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Immature cell monolayers are vulnerable to tensile strain, which can cause severe diseases including fibrosis, cancer, and atherosclerosis. However, little is known about how immature cell monolayers respond to tensile strain. To study the effect of tensile strain on the morphology of cells in immature monolayers, we developed a miniaturized stretching device that allows stable real-time imaging of changes in cell shape induced by uniaxial static stretching of up to 20%. Upon stretching, the shape of cells in the immature (2 day-cultured) monolayer changed dynamically, unlike in the mature (7 day-cultured) monolayer. Dynamic changes in the immature cell monolayers induced by tensile strain can be explained by the increase in di-phosphorylated myosin light chain (pp-MLC), which initiates actomyosin contraction. We suggest that our stretching-observation system can be used to investigate the morphological response of epithelial cell monolayers to tensile strain.

Human papillomavirus (HPV) causes cervical cancer, anal cancer, vulvar cancer, vaginal cancer, penile cancer, and oropharyngeal cancer. Squamous cell carcinoma (SCC) in the genital region in particular is recognized to be caused by HPV infection, and intraepithelial lesions of the penis and vulva are termed penile intraepithelial neoplasia and vulvar intraepithelial neoplasia, respectively. Although SCC of the nail apparatus is recognized as being associated with high-risk HPVs, it is not well-known in general medicine, and its analysis has been insufficient. In this article, we reviewed 136 cases of HPV-associated nail SCC and SCC in situ and delineated their clinical characteristics. We found that half of the cases were high-risk HPV-associated. Almost all of the types were high-risk α-HPVs. This disease had a male dominance and left hand digit 3 and right hand digits 1-3 were typically affected. In this review, 24% of the cases of nail SCC had a history of other HPV-associated diseases, suggesting the possibility of genitodigital transmission. We propose that nail SCC is a hidden high-risk HPV-associated reservoir and should be recognized as a sexually transmitted infection.

Claudins are cell-cell adhesion molecules located at the tight junctions (TJs) between cells in epithelial cell sheets. The claudin family in mammals consists of 27 four-transmembrane domain proteins. Claudins are responsible for the paracellular barrier function of TJs, and in some cases confer paracellular channel functions to the paracellular barriers of TJs. Based on recent breakthroughs in the molecular structure of claudins, the hypothetical 'antiparallel double row model' was proposed, which suggests how claudins polymerize in a linear fashion and form TJ strands with paracellular barrier and channel functions. Meanwhile, ongoing studies at the cell and tissue levels are clarifying how the paracellular barrier and/or channel functions of claudin-based TJs, which are both robust and flexible, organize various biological systems.

Background & Aims Most cholesterol gallstones have a core consisting of inorganic and/or organic calcium salts, although the mechanisms of core formation are poorly understood. We examined whether the paracellular permeability of ions at hepatic tight junctions is involved in the core formation of cholesterol gallstones, with particular interest in the role of phosphate ion, a common food additive and preservative. Methods We focused on claudin-3 (Cldn3), a paracellular barrier-forming tight junction protein whose expression in mouse liver decreases with age. Since Cldn3-knockout mice exhibited gallstone diseases, we used them to assess the causal relationship between paracellular phosphate ion permeability and the core formation of cholesterol gallstones. Results In the liver of Cldn3-knockout mice, the paracellular phosphate ion permeability through hepatic tight junctions was significantly increased, resulting in calcium phosphate core formation. Cholesterol overdose caused cholesterol gallstone disease in these mice. Conclusion We revealed that in the hepatobiliary system, Cldn3 functions as a paracellular barrier for phosphate ions, to help maintain biliary ion homeostasis. We provide in vivo evidence that elevated phosphate ion concentrations play a major role in the lifestyle- and age-related risks of developing cholesterol gallstone disease under cholesterol overdose. Lay summary Herein, we reveal a new mechanism for cholesterol gallstone formation, in which increased paracellular phosphate ion permeability across hepatobiliary epithelia causes calcium phosphate core formation and cholesterol gallstones. Thus, altered phosphate ion metabolism under cholesterol overdose plays a major role in the lifestyle- and age-related risks of developing cholesterol gallstone disease.

Melatonin (MLT), a hormone secreted from the pineal gland, is recognized as a potential candidate for stimulation of bone regeneration. However, because of its hydrophobicity, the administration of MLT to stimulate bone regeneration is difficult. In this study, an inclusion complex of MLT with 2-hydroxypropyl β-cyclodextrin (HP-β-CD) was prepared to improve the water solubility, and the osteogenic differentiation ability of the inclusion complex was investigated in MC3T3-E1 cells. The formation of HP-β-CD/MLT inclusion complex was confirmed by 1H and 13C nuclear magnetic resonance spectroscopy and wide-angle X-ray diffraction. The water solubility of MLT increased linearly upon addition of HP-β-CD because of the formation of the inclusion complex. Additionally, treatment of the cells with HP-β-CD/MLT inclusion complex showed higher uptake amount of MLT than that treated with free MLT. In addition, treatment of MC3T3-E1 cells with HP-β-CD/MLT inclusion complex increased alkaline phosphatase activity and mineralized matrix deposition, compared to that in free MLT-treated and untreated cells. Furthermore, cells treated with HP-β-CD/MLT inclusion complex exhibited higher expression levels of osteogenic differentiation genes than those in the untreated and free MLT-treated cells. Accordingly, these results suggested that inclusion complexation of MLT with HP-β-CD would be a potential formulation for bone regeneration because of its improved solubility and enhanced osteogenic differentiation efficiency.

Autophagy plays a pivotal role in the development and prevention of numerous diseases, and the induction of autophagy is regarded as a potential therapeutic approach for intractable diseases. In this study, the induction of autophagy by methylated β-cyclodextrins (Me-β-CDs)-threaded acid-labile polyrotaxane (Me-PRX) that can release the threaded Me-β-CDs in response to acidic pH in lysosomes was investigated. We hypothesized that the Me-β-CDs released from the Me-PRX interact with the membrane of organelles and cause autophagy. The Me-PRX preferentially accumulated in endoplasmic reticulum (ER) and caused ER stress, which was confirmed by gene expression analysis and the expression of an ER stress-marker protein. Accompanying the ER stress, cells treated with Me-PRX showed autophagy, which was not observed in cells treated with non-labile Me-PRX, other chemically modified PRXs, or free Me-β-CD. Furthermore, the Me-PRX treatment induced autophagic cell death and caused cell death even in apoptosis-resistant cells. Overall, this study demonstrates that the acid-labile Me-PRX induces ER stress-mediated autophagic cell death, and the Me-PRX would be a promising candidate to induce effective cell death in apoptosis-resistant malignant tumors.

Niemann-Pick type C (NPC) disease is a fatal metabolic disorder characterized by the lysosomal accumulation of cholesterol. Although 2-hydroxypropyl β-cyclodextrin (HP-β-CD) promotes the excretion of cholesterol and prolongs the life span in animal models of NPC disease, it requires extremely high dose. We developed acid-labile β-CD-based polyrotaxanes (PRXs) comprising multiple β-CDs threaded along a polymer chain capped with acid-cleavable stopper molecules for potentiating therapeutic efficacy of β-CD in NPC disease. The acid-labile PRXs dissociate under the acidic lysosomes and release threaded β-CDs in lysosomes, which promotes cholesterol excretion in NPC disease model cells at lower concentration than HP-β-CD. In this study, the therapeutic effect of the PRXs in a mouse model of NPC disease was investigated. Weekly administration of the PRXs significantly prolonged the life span and suppressed neurodegeneration in mice, even at a dose of 500mg/kg, a markedly lower dose than previously reported for HP-β-CD. Detailed analysis of tissue cholesterol revealed that PRX treatment markedly suppressed the tissue accumulation of cholesterol in the NPC mouse model, but did not alter cholesterol content in wild-type mice. Acid-labile PRX is therefore a promising candidate for potentiating the efficacy of β-CD in the treatment of NPC disease.

Dipole layers can be formed at epitaxial interfaces of perovskite oxides by intentionally inserting charged atomic layers. Perovskite oxides such as SrTiO3 (STO) can provide dielectrics with very high dielectric constants, but their bandgaps are so small that the suppression of leakage current is one of the critical issues when they are applied for DRAM capacitors. However, their conduction band offset will be manipulated if we can control the dipole layer formation. In this study, we investigated the opportunities of band alignment manipulation using the interface dipole effect especially focusing on the number of charge-introducing atomic layers. STO/LaAlO3 (LAO)/SrRuO3(SRO) stacks were fabricated by pulsed laser deposition method, to demonstrate the modulation of the band alignment at STO/SRO interface as large as ± 0.5 eV, when we change the number of inserted atomic layers of LAO.

Pneumothorax is a possible complication of tracheostomy. We report a rare case of bilateral pneumothoraces resulting from tracheostomy in an advanced laryngeal cancer patient. A 59-year-old man was referred to our clinic for evaluation and treatment of laryngeal tumor. Laryngeal endoscopy showed limited movement of bilateral vocal cords, and computed tomography revealed a tumor lesion extending from the vocal cords to the subglottic area. Three days after the first visit, the patient developed respiratory difficulty, and we elected to perform emergency tracheostomy for airway management. Immediately after the start of the procedure, he began hyperventilating, and complained of respiratory discomfort and chest pain. We then recognized a mediastinal air leak, and we suspected pneumothorax resulting from the tracheostomy. Chest X-ray showed bilateral pneumothoraces; therefore, we inserted bilateral chest drainage tubes, which stabilized his respiratory condition. We speculated that the pathogenesis of the bilateral pneumothoraces was weakened alveolar walls secondary to long-term smoking, and a significant rise in airway pressure because of airway constriction by the neck-extended position and hyperventilation, during tracheostomy.

Genome-wide association studies and subsequent deep sequencing analysis have identified susceptible loci for inflammatory bowel diseases (IBDs) including ulcerative colitis (UC). A gene encoding RING finger protein 186 (RNF186) is located within UC-susceptible loci. However, it is unclear whether RNF186 is involved in IBD pathogenesis. Here, we show that RNF186 controls protein homeostasis in colonic epithelia and regulates intestinal inflammation. RNF186, which was highly expressed in colonic epithelia, acted as an E3 ligase mediating polyubiquitination of its substrates. Permeability of small organic molecules was augmented in the intestine of Rnf186-/- mice. Increased expression of several RNF186 substrates, such as occludin, was found in Rnf186-/- colonic epithelia. The disturbed protein homeostasis in Rnf186-/- mice correlated with enhanced endoplasmic reticulum (ER) stress in colonic epithelia and increased sensitivity to intestinal inflammation after dextran sulfate sodium (DSS) treatment. Introduction of an UC-associated Rnf186 mutation led to impaired E3 ligase activity and increased sensitivity to DSS-induced intestinal inflammation in mice. Thus, RNF186 maintains gut homeostasis by controlling ER stress in colonic epithelia.

Five series of acylated polyrotaxanes (PRXs) with different acyl groups (acetyl, propionyl, butyryl, valeryl, and benzoyl) were synthesized to investigate their solubility in aqueous solutions and the formation and stability of self-assembled nanoparticles. Acetylated PRXs (Ac-PRXs), propionylated PRXs (Pr-PRXs), and butyrylated PRXs (Bu-PRXs) dissolved in water, and yielded transparent solutions at a low degree of substitution, whereas nanoparticle formation was observed when the degree of substitution exceeded the threshold values (34% for Ac-PRXs, 18% for Pr-PRXs and 11% for Bu-PRXs). Pr-PRX and Bu-PRX nanoparticles exhibited low critical micelle concentration compared to Ac-PRXs. Valeryl or benzoyl group-modified PRXs precipitated in aqueous solutions due to their strong hydrophobicity. The loading efficiency of hydrophobic drugs in Pr-PRX nanoparticles improved significantly compared to those in Ac-PRX nanoparticles. Collectively, the moderate hydrophobicity of the acyl groups is optimal for the formation of stable self-assembled nanoparticles in aqueous solutions and efficient encapsulation of hydrophobic drugs.

Noninvasive low-level laser therapy (LLLT) is neuroprotective, but the mechanism of this effect is not fully understood. In this study, the use of LLLT as a novel treatment for noise-induced hearing loss (NIHL) is investigated. Sprague-Dawley rats were exposed to intense noise and their right ears were irradiated with an 808nm diode laser at an output power density of 110 or 165mW/cm(2) for a 30min period for 5 consecutive days. Measurement of the auditory brainstem response revealed an accelerated recovery of auditory function in the groups treated with LLLT compared with the non-treatment group at days 2, 4, 7 and 14 after noise exposure. Morphological observations also revealed a significantly higher outer hair cell survival rate in the LLLT groups. Immunohistochemical analyses for inducible nitric oxide synthase (iNOS) and cleaved caspase-3 were used to examine oxidative stress and apoptosis. Strong immunoreactivities were observed in the inner ear tissues of the non-treatment group, whereas these signals were decreased in the LLLT group at 165mW/cm(2) power density. Our findings suggest that LLLT has cytoprotective effects against NIHL via the inhibition of iNOS expression and apoptosis.

Background Forest management strategies such as thinning have long been used to enhance ecosystem functions, especially in plantations. Thinning in plantations with high deer density, however, may not yield a desired increase in understory vegetation because deer graze on germinating plants after thinning. Here, we examine the changes in understory vegetation after thinning in plantations that have been overgrazed by sika deer to provide insight into the effects of thinning on ecosystem functions such as soil conservation and biological diversity. Methods We conducted our survey in the Tanzawa Mountains of eastern Japan. We surveyed the change in understory vegetation within and outside of three deer exclosures on a single slope with three levels of understory vegetation cover: sparse (1%, exclosure “US”), moderate (30%, exclosure “MM”), and dense (80%, exclosure “LD”) over 10 years after a 30% thinning of an old-growth cedar and cypress plantation which was overgrazed by sika deer. Results Understory vegetation cover, biomass and species richness increased within and outside the “US” and “MM” exclosures after thinning, and biomass was greater within than outside the exclosures at 10 years after thinning. Unpalatable species dominated both “US” and “MM” exclosures before thinning, and trees and shrubs dominated within the exclosures over time after thinning. In contrast, unpalatable, grazing-tolerant, perennial, and annual species increased outside the “US” and “MM” exclosures. No noticeable changes were observed within and outside the “LD” exclosure when compared with the “US” and “MM” exclosures. Conclusions Our results suggest that thinning a stand by 30% based on volume resulted in an increase in understory vegetation cover mainly composed of both unpalatable and grazing-tolerant species in a plantation forest where understory vegetation is sparse or moderate and sika deer density is high. We emphasize that establishing deer exclosures or controlling deer is essential to maintaining similar understory vegetation both within and outside exclosures.

Claudin was first identified as a four-transmembrane protein in the tight junctions (TJs) between epithelial cells. The claudin family has 27 members, which are specifically expressed depending on the epithelial cell type. Accumulating evidence has revealed that claudins are responsible for the paracellular barrier that prevents molecules from passing through epithelial cell sheets. In addition, the extracellular domains of some claudins enable them to act as a permselective paracellular channel for specific molecules, including ions and/or non-ionic solutes. Recent studies using claudin knockout mice revealed that the loss of claudins' specific paracellular barrier and/or channel functions affects specific biological functions and leads to pathological states. In this review, considering recent findings in vivo, we describe how, sometimes in concert with canonical transporters and channels, the paracellular barrier and channel functions of claudins sophisticatedly organize biological systems.

To achieve successful delivery of siRNA therapeutics, cytocleavable cationic polyrotaxanes (PRXs) composed of N,N-dimethylaminoethyl (DMAE) group-modified α-cyclodextrins (CDs) that were threaded onto a poly(ethylene glycol) (PEG) axis and capped with a bulky stopper using cytocleavable disulfide linkages (DMAE-PRX) were utilized as an siRNA carrier. DMAE-PRXs with various numbers of threading CDs and modified DMAE groups were synthesized, and the physicochemical properties, cellular internalization, and gene silencing activity of DMAE-PRX/siRNA were investigated to elucidate the relationship between its supramolecular structure and its function. When the numbers of modified DMAE groups were increased, the DMAE-PRXs formed closely associated polyplexes with siRNA and increased their polyanion exchange resistance. Additionally, the DMAE-PRXs with 52 threading CDs (52CD-PRXs) showed greater binding capabilities with siRNA and greater resistance to polyanion competition than 31CD-PRXs, indicating that the highly CD-threaded PRX structure in the 52CD-PRXs is superior in forming stable polyplexes with siRNA. Indeed, 52CD-PRX/siRNA showed greater intracellular uptake of siRNA than 31CD-PRX/siRNA with comparable numbers of DMAE groups. 52CD-PRX/siRNA successfully induced gene silencing of a targeted luciferase expressed in human cervical carcinoma without marked cytotoxicity and non-specific gene silencing. Although the gene silencing activities of DMAE-PRX/siRNA were comparable to those of linear poly(ethylenimine) (L-PEI), L-PEI showed cytotoxicity and non-specific gene silencing. Additionally, DMAE-PRXs with cytocleavable capabilities were found to enhance gene silencing, in comparison with non-cleavable DMAE-PRX. Thus, the cytocleavable cationic PRXs are suggested to be attractive supermolecules for the delivery of therapeutic siRNAs.

Multicellular neuronal activities should be investigated to reveal the dynamics of the neuronal circuit. Optical recording from neuronal populations is suitable for recording multicellular activities. We fabricated the prototype of the micro-imaging probe in combination with a gradient index lens and image fiber. This probe has a smaller diameter than traditional probes. We found an optimal optical configuration for maximizing the efficiency of the imaging probe. Using this optical configuration with the prototype of the imaging probe, the fluorescence images were captured from neurons expressing green fluorescent protein in a cerebellar block preparation, and the calcium-dependent images were sampled in a mouse brain slice preparation. Our optical system would facilitate the in vivo imaging studies with less invasive manners using thinner optic fiber than previously made.

Tight junctions (TJs) connect epithelial cells and form a semipermeable barrier that only allows selective passage of ions and solutes across epithelia. Here we show that mice lacking EpCAM, a putative cell adhesion protein frequently overexpressed in human cancers, manifest intestinal barrier defects and die shortly after birth as a result of intestinal erosion. EpCAM was found to be highly expressed in the developing intestinal epithelium of wild-type mice and to localize to cell–cell junctions including TJs. Claudin-7 colocalized with EpCAM at cell–cell junctions, and the two proteins were found to associate with each other. Claudins 2, 3, 7, and 15 were down-regulated in the intestine of EpCAM mutant mice, with claudin-7 being reduced to undetectable levels. TJs in the mutant intestinal epithelium were morphologically abnormal with the network of TJ strands scattered and dispersed. Finally, the barrier function of the intestinal epithelium was impaired in the mutant animals. These results suggest that EpCAM contributes to formation of intestinal barrier by recruiting claudins to cell–cell junctions.

Cell cultivation on the surface of microcarriers in stirred suspension is an essential method for the large-scale culture of anchorage-dependent cells. For applying this method to the field of cell therapy and for obtaining a large number of intact cells, non-invasive cell harvest without proteolytic enzyme treatment is an advantageous method. In this regard, temperature-responsive microcarriers that bearing poly(N-isopropylacrylamide) (PIPAAm)-grafted chains on the outermost surface were developed for harvesting cultured cells by temperature alteration. PIPAAm-grafted beads with the various grafted amount of PIPAAm and various bead diameters were synthesized for optimizing cell proliferation and thermally-induced detachment on the surface. The chinese hamster ovary (CHO-K1) cells adhered on the surface of all PIPAAm-grafted beads at 37 °C, while the adhering cells were found to detach themselves from the surfaces at 20 °C. The efficiency of thermally-induced cell detachment increased with increasing the grafted amount of PIPAAm and the diameter of bead. An efficient cell proliferation on bead surfaces in stirred suspension culture and subsequent thermally-induced cell detachment were achieved by the precise regulation of both the grafted amount of PIPAAm and diameter of bead. The temperature-responsive microcarriers exhibiting temperature-dependent cell adhesion and detachment will be an attractive candidate for the large-scale cell culture of therapeutic cells.

The biocompatibility and body distribution of PEGylated polyamine nanogels composed of chemically cross-linked poly(2-N,N-(diethylamino)ethyl methacrylate) (PEAMA) gel cores surrounded by poly(ethylene glycol) (PEG) chains were investigated to evaluate their feasibility as drug nanocarriers for systemic administration. PEGylated nanogels with different cross-linking densities (1, 2, and 5 mol.%) were prepared to evaluate their biocompatibilities by in vitro cytotoxicity assay, hemolysis assay, and in vivo acute toxicity assay. The toxic effect of the PEGylated nanogels derived from polyamine gel cores was significantly reduced when the cross-linking density was increased, and those with a cross-linking density of 5 mol.% showed a remarkably high median lethal dose (LD50) value >200 mg kg−1,despite the abundance of amino groups in the core. One hour after intravenous injection the PEGylated nanogels were found to have been eliminated from the systemic circulation, and less than 1% of the injected dose (ID) remained in the bloodstream. To improve the blood circulation time by increasing the surface PEG density of the PEGylated nanogels post-PEGylation of the PEGylated nanogels (via the Menschutkin reaction between tertiary amines of the PEAMA gel core and bromobenzyl-terminated short PEG) was carried out. A biodistribution study of these post-PEGylated nanogels revealed that the blood circulation time of the nanogels was definitely prolonged as the PEG content was increased. Therefore, the precise design of PEGylated nanogels with increased cross-linking densities in their polyamine gel cores and increased surface PEG densities seems promising for systemic applications.

Background & Aims In the small intestine, the paracellular transport of Na + is thought to be critical for luminal Na + -homeostasis and the transcellular absorption of nutrients by Na + -driven transporters. Na + is supplied to the intestinal lumen from the submucosa and serum through tight junctions, which form a paracellular barrier between the cells of epithelial sheets. However, the molecular basis for this paracellular transport of Na + is not well understood. Here, we examined this mechanism by performing loss-of-function studies of claudin-2 and claudin-15, two tight-junctional membrane proteins that are specifically and age-dependently expressed in the villi and/or crypts of small intestinal epithelia. Methods Knockout mice for claudin-2 or claudin-15 were subjected to histologic, cell biologic, electrophysiologic, and physiologic analyses. Results Examination of the knockout mice revealed that both claudin-2 and claudin-15 play crucial roles in the transepithelial paracellular channel-like permselectivity for extracellular monovalent cations, particularly Na + , in infants and adults. Especially in Cldn15 −/− adults, the luminal Na + concentration in the small intestine measured directly in vivo was abnormally low, and glucose absorption was impaired, as assessed by the oral glucose tolerance test and estimation of unabsorbed glucose. Conclusions We propose that the "Na + -leaky" claudin-15 is indispensable in vivo for the paracellular Na + permeability, luminal Na + -homeostasis, and efficient glucose absorption in the small intestine, but claudin-2 is indispensable for only the first of these functions. Claudin-15 knockout leads to Na + deficiency and glucose malabsorption in the mouse adult small intestine.

ERM (ezrin/radixin/moesin) proteins are organizers of apical actin cortical layer in general. We previously reported that the knockout of radixin resulted in Rdx − / − mice with displacement/loss of the canalicular transporter Mrp2, giving rise to Dubin–Johnson syndrome-like conjugated hyperbilirubinemia in the mixed genetic background (C57BL/6-129/Sv) (Kikuchi, et al. (2002) Nature Genetics 31, 320–325). However, when these mice were kept under mixed genetic background for years (late mixed backgrounds; LMB), the conjugated hyperbilirubinemia gradually became inconspicuous, while evidence of liver injury increased. We examined the effect of genetic background by backcrossing LMB Rdx − / − mice to C57BL/6 and 129/Sv wild type mice with the result that the Rdx − / − congenic mice regained hyperbilirubinemia with reduced hepatocellular damage. As revealed by immunofluorescence and western blots, the localization/expression of apical transporters, Mrp2, CD26, P-gps, and Bsep were not influenced by backcrossing, though those of a basolateral transporter, Mrp3, were strikingly increased by backcrossing.

A series of 1beta-methylcarbapenems bearing an (imidazo[5,1-b]thiazolium-6-yl)methyl moiety, a 5,5-fused heterobicycle, at the C-2 position was synthesized and evaluated for in vitro antibacterial activities. CP0569 (1r) and its analogues showed potent antibacterial activities against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), and Gram-negative bacteria, including Pseudomonas aeruginosa. Moreover, CP0569 (1r) exhibited stronger antibacterial activity against MRSA and higher resistance to renal dehydropeptidase-1 (DHP-1) than any currently marketed carbapenems, that is, imipenem (IPM), panipenem (PAPM), and meropenem (MEPM).

Diarrhea is a host response to enteric pathogens, but its impact on pathogenesis remains poorly defined. By infecting mice with the attaching and effacing bacteria Citrobacter rodentium, we defined the mechanisms and contributions of diarrhea and intestinal barrier loss to host defense. Increased permeability occurred within 2 days of infection and coincided with IL-22-dependent upregulation of the epithelial tight junction protein claudin-2. Permeability increases were limited to small molecules, as expected for the paracellular water and Na+ channel formed by claudin-2. Relative to wild-type, claudin-2-deficient mice experienced severe disease, including increased mucosal colonization by C. rodentium, prolonged pathogen shedding, exaggerated cytokine responses, and greater tissue injury. Conversely, transgenic claudin-2 overexpression reduced disease severity. Chemically induced osmotic diarrhea reduced colitis severity and C. rodentium burden in claudin-2-deficient, but not transgenic, mice, demonstrating that claudin-2-mediated protection is the result of enhanced water efflux. Thus, IL-22-induced claudin-2 upregulation drives diarrhea and pathogen clearance.

The function of ERM (ezrin/radixin/moesin) proteins as general cross-linkers between actin filaments and plasma membranes is regulated downstream of Rho, through the transition between active and inactive forms. To directly examine the conformational change between the active and inactive forms of ERM proteins, we applied low-angle rotary-shadowing electron microscopy to the radixin molecules, wild-type, T564A-non-phosphorylated-type, and T564E-phosphorylated-type, since most of the active forms are reportedly stabilized in cells by the C-terminal threonine phosphorylation. As a result, the T564A- and wild-type radixin molecules yielded the globular closed forms, ∼8-14 nm in diameter, with some striations on their surfaces. In contrast, the T564E-radixin molecules tended to take elongated open forms, in which two globular structures measuring ∼8 nm and ∼5 nm in diameter were associated with both ends of the filamentous structures. The filamentous structure took either a ∼20-25 nm-long straight course or a folded course. Taken together with the biochemical and the crystal structural results obtained to date, the closed and open forms represent the inactive and active forms of radixin as cross-linkers between actin filaments and plasma membranes.

A series of cephalosporin derivatives with various bicyclic heterocycles at the C-3 position was synthesized and evaluated for antibacterial activity. Among them CP0467 (3a) showed excellent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) (MIC90 = 6.25 microg/mL), and extremely high affinity for the penicillin binding protein 2' of MRSA (I50 = 0.49 microg/mL). Furthermore, 3a showed a long-acting pharmacokinetic profile in mice (AUC(infinity) = 482.3 microg/h/mL and T(1/2) = 1.9 h).

Secretion of insulin from pancreatic the s-cells and a mouse insulinoma cell line MIN6 with glucose stimulation results in an exocytotic reaction through membrane depolarization and an increase of intracellular Ca 2+ concentration ([Ca 2+ ] i ). To investigate the relationship between membrane depolarization and intracellular calcium mobilization in a mouse insulinoma cell line, MIN6, we developed a simultaneous double-probe imaging video-microscope system, which could simultaneously detect two different kinds of fluorescent signals from a single cell. When MIN6 cells, which retain glucose-induced insulin secretion like pancreatic s-cells, were stimulated by increasing the extracellular concentration of glucose from 5 to 25 mM, the membrane potential was quickly increased after the stimulation, but elevation of [Ca 2+ ] i was observed ca. 40 s after the increase in membrane potential. In the case of high K + -stimulation, such a time lag was not observed. When the extracellular concentration of glucose was increased stepwise from 5 to 25 mM, the number of cells which showed change of both membrane potential and [Ca 2+ ] i was increased, indicating that MIN6 cells show the so-called all-or-none-type of response. It was also found that cells which formed colonies responded to glucose stimulus. This method would be useful not only for investigating the detailed relationship between membrane depolarization and intracellular calcium mobilization in MIN6 cells as well as in the pancreatic s-cells, but also for studying the signaling mechanisms in many kinds of cells.

The solute trapping procedure onto a bovine serum albumin (BSA)-coated ODS pre-column was directly observed with a chromato-videoscope. The fluorescence image of dansyl (DNS) amino acids along the column was detected by a video camera and the images were digitized and analyzed as densitograms by an image processor. DNS-amino acids were retained at the top of pre-column and formed a distinctive band when phosphate-buffered saline (PBS) was used as a mobile phase. However, the shape of the solute bands was affected by the presence of BSA in the sample solution and also by the flow rate.

The determination of dissolved oxygen in water samples was performed by capillary electrophoresis. This was based on the oxidation of pyrogallol in alkaline media by dissolved oxygen. The oxygen sample was injected into the capillary electrophoresis system consisting of phosphate buffer at pH 5.6 and 0.5 M pyrogallol. Electrophoretic mixing of sample and reagent was achieved during the electrophoretic run and the reaction product was monitored at 440 nm. The assay procedure was optimized with respect to the carrier electrolyte concentration and pH, pyrogallol concentration, sample pH, and modes of operation of the applied voltage. As little as 20 ppb of the dissolved oxygen could be determined by the proposed method within 10 min. The method was successfully applied for the determination of dissolved oxygen in river and drinking water.

The determination of seven food additive dyestuffs was investigated by capillary electrophoresis. When β-cyclodextrin was introduced into the carrier electrolyte, the apparent mobility was increased, leading to 9.5–39% lower migration times due to the increase in the solute's mass after inclusion complex formation. The reproducibility and peak shape were improved because interaction between the solute and the capillary wall was alleviated. The effects of β-cyclodextrin on the migration time, elution order, peak shape and reproducibility of food additive dyestuffs are discussed in terms of providing a considerable advantage for determining organic anions by capillary electrophoresis. Sequential injection of dyestuffs and β-cyclodextrin into a capillary electrophoresis column was found to be a simple and rapid method for a qualitative comparative study of inclusion complexation phenomena.

Background & Aims Bile formation and secretion are essential functions of the hepatobiliary system. Bile flow is generated by transepithelial transport of water and ionic/nonionic solutes via transcellular and paracellular pathways that is mainly driven by osmotic pressure. We examined the role of tight junction–based paracellular transport in bile secretion. Claudins are cell–cell adhesion molecules in tight junctions that create the paracellular barrier. The claudin family has 27 reported members, some of which have paracellular ion- and/or water-channel–like functions. Claudin 2 is a paracellular channel-forming protein that is highly expressed in hepatocytes and cholangiocytes; we examined the hepatobiliary system of claudin 2 knockout ( Cldn2 −/− ) mice. Methods We collected liver and biliary tissues from Cldn2 −/− and Cldn2 +/+ mice and performed histologic, biochemical, and electrophysiologic analyses. We measured osmotic movement of water and/or ions in Cldn2 −/− and Cldn2 +/+ hepatocytes and bile ducts. Mice were placed on lithogenic diets for 4 weeks and development of gallstone disease was assessed. Results The rate of bile flow in Cldn2 −/− mice was half that of Cldn2 +/+ mice, resulting in significantly more concentrated bile in livers of Cldn2 −/− mice. Consistent with these findings, osmotic gradient-driven water flow was significantly reduced in hepatocyte bile canaliculi and bile ducts isolated from Cldn2 −/− mice, compared with Cldn2 +/+ mice. After 4 weeks on lithogenic diets, all Cldn2 −/− mice developed macroscopically visible gallstones; the main component of the gallstones was cholesterol (>98%). In contrast, none of the Cldn2 +/+ mice placed on lithogenic diets developed gallstones. Conclusions Based on studies of Cldn2 −/− mice, claudin 2 regulates paracellular ion and water flow required for proper regulation of bile composition and flow. Dysregulation of this process increases susceptibility to cholesterol gallstone disease in mice.

Background & Aims: Claudins, the major components of tight junction (TJ) strands, which form paracellular barriers, consist of 24 family members, the combination of which determines the properties of TJ-based paracellular barriers. Here, we generated claudin-15–deficient ( Cldn15 −/− ) mice to examine the ubiquitously expressed functions of claudin-15. Methods: We generated Cldn15 −/− mice by the conventional gene-targeting strategy. Because the upper small intestine was enlarged in Cldn15 −/− mice, we analyzed the phenotype from various angles regarding histology, physiology, and cell biology. Results: Cldn15 −/− mice were born and grew normally with an enlarged upper small intestinal phenotype, megaintestine. Deficiency of claudin-15 did not cause a compensatory increase in the background expression of other types of claudins, claudin-1, -2, -3, -4, -7, -12, -18, -20, and -23, in the small intestine. Cldn15 −/− mice showed enhanced proliferation of normal cryptic cells after weaning without diseased states such as polyps or cancer, resulting in megaintestine, in which the upper small intestine was approximately 2 times larger than normal in length and diameter. The number of transit-amplifying cells in crypts increased ∼2-fold. Freeze-fracture electron microscopy revealed that deficiency of claudin-15 decreased the number of TJ strands, although the electric conductance was decreased in distal segments in Cldn15 −/− jejunum, as compared with Cldn15 +/+ littermates. Conclusions: Based on the specific roles of claudins in paracellular barrier formation without any direct role in cell proliferation, as previously shown in cultured epithelial cells, we propose that claudin-15–based formation of TJs to organize the microenvironment including ion conductance is important for normal-sized morphogenesis of the small intestine.

A new indole-N-glycoside, neosidomycin (I), is produced by a strain belonging to Streptomyces hygroscopicus. The structure of I has been deduced from physico-chemical data obtained using the natural compound, its derivatives and products of degradation reactions.