Effects of a chitosan intake on the fecal excretion of dioxins and fat in rats

We evaluated the effects of the intake of various dietary fibers on the fecal excretion of dioxins in rats. The rats were fed five types of dietary fiber diets, including a chitosan diet and control diet, for 20 d and then dioxins (120 ng/rat) were orally administered on day 15. The excretion of fecal dioxins was significantly higher in the chitosan group than in the control group, and dioxin excretion was positively correlated with fecal fat excretion. A comparison of the different types of chitosan showed that the efficacy of chitosan for fecal fat excretion was partly related to its viscosity. The chitosan intake promoted fecal dioxin excretion when the rats were exposed to highly toxic dioxins, and this excretion of fecal dioxins was related to the fecal fat excretion, suggesting that chitosan might be useful for reducing the adverse effects caused by lipophilic xenobiotics.     

An extremely low frequency magnetic field attenuates insulin secretion from the insulinoma cell line, RIN-m

In this study, we investigated the effects of exposure to an extremely low frequency magnetic field (ELFMF) on hormone secretion from an islet derived insulinoma cell line, RIN-m. We stimulated RIN-m cells to secrete insulin under exposure to an ELFMF, using our established system for the exposure of cultured cells to an ELFMF at 5 mT and 60 Hz, or under sham exposure conditions for 1 h and observed the effects. In the presence of a depolarizing concentration of potassium (45 mM KCl), exposure to ELFMF significantly attenuated insulin release from RIN-m cells, compared to sham exposed cells. Treatment with nifedipine reduced the difference in insulin secretion between cells exposed to an ELFMF and sham exposed cells. The expression of mRNA encoding synaptosomal associated protein of 25 kDa (SNAP-25) and synaptotagmin 1, which play a role in exocytosis in hormone secretion and influx of calcium ions, decreased with exposure to an ELFMF in the presence of 45 mM KCl. These results suggest that exposure to ELFMF attenuates insulin secretion from RIN-m cells by affecting calcium influx through calcium channels.     

Induction of autophagy via innate bacterial recognition

Macroautophagy (referred to hereafter as autophagy) functions not only in self-digestion, but also in the killing and degradation of infectious pathogens in in vitro-cultured cells. Based on genetic manipulations of both the host, Drosophila and pathogen, Listeria monocytogenes, we recently reported that L. monocytogenes-induced autophagy is dependent on the recognition of the pathogen by the Drosophila pattern recognition protein, PGRP-LE. Autophagy and PGRP-LE are crucial for inhibition of the intracellular growth of bacteria in hemocytes, the target cells of L. monocytogenes infection in vivo. The importance of autophagy in the resistance of Drosophila against L. monocytogenes is further indicated in in vivo survival experiments. The signaling pathway(s) that induces autophagy by PGRP-LE is independent of the known immune signaling pathways, suggesting that another unidentified pathway(s) is involved. The results of the present study demonstrate that the induction of autophagy, as an innate immune response targeting intracellular pathogens, is activated by intracellular sensors through unidentified pathways.     

Interaction of D-lactate dehydrogenase protein 2 (Dld2p) with F-actin: implication for an alternative function of Dld2p

D-Lactate dehydrogenase protein 2 [Yeast 15 (1999) 1377; Biochem. Biophys. Res. Commun. 295 (2002) 910] was initially identified as the actin interacting protein 2 (Aip2p) using a two-hybrid screen to search for proteins that interact with actin [Nat. Struct. Biol. 2 (1995) 28], but no other evidence indicating an interaction between Aip2p and actin cytoskeleton has been reported so far. During our search for the protein conformation modifying activity, we serendipitously identified Aip2p isolated from Saccharomyces cerevisiae as exhibiting an interaction with F-actin both in vitro and in vivo. Incubation with Aip2p facilitated the formation of the circular form of F-actin in vitro, which exhibited an aberrant trypsin susceptibility. Overexpression of Aip2p induced multi-buds in yeast cells, whereas reduced expression interfered with the formation of the cleavage furrow for the cell division, which was rescued by the introduction of wild-type Aip2p. While Aip2p-treated F-actin in the circular form was negligibly stained by rhodamine-labeled phalloidin (rhodamine-phalloidin) in vitro, rhodamine-phalloidin staining profiles in actin interacting protein 2 gene (AIP2)-modified cells suggested a correlation between the conformation of F-actin and the expression of Aip2p in vivo. AIP2-deleted cells became sensitive to osmotic conditions, a hallmark of actin dysfunction. Finally, immunoprecipitation of yeast cells using anti-Aip2p antibody demonstrated that Aip2p associates with actin. These properties suggest that Aip2p may interact with F-actin in vivo and play an important role in the yeast cell morphology.     

Antibacterial action of L-amino acid oxidase from the skin mucus of rockfish Sebastes schlegelii

L-amino acid oxidase (LAO) shows broadly antibacterial activity against Gram-positive and Gram-negative bacteria by H(2)O(2) generated in the oxidative process of L-amino acids. However, LAO (termed SSAP) isolated from the rockfish Sebastes schlegelii skin mucus acted selectively on Gram-negative bacteria. Therefore, this study was undertaken to clarify the antibacterial action of SSAP as compared with H(2)O(2). SSAP inhibited potently the growth of Aeromonas salmonicida, Photobacterium damselae subsp. piscicida and Vibrio parahaemolyticus with a minimum inhibitory concentration (MIC) of 0.078, 0.16 and 0.63 microg/mL, respectively. H(2)O(2) inhibited the growth of both Gram-positive and Gram-negative bacteria with an MIC ranging from 0.31 to 2.5 mM. When SSAP was incubated with P. damselae subsp. piscicida and Escherichia coli, SSAP was demonstrated to bind to P. damselae subsp. piscicida but not to E. coli by Western blotting and LAO activity measurement. These results show that the bacteria binding activity may be involved in the bacterial cell selectivity of SSAP. Electron microscopic observation of A. salmonicida, P. damselae subsp. piscicida and V. parahaemolyticus revealed that the treatments with SSAP and H(2)O(2) induced cell surface damage to A. salmonicida, remarkable elongation of P. damselae subsp. piscicida bodies and pores into V. parahaemolyticus cells.     

Expression of taste signal transduction molecules in the caecum of common marmosets

The extraoral presence of taste signal transduction proteins has recently been reported in rodents and humans. Here, we report for the first time the presence of these signal transduction proteins in the caecum of a non-human primate, the common marmoset. Quantitative RT-PCR data on the gene expression of taste signal transduction molecules (gustducin and TRPM5) in common marmosets suggested high expression in the caecum, which was not observed in other non-human primates. Immunohistochemical analysis confirmed the specific presence of gustducin and taste receptors in marmoset caecal cells. These results may relate to the specific feeding behaviour of marmosets, which consume plant exudates, primarily gums.     

Afadin: A Novel Actin Filament–binding Protein with One PDZ Domain Localized at Cadherin-based Cell-to-Cell Adherens Junction

A novel actin filament (F-actin)–binding protein with a molecular mass of ~205 kD (p205), which was concentrated at cadherin-based cell-to-cell adherens junction (AJ), was isolated and characterized. p205 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. p205 was a protein of 1,829 amino acids (aa) with a calculated molecular mass of 207,667 kD. p205 had one F-actin–binding domain at 1,631–1,829 aa residues and one PDZ domain at 1,016– 1,100 aa residues, a domain known to interact with transmembrane proteins. p205 was copurified from rat brain with another protein with a molecular mass of 190 kD (p190). p190 was a protein of 1,663 aa with a calculated molecular mass of 188,971 kD. p190 was a splicing variant of p205 having one PDZ domain at 1,009–1,093 aa residues but lacking the F-actin–binding domain. Homology search analysis revealed that the aa sequence of p190 showed 90% identity over the entire sequence with the product of the AF-6 gene, which was found to be fused to the ALL-1 gene, known to be involved in acute leukemia. p190 is likely to be a rat counterpart of human AF-6 protein. p205 bound along the sides of F-actin but hardly showed the F-actin–cross-linking activity. Northern and Western blot analyses showed that p205 was ubiquitously expressed in all the rat tissues examined, whereas p190 was specifically expressed in brain. Immunofluorescence and immunoelectron microscopic studies revealed that p205 was concentrated at cadherin-based cell-to-cell AJ of various tissues. We named p205 l-afadin (a large splicing variant of AF-6 protein localized at adherens junction) and p190 s-afadin (a small splicing variant of l-afadin). These results suggest that l-afadin serves as a linker of the actin cytoskeleton to the plasma membrane at cell-to-cell AJ.     

Methylmalonate-semialdehyde dehydrogenase is induced in auxin-stimulated and zinc-stimulated root formation in rice

Proteins induced in rice by auxin and zinc were determined by proteome analysis. Cultured suspension cells of rice were treated with 2,4-dichlorophenoxyacetic acid and ZnSO₄ and then proteins were separated by two-dimensional polyacrylamide gel electrophoresis; seven proteins were found to be induced by auxin and zinc. Of these seven, methylmalonate-semialdehyde dehydrogenase (MMSDH) was elevated by treatment with auxin alone. MMSDH was detected in cultured suspension cells, root and leaf sheath, but not in leaf blades. MMSDH responded to auxin and gibberellin, but did not respond to brassinolide and cytokinin. Furthermore, the amount of MMSDH in slr1, a constitutive gibberellin response mutant, was 2-fold that of wild type. MMSDH mRNA and protein were stimulated in root formation induced by auxin and/or zinc over a 4-week period. These results suggest that MMSDH may be necessary for root formation in rice induced by auxin and/or zinc.Abbreviations BA 6-Benzylaminopurine - BL Brassinolide - CBB Coomassie Brilliant Blue - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA Gibberellic acid - IAA Indole-3-acetic acid - MALDI-TOF MS Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry - MMSDH Methylmalonate-semialdehyde dehydrogenase - 2D-PAGE Two-dimensional polyacrylamide gel electrophoresis - PVDF Polyvinylidene difluoride     

Tropomyosin inhibits ADF/cofilin-dependent actin filament dynamics

Tropomyosin binds to actin filaments and is implicated in stabilization of actin cytoskeleton. We examined biochemical and cell biological properties of Caenorhabditis elegans tropomyosin (CeTM) and obtained evidence that CeTM is antagonistic to ADF/cofilin-dependent actin filament dynamics. We purified CeTM, actin, and UNC-60B (a muscle-specific ADF/cofilin isoform), all of which are derived from C. elegans, and showed that CeTM and UNC-60B bound to F-actin in a mutually exclusive manner. CeTM inhibited UNC-60B-induced actin depolymerization and enhancement of actin polymerization. Within isolated native thin filaments, actin and CeTM were detected as major components, whereas UNC-60B was present at a trace amount. Purified UNC-60B was unable to interact with the native thin filaments unless CeTM and other associated proteins were removed by high-salt extraction. Purified CeTM was sufficient to restore the resistance of the salt-extracted filaments from UNC-60B. In muscle cells, CeTM and UNC-60B were localized in different patterns. Suppression of CeTM by RNA interference resulted in disorganized actin filaments and paralyzed worms in wild-type background. However, in an ADF/cofilin mutant background, suppression of CeTM did not worsen actin organization and worm motility. These results suggest that tropomyosin is a physiological inhibitor of ADF/cofilin-dependent actin dynamics.     

Increased nitric oxide production by neutrophils from patients with chronic granulomatous disease on trimethoprim-sulfamethoxazole.

Chronic granulomatous disease (CGD) is an inherited disease characterized by severe and recurrent bacterial and fungal infections. Phagocytic cells of CGD patients are unable to produce superoxide anion, and their efficiency in bacterial killing is significantly impaired. In these patients, the prophylactic and therapeutic validity of a long-term use of trimethoprim-sulfamethoxazole (TMP-SMX) has been well established. However a role of nitric oxide (NO) produced by phagocytic cells from CGD patients is unknown, and the mechanism of TMP-SMX in CGD is unclear. We have directly measured NO production in whole human blood by using 4,5-diaminofluorescein as a novel fluorescent indicator for intracellular NO. Intracellular NO production of gated neutrophils increased time dependently when stimulated by lipopolysaccharide (LPS) and calcium ionophore. Although all polymorphonuclear leukocyte (PMN) specimens from patients with CGD failed to generate hydrogen peroxide, NO production by CGD PMNs was significantly increased compared with that of control PMNs (p<0.05). TMP-SMX with LPS significantly increased compared with LPS-stimulated samples at clinical (n=5, p<0.05) and 10-fold clinical concentrations (n=5, p<0.01). TMP-SMX with LPS in CGD PMNs significantly increased the production of NO in comparison with the LPS stimulation at 10-fold clinical concentrations (n=5, p<0.05). In conclusion, our data indicate the possibility that NO production by neutrophils from patients with CGD treated with TMP-SMX has a role of bactericidal activity instead of O(2)(-) in host defense mechanism.