Mechanoreception in motile flagella of Chlamydomonas

Ciliates and flagellates temporarily swim backwards on collision by generating a mechanoreceptor potential. Although this potential has been shown to be associated with cilia in Paramecium, the molecular entity of the mechanoreceptor has remained unknown. Here we show that Chlamydomonas cells express TRP11, a member of the TRP (transient receptor potential) subfamily V, in the proximal region of the flagella, and that suppression of TRP11 expression results in loss of the avoiding reaction. The results indicate that Chlamydomonas flagella exhibit mechanosensitivity, despite constant motility, by localizing the mechanoreceptor in the proximal region, where active bending is restricted.     

Taxonomic relationships within the pan-oriental narrow-mouth toad Microhyla ornata as revealed by mtDNA analysis (Amphibia, Anura, Microhylidae)

A molecular phylogenetic survey was conducted using mtDNA sequences of 12S and 16S rRNA, and cyt-b genes to examine taxonomic relationships among populations of the Pan-Oriental microhylid, Microhyla ornata, from India, Bangladesh, Thailand, Laos, China, Taiwan, and the Ryukyu Archipelago of Japan. Two discrete clades are recognized within this species, one consisting of populations from India and Bangladesh, and the other encompassing the remaining populations. In the latter clade, populations from the Ryukyu Archipelago are clearly split from the rest (populations from Taiwan and the continent) with considerable degrees of genetic differentiations. Each of the three lineages is judged to represent a good species, and the name Microhyla ornata is restricted to the South Asian populations. For the populations from Taiwan and a wide region from China to Southeast Asia, the name Microhyla fissipes should be applied, whereas the Ryukyu populations are most appropriately referred to as Microhyla okinavensis, although further substantial genetic differentiations are recognized among some island group populations within this last species.     

Hyaluronan fragments induce endothelial cell differentiation in a CD44- and CXCL1/GRO1-dependent manner

Hyaluronan is a glycosaminoglycan of the extracellular matrix. In tumors and during chronic inflammatory diseases, hyaluronan is degraded to smaller fragments, which are known to stimulate endothelial cell differentiation. In this study, we have compared the molecular mechanisms through which hyaluronan dodecasaccharides (HA12), and the known angiogenic factor, fibroblast growth factor 2 (FGF-2), induce capillary endothelial cell sprouting in a three-dimensional collagen gel. The gene expression profiles of unstimulated and HA12- or FGF-2-stimulated endothelial cells were compared using a microarray analysis approach. The data revealed that both FGF-2 and HA12 promoted endothelial cell morphogenesis in a process depending on the expression of ornithine decarboxylase (Odc) and ornithine decarboxylase antizyme inhibitor (Oazi) genes. Among the genes selectively up-regulated in response to HA12 was the chemokine CXCL1/GRO1 gene. The notion that the induction of CXCL1/GRO1 is of importance for HA12-induced endothelial cell sprouting was supported by the fact that morphogenesis was inhibited by antibodies specifically neutralizing the CXCL1/GRO1 protein product. HA12-stimulated endothelial cell differentiation was exerted via binding to CD44 since it was inhibited by antibodies blocking CD44 function. Our data show that hyaluronan fragments and FGF-2 affect endothelial cell morphogenesis by the induction of overlapping but also by distinct sets of genes.     

Inhibitory effects of trientine, a copper-chelating agent, on induction of DNA strand breaks in hepatic cells of Long-Evans Cinnamon rats

Effects of treatment with trientine, a specific copper-chelating agent, on accumulation of copper and induction of DNA strand breaks were investigated in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson's disease. Copper accumulated in the livers of LEC rats in an age-dependent manner from 4 to 13 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, hepatic copper contents did not increase and were maintained at the same levels as those in 10-week-old LEC rats. When the amounts of DNA single-strand breaks (SSBs) were estimated by a comet assay, SSBs of DNA were induced in a substantial population of LEC rat hepatic cells around 8 weeks of age and the amounts of SSBs increased in an age-dependent manner from 8 to 15 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, the observed number of cells with DNA damage decreased dramatically, suggesting that induction of SSBs of DNA was inhibited and/or SSBs were repaired during the period of treatment with trientine. The results show that treatment of LEC rats with trientine decreases the number of DNA strand breaks observed, although copper contents remain high in the liver.     

Lineage-specific cell disruption in living mice by Cre-mediated expression of diphtheria toxin A chain

We have established a transgenic mouse line in which floxed neomycin resistant cassette was inserted between the CAG promoter and EGFP. When these transgenic mice were mated with Cre-expressing transgenic animals, the offspring obtained were fluorescent green. We then established a transgenic mouse line in which EGFP in the above construct was replaced by diphtheria toxin A chain (DT). When the latter transgenic mice were mated with mice expressing Cre restricted to germ cells, we obtained healthy but sterile offspring due to a disruption of germ line cells by DT expression. We predict that this strategy will be useful for the construction of new animal models for human diseases, featuring a variety of missing cell lineages produced by disruption with DT.     

Molecular cloning and tissue-specific expression analysis of mouse spinesin, a type II transmembrane serine protease 5

We have previously reported novel serine proteases isolated from cDNA libraries of the human and mouse central nervous system (CNS) by PCR using degenerate oligodeoxyribonucleotide primers designed on the basis of the serine protease motifs, AAHC and DSGGP. Here we report a newly isolated serine protease from the mouse CNS. This protease is homologous (77.9% identical) to human spinesin type II transmembrane serine protease 5. Mouse spinesin (m-spinesin) is also composed of (from the N-terminus) a short cytoplasmic domain, a transmembrane domain, a stem region containing a scavenger-receptor-like domain, and a serine protease domain, as is h-spinesin. We also isolated type 1, type 2, and type 3 variant cDNAs of m-spinesin. Full-length spinesin (type 4) and type 3 contain all the domains, whereas type 1 and type 2 variants lack the cytoplasmic, transmembrane, and scavenger-receptor-like domains. Subcellular localization of the variant forms was analyzed using enhanced green fluorescent protein (EGFP) fusion proteins. EGFP-type 4 fusion protein was predominantly localized to the ER, Golgi apparatus, and plasma membrane, whereas EGFP-type 1 was localized to the cytoplasm, reflecting differential classification of m-spinesin variants into transmembrane and cytoplasmic types. We analyzed the distribution of m-spinesin variants in mouse tissues, using RT-PCR with variant-specific primer sets. Interestingly, transmembrane-type spinesin, types 3 and 4, was specifically expressed in the spinal cord, whereas cytoplasmic type, type 1, was expressed in multiple tissues, including the cerebrum and cerebellum. Therefore, m-spinesin variants may have distinct biological functions arising from organ-specific variant expression.     

Expression of genes for gelatinases and tissue inhibitors of metalloproteinases in periodontal tissues during orthodontic tooth movement

Orthodontic tooth movement progresses by a combination of periodontal ligament (PDL) tissue and alveolar bone remodeling processes. Besides the remodeling of alveolar bone around the moving teeth, the major extracellular matrix (ECM) components of PDLs, collagens, are degenerated, degraded, and restructured. Matrix metalloproteinases (MMPs) and their specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs), act in a co-ordinated fashion to regulate the remodeling of periodontal tissues. We hypothesized that the expression levels of the genes for MMP-2, MMP-9, and TIMPs 1–3 are increased transiently in the periodontal tissue during orthodontic tooth movement. To test this hypothesis, we employed an animal model of tooth movement using rats, as well as in situ hybridization to analyze the expression levels of Mmp-2, Mmp-9, and Timps 1-3. The expression levels of these genes increased transiently in cells of periodontal tissues, which include cementoblasts, fibroblasts, osteoblasts, and osteoclasts, at the compression side of the moving teeth. The transient increases in gene expression at the tension side were mainly limited to osteoblasts and cementoblasts. In conclusion, the expression levels of Mmp-2, Mmp-9, and Timps 1-3 increase transiently during orthodontic tooth movement at both the tension and compression sides. The expression of these genes is regulated differentially in the periodontal tissue of the tension side and compression side. This altered pattern of gene expression may determine the rate and extent of remodeling of the collagenous ECM in periodontal tissues during orthodontic tooth movement.     

The Activity of Hyaluronan Synthase 2 Is Regulated by Dimerization and Ubiquitination

Hyaluronan is a component of the extracellular matrix, which affects tissue homeostasis. In this study, we investigated the regulatory mechanisms of one of the hyaluronan-synthesizing enzymes, HAS2. Ectopic expression of Flag- and 6myc-HAS2 in COS-1 cells followed by immunoprecipitation and immunoblotting revealed homodimers; after co-transfection with Flag-HAS3, also heterodimers were seen. Furthermore, the expressed HAS2 was ubiquitinated. We identified one acceptor site for ubiquitin on lysine residue 190. Mutation of this residue led to inactivation of the enzymatic activity of HAS2. Interestingly, K190R-mutated HAS2 formed dimers with wt HAS2 and quenched the activity of wt HAS2, thus demonstrating a functional role of the dimeric configuration.     

Exploring amino acids responsible for the temperature profile of glycoside hydrolase family 45 endoglucanase EGL3 from Humicola grisea

EGL3 and RCE1 are glycoside hydrolase family 45 endoglucanases isolated from Humicola grisea and Rhizopus oryzae respectively. The amino acid sequences of the two endoglucanases are homologous; on the other hand, the optimum temperature of EGL3 is higher than that of RCE1. In this study, four chimeric endoglucanases, named ER1, ER2, ER3 and ER4, in which one of four sequential amino acid regions of the EGL3 catalytic domain (CAD) was replaced by the corresponding RCE1 amino acids, were constructed to explore the region responsible for the EGL3 temperature profile. Then their temperature profiles were compared with that of the recombinant EGL3. Replacement of the N-terminal region of EGL3 with that of RCE1 caused the EGL3 temperature profile to shift to a lower temperature. These results suggest that the N-terminal amino acids of the EGL3 are responsible for the EGL3 temperature profile.     

Amino acid regions of family 45 endoglucanases involved in cotton defibrillation and in resistance to anionic surfactants and oxidizing agents

In the detergent industry, fungal endoglucanases are used to release microfibrils from the surfaces of dyed cellulosic fabrics to enhance color brightness. Family 45 endoglucanase (glycoside hydrolase family 45, GH45) EGL3 from Humicola grisea is more resistant to anionic surfactants and oxidizing agents than family 45 endoglucanase RCE1 from Rhizopus oryzae, while in the present study, a catalytic domain of RCE1 had higher defibrillation activity on dyed cotton fabrics than did that of EGL3. To identify the amino acid regions involved in these properties, we compared the characteristics of RCE1, EGL3, and three chimeric endoglucanases, in which each of the three regions of the catalytic domain of EGL3 was replaced by the corresponding region of the catalytic domain of RCE1. Amino acids in the N-terminal region were involved in resistance to anionic surfactants and oxidizing agents. Furthermore, amino acids in the region adjacent to the N-terminal region were involved in releasing microfibrils and in binding to dyed cotton fabrics, indicating that the binding of the amino acids in this region might be important in the release of microfibrils from dyed cotton fabrics.