Enzyme electrode formed by evaporative concentration and its performance characterization

A highly concentrated immobilized enzyme layer was formed on a small working electrode, and the behavior of the electrode as an amperometric sensor was examined. To this end, a super-hydrophobic layer was formed in an area other than the sensitive area by using polytetrafluoroethylene (PTFE) beads. A small droplet of an enzyme solution containing glucose oxidase (GOD) and bovine serum albumin (BSA) was placed on the sensitive area, concentrated by evaporation, and crosslinked with glutaraldehyde. With the same enzyme activity per unit area, the current density increased with smaller working electrodes. Also, the current density increased with higher enzyme loadings up to a limiting value. In addition, the linear range of the calibration plot was expanded to higher glucose concentrations. The enzyme electrode fabricated by the novel method was incorporated in a micro-flow channel. Compared with large enzyme electrodes with the same enzyme activity per unit area, smaller electrodes showed a significant increase in the current density and a decrease in the flow dependence. The conversion efficiency could be improved by narrowing the flow channel and increasing the number of electrodes, which was comparable with a large electrode placed in a shallow flow channel.     

Determination of growth inhibitory action point of interferon gamma on WISH cells in cell cycle progression and the window of responsiveness of the cells to the interferon

We had earlier shown that human foetal epithelial cells (WISH), growth-inhibited by interferon gamma (IFNgamma), were reversibly detained at a point prior to DNA synthesis. In the present study, we determined the window of action of IFNgamma in the G1 phase duration and the exact point of detention of WISH cells in cell cycle progression with respect to the known points of detention by the inhibitors of DNA replication initiation (aphidicolin and carbonyl diphosphonate) and of activation of replication protein A (6-dimethylaminopurine), of which RPA activation being the earlier event compared to DNA replication initiation in cell cycle progression. WISH cells, which were released from IFNgamma-induced arrest, permeabilised and exposed independently to these inhibitors show that IFNgamma detains WISH cells prior to initiation of DNA synthesis. Further, exposure of IFNalpha-synchronized (at G0/G1) or mimosine-synchronized (at G1/S) WISH cells to IFNgamma, which was added at different time points post-release from the synchronizing agent, showed that the cells were promptly responsive to the growth inhibitory action of IFNgamma only during the first 11h in G1 phase. Taken together, these results suggest that IFNgamma inhibits growth of WISH cells by detaining them at a point prior to initiation of DNA synthesis and that the IFN acts within the first 11h in G1 phase of the cell cycle.     

Gadd45alpha does not modulate the carboplatin or 5-fluorouracil-induced apoptosis in human papillomavirus-positive cells

Gadd45alpha is shown to be induced by a wide spectrum of DNA-damaging agents and implicated in negative regulation of cell growth by causing G2-M arrest or induction of apoptosis. In the present study, we explored the involvement of p53 in the promoter activation of Gadd45alpha as well as the role of Gadd45alpha in carboplatin (Carb) or 5-fluorouracil (5-FU)-induced apoptosis in human papillomavirus virus (HPV)-positive HEp-2 and HeLa cells. We report that Carb or 5-FU upregulate Gadd45alpha and p53 in both these cells. Transient transfection of chloramphenicol acetyl transferase (CAT)-reporter construct driven by Gadd45alpha promoter clearly indicated that Gadd45alpha upregulation was mediated through activation of its promoter. Inhibition of p53 function by dominant-negative-p53 expression partially suppressed the activation of Gadd45alpha promoter. Further, the induction of apoptosis was assessed by detection of poly (ADP-ribose) polymerase (PARP) cleavage by Western blot analysis. Inhibition of upregulated Gadd45alpha expression by antisense expression vector did not modulate the Carb or 5-FU-induced apoptosis. Overall, we conclude that Gadd45alpha promoter activation partially depends on p53 function in HPV-positive cells. Moreover, Gadd45alpha protein does not modulate Carb or 5-FU-induced apoptosis in these cells.     

Dietary Intakes and Health Risk of Toxic and Essential Heavy Metals through the Food Chain in Agricultural,Industrial, and Coal Mining Areas of Northern India

Minerals can enter the food chain through industrial and mining activities. Soil-to-vegetable transfer is higher than soil-to-cereal, but human consumption of metals is attributable to balanced diets containing both vegetables and cereals and drinking water. However, the impact of location on intakes of metals from predominantly cereal-based Indian diets is not clear. Hence, the present study was undertaken in selected Agricultural, Industrial, and Coal Mining Areas (AA, IA, CMA) around the Allahabad District in Northern India to compare transfer of toxic metals, Pb, Cd, Cr and essential metals, Fe, Zn, Cu, Co in soil and water to common crops: cereals (rice, wheat, maize) and vegetables (spinach, potato), and to assess Daily Intake of Metal (DIM) and consequent Health Risk Index (HRI). The overall content of all metals, except Cu, in water, soils, and crops followed the pattern CMA > IA > AA. Transfer factors (TFs) followed the sequence spinach > potato > cereals. Quantitatively, however, cereals contribute maximally to a balanced diet, so DIM and HRI were higher from cereals than vegetables. Even though spinach had the highest TFs, cereals contributed maximally to HRI. CMA had the highest metal content so locally grown cereals contributed significantly to intake of both toxic and essential metals.     

Coordinated methyl-lysine erasure: structural and functional linkage of a Jumonji demethylase domain and a reader domain

Both components of chromatin (DNA and histones) are subjected to dynamic postsynthetic covalent modifications. Dynamic histone lysine methylation involves the activities of modifying enzymes (writers), enzymes removing modifications (erasers), and readers of the epigenetic code. Known histone lysine demethylases include flavin-dependent monoamine oxidase lysine-specific demethylase 1 and α-ketoglutarate-Fe(II)-dependent dioxygenases containing Jumonji domains. Importantly, the Jumonji domain often associates with at least one additional recognizable domain (reader) within the same polypeptide that detects the methylation status of histones and/or DNA. Here, we summarize recent developments in characterizing structural and functional properties of various histone lysine demethylases, with emphasis on a mechanism of crosstalk between a Jumonji domain and its associated reader module(s). We further discuss the role of recently identified Tet1 enzyme in oxidizing 5-methylcytosine to 5-hydroxymethylcytosine in DNA.     

Mechanoluminescence properties of gamma-ray-irradiated BaSO4:Eu phosphors

BaSO(4) activated with various concentrations of Eu were prepared by solid-state reaction technique. Thermoluminescence (TL) and mechanoluminescence (ML) of γ-ray-irradiated BaSO(4):Eu(2)O(3) phosphors were recorded. In the TL glow curve of the phosphor a single peak at 170°C was observed. The TL of the phosphors were also recorded after deforming the phosphors by dropping a piston of mass 0.4 kg onto them with different impact velocities. TL intensity (after deformation) decreased with increasing the impact velocity. In the ML intensity vs time curve two peaks were observed. ML intensity increased with increasing impact velocity of the piston and the time corresponding to peak ML intensity shifted to a shorter time value. ML intensity decreased drastically when it was recorded after annealing the sample at 170°C. The BaSO(4) phosphors activated with 0.1 mol% of Eu(2)O(3) showed optimum TL and ML. The photoluminescence emission spectrum of the sample showed that Eu enters as Eu(2+) ion in host lattice.     

Insect feeding deterrent and growth inhibitory activities of scopoletin isolated from Artemisia annua against Spilarctia obliqua (Lepidoptera: Noctuidae)

Abstract Artemisia annua (Asteraceae) is well known for its antimalarial activities due to presence of the compound artemisinin. We isolated a methoxy coumarin from the stem part of A. annua and confirmed its identity as scopoletin through mass spectral data. The structure was established from ¹H‐nuclear magnetic resonance (NMR), ¹³C‐NMR. The compound scopoletin was evaluated for its feeding deterrence and growth inhibitory potential against a noxious lepidopteran insect, Spilartctia obliqua Walker. Scopoletin gave FD₅₀ (feeding deterrence of 50%) value of 96.7 μg/g diet when mixed into artificial diet. S. obliqua larvae (12‐day‐old) exposed to the highest concentration (250 μg/g diet) of scopoletin showed 77.1% feeding‐deterrence. In a growth inhibitory assay, scopoletin provided 116.9% growth inhibition at the highest dose of 250 μg/g diet with a GI₅₀ (growth inhibition of 50%) value of 20.9 μg/g diet. Statistical analysis showed a concentration‐dependent dose response relationship toward both feeding deterrent and growth inhibitory activities. Artemisinin is found mainly in the leaves of A. annua and not in the stems, which are typically discarded as waste. Therefore identification of scopoletin in stems of A. annua may be important as a source of this material for pest control.