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The primary structure of the second transmembrane (M2) segment of resistant to dieldrin (RDL), an ionotropic γ-aminobutyric acid receptor (GABAR) subunit, and the structure-function relationships in RDL are well conserved among insect species. An amino acid substitution at the 2' position in the M2 segment (Ala to Ser or Gly) confers resistance to non-competitive antagonists (NCAs) of GABARs. Here, a cDNA encoding RDL was cloned from the two-spotted spider mite Tetranychus urticae Koch. Unlike insect homologs, native TuRDL has His at the 2' position (H305) and Ile at 6' (I309) in the M2 segment and is insensitive to NCAs. Single and multiple mutations were introduced in the M2 segment of TuRDL, and the mutant proteins were expressed in Xenopus oocytes and examined for the restoration of sensitivity to NCAs. The sensitivity of a double mutant (H305A and I309T in the M2 segment) was greatly increased but was still considerably lower than that of insect RDLs. We therefore constructed chimeric RDLs consisting of TuRDL and Drosophila melanogaster RDL and examined their sensitivities to NCAs. The results show that the N-terminal region containing the Cys-loop as well as the M2 segment confers functional specificity; thus, our current understanding of the mechanism underlying NCA binding to GABARs requires reappraisal., (© 2020 International Society for Neurochemistry.)

Abnormal metabotropic glutamate receptor (mGluR) activity could cause brain disorders; however, its regulation has not yet been fully understood. Here, we report that protein kinase N1 (PKN1), a protein kinase expressed predominantly in neurons in the brain, normalizes group 1 mGluR function by upregulating a neuronal glutamate transporter, excitatory amino acid transporter 3 (EAAT3), and supports silent synapse activation. Knocking out PKN1a, the dominant PKN1 subtype in the brain, unmasked abnormal input-nonspecific mGluR-dependent long-term depression (mGluR-LTD) and AMPA receptor (AMPAR) silencing in the developing hippocampus. mGluR-LTD was mimicked by inhibiting glutamate transporters in wild-type mice. Knocking out PKN1a decreased hippocampal EAAT3 expression and PKN1 inhibition reduced glutamate uptake through EAAT3. Also, synaptic transmission was immature; there were more silent synapses and fewer spines with shorter postsynaptic densities in PKN1a knockout mice than in wild-type mice. Thus, PKN1 plays a critical role in regulation of synaptic maturation by upregulating EAAT3 expression.

Lateral root (LR) formation in Arabidopsis thaliana is initiated by asymmetric division of founder cells, followed by coordinated cell proliferation and differentiation for patterning new primordia. The sequential developmental processes of LR formation are triggered by a localized auxin response. LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16), an auxin-inducible transcription factor, is one of the key regulators linking auxin response in LR founder cells to LR initiation. We identified key genes for LR formation that are activated by LBD16 in an auxin-dependent manner. LBD16 targets identified include the transcription factor gene PUCHI, which is required for LR primordium patterning. We demonstrate that LBD16 activity is required for the auxin-inducible expression of PUCHI. We show that PUCHI expression is initiated after the first round of asymmetric cell division of LR founder cells and that premature induction of PUCHI during the preinitiation phase disrupts LR primordium formation. Our results indicate that LR initiation requires the sequential induction of transcription factors LBD16 and PUCHI., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

In plants, the position of lateral roots (LRs) depends on initiation sites induced by auxin. The domain of high auxin response responsible for LR initiation stretches over several cells, but only a pair of pericycle cells (LR founder cells) will develop into LRs. In this work, we identified a signaling cascade controlling LR formation through lateral inhibition. It comprises a peptide hormone TARGET OF LBD SIXTEEN 2 (TOLS2), its receptor RLK7, and a downstream transcription factor PUCHI. TOLS2 is expressed at the LR founder cells and inhibits LR initiation. Time-lapse imaging of auxin-responsive DR5:LUCIFERASE reporter expression revealed that occasionally two pairs of LR founder cells are specified in close proximity even in wild-type and that one of them exists only transiently and disappears in an RLK7-dependent manner. We propose that the selection of LR founder cells by the peptide hormone-receptor cascade ensures proper LR spacing., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Knock-in mice lacking PKN1 kinase activity were generated by introducing a T778A point mutation in the catalytic domain. PKN1[T778A] mutant mice developed to adulthood without apparent external abnormalities, but exhibited lower T and B lymphocyte counts in the peripheral blood than those of wild-type (WT) mice. T and B cell development proceeded in an apparently normal fashion in bone marrow and thymus of PKN1[T778A] mice, however, the number of T and B cell counts were significantly higher in the lymph nodes and spleen of mutant mice in those of WT mice. After transfusion into WT recipients, EGFP-labelled PKN1[T778A] donor lymphocytes were significantly less abundant in the peripheral circulation and more abundant in the spleen and lymph nodes of recipient mice compared with EGFP-labelled WT donor lymphocytes, likely reflecting lymphocyte sequestration in the spleen and lymph nodes in a cell-autonomous fashion. PKN1[T778A] lymphocytes showed significantly lower chemotaxis towards chemokines and sphingosine 1-phosphate (S1P) than WT cells in vitro. The biggest migration defect was observed in response to S1P, which is essential for lymphocyte egress from secondary lymphoid organs. These results reveal a novel role of PKN1 in lymphocyte migration and localization.

PKN, a conserved family member related to PKC, was the first protein kinase identified as a target of the small GTPase Rho. PKN is involved in various functions including cytoskeletal arrangement and cell adhesion. Furthermore, the enrichment of PKN3 mRNA in some cancer cell lines as well as its requirement in malignant prostate cell growth suggested its involvement in oncogenesis. Despite intensive research efforts, physiological as well as pathological roles of PKN3 in vivo remain elusive. Here, we generated mice with a targeted deletion of PKN3. The PKN3 knockout (KO) mice are viable and develop normally. However, the absence of PKN3 had an impact on angiogenesis as evidenced by marked suppressions of micro-vessel sprouting in ex vivo aortic ring assay and in vivo corneal pocket assay. Furthermore, the PKN3 KO mice exhibited an impaired lung metastasis of melanoma cells when administered from the tail vein. Importantly, PKN3 knock-down by small interfering RNA (siRNA) induced a glycosylation defect of cell-surface glycoproteins, including ICAM-1, integrin β1 and integrin α5 in HUVECs. Our data provide the first in vivo genetic demonstration that PKN3 plays critical roles in angiogenesis and tumor metastasis, and that defective maturation of cell surface glycoproteins might underlie these phenotypes.

Since its discovery in 1923, the biology of photoperiodism remains a mystery in many ways. We sought the link connecting the circadian system to an endocrine switch, using Antheraea pernyi. PER-, CLK- and CYC-ir were co-expressed in two pairs of dorsolateral neurons of the protocerebrum, suggesting that these are the circadian neurons that also express melatonin-, NAT- and HIOMT-ir. The results suggest that a melatonin pathway is present in the circadian neurons. Melatonin receptor (MT2 or MEL-1B-R)-ir in PTTH-ir neurons juxtaposing clock neurons suggests that melatonin gates PTTH release. RIA showed a melatonin rhythm with a peak four hours after lights off in adult brain both under LD16:8 (LD) and LD12:12 (SD), and both the peak and the baseline levels were higher under LD than SD, suggesting a photoperiodic influence. When pupae in diapause were exposed to 10 cycles of LD, or stored at 4 °C for 4 months under constant darkness, an increase of NAT activity was observed when PTTH released ecdysone. DNA sequence upstream of nat contained E-boxes to which CYC/CLK could bind, and nat transcription was turned off by clk or cyc dsRNA. dsRNA(NAT) caused dysfunction of photoperiodism. dsRNA(PER) upregulated nat transcription as anticipated, based on findings in the Drosophila melanogaster circadian system. Transcription of nat, cyc and clk peaked at ZT12. RIA showed that dsRNA(NAT) decreased melatonin while dsRNA(PER) increased melatonin. Thus nat, a clock controlled gene, is the critical link between the circadian clock and endocrine switch. MT-binding may release PTTH, resulting in termination of diapause. This study thus examined all of the basic functional units from the clock: a photoperiodic counter as an accumulator of mRNA(NAT), to endocrine switch for photoperiodism in A. pernyi showing this system is self-complete without additional device especially for photoperiodism.

Repetitive maternal deprivation (MD) of neonatal rats during early life is known as one of the strongest stressors to pre-weaned animals. There is increasing evidence that the cerebellum is involved in cognition and emotion. In the present study, we examined how neurotrophic factors and myelin-associated molecules and their receptors (NGF, BDNF, OMgp, TrkA, TrkB, p75 NTR, and NgR) in the cerebellum are affected by early postnatal maternal separation. Rat pups were separated from their mothers for 3h/day during postnatal days (PND) 10-15. At PND 16 and 30, the levels of mRNA and protein in the cerebellum were determined using real-time PCR and Western blot analysis. Cerebellar mRNA and protein levels of BDNF, TrkB, and OMgp were significantly increased in MD rats at PND 16. However, by PND 30 these variables normalized to control levels. In contrast, the levels of mRNA and protein for NGF, TrkA, p75 NTR, and NgR were unchanged at both ages examined. Transient enhancement of neurotrophic system and myelin-associated molecule expression may cause interference of normal development of the cerebellum such as precocious myelination, which may lead to functional and cognitive deficits later in life., (© 2013.)

Tobacco cytochrome P450 (CYP) 71AH11 metabolized the herbicide chlorotoluron, and its mRNA level was increased in tobacco culture cells by the treatment of 2,4-D. In order to clarify molecular mechanisms of induced gene expression of CYP71AH11 by herbicide treatment, a 1574-bp 5'-flanking region of CYP71AH11 was cloned, ligated to the reporter β-glucuronidase (GUS) gene, and then transformed into tobacco plants. The GUS activity in the transgenic tobacco plants was highly induced by bromoxynil treatment, followed by 2,4-D. Chlorotoluron was slightly increased the GUS activity. The bromoxynil-increased GUS activity was partially repressed by the antioxidants, suggesting that reactive oxygen species may be involved in activation of the 5'-flanking region of CYP71AH11 by bromoxynil treatment. Deletion and mutation assays showed that the region CD (-1281 to -770bp from the start codon of CYP71AH11) was important, but not sufficient, for response to bromoxynil. Electrophoretic mobility shift assays and southwestern blotting revealed that the sequences AAAAAG, and GAACAAAC and GAAAATTC in the CD region were important for interaction to the nuclear proteins of <30 and ≈75 kDa, respectively. Particularly, interaction between AAAAAG and <30 kDa proteins was increased by bromoxynil treatment. These results gave a cue for understanding the bromoxynil-induced gene expression of CYP71AH11, which may contribute to herbicide tolerance and selectivity in crop plants., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Cytochromes b561, novel transmembrane electron transport proteins residing in eukaryotic cells, have a number of common features including six transmembrane α-helices and two heme ligation sites. Our recent studies on recombinant Zea mays cytochrome b561 suggested that concerted proton/electron transfer mechanism was functioning in plant cytochromes b561 as well and that conserved Lys(83) on a cytosolic loop had important roles for ascorbate-binding and a succeeding electron transfer. In the present study, we conducted site-directed mutagenesis analyses on conserved Arg(72) and Tyr(71). Removal of a positive charge at Arg(72) did not affect significantly on the final heme reduction level with ascorbate as reductant. However, characteristic pH-dependent initial time-lag upon electron acceptance from ascorbate was completely lost for R72A and R72E mutants. Substitution of Tyr(71) with Ala or Phe affected both on the final heme reduction level and on the pH-dependent initial time-lag, causing acceleration of the electron transfer. These observations were interpreted as existence of specific interactions of Tyr(71) and Arg(72) with ascorbate. However, their mechanistic roles were distinctly different from that of Lys(83), as exemplified by K83A/Y71A double mutant, and might be related for expelling of monodehydroascorbate radical from the substrate-binding site to prevent a back-flow of electrons., (Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Victorin, the host-selective toxin produced by the fungus Cochliobolus victoriae, induces programmed cell death (PCD) in victorin-sensitive oat lines with characteristic features of animal apoptosis, such as mitochondrial permeability transition, chromatin condensation, nuclear DNA laddering and rRNA/mRNA degradation. In this study, we characterized a calcium-binding protein, namely AsALG-2, which might have a role in the victorin-induced PCD. AsALG-2 is homologous to the Apoptosis-Linked Gene ALG-2 identified in mammalian cells. Northern blot analysis revealed that the accumulation of AsALG-2 transcripts increased during victorin-induced PCD, but not during necrotic cell death. Salicylic acid, chitosan and chitin strongly activated the expression of general defence response genes, such as PR-10; however, neither induced cell death nor the accumulation of AsALG-2 mRNA. Pharmacological studies indicated that victorin-induced DNA laddering and AsALG-2 expression were regulated through similar pathways. The calcium channel blocker, nifedipine, moderately inhibited the accumulation of AsALG-2 mRNA during cell death. Trifluoperazine (calmodulin antagonist) and K252a (serine-threonine kinase inhibitor) reduced the victorin-induced phytoalexin accumulation, but did not prevent the victorin-induced DNA laddering or accumulation of AsALG-2 mRNA. Taken together, our investigations suggest that there is a calcium-mediated signalling pathway in animal and plant PCD in common., (© 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.)

Previously, the specific antibody-mediated persorption of antigenic molecules and particulates from the small-intestinal lumen into the peripheral blood was clarified in rats, but the intermediation of the receptor for the specific antibodies was not. In this study, the existence of receptor for the specific antibody was experimentally examined in the rat small intestine. Glutaraldehyde-fixed sheep erythrocytes (SEs) coated by Fc-fragments of IgG (IgG-Fc), (Fab')(2)-fragments of IgG (IgG-Fab) or bovine serum albumin (BSA), were injected into 3 jejunal loops each 2 cm in length in non-orally pre-immunized rats, respectively. Thirty minutes after the injection, IgG-Fc-coated SEs were significantly more engulfed by villous columnar epithelial cells than Fab- or BSA-coated SEs. The most frequent absorption sites were the intestinal villous apices. The IgG-Fc-coated SEs were adhered to the striated borders and were engulfed by villous columnar epithelial cells. IgG-Fc-coated SEs passing through the epithelial cells were also detected in the subepithelial blood capillaries just beneath the villous epithelium, but not in the connective tissue and the lymph vessels. These findings suggest that the absorption of luminal antigenic particulates is probably mediated by the Fc-receptor, and that the absorbed antigenic particulates are directly transferred to the hepatic portal blood by passing through the endothelium of the subepithelial blood capillaries.

Novel trigonal DNA-carbohydrate conjugates were prepared and evaluated to explore efficient carbohydrate-lectin interactions. Carbohydrate-modified oligonucleotides were enzymatically prepared, then hybridized to form 3-way junction DNAs. The thermal stabilities of the junctions were assessed by UV melting analysis and formation of constructs was confirmed by gel electrophoresis. Fluorescence titration assays revealed that the trigonal DNA-carbohydrate conjugates exhibit high affinity to lectins depending on the distribution of carbohydrates presented in each arm. These results suggest that self-assembled 3-way DNA architectures could offer a useful platform for controlling the spatial distribution of carbohydrates on conjugates and achieving more efficient molecular recognition., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Gamma-amino butyric acid (GABA) is a component of pharmaceuticals, functional foods, and the biodegradable plastic polyamide 4. Here, we report a simple and robust system to produce GABA from glucose using the recombinant Corynebacterium glutamicum strain GAD, which expresses GadB, a glutamate decarboxylase encoded by the gadB gene of Escherichia coli W3110. As confirmed by HPLC analysis, GABA fermentation by C. glutamicum GAD cultured at 30°C in GABA Production 1 (GP1) medium containing 50 g/L glucose without the addition of glutamate yielded 8.07 ± 1.53 g/L extracellular GABA after 96 h. Addition of 0.1mM pyridoxal 5'-phosphate (PLP) was found to enhance the production of GABA, whereas Tween 40 was unnecessary for GABA fermentation. Using the optimized GABA Production 2 (GP2) medium, which contained 50 g/L glucose and 0.1mM PLP, fermentation was performed in a flask at 30°C with 10% (v/v) seed culture of C. glutamicum GAD. GABA was produced in the culture supernatant with a yield of 12.37 ± 0.88 g/L after 72 h with a space-time yield of 0.172 g/L/h, which is the highest yield obtained to date for GABA from fermentation with glucose as a main carbon source., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Expression of mRNAs and proteins of ZO-1 and occludin was analyzed in pig oocytes and parthenogenetic diploid embryos during preimplantation development using real-time RT-PCR, western blotting and immunocytochemistry. All germinal vesicle (GV) and metaphase (M)II oocytes and preimplantation embryos expressed mRNAs and proteins of ZO-1 and occludin. mRNA levels of both ZO-1 and occludin decreased significantly from GV to MII, but increased at the 2-cell stage followed by temporal decrease during the early and late 4-cell stages. Then, both mRNAs increased after compaction. Relative concentration of zo1α- was highest in 2-cell embryos, while zo1α+ was expressed from the morula stage. Occludin expression greatly increased after the morula stage and was highest in expanded blastocysts. Western blotting analysis showed constant expression of ZO-1α- throughout preimplantation development and limited translation of ZO-1α+ from the blastocysts, and species-specific expression pattern of occludin. Immunocytochemistry analysis revealed homogeneous distribution of ZO-1 and occludin in the cytoplasm with moderately strong fluorescence in the vicinity of the contact region between blastomeres, around the nuclei in the 2-cell to late 4-cell embryos, and clear network localization along the cell-boundary region in embryos after the morula stage. Present results show that major TJ proteins, ZO-1 and occludin are expressed in oocytes and preimplantation embryos, and that ZO-1α+ is transcribed by zygotic gene activation and translated from early blastocysts with prominent increase of occludin at the blastocyst stage.

Coplanar polychlorinated biphenyls (Co-PCBs) consisting of non-ortho and mono-ortho-chlorinated PCBs are dioxin-like compounds and cause wide contamination in the environment. To monitor Co-PCB residues, it was attempted to establish an enzyme-linked immunosorbent assay (ELISA) with monoclonal and recombinant antibodies selective to Co-PCBs. When 3,3',5,5'-tetrachlorobiphenoxybutyric acid (PCBH)-keyhole limpet hemocyanin conjugate was immunized into mice, two monoclonal antibodies, Mab-0217 and Mab-4444, were obtained. 3,3',5,5'-Tetrachlorobiphenyl (PCB80) was determined with an IC(50) value of 2.6 and 0.46 ng mL(-1) in ELISA based on Mab-0217 and Mab-4444, respectively. Mab-4444 cross-reacted with Co-PCB congeners, except for PCB77 and PCB81. Mab-0217 reacted with PCB80 and cross-reacted with PCB111. A single-chain variable fragment (scFv) antibody derived from Mab-4444 was produced in recombinant Escherichia coli cells. The scFv antibody showed nearly the same sensitivity toward PCBH as the parent monoclonal antibody in ELISA. These results clearly suggested that Mab-4444 and its scFv antibodies were suitable for monitoring the representative congeners of Co-PCBs.

Protein dynamics of human adult hemoglobin (HbA) upon ligand photolysis of oxygen (O(2)) and carbon monoxide (CO) was investigated using time-resolved resonance Raman (TR(3)) spectroscopy. The TR(3) spectra of the both photoproducts at 1-ns delay differed from that of the equilibrium deligated form (deoxy form) in the frequencies of the iron-histidine stretching [ν(Fe-His)] and methine wagging (γ(7)) modes, and the band intensity of pyrrole stretching and substituent bending (ν(8)) modes. Spectral changes of the O(2) photoproduct in the submicrosecond region were faster than those of the CO photoproduct, indicating that the structural dynamics following the photodissociation is ligand dependent for HbA. In contrast, no ligand dependence of the dynamics was observed for myoglobin, which has a structure similar to that of the subunit of HbA. The structural dynamics and relevance to the functionality of HbA also are discussed., (© 2012 American Chemical Society)

Aquatic humic substances (AHSs) are major constituents of dissolved organic matter (DOM) in freshwater, where they perform a number of important ecological and geochemical functions, yet no method exists for quantifying all AHSs. We have developed a method for the quantitative analysis of AHSs based on their carbon concentration. Our approach includes: (1) the development of techniques for clear-water samples with low AHS concentrations, which normally complicate quantification; (2) avoiding carbon contamination in the laboratory; and (3) optimizing the AHS adsorption conditions.

A taxonomic study of the genus Padina from Japan, Southeast Asia, and Hawaii based on morphology and gene sequence data (rbcL and cox3) resulted in the recognition of four new species, that is, Padina macrophylla and Padina ishigakiensis from Ryukyu Islands, Japan; Padina maroensis from Hawaii; and Padina usoehtunii from Myanmar and Thailand. All species are bistratose and morphologically different from one another as well as from any known taxa by a combination of characters relating to degree of calcification; the structure, position, and arrangement of hairlines (HLs) and reproductive sori; and the presence or absence of rhizoid-like groups of hairs and an indusium. Molecular phylogenetic analyses demonstrated a close relationship between P. ishigakiensis, P. macrophylla, P. maroensis, and Padina australis Hauck. The position of P. usoehtunii, however, was not fully resolved, being either sister to a clade comprising the other three new species and P. australis in the rbcL tree or more closely related to a clade comprising several other recently described species in the cox3 tree. The finding of the four new species demonstrates high species diversity particularly in southern Japan. The following characters were first recognized here to be useful for species delimitation: the presence or absence of small rhizoid-like groups of hairs on the thallus surface, structure and arrangement of HLs on both surfaces either alternate or irregular, and arrangement of the alternating HLs between both surfaces in equal or unequal distance. The evolutionary trajectory of these and six other morphological characters used in species delineation was traced on the phylogenetic tree., (© 2011 Phycological Society of America.)

High performance size-exclusion chromatography (HPSEC) is useful for the molecular size separation of soil humic acids (HAs), but there is no method available for various HAs with different chemical properties. In this paper the authors propose a new preparative HPSEC method for various soil HAs. Three soil HAs with different chemical properties were fractionated by a Shodex OHpak SB-2004 HQ column with 10mM sodium phosphate buffer (pH 7.0)/acetonitrile (3:1, v/v) as an eluent. The HAs eluted within a reasonable column range time (12-25 min) without peak tailing. Preparative HPSEC chromatograms of these HAs indicated that non-size-exclusion effects were suppressed. The separated fractions were analyzed by HPSEC to determine their apparent molecular weights. These decreased sequentially from fraction 1 to fraction 10, suggesting that the HAs had been separated by their molecular size. The size-separated fractions of the soil HA were mixed to compare them with unfractionated HA. The analytical HPSEC chromatogram of the mixed HA was almost identical to that of the unfractionated HA. It appears that the HAs do not adsorb specifically to the column during preparative HPSEC. Our preparative HPSEC method allows for rapid and reproducible separation of various soil HAs by molecular size., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The hypothesis that apoptotic factors play some roles in the denucleation of erythroblasts has been confirmed by the immunohistological detection of both phosphatidylserine and thrombospondin as phagocytosis-inducing factors in general apoptotic events. Both phosphatidylserine and thrombospondin were detected on the surface of cell membrane of mature erythroblasts, while thrombospondin was also detected in more immature erythroblasts. The intensities of their immune reactions increased as the erythroids matured. During denucleation, the positivities of both phosphatidylserine and thrombospondin were restricted on the surface of the cell membrane surrounding the protruding nuclei. Thus, the apoptotic process involves denucleation of erythroblasts and phosphatidylserine, and thrombospondin acts as phagocytosis-inducing factors in the denucleation event.

Five hundred and fifty fish samples from various stages in the course of distribution in Hyogo Prefecture (209 retailed in super markets, 173 obtained from fishery cooperatives at a harbor, 91 caught by trawling and 77 caught by rod fishing) were examined for contamination with Staphylococcus aureus (S. aureus). S. aureus was detected in 41 (19.6%) of the retail fish samples and 46 (26.6%) of the samples from the fishery cooperatives. No S. aureus was isolated from the live fish (91 trawled and 77 fished by rod). With regard to the retail fish, the contamination rate of processed fish (26.0%) was significantly higher than that of unprocessed fish (14.2%). For 88 samples, the efficacy of the selective medium was compared using Baird-Parker agar and mannitol salt agar supplemented with egg yolk (MSEY agar) by the direct plate and enrichment culture methods. Using the direct culture method, the S. aureus positive rate with the Baird-Parker agar (30.7%) was significantly higher (P<0.01) than that with the MSEY agar (6.8%). The enrichment culture method remarkably raised the S. aureus detection rate. Seventy-eight (85.7%) of 91 isolates belonged to the human ecovar. Sixty-two (68.1%) of the 91 isolates had some enterotoxin genes, including 44 (48.4%) with the sea gene. These data showed that the fish were contaminated with S. aureus after landing and that Baird-Parker agar had an advantage in detecting S. aureus with a direct plate culture.

Surfaces of the most luminal positions of mucosae are fundamental settlement sites of indigenous bacteria throughout the rat alimentary tract. In these positions, also epithelial cell-shedding sites, the special sugar expression in the glycocalyx is very important as it provides possible ligands of bacterial lectins for attachment to epithelial cells. Therefore, the sugar expression in glycocalyx of epithelial cells was lectin-histochemically surveyed using 21 lectins throughout the rat alimentary tract. From the tongue to the nonglandular part of the stomach, α-D-Man, α-D-Glc and α-D-GalNAc were detected on the surface of the keratinized stratified squamous epithelium. In the glandular part of the stomach, α-D-Man, β-D-Gal-4GlcNAc, D-Gal, D-GalNAc, D-GlcNAc, α-L-Fuc- α-D-Gal-β(1-4)GlcNAc and bisected triantennary N-glycans were detected on the surface of gastric superficial epithelial cells. From the duodenum to the ileum, (GlcNAc)(2-4) was expressed exclusively on the epithelial cells in the apical portions of the intestinal villi. From the cecum to the rectum, α-D-Man, β-D-Gal-4GlcNAc, D-Gal, D-GalNAc, α-D-Gal(1-3)D-GalNAc, (GalNAc)(n) and NeuNAc were expressed on the intestinal superficial epithelial cells. These results suggest that special sugars are expressed on the most luminal portions of mucosae as exclusive epithelial cell-shedding sites, and that sugar expression differs among the various segments of the alimentary tract. These site differences might reflect differences in resident bacterial species in the rat alimentary tract.