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.     

Effects of specific signal transduction inhibitors on increased permeability across rat endothelial monolayers induced by neuropeptide Y or VEGF

Neuropeptide Y (NPY) elevates the permeability of cultured rat aortic endothelial cells (RAECs) in monolayer cultures under hypoxic conditions (5% O(2)) possibly by binding to the NPY Y(3) receptor. The present study evaluated the effects of NPY compared to vascular endothelial growth factor (VEGF). RAECs were cultured on the upper chamber base of a double-chamber culture system, FITC-labeled albumin was introduced into the chamber, and permeation into the lower chamber was measured. Treatment was with 3 x 10(-7) M NPY or 10(-7) g/ml VEGF for 2 h along with specific inhibitors. The VEGF receptor-2 tyrosine kinase inhibitor tyrphostin SU-1498 and the protein kinase C inhibitor bis-indolylmaleimide I (GF-109203X) suppressed the VEGF-induced increase in monolayer permeability but not that caused by NPY. Furthermore, although the action of NPY was blocked in a concentration-dependent manner by phospholipase C inhibitor 1-(6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl)-1H-pyrrole-2,5-dione (U-73122), it was less sensitive than VEGF. However, the effects of both NPY and VEGF on the permeability of the RAEC monolayer were blocked with equal concentration dependence by STI571 (imatinib mesylate), which is an inhibitor of Abl tyrosine kinase in the nucleus and/or cytoplasm. The myosin light-chain kinase inhibitor 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine HCl (ML-9) suppressed both NPY- and VEGF-induced increment in permeability by approximately 70%, whereas the calmodulin-dependent kinase inhibitor DY-9760e could decrease to below the baseline. These results indicate that the NPY Y(3)-receptor subtype is specifically linked to the effects of STI571 on endothelial cells, and that NPY, a sympathetic coneurotransmitter, may increase vascular permeability in association with altered intracellular or nuclear signal transduction.     

Possible involvement of a cyclic AMP-dependent mechanism in PACAP-induced proliferation and ERK activation in astrocytes

In cultured astrocytes, PACAP activates extracellular signal-regulated kinase (ERK) and induces cell proliferation at picomolar concentrations. Here, we examined the role of cyclic AMP signaling underlying the effects of PACAP. PACAP38 induced accumulation of cyclic AMP in astrocytes at concentrations as low as 10(-12)M. PACAP38 (10(-12)-10(-9)M)-stimulated cell proliferation was completely abolished by the cyclic AMP antagonist Rp-cAMP, whereas the protein kinase A (PKA) inhibitor H89 had no effect. This PACAP38-mediated effect was also abolished by the ERK kinase inhibitor PD98059, suggesting the involvement of ERK in PACAP-induced proliferation. PACAP38 (10(-12)M)-stimulated phosphorylation of ERK lasted for at least 60 min. This effect was completely abolished by Rp-cAMP but not by H89. Dibutyryl cyclic AMP maximally stimulated the incorporation of thymidine and activation of ERK at 10(-10)M. These results suggest that PACAP-mediated stimulation of ERK activity and proliferation of astrocytes may involve a cyclic AMP-dependent, but PKA-independent, pathway.     

Element analysis of the Polysphondylium pallidum gp64 promoter

gp64 mRNA in Polysphondylium pallidum is expressed extensively during vegetative growth, and begins to rapidly decrease at the onset of development. To examine this unique regulation, 5' deletion analysis of the gp64 promoter was undertaken, and two growth-phase activated elements have been found: a food-dependent, upstream regulatory region (FUR, -222 to -170) and a vegetatively activated, downstream region (VAD, -110 to -63). Here we concentrate our analysis on an A1 and A2 sequences in the FUR region: A1 consists of a GATTTTTTTA sequence called a corresponding sequence and A2 consists of the direct repeat TTTGTTGTG. The cells carrying a combined construct of A1 and A2 acted synergistically in a reporter activity. A point mutation analysis in A1 indicates that a G residue is required for the activation of A1. From analyses of promoter regulation in a liquid or a solid medium, the promoter activity of the cells fed on bacteria in A-medium (axenic medium for Polysphondylium) or grown in A-medium alone was only one fourth of that of the cells fed on bacteria. By the gel retardation, we detected a protein bound to the A1 sequence.     

Ex vivo characterization of γδ T-cell repertoire in patients after adoptive transfer of Vγ9Vδ2 T cells expressing the interleukin-2 receptor β-chain and the common γ-chain

Background aimsAdoptive immunotherapy is emerging as a potent anti-tumor treatment modality; Vγ9Vδ2 T cells may represent appropriate agents for such cancer immunotherapy. To improve the currently limited success of Vγ9Vδ2 T-cell–based immunotherapy, we examined the in vivo dynamics of these adoptively-transferred cells and hypothesized that interleukin (IL)-15 is the potential factor for Vγ9δ2 T cell in vivo survival.MethodsWe conducted a clinical trial of adoptive Vγ9Vδ2 T-cell transfer therapy in six colorectal cancer patients who received pulmonary metastasectomy. Patients' peripheral blood mononuclear cells were stimulated with zoledronate (5 μmol/L) and IL-2 (1000 IU/mL) for 14 d. Harvested cells, mostly Vγ9Vδ2 T cells, were given intravenously weekly without additional IL-2 eight times in total. The frequency, phenotype and common γ-chain cytokine receptor expression of Vγ9Vδ2 T cells in peripheral blood was monitored by flow cytometry at each time point during treatment and 4 and 12 weeks after the last administration.ResultsAdoptively transferred Vγ9Vδ2 T cells expanded well without exogenous IL-2 administration or lymphodepleting preconditioning. They maintained effector functions in terms of interferon-γ secretion and prompt release of cytotoxic granules in response to PMA/ionomycin or isopentenyl pyrophosphate–positive cells. Because they are IL-2Rα^?IL-7Rα^?IL-15Rα^?IL-2Rβ⁺γ_c⁺, it is likely that IL-2 or IL-15 is required for their maintenance.ConclusionsThe persistence of large numbers of functionally active adoptively transferred Vγ9Vδ2 T cells in the absence of exogenous IL-2 implies that an endogenous factor, such as IL-15 transpresentation, is adequate to support these cells in vivo.     

Sequence variation in the Tbx4 gene in marine mammals

The amino-acid sequences of the T-domain region of the Tbx4 gene, which is required for hindlimb development, are 100% identical in humans and mice. Cetaceans have lost most of their hindlimb structure, although hindlimb buds are present in very early cetacean embryos. To examine whether the Tbx4 gene has the same function in cetaceans as in other mammals, we analyzed Tbx4 sequences from cetaceans, dugong, artiodactyls and marine carnivores. A total of 39 primers were designed using human and dog Tbx4 nucleotide sequences. Exons 3, 4, 5, 6, 7, and 8 of the Tbx4 genes from cetaceans, artiodactyls, and marine carnivores were sequenced. Non-synonymous substitution sites were detected in the T-domain regions from some cetacean species, but were not detected in those from artiodactyls, the dugong, or the carnivores. The C-terminal regions contained a number of non-synonymous substitutions. Although some indels were present, they were in groups of three nucleotides and therefore did not cause frame shifts. The dN/dS values for the T-domain and C-terminal regions of the cetacean and artiodactylous Tbx4 genes were much lower than 1, indicating that the Tbx4 gene maintains it function in cetaceans, although full expression leading to hindlimb development is suppressed.     

Phylogenetic relationships among cetaceans revealed by Y-chromosome sequences

The Y chromosome has recently come into the spotlight as a new and efficient genetic marker for tracing paternal lineages. We reconstructed cetacean phylogeny using a 1.7-kbp fragment of the non-recombining Y chromosome (NRY), including the SRY gene and a flanking non-coding region. The topology of the Y-chromosome tree is robust to various methods of analysis and exhibits high branch-support values, possibly due to the absence of recombination, small effective population size, and low homoplasy. The Y-chromosome tree indicates monophyly of each suborder, Mysticeti and Odontoceti, with high branch support values (BS> or =86%; PP> or =98%). In the Odontoceti clade, three superfamilies, Physeteroidea, Ziphioidea, and Delphinoidea, diverged soon after the split between Mysticeti and Odontoceti. Our analysis allows resolution of this rapid radiation and indicates that Physeteroidea is basal in the Odontoceti clade (BS, 99%; PP, 100%; MBS, 61%). The major split within the superfamily Delphinoidea is between the Delphinidae clade and the Monodontidae+ Phocoenidae clade. The phylogenetic relationships among delphinid species are ambiguous, probably because of the rapid radiation of this family. In the Mysticeti clade, the first major split is between Balaenidae and Balaenopteridae; within Balaenopteridae, a Balaenoptera acutorostrata+B. bonaerensis (minke whales) clade forms a sister clade with the other balaenopterid species. Megaptera novaeangliae is nested within Balaenoptera, making the latter paraphyletic. The low homoplasy exhibited by the Y-chromosome data presented here suggests that an extended data set incorporating longer sequences would provide better resolution of cetacean lower-level pylogeny.     

Analysis of invariant sequences in 266 complete genomes

To date, the complete genome sequences of more than 250 organisms have been determined. This information can now be used to determine whether there exist any invariant sequences that are conserved among all organisms, from bacteria to plants, animals, and humans. The existence of invariant sequences would strongly suggest that these sequences have been inherited unchanged from the last common ancestor of all life, and that they have essential functions. We have developed a new software program to identify invariant sequences conserved among the currently sequenced genomes and applied this analysis to the complete genome sequences of 266 organisms. We have identified 3 invariant DNA sequences longer than or equal to 11 bp and 6 invariant amino acid sequences longer than or equal to 6 aa. The longest invariant DNA sequence, AAGTCGTACAAGGT (15 bp), was found in the 16S/18S rRNA gene. Two 8 aa sequences, GHVDHGKT in IF2 and EF-Tu and DTPGHVDF in EF-G, were the longest invariant amino acid sequences detected. These sequences could be essential elements from the genome of the last common ancestor and may have remained unchanged throughout evolution.     

Problem-based learning is suitable for the curriculum of “Sleep disorders and disease” for students in dentistry

Sleep and Biological Rhythms -     

Clinical significance and management of insomnia

Sleep and Biological Rhythms - Several epidemiological studies have shown that more than 20% of the general adult population has symptoms of insomnia, but only about 5% of them are receiving...