Decreased expression of VE-cadherin and claudin-5 and increased phosphorylation of VE-cadherin in vascular endothelium in nasal polyps

VE-cadherin and claudin-5 are major components of adherens and tight junctions of vascular endothelial cells and a decrease in their expression and an increase in the tyrosine-phosphorylation of VE-cadherin are associated with an increase in endothelial paracellular permeability. To clarify the mechanism underlying the development of edema in nasal polyps, we studied these molecules in polyp microvessels. Normal inferior turbinate mucosal tissues and nasal polyps from patients treated with or without glucocorticoid were stained for VE-cadherin or claudin-5 and CD31 by a double-immunofluorescence method and the immunofluorescence intensities were graded 1–3 with increasing intensity. To correct for differences in fluorescence intensity attributable to a different endothelial area being exposed in a section or to the thickness of a section, the relative immunofluorescence intensity was estimated by dividing the grade of VE-cadherin or claudin-5 by that of CD31 in each microvessel. Tyrosine-phosphorylation of VE-cadherin was examined by Western blot analysis. The relative intensities of VE-cadherin and claudin-5 in the CD31-positive microvessels significantly decreased in the following order; inferior turbinate mucosa, treated polyps and untreated polyps. The ratio of tyrosine-phosphorylated VE-cadherin to VE-cadherin was significantly higher in untreated polyps than in the inferior turbinate mucosa and treated polyps, between which no significant difference in the ratio was seen. Thus, in nasal polyps, the barrier function of endothelial adherens and tight junctions is weakened, although glucocorticoid treatment improves this weakened barrier function.     

Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells

Combinations of histone modifications have significant biological roles, such as maintenance of pluripotency and cancer development, but cannot be analyzed at the single cell level. Here, we visualized a combination of histone modifications by applying the in situ proximity ligation assay, which detects two proteins in close vicinity (∼30 nm). The specificity of the method [designated as imaging of a combination of histone modifications (iChmo)] was confirmed by positive signals from H3K4me3/acetylated H3K9, H3K4me3/RNA polymerase II and H3K9me3/H4K20me3, and negative signals from H3K4me3/H3K9me3. Bivalent modification was clearly visualized by iChmo in wild-type embryonic stem cells (ESCs) known to have it, whereas rarely in Suz12 knockout ESCs and mouse embryonic fibroblasts known to have little of it. iChmo was applied to analysis of epigenetic and phenotypic changes of heterogeneous cell population, namely, ESCs at an early stage of differentiation, and this revealed that the bivalent modification disappeared in a highly concerted manner, whereas phenotypic differentiation proceeded with large variations among cells. Also, using this method, we were able to visualize a combination of repressive histone marks in tissue samples. The application of iChmo to samples with heterogeneous cell population and tissue samples is expected to clarify unknown biological and pathological significance of various combinations of epigenetic modifications.     

Mitochondria‐type GPAT is required for mitochondrial fusion

Glycerol‐3‐phosphate acyltransferase (GPAT) is involved in the first step in glycerolipid synthesis and is localized in both the endoplasmic reticulum (ER) and mitochondria. To clarify the functional differences between ER‐GPAT and mitochondrial (Mt)‐GPAT, we generated both GPAT mutants in C. elegans and demonstrated that Mt‐GPAT is essential for mitochondrial fusion. Mutation of Mt‐GPAT caused excessive mitochondrial fragmentation. The defect was rescued by injection of lysophosphatidic acid (LPA), a direct product of GPAT, and by inhibition of LPA acyltransferase, both of which lead to accumulation of LPA in the cells. Mitochondrial fragmentation in Mt‐GPAT mutants was also rescued by inhibition of mitochondrial fission protein DRP‐1 and by overexpression of mitochondrial fusion protein FZO‐1/mitofusin, suggesting that the fusion/fission balance is affected by Mt‐GPAT depletion. Mitochondrial fragmentation was also observed in Mt‐GPAT‐depleted HeLa cells. A mitochondrial fusion assay using HeLa cells revealed that Mt‐GPAT depletion impaired mitochondrial fusion process. We postulate from these results that LPA produced by Mt‐GPAT functions not only as a precursor for glycerolipid synthesis but also as an essential factor of mitochondrial fusion.     

Alarm call discrimination in a social rodent: adult but not juvenile degu calls induce high vigilance

Many social animals develop vocal communications to send and receive information efficiently in a group. In alarm communication, call recipients in a social group evaluate alarm calls, enhancing their probability of survival in the face of predatory threats. Calls from naïve and younger group members might be less evocative, in terms of rendering group members vigilant, than calls from more experienced adults because adults are generally more reliable. It remains uncertain, however, what acoustic characteristics render an alarm call reliable. Here, we report that adult degus, Octodon degus (Rodentia, Octodontidae), produced an alarm with a frequency-modulated (FM) syllable, accompanied by low bandwidth and entropy, to evoke a high-vigilance response amongst receivers. Unlike adults, subadult degus did not emit the FM syllable in the warning context, and their call without the FM syllable evoked less vigilance than the adult alarm. We suggest that the FM structure of the adult-produced syllable serves as the primary feature characterizing a reliable alarm call. Our results are consistent with those found in other social rodents, e.g., ground squirrels and gerbils, also produce FM alarm calls in high-urgency situations supports the importance of the FM syllable in alarm communication.     

Organization of cortical processing for facial movements during licking in cats

We proposed that cortical organization for the execution of adequate licking in cats was processed under the control of two kinds of affiliated groups for face and jaw & tongue movements (Hiraba H, Sato T. 2005A. Cerebral control of face, jaw, and tongue movements in awake cats: Changes in regional cerebral blood flow during lateral feeding Somatosens Mot Res 22:307–317). We assumed the cortical organization for face movements from changes in MRN (mastication-related neuron) activities recorded at area M (motor cortex) and orofacial behaviors after the lesion in the facial SI (facial region in the primary somatosensory cortex). Although we showed the relationship between facial SI (area 3b) and area M (area 4δ), the property of area C (area 3a) was not fully described. The aim of this present study is to investigate the functional role of area C (the anterior part of the coronal sulcus) that transfers somatosensory information in facial SI to area M, as shown in a previous paper (Hiraba H. 2004. The function of sensory information from the first somatosensory cortex for facial movements during ingestion in cats Somatosens Mot Res 21:87--97). We examined the properties of MRNs in area C and changes in orofacial behaviors after the area C or area M lesion. MRNs in area C had in common RFs in the lingual, perioral, and mandibular parts, and activity patterns of MRNs showed both post- and pre-movement types. Furthermore, cats with the area C lesion showed similar disorders to cats with the area M lesion, such as the dropping of food from the contralateral mouth, prolongation of the period of ingestion and mastication, and so on. From these results, we believe firmly the organization of unilateral cortical processing in facial SI, area C, and area M for face movements during licking.     

Involvement of gangliosides in the process of Cbp/PAG phosphorylation by Lyn in developing cerebellar growth cones

The association of gangliosides with specific proteins in the central nervous system was examined by coimmunoprecipitation with an anti‐ganglioside antibody. The monoclonal antibody to the ganglioside GD3 (R24) immunoprecipitated the Csk (C‐terminal src kinase)‐binding protein (Cbp). Sucrose density gradient analysis showed that Cbp of rat cerebellum was detected in detergent‐resistant membrane (DRM) raft fractions. R24 treatment of the rat primary cerebellar cultures induced Lyn activation and tyrosine phosphorylation of Cbp. Treatment with anti‐ganglioside GD1b antibody also induced tyrosine phosphorylation. Furthermore, over‐expressions of Lyn and Cbp in Chinese hamster ovary (CHO) cells resulted in tyrosine 314 phosphorylation of Cbp, which indicates that Cbp is a substrate for Lyn. Immunoblotting analysis showed that the active form of Lyn and the Tyr314‐phosphorylated form of Cbp were highly accumulated in the DRM raft fraction prepared from the developing cerebellum compared with the DRM raft fraction of the adult one. In addition, Lyn and the Tyr314‐phosphorylated Cbp were highly concentrated in the growth cone fraction prepared from the developing cerebellum. Immunoelectron microscopy showed that Cbp and GAP‐43, a growth cone marker, are localized in the same vesicles of the growth cone fraction. These results suggest that Cbp functionally associates with gangliosides on growth cone rafts in developing cerebella.     

EXTL2, a Member of the EXT Family of Tumor Suppressors,Controls Glycosaminoglycan Biosynthesis in a Xylose Kinase-dependent Manner

Mutant alleles of EXT1 or EXT2, two members of the EXT gene family, are causative agents in hereditary multiple exostoses, and their gene products function together as a polymerase in the biosynthesis of heparan sulfate. EXTL2, one of three EXT-like genes in the human genome that are homologous to EXT1 and EXT2, encodes a transferase that adds not only GlcNAc but also N-acetylgalactosamine to the glycosaminoglycan (GAG)-protein linkage region via an α1,4-linkage. However, both the role of EXTL2 in the biosynthesis of GAGs and the biological significance of EXTL2 remain unclear. Here we show that EXTL2 transfers a GlcNAc residue to the tetrasaccharide linkage region that is phosphorylated by a xylose kinase 1 (FAM20B) and thereby terminates chain elongation. We isolated an oligosaccharide from the mouse liver, which was not detected in EXTL2 knock-out mice. Based on structural analysis by a combination of glycosidase digestion and 500-MHz ¹H NMR spectroscopy, the oligosaccharide was found to be GlcNAcα1-4GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate), which was considered to be a biosynthetic intermediate of an immature GAG chain. Indeed, EXTL2 specifically transferred a GlcNAc residue to a phosphorylated linkage tetrasaccharide, GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate). Remarkably, the phosphorylated linkage pentasaccharide generated by EXTL2 was not used as an acceptor for heparan sulfate or chondroitin sulfate polymerases. Moreover, production of GAGs was significantly higher in EXTL2 knock-out mice than in wild-type mice. These results indicate that EXTL2 functions to suppress GAG biosynthesis that is enhanced by a xylose kinase and that the EXTL2-dependent mechanism that regulates GAG biosynthesis might be a “quality control system” for proteoglycans.     

Dimorphisms of the proteasome subunit beta type 8 gene (PSMB8) of ectothermic tetrapods originated in multiple independent evolutionary events

The proteasome subunit beta type 8 gene (PSMB8) encodes one of the beta subunits of the immunoproteasome responsible for the generation of peptides presented by major histocompatibility complex class I molecules. Dimorphic alleles of the PSMB8 gene, termed A and F types, based on the deduced 31st amino acid residue of the mature protein have been reported from various vertebrates. Phylogenetic analysis revealed the presence of dichotomous ancient lineages, one comprising the F-type PSMB8 of basal ray-finned fishes, and the other comprising the A-type PSMB8 of these animals and both the F- and A-type PSMB8 of Xenopus and acanthopterygians, indicating that evolutionary history of the PSMB8 dimorphism was not straightforward. We analyzed the PSMB8 gene of five reptile and one amphibian species and found both the A and F types from all six. Phylogenetic analysis indicated that the PSMB8 F type was apparently regenerated from the PSMB8 A type at least five times independently during tetrapod evolution. Genomic typing of wild individuals of geckos and newts indicated that the frequencies of the A- and F-type alleles are not highly biased in these species. Phylogenetic analysis of each exon of the reptile PSMB8 gene suggested interallelic sequence homogenization as a possible evolutionary mechanism for the apparent recurrent regeneration of PSMB8 dimorphism in tetrapods. An extremely strong balancing selection acting on PSMB8 dimorphism was implicated in an unprecedented pattern of allele evolution.     

Vegetation and fire history with their implication for climatic change and fire events since the last deglacial in the Aso Valley, central Kyushu, southwestern Japan: new pollen and charcoal data

To better understand the response of forest vegetation to climate and fire regimes with reference to human activities over the last deglacial period in the Aso Caldera, central Kyushu, southwestern Japan, a 33.9 m long sediment core was examined in order to reconstruct the vegetational and fire history using pollen and charcoal analyses. The results show that a cool temperate broad-leaved deciduous forest, dominated by Quercus (deciduous oaks) with Carpinus and Fagus, prevailed in the Aso Valley from ca. 14.6 ka cal. b.p., indicating warming since the last glacial period. The landscape was presumably covered by a mosaic of deciduous Quercus forests and terrestrial Artemisia communities. Around 12.8–11.7 ka cal. b.p., Quercus dominated the forest and fires occurred frequently. Co-expansion of distinctive Ulmus–Zelkova and Celtis–Aphananthe forests coupled with a progressive retreat of Quercus in the early Holocene could reflect a strengthening of the East Asian summer monsoon under mild and humid climate conditions. Around 8 ka cal. b.p., significant increases in Cyclobalanopsis (evergreen oaks), Castanopsis/Castanea and Podocarpus indicate a further warming, in particular an increased winter temperature. Warm temperate lucidophyllous forests, dominated by Cyclobalanopsis, flourished after 7.3 ka cal. b.p., probably corresponding to the “Holocene Climatic Optimum” interval. Progressive expansion of Quercus at the expense of Cyclobalanopsis began around 6.4 ka cal. b.p. and paralleled an increase in charcoal until ca. 4.8 ka cal. b.p.; this could be evidence of fire disturbance induced by the early-middle Jomon people. The disturbed evergreen forest experienced a temporary recovery but then opened again from 3.6 ka cal. b.p. due to extensive fire deforestation, as suggested by the high charcoal levels during this time. Human exploitation and buckwheat (Fagopyrum) agriculture may have contributed to the opening of the forest, which allowed secondary forests (primarily Pinus and Quercus) and herbaceous communities (mainly Poaceae) to spread. These results are discussed in comparison with other high-resolution pollen data from western Japan to better elucidate the vegetation and fire history over the last deglacial in the Aso Caldera.