Dual Mechanisms of Ca(2+) Increases Elicited by N-methyl-D-aspartate in Immature and Mature Cultured Cortical Neurons

Journal of Neuroscience Research. Jan, 2002  |  Pubmed ID: 11782971

Cortical primary cultures were loaded with the fluorescent indicator fluo-3 for assessment of intracellular-free Ca(2+) ions with the aid of a confocal laser-scanning microscope. The addition of N-methyl-D-aspartic acid (NMDA) markedly increased the number of fluorescent cells in a manner sensitive to prevention by both an NMDA channel blocker and MgCl(2). In the absence of added MgCl(2), NMDA induced a sustained increase in the number of fluorescent cells with a transient increase by KCl in cells cultured for 3 days in vitro (DIV). Both nifedipine and dantrolene were more potent in preventing the increase by NMDA in cortical preparations cultured for 9 DIV than those for 3 DIV. These results suggest that activation of NMDA receptors may lead to a sustained increase in intracellular-free Ca(2+) concentrations in immature cultured neurons, in a manner less dependent on the influx through L-type voltage-dependent channels as well as the release from intracellular stores than in mature neurons.

Essential Role of S-adenosylmethionine Decarboxylase in Mouse Embryonic Development

Genes to Cells : Devoted to Molecular & Cellular Mechanisms. Jan, 2002  |  Pubmed ID: 11856372

S-Adenosylmethionine decarboxylase (AdoMetDC) is one of the key enzymes involved in the biosynthesis of spermidine and spermine, which are essential for normal cell growth. To examine the role of polyamines in embryogenesis, we carried out targeted disruption of the mouse Amd1 gene, encoding AdoMetDC, to generate mice that can not synthesize spermidine and spermine.

The Polarized Epithelia-specific Mu 1B-adaptin Complements Mu 1A-deficiency in Fibroblasts

EMBO Reports. May, 2002  |  Pubmed ID: 11964383

The heterotetrameric AP-1A adaptor complex of clathrin-coated vesicles is ubiquitously expressed. The mu 1-adaptin subunit of the complex exists as the ubiquitous mu 1A and the polarized epithelia-specific mu 1B, which are 80% identical. In polarized epithelia, mu 1B is incorporated into the AP-1B complex, which is required for basolateral plasma membrane sorting of the low-density lipoprotein receptor. Binding of AP-1B to subdomains of the trans-Golgi network (TGN) appears to be part of the mechanism by which protein sorting is mediated. We expressed mu 1B in mu 1A-deficient fibroblasts to test for mu 1B function in non-polarized cells. AP-1B complexes were formed and bound to the TGN and to endosomes. Moreover, AP-1B restored the AP-1A-dependent sorting of mannose 6-phosphate receptors between endosomes and the TGN. This demonstrates that mu 1A and mu 1B do have overlapping sorting functions and indicates that AP-1A and AP-1B mediate protein sorting along parallel pathways between the TGN and endosomes in polarized epithelia.

Differential Recognition of Tyrosine-based Basolateral Signals by AP-1B Subunit Mu1B in Polarized Epithelial Cells

Molecular Biology of the Cell. Jul, 2002  |  Pubmed ID: 12134076

To investigate the importance of tyrosine recognition by the AP-1B clathrin adaptor subunit mu1B for basolateral sorting of integral membrane proteins in polarized epithelial cells, we have produced and characterized a mutant form of mu1B. The mutant (M-mu1B) contains alanine substitutions of each of the four conserved residues, which in the AP-2 adaptor subunit micro2 are critical for interacting with tyrosine-based endocytosis signals. We show M-mu1B is defective for tyrosine binding in vitro, but is nevertheless incorporated into AP-1 complexes in transfected cells. Using LLC-PK1 cells expressing either wild type or M-mu1B, we find that there is inefficient basolateral expression of membrane proteins whose basolateral targeting signals share critical tyrosines with signals for endocytosis. In contrast, membrane proteins whose basolateral targeting signals are distinct from their endocytosis signals (transferrin and low-density lipoprotein receptors) accumulate at the basolateral domain normally, although in a manner that is strictly dependent on mu1B or M-mu1B expression. Our results suggest that mu1B interacts with different classes of basolateral targeting signals in distinct ways.

Neuronal Leucine-rich Repeat Protein-3 Amplifies MAPK Activation by Epidermal Growth Factor Through a Carboxyl-terminal Region Containing Endocytosis Motifs

The Journal of Biological Chemistry. Nov, 2002  |  Pubmed ID: 12297494

Neuronal leucine-rich repeat protein-3 (NLRR-3) belongs to the LRR superfamily. Expression of rat NLRR-3 gene isolated from c-Ha-ras transgenic rat tumor is regulated mainly through the Ras-MAPK signaling pathway. NLRR-3 was found to enhance phosphorylation of MAPK when COS-7 cells were transfected with NLRR-3 and stimulated with a low concentration (0.01 ng/ml) of epidermal growth factor (EGF), but the amplification of MAPK phosphorylation by NLRR-3 was no longer observed when the carboxyl-terminal 30 amino acid stretch containing clathrin-mediated endocytosis motifs was deleted. A green fluorescent protein-tagged NLRR-3 localized at the plasma membrane was efficiently internalized in COS-7 cells, but internalization of a carboxyl-terminal-deleted version (NLRRDeltaC) was less efficient. The presence of clathrin-adaptor protein complexes containing NLRR-3 in brain lysate was confirmed by immunoprecipitation and glutathione S-transferase pull-down experiments, and affinity column chromatography revealed that the carboxyl-terminal region of NLRR-3 interacts with beta-adaptin. We propose that NLRR-3 potentiates Ras-MAPK signaling by facilitating internalization of EGF in clathrin-coated vesicles.

Blockade by Ferrous Iron of Ca2+ Influx Through N-methyl-D-aspartate Receptor Channels in Immature Cultured Rat Cortical Neurons

Journal of Neurochemistry. Oct, 2002  |  Pubmed ID: 12358723

Rat cortical neurons cultured for 3 days in vitro were loaded with the fluorescent indicator fluo-3 for assessment of intracellular free calcium ion (Ca2+) concentrations with the aid of a confocal laser-scanning microscope. In the absence of added MgCl2, the addition of NMDA induced a rapid but sustained increase in the number of fluorescent neurons in a concentration-dependent manner at a concentration range of 1-100 micro m with the increase by KCl being transient. The addition of FeCl2, but not FeCl3, markedly inhibited the increase by NMDA in a reversible manner at concentrations of 10-200 micro m, without affecting that by KCl. Extensive analyses revealed clear differentiation between inhibitions by ferrous iron and other channel blockers known to date. The inhibition by FeCl2 was completely prevented by the addition of two different iron chelators. Exposure to NMDA alone did not lead to cell death in immature cultured neurons, however, while further addition of FeCl2 invariably induced neuronal cell death 24 h after exposure. These results give support to our previous proposal that NMDA receptor complex may contain a novel site sensitive to blockade by ferrous iron in rat brain.

Recording Site Dependence of the Neuronal Spiking Statistics

Bio Systems. Oct-Dec, 2002  |  Pubmed ID: 12459306

Spiking characteristics of neurons in the middle temporal (MT) area and the medial superior temporal (MST) area in the visual cortex of a monkey are compared with the ones in the principal sulcus (PS) area in the prefrontal cortex. The comparison is based on the basis of three inter-spike interval statistical measures: the coefficient of variation (CV), the skewness coefficient (SK) and the correlation coefficient of consecutive intervals (COR). Even for the spike sequences recorded from the same neuron, three coefficients computed from 100 intervals do not always exhibit similar values, but distribute rather widely. The distribution of three coefficients obtained from a single neuron in the MST area does not largely deviate from the distribution obtained from multiple neurons in MT and MST areas. Those distributions, however, largely deviate from the distribution obtained from neurons in the PS area. In this way, the distribution of those statistical coefficients reflects the nature of the recording site.

Alternative Splicing Regulates the Subcellular Localization of Divalent Metal Transporter 1 Isoforms

Molecular Biology of the Cell. Dec, 2002  |  Pubmed ID: 12475959

Divalent metal transporter 1 (DMT1) is responsible for dietary-iron absorption from apical plasma membrane in the duodenum and iron acquisition from the transferrin cycle endosomes in peripheral tissues. Two isoforms of the DMT1 transcript generated by alternative splicing of the 3' exons have been identified in mouse, rat, and human. These isoforms can be distinguished by the different C-terminal amino acid sequences and by the presence (DMT1A) or absence (DMT1B) of an iron response element located in the 3' untranslated region of the mRNA. However, it has been still unknown whether the structural differences between the two DMT1 isoforms is functionally important. Here, we report that each DMT1 isoform exhibits a differential cell type-specific expression patterns and distinct subcellular localizations. DMT1A is predominantly expressed by epithelial cell lines, whereas DMT1B is expressed by the blood cell lines. In HEp-2 cells, GFP-tagged DMT1A is localized in late endosomes and lysosomes, whereas GFP-tagged DMT1B is localized in early endosomes. Using site-directed mutagenesis, a Y(555)XLXX sequence in the cytoplasmic tail of DMT1B has been identified as an important signal sequence for the early endosomal-targeting of DMT1B. In polarized MDCK cells, GFP-tagged DMT1A and DMT1B are localized in the apical plasma membrane and their respective specific endosomes. Disruption of the N-glycosylation sites in each of the DMT1 isoforms affects their polarized distribution into the apical plasma membrane but not their correct endosomal localization. Our data indicate that the cell type-specific expression patterns and the distinct subcellular localizations of two DMT1 isoforms may be involved in the different iron acquisition steps from the subcellular membranes in various cell types.

Solid-phase Synthesis of Naltrindole Derivatives Using Fischer Indole Synthesis Based on One-pot Release and Cyclization Methodology

Organic Letters. Apr, 2003  |  Pubmed ID: 12688708

[reaction: see text] We describe a new approach for the solid-phase synthesis of indoles 1 that involves a one-pot release and cyclization reaction of a solid-supported hydrazone through a Wang-type linker. Using this solid-phase methodology, we accomplished the synthesis of 40 naltrindole derivatives.

Identification of a Five-pass Transmembrane Protein Family Localizing in the Golgi Apparatus and the ER

Biochemical and Biophysical Research Communications. Dec, 2003  |  Pubmed ID: 14680843

A family of five-pass transmembrane proteins (FinGERs) were identified from the protein sequence database. The family includes yeast Yip1p, Yip4p, Yip5p, and Yif1p, and also their plant, insects, nematode, and mammalian homologues, suggesting their conserved function in a broad range of species. Eight family members were found in human. Multiple sequence alignment revealed three regions conserved among all family members. All of the human family members were expressed widely in various tissues. The human proteins were localized in and around the Golgi apparatus and may also be in the ER to some extent. The Golgi apparatus was fragmented by overexpression of the five of the family members. Some of the members were found to interact by yeast two-hybrid analysis, suggesting the formation of a complex. These results suggest that FinGERs function in maintenance of the Golgi structure and/or transport between the ER and the Golgi apparatus.

Membrane Traffic in the Post-Golgi Network: Toward a Better Understanding of the Higher Order Functioning Systems

Cell Structure and Function. Oct, 2003  |  Pubmed ID: 14745132

Adaptor Protein Complexes As the Key Regulators of Protein Sorting in the Post-Golgi Network

Cell Structure and Function. Oct, 2003  |  Pubmed ID: 14745134

Adaptor protein (AP) complexes are cytosolic heterotetramers that mediate the sorting of membrane proteins in the secretory and endocytic pathways. AP complexes are involved in the formation of clathrin-coated vesicles (CCVs) by recruiting the scaffold protein, clathrin. AP complexes also play a pivotal role in the cargo selection by recognizing the sorting signals within the cytoplasmic tail of integral membrane proteins. Six distinct AP complexes have been identified. AP-2 mediates endocytosis from the plasma membrane, while AP-1, AP-3 and AP-4 play a role in the endosomal/lysosomal sorting pathways. Moreover, tissue-specific sorting events such as the basolateral sorting in polarized epithelial cells and the biogenesis of specialized organelles including melanosomes and synaptic vesicles are also regulated by members of AP complexes. The application of a variety of methodologies have gradually revealed the physiological role of AP complexes.

Dynamics of Golgi Matrix Proteins After the Blockage of ER to Golgi Transport

Journal of Biochemistry. Feb, 2004  |  Pubmed ID: 15047722

When the ER to Golgi transport is blocked by a GTP-restricted mutant of Sar1p (H79G) in NRK-52E cells, most Golgi resident proteins are transported back into the ER. In contrast, the cis-Golgi matrix proteins GM130 and GRASP65 are retained in punctate cytoplasmic structures, namely Golgi remnants. Significant amounts of the medial-Golgi matrix proteins golgin-45, GRASP55 and giantin are retained in the Golgi remnants, but a fraction of these proteins relocates to the ER. Golgin-97, a candidate trans-Golgi network matrix protein, is retained in Golgi remnant-like structures, but mostly separated from GM130 and GRASP65. Interestingly, most Sec13p, a COPII component, congregates into larger cytoplasmic clusters soon after the microinjection of Sar1p(H79G), and these move to accumulate around the Golgi apparatus. Sec13p clusters remain associated with Golgi remnants after prolonged incubation. Electron microscopic analysis revealed that Golgi remnants are clusters of larger vesicles with smaller vesicles, many of which are coated. GM130 is mainly associated with larger vesicles and Sec13p with smaller coated vesicles. The Sec13p clusters disperse when p115 binding to the Golgi apparatus is inhibited. These results suggest that cis-Golgi matrix proteins resist retrograde transport flow and stay as true residents in Golgi remnants after the inhibition of ER to Golgi transport.

Reduction of SNAP25 in Acid Secretion Defect of Foxl1-/- Gastric Parietal Cells

Biochemical and Biophysical Research Communications. Jul, 2004  |  Pubmed ID: 15240114

Foxl1 is a winged helix transcription factor expressed in the mesenchyme of the gastrointestinal tract. In the absence of Foxl1, parietal cells fail to secrete gastric acid in response to various secretagogue stimuli including cAMP. A marked decrease in H+,K(+)-ATPase expression was observed even though a substantial number of parietal cells still existed in Foxl1-deficient mice. Ultrastructural analysis suggested that the gastric acid secretion defect in Foxl1-deficient mice is mainly due to impairment in the fusion of cytoplasmic tubulovesicular structures to the apical canalicular plasma membrane. Among the molecules involved in the membrane fusion event, only SNAP25 showed a significant decrease in mRNA expression, which likely caused the impairment in acid secretion from parietal cells in Foxl1-deficient mice, with the reduction in H+,K(+)-ATPase expression contributing to additional effect.

Retinal Target Cells of the Centrifugal Projection from the Isthmo-optic Nucleus

The Journal of Comparative Neurology. Aug, 2004  |  Pubmed ID: 15248195

Although the avian retina has long been known to receive projection from a midbrain nucleus, the isthmo-optic nucleus (ION), the output of its target cells has remained obscure. We labeled the isthmo-optic (IO) terminals in the Japanese quail retina, by using anterograde transport of fluorescent tracer injected into the ION, and then labeled target cells for these terminals by means of intracellular tracer injection under direct microscopic observation. Somata of the IO target cells (IOTCs) lie in the innermost zone of the inner nuclear layer of the ventral half of the retina and have no dendrites but an axon. The axons run in the inner plexiform layer (IPL) for up to 6 mm and terminate densely in a round or elliptical terminal field, about 90-290 microm in diameter, of the outermost zone of the IPL. Longer axons (> 2 mm) extend dorsally, but shorter ones (< 1 mm) project ventrally or horizontally, so the terminals are distributed widely in both dorsal and ventral halves of the retina. The IOTCs cannot be classified into any of the five conventional major classes of retinal cells, including amacrine cells, and are thought to be "slave" neurons whose output is controlled by the neurons in the brain. Topographic separation between input to and output from the IOTCs by the axons might be essential for the overall topographic organization of the centrifugal visual system in birds.

Defective Function of GABA-containing Synaptic Vesicles in Mice Lacking the AP-3B Clathrin Adaptor

The Journal of Cell Biology. Oct, 2004  |  Pubmed ID: 15492041

AP-3 is a member of the adaptor protein (AP) complex family that regulates the vesicular transport of cargo proteins in the secretory and endocytic pathways. There are two isoforms of AP-3: the ubiquitously expressed AP-3A and the neuron-specific AP-3B. Although the physiological role of AP-3A has recently been elucidated, that of AP-3B remains unsolved. To address this question, we generated mice lacking mu3B, a subunit of AP-3B. mu3B-/- mice suffered from spontaneous epileptic seizures. Morphological abnormalities were observed at synapses in these mice. Biochemical studies demonstrated the impairment of gamma-aminobutyric acid (GABA) release because of, at least in part, the reduction of vesicular GABA transporter in mu3B-/- mice. This facilitated the induction of long-term potentiation in the hippocampus and the abnormal propagation of neuronal excitability via the temporoammonic pathway. Thus, AP-3B plays a critical role in the normal formation and function of a subset of synaptic vesicles. This work adds a new aspect to the pathogenesis of epilepsy.

Interaction of a Farnesylated Protein with Renal Type IIa Na/Pi Co-transporter in Response to Parathyroid Hormone and Dietary Phosphate

The Biochemical Journal. Feb, 2004  |  Pubmed ID: 14558883

Treatment with PTH (parathyroid hormone) or a high-P(i) diet causes internalization of the type IIa sodium-dependent phosphate (Na/P(i) IIa) co-transporter from the apical membrane and its degradation in the lysosome. A dibasic amino acid motif (KR) in the third intracellular loop of the co-transporter is essential for protein's PTH-induced retrieval. To elucidate the mechanism of internalization of Na/P(i) IIa, we identified the interacting protein for the endocytic motif by yeast two-hybrid screening. We found a strong interaction of the Na/P(i) IIa co-transporter with a small protein known as the PEX19 (human peroxisomal farnesylated protein; PxF, Pex19p). PEX19 can bind to the KR motif, but not to a mutant with this motif replaced with NI residues. PEX19 is highly expressed in mouse and rat kidney. Western blot analysis indicates that PEX19 is located in the cytosolic and brush-border membrane fractions (microvilli and the subapical component). Overexpression of PEX19 stimulated the endocytosis of the Na/P(i) IIa co-transporter in opossum kidney cells in the absence of PTH. In conclusion, the present study indicates that PEX19 may be actively involved in controlling the internalization and trafficking of the Na/P(i) IIa co-transporter.

Oral Administration of Bifidobacterium Bifidum G9-1 Suppresses Total and Antigen Specific Immunoglobulin E Production in Mice

Biological & Pharmaceutical Bulletin. Aug, 2005  |  Pubmed ID: 16079493

Recent studies have suggested that oral bacteriotherapy with probiotics might be useful in the management of allergic diseases. We investigated the effect of oral administration of Bifidobacterium bifidum G9-1 (BBG9-1) on immunoglobulin (Ig) E production in BALB/c mice. Live BBG9-1 was orally administered to mice for 2 weeks from 1 week before ovalbumin (OVA)-immunization. The treatment of BBG9-1 significantly reduced serum total IgE level. In addition, BBG9-1 significantly and largely reduced the serum level of OVA-specific IgE without lowering of the specific IgG1 and increasing of the specific IgG2a. We also examined T helper type (Th) 1 and Th2 cytokine production from OVA-immunized splenocytes by restimulation with OVA in vitro. Productions of interferon (IFN)-gamma, interleukin (IL)-4 and IL-5 from the splenocytes of mice given BBG9-1 were weaker than those of control mice. We conclude that oral administration of BBG9-1 selectively and powerfully suppresses total and antigen specific IgE production in mice. It is suggested that BBG9-1 is useful for the prophylactic treatment in IgE-dependent allergic diseases.

Magnetic Field Generation from Cosmological Perturbations

Physical Review Letters. Sep, 2005  |  Pubmed ID: 16197062

In this Letter, we discuss the generation of magnetic field from cosmological perturbations. We consider the evolution of three component plasma (electron, proton, and photon) evaluating the collision term between electrons and photons up to the second order. The collision term is shown to induce electric current, which then generates magnetic field. There are three contributions, two of which can be evaluated from the first-order quantities, while the other one is fluid vorticity, which is purely second order. We estimate the magnitudes of the former contributions and show that the amplitude of the produced magnetic field is about approximately 10(-19) G at 10 Mpc comoving scale at the decoupling. Compared to astrophysical and inflationary mechanisms for seed-field generation, our study suffers from much less ambiguities concerning unknown physics and/or processes.

Clathrin Adaptor AP-2 is Essential for Early Embryonal Development

Molecular and Cellular Biology. Nov, 2005  |  Pubmed ID: 16227583

The heterotetrameric adaptor protein (AP) complexes AP-1, AP-2, AP-3, and AP-4 play key roles in transport vesicle formation and cargo sorting in post-Golgi trafficking pathways. Studies on cultured mammalian cells have shown that AP-2 mediates rapid endocytosis of a subset of plasma membrane receptors. To determine whether this function is essential in the context of a whole mammalian organism, we carried out targeted disruption of the gene encoding the mu2 subunit of AP-2 in the mouse. We found that mu2 heterozygous mutant mice were viable and had an apparently normal phenotype. In contrast, no mu2 homozygous mutant embryos were identified among blastocysts from intercrossed heterozygotes, indicating that mu2-deficient embryos die before day 3.5 postcoitus (E3.5). These results indicate that AP-2 is indispensable for early embryonic development, which might be due to its requirement for cell viability.

Distinct Gene Expression Profiles Characterize Cellular Phenotypes of Follicle-associated Epithelium and M Cells

DNA Research : an International Journal for Rapid Publication of Reports on Genes and Genomes. 2005  |  Pubmed ID: 16303744

Follicle-associated epithelium (FAE) covering Peyer's patches contains specialized epithelial M cells that take up ingested macromolecules and microorganisms from the lumen of the gut by transcytosis. Using high-density oligonucleotide microarrays, we analyzed the gene expression profiles of FAE and M cells in order to characterize their cellular phenotypes. The microarray data revealed that, among approximately 14,000 genes, 409 were expressed in FAE at twofold or higher levels compared to the intestinal epithelial cells (IECs) of the villi. These included genes involved in membrane traffic, host defense and transcriptional regulation, as well as uncharacterized genes. Real-time PCR and in situ hybridization analyses identified three molecules, ubiquitin D (Ub-D), tumor necrosis factor receptor superfamily 12a (TNFRsf12a), and transmembrane 4 superfamily 4 (Tm4sf4), which were predominantly distributed throughout FAE, but were expressed little, if at all, in IECs. By contrast, transcripts of secretory granule neuroendocrine protein 1 (Sgne-1) were scattered in FAE, and were co-localized with Ulex europaeus agglutinin-1 (UEA-1)-positive cells. This clearly suggests that expression of Sgne-1 in the gut is specific to M cells. Such a unique pattern of gene expression distinguishes FAE and M cells from IECs, and may reflect their cellular phenotype(s) associated with specific functional features.

The Membrane-bound Chemokine CXCL16 Expressed on Follicle-associated Epithelium and M Cells Mediates Lympho-epithelial Interaction in GALT

Journal of Immunology (Baltimore, Md. : 1950). Jan, 2006  |  Pubmed ID: 16365394

The recently identified CXCL16 has dual functions as a transmembrane adhesion molecule and a soluble chemokine. In this study we found that CXCL16 mRNA and protein were expressed constitutively on the follicle-associated epithelium covering Peyer's patches (PPs), isolated lymphoid follicles, and cecal patches, but minimally on the villous epithelium in the murine gastrointestinal tract. The CXCL16 receptor CXCR6/Bonzo was constitutively expressed on subpopulations of CD4+ and CD8+ T cells isolated from PPs. The expression of CXCR6/Bonzo on the PP T cells was up-regulated after stimulation with anti-CD3 and anti-CD28 mAbs. The activated PP T cells showed chemotactic migration in response to the soluble N-terminal chemokine domain of CXCL16. Furthermore, the activated PP T cells selectively adhered to cells expressing murine CXCL16. To determine the physiological role of CXCL16 in GALT, we first carefully analyzed T cell distribution in PPs. T cells localized not only in the interfollicular region but also at a lesser frequency in the subepithelial dome (SED) and in the germinal center of lymphoid follicles. Consistently, the majority of the adoptive transferred activated T cells migrated into the SED and the interfollicular region. However, the neutralization of CXCL16 specifically reduced the migration of the adoptive, transferred, activated T cells into the SED of PPs. These data suggest that CXCL16 expressed on the follicle-associated epithelium plays an important role in the recruitment and retention of activated T cells in the SED and should, at least partially, be responsible for lymphocyte compartmentalization in GALT.

Cosmological Magnetic Field: a Fossil of Density Perturbations in the Early Universe

Science (New York, N.Y.). Feb, 2006  |  Pubmed ID: 16400110

The origin of the substantial magnetic fields that are found in galaxies and on even larger scales, such as in clusters of galaxies, is yet unclear. If the second-order couplings between photons and electrons are considered, then cosmological density fluctuations, which explain the large-scale structure of the universe, can also produce magnetic fields on cosmological scales before the epoch of recombination. By evaluating the power spectrum of these cosmological magnetic fields on a range of scales, we show here that magnetic fields of 10(-18.1) gauss are generated at a 1-megaparsec scale and can be even stronger at smaller scales (10(-14.1) gauss at 10 kiloparsecs). These fields are large enough to seed magnetic fields in galaxies and may therefore have affected primordial star formation in the early universe.

Foxl1-deficient Mice Exhibit Aberrant Epithelial Cell Positioning Resulting from Dysregulated EphB/EphrinB Expression in the Small Intestine

American Journal of Physiology. Gastrointestinal and Liver Physiology. Jul, 2006  |  Pubmed ID: 16469829

The winged helix transcription factor Foxl1, expressed in the gut mesenchyme, regulates epithelial cell proliferation and differentiation through the Wnt/beta-catenin pathway. To better understand the role of Foxl1 in epithelial morphogenesis, we examined the tissue structure and positioning of epithelial cells in the small intestine of Foxl1-deficient mice. The small intestine of Foxl1-deficient mice manifested aberrant crypt structure, including widely distributed Paneth cells, which coincided with the ectopic and increased expression of EphB2 and EphB3, which are key regulators of epithelial cell positioning. Furthermore, real-time quantitative PCR indicated that a subset of Wnt family genes was highly expressed in the gut mesenchyme of Foxl1-deficient mice compared with that of wild-type mice. Such an increase in Wnt expression was remarkable in the mesenchyme, where the aberrant Paneth cell positioning was observed by in situ hybridization. Foxl1 plays an important role in the maintenance of crypt architecture and epithelial cell positioning through the mesenchymal-epithelial interaction in the small intestine. This interaction is essential for the normal regulation of the Wnt/beta-catenin pathway and the subsequent EphB/EphrinB expression.

[Epithelial Cells As Sentinels in Mucosal Immune Barrier]

Nihon Rinshō Men'eki Gakkai Kaishi = Japanese Journal of Clinical Immunology. Feb, 2006  |  Pubmed ID: 16505599

The mucosal surface of the body is exposed to a vast array of exogenous antigens and microorganisms. Epithelial cells evoke minimal immune response to food ingredients and commensal bacteria, while they release an array of antimicrobial peptides and CXC chemokines in response to bacterial invasion or inflammatory stimuli. The mucosal antigens are transported from the gut lumen to organized lymphoid follicles by specialized epithelial M cells residing in follicle-associated epithelium (FAE). An alternative pathway of antigen uptake with neonatal Fc receptor (FcRn) is also reported. Furthermore, intestinal dendritic cells underneath epithelium directly take up luminal antigens, where epithelial fractalkine expression plays a critical role in the guidance of dendrite extrusion. Epithelial cells express polymeric Ig receptor (pIgR) that is essential for the luminal secretion of dimeric IgA produced in the lamina propria. Furthermore, soluble factors released by mucosal epithelial cells condition dendritic cells, which in turn promote Th2 response. These multiple lines of evidence clearly suggest the significant role of epithelial cells at the front line of mucosal immune defense.

Overview: Membrane Traffic in Multicellular Systems: More Than Just a Housekeeper

Journal of Biochemistry. Jun, 2006  |  Pubmed ID: 16788043

Membrane traffic is a fundamental cellular function by which molecules are transported between organelles in the post-Golgi network. Accumulating evidence supports the notion that membrane traffic is not only indispensable for normal cellular function and maintenance of cellular viability by playing housekeeping roles, but also critical for various functions characteristic of multicellular organisms. This Minireview series will focus on the latter aspects of membrane traffic. The topics discussed are: the pathophysiological impact of clathrin-associated adaptor protein (AP) complexes, the significance of membrane traffic in Alzheimer's disease, regulated exocytosis of insulin, secretory lysosomes in immune response, exosomes in physiology and pathology, viral and mammalian ubiquitin ligases modulating immune response, membrane traffic of bacterial toxins, and containment of bacterial infection by autophagy.

Physiological Roles of Clathrin Adaptor AP Complexes: Lessons from Mutant Animals

Journal of Biochemistry. Jun, 2006  |  Pubmed ID: 16788044

Clathrin-associated adaptor protein (AP) complexes play a key role in the transport of proteins, by regulating the formation of transport vesicles as well as cargo selection, between organelles of the post-Golgi network, namely, the trans-Golgi network (TGN), endosomes, lysosomes and the plasma membrane. Evidence has been accumulating for the physiological importance of AP complexes. Deficiency in AP-1A or AP-2 results in embryonic lethality in mice, indicating that these AP complexes are essential for normal development of embryos in mammals. In contrast, mutations in the genes encoding subunits of AP-3A cause an autosomal recessive disorder, Hermansky-Pudlak syndrome in human and its disease models in mice. Knockout mice for the neuron-specific AP-3B suffer from epileptic seizure. Further studies on the physiological and pathological aspects of AP complexes will not only be beneficial for better understanding of developmental biology and medical sciences, but also deepen our insight into the molecular mechanisms of vesicular traffic.

CD300 Antigen Like Family Member G: A Novel Ig Receptor Like Protein Exclusively Expressed on Capillary Endothelium

Biochemical and Biophysical Research Communications. Sep, 2006  |  Pubmed ID: 16876123

We report the characteristics of CD300LG, a member of the CD300 antigen like family. Its genomic structure is similar in both mouse and human, and at least four isoforms exist in both species. The amino acid sequence of the immunoglobulin (Ig) V like domain of CD300LG showed approximately 35% identity to those of the polymeric Ig receptor (pIgR) and Fcalpha/muR. Interestingly, mouse CD300LG proteins were uniquely expressed on capillary endothelium. Immunoelectron microscopy revealed that mouse CD300LG is localized on both apical and basolateral plasma membranes, as well as on intracellular vesicular structures, in the capillary endothelium. Transcytosis assays using polarized MDCK epithelial cells showed that CD300LG could be transcytosed bidirectionally. Furthermore, CD300LG exogenously expressed on HeLa cells could take up IgA2 and IgM, but not IgG. These results suggest that CD300LG might play an important role in molecular traffic across the capillary endothelium.

CMRF-35-like Molecule-5 Constitutes Novel Paired Receptors, with CMRF-35-like Molecule-1, to Transduce Activation Signal Upon Association with FcRgamma

International Immunology. Oct, 2006  |  Pubmed ID: 16940041

The murine CMRF-35-like molecule (CLM) family belongs to the Ig superfamily, and consists of nine mapped genes. Here we report that CLM-5 and CLM-1 constitute novel paired Ig-like receptors (PIRs). CLM-1 and CLM-5 genes are encoded in close vicinity on chromosome 11. CLM-1 has been reported to possess immunoreceptor tyrosine-based inhibitory motifs in its cytoplasmic region and to inhibit the differentiation of osteoclast. CLM-5 has an Ig domain that is highly homologous with that of CLM-1 in its extracellular domain, and possesses a negatively charged residue in its transmembrane domain. CLM-5, like CLM-1, is expressed in myeloid cells, such as dendritic cells, macrophages and granulocytes. We also show that CLM-5 interacts with FcRgamma to be expressed on the plasma membrane and that the cross-linking of CLM-5 leads to tyrosine phosphorylation of proteins, including FcRgamma, in the monocyte/macrophage cell line. Taken together, these characteristics suggest that CLM-1 and CLM-5 constitute novel PIRs and that CLM-5 may transduce activation signals as the activating receptor in myeloid cells.

Clathrin-associated Adaptor Protein Complexes

Journal of Cell Science. Sep, 2006  |  Pubmed ID: 16959901

Visualization of the Post-Golgi Trafficking of Multiphoton Photoactivated Transferrin Receptors

Cell Structure and Function. 2006  |  Pubmed ID: 17072087

Newly synthesized membrane proteins are sorted in the trans-Golgi network (TGN) on the basis of sorting signals carried in their cytoplasmic domains and delivered to their final destinations in the secretory and endocytic pathways. Although previous studies have suggested the involvement of early endosomes in the biosynthetic pathway of transmembrane proteins, the precise trafficking routes followed by the newly synthesized plasma membrane proteins, such as transferrin receptors (TfRs), after exit from the TGN remain unclear. In this report, first, we demonstrated the advantages of photoactivating PA-GFP, a variant of the Aequorea victoria green fluorescent protein (GFP), with multiphoton laser light rather than single-photon laser light, in terms of photoactivation efficiency and spatial resolution. We then applied the multiphoton photoactivation technique to selectively photoactivate the TfR tagged with PA-GFP (PA-GFP-TfR) at the TGN, and monitored the movement of the photoactivated PA-GFP-TfR in live cells. We observed that the PA-GFP-TfR photoactivated at the TGN are transported to the Tfn(+)EEA1(+) endosomal compartments after exiting the TGN. These data support the notion that early endosomes can serve as a sorting station for not only internalized plasma membrane proteins in the endocytic pathway but also newly synthesized membrane proteins in the post-Golgi secretory pathway.

Affinity Identification of Delta-opioid Receptors Using Latex Nanoparticles

Bioorganic & Medicinal Chemistry Letters. Jan, 2006  |  Pubmed ID: 16216499

Three types of latex nanoparticles carrying naltrindole (NTI) derivatives were synthesized as probes for the affinity isolation of their binding proteins including the delta-opioid receptor. The effect of the attachment of NTI to different positions on the linker was investigated. Only latex nanoparticles in which the NTI derivative was linked through the phenol group were useful for isolating the recombinant delta-opioid receptor solubilized from CHO cell membrane. These latex nanoparticles could be a useful tool for investigations of the pharmacological activity of NTI.

Ischemia Promotes Calpain-mediated Degradation of P120-catenin in SH-SY5Y Cells

Biochemical and Biophysical Research Communications. Feb, 2007  |  Pubmed ID: 17196166

p120-catenin contributes to the cadherin-mediated adhesion and aggregation of cells. mu-Calpain was activated and p120-catenin was degraded after 36 h of ischemia in differentiated SH-SY5Y cells. Calpain inhibitors Cbz-Val-Phe-H (MDL28170, 20 microM) and N-acetyl-leucyl-leucyl-norleucinal (ALLN, 20 microM) increased the levels of dephosphorylated p120-catenin, aggregation, and cell survival as detected by reduced LDH release in ischemic cells. However, a proteasome inhibitor lactacystin had no such effects. This is the first report of the calpain-mediated degradation of p120-catenin and an association between the level of dephosphorylated p120-catenin and cell aggregation in ischemic neuronal cells.

Mutation Screening of AP3M2 in Japanese Epilepsy Patients

Brain & Development. Sep, 2007  |  Pubmed ID: 17293072

Evidence that some types of epilepsies show strong genetic predisposition has been well documented. AP3M2 is considered to be an epileptogenic gene because AP3M2 knockout mice exhibit symptoms of spontaneous epileptic seizures. In order to investigate whether the AP3M2 gene causes susceptibility to epilepsy, we performed mutation screening of the genomic DNA of 190 patients with six epilepsy types; this screening involved all the 9 exons and the relevant exon-intron boundaries of AP3M2. Although neither missense nor nonsense mutations were detected, we identified 21 sequence variations, of which 16 variations were novel. Of the 21 variations, 11 were detected in 5' and 3' UTRs, while the remaining variations were detected in introns. Although the present study failed to identify the possible AP3M2 mutations that may cause epilepsy, our results suggest that some AP3M2 mutations still remain candidates for unmapped disorders including epilepsy, febrile seizure, and other neuronal developmental disorders associated with functional abnormalities of GABAergic transmission.

Splenic CD19-CD35+B220+ Cells Function As an Inducer of Follicular Dendritic Cell Network Formation

Blood. Aug, 2007  |  Pubmed ID: 17519390

Follicular dendritic cells (FDCs) form a reticular FDC network in the lymphoid follicle that is essential for the retention and presentation of native antigens in the form of antigen-antibody immune complexes (ICs) to B cells during secondary immune response. Although the presence of migrating precursors of FDCs has been hypothesized, their entity has not been elucidated. Here we report the identification of murine splenic CD19(-)CD11c(-)CD35(+)B220(+) cells as an inducer of FDC network formation. We demonstrated that CD19(-)-CD11c(-)CD35(+)B220(+) cells, together with stromal cells, had the remarkable ability to form lymphoid-follicle-like structures that contained B220(+)FDC-M1(+) reticular cells originally derived from CD19(-)-CD11c(-)CD35(+)B220(+) cells in the CD35(+) reticulum. Our results indicate that CD19(-)CD11c(-)CD35(+)B220(+) cells function as an inducer of FDC network formation and that the interaction between CD19(-)CD11c(-)CD35(+)B220(+) cells and stromal cells is required to initiate lymphoid follicle formation.

Mast Cells Appearing in Long-term Skeletal Muscle Cell Cultures of Rat

Anatomical Record (Hoboken, N.J. : 2007). Nov, 2007  |  Pubmed ID: 17853403

Mast cells are known to be involved in type I allergy and to be localized in almost all tissues in the body. However, they have slightly different properties depending on their tissue of residence. Although mast cells are found in skeletal muscle tissue, there have been no reports of their appearance in cultured skeletal muscles. We report here that mast cells appear in long-term cultures of skeletal muscles from neonatal rats and rat fetuses. When muscle cells were disseminated and cultured in minimum essential medium with 10% fetal calf serum and 10% horse serum, oval cells containing large granules started to appear on myotube sheets at 5 days of culture. These oval cells continued to proliferate for 2-3 months, and showed immunoreactivity for histamine, tryptase, Fc(epsilon)RI, and c-kit. They showed metachromatic staining with 0.5% toluidine blue at pH 0.5 and were stained with both Alcian blue and safranin. Biochemically measured histamine content per dish was significantly higher in 2-month than in 5-day culture. From these results, we concluded that these oval cells were mast cells. Because proteases from mast cells have been reported previously to affect myoblast proliferation, the present findings suggest that there may be some interaction between mast cells and muscle cell proliferation or differentiation. The present finding that mast cells are easily obtained from ordinary skeletal muscle cultures provides a useful method for the study of the diverse functions of mast cells.

Construction of an Open-access Database That Integrates Cross-reference Information from the Transcriptome and Proteome of Immune Cells

Bioinformatics (Oxford, England). Nov, 2007  |  Pubmed ID: 17893089

Although a huge amount of mammalian genomic data does become publicly available, there are still hurdles for biologists to overcome before such data can be fully exploited. One of the challenges for gaining biological insight from genomic data has been the inability to cross-reference transcriptomic and proteomic data using a single informational platform. To address this, we constructed an open-access database that enabled us to cross-reference transcriptomic and proteomic data obtained from immune cells.

Psg18 is Specifically Expressed in Follicle-associated Epithelium

Cell Structure and Function. 2007  |  Pubmed ID: 17984568

Pregnancy-specific glycoproteins (Psgs) secreted by the placenta regulate the immune system to ensure the survival of the fetal allograft by inducing IL-10, an anti-inflammatory cytokine. However, it is unknown whether Psgs are involved in more general aspects of immune response other than maternal immunity. Here, we report that Psg18 is highly expressed in the follicle-associated epithelium (FAE) overlaying Peyer's patches (PPs). Bioinformatics analysis with Reference Database for Immune Cells (RefDIC) as well as RT-PCR data demonstrated that Psg18 is exclusively expressed in FAE in adult mice, in contrast to other Psg family members that are either not expressed or only slightly expressed in FAE. Psg18 expression was observed in FAE of germ-free-conditioned mice, and was slightly upregulated after bacterial inoculation. In situ hybridization analysis revealed that Psg18 is widely expressed throughout FAE. Furthermore, Psg18 protein is deposited on the extracellular matrix in the subepithelial dome beneath FAE, where antigen-presenting cells accumulate. These results suggest that Psg18 is an FAE-specific marker protein that could promote interplay between FAE and immune cells in mucosa-associated lymphoid tissues.

Construction of a Biological Tissue Model Based on a Single-cell Model: a Computer Simulation of Metabolic Heterogeneity in the Liver Lobule

Artificial Life. 2008  |  Pubmed ID: 18171128

An enormous body of information has been obtained by molecular and cellular biology in the last half century. However, even these powerful approaches are not adequate when it comes to higher-level biological structures, such as tissues, organs, and individual organisms, because of the complexities involved. Thus, accumulation of data at the higher levels supports and broadens the context for that obtained on the molecular and cellular levels. Under such auspices, an attempt to elucidate mesoscopic and macroscopic subjects based on plentiful nanoscopic and microscopic data is of great potential value. On the other hand, fully realistic simulation is impracticable because of the extensive cost entailed and enormous amount of data required. Abstraction and modeling that balance the dual requirements of prediction accuracy and manageable calculation cost are of great importance for systems biology. We have constructed an ammonia metabolism model of the hepatic lobule, a histological component of the liver, based on a single-hepatocyte model that consists of the biochemical kinetics of enzymes and transporters. To bring the calculation cost within reason, the porto-central axis, which is an elemental structure of the lobule, is defined as the systems biological unit of the liver, and is accordingly modeled. A model including both histological structure and position-specific gene expression of major enzymes largely represents the physiological dynamics of the hepatic lobule in nature. In addition, heterogeneous gene expression is suggested to have evolved to optimize the energy efficiency of ammonia detoxification at the macroscopic level, implying that approaches like this may elucidate how properties at the molecular and cellular levels, such as regulated gene expression, modify higher-level phenomena of multicellular tissue, organs, and organisms.

Comparative Genome Analysis of Lactobacillus Reuteri and Lactobacillus Fermentum Reveal a Genomic Island for Reuterin and Cobalamin Production

DNA Research : an International Journal for Rapid Publication of Reports on Genes and Genomes. Jun, 2008  |  Pubmed ID: 18487258

Lactobacillus reuteri is a heterofermentative lactic acid bacterium that naturally inhabits the gut of humans and other animals. The probiotic effects of L. reuteri have been proposed to be largely associated with the production of the broad-spectrum antimicrobial compound reuterin during anaerobic metabolism of glycerol. We determined the complete genome sequences of the reuterin-producing L. reuteri JCM 1112(T) and its closely related species Lactobacillus fermentum IFO 3956. Both are in the same phylogenetic group within the genus Lactobacillus. Comparative genome analysis revealed that L. reuteri JCM 1112(T) has a unique cluster of 58 genes for the biosynthesis of reuterin and cobalamin (vitamin B(12)). The 58-gene cluster has a lower GC content and is apparently inserted into the conserved region, suggesting that the cluster represents a genomic island acquired from an anomalous source. Two-dimensional nuclear magnetic resonance (2D-NMR) with (13)C(3)-glycerol demonstrated that L. reuteri JCM 1112(T) could convert glycerol to reuterin in vivo, substantiating the potential of L. reuteri JCM 1112(T) to produce reuterin in the intestine. Given that glycerol is shown to be naturally present in feces, the acquired ability to produce reuterin and cobalamin is an adaptive evolutionary response that likely contributes to the probiotic properties of L. reuteri.

Comprehensive Gene Expression Profiling of Peyer's Patch M Cells, Villous M-like Cells, and Intestinal Epithelial Cells

Journal of Immunology (Baltimore, Md. : 1950). Jun, 2008  |  Pubmed ID: 18523247

Separate populations of M cells have been detected in the follicle-associated epithelium of Peyer's patches (PPs) and the villous epithelium of the small intestine, but the traits shared by or distinguishing the two populations have not been characterized. Our separate study has demonstrated that a potent mucosal modulator cholera toxin (CT) can induce lectin Ulex europaeus agglutinin-1 and our newly developed M cell-specific mAb NKM 16-2-4-positive M-like cells in the duodenal villous epithelium. In this study, we determined the gene expression of PP M cells, CT-induced villous M-like cells, and intestinal epithelial cells isolated by a novel approach using FACS. Additional mRNA and protein analyses confirmed the specific expression of glycoprotein 2 and myristoylated alanine-rich C kinase substrate (MARCKS)-like protein by PP M cells but not CT-induced villous M-like cells. Comprehensive gene profiling also suggested that CT-induced villous M-like cells share traits of both PP M cells and intestinal epithelial cells, a finding that is supported by their unique expression of specific chemokines. The genome-wide assessment of gene expression facilitates discovery of M cell-specific molecules and enhances the molecular understanding of M cell immunobiology.

Barriers to Vaccination Among Japanese Medical Students: Focus Group Interviews

Pediatrics International : Official Journal of the Japan Pediatric Society. Jun, 2008  |  Pubmed ID: 18533941

To date, medical schools and clinical training hospitals in Japan that require students to show immunity for measles, mumps, rubella, varicella (chickenpox), and hepatitis B prior to the commencement of residency are limited.

Aberrant Trafficking of the High-affinity Choline Transporter in AP-3-deficient Mice

The European Journal of Neuroscience. Jun, 2008  |  Pubmed ID: 18554297

The high-affinity choline transporter (CHT) is expressed in cholinergic neurons and efficiently transported to axon terminals where it controls the rate-limiting step in acetylcholine synthesis. Recent studies have shown that the majority of CHT is unexpectedly localized on synaptic vesicles (SV) rather than the presynaptic plasma membrane, establishing vesicular CHT trafficking as a basis for activity-dependent CHT regulation. Here, we analyse the intracellular distribution of CHT in the adaptor protein-3 (AP-3)-deficient mouse model mocha. In the mocha mouse, granular structures in cell bodies are intensely labelled with CHT antibody, indicating possible deficits in CHT trafficking from the cell body to the axon terminal. Western blot analyses reveal that CHT on SV in mocha mice is decreased by 30% compared with wild-type mice. However, no significant difference in synaptosomal choline uptake activity is detected, consistent with the existence of a large reservoir pool for CHT. To further characterize CHT trafficking, we established a PC12D-CHT cell line. In this line, CHT is found associated with a subpopulation of synaptophysin-positive synaptic-like microvesicles (SLMV). The amounts of CHT detected on SLMV are greatly reduced by treating the cell with agents that halt AP-dependent membrane trafficking. These results demonstrate that APs have important functions for CHT trafficking in neuronal cells.

Design and Synthesis of a Metabolically Stable and Potent Antitussive Agent, a Novel Delta Opioid Receptor Antagonist, TRK-851

Bioorganic & Medicinal Chemistry. Sep, 2008  |  Pubmed ID: 18701308

We have previously reported on antitussive effect of (5R,9R,13S,14S)-17-cyclopropylmethyl-6,7-didehydro-4,5-epoxy-5',6'-dihydro-3-methoxy-4'H-pyrrolo[3,2,1-ij]quinolino[2',1':6,7]morphinan-14-ol(1b) methanesulfonate (TRK-850), a selective delta opioid receptor antagonist which markedly reduced the number of coughs in a rat cough model. We designed TRK-850 based on naltrindole (NTI), a typical delta opioid receptor antagonist, to improve its permeability through the blood-brain barrier by introducing hydrophobic moieties to NTI. The ED(50) values of NTI and compound 1b by intraperitoneal injections were 104 microg/kg and 2.07 microg/kg, respectively. This increased antitussive potency probably resulted from the improved brain exposure of compound 1b. However, 1b was extremely unstable toward metabolism by cytochrome P450. In this study, we designed and synthesized compound 1b derivatives to improve the metabolic instability, which resulted in affording highly potent and metabolically stable oral antitussive agent (5R,9R,13S,14S)-17-cyclopropylmethyl-6,7-didehydro-4,5-epoxy-8'-fluoro-5',6'-dihydro-4'H-pyrrolo[3,2,1-ij]quinolino[2',1':6,7]morphinan-3,14-diol (1c) methanesulfonate (TRK-851).

Activation-induced Cytidine Deaminase Deficiency Causes Organ-specific Autoimmune Disease

PloS One. 2008  |  Pubmed ID: 18716662

Activation-induced cytidine deaminase (AID) expressed by germinal center B cells is a central regulator of somatic hypermutation (SHM) and class switch recombination (CSR). Humans with AID mutations develop not only the autosomal recessive form of hyper-IgM syndrome (HIGM2) associated with B cell hyperplasia, but also autoimmune disorders by unknown mechanisms. We report here that AID-/- mice spontaneously develop tertiary lymphoid organs (TLOs) in non-lymphoid tissues including the stomach at around 6 months of age. At a later stage, AID-/- mice develop a severe gastritis characterized by loss of gastric glands and epithelial hyperplasia. The disease development was not attenuated even under germ-free (GF) conditions. Gastric autoantigen -specific serum IgM was elevated in AID-/- mice, and the serum levels correlated with the gastritis pathological score. Adoptive transfer experiments suggest that autoimmune CD4+ T cells mediate gastritis development as terminal effector cells. These results suggest that abnormal B-cell expansion due to AID deficiency can drive B-cell autoimmunity, and in turn promote TLO formation, which ultimately leads to the propagation of organ-specific autoimmune effector CD4+ T cells. Thus, AID plays an important role in the containment of autoimmune diseases by negative regulation of autoreactive B cells.

Rats Harboring S284L Chrna4 Mutation Show Attenuation of Synaptic and Extrasynaptic GABAergic Transmission and Exhibit the Nocturnal Frontal Lobe Epilepsy Phenotype

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Nov, 2008  |  Pubmed ID: 19020039

Mutations of genes encoding alpha4, beta2, or alpha2 subunits (CHRNA4, CHRNB2, or CHRNA2, respectively) of nAChR [neuronal nicotinic ACh (acetylcholine) receptor] cause nocturnal frontal lobe epilepsy (NFLE) in human. NFLE-related seizures are seen exclusively during sleep and are characterized by three distinct seizure phenotypes: "paroxysmal arousals," "paroxysmal dystonia," and "episodic wandering." We generated transgenic rat strains that harbor a missense mutation S284L, which had been identified in CHRNA4 in NFLE. The transgenic rats were free of biological abnormalities, such as dysmorphology in the CNS, and behavioral abnormalities. The mRNA level of the transgene (mutant Chrna4) was similar to the wild type, and no distorted expression was detected in the brain. However, the transgenic rats showed epileptic seizure phenotypes during slow-wave sleep (SWS) similar to those in NFLE exhibiting three characteristic seizure phenotypes and thus fulfilled the diagnostic criteria of human NFLE. The therapeutic response of these rats to conventional antiepileptic drugs also resembled that of NFLE patients with the S284L mutation. The rats exhibited two major abnormalities in neurotransmission: (1) attenuation of synaptic and extrasynaptic GABAergic transmission and (2) abnormal glutamate release during SWS. The currently available genetically engineered animal models of epilepsy are limited to mice; thus, our transgenic rats offer another dimension to the epilepsy research field.

[Membrane Traffic: Overview]

Tanpakushitsu Kakusan Koso. Protein, Nucleic Acid, Enzyme. Dec, 2008  |  Pubmed ID: 21162329

[Physiological Roles of Clathrin-associated Adaptor Protein (AP) Complexes]

Tanpakushitsu Kakusan Koso. Protein, Nucleic Acid, Enzyme. Dec, 2008  |  Pubmed ID: 21038602

Lack of Potassium Current in W309R Mutant KCNQ3 Channel Causing Benign Familial Neonatal Convulsions (BFNC)

Epilepsy Research. Mar, 2009  |  Pubmed ID: 19167866

BFNC is an autosomal dominant epileptic disorder caused by mutations of KCNQ2 or KCNQ3 potassium channel gene. W309R missense mutation in KCNQ3 gene was previously reported in a family with BFNC. In this study, potassium currents were recorded from HEK293 cells expressing both W309R mutant KCNQ3 and wild type KCNQ2 channels. We found a lack of potassium current in W309R mutant KCNQ3 and KCNQ2 channels, which can explain the hyper-excitability of CNS in patients with BFNC.

Non-visually Evoked Activity of Isthmo-optic Neurons in Awake, Head-unrestrained Quail

Experimental Brain Research. Experimentelle Hirnforschung. Expérimentation Cérébrale. Apr, 2009  |  Pubmed ID: 19183972

Changes in the internal state of the brain may modulate retinal function. In birds, most neurons in the isthmo-optic (IO) nucleus project their axons topographically into the contralateral retina, and activity in IO neurons enhances visual responses of retinal ganglion cells in the target retinal region. To elucidate the significance of this pathway, we recorded spikes of IO neurons in four awake Japanese quail using an implanted electrode assembly while recording unrestrained head movements. The IO neurons fired passively in response to visual stimuli in receptive fields and non-visually without visual stimuli or eye-head movements. Non-visually evoked activity was observed in the middle of eye-head fixation, as well as at about 200 ms before the onset of head saccades. Intensity of activity before onset of head saccades depended on the direction of motion of subsequent head saccades. Local retinal output may be enhanced by centrifugal signals before gaze shifts.

Raman Studies of Methane-ethane Hydrate Metastability

The Journal of Physical Chemistry. A. Mar, 2009  |  Pubmed ID: 19209919

The interconversion of methane-ethane hydrate from metastable to stable structures was studied using Raman spectroscopy. sI and sII hydrates were synthesized from methane-ethane gas mixtures of 65% or 93% methane in ethane and water, both with and without the kinetic hydrate inhibitor, poly(N-vinylcaprolactam). The observed faster structural conversion rate in the higher methane concentration atmosphere can be explained in terms of the differences in driving force (difference in chemical potential of water in sI and sII hydrates) and kinetics (mass transfer of gas and water rearrangement). The kinetic hydrate inhibitor increased the conversion rate at 65% methane in ethane (sI is thermodynamically stable) but retards the rate at 93% methane in ethane (sII is thermodynamically stable), implying there is a complex interaction between the polymer, water, and hydrate guests at crystal surfaces.

Evaluation and Characterization of Bacterial Metabolic Dynamics with a Novel Profiling Technique, Real-time Metabolotyping

PloS One. 2009  |  Pubmed ID: 19287504

Environmental processes in ecosystems are dynamically altered by several metabolic responses in microorganisms, including intracellular sensing and pumping, battle for survival, and supply of or competition for nutrients. Notably, intestinal bacteria maintain homeostatic balance in mammals via multiple dynamic biochemical reactions to produce several metabolites from undigested food, and those metabolites exert various effects on mammalian cells in a time-dependent manner. We have established a method for the analysis of bacterial metabolic dynamics in real time and used it in combination with statistical NMR procedures.

New Approach for M-cell-specific Molecules Screening by Comprehensive Transcriptome Analysis

DNA Research : an International Journal for Rapid Publication of Reports on Genes and Genomes. Aug, 2009  |  Pubmed ID: 19675110

A minor population of M cells within the follicle-associated epithelium (FAE) of intestinal Peyer's patches (PPs) serves as a major portal for entry of exogenous antigens. Characterization of the mammalian M cells, including identification of M-cell surface molecules used for bacterial uptake, has been hampered by their relative rarity. In contrast, M cells constitute virtually all of the FAE cells in the avian bursa of Fabricius. We therefore performed comparative gene expression profiling of chicken and murine FAE to identify commonly expressed genes by M cells in both species. The comprehensive transcriptome analysis revealed that 28 genes were commonly up-regulated in FAE from both species. In situ hybridization revealed that annexin A10 (Anxa10) mRNA was scattered in FAE, and co-localized with Ulex europaeus agglutinin-1 binding to M cells. Whole-mount immunostaining also revealed that cellular prion protein (PrP(C)) was expressed on the luminal side of the apical plasma membrane of M cells, and co-localized with grycoprotein 2 that recognizes only M cells in murine PP. Our findings identify new M-cell-specific molecules through using comprehensive transcriptome analysis. These conserved molecules in M cells of mice and chickens may play essential roles in M-cell function and/or differentiation.

AP-1 and KIF13A Coordinate Endosomal Sorting and Positioning During Melanosome Biogenesis

The Journal of Cell Biology. Oct, 2009  |  Pubmed ID: 19841138

Specialized cell types exploit endosomal trafficking to deliver protein cargoes to cell type-specific lysosome-related organelles (LROs), but how endosomes are specified for this function is not known. In this study, we show that the clathrin adaptor AP-1 and the kinesin motor KIF13A together create peripheral recycling endosomal subdomains in melanocytes required for cargo delivery to maturing melanosomes. In cells depleted of AP-1 or KIF13A, a subpopulation of recycling endosomes redistributes to pericentriolar clusters, resulting in sequestration of melanosomal enzymes like Tyrp1 in vacuolar endosomes and consequent inhibition of melanin synthesis and melanosome maturation. Immunocytochemistry, live cell imaging, and electron tomography reveal AP-1- and KIF13A-dependent dynamic close appositions and continuities between peripheral endosomal tubules and melanosomes. Our results reveal that LRO protein sorting is coupled to cell type-specific positioning of endosomes that facilitate endosome-LRO contacts and are required for organelle maturation.

Identity of the Elusive IgM Fc Receptor (FcmuR) in Humans

The Journal of Experimental Medicine. Nov, 2009  |  Pubmed ID: 19858324

Although Fc receptors (FcRs) for switched immunoglobulin (Ig) isotypes have been extensively characterized, FcR for IgM (FcmuR) has defied identification. By retroviral expression and functional cloning, we have identified a complementary DNA (cDNA) encoding a bona fide FcmuR in human B-lineage cDNA libraries. FcmuR is defined as a transmembrane sialoglycoprotein of approximately 60 kD, which contains an extracellular Ig-like domain homologous to two other IgM-binding receptors (polymeric Ig receptor and Fcalpha/muR) but exhibits an exclusive Fcmu-binding specificity. The cytoplasmic tail of FcmuR contains conserved Ser and Tyr residues, but none of the Tyr residues match the immunoreceptor tyrosine-based activation, inhibitory, or switch motifs. Unlike other FcRs, the major cell types expressing FcmuR are adaptive immune cells, including B and T lymphocytes. After antigen-receptor ligation or phorbol myristate acetate stimulation, FcmuR expression was up-regulated on B cells but was down-modulated on T cells, suggesting differential regulation of FcmuR expression during B and T cell activation. Although this receptor was initially designated as Fas apoptotic inhibitory molecule 3, or TOSO, our results indicate that FcmuR per se has no inhibitory activity in Fas-mediated apoptosis and that such inhibition is only achieved when anti-Fas antibody of an IgM but not IgG isotype is used for inducing apoptosis.

Random Walker Test: a Computerized Alternative to the Road-Map Test

Behavior Research Methods. Nov, 2009  |  Pubmed ID: 19897833

The Road-Map Test (RMT) is a popular neurological assessment of left-right orientation, using a simplified road map. Inspired by the RMT, we developed a new computerized navigation test, the Random Walker Test (RWT), for further quantitative assessment of left-right orientation ability. RWT provides verbal or nonverbal instructions for the direction (left, right, or front) in which to proceed, and participants must judge the spatially correct direction. Perspectives rotate by 90 degrees during navigation. Verbal judgments demand verbal-to-spatial mapping of left/right/front and, if necessary, egocentric perspective rotation. Using the RWT, we evaluated the left-right orientation of normal male participants. The RWT reliably recorded the response times and error rates for participant performance and also revealed egocentric perspective rotation as an unreliable mental process with large intra- and interpersonal variability. These results indicate that the RWT may be useful in investigating left-right orientation and/or egocentric perspective rotation in both normal participants and neuropathological patients.

Uptake Through Glycoprotein 2 of FimH(+) Bacteria by M Cells Initiates Mucosal Immune Response

Nature. Nov, 2009  |  Pubmed ID: 19907495

The mucosal immune system forms the largest part of the entire immune system, containing about three-quarters of all lymphocytes and producing grams of secretory IgA daily to protect the mucosal surface from pathogens. To evoke the mucosal immune response, antigens on the mucosal surface must be transported across the epithelial barrier into organized lymphoid structures such as Peyer's patches. This function, called antigen transcytosis, is mediated by specialized epithelial M cells. The molecular mechanisms promoting this antigen uptake, however, are largely unknown. Here we report that glycoprotein 2 (GP2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for mucosal antigens. Recombinant GP2 protein selectively bound a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane. Consistently, these bacteria were colocalized with endogenous GP2 on the apical plasma membrane as well as in cytoplasmic vesicles in M cells. Moreover, deficiency of bacterial FimH or host GP2 led to defects in transcytosis of type-I-piliated bacteria through M cells, resulting in an attenuation of antigen-specific immune responses in Peyer's patches. GP2 is therefore a previously unrecognized transcytotic receptor on M cells for type-I-piliated bacteria and is a prerequisite for the mucosal immune response to these bacteria. Given that M cells are considered a promising target for oral vaccination against various infectious diseases, the GP2-dependent transcytotic pathway could provide a new target for the development of M-cell-targeted mucosal vaccines.

M-Sec Promotes Membrane Nanotube Formation by Interacting with Ral and the Exocyst Complex

Nature Cell Biology. Dec, 2009  |  Pubmed ID: 19935652

Cell-cell communication is essential for the development and homeostasis of multicellular organisms. Recently, a new type of cell-cell communication was discovered that is based on the formation of thin membranous nanotubes between remote cells. These long membrane tethers, termed tunneling nanotubes (TNTs), form an intercellular conduit and have been shown to enable the transport of various cellular components and signals. However, the molecular basis for TNT formation remains to be elucidated. Here we report that a mammalian protein, M-Sec, induces de novo formation of numerous membrane protrusions extending from the plasma membrane, some of which tether onto adjacent cells and subsequently form TNT-like structures. Depletion of M-Sec by RNA interference (RNAi) greatly reduced endogenous TNT formation as well as intercellular propagation of a calcium flux in a macrophage cell line. Furthermore, blockage of the interaction of M-Sec with Ral and the exocyst complex, which serves as a downstream effector of Ral, attenuated the formation of membrane nanotubes. Our results reveal that M-Sec functions as a key regulator of membrane nanotube formation through interaction with the Ral-exocyst pathway.

Identification of TOSO/FAIM3 As an Fc Receptor for IgM

International Immunology. Mar, 2010  |  Pubmed ID: 20042454

Fc receptors specifically bind to the Fc region of Igs to mediate the unique functions to each class of Igs. To identify a novel Fc receptor for IgM, we searched expressed sequence tag database for molecules containing Ig domains with homology to those of known Fc receptors for IgM, Fcalpha/muR and polymeric Ig receptor. As a result, we identified TOSO/Fas apoptotic inhibitory molecule 3 (FAIM3) as a possible Fc receptor for IgM. HeLa cells transfected with a TOSO/FAIM3-expression vector bound to IgM but not IgG and were able to internalize IgM-conjugated beads but not IgG-conjugated beads, suggesting that TOSO/FAIM3 is indeed a receptor for IgM (FcmuR). FcmuR protein was expressed predominantly on B-lineage cells; expression of the Fcmr transcripts was observed from the pre-B-cell stage and maintained thereafter during B-cell development. These results identify TOSO/FAIM3 as a receptor for IgM and suggest that FcmuR may serve as an uptake receptor for IgM-opsonized antigens by B cells.

Towards a Green Hydrate Inhibitor: Imaging Antifreeze Proteins on Clathrates

PloS One. 2010  |  Pubmed ID: 20161789

The formation of hydrate plugs in oil and gas pipelines is a serious industrial problem and recently there has been an increased interest in the use of alternative hydrate inhibitors as substitutes for thermodynamic inhibitors like methanol. We show here that antifreeze proteins (AFPs) possess the ability to modify structure II (sII) tetrahydrofuran (THF) hydrate crystal morphologies by adhering to the hydrate surface and inhibiting growth in a similar fashion to the kinetic inhibitor poly-N-vinylpyrrolidone (PVP). The effects of AFPs on the formation and growth rate of high-pressure sII gas mix hydrate demonstrated that AFPs are superior hydrate inhibitors compared to PVP. These results indicate that AFPs may be suitable for the study of new inhibitor systems and represent an important step towards the development of biologically-based hydrate inhibitors.

New Monitoring Approach for Metabolic Dynamics in Microbial Ecosystems Using Stable-isotope-labeling Technologies

Journal of Bioscience and Bioengineering. Jul, 2010  |  Pubmed ID: 20541122

We have developed a new approach for monitoring the metabolic dynamics in microbial ecosystems using a combination of DNA fingerprinting and metabolome analysis based on stable-isotope-labeling technologies. Stable-isotope probing of DNA (DNA-SIP) has been used previously for the evaluation of cross-feeding in microbial communities. For the development and validation of our monitoring approach, fecal microbiota were analyzed with stable-isotope-labeled glucose used as the sole carbon source. In order to link the metabolic information and the microbial variability, we performed metabolic-microbial correlation analysis based on nuclear magnetic resonance (NMR) profiles and denaturing gradient gel electrophoresis (DGGE) fingerprints, which successfully identified the glucose-utilizing bacteria and their related extracellular metabolites. Moreover, our approach revealed information regarding the carbon flux, in that the "first" wave of extracellular metabolites secreted by the glucose-utilizing bacteria were incorporated into the "secondary" group of substrate-utilizing bacteria, and that this "secondary" group further produced their own secondary metabolized substrates. Thus, this approach is a powerful tool for monitoring the metabolic dynamics in microbial ecosystems and allows for the tracking of the carbon flux within a microbial community.

Interaction of Antifreeze Proteins with Hydrocarbon Hydrates

Chemistry (Weinheim an Der Bergstrasse, Germany). Sep, 2010  |  Pubmed ID: 20623806

Recombinant antifreeze proteins (AFPs), representing a range of activities with respect to ice growth inhibition, were investigated for their abilities to control the crystal formation and growth of hydrocarbon hydrates. Three different AFPs were compared with two synthetic commercial inhibitors, poly-N-vinylpyrrolidone (PVP) and HIW85281, by using multiple approaches, which included gas uptake, differential scanning calorimetry (DSC) temperature ramping, and DSC isothermal observations. A new method to assess the induction period before heterogeneous nucleation and subsequent hydrate crystal growth was developed and involved the dispersal of water in the pore space of silica gel beads. Although hydrate nucleation is a complex phenomenon, we have shown that it can now be carefully quantified. The presence of AFPs delayed crystallization events and showed hydrate growth inhibition that was superior to that of one of the benchmark commercial inhibitors, PVP. Nucleation and growth inhibition were shown to be independent processes, which indicates a difference in the mechanisms required for these two inhibitory actions. In addition, there was no apparent correlation between the assayed activities of the three AFPs toward hexagonal ice and the cubic structure II (sII) hydrate, which suggests that there are distinctive differences in the protein interactions with the two crystal surfaces.

M-Sec: Emerging Secrets of Tunneling Nanotube Formation

Communicative & Integrative Biology. May, 2010  |  Pubmed ID: 20714400

Tunneling nanotubes (TNT) are the latest addition to the array of strategies used for intercellular signaling. TNTs are continuous conduits of the plasma membrane that allow direct physical connection of plasma membranes and cytosol among remote cells. They are important for intercellular communication by mediating exchange of cellular components as well as signal transduction molecules. Despite ample evidence suggesting the pathophysiological importance of TNTs, virtually nothing is known about the molecular basis for their formation. With the lack of specific TNT markers, their study has relied solely on morphological analyses, and the precise identity of TNT and TNTlike structures have been difficult to define. We have now shown that M-Sec is a TNT marker and a central factor for TNT formation. In cooperation with the RalA small GTPase and the exocyst complex, M-Sec can induce the formation of functional TNTs, indicating that the remodeling of the actin cytoskeleton is involved in M-Sec-mediated TNT formation. Discovery of the role of M-Sec will accelerate our understanding of TNTs, both at the molecular and physiological levels.

Symmetric Stretching Vibration of CH4 in Clathrate Hydrate Structures

Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry. Oct, 2010  |  Pubmed ID: 20718071

Glycoprotein 2 (GP2): Grabbing the FimH Bacteria into M Cells for Mucosal Immunity

Gut Microbes. Nov-Dec, 2010  |  Pubmed ID: 21468225

Membranous (M) cells are specialized epithelial antigen-transporting cells scattered in the follicle-associated epithelium covering the gut lymphoid follicles such as Peyer's patches. Although the importance of M cells as a main portal for luminal antigens has long been recognized, molecular mechanisms for M-cell antigen uptake has remained largely elusive. We have recently found that glycoprotein 2 (GP2) is exclusively expressed on M cells among intestinal epithelial cells and serves as an uptake receptor for a subset of commensal and pathogenic bacteria. GP2 interacts with FimH, a major component of the type 1 pilus on the outer membrane of a subset of gram-negative enterobacilli such as E. coli and Salmonella enterica. Furthermore, GP2-FimH interaction is necessary for efficient uptake of FimH(+) bacteria by M cells and subsequent bacteria-specific mucosal immune responses. Pancreatic GP2 may also be involved in innate immunity by 'opsonization' of FimH(+) bacteria to facilitate their egestion in feces as well as translocation across the intestinal epithelium.

Dynamic Omics Approach Identifies Nutrition-mediated Microbial Interactions

Journal of Proteome Research. Feb, 2011  |  Pubmed ID: 21058740

"Omics" studies reported to date have dealt with either thoroughly characterized single species or poorly explored meta-microbial communities. However, these techniques are capable of producing highly informative data for the analysis of interactions between two organisms. We examined the bacterial interaction between Escherichia coli O157:H7 (O157) and Bifidobacterium longum (BL) as a pathogenic-commensal bacterial model creating a minimum microbial ecosystem in the gut using dynamic omics approaches, consisting of improved time-lapse 2D-nuclear magnetic resonance (NMR) metabolic profiling, transcriptomic, and proteomic analyses. Our study revealed that the minimum ecosystem was established by bacterial adaptation to the changes in the extracellular environment, primarily by O157, but not by BL. Additionally, the relationship between BL and O157 could be partially regarded as that between a producer and a consumer of nutrients, respectively, especially with regard to serine and aspartate metabolism. Taken together, our profiling system can provide a new insight into the primary metabolic dynamics in microbial ecosystems.

Bifidobacterium Kashiwanohense Sp. Nov., Isolated from Healthy Infant Faeces

International Journal of Systematic and Evolutionary Microbiology. Nov, 2011  |  Pubmed ID: 21148680

Strains HM2-1 and HM2-2(T) were isolated from the faeces of a healthy infant and were characterized by determining their phenotypic and biochemical features and phylogenetic positions based on partial 16S rRNA gene sequence analysis. They were Gram-positive, obligately anaerobic, non-spore-forming, non-gas-producing, and catalase-negative non-motile rods. They did not grow at 15 or 45 °C in anaerobic bacterial culture medium, and their DNA G+C content was in the range 56-59 mol%. In enzyme activity tests, strains HM2-1 and HM2-2(T) were positive for α/β-galactosidases and α/β-glucosidases but negative for β-glucuronidase and cystine arylamidase. An analysis of the cell-wall composition of strains HM2-1 and HM2-2(T) revealed the presence of glutamic acid, alanine and lysine. The presence of fructose-6-phosphate phosphoketolase shows that isolates HM2-1 and HM2-2(T) are members of the genus Bifidobacterium. These two isolates belong to the same species of the genus Bifidobacterium. Strain HM2-2(T) was found to be related to Bifidobacterium catenulatum JCM 1194(T) (97.4 % 16S rRNA gene sequence identity: 1480/1520 bp), Bifidobacterium pseudocatenulatum JCM 1200(T) (97.2 %: 1472/1514 bp), Bifidobacterium dentium ATCC 27534(T) (96.7 %: 1459/1509 bp) and Bifidobacterium angulatum ATCC 27535(T) (96.5 %: 1462/1515 bp). The predominant cellular fatty acids of strains HM2-1 and HM2-2(T) were 16 : 0 and 18 : 1ω9c, with proportions greater than 18 % of the total. Phylogenetic analyses involving phenotypic characterization, DNA-DNA hybridization and partial 16S rRNA gene sequencing proves that the strains represent a novel species of the genus Bifidobacterium, for which the name Bifidobacterium kashiwanohense sp. nov. is proposed. The type strain is HM2-2(T) ( = JCM 15439(T) = DSM 21854(T)).

SP600125 Inhibits Cap-dependent Translation Independently of the C-Jun N-terminal Kinase Pathway

Cell Structure and Function. 2011  |  Pubmed ID: 21263197

We investigated the effects of SP600125 (formerly called c-Jun N-terminal kinase (JNK) inhibitor II) on translation using cultured mouse cells. SP600125 (50 µM) treatment rapidly repressed overall protein synthesis, accompanied by a reduction in the mRNAs for housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase in the polysomal fraction. SP600125 decreased polysomes with a concomitant increase in free ribosomal subunits in the cytoplasm, suggesting that global translation was inhibited at the initiation step. A reporter analysis using exogenous mRNAs showed that SP600125 inhibited cap-dependent but not internal ribosome entry site-dependent translation. SP600125 significantly attenuated phosphorylation of components in the mTOR pathway, which is responsible for cap-dependent translation. In contrast to SP600125, short hairpin RNAs for JNK1 and JNK2 failed to affect overall protein synthesis. Collectively, SP600125 inhibits cap-dependent translation, independent of the JNK pathway.

Bifidobacteria Can Protect from Enteropathogenic Infection Through Production of Acetate

Nature. Jan, 2011  |  Pubmed ID: 21270894

The human gut is colonized with a wide variety of microorganisms, including species, such as those belonging to the bacterial genus Bifidobacterium, that have beneficial effects on human physiology and pathology. Among the most distinctive benefits of bifidobacteria are modulation of host defence responses and protection against infectious diseases. Nevertheless, the molecular mechanisms underlying these effects have barely been elucidated. To investigate these mechanisms, we used mice associated with certain bifidobacterial strains and a simplified model of lethal infection with enterohaemorrhagic Escherichia coli O157:H7, together with an integrated 'omics' approach. Here we show that genes encoding an ATP-binding-cassette-type carbohydrate transporter present in certain bifidobacteria contribute to protecting mice against death induced by E. coli O157:H7. We found that this effect can be attributed, at least in part, to increased production of acetate and that translocation of the E. coli O157:H7 Shiga toxin from the gut lumen to the blood was inhibited. We propose that acetate produced by protective bifidobacteria improves intestinal defence mediated by epithelial cells and thereby protects the host against lethal infection.

Salmonella Septic Arthritis Following Total Knee Arthroplasty for Rheumatoid Arthritis in a Patient Receiving Etanercept

Journal of Orthopaedic Science : Official Journal of the Japanese Orthopaedic Association. Mar, 2011  |  Pubmed ID: 21301900

[Biology of M Cells, a Unique Subset of Intestinal Epithelial Cells]

Seikagaku. The Journal of Japanese Biochemical Society. Jan, 2011  |  Pubmed ID: 21404619

CCR6hiCD11c(int) B Cells Promote M-cell Differentiation in Peyer's Patch

International Immunology. Apr, 2011  |  Pubmed ID: 21422150

M cells are responsible for uptake of mucosal antigens in Peyer's patches (PPs). Differentiation of M cells is thought to be induced by interactions between follicle-associated epithelium and PP cells; however, it remains elusive what types of immune cells function as M-cell inducers. Here, we attempted to identify the cells that serve as an M-cell inducer in PP. We found that a unique B-cell subset characterized by CCR6(hi)CD11c(int) resided in the subepithelial dome (SED) in mouse PP. CCR6(hi)CD11c(int) B cells showed chemotactic migration in response to CCL20. Furthermore, this unique B-cell subset substantially decreased in PP of CCR6-deficient mice, indicating that the SED localization of CCR6(hi)CD11c(int) B cells is most likely regulated by the CCL20-CCR6 system. Concomitantly, CCR6 deficiency caused remarkable decrement of M cells. Moreover, adoptive transfer of CCR6(hi)CD11c(int) B cells from wild-type mice restored the M-cell decrement in CCR6-deficient mice. Collectively, the spatial regulation of CCR6(hi)CD11c(int) B cells via the CCL20-CCR6 system may play a vital role in M-cell differentiation in mice.

Morphological and Compositional Characterization of Self-preserved Gas Hydrates by Low-vacuum Scanning Electron Microscopy

Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry. Jun, 2011  |  Pubmed ID: 21509926

The Epithelia-specific Membrane Trafficking Factor AP-1B Controls Gut Immune Homeostasis in Mice

Gastroenterology. Aug, 2011  |  Pubmed ID: 21669204

Epithelial cells that cover the intestinal mucosal surface maintain immune homeostasis and tolerance in the gastrointestinal tract. However, little is known about the molecular mechanisms that regulate epithelial immune functions. Epithelial cells are distinct in that they are highly polarized; this polarity is, at least in part, established by the epithelium-specific polarized sorting factor adaptor protein (AP)-1B. We investigated the role of AP-1B-mediated protein sorting in the maintenance of gastrointestinal immune homeostasis.

Dissociation Behavior of C2H6 Hydrate at Temperatures Below the Ice Point: Melting to Liquid Water Followed by Ice Nucleation

The Journal of Physical Chemistry. A. Aug, 2011  |  Pubmed ID: 21744826

The dissociation of C(2)H(6) hydrate particles by slow depressurization at temperatures slightly below the ice melting point was studied using optical microscopy and Raman spectroscopy. Visual observations and Raman measurements revealed that ethane hydrates can be present as a metastable state at pressures lower than the dissociation pressures of the three components: ice, hydrate, and free gas. However, they decompose into liquid water and gas phases once the system pressure drops to the equilibrium boundary for supercooled water, hydrate, and free gas. Structural analyses of obtained Raman spectra indicate that structures of the metastable hydrates and liquid water from the hydrate decay are fundamentally identical to those of the stable hydrates and supercooled water without experience of the hydration. These results imply a considerably high energy barrier for the direct hydrate-to-ice transition. Water solidification, probably induced by dynamic nucleation, was also observed during melting.

Complete Genome Sequences of Rat and Mouse Segmented Filamentous Bacteria, a Potent Inducer of Th17 Cell Differentiation

Cell Host & Microbe. Sep, 2011  |  Pubmed ID: 21925114

Segmented filamentous bacteria (SFB) are noncultivable commensals inhabiting the gut of various vertebrate species and have been shown to induce Th17 cells in mice. We present the complete genome sequences of both rat and mouse SFB isolated from SFB-monocolonized hosts. The rat and mouse SFB genomes each harbor a single circular chromosome of 1.52 and 1.59 Mb encoding 1346 and 1420 protein-coding genes, respectively. The overall nucleotide identity between the two genomes is 86%, and the substitution rate was estimated to be similar to that of the free-living E. coli. SFB genomes encode typical genes for anaerobic fermentation and spore and flagella formation, but lack most of the amino acid biosynthesis enzymes, reminiscent of pathogenic Clostridia, exhibiting large dependency on the host. However, SFB lack most of the clostridial virulence-related genes. Comparative analysis with clostridial genomes suggested possible mechanisms for host responses and specific adaptations in the intestine.

Structures of Hydrocarbon Hydrates During Formation with and Without Inhibitors

The Journal of Physical Chemistry. A. Feb, 2012  |  Pubmed ID: 22185460

The formation of hydrates from a methane-ethane-propane mixture is more complex than with single gases. Using nuclear magnetic resonance (NMR) and high-pressure powder X-ray diffraction (PXRD), we have investigated the structural properties of natural gas hydrates crystallized in the presence of kinetic hydrate inhibitors (KHIs), two commercial inhibitors and two biological ice inhibitors, or antifreeze proteins (AFPs). NMR analyses indicated that hydrate cage occupancy was at near saturation for controls and most inhibitor types. Some exceptions were found in systems containing a new commercial KHI (HIW85281) and a recombinant plant AFP, suggesting that these two inhibitors could impact the kinetics of cavity formation. NMR analysis confirmed that the hydrate composition varies during crystal growth by kinetic effects. Strikingly, the coexistence of both structures I (sI) and II (sII) were observed in NMR spectra and PXRD profiles. It is suggested that sI phases may form more readily from liquid water. Real time PXRD monitoring showed that sI hydrates were less stable than sII crystals, and there was a conversion to the stable phase over time. Both commercial KHIs and AFPs had an impact on hydrate metastability, but transient sI PXRD intensity profiles indicated significantly different modes of interaction with the various inhibitors and the natural gas hydrate system.

Epithelial Cell-Intrinsic Notch Signaling Plays an Essential Role in the Maintenance of Gut Immune Homeostasis

Journal of Immunology (Baltimore, Md. : 1950). Jan, 2012  |  Pubmed ID: 22279105

Intestinal epithelial cells (IECs) have important functions as the first line of defense against diverse microorganisms on the luminal surface. Impaired integrity of IEC has been implicated in increasing the risk for inflammatory disorders in the gut. Notch signaling plays a critical role in the maintenance of epithelial integrity by regulating the balance of secretory and absorptive cell lineages, and also by facilitating epithelial cell proliferation. We show in this article that mice harboring IEC-specific deletion of Rbpj (RBP-J(ΔIEC)), a transcription factor that mediates signaling through Notch receptors, spontaneously develop chronic colitis characterized by the accumulation of Th17 cells in colonic lamina propria. Intestinal bacteria are responsible for the development of colitis, because their depletion with antibiotics prevented the development of colitis in RBP-J(ΔIEC) mice. Furthermore, bacterial translocation was evident in the colonic mucosa of RBP-J(ΔIEC) mice before the onset of colitis, suggesting attenuated epithelial barrier functions in these mice. Indeed, RBP-J(ΔIEC) mice displayed increase in intestinal permeability after rectal administration of FITC-dextran. In addition to the defect in physical barrier, loss of Notch signaling led to arrest of epithelial cell turnover caused by downregulation of Hes1, a transcriptional repressor of p27(Kip1) and p57(Kip2). Thus, epithelial cell-intrinsic Notch signaling ensures integrity and homeostasis of IEC, and this mechanism is required for containment of intestinal inflammation.

AP1B Plays an Important Role in Intestinal Tumorigenesis with the Truncating Mutation of an APC Gene

International Journal of Cancer. Journal International Du Cancer. Mar, 2012  |  Pubmed ID: 21484796

Recent evidence has suggested that carcinoma is accompanied by the loss of cell polarity. An epithelial cell-specific form of the AP-1 clathrin adaptor complex, AP1B, is involved in the polarized transport of membrane proteins to the basolateral surface of epithelial cells. In our study, we investigated whether AP1B is involved in intestinal tumorigenesis. The cellular polarity of intestinal tumor cells was examined using APC(Min/+) mice as an in vivo model and SW480 cells with a truncating mutation in the adenomatous polyposis coli (APC) gene as an in vitro model by confocal microscopy. Next, the expression of AP1B in intestinal tumor cells was examined by real-time polymerase chain reaction (PCR) and Western blotting. The localization of β-catenin and the expression of AP1B in the tumor tissue of patients with colorectal cancer were evaluated by confocal microscopy and real-time PCR, respectively, and the relationships among cell polarity, AP1B expression and intestinal tumorigenesis were examined. Cellular polarity was lost in intestinal tumor cells, and the expression of AP1B was downregulated. In addition, the reduction in the expression level of AP1B correlated with the nuclear localization of β-catenin in human colorectal cancer. Our study indicates the close associations between AP1B, intestinal tumorigenesis and mutations in the APC gene. This is the first report to reveal the relationships among AP1B, cellular polarity and intestinal tumorigenesis, and achieving a detailed understanding of AP1B will hopefully lead to discovery of therapeutic targets and novel biomarkers for intestinal cancer.