Three species of otter can be found throughout Malay Peninsula: Aonyx cinereus, Lutra sumatrana, and Lutrogale perspicillata. In this study, we focused on the A. cinereus population that ranges from the southern and the east coast to the northern regions of Malay Peninsula up to southern Thailand to review the relationships between the populations based on the mitochondrial D-loop region. Forty-eight samples from six populations were recognized as Johor, Perak, Terengganu, Kelantan, Ranong, and Thale Noi. Among the 48 samples, 33 were identified as A. cinereus, seven as L. sumatrana, and eight as L. perspicillata. Phylogenetically, two subclades formed for A. cinereus. The first subclade grouped all Malay Peninsula samples except for samples from Kelantan, and the second subclade grouped Kelantan samples with Thai sample. Genetic distance analysis supported the close relationships between Thai and Kelantan samples compared to the samples from Terengganu and the other Malaysian states. A minimum-spanning network showed that Kelantan and Thailand formed a haplogroup distinct from the other populations. Our results show that Thai subspecies A. cinereus may have migrated to Kelantan from Thai mainland. We also suggest the classification of a new subspecies from Malay Peninsula, the small-clawed otter named A. cinereus kecilensis.

The study shows constitutive activation of the Notch pathway in various types of malignancies. However, it remains unclear how the Notch pathway is involved in the pathogenesis of osteosarcoma. We investigated the expression of the Notch pathway molecules in osteosarcoma biopsy specimens and examined the effect of Notch pathway inhibition. Real-time PCR revealed overexpression of Notch2, Jagged1, HEY1, and HEY2. On the other hand, Notch1 and DLL1 were downregulated in biopsy specimens. Notch pathway inhibition using gamma-secretase inhibitor and CBF1 siRNA slowed the growth of osteosarcomas in vitro. In addition, gamma-secretase inhibitor-treated xenograft models exhibited significantly slower osteosarcoma growth. Cell cycle analysis revealed that gamma-secretase inhibitor promoted G1 arrest. Real-time PCR and western blot revealed that gamma-secretase inhibitor reduced the expression of accelerators of the cell cycle, including cyclin D1, cyclin E1, E2, and SKP2. On the other hand, p21(cip1) protein, a cell cycle suppressor, was upregulated by gamma-secretase inhibitor treatment. These findings suggest that inhibition of Notch pathway suppresses osteosarcoma growth by regulation of cell cycle regulator expression and that the inactivation of the Notch pathway may be a useful approach to the treatment of patients with osteosarcoma.

The Antarctic continental margin supplies the densest bottom water to the global abyss. From the late twentieth century, an acceleration in the long-term freshening of Antarctic Bottom Waters (AABW) has been detected in the Australian-Antarctic Basin. Our latest hydrographic observations reveal that, in the late 2010s, the freshening trend has reversed broadly over the continental slope. Near-bottom salinities in 2018-2019 were higher than during 2011-2015. Along 170° E, the salinity increase between 2011 and 2018 was greater than that observed in the west. The layer thickness of the densest AABW increased during the 2010s, suggesting that the Ross Sea Bottom Water intensification was a major source of the salinity increase. Freshwater content on the continental slope decreased at a rate of 58 ± 37 Gt/a in the near-bottom layer. The decadal change is very likely due to changes in Ross Sea shelf water attributable to a decrease in meltwater from West Antarctic ice shelves for the corresponding period.

To examine the effects of oxidants on the airway epithelial barrier functions, human tracheal epithelial cells were cultured on porous filter membrane. Glucose oxidase (GO; 10 U/ml), hydrogen peroxide (H2O2; 4 x 10(-3) M), and xanthine (5 x 10(-4) M) plus xanthine oxidase (20 mU/ml) (X-XO) significantly increased electrical conductance across epithelial membrane (G), short-circuit current (Isc) measured with Ussing's chamber methods, and [3H]mannitol flux through the cultured epithelium. Increases in G and Isc induced by oxidants were significantly inhibited by catalase (1,000 U/ml) and the protein kinase C inhibitor staurosporine (10(-7) M), but superoxide dismutase (SOD; 100 U/ml) was without effect. GO, H2O2, and X-XO inhibited the epithelial cell growth, [3H]thymidine incorporation by the cells, and epithelial repair of artificially produced focal epithelial defects (1-2 mm diam) on plastic vessels. Catalase also inhibited effects induced by oxidants on cell growth and proliferation. These results suggest that oxidants reduce tracheal epithelial barrier functions by damaging tight junctions and inhibiting cell proliferation, and these effects of oxidants on epithelial cells may be mediated by H2O2 rather than superoxide anion and by activation of protein kinase C.

Claudins, comprising a multigene family, constitute tight junction (TJ) strands. Clostridium perfringens enterotoxin (CPE), a single approximately 35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half. We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4. C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2. In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface. In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation. At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased. In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner. These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.

Atypical trajectory of brain growth in autism spectrum disorders (ASDs) has been recognized as a potential etiology of an atypical course of behavioral development. Numerous neuroimaging studies have focused on childhood to investigate atypical age-related change of brain structure and function, because it is a period of neuron and synapse maturation. Recent studies, however, have shown that the atypical age-related structural change of autistic brain expands beyond childhood and constitutes neural underpinnings for lifelong difficulty to behavioral adaptation. Thus, we examined effects of aging on neurochemical aspects of brain maturation using 3-T proton magnetic resonance spectroscopy ((1)H-MRS) with single voxel in the medial prefrontal cortex (PFC) in 24 adult men with non-medicated high-functioning ASDs and 25 age-, IQ- and parental-socioeconomic-background-matched men with typical development (TD). Multivariate analyses of covariance demonstrated significantly high N-acetylaspartate (NAA) level in the ASD subjects compared with the TD subjects (F=4.83, P=0.033). The low NAA level showed a significant positive correlation with advanced age in the TD group (r=-0.618, P=0.001), but was not evident among the ASD individuals (r=0.258, P=0.223). Fisher's r-to-z transformation showed a significant difference in the correlations between the ASD and TD groups (Z=-3.23, P=0.001), which indicated that the age-NAA relationship was significantly specific to people with TD. The current (1)H-MRS study provided new evidence that atypical age-related change of neurochemical aspects of brain maturation in ASD individuals expands beyond childhood and persists during adulthood.

Fetal fibroblast cell culture from cotyledons of bovine placenta and animal experiments close to term were used to elucidate afterbirth release and factors missing in the signal transduction mechanism for retained fetal membranes (RFM) after delivery. In cell culture the addition of arachidonic acid (Ara) to the medium caused rapid release to free floating cell in the culture dish, accompanied by matrix metalloproteinase (MMP) activation, being consistent with previous in vivo observations, where a relation between MMP and fetal membrane release had been shown. Ara-induced cell floating was not inhibited by the addition of cyclooxygenase (COX) inhibitor, and not induced by the addition of PGF2α or PGE2 to replace Ara, while 12-lipoxygenase (12-LOX) metabolite of Ara, 12-oxo-eicosatetraenoic acid (12-oxoETE), strongly induced cell floating. In the animal experiments, 12-oxoETE injection to delivery-induced cows (n = 6) using prostaglandin (PG) and dexamethazone resulted in rapid release of fetal membranes. In cows with natural calf delivery, a 12-oxoETE peak (11.7-16.8 ng/ml) was observed in maternal blood plasma prior to release of fetal membranes. This investigation thus gives new indications for that the mediator for fetal membrane release is 12-oxoETE and not PG.

In tight junctions (TJs), TJ strands are associated laterally with those of adjacent cells to form paired strands to eliminate the extracellular space. Claudin-1 and -2, integral membrane proteins of TJs, reconstitute paired TJ strands when transfected into L fibroblasts. Claudins comprise a multigene family and more than two distinct claudins are coexpressed in single cells, raising the questions of whether heterogeneous claudins form heteromeric TJ strands and whether claudins interact between each of the paired strands in a heterophilic manner. To answer these questions, we cotransfected two of claudin-1, -2, and -3 into L cells, and detected their coconcentration at cell-cell borders as elaborate networks. Immunoreplica EM confirmed that distinct claudins were coincorporated into individual TJ strands. Next, two L transfectants singly expressing claudin-1, -2, or -3 were cocultured and we found that claudin-3 strands laterally associated with claudin-1 and -2 strands to form paired strands, whereas claudin-1 strands did not interact with claudin-2 strands. We concluded that distinct species of claudins can interact within and between TJ strands, except in some combinations. This mode of assembly of claudins could increase the diversity of the structure and functions of TJ strands.

We generated a series of knockin mouse lines, in which the cytokine receptor gp130-dependent STAT3 and/or SHP2 signals were disrupted, by replacing the mouse gp130 gene with human gp130 mutant cDNAs. The SHP2 signal-deficient mice (gp130F759/F759 were born normal but displayed splenomegaly and lymphadenopathy and an enhanced acute phase reaction. In contrast, the STAT3 signal-deficient mice (gp130FXQ/FXXQ) died perinatally, like the gp130-deficient mice (gp130D/D). The gp130F759/F759 mice showed prolonged gp130-induced STAT3 activation, indicating a negative regulatory role for SHP2. Th1-type cytokine production and IgG2a and IgG2b production were increased in the gp130F759/F759 mice, while they were decreased in the gp130FXXQ/FXXQ immune system. These results indicate that the balance of positive and negative signals generated through gp130 regulates the immune responses.

The expression of a winged-helix transcription factor, Foxa2/HNF3beta, is essential for development of the node and the notochord. We examined the node/notochord enhancer of mouse Foxa2 for sequence motifs conserved across vertebrate species. We cloned Foxa2 genes from chicken and fish, and identified the respective node/notochord enhancers that were active in transgenic mouse embryos. Comparison of the sequences of the enhancers revealed three evolutionally conserved sequence motifs, CS1, CS2 and CS3. Mutational analysis of the mouse enhancer indicated that CS3 is indispensable for gene expression in the node and the notochord, while CS1 and CS2 are required to augment enhancer activity. These motifs do not correspond to the consensus binding sequences of transcription factors known to be involved in node/notochord development.

There are two strains of MDCK cells, MDCK I and II. MDCK I cells show much higher transepithelial electric resistance (TER) than MDCK II cells, although they bear similar numbers of tight junction (TJ) strands. We examined the expression pattern of claudins, the major components of TJ strands, in these cells: claudin-1 and -4 were expressed both in MDCK I and II cells, whereas the expression of claudin-2 was restricted to MDCK II cells. The dog claudin-2 cDNA was then introduced into MDCK I cells to mimic the claudin expression pattern of MDCK II cells. Interestingly, the TER values of MDCK I clones stably expressing claudin-2 (dCL2-MDCK I) fell to the levels of MDCK II cells (>20-fold decrease). In contrast, when dog claudin-3 was introduced into MDCK I cells, no change was detected in their TER. Similar results were obtained in mouse epithelial cells, Eph4. Morphometric analyses identified no significant differences in the density of TJs or in the number of TJ strands between dCL2-MDCK I and control MDCK I cells. These findings indicated that the addition of claudin-2 markedly decreased the tightness of individual claudin-1/4-based TJ strands, leading to the speculation that the combination and mixing ratios of claudin species determine the barrier properties of individual TJ strands.

Allergen-activated T cells secrete several kinds of bioactive lymphokines such as IL2, IL3, IL4, IL5 and IFN-gamma. They function as helpers in IgE production involved in immediate type hypersensitivity and/or effector cells in delayed type hypersensitivity in allergic patients. The acquisition of interleukin 2 (IL2) responsiveness by specific antigen-stimulated cells is generally an essential event for the induction of specific immunological phenomena. To investigate the immunological changes in asthmatic children in remission, the induction of IL2-responsiveness and production by Df (Dermatophagoides farinae)-stimulated patient lymphocytes, and Df-induced IFN-gamma production by patient lymphocytes were evaluated. The patients were divided into 3 groups. The remission group (I) consisted of those patients who had had no or only a few asthmatic attacks for more than 2 years without medication. The group of active asthma were divided into 2 groups according to attack frequency and severity (II, partial remission; III, active asthma). IL2 responsiveness and production by Df-stimulated lymphocytes from group II and III were increased. As symptoms improved, the extent of the response subsided to a level comparable to that of normal individuals (group III). IFN-gamma production by Df-stimulated lymphocytes from patients with active asthma was lower than that of normal lymphocytes. In contrast, lymphocytes from patients in complete remission group (I) induced far greater IFN-gamma generation than those from normal and group II and III patients in a Df antigen-dependent manner, which might downregulate Df-induced hyperreactivity for Df-mediated allergic response.(ABSTRACT TRUNCATED AT 250 WORDS)

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

To clarify whether the effect of sulfonylurea on glucagon secretion is directly on the pancreatic A cell, we examined changes produced by gliclazide in glucagon (IRG), insulin (IRI) and somatostatin (IRS) release from the isolated perfused rat pancreas. Under 5 mM glucose infusion, IRI and IRS were increased by gliclazide in a dose-dependent manner, but IRG was unchanged. When 20 mM arginine was infused to stimulate glucagon secretion, both IRI and IRG increased markedly in a biphasic fashion and IRS increased slightly. The administration of gliclazide at the time of second phase response of IRG, IRI and IRS increased further and IRG decreased at every dose used. Insulin administration to the control and streptozotocin-treated rat pancreas did not change arginine-induced IRG secretion. Gliclazide-induced glucagon suppression was also observed in streptozotocin-diabetic rat pancreas. The amount of administered somatostatin required for inhibiting glucagon secretion was higher than the maximal level obtained from endogenous secretion of somatostatin after gliclazide. Neither cysteamine treatment alone (somatostatin-depleted) nor combined with streptozotocin-treatment (combined depletion of somatostatin and insulin) changed gliclazide-induced glucagon suppression. Thus, it is concluded that suppression of glucagon is induced by sulfonylurea itself.

Antihistamine induced cognitive decline was evaluated using positron emission tomography (PET) measurement of histamine H1 receptor (H1R) occupancy and regional cerebral blood flow (rCBF). Cognitive performance in attention-demanding task deteriorated dose-dependently and the effects were statistically significant after the treatment of 2 mg of d-chlorpheniramine. There was no significant change in subjective sleepiness in the same dose. The regional blockade of H1R was observed mainly in the frontal, temporal and anterior cingulate cortices, and the intravenous administration of d-chlorpheniramine as a therapeutic dose (2 mg) blocked over 60% of H1R in the frontal cortices. The results from activation study using visual discrimination tasks demonstrated that enhanced activity in the right prefrontal and anterior cingulate cortices as well as a decreased activity in the left temporal and frontal cortices and midbrain after the treatment of d-chlorpheniramine. There were no changes in global CBF for the subjects treated with 2 mg d-chlorpheniramine (pre; 44.8+/-3.3 ml dl(-1) min(-1) vs post; 44.4+/-4.7 ml dl(-1) min(-1)). The results indicated that the attention system of human brain could be altered by therapeutic doses of H1R antagonists. These findings provide the information as to the potential risk of antihistamines in our daily activities. British Journal of Pharmacology (2000) 129, 115 - 123

At tight junctions (TJs), claudins with four transmembrane domains are incorporated into TJ strands. Junctional adhesion molecule (JAM), which belongs to the immunoglobulin superfamily, is also localized at TJs, but it remains unclear how JAM is integrated into TJs. Immunoreplica electron microscopy revealed that JAM showed an intimate spatial relationship with TJ strands in epithelial cells. In L fibroblasts expressing exogenous JAM, JAM was concentrated at cell-cell adhesion sites, where there were no strand-like structures, but rather characteristic membrane domains free of intramembranous particles were detected. These domains were specifically labeled with anti-JAM polyclonal antibody, suggesting that JAM forms planar aggregates through their lateral self-association. Immunofluorescence microscopy and in vitro binding assays revealed that ZO-1 directly binds to the COOH termini of claudins and JAM at its PDZ1 and PDZ3 domains, respectively. Furthermore, another PDZ-containing polarity-related protein, PAR-3, was directly bound to the COOH terminus of JAM, but not to that of claudins. These findings led to a molecular architectural model for TJs: small aggregates of JAM are tethered to claudin-based strands through ZO-1, and these JAM aggregates recruit PAR-3 to TJs. We also discuss the importance of this model from the perspective of the general molecular mechanisms behind the recruitment of PAR proteins to plasma membranes.

We identified new gene fusions in patients with lung cancer harboring the kinase domain of the NTRK1 gene that encodes the high-affinity nerve growth factor receptor (TRKA protein). Both the MPRIP-NTRK1 and CD74-NTRK1 fusions lead to constitutive TRKA kinase activity and are oncogenic. Treatment of cells expressing NTRK1 fusions with inhibitors of TRKA kinase activity inhibited autophosphorylation of TRKA and cell growth. Tumor samples from 3 of 91 patients with lung cancer (3.3%) without known oncogenic alterations assayed by next-generation sequencing or fluorescence in situ hybridization demonstrated evidence of NTRK1 gene fusions.

Occludin is the only known integral membrane protein of tight junctions (TJs), and is now believed to be directly involved in the barrier and fence functions of TJs. Occludin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the occludin gene. When these cells were subjected to suspension culture, they aggregated to form simple, and then cystic embryoid bodies (EBs) with the same time course as EB formation from wild-type ES cells. Immunofluorescence microscopy and ultrathin section electron microscopy revealed that polarized epithelial (visceral endoderm-like) cells were differentiated to delineate EBs not only from wild-type but also from occludin-deficient ES cells. Freeze fracture analyses indicated no significant differences in number or morphology of TJ strands between wild-type and occludin-deficient epithelial cells. Furthermore, zonula occludens (ZO)-1, a TJ-associated peripheral membrane protein, was still exclusively concentrated at TJ in occludin-deficient epithelial cells. In good agreement with these morphological observations, TJ in occludin-deficient epithelial cells functioned as a primary barrier to the diffusion of a low molecular mass tracer through the paracellular pathway. These findings indicate that there are as yet unidentified TJ integral membrane protein(s) which can form strand structures, recruit ZO-1, and function as a barrier without occludin.

Frequency of FGFR2 amplification, its clinicopathological features, and the results of high-throughput screening assays in a large cohort of gastric clinical samples remain largely unclear.

Previous work has shown that high body mass index (BMI) is associated with low grey matter volume. However, evidence on the relationship between waist circumference (WC) and brain volume is relatively scarce. Moreover, the influence of mild obesity (as indexed by WC and BMI) on brain volume remains unclear. This study explored the relationships between WC and BMI and grey matter volume in a large sample of Japanese adults.