Co-expression pattern of Shh with Prox1 and that of Nkx2.2 with Mash1 in mouse taste bud

In mammals, taste buds are maintained by continuous turnover of cells, even in adulthood. Cell proliferation and differentiation continue to produce taste cells, which express various genes related to taste reception. We found the co-expression of Sonic hedgehog (Shh) with Prox1 and that of Nkx2.2 with Mash1 in adult mouse taste buds. Whereas Prox1was expressed strongly in cells in the basal region of mouse taste buds where Shh was co-expressed, it was expressed weakly in almost all taste bud cells lacking Shh expression. At 0.5 day after birth, when taste cells have not yet differentiated, the expressions of Shh and Prox1 completely overlapped in the epithelium of circumvallate papillae. Nkx2.2 was observed in cells expressing Mash1, but not in cells expressing genes related to taste reception, such as gustducin and T1R3. Almost all fusiform cells expressing Mash1 co-expressed Nkx2.2, while the majority of round cells expressing Mash1 in the basal region of taste buds lacked Nkx2.2 expression.     

Magnesium-deficient medium enhances NO production in alveolar macrophages isolated from rats

Magnesium deficiency has been shown to increase nitric oxide (NO) levels in plasma and to aggravate endotoxin lethality. The present study was performed to examine the effects of magnesium (Mg(2+))-deficient culture medium, with and without endotoxin (LPS), on NO release and inducible NOS (iNOS) mRNA levels in alveolar macrophages isolated from rats. Decreasing the Mg(2+) concentration in the culture medium from 0.39 mM (normal-Mg(2+) medium) to 0.021 mM (Mg(2+)-deficient medium) increased NO release from alveolar macrophages for 2 h. However, LPS stimulation in Mg(2+)-deficient medium had little effect on NO release. The increased NO release in Mg(2+)-deficient medium was suppressed completely by L-NAME and aminoguanidine. Dexamethasone, pyrrolidine dithiocarbamate and curcumin strongly inhibited NO release. Verapamil, U73122, TMB-8 and W-7 had no significant effect on NO release induced by Mg(2+) deficiency. Preculture of macrophages with Mg(2+)-deficient medium for 22 h markedly increased NO release and iNOS mRNA levels for a further 2 h; these increments were suppressed completely by curcumin. These results suggest that Mg(2+) deficiency enhances NO production via iNOS by alveolar macrophages. In this experimental condition, we can not suggest that NO production from alveolar macrophage plays an essential role in the pathogenesis of enhanced endotoxin lethality in Mg-deficient rats.     

Organization of the lipoprotein lipase gene of red sea bream Pagrus major

Lipoprotein lipase (LPL) is a key enzyme of lipid deposition and metabolism. To investigate the mechanism of lipid deposition in fish, as a first step, we have characterized the LPL gene of a marine teleost red sea bream Pagrus major by cDNA and genomic structure analysis. The red sea bream LPL gene encodes 511 amino acids and spans approximately 6.3 kb of the genome. The coding region is organized into ten exons and nine introns. In comparison with the LPL of other animals, the deduced amino acid sequence shows a high degree of similarity with a conservation of functional domains, e.g. catalytic triad, N-glycosylation sites, lipid and heparin binding regions. The 1.1 kb of 5′ flanking region contains two CCAAT, sequences homologous to Oct-I site and response elements for hormones including glucocorticoid, insulin and thyroid hormone. The results of the present study will facilitate further study of the function and regulation of the LPL in non-mammalian vertebrates.     

Cigarette smoke-induced pulmonary inflammation is attenuated in CD69-deficient mice

Cluster of differentiation 69 (CD69) has been identified as a lymphocyte early activation marker, and recent studies have indicated that CD69 mediates intracellular signals and plays an important role in various inflammatory diseases. Cigarette smoke (CS) is a strong proinflammatory stimulus that induces the release of proinflammatory mediators by recruiting macrophages and neutrophils into the lung tissue, and is one of the main risk factors for a number of chronic diseases. However, the potential role of CD69 in CS-induced pulmonary inflammation has not been determined. To address to this question, CD69-deficient (KO) and wild-type (WT) mice were subjected to CS-induced acute pulmonary inflammation. After the exposure with CS, the expression of CD69 in the lung of WT mice was significantly induced, it was predominantly observed in macrophages. In conjunction with this phenomenon, neutrophil and macrophage cell counts, and expression of several cytokines were significantly higher in the bronchoalveolar lavage fluid (BALF) of CS-exposed WT mice compared with air-exposed WT mice. Likewise, the CS-induced accumulation of inflammatory cells and cytokines expression were significantly lower in CD69-KO mice than in WT mice. These results suggest that CD69 on macrophages is involved in CS-induced acute pulmonary inflammation.     

Role of fibulin-5 in metastatic organ colonization

The tumor microenvironment is now recognized as a major factor in determining the survival and growth of disseminated tumor cells at potential metastatic sites. Tumor cells send signals to stroma cells and stimulate them to produce factors that in turn create favorable conditions for tumor cell metastasis. Activated fibroblasts constitute an important component of the tumor-associated stroma. We have previously shown that S100A4 protein produced by stromal fibroblasts in the primary tumor stimulates metastasis formation. Here we show that activated fibroblasts also stimulate the formation of metastases independently of S100A4 expression during organ colonization. To identify genes that could potentially interfere with fibroblast-driven metastasis, we used gene expression profiling of S100A4-deficient fibroblasts treated with and without tumor cell-conditioned media. Five differentially expressed genes encoding cell surface and secreted proteins with potential metastasis-modulating activity were selected. Expression of lymphocyte antigen 6 complex (Ly6c) and matrix metalloproteinase 3 (Mmp3) was upregulated in fibroblasts in response to tumor-conditioned medium, whereas expression of cadherin-16 (Cdh16), Ccn2, and fibulin-5 (Fbln5) was downregulated. Further analysis showed that Fibulin-5 is able to suppress the metastatic colonization of lungs and liver. Additional studies suggest a mechanism in which Fibulin-5 suppresses metastasis formation by inhibiting production of matrix metalloproteinase 9 (MMP9) and reducing the invasive behavior of fibroblasts. Together our data are consistent with the notion that tumors secrete factors that downregulate expression of Fbln5 in fibroblasts at sites of metastatic colonization, in turn upregulating Mmp9 expression and stimulating metastatic organ colonization.     

Antiviral inhibition targeting the HCMV kinase pUL97 requires pUL27-dependent degradation of Tip60 acetyltransferase and cell-cycle arrest

Infection with the β-herpesvirus human cytomegalovirus (HCMV) is lifelong, causing limited disease in healthy adults, but life threatening in immunocompromised individuals. The viral kinase pUL97, a functional ortholog of cellular cyclin-dependent kinases (CDKs), is critical for HCMV replication and a target for antiviral drug development. Upon kinase inhibition, drug-resistant strains emerge with mutations in UL27, an HCMV gene of unknown function. Using a proteomics approach, we discovered that pUL27 is necessary and sufficient to degrade Tip60, a host acetyltransferase and interacting partner of HIV Tat. Consistent with this, the expression of Tat restored antiviral inhibition of an otherwise resistant HCMV strain. The functional consequence of Tip60 degradation was the induction of the CDK inhibitor p21(Waf1/Cip1) and cell-cycle arrest, representing changes necessary for the antiviral effects of pUL97 inhibition. Consequently, either increasing p21(Waf1/Cip1) expression or decreasing Tip60 levels improved the antiviral activity of the HCMV kinase inhibitor maribavir.     

Simple autogeneic feeder cell preparation for pluripotent stem cells

Mouse embryonic fibroblasts (MEFs) are the most commonly used feeder cells for pluripotent stem cells. However, autogeneic feeder (AF) cells have several advantages such as no xenogeneic risks and reduced costs. In this report, we demonstrate that common marmoset embryonic stem (cmES) cells can be maintained on common marmoset AF (cmAF) cells. These cmES cells were maintained on cmAF cells for 6 months, retaining their morphology, normal karyotype, and expression patterns for the pluripotent markers Oct-3/4, Nanog, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81, as well as their ability to differentiate into cardiac and neural cells. Antibody array analysis revealed equivalent protein expression profiles between cmES cells maintained on cmAF cells and MEFs. In addition, similarly prepared human embryonic stem (hES) and induced pluripotent stem (hiPS) cell-derived AF cells supported the growth of and maintained the morphology and pluripotent marker expressions of hES and hiPS cells, respectively. DNA microarray analysis revealed that these hES and hiPS cells had mRNA expression profiles similar to those of hES and hiPS cells maintained on MEFs, respectively. Taken together, these findings imply that AF cells can replace MEFs in the routine maintenance of primate pluripotent stem cells.     

Comparative studies of genome-wide maps of nucleosomes between deletion mutants of elp3 and hos2 genes of Saccharomyces cerevisiae

In order to elucidate the influence of histone acetylation upon nucleosomal DNA length and nucleosome position, we compared nucleosome maps of the following three yeast strains; strain BY4741 (control), the elp3 (one of histone acetyltransferase genes) deletion mutant, and the hos2 (one of histone deactylase genes) deletion mutant of Saccharomyces cerevisiae. We sequenced mononucleosomal DNA fragments after treatment with micrococcal nuclease. After mapping the DNA fragments to the genome, we identified the nucleosome positions. We showed that the distributions of the nucleosomal DNA lengths of the control and the hos2 disruptant were similar. On the other hand, the distribution of the nucleosomal DNA lengths of the elp3 disruptant shifted toward shorter than that of the control. It strongly suggests that inhibition of Elp3-induced histone acetylation causes the nucleosomal DNA length reduction. Next, we compared the profiles of nucleosome mapping numbers in gene promoter regions between the control and the disruptant. We detected 24 genes with low conservation level of nucleosome positions in promoters between the control and the elp3 disruptant as well as between the control and the hos2 disruptant. It indicates that both Elp3-induced acetylation and Hos2-induced deacetylation influence the nucleosome positions in the promoters of those 24 genes. Interestingly, in 19 of the 24 genes, the profiles of nucleosome mapping numbers were similar between the two disruptants.     

The reliability and validity of the Japanese version of the Levels of Emotional Awareness Scale (LEAS-J)

Background  

The Levels of Emotional Awareness Scale (LEAS) was developed to assess five levels of emotional awareness: bodily sensations, action tendencies, single emotions, blends of emotion, and combinations of blends. It is a paper and pencil performance questionnaire that presents 20 emotion-evoking scenes. We developed a Japanese version of the LEAS (LEAS-J), and its reliability and validity were examined.