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.     

Effects of Acifluorfen on Endogenous Antioxidants and Protective Enzymes in Cucumber (Cucumis sativus L.) Cotyledons

The herbicide acifluorfen (2-chloro-4-(trifluoromethyl)phenoxy-2-nitrobenzoate) causes strong photooxidative destruction of pigments and lipids in sensitive plant species. Antioxidants and oxygen radical scavengers slow the bleaching action of the herbicide. The effect of acifluorfen on glutathione and ascorbate levels in cucumber (Cucumis sativus L.) cotyledon discs was investigated to assess the relationship between herbicide activity and endogenous antioxidants. Acifluorfen decreased the levels of glutathione and ascorbate over 50% in discs exposed to less than 1.5 hours of white light (450 microeinsteins per square meter per second). Coincident increases in dehydroascorbate and glutathione disulfide were not observed. Acifluorfen also caused the rapid depletion of ascorbate in far-red light grown plants which were photosynthetically incompetent.

Glutathione reductase, dehydroascorbate reductase, superoxide dismutase, ascorbate oxidase, ascorbate free radical reductase, peroxidase, and catalase activities rapidly decreased in acifluorfen-treated tissue exposed to white light. None of the enzymes were inhibited in vitro by the herbicide. Acifluorfen causes irreversible photooxidative destruction of plant tissue, in part, by depleting endogenous antioxidants and inhibiting the activities of protective enzymes.

     

Spectroscopic detection of transient thiamin diphosphate-bound intermediates on benzoylformate decarboxylase

Thiamin diphosphate (ThDP)-dependent enzymes catalyze a range of transformations, such as decarboxylation and ligation. We report a novel spectroscopic assay for detection of some of the ThDP-bound intermediates produced on benzoylformate decarboxylase. Benzoylformate decarboxylase was mixed with its alternate substrate p-nitrobenzoylformic acid on a rapid-scan stopped-flow instrument, resulting in formation of three absorbing species (lambda(max) in parentheses): I(1) (a transient at 620 nm), I(2) (a transient at 400 nm), and I(3) (a stable absorbance with lambda(max) > 730 nm). Analysis of the kinetics of the two transient species supports a model in which a noncovalent complex of the substrate and the enzyme is converted to the first covalent intermediate I(1); the absorbance corresponding to I(1) is probably a charge-transfer band arising from the interaction of the thiamin diphosphate-p-nitrobenzoylformic acid covalent adduct (2-p-nitromandelylThDP) and the enzyme. The rate of disappearance of I(1) parallels the rate of formation of I(2). Chemical models suggest the lambda(max) of I(2) (near 400 nm) to be appropriate to the enamine, a key intermediate in ThDP-dependent reactions resulting from the decarboxylation of the thiamin diphosphate-p-nitrobenzoylformic acid covalent adduct. Therefore, the rate of disappearance of I(1) and/or the appearance of I(2) directly measure the rate of decarboxylation. A relaxation kinetic treatment of the pre-steady-state kinetic data also revealed a hitherto unreported facet of the mechanism, alternating active-sites reactivity. Parallel studies of the His70Ala BFD active-site variant indicate that it cannot form the complex reported by the charge-transfer band (I(1)) at the level of the wild-type protein.     

Structures of manganese(II) complexes with ATP, ADP, and phosphocreatine in the reactive central complexes with creatine kinase: electron paramagnetic resonance studies with oxygen-17-labeled ligands

Coordination of Mn(II) to the phosphate groups of the substrates and products in the central complexes of the creatine kinase reaction mixture has been investigated by electron paramagnetic resonance (EPR) spectroscopy with regiospecifically 17O-labeled substrates. The EPR pattern for the equilibrium mixture is a superposition of spectra for the two central complexes, and this pattern differs from those observed for the ternary enzyme-Mn(II)-nucleotide complexes and from that for the dead-end complex enzyme-Mn(II)ADP-creatine. In order to identify those signals that are associated with each of the central complexes of the equilibrium mixture, spectra were obtained for a complex of enzyme, Mn(II)ATP, and a nonreactive analogue of creatine, 1-(carboxymethyl)-2-iminoimidazolidin-4-one, which is a newly synthesized competitive inhibitor. This inhibitor permits an unobstructed view of the EPR spectrum for Mn(II)ATP in the closed conformation of the active site. The EPR spectrum for this nonreactive complex with Mn(II)ATP matches one subset of signals in the spectrum for the equilibrium mixture, i.e., those due to the enzyme-Mn(II)-ATP-creatine complex. Chemical quenching of the samples followed by chromatographic assays for both ATP and ADP indicates that the enzyme-Mn(II)ADP-phosphocreatine and the enzyme-Mn(II)ATP-creatine complexes are present in a ratio of approximately 0.7 to 1. A similar value for the equilibrium constant for enzyme-bound substrates is obtained directly from the EPR spectrum for the equilibrium mixture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective silencing of cell communication influences anteroposterior pattern formation in C. elegans

In C. elegans males, laterally located V cells generate a simple pattern of anterior alae (cuticular ridges) and posterior rays (mating sensilla). We have found that this pattern is generated, at least in part, by the selective interruption of cell-cell interactions. In anterior V cells, lineages leading to the production of alae are induced by cell interactions. These cell interactions are inhibited in specific posterior V cells by the activity of the gene pal-1, which allows these cells to generate rays instead of alae. The activities of cell signals and pal-1 appear to influence V cell fates by determining the state of a developmental switch that involves two homeotic genes, lin-22 and mab-5.     

4-Oxalocrotonate tautomerase, an enzyme composed of 62 amino acid residues per monomer.

The xylH gene encoding 4-oxalocrotonate tautomerase (4-OT) has been located on a subclone of the Pseudomonas putida mt-2 TOL plasmid pWW0 and inserted into an Escherichia coli expression vector. Several of the genes of the metafission pathway encoded by pWW0 have been cloned in E. coli, but the overexpression of their gene products has met with limited success. By utilizing the E. coli alkaline phosphatase promoter (phoA) coupled with the proper positioning of a ribosome-binding region, we are able to express functional 4-OT in yields of at least 10 mg of pure enzyme/liter of culture. 4-OT has been previously characterized and shown to be an extremely efficient catalyst (Whitman, C. P., Aird, B. A., Gillespie, W. R., and Stolowich, N. J. (1991) J. Am. Chem. Soc. 113, 3154-3162). Kinetic and physical characterization of the E. coli-expressed protein show that it is identical with that of the 4-OT isolated from P. putida. The functional unit is apparently a pentamer of identical subunits, each consisting of only 62 amino acid residues. This is the smallest enzyme subunit reported to date. The amino acid sequence, determined in part from automated Edman degradation and also deduced from the primary sequence of xylH, did not show homology with any of the sequences in the current data bases nor with any of the sequences of enzymes that catalyze similar reactions. We propose that the active site of 4-OT may be established by an overlap of subunits and comprised of amino acid residues belonging to several, if not all, of the subunits.     

A novel locus for autosomal dominant nonsyndromic hearing loss, DFNA13, maps to chromosome 6p.

Nonsyndromic hearing loss (NSHL) is the most common type of hearing impairment in the elderly. Environmental and hereditary factors play an etiologic role, although the relative contribution of each is unknown. To date, 39 NSHL genes have been localized. Twelve produce autosomal dominant hearing loss, most frequently postlingual in onset and progressive in nature. We have ascertained a large, multigenerational family in which a gene for autosomal dominant NSHL is segregating. Affected individuals experience progressive hearing loss beginning in the 2d-4th decades, eventually making the use of amplification mandatory. A novel locus, DFNA13, was identified on chromosome 6p; the disease gene maps to a 4-cM interval flanked by D6S1663 and D6S1691, with a maximum two-point LOD score of 6.409 at D6S299.     

Monitoring for occupational exposure to 4,4′-methylenebis(2-chloroaniline) by gas chromatographic-mass spectrometric analysis of haemoglobin adducts, blood, plasma and urine

The feasibility of using plasma, blood and haemoglobin adducts for monitoring occupational exposure to the suspected human carcinogen 4,4′-methylenebis(2-chloroaniline) (MOCA) was investigated. A method utilising capillary gas chromatography-negative-ion chemical-ionisation mass spectrometry (GC-MS) for the determination of pentafluoropropionyl (PFP) derivatives of MOCA, released by alkaline hydrolysis from protein adducts and conjugates, was both sensitive and selective. When selected ion monitoring was used, sub-femtomole amounts of PFP-MOCA could be measured. The detection limit for haemoglobin adducts of MOCA was below 10 fmol/g Hb, well below the levels found for occupationally exposed individuals. Capillary GC with electron-capture detection also had the required sensitivity for the determination of MOCA in blood and urine of five individuals who were exposed to MOCA during the manufacture of polyurethane elastomers were determined by the GC-MS method. The MOCA concentrations for the various blood fractions and urine were within the following ranges: haemoglobin adducts, 0.73–43.3 pmol MOCA/g Hb; plasma alkaline hydrolysate, 0.05–22.0 nmol/l; whole blood, 0.13–17.4nmol/l; urine, 4.5–2390 nmol/l. Because the products of MOCA in the blood reflect metabolic activation of MOCA and integrate exposure over a period of weeks, the use of blood samples for monitoring exposure to MOCA offers advantages over the currently used urinary MOCA measurements.