Genetic structure of the critically endangered Red‐headed Wood Pigeon Columba janthina nitens and its implications for the management of threatened island populations

The Red‐headed Wood Pigeon Columba janthina nitens is endemic to the Ogasawara Islands, an oceanic island chain located 1000 km south of the main islands of Japan. The subspecies is at high risk of extinction because of its small population size and restricted habitat range. We undertook genetic analyses of this pigeon using sequences of a portion of the mitochondrial control region and five microsatellite markers to estimate the genetic characteristics of two wild populations from the Bonin and Volcano Islands, as well as one captive breeding population. The genetic diversity of the wild individuals was exceptionally low in both the mitochondria (nucleotide diversity = 0.00105) and at the microsatellite (3.2 alleles per locus and H_E = 0.12) loci. Higher numbers of microsatellite genotypes were observed in the Volcano Islands population than in the Bonin Islands population, which may be because of the relatively low impact of human disturbance. The most common mitochondrial haplotypes and microsatellite alleles observed in the two wild populations were completely fixed in the captive population. Our results suggest that the genetic diversity of the captive population needs to be increased. However, introduction of a wild individual into a captive population can lead to a decreased genetic diversity in the wild population and therefore should be done with caution. The genetic differentiation between the Bonin and the Volcano island groups was low, and the populations of the two island groups should be regarded as a single evolutionarily significant unit. However, special consideration is required for habitat conservation in the Volcano Islands, which may be functioning as a sanctuary for the Red‐headed Wood Pigeon. For the long‐term conservation of threatened bird species that live on remote oceanic islands, determination of management units considering gene flow caused by their flying capacity and maintenance of genetically suitable wild and captive populations are essential.     

Inhibition of estrogen signaling through depletion of estrogen receptor alpha by ursolic acid and betulinic acid from Prunella vulgaris var. lilacina

Extracts of Prunella vulgaris have been shown to exert antiestrogenic effects. To identify the compounds responsible for these actions, we isolated the constituents of P. vulgaris and tested their individual antiestrogenic effects. Rosmarinic acid, caffeic acid, ursolic acid (UA), oleanolic acid, hyperoside, rutin and betulinic acid (BA) were isolated from the flower stalks of P. vulgaris var. lilacina Nakai (Labiatae). Among these constituents, UA and BA showed significant antiestrogenic effects, measured as a decrease in the mRNA level of GREB1, an estrogen-responsive protein; the effects of BA were stronger than those of UA. UA and BA were capable of suppressing estrogen response element (ERE)-dependent luciferase activity and expression of estrogen-responsive genes in response to exposure to estradiol, further supporting the suppressive role of these compounds in estrogen-induced signaling. However, neither UA nor BA was capable of suppressing estrogen signaling in cells ectopically overexpressing estrogen receptor α (ERα). Furthermore, both mRNA and protein levels of ERα were reduced by treatment with UA or BA, suggesting that UA and BA inhibit estrogen signaling by suppressing the expression of ERα. Interestingly, both compounds enhanced prostate-specific antigen promoter activity. Collectively, these findings demonstrate that UA and BA are responsible for the antiestrogenic effects of P. vulgaris and suggest their potential use as therapeutic agents against estrogen-dependent tumors.     

Characterization of germin-like protein with polyphenol oxidase activity from Satsuma mandarine

Polyphenol oxidases (PPOs) catalyzing the oxygen dependent oxidation of phenols to quinones are ubiquitously distributed in plants and are assumed to be involved in plant defense against pests and pathogens. A protein with high PPO activity was identified in Satsuma mandarine, extracted with Tris–HCl buffer, purified by salt precipitation and column chromatography, and characterized by mass spectrometry as germin-like protein (GLP), which belongs to pathogenesis related protein (PR) family. In the present study, the structure and enzymatic properties of GLP were characterized using spectroscopy methods. Based on native PAGE analysis, the molecular weight of GLP was estimated to be 108 kDa and GLP was identified as a pentamer containing five subunits of 22 kDa. The optimum pH and temperature for PPO catalyzing activity of GLP was 6.5 and 65 °C, respectively. Kinetic constants were 0.0365 M and 0.0196 M with the substrates catechol and pyrogallol, respectively. The structural characterization of GLP provided better insights into the regions responsible for its PPO activity.     

Structural characterization and interaction of periostin and bone morphogenetic protein for regulation of collagen cross-linking

Periostin appears to be a unique extracellular protein secreted by fibroblasts that is upregulated following injury to the heart or changes in the environment. Periostin has the ability to associate with other critical extracellular matrix (ECM) regulators such as TGF-β, tenascin, and fibronectin, and is a critical regulator of fibrosis that functions by altering the deposition and attachment of collagen. Periostin is known to be highly expressed in carcinoma cells, but not in normal breast tissues. The protein has a structural similarity to insect fasciclin-1 (Fas 1) and can be induced by transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP)-2. To investigate the molecular interaction of periostin and bone morphogenetic protein, we modeled these three-dimensional structures and their binding sites. We demonstrated direct interaction between periostin and BMP1/2 in vitro using several biochemical and biophysical assays. We found that the structures of the first, second, and fourth Fas1 domains in periostin are similar to that of the fourth Fas 1 domain of TGFBIp. However, the structure of the third Fas 1 domain in periostin is different from those of the first, second, and fourth Fas1 domains, while it is similar to the NMR structure of Fasciclin-like protein from Rhodobacter sphaeroides. These results will useful in further functional analysis of the interaction of periostin and bone morphogenetic protein.     

Inhibition of HAS2 induction enhances the radiosensitivity of cancer cells via persistent DNA damage

Hyaluronan synthase 2 (HAS2), a synthetic enzyme for hyaluronan, regulates various aspects of cancer progression, including migration, invasion and angiogenesis. However, the possible association of HAS2 with the response of cancer cells to anticancer radiotherapy, has not yet been elucidated. Here, we show that HAS2 knockdown potentiates irradiation-induced DNA damage and apoptosis in cancer cells. Upon exposure to radiation, all of the tested human cancer cell lines exhibited marked (up to 10-fold) up-regulation of HAS2 within 24 h. Inhibition of HAS2 induction significantly reduced the survival of irradiated radioresistant and -sensitive cells. Interestingly, HAS2 depletion rendered the cells to sustain irradiation-induced DNA damage, thereby leading to an increase of apoptotic death. These findings indicate that HAS2 knockdown sensitizes cancer cells to radiation via persistent DNA damage, further suggesting that the irradiation-induced up-regulation of HAS2 contributes to the radioresistance of cancer cells. Thus, HAS2 could potentially be targeted for therapeutic interventions aimed at radiosensitizing cancer cells.     

Death domain complex of the TNFR-1, TRADD,and RIP1 proteins for death-inducing signaling

Apoptosis can be induced by an extrinsic pathway involving the ligand-mediated activation of death receptors such as tumor necrosis factor receptor-1 (TNFR-1). TNFR-1-associated death domain (TRADD) protein is an adapter molecule that bridges the interaction between TNFR-1 and receptor-interacting serine/threonine-protein kinase 1 (RIP1). However, the molecular mechanism of the complex formation of these proteins has not yet been identified. Here, the binding among TNFR-1, TRADD, and RIP1 was identified using a GST pull-down assay and Biacore biosensor experiment. This study showed that structural characterization and formation of the death-signaling complex could be predicted using TNFR-1, TRADD, and RIP1. In addition, we found that the structure-based mutations of TNFR-1 (P367A and P368A), TRADD (F266A), and RIP1 (M637A and R638A) disrupted formation of the death domain (DD) complex and prevented stable interactions among those DDs.