Cannabinoids Facilitate the Swallowing Reflex Elicited by the Superior Laryngeal Nerve Stimulation in Rats

Cannabinoids have been reported to be involved in affecting various biological functions through binding with cannabinoid receptors type 1 (CB1) and 2 (CB2). The present study was designed to investigate whether swallowing, an essential component of feeding behavior, is modulated after the administration of cannabinoid. The swallowing reflex evoked by the repetitive electrical stimulation of the superior laryngeal nerve in rats was recorded before and after the administration of the cannabinoid receptor agonist, WIN 55-212-2 (WIN), with or without CB1 or CB2 antagonist. The onset latency of the first swallow and the time intervals between swallows were analyzed. The onset latency and the intervals between swallows were shorter after the intravenous administration of WIN, and the strength of effect of WIN was dose-dependent. Although the intravenous administration of CB1 antagonist prior to intravenous administration of WIN blocked the effect of WIN, the administration of CB2 antagonist did not block the effect of WIN. The microinjection of the CB1 receptor antagonist directly into the nucleus tractus solitarius (NTS) prior to intravenous administration of WIN also blocked the effect of WIN. Immunofluorescence histochemistry was conducted to assess the co-localization of CB1 receptor immunoreactivity to glutamic acid decarboxylase 67 (GAD67) or glutamate in the NTS. CB1 receptor was co-localized more with GAD67 than glutamate in the NTS. These findings suggest that cannabinoids facilitate the swallowing reflex via CB1 receptors. Cannabinoids may attenuate the tonic inhibitory effect of GABA (gamma-aminobuteric acid) neurons in the central pattern generator for swallowing.     

Crystal structure of Aspergillus niger isopullulanase, a member of glycoside hydrolase family 49

An isopullulanase (IPU) from Aspergillus niger ATCC9642 hydrolyzes α-1,4-glucosidic linkages of pullulan to produce isopanose. Although IPU does not hydrolyze dextran, it is classified into glycoside hydrolase family 49 (GH49), major members of which are dextran-hydrolyzing enzymes. IPU is highly glycosylated, making it difficult to obtain its crystal. We used endoglycosidase H_f to cleave the N-linked oligosaccharides of IPU, and we here determined the unliganded and isopanose-complexed forms of IPU, both solved at 1.7-Å resolution. IPU is composed of domains N and C joined by a short linker, with electron density maps for 11 or 12 N-acetylglucosamine residues per molecule. Domain N consists of 13 β-strands and forms a β-sandwich. Domain C, where the active site is located, forms a right-handed β-helix, and the lengths of the pitches of each coil of the β-helix are similar to those of GH49 dextranase and GH28 polygalacturonase. The entire structure of IPU resembles that of a GH49 enzyme, Penicillium minioluteum dextranase (Dex49A), despite a difference in substrate specificity. Compared with the active sites of IPU and Dex49A, the amino acid residues participating in subsites + 2 and + 3 are not conserved, and the glucose residues of isopanose bound to IPU completely differ in orientation from the corresponding glucose residues of isomaltose bound to Dex49A. The shape of the catalytic cleft characterized by the seventh coil of the β-helix and a loop from domain N appears to be critical in determining the specificity of IPU for pullulan.     

Expression pattern of <Emphasis Type="Italic">serine protease inhibitor kazal type 3 (Spink3)</Emphasis> during mouse embryonic development

Recent evidence shows that the serine protease inhibitor Kazal type 3 (Spink3) has more diverse functions than expected. To gain insight into its function, we analyzed the spatiotemporal expression profile of Spink3, using in situ hybridization (ISH) and a Spink3 ( +/lacZ ) knock-in mouse, in which lacZ was inserted into the Spink3 locus. Spink3 ( lacZ ) expression was first observed in the foregut, midgut, hindgut and the forebrain/midbrain junction region at 9.5 days post coitus (dpc). In the pancreas, Spink3 mRNA was detected at 11.5 dpc, before formation of the typical shape of the exocrine structure of the pancreas. Acinar cell expression was clearly identified by 13.5 dpc. After differentiation of the intestinal tract, Spink3 ( lacZ ) expression was observed in the large intestine at 11.5 dpc, followed by expression in the small intestine at 13.5 dpc, before appearance of intestinal digestive enzymes. Spink3 mRNA and Spink3 ( lacZ ) activity were also detected in other tissues, including the mesonephric tubules and the urogenital ridge at 11.5 dpc, the genital swelling at 13.5 dpc, the ductus epididymis at 17.5 dpc, and the seminal vesicle at 8 weeks. These data suggest that Spink3 may play important roles in proliferation and/or differentiation of various cell types during development.     

Construction of novel single-cell screening system using a yeast cell chip for nano-sized modified-protein-displaying libraries

A novel screening system using a microchamber array chip was developed for construction of combinatorial nano-sized protein libraries in combination with yeast cell surface engineering. It is possible to place a single yeast cell into each microchamber, to observe its behavior, and to pick up the target cell. The microchamber array chip is referred to as a “yeast cell chip.” A single EGFP-displaying yeast cell could be detected, picked up by a micro-manipulator, and cultivated on agar medium. Furthermore, a catalytic reaction, the hydrolysis of fluorescein dioctanate, by a single yeast cell displaying Rhizopus oryzae lipase (ROL) was carried out in one microchamber. The ROL-encoding gene in a single ROL-displaying cell was amplified by PCR. These results demonstrate that this yeast cell chip in combination with cell surface engineering could be used as a tool in a high-throughput screening system not only for a single living cell and a whole-cell catalyst with a nano-sized protein cluster but also for modified nano-sized and functional protein molecules from protein libraries on the cell surface.     

Molecular mechanisms for Kv1.3 potassium channel current inhibition by CD3/CD28 stimulation in Jurkat T cells

In T lymphocyte, activation of Kv1.3 channel, the major voltage-dependent K⁺ channel, is an essential step for cell proliferation in immune responses. Here, effects of anti-CD3 and anti-CD28 antibodies on Kv1.3 current were examined in three types of human T lymphocyte derived cell lines, Jurkat E6-1, p56lck-kinase deficient mutant JCaM.1, and CD45-phosphatase deficient mutant J45.01. Kv1.3 current was partly reduced by CD3 stimulation and more strongly by addition of anti-CD28 antibody in E6-1. In JCaM.1, Kv1.3 current responses to anti-CD28/CD3 antibodies were similar to those in E6-1. In J45.01, CD3 stimulation partly inhibited Kv1.3 current, but the additive reduction by CD28 stimulation was not significant. The inhibition of tyrosine phosphatase in E6-1 abolished the additional inhibition by anti-CD28 antibody in a similar manner as in J45.01. In conclusion, the stimulation of CD28 in addition to CD3 strongly inhibits Kv1.3 current and this additive inhibition is mediated by CD45 activation.     

Excitation and deexcitation processes of Eu(III) benzo-15-crown-5 complex studied on comparing with its first coordination sphere

The coordination sphere and the deexcitation mechanism of the Eu(III) benzo-15-crown-5 complex, Eu(B15C5), were studied with references of the Eu(III) complexes with a similar coordination sphere; the dibenzo-18-crown-6 complex, Eu₃(B₂18C6)₂, and the cryptand[2.2.1] complex, Eu([2.2.1]). NMR spectroscopy reveals that the Eu(B15C5) complex is quite stable in acetonitrile solution whereas only 40% of the Eu(III) ion forms the complex in the equimolar Eu(NO₃)₃ and B₂18C6 acetonitrile solution. The coordination sphere of the Eu(III) complexes in acetonitrile solutions were also discussed by the degenerate ⁷F₀→⁵D₀ transition energy levels. The Eu(B15C5) have a negative shift compared with the europium(III) nitrate in acetonitrile and it is explained by the coordination of both nitrate ions and the crown ether ligand. Energy transfer from the n–π* excited state located in the catechol structure to the central europium ion was first observed as the sensitized luminescence of ⁵D₀→⁷F_J. The excited state lifetime of the Eu(B15C5) complex was first determined as 201 μs in the present study.     

Syk expression and novel function in a wide variety of tissues.

Syk protein-tyrosine kinase has been implicated in a variety of hematopoietic cell responses, in particular immunoreceptor signaling events that mediate diverse cellular responses including proliferation, differentiation, and phagocytosis. On the other hand, Syk exhibits a more widespread expression pattern in nonhematopoietic cells like fibroblasts, epithelial cells, breast tissue, hepatocytes, neuronal cells, and vascular endothelial cells and has been shown to be functionally important on these cell types. Thus, Syk appears to play a general physiological function in a wide variety of cells. In this article, we briefly review the current literature regarding the expression and novel function of Syk in various cells and tissues.     

Isolation and identification of a light-induced growth inhibitor in diffusates from blue light-illuminated oat (Avena sativa L.) coleoptile tips

The amounts of two growth inhibitors in diffusates from illuminatedhalves of phototropically stimulated oat (Avena sativa L.)coleoptile tips were larger than those from shaded halves. The less polarinhibitor was isolated from diffusates from oat coleoptile tips illuminatedwithblue light, and identified as uridine from ¹H NMR spectrum. Thedistribution of endogenous uridine in diffusates from the illuminated andshadedsides of coleoptile tips unilaterally exposed to blue light for 3, causing a first positive phototropic curvature, and fromdark-control tips, was determined using a physicochemical assay. The uridineconcentration was significantly higher in the diffusates from the illuminatedside than in those from the shaded side and the dark-control. Uridine inhibitedthe growth of etiolated oat coleoptile tips at concentrations of 30 and above. These results suggest that uridine plays a role inthe phototropism of oat coleoptiles.     

Effects of head shape variation on growth, metamorphosis and survivorship in larval salamanders (Hynobius retardatus)

The effects of head shape variation on growth and metamorphosis in larval salamander (Hynobius retardatus) were examined by a laboratory experiment and a field experiment. In the laboratory experiment, each larva was fed equal amounts and was prevented from accessing others in both the solitary and group treatments, although chemical cues could be transmitted through water in the group treatment. The relative head width of larvae became larger in the group treatment during the early periods but having a large head width did not finally influence growth rate and days for metamorphosis. In the field experiment, larvae were allowed to contact each other directly in two density conditions. The enlarged relative head width was linked to high growth rate in the high-density treatment but not in the low-density treatment. The larval body size distribution in the high-density condition tended to be smaller, and there was a small proportion of large-sized individuals with a broad head width. Moreover, the small number of large larvae metamorphosed much earlier than the others. The mortality of larvae in high-density conditions was much higher than that in the low-density treatments. This would be a consequence of cannibalism in the high-density condition. From the experimental results obtained, it is argued that for the larvae of H. retardatus having a large head is an adaptive tactic that maximizes fitness, particularly in temporary ponds with an unpredictable environment and in crowded conditions.