See globally, spike locally: oscillations in a retinal model encode large visual features

We show that coherent oscillations among neighboring ganglion cells in a retinal model encode global topological properties, such as size, that cannot be deduced unambiguously from their local, time-averaged firing rates. Whereas ganglion cells may fire similar numbers of spikes in response to both small and large spots, only large spots evoke coherent high frequency oscillations, potentially allowing downstream neurons to infer global stimulus properties from their local afferents. To determine whether such information might be extracted over physiologically realistic spatial and temporal scales, we analyzed artificial spike trains whose oscillatory correlations were similar to those measured experimentally. Oscillatory power in the upper gamma band, extracted on single-trials from multi-unit spike trains, supported good to excellent size discrimination between small and large spots, with performance improving as the number of cells and/or duration of the analysis window was increased. By using Poisson distributed spikes to normalize the firing rate across stimulus conditions, we further found that coincidence detection, or synchrony, yielded substantially poorer performance on identical size discrimination tasks. To determine whether size encoding depended on contiguity independent of object shape, we examined the total oscillatory activity across the entire model retina in response to random binary images. As the ON-pixel probability crossed the percolation threshold, which marks the sudden emergence of large connected clusters, the total gamma-band activity exhibited a sharp transition, a phenomena that may be experimentally observable. Finally, a reanalysis of previously published oscillatory responses from cat ganglion cells revealed size encoding consistent with that predicted by the retinal model.     

Determinants of substrate specificity in KdcA, a thiamin diphosphate-dependent decarboxylase

Thiamin diphosphate-dependent decarboxylases catalyze the non-oxidative decarboxylation of 2-keto carboxylic acids. Although they display relatively low sequence similarity, and broadly different range of substrates, these enzymes show a common homotetrameric structure. Here we describe a kinetic characterization of the substrate spectrum of a recently identified member of this class, the branched chain 2-keto acid decarboxylase (KdcA) from Lactococcus lactis. In order to understand the structural basis for KdcA substrate recognition we developed a homology model of its structure. Ser286, Phe381, Val461 and Met358 were identified as residues that appeared to shape the substrate binding pocket. Subsequently, site-directed mutagenesis was carried out on these residues with a view to converting KdcA into a pyruvate decarboxylase. The results show that the mutations all lowered the Km value for pyruvate and both the S286Y and F381W variants also had greatly increased values of k(cat) with pyruvate as a substrate.     

Exploring the role of the active site cysteine in human muscle creatine kinase

All known guanidino kinases contain a conserved cysteine residue that interacts with the non-nucleophilic eta1-nitrogen of the guanidino substrate. Site-directed mutagenesis studies have shown that this cysteine is important, but not essential for activity. In human muscle creatine kinase (HMCK) this residue, Cys283, forms part of a conserved cysteine-proline-serine (CPS) motif and has a pKa about 3 pH units below that of a regular cysteine residue. Here we employ a computational approach to predict the contribution of residues in this motif to the unusually low cysteine pKa. We calculate that hydrogen bonds to the hydroxyl and to the backbone amide of Ser285 would both contribute approximately 1 pH unit, while the presence of Pro284 in the motif lowers the pKa of Cys283 by a further 1.2 pH units. Using UV difference spectroscopy the pKa of the active site cysteine in WT HMCK and in the P284A, S285A, and C283S/S285C mutants was determined experimentally. The pKa values, although consistently about 0.5 pH unit lower, were in broad agreement with those predicted. The effect of each of these mutations on the pH-rate profile was also examined. The results show conclusively that, contrary to a previous report (Wang et al. (2001) Biochemistry 40, 11698-11705), Cys283 is not responsible for the pKa of 5.4 observed in the WT V/K(creatine) pH profile. Finally we use molecular dynamics simulations to demonstrate that, in order to maintain the linear alignment necessary for associative inline transfer of a phosphoryl group, Cys283 needs to be ionized.     

Template switching within exons 3 and 4 of KV11.1 (HERG) gives rise to a 5' truncated cDNA

K(V)11.1 (HERG) channels contribute to membrane potential in a number of excitable cell types. We cloned a variant of K(V)11.1 from human jejunum containing a 171 bp deletion spanning exons 3 and 4. Expression of a full-length cDNA clone containing this deletion gave rise to protein that trafficked to the cell membrane and generated robust currents. The deletion occurred in a G/C-rich region and identical sequence elements of UGGUGG were located at the deletion boundaries. In recent studies these features have been implicated to cause deletions via template switching during cDNA synthesis. To examine this possibility we compared cDNAs from human brain, heart, and jejunum synthesized at lower (42 degrees C) and higher temperatures (70 degrees C). The 171 bp deletion was absent at the higher temperature. Our results suggest that the sequence and secondary structure of mRNA in the G/C rich region leads to template switching producing a cDNA product with a 171 bp deletion.     

A multiplexed protein microarray for the simultaneous serodiagnosis of human immunodeficiency virus/hepatitis C virus infection and typing of whole blood

All donor blood samples must be tested pretransfusion to determine the donor blood type. Standard testing protocols require that assays be performed for important bloodborne pathogens such as hepatitis C, syphilis, hepatitis B, and human immunodeficiency virus. We have demonstrated proof of the concept that a protein microarray can type whole blood and detect antibody to significant pathogens simultaneously from the same donor blood sample. The data collected demonstrate the ability of the array to accurately type blood samples while also detecting the presence of antibodies against both human immunodeficiency virus and hepatitis C virus. In conclusion, we have successfully developed a platform capable of typing human whole blood samples, while at the same time testing for the presence of antibodies specific for human immunodeficiency virus/hepatitis C virus. The major benefits of this system are its amenability to expansion with additional assays, for example, rhesus typing and syphilis and/or hepatitis B virus detection, and also the adaptability of the assay to higher-throughput analysis, currently 16 individual samples per slide, but readily expandable to a 96-well format.     

Conservation of biodiversity in the Arabuko Sokoke Forest, Kenya

Using an economic approach to provide a rationale for rainforestconservation has been a popular exercise in recent years. This paper uses suchan approach to assess the net value of the Arabuko Sokoke Forest in Kenya. Theeconomic benefits associated with the forest derived by local and globalpopulations are estimated by combining evidence from existing studies and theresults of a contingent valuation study carried out by the authors. Thesebenefits are set against the cost of preserving the forest to the Kenyan ForestDepartment. Even when the opportunity cost of the forest land is omitted fromthe costs of forest preservation, and when the revenues generated from theGlobal Environment Facility (GEF) funded project are included, the costs offorest conservation outweigh the benefits. It is only when non-use andexistencevalues are included (which are not realised by the Kenyan population) that theforest benefits exceed the costs. The paper concludes by arguing that, althoughsome projects within the Arabuko Sokoke Forest have been successful incapturingsome of the economic value associated with the forest, more needs to be done todesign additional capture mechanisms so that a greater proportion of the globalbenefit of the forest can be realised by local populations and localgovernments.