potential functional CGIs in other mammalian genomes, as the CpG dinucleotide contents and cumulative mutual information distributions are almost the same among six mammalian genomes in our analysis. It is a reliable quantitative tool for the identification of functional CGIs from bulk genomes and helps in understanding the relationships between genomic functional elements and epigenomic modifications.     

An approach to identifying novel substrates of bacterial arylamine N-acetyltransferases

Arylamine N-acetyltransferases (NATs) catalyse the acetylation of arylamine, arylhydrazine and arylhydroxylamine substrates by acetyl Coenzyme A. NAT has been discovered in a wide range of eukaryotic and prokaryotic species. Although prokaryotic NATs have been implicated in xenobiotic metabolism, to date no endogenous role has been identified for the arylamine N-acetyl transfer reaction in prokaryotes. Investigating the substrate specificity of these enzymes is one approach to determining a possible endogenous role for prokaryotic NATs. We describe an accurate and efficient assay for NAT activity that is suitable for high-throughput screening of potential NAT ligands. This assay has been utilised to identify novel substrates for pure NAT from Salmonella typhimurium and Mycobacterium smegmatis which show a relationship between the lipophilicity of the arylamine and its activity as a substrate. The lipophilic structure/activity relationship observed is proposed to depend on the topology of the active site using docking studies of the crystal structures of these NAT isoenzymes. The evidence suggests an endogenous role of NAT in the protection of bacteria from aromatic and lipophilic toxins.     

In silico multi-filter screening approaches for developing novel beta-secretase inhibitors

A large database of chemical structures was screened for potential inhibitors of β-secretase was carried out using in silico multi-filter techniques. Substructure screening, computer-aided ligand docking, binding free energy calculations, and partial interaction energy analyses were performed successively to identify chemical compounds which could serve as different scaffolds from known β-secretase inhibitors for future drug design. We showed that our in silico multi-filter screening retrieved all known inhibitors from the compound database investigated, which suggests that the other compounds identified as inhibitors by this computerized screening process are potential β-secretase inhibitors.     

Representing genetic variation as continuous surfaces: an approach for identifying spatial dependency in landscape genetic studies

Landscape genetics, an emerging field integrating landscape ecology and population genetics, has great potential to influence our understanding of habitat connectivity and distribution of organisms. Whereas typical population genetics studies summarize gene flow as pairwise measures between sampling localities, landscape characteristics that influence population genetic connectivity are often continuously distributed in space. Thus, there are currently gaps in both the ability to analyze genotypic data in a continuous spatial context and our knowledge of expected of landscape genetic structure under varying conditions. We present a framework for generating continuous “genetic surfaces”, evaluate their statistical properties, and quantify statistical behavior of landscape genetic structure in a simple landscape. We simulated microsatellite genotypes under varying parameters (time since vicariance, migration, effective population size) and used ancestry (q) values from STRUCTURE to interpolate a genetic surface. Using a spatially adjusted Pearson's correlation coefficient to test the significance of landscape variable(s) on genetic structure we were able to detect landscape genetic structure on a contemporary time scale (≥5 generations post vicariance, migration probability ≤0.10) even when population differentiation was minimal (F_ST≥0.00015). We show that genetic variation can be significantly correlated with geographic distance even when genetic structure is due to landscape variable(s), demonstrating the importance of testing landscape influence on genetic structure. Finally, we apply genetic surfacing to analyze an empirical dataset of black bears from northern Idaho USA. We find black bear genetic variation is a function of distance (autocorrelation) and habitat patch (spatial dependency), consistent with previous results indicating genetic variation was influenced by landscape by resistance. These results suggest genetic surfaces can be used to test competing hypotheses of the influence of landscape characteristics on genetic structure without delineation of categorical groups.     

The quartz crystal microbalance as a novel means to study cell-substrate interactions In situ

The quartz crystal microbalance (QCM) was first introduced as a mass sensor in gas phase and in vacuum. Since oscillator circuits capable of exciting shear vibrations of quartz resonators under liquid loading have been developed, the QCM became accepted as a new, powerful technique to follow adsorption processes at solid-liquid interfaces in chemical and biological research. Lately, the QCM technique has attracted considerable interest as a novel means to monitor cell-substrate interactions of mammalian cells in vitro. Because the establishment and modulation of cell-substrate contacts is important for many physiological processes, and potent techniques to measure these interactions noninvasively are rare, the present review highlights applications of the QCM technique in this field. The suitability of the QCM device to monitor attachment and spreading of mammalian cells in real time has been well established. The QCM response is dependent on the individual cell type that is examined. In order to identify the sources for these cell-type-specific results of QCM readings, and to understand the information content of the signal, attempts have been made to decompose the overall QCM response into subcellular contributions. The aforementioned subjects, together with a condensed introduction into the QCM technology, are included in this article.     

In situ hydrogen consumption kinetics as an indicator of subsurface microbial activity

There are few methods available for broadly assessing microbial community metabolism directly within a groundwater environment. In this study, hydrogen consumption rates were estimated from in situ injection/withdrawal tests conducted in two geochemically varying, contaminated aquifers as an approach towards developing such a method. The hydrogen consumption first-order rates varied from 0.002 nM h(-1) for an uncontaminated, aerobic site to 2.5 nM h(-1) for a contaminated site where sulfate reduction was a predominant process. The method could accommodate the over three orders of magnitude range in rates that existed between subsurface sites. In a denitrifying zone, the hydrogen consumption rate (0.02 nM h(-1)) was immediately abolished in the presence of air or an antibiotic mixture, suggesting that such measurements may also be sensitive to the effects of environmental perturbations on field microbial activities. Comparable laboratory determinations with sediment slurries exhibited hydrogen consumption kinetics that differed substantially from the field estimates. Because anaerobic degradation of organic matter relies on the rapid consumption of hydrogen and subsequent maintenance at low levels, such in situ measures of hydrogen turnover can serve as a key indicator of the functioning of microbial food webs and may be more reliable than laboratory determinations.     

A novel approach to identifying regulatory motifs in distantly related genomes

Although proven successful in the identification of regulatory motifs, phylogenetic footprinting methods still show some shortcomings. To assess these difficulties, most apparent when applying phylogenetic footprinting to distantly related organisms, we developed a two-step procedure that combines the advantages of sequence alignment and motif detection approaches. The results on well-studied benchmark datasets indicate that the presented method outperforms other methods when the sequences become either too long or too heterogeneous in size.