The flavonoid galangin inhibits the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia

The flavonoid galangin inhibits the partially purified metallo-beta-lactamase from Stenotrophomonas maltophilia. The effect was not reversed by the addition of ZnCl(2) suggesting that the inhibitory effect is not related to metal chelation. The flavonoid quercetin also has some inhibitory effect against the enzyme. Using the crystal structure of the enzyme, a molecular modelling study predicts a possible orientation of galangin at the active site of the enzyme.     

Axon navigation in the mammalian primary olfactory pathway: where to next?

The process of establishing long-range neuronal connections can be divided into at least three discrete steps. First, axons need to be stimulated to grow and this growth must be towards appropriate targets. Second, after arriving at their target, axons need to be directed to their topographically appropriate position and in some cases, such as in cortical structures, they must grow radially to reach the correct laminar layer. Third, axons then arborize and form synaptic connections with only a defined subpopulation of potential post-synaptic partners. Attempts to understand these mechanisms in the visual system have been ongoing since pioneer studies in the 1940s highlighted the specificity of neuronal connections in the retino-tectal pathway. These classical systems-based approaches culminated in the 1990s with the discovery that Eph-ephrin repulsive interactions were involved in topographical mapping. In marked contrast, it was the cloning of the odorant receptor family that quickly led to a better understanding of axon targeting in the olfactory system. The last 10 years have seen the olfactory pathway rise in prominence as a model system for axon guidance. Once considered to be experimentally intractable, it is now providing a wealth of information on all aspects of axon guidance and targeting with implications not only for our understanding of these mechanisms in the olfactory system but also in other regions of the nervous system.     

Comparative growth analysis of the facultative anaerobes Bacillus subtilis,Bacillus licheniformis,and Escherichia coli

Bacillus subtilis anaerobic respiration and fermentative growth capabilities were compared to two other facultative anaerobes, Bacillus licheniformis and Escherichia coli. In glycerol defined medium, B. subtilis grew with nitrate, but not nitrite or fumarate, while B. licheniformis grew with nitrate or fumarate, but not nitrite. Growth of E. coli occurred in glycerol defined medium with either nitrate, nitrite, or fumarate. In order to grow by fermentation, B. subtilis required both glucose and pyruvate, while B. licheniformis and E. coli were capable of using either glucose or pyruvate.     

Nuclear localization and DNA interaction of protein disulfide isomerase ERp57 in mammalian cells

Protein disulfide isomerase ERp57 is localized predominantly in the endoplasmic reticulum, but is also present in the cytosol and, according to preliminary evidence, in the nucleus of avian cells. Conclusive evidence of its nuclear localization and of its interaction with DNA in vivo in mammalian cells is provided here on the basis of DNA-protein cross-linking experiments performed with two different cross-linking agents on viable HeLa and 3T3 cells. Nuclear ERp57 could also be detected by immunofluorescence in HeLa cells, where it showed an intracellular distribution clearly different from that of an homologous protein, located exclusively in the endoplasmic reticulum. Mammalian ERp57 resembles the avian protein in its recognition of S/MAR-like DNA sequences and in its association with the nuclear matrix. It can be hypothesized that ERp57, which is known to associate with other proteins, in particular STAT3 and calreticulin, may contribute to their nuclear import, DNA binding, or other functions that they fulfil inside the nucleus.     

fRFLP and fAFLP: medium-throughput genotyping by fluorescently post-labeling restriction digestion

Genome-scale studies of population structure and high-resolution mapping of genetically complex traits both require techniques for accurately and efficiently genotyping large numbers of polymorphic sites in multiple individuals. Many high-throughput genotyping technologies require the purchase of expensive equipment or consumables and are therefore out of reach of some individual research laboratories. Conversely, less expensive technologies are often labor intensive so that the effort involved in typing large numbers of samples or polymorphic sites is prohibitive. Here we present a method of fluorescently post-labeling restriction digestion using standard dye-terminator sequencing chemistry so that RFLP and AFLP products can be visualized on an automated sequencer This labeling method is efficient, inexpensive, easily multiplexed, and requires no unusual equipment or reagents, thus striking a balance between cost and throughput that should be appropriate for many research groups and core facilities.     

Membrane biochips

Membrane-bound proteins represent the single most important class of drug targets. This article discusses the issues surrounding fabrication of membrane-protein microarrays by conventional robotic pin printing techniques. Ligand binding selectivity and specificity to G protein-coupled receptor (GPCR) microarrays are presented. The potential applications of these arrays for drug screening are discussed.     

Use of at-line and in-situ near-infrared spectroscopy to monitor biomass in an industrial fed-batch Escherichia coli process

One of the key goals in bioprocess monitoring is to achieve real-time knowledge of conditions within the bioreactor, i.e., in-situ. Near-infrared spectroscopy (NIRS), with its ability to carry out multi-analyte quantification rapidly with little sample presentation, is potentially applicable in this role. In the present study, the application of NIRS to a complex, fed-batch industrial E. coli (RV308/PHKY531) process was investigated. This process undergoes a series of temperature changes and is vigorously agitated and aerated. These conditions can pose added challenges to in-situ NIRS. Using the measurement of a key analyte (biomass) as an illustration, the details of the relationship between the at-line and in-situ use of NIRS are considered from the viewpoint of both theory and practical application. This study shows that NIRS can be used both at-line and in-situ in order to achieve good predictive models for biomass. There are particular challenges imposed by in-situ operation (loss of wavelength regions and noise) which meant the need for signal optimisation studies. This showed that whilst the at-line modelling process may provide some useful information for the in-situ process, there were distinct differences. This study shows that the in-situ use of NIRS in a highly challenging matrix (similar to those encountered in current industrial practice) is possible, and thus extends previous works in the area.     

X-linked cone-rod dystrophy (locus COD1): identification of mutations in RPGR exon ORF15

X-linked cone-rod dystrophy (COD1) is a retinal disease that primarily affects the cone photoreceptors; the disease was originally mapped to a limited region of Xp11.4. We evaluated the three families from our original study with new markers and clinically reassessed all key recombinants; we determined that the critical intervals in families 2 and 3 overlapped the RP3 locus and that a status change (from affected to probably unaffected) of a key recombinant individual in family 1 also reassigned the disease locus to include RP3 as well. Mutation analysis of the entire RPGR coding region identified two different 2-nucleotide (nt) deletions in ORF15, in family 2 (delAG) and in families 1 and 3 (delGG), both of which result in a frameshift leading to altered amino acid structure and early termination. In addition, an independent individual with X-linked cone-rod dystrophy demonstrated a 1-nt insertion (insA) in ORF15. The presence of three distinct mutations associated with the same disease phenotype provides strong evidence that mutations in RPGR exon ORF15 are responsible for COD1. Genetic heterogeneity was observed in three other families, including the identification of an in-frame 12-nt deletion polymorphism in ORF15 that did not segregate with the disease in one of these families.     

Residues in the epsilon subunit of the nicotinic acetylcholine receptor interact to confer selectivity of waglerin-1 for the alpha-epsilon subunit interface site

Waglerin-1 (Wtx-1) is a 22-amino acid peptide that competitively antagonizes muscle nicotinic acetylcholine receptors (nAChRs). Previous work demonstrated that Wtx-1 binds to mouse nAChRs with higher affinity than receptors from rats or humans, and distinguished residues in alpha and epsilon subunits that govern the species selectivity. These studies also showed that Wtx-1 binds selectively to the alpha-epsilon binding site with significantly higher affinity than to the alpha-delta binding site. Here we identify residues at equivalent positions in the epsilon, gamma, and delta subunits that govern Wtx-1 selectivity for one of the two binding sites on the nAChR pentamer. Using a series of chimeric and point mutant subunits, we show that residues Gly-57, Asp-59, Tyr-111, Tyr-115, and Asp-173 of the epsilon subunit account predominantly for the 3700-fold higher affinity of the alpha-epsilon site relative to that of the alpha-gamma site. Similarly, we find that residues Lys-34, Gly-57, Asp-59, and Asp-173 account predominantly for the high affinity of the alpha-epsilon site relative to that of the alpha-delta site. Analysis of combinations of point mutations reveals that Asp-173 in the epsilon subunit is required together with the remaining determinants in the epsilon subunit to achieve Wtx-1 selectivity. In particular, Lys-34 interacts with Asp-173 to confer high affinity, resulting in a DeltaDeltaG(INT) of -2.3 kcal/mol in the epsilon subunit and a DeltaDeltaG(INT) of -1.3 kcal/mol in the delta subunit. Asp-173 is part of a nonhomologous insertion not found in the acetylcholine binding protein structure. The key role of this insertion in Wtx-1 selectivity indicates that it is proximal to the ligand binding site. We use the binding and interaction energies for Wtx-1 to generate structural models of the alpha-epsilon, alpha-gamma, and alpha-delta binding sites containing the nonhomologous insertion.