Identification of a novel actin-related protein in Tetrahymena cilia

Actin is an ancient cytoskeletal protein that plays many essential roles in cell motility. In eukaryotes, its gene belongs to a highly conserved gene family, while the protein is also a member of an actin superfamily comprising various kinds of actin-related proteins (Arps). A ciliate, Tetrahymena, has a unique conventional actin. Data from the TIGR Tetrahymena genome project and our own research suggest the existence of 12 actin-like sequences: four conventional actins, two of Arp4, one each of Arp1, Arp2, Arp3, Arp5, and Arp6, and a novel actin-related protein, tArp. We cloned the entire cDNA sequences of Tetrahymena Arp2 (tArp2), Tetrahymena Arp3 (tArp3), and tArp for the work described herein. In phylogenetic analyses, tArp was not included in any Arp subfamily. Unlike other known Arps, tArp localizes in cilia, and its expression was upregulated after deciliation. To see the precise localization of tArp, cilia were fractionated and analyzed using specific antibodies. tArp was observed preferentially in the "outer-doublet" fraction, while actin was found in the "crude-dynein" fraction. In immunoelectron microscopy, most of the gold particles were found either on the outer-doublet or central-pair microtubules. These results suggest that tArp is a ciliary component and that it has a unique function in the formation and maintenance of cilia.     

Solution structure and binding specificity of FBP11/HYPA WW domain as Group-II/III

The Group-II/III WW domains bind Pro-rich sequences, the most frequent protein motif found in eucaryotic genomes. We have proposed that the Group-II and -III WW domains be merged into a larger group because the members of each group have relatively wide specificity and bind to the common ligands [Kato et al., J Biol Chem 2004;279:31833-31841]. We have also proposed that Group-II/III has a common surface patch, the XP2 groove, to bind the ligands. The first WW domain of FBP11/HYPA is one of the Group-II/III WW domains. The solution structure of the 26 residue-long converged region exhibits an antiparallel triple stranded beta-sheet with a small hydrophobic core. The WW domain of FBP11/HYPA has both XP and XP2 grooves on its surface. Ligand titration by 1H-15N HSQC NMR spectra revealed that the WW domain of FBP11/HYPA binds all the peptides with the PL, PP, and PR motifs. The profile patterns of chemical shift perturbation were quite similar among the spectra titrated with all three ligands. In addition, the titration significantly shifts the signals of the residues that compose the XP2 groove. All these findings suggest the functional importance of the XP2 groove and group definition of Group-II/III of the WW domains.     

Activation of genes for growth factor and cytokine pathways late in chondrogenic differentiation of ATDC5 cells

The mouse embryonal carcinoma cell line ATDC5 provides an excellent model system for chondrogenesis in vitro. To understand better the molecular mechanisms of endochondral bone formation, we investigated gene expression profiles during the differentiation course of ATDC5 cells, using an in-house microarray harboring full-length-enriched cDNAs. For 28 days following chondrogenic induction, 507 genes were up- or down-regulated at least 1.5-fold. These genes were classified into five clusters based on their expression patterns. Genes for growth factor and cytokine pathways were significantly enriched in the cluster characterized by increases in expression during late stages of chondrocyte differentiation. mRNAs for decorin and osteoglycin, which have been shown to bind to transforming growth factors-beta and bone morphogenetic proteins, respectively, were found in this cluster and were detected in hypertrophic chondrocytes of developing mouse bones by in situ hybridization analysis. Taken together with assigned functions of individual genes in the cluster, interdigitated interaction between a number of intercellular signaling molecules is likely to take place in the late chondrogenic stage for autocrine and paracrine regulation among chondrocytes, as well as for chemoattraction and stimulation of progenitor cells of other lineages.     

The human HYMAI/PLAGL1 differentially methylated region acts as an imprint control region in mice

Imprinting centers (IC) can be defined as cis-elements that are recognized in the germ line and are epigenetically modified to bring about the full imprinting program in a somatic cell. Two paternally expressed human genes, HYMAI and PLAGL1 (LOT1/ZAC), are located within human chromosome 6q24. Within this region lies a 1-kb CpG island that is differentially methylated in somatic cells, unmethylated in sperm, and methylated in mature oocytes in mice, characteristic features of an IC. Loss of methylation of the homologous region in humans is observed in patients with transient neonatal diabetes mellitus and hypermethylation is associated with a variety of cancers, suggesting that this region regulates the expression of one or more key genes in this region involved in these diseases. We now report that a transgene carrying the human HYMAI/PLAGL1 DMR was methylated in the correct parent-origin-specific manner in mice and this was sufficient to confer imprinted expression from the transgene. Therefore, we propose that this DMR functions as the IC for the HYMAI/PLAGL1 domain.     

Periodontopathic bacterial endotoxin-induced tumor necrosis factor alpha production was inhibited by exercise in mice

The effects of exercise on serum interleukin-6 and tumor necrosis factor alpha levels were investigated using mice. Five-week-old female BALB/c mice (Th2-biased) and C57BL/6 mice (Th1-biased) were divided into exercise and control groups. The exercise group was exercised in a rotating basket type treadmill for 1 h (5 r.p.m.). Blood was collected and the serum was separated immediately after exercise. The serum interleukin-6 and tumor necrosis factor alpha levels were measured using an Endogen ELISA kit. Exercise significantly increased the serum interleukin-6 level in the two strains of mice (P<0.05 and P<0.01). The tumor necrosis factor alpha level was decreased in the exercise group. Next, periodontopathic bacterial endotoxin (lipopolysaccharide) was administered after exercise, and the effects of exercise on the lipopolysaccharide-induced serum interleukin-6 and tumor necrosis factor alpha levels were investigated. Exercise inhibited lipopolysaccharide-induced tumor necrosis factor alpha production, suggesting it has a defensive action against endotoxin shock.