A genetic analysis of the Sakishima islanders reveals no relationship with Taiwan aborigines but shared ancestry with Ainu and main‐island Japanese

The Sakishima islands are members of the Ryukyu island chain, stretching from the southwestern tip of the Japanese archipelago to Taiwan in the East China Sea. Archaeological data indicate cultural similarities between inhabitants of prehistoric Sakishima and Neolithic Taiwan. Recent studies based on tooth crown traits show remarkably high inter‐island diversity among Ryukyu islanders, suggesting that the Sakishima islanders might have genetic backgrounds distinct from main‐island Okinawa people. To investigate the genetic diversity of the Ryukyu islanders, we analyzed mtDNA, Y chromosome, and autosomal short tandem repeat loci in a sample of main‐island Okinawa people and Sakishima (Miyako and Ishigaki) islanders whose participated in a previous study of tooth crown morphology. Our phylogenetic analysis of maternal (mtDNA) and paternal (Y chromosome) lineages shows that the Sakishima islanders are more closely related to people from the Japanese archipelago than to Taiwan aborigines. Miyako islanders and the Hokkaido Ainu have the first and second highest frequencies (respectively) of the Y‐chromosomal Alu‐insertion polymorphism, which is a presumable Jomon marker. Genetic diversity statistics show no evidence of demographic reduction or of extreme isolation in each island's population. Thus, we conclude that 1) Neolithic expansion from Taiwan did not contribute to the gene pool of modern Sakishima islanders, 2) male‐lineage of the Ryukyu islanders likely shares a common ancestor with the Hokkaido Ainu who are presumably direct descendants of the Jomon people, and 3) frequent reciprocal gene flow among islands has masked the trace of common ancestry in the Ryukyu island chain. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc.     

Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei

The trypanosome alternative oxidase (TAO) functions in the African trypanosomes as a cytochrome-independent terminal oxidase, which is essential for their survival in the mammalian host and as it does not exist in the mammalian host is considered to be a promising drug target for the treatment of trypanosomiasis. In the present study, recombinant TAO (rTAO) overexpressed in a haem-deficient Escherichia coli strain has been solubilized from E. coli membranes and purified to homogeneity in a stable and highly active form. Analysis of bound iron detected by inductively coupled plasma-mass spectrometer (ICP-MS) reveals a stoichiometry of two bound iron atoms per monomer of rTAO. Confirmation that the rTAO was indeed a diiron protein was obtained by EPR analysis which revealed a signal, in the reduced forms of rTAO, with a g-value of 15. The kinetics of ubiquiol-1 oxidation by purified rTAO showed typical Michaelis-Menten kinetics (K_m of 338 μM and V_max of 601 μmol/min/mg), whereas ubiquinol-2 oxidation showed unusual substrate inhibition. The specific inhibitor, ascofuranone, inhibited the enzyme in a mixed-type inhibition manner with respect to ubiquinol-1.     

Genetic immunization based on the ubiquitin-fusion degradation pathway against Trypanosoma cruzi

Cytotoxic CD8⁺ T cells are particularly important to the development of protective immunity against the intracellular protozoan parasite, Trypanosoma cruzi, the etiological agent of Chagas disease. We have developed a new effective strategy of genetic immunization by activating CD8⁺ T cells through the ubiquitin-fusion degradation (UFD) pathway. We constructed expression plasmids encoding the amastigote surface protein-2 (ASP-2) of T. cruzi. To induce the UFD pathway, a chimeric gene encoding ubiquitin fused to ASP-2 (pUB-ASP-2) was constructed. Mice immunized with pUB-ASP-2 presented lower parasitemia and longer survival period, compared with mice immunized with pASP-2 alone. Depletion of CD8⁺ T cells abolished protection against T. cruzi in mice immunized with pUB-ASP-2 while depletion of CD4⁺ T cells did not influence the effective immunity. Mice deficient in LMP2 or LMP7, subunits of immunoproteasomes, were not able to develop protective immunity induced. These results suggest that ubiquitin-fused antigens expressed in antigen-presenting cells were effectively degraded via the UFD pathway, and subsequently activated CD8⁺ T cells. Consequently, immunization with pUB-ASP-2 was able to induce potent protective immunity against infection of T. cruzi.     

Tea catechins reduce inflammatory reactions via mitogen-activated protein kinase pathways in toll-like receptor 2 ligand-stimulated dental pulp cells

AimsIn this study, we evaluated whether catechins could inhibit the expression of pro-inflammatory mediators induced by dental caries-related bacteria, Streptococci, or pathogen-associated molecular patterns (PAMPs) stimulation in human dental pulp fibroblasts (HDPF). We further determined the mechanisms of the anti-inflammatory activity of catechins.Main methodsStreptococci or PAMP-stimulated HDPF were treated with catechin, and then the expression and production of pro-inflammatory mediators were determined by RT-PCR and ELISA. Furthermore, the signal transduction pathways activated with toll-like receptor (TLR)2 ligand were assessed by Immunoblot and ELISA using blocking assay with specific inhibitors.Key findingsIncreased expressions of pro-inflammatory mediators are found in inflamed dental pulp, especially in HDPF. We recently reported that dental pulpal innate immune responses may mainly result from the predominantly-expressed TLR2 signaling. Catechins, polyphenolic compounds in green tea, exert protective and healing effects through multiple mechanisms, including antioxidative and anti-inflammatory effects. However, there are no reports concerning the effects of catechins on dental pulp. In this study, we demonstrated that the up-regulated expressions of IL-8 or PGE₂ in Streptococci or PAMP-stimulated HDPF were inhibited by catechins, (?)-epicatechin gallate (ECG) and (?)-epigallocatechin gallate (EGCG). In TLR2 ligand-stimulated HDPF, specific inhibitors of extracellular signal regulated kinase (ERK)1/2, p38, c-jun NH₂-terminal kinase (SAP/JNK), NF-κB or catechins markedly reduced the level of pro-inflammatory mediators and the phosphorylation of these signal transduction molecules was suppressed by catechins.SignificanceThese findings suggest that catechins might be useful therapeutically as an anti-inflammatory modulator of dental pulpal inflammation.     

Gravity-Induced Modifications to Development in Hypocotyls of Arabidopsis Tubulin Mutants

We investigated the roles of cortical microtubules in gravity-induced modifications to the development of stem organs by analyzing morphology and orientation of cortical microtubule arrays in hypocotyls of Arabidopsis (Arabidopsis thaliana) tubulin mutants, tua3(D205N), tua4(S178Δ), and tua6(A281T), cultivated under 1g and hypergravity (300g) conditions. Hypocotyls of tubulin mutants were shorter and thicker than the wild type even at 1g, and hypergravity further suppressed elongation and stimulated expansion. The degree of such changes was clearly smaller in tubulin mutants, in particular in tua6. Hypocotyls of tubulin mutants also showed either left-handed or right-handed helical growth at 1g, and the degree of twisting phenotype was intensified under hypergravity conditions, especially in tua6. Hypergravity induced reorientation of cortical microtubules from transverse to longitudinal directions in epidermal cells of wild-type hypocotyls. In tubulin mutants, especially in tua6, the percentage of cells with longitudinal microtubules was high even at 1g, and it was further increased by hypergravity. The twisting phenotype was most obvious at cells 10 to 12 from the top, where reorientation of cortical microtubules from transverse to longitudinal directions occurred. Moreover, the left-handed helical growth mutants (tua3 and tua4) had right-handed microtubule arrays, whereas the right-handed mutant (tua6) had left-handed arrays. There was a close correlation between the alignment angle of epidermal cell files and the alignment of cortical microtubules. Gadolinium ions, blockers of mechanosensitive ion channels (mechanoreceptors), suppressed the twisting phenotype in tubulin mutants under both 1g and 300g conditions. Microtubule arrays in tubulin mutants were oriented more transversely by gadolinium treatment, irrespective of gravity conditions. These results support the hypothesis that cortical microtubules play an essential role in maintenance of normal growth phenotype against the gravitational force, and suggest that mechanoreceptors are involved in modifications to morphology and orientation of microtubule arrays by 1g gravity and hypergravity in tubulin mutants.The direction of cell expansion is important for determining the shape of whole plant body. Cortical microtubules are assumed to be responsible for anisotropic expansion of plant cells (Wasteneys and Galway, 2003; Lloyd and Chan, 2004; Mathur, 2004; Baskin, 2005; Paredez et al., 2008). The prevailing view is that cortical microtubule arrays direct or constrain the movement of the cellulose synthase complexes and thus align nascent cellulose microfibrils in the same direction in the innermost layer of the cell wall (Baskin, 2001), although some other mechanisms may also be involved (Baskin, 2001; Sugimoto et al., 2003; Wasteneys, 2004).It is evident that orientation of cortical microtubules plays an essential role in creating the distinct shape of higher plant organs, even if there is uncertainty over the mechanism by which microtubules influence morphogenesis. The importance of cortical microtubule arrays for anisotropic growth has been documented by pharmacological studies and experiments with helical growth mutants of Arabidopsis (Arabidopsis thaliana). Mutants on α- and β-tubulins as well as microtubule-associated proteins show either left-handed or right-handed helical growth (Thitamadee et al., 2002; Nakajima et al., 2004; Sedbrook et al., 2004; Shoji et al., 2004). The rapidly elongating cells of these mutants skew consistently either to the right or to the left and exhibit cortical microtubule arrays that form shallow helices with fixed handedness (Thitamadee et al., 2002; Abe and Hashimoto, 2005; Ishida et al., 2007). Cortical microtubule arrays in the left-handed helical growth mutants form right-handed helix, whereas those in right-handed helical growth mutants form left-handed helix (Thitamadee et al., 2002; Abe and Hashimoto, 2005; Ishida et al., 2007). These results indicate that dysfunctional cortical microtubules are arranged in helical arrays and affect the direction of cell expansion.The gravitational force is one of the environmental factors that determine the plant body shape. Under hypergravity conditions produced by centrifugation, plants generally have a shorter and thicker body (Soga et al., 2006). Namely, hypergravity modifies growth anisotropy. In Arabidopsis hypocotyls, the expression of most α- and β-tubulin genes was up-regulated by hypergravity (Yoshioka et al., 2003; Matsumoto et al., 2007). In protoplasts of Brassica hypocotyls, hypergravity stimulated the regeneration of cortical microtubules into parallel arrays (Skagen and Iversen, 1999), and in azuki bean (Vigna angularis) epicotyls it increased the percentage of cells with longitudinal cortical microtubules (Soga et al., 2006). The reorientation of cortical microtubules from transverse to longitudinal directions may be involved in modifications by hypergravity to growth anisotropy.The aim of this study was to clarify the roles of cortical microtubules in gravity-induced modifications to development of stem organs. For this purpose, we examined the changes in growth, morphology, and orientation of cortical microtubule arrays in hypocotyls of Arabidopsis amino acid substitution mutants in α-tubulin structure, tua3, tua4, and tua6, grown under 1g and 300g conditions. We have reported the possible involvement of mechanosensitive ion channels (mechanoreceptors) in hypergravity-induced modifications to growth and cell wall properties (Soga et al., 2004, 2005, 2006). Thus, we also examined the effect of blockers of mechanoreceptors on helical growth and orientation of cortical microtubule arrays in the tubulin mutants.     

Silicon enhances growth independent of silica deposition in a low-silica rice mutant, lsi1

To examine whether silica bodies are essential for silicon-enhanced growth of rice seedlings, we investigated the response of rice, Oryza sativa L., to silicon treatment. Silicic acid treatment markedly enhanced the SPAD (soil plant analytical development) values of leaf blades and the growth and development of leaves and lateral roots in cvs. Hinohikari and Oochikara, and a low-silicon mutant, lsi1. Combination of ethanol–benzene displacement and staining with crystal violet lactone enabled more detailed histochemical analysis to visualize silica bodies in the epidermis under bright-field microscopy. Supply of silicon induced the development of motor cells and silica bodies in epidermal cells in Hinohikari and Oochikara but not or marginal in lsi1. X-ray analytical microscopy detected silicon specifically in the leaf sheath, the outermost part of the stem, and the leaf blade midrib, suggesting that silicon is distributed to tissues involved in maintaining rigidity of the plant to prevent lodging, rather than being passively deposited in growing tissues. Silicon supplied at high dose accumulated in all rice seedlings and enhanced growth and SPAD values with or without silica body formation. Silicon accumulated in the cell wall may play an important physiological role different from that played by the silica deposited in the motor cell and silica bodies.     

Declining expression of a single epithelial cell‐autonomous gene accelerates age‐related thymic involution

Age‐related thymic involution may be triggered by gene expression changes in lymphohematopoietic and/or nonhematopoietic thymic epithelial cells (TECs). The role of epithelial cell‐autonomous gene FoxN1 may be involved in the process, but it is still a puzzle because of the shortage of evidence from gradual loss‐of‐function and exogenous gain‐of‐function studies. Using our recently generated loxP‐floxed‐FoxN1(fx) mouse carrying the ubiquitous CreER^T (uCreER^T) transgene with a low dose of spontaneous activation, which causes gradual FoxN1 deletion with age, we found that the uCreER^T‐fx/fx mice showed an accelerated age‐related thymic involution owing to progressive loss of FoxN1⁺ TECs. The thymic aging phenotypes were clearly observable as early as at 3–6 months of age, resembling the naturally aged (18–22‐month‐old) murine thymus. By intrathymically supplying aged wild‐type mice with exogenous FoxN1‐cDNA, thymic involution and defective peripheral CD4⁺ T‐cell function could be partially rescued. The results support the notion that decline of a single epithelial cell‐autonomous gene FoxN1 levels with age causes primary deterioration in TECs followed by impairment of the total postnatal thymic microenvironment, and potentially triggers age‐related thymic involution in mice.