Cloning and expression of a human ATP-citrate lyase cDNA.

A full-length cDNA clone of 4.3 kb encoding the human ATP-citrate lyase enzyme has been isolated by screening a human cDNA library with the recently isolated rat ATP-citrate lyase cDNA clone [Elshourbagy et al. (1990) J. Biol. Chem. 265, 1430]. Nucleic-acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1105 amino acids in length with a calculated molecular mass of 121,419 Da. Comparison of the human and rat ATP-citrate lyase cDNA sequences reveals 96.3% amino acid identity throughout the entire sequence. Further sequence analysis identified the His765 catalytic phosphorylation site, the ATP-binding site, as well as the CoA binding site. The human ATP-citrate lyase cDNA clone was subcloned into a mammalian expression vector for expression in African green monkey kidney cells (COS) and Chinese hamster ovary cells (CHO) cells. Transfected COS cells expressed detectable levels of an enzymatically active recombinant ATP-citrate lyase enzyme. Stable, amplified expression of ATP-citrate lyase in CHO cells as achieved by using coamplification with dihydrofolate reductase. Resistant cells expressed high levels of enzymatically active ATP-citrate lyase (3 pg/cell/d). Site-specific mutagenesis of His765----Ala diminishes the catalytic activity of the expressed ATP-citrate lyase protein. Since catalysis of ATP-citrate lyase is postulated to involve the formation of phosphohistidine, these results are consistent with the pattern of earlier observations of the significance of the histidine residue in catalysis of the human ATP-citrate lyase.     

DNA deoxyribophosphodiesterase.

A previously unrecognized enzyme acting on damaged termini in DNA is present in Escherichia coli. The enzyme catalyses the hydrolytic release of 2-deoxyribose-5-phosphate from single-strand interruptions in DNA with a base-free residue on the 5' side. The partly purified protein appears to be free from endonuclease activity for apurinic/apyrimidinic sites, exonuclease activity and DNA 5'-phosphatase activity. The enzyme has a mol. wt of approximately 50,000-55,000 and has been termed DNA deoxyribophosphodiesterase (dRpase). The protein presumably is active in DNA excision repair to remove a sugar-phosphate residue from an endonucleolytically incised apurinic/apyrimidinic site, prior to gap filling and ligation.     

Characterization of Na+/K+/Cl- cotransport in cultured HT29 human colonic adenocarcinoma cells

A Na+/K+/Cl- cotransport pathway has been examined in the HT29 human colonic adenocarcinoma cell line using 86Rb as the K congener. Ouabain-resistant bumetanide-sensitive (OR-BS) K+ influx in attached HT29 cells was 17.9 +/- 0.9 nmol/min per mg protein at 25 degrees C. The identity of this pathway as a Na+/K+/Cl- cotransporter has been deduced from the following findings: (a) OR-BS K+ influx ceased if the external Cl- (Cl-o) was replaced by NO3- or the external Na+ (Na+o) by choline; (b) neither OR-BS 24Na+ nor 36Cl- influx was detectable in the absence of external K+ (K+o); and (c) concomitant measurements of 86Rb+, 22Na+, and 36Cl- influx indicated that the stoichiometry of the cotransport system approached a ratio of 1N+:1K+:2Cl-. In addition, OR-BS K+ influx was exquisitely sensitive to cellular ATP levels. Depletion of the normal ATP content of 35-40 nmol/mg protein to 10-15 nmol/mg protein, a concentration at which the ouabain-sensitive K+ influx was unaffected, completely abolished K+ cotransport. OR-BS K+ influx was slightly reduced by the divalent cations Ca2+, Ba2+, Mg2+ and Mn2+. Although changes in cell volume, whether shrinking or swelling, did not influence OR-BS K+ influx, ouabain-sensitive K+ influx was activated by cell swelling. As in T84 cells, we found that the OR-BS K+ influx in HT29 cells was stimulated by exogenous cyclic AMP analogues and by augmented cyclic AMP content in response to vasoactive intestinal peptide, forskolin, norepinephrine and forskolin or prostaglandin E1.     

Equivalence of microbial biomass measures based on membrane lipid and cell wall components,adenosine triphosphate,and direct counts in subsurface aquifer sediments

An uncontaminated subsurface aquifer sediment contains a sparse microbial community consisting primarily of coccobacillary bacteria of relatively uniform size which can be counted directly with appropriate staining. The morphological simplicity and the relatively decreased cell numbers, when compared with surface soils and sediments, make the subsurface an ideal natural community with which to compare the utility of chemical measures of microbial biomass to direct microscopic counts. The membrane phospholipids (estimated as the polar lipid fatty acids, the lipid phosphate, and phosopholipid glycerol phosphate), lipopolysaccharide lipid A (estimated as the LPS hydroxy fatty acids), cell walls (estimated as the muramic acid), and adenosine triphosphate all give essentially identical estimates of cell numbers and dry weight as the direct counts, using conversion factors determined on subsurface microorganism monocultures. Assays of microbial cell components are thus validated by comparison with the classical direct count in at least one soil/sediment.     

Metabolism of nitropolycyclic aromatic hydrocarbons by human intestinal microflora

Anaerobic bacterial suspensions from human and rat feces and intestinal contents, and pure cultures of anaerobic bacteria metabolized 1-nitropyrene and 6-nitrobenzo[a]pyrene to 1-aminopyrene and 6-aminobenzo[a]pyrene, respectively. The metabolites were isolated by reversed-phase high performance liquid chromatography and identified by comparison of their chromatographic and mass spectral properties with those of authentic compounds. The results suggest that anaerobic intestinal bacteria could play a significant role in the metabolism of potentially carcinogenic nitropolycyclic aromatic hydrocarbons.     

Mechanism of Na+ Insertion in Alkali Vanadates and Its Influence on Battery Performance

Sodium‐ion batteries may become an alternative to the widespread lithium‐ion technology due to cost and kinetic advantages provided that cyclability is improved. For this purpose, the interplay between electrochemical and structural processes is key and is demonstrated in this work for Na_2.46V₆O₁₆ (NVO) and Li_2.55V₆O₁₆ employing operando synchrotron X‐ray diffraction. When NVO is cycled between 4.0 and 1.6 V, Na‐ions reversibly occupy two crystallographic sites, which results in remarkable cyclability. Upon discharge to 1.0 V, however, Na‐ions occupy also interstitial sites, inducing irreversible structural change with some loss of crystallinity concomitant with a decrease in capacity. Capacity fading increases with the ionic radius of the alkali ions (K⁺ > Na⁺ > Li⁺), suggesting that smaller ions stabilize the structure. This correlation of structural variation and electrochemical performance suggests a route toward improving cycling stability of a sodium‐ion battery. Its essence is a minor Li⁺‐retention in the A_2+xV₆O₁₆ structure. Even though the majority of Li‐ions are replaced by the abundant Na⁺, the residual Li‐ions (≈10%) are sufficient to stabilize the layered structure, diminishing the irreversible structural damage. These results pave the way for further exploitation of the role of small ions in lattice stabilization that increases cycling performance.     

Estimating Tropical Forest Structure Using a Terrestrial Lidar

Forest structure comprises numerous quantifiable biometric components and characteristics, which include tree geometry and stand architecture. These structural components are important in the understanding of the past and future trajectories of these biomes. Tropical forests are often considered the most structurally complex and yet least understood of forested ecosystems. New technologies have provided novel avenues for quantifying biometric properties of forested ecosystems, one of which is LIght Detection And Ranging (lidar). This sensor can be deployed on satellite, aircraft, unmanned aerial vehicles, and terrestrial platforms. In this study we examined the efficacy of a terrestrial lidar scanner (TLS) system in a tropical forest to estimate forest structure. Our study was conducted in January 2012 at La Selva, Costa Rica at twenty locations in a predominantly undisturbed forest. At these locations we collected field measured biometric attributes using a variable plot design. We also collected TLS data from the center of each plot. Using this data we developed relative vegetation profiles (RVPs) and calculated a series of parameters including entropy, Fast Fourier Transform (FFT), number of layers and plant area index to develop statistical relationships with field data. We developed statistical models using a series of multiple linear regressions, all of which converged on significant relationships with the strongest relationship being for mean crown depth (r² = 0.88, p < 0.001, RMSE = 1.04 m). Tree density was found to have the poorest significant relationship (r² = 0.50, p < 0.01, RMSE = 153.28 n ha^-1). We found a significant relationship between basal area and lidar metrics (r² = 0.75, p < 0.001, RMSE = 3.76 number ha^-1). Parameters selected in our models varied, thus indicating the potential relevance of multiple features in canopy profiles and geometry that are related to field-measured structure. Models for biomass estimation included structural canopy variables in addition to height metrics. Our work indicates that vegetation profiles from TLS data can provide useful information on forest structure.     

Personality Type Influences Attentional Bias in Individuals with Chronic Back Pain

Attentional biases reflect an individual’s selective attention to salient stimuli within their environment, for example an experience of back pain. Eysenck suggests that different personality types show different attentional biases to threatening information. This study is the first to test Eysenck’s theory within a chronic back pain population by investigating the attentional biases of four different personality types using a back pain specific dot-probe paradigm. Participants were 70 volunteers (45 female) recruited from a back rehabilitation program at an NHS Trust. The four groups were selected on their trait anxiety and defensiveness scores: defensive high-anxious; high-anxious; repressor and non-extreme. Participants completed a dot probe task comprising 20 practice trials and 250 experimental trials. The experimental trials contained 100 threat-neutral pairs, 100 positive-neutral pairs and 50 neutral-neutral image pairings. The threat images were taken from the Photograph Series of Daily Activities (PHODA) and the neutral and positive images from the International Affective Picture System (IAPS) image bank. The results provided partial support for Eysenck’s theory; defensive high-anxious individuals showed an attentional bias for threatening information compared to high-anxious individuals who demonstrated no bias. Repressors showed an avoidant bias to threatening images and an attentional bias to positive stimuli relative to neutral images. The clear difference in responses demonstrated by high-anxious individuals who vary in defensiveness highlight the need for separate investigation of these heterogeneous groups and help to explain the cognitive processes of defensive high-anxious individuals within a pain population. The demonstration of an attentional bias in this group to threatening information could explain why defensive high-anxious individuals are more likely to re-present for treatment.     

A new technique for retarding fading of fluorescence: DPX-BME

The antioxidant beta-mercaptoethanol (BME) used in conjunction with the permanent mountant DPX (DPX-BME) retarded fluorescent fading of mithramycin, acridine orange and Hoechst 33258 stained chicken erythrocytes, each to a varying degree. The initial fluorescence of all dyes examined was more intense with DPX-BME than with DPX alone. Specimens mounted in DPX-BME showed strong fluorescence and excellent morphology; if kept in the dark, they could be stored indefinitely without deterioration. Retarding fading of fluorescence with DPX-BME faciliated quantitation of DNA using fluorescence cytophotometry.