Correlation between the optimal growth pressures of four Shewanella species and the stabilities of their cytochromes c 5

Shewanella species live widely in deep-sea and shallow-water areas, and thus grow piezophilically and piezosensitively. Piezophilic and psychrophilic Shewanella benthica cytochrome c ₅ (SB cytc ₅) was the most stable against guanidine hydrochloride (GdnHCl) and thermal denaturation, followed by less piezophilic but still psychrophilic Shewanella violacea cytochrome c ₅ (SV cytc ₅). These two were followed, as to stability level, by piezosensitive and mesophilic Shewanella amazonensis cytochrome c ₅ (SA cytc ₅), and piezosensitive and psychrophilic Shewanella livingstonensis cytochrome c ₅ (SL cytc ₅). The midpoint GdnHCl concentrations of SB cytc ₅, SV cytc ₅, SL cytc ₅, and SA cytc ₅ correlated with the optimal growth pressures of the species, the correlation coefficient value being 0.93. A similar trend was observed for thermal denaturation. Therefore, the stability of each cytochrome c ₅ is related directly to its host’s optimal growth pressure. Phylogenetic analysis indicated that Lys-37, Ala-41, and Leu-50 conserved in piezosensitive SL cytc ₅ and SA cytc ₅ are ancestors of the corresponding residues in piezophilic SB cytc ₅ and SV cytc ₅, Gln, Thr, and Lys, respectively, which might have been introduced during evolution on adaption to environmental pressure. The monomeric Shewanella cytochromes c ₅ are suitable tools for examining protein stability with regard to the optimal growth pressures of the source species.     

Natural Hybridization and Introgression between Ligularia cymbulifera and L. tongolensis (Asteraceae,Senecioneae) in Four Different Locations

Natural hybridization has been considered to represent an important factor influencing the high diversity of the genus Ligularia Cass. in the Hengduan Mountains, China. Natural hybridization has been confirmed to occur frequently in Ligularia. To date, however, it has been demonstrated only within a single population. In this paper, we present evidence of natural hybridization in Ligularia from four different locations. The internal transcribed spacer (ITS) region of the nuclear ribosomal DNA and three chloroplast intergenic spacers (trnK-rps16, trnL-rpl32 and trnQ-5''rps16) of 149 accessions of putative hybrids and their putative parents (L. cymbulifera and L. tongolensis) were analyzed for evidence of hybridization. The ITS data clearly distinguished two putative parental species and sympatric L. vellerea and supported the hypothesis that those morphological intermediates were products of natural hybridization between L. cymbulifera and L. tongolensis. Moreover, several identified morphological parents were actual introgressed products. Because of hybridization and introgression, chloroplast DNA sequences generated a poorly resolved network. The present results indicate that varying degrees of hybridization and introgression occur differently depending on the habitat context. We conclude that gene flow caused by natural hybridization in Ligularia indeed plays an important role in the species diversity.     

Prediction Models Discriminating between Nonlocomotive and Locomotive Activities in Children Using a Triaxial Accelerometer with a Gravity-removal Physical Activity Classification Algorithm

The aims of our study were to examine whether a gravity-removal physical activity classification algorithm (GRPACA) is applicable for discrimination between nonlocomotive and locomotive activities for various physical activities (PAs) of children and to prove that this approach improves the estimation accuracy of a prediction model for children using an accelerometer. Japanese children (42 boys and 26 girls) attending primary school were invited to participate in this study. We used a triaxial accelerometer with a sampling interval of 32 Hz and within a measurement range of ±6 G. Participants were asked to perform 6 nonlocomotive and 5 locomotive activities. We measured raw synthetic acceleration with the triaxial accelerometer and monitored oxygen consumption and carbon dioxide production during each activity with the Douglas bag method. In addition, the resting metabolic rate (RMR) was measured with the subject sitting on a chair to calculate metabolic equivalents (METs). When the ratio of unfiltered synthetic acceleration (USA) and filtered synthetic acceleration (FSA) was 1.12, the rate of correct discrimination between nonlocomotive and locomotive activities was excellent, at 99.1% on average. As a result, a strong linear relationship was found for both nonlocomotive (METs = 0.013×synthetic acceleration +1.220, R² = 0.772) and locomotive (METs = 0.005×synthetic acceleration +0.944, R² = 0.880) activities, except for climbing down and up. The mean differences between the values predicted by our model and measured METs were −0.50 to 0.23 for moderate to vigorous intensity (>3.5 METs) PAs like running, ball throwing and washing the floor, which were regarded as unpredictable PAs. In addition, the difference was within 0.25 METs for sedentary to mild moderate PAs (<3.5 METs). Our specific calibration model that discriminates between nonlocomotive and locomotive activities for children can be useful to evaluate the sedentary to vigorous PAs intensity of both nonlocomotive and locomotive activities.     

Temporal Alterations in the Secretome of the Selective Ligninolytic Fungus Ceriporiopsis subvermispora during Growth on Aspen Wood Reveal This Organism's Strategy for Degrading Lignocellulose

The white-rot basidiomycetes efficiently degrade all wood cell wall polymers. Generally, these fungi simultaneously degrade cellulose and lignin, but certain organisms, such as Ceriporiopsis subvermispora, selectively remove lignin in advance of cellulose degradation. However, relatively little is known about the mechanism of selective ligninolysis. To address this issue, C. subvermispora was grown in liquid medium containing ball-milled aspen, and nano-liquid chromatography-tandem mass spectrometry was used to identify and estimate extracellular protein abundance over time. Several manganese peroxidases and an aryl alcohol oxidase, both associated with lignin degradation, were identified after 3 days of incubation. A glycoside hydrolase (GH) family 51 arabinofuranosidase was also identified after 3 days but then successively decreased in later samples. Several enzymes related to cellulose and xylan degradation, such as GH10 endoxylanase, GH5_5 endoglucanase, and GH7 cellobiohydrolase, were detected after 5 days. Peptides corresponding to potential cellulose-degrading enzymes GH12, GH45, lytic polysaccharide monooxygenase, and cellobiose dehydrogenase were most abundant after 7 days. This sequential production of enzymes provides a mechanism consistent with selective ligninolysis by C. subvermispora.     

Identification and Characterization of Receptors for Ion Transport Peptide (ITP) and ITP-like (ITPL) in the Silkworm Bombyx mori

Ion transport peptide (ITP) and its alternatively spliced variant, ITP-like (ITPL), are insect peptides that belong to the crustacean hyperglycemic hormone family. These peptides modulate the homeostatic mechanisms for regulating energy metabolism, molting, and reproduction and are specifically conserved in ecdysozoans. Many of the details of the molecular mechanisms by which crustacean hyperglycemic hormone family peptides exert pleiotropy remain to be elucidated, including characterization of their receptors. Here we identified three Bombyx mori orphan neuropeptide G protein-coupled receptors (BNGRs), BNGR-A2, -A24, and -A34, as receptors for ITP and ITPL (collectively referred to as ITPs). BNGR-A2 and -A34 and BNGR-A24 respond to recombinant ITPs, respectively, with EC₅₀ values of 1.1–2.6 × 10⁻⁸ m, when expressed in a heterologous expression system. These three candidate BNGRs are expressed at larval B. mori tissues targeted by ITPs, with cGMP elevation observed after exposure to recombinant ITPs. ITPs also increased the cGMP level in B. mori ovary-derived BmN cells via membrane-bound and soluble guanylyl cyclases. The simultaneous knockdown of bngr-A2 and -A34 significantly decreased the response of BmN cells to ITP, whereas knockdown of bngr-A24 led to decreased responses to ITPL. Conversely, transient expression of bngr-A24 potentiated the response of BmN cells to ITPL. An in vitro binding assay showed direct interaction between ITPs and heterologously expressed BNGRs in a ligand-receptor-specific manner. Taken together, these data demonstrate that BNGR-A2 and -A34 are ITP receptors and that BNGR-A24 is an ITPL receptor in B. mori.     

Microbial fluorescence sensing for human neurotensin receptor type 1 using Gα-engineered yeast cells

Neurotensin receptor type-1 (NTSR1) is a member of the G-protein-coupled receptor (GPCR) family. The natural ligand of NTSR1 is neurotensin (NT), a neuromodulator of the central nervous system. Because NT is also involved in many oncogenic actions, the signaling mediator NTSR1 is a significant molecular target in medicinal and therapeutic fields. In the current study, we constructed a fluorescence-based microbial yeast biosensor that can monitor the activation of human NTSR1 signaling responding to its agonist. To increase the sensitivity of the biosensor, a yeast strain with the green fluorescent protein (GFP) reporter gene was genetically engineered to enhance binding with human NTSR1 expressed on the membrane. Following previous reports, the 5 carboxy-terminal amino acid residues of the guanine nucleotide binding protein α-subunit (Gα) in yeast Gpa1p were substituted with the equivalent human Gα_q sequences (Gpa1/Gα_q transplant). After optimizing the assay conditions, the Gα-engineered yeast demonstrated significantly improved sensing for NTSR1 signaling. Because detection using a GFP fluorescence reporter considerably simplifies the measurement procedure, this microbial fluorescence sensor holds promise for use in the diagnosis of NTSR1-associated diseases and the development of agonists.     

Construction of protein-modified TiO2 nanoparticles for use with ultrasound irradiation in a novel cell injuring method

Recently, our group discovered an alternative titanium dioxide (TiO₂) activation method that uses ultrasound irradiation (US/TiO₂) instead of ultraviolet irradiation. The pre-S1/S2 protein from hepatitis B virus, which recognizes liver cells, was immobilized to the surface of TiO₂ nanoparticles using an amino-coupling method. The ability of the protein-modified TiO₂ nanoparticles to recognize liver cells was confirmed by surface plasmon resonance analysis and immuno-staining analyses. After uptake of TiO₂ nanoparticles by HepG2 cancer cells, the cells were injured using this US/TiO₂ method; significant cell injury was observed at an ultrasound irradiation intensity of 0.4 W/cm². Together with these results, this strategy could be applied to new cell injuring systems that use ultrasound irradiation in place of photodynamic therapy in the near future.     

Affibody-displaying bionanocapsules for specific drug delivery to HER2-expressing cancer cells

A novel HER2-targeted carrier was developed using bionanocapsules (BNCs). Bionanocapsules (BNCs) are 100-nm hollow nanoparticles composed of the l-protein of hepatitis B virus surface antigen. An affibody of HER2 was genetically displayed on the BNC surface (Z_HER2-BNC). For the investigation of binding affinity, Z_HER2-BNC was incubated with the cancer cell lines SK-BR-3 (HER2 positive), and MDA-MB-231 (HER2 negative). For analysis of HER2 targeting specificity, Z_HER2-BNC or Z_WT-BNC (without affibody) was incubated with both SK-BR-3 and MDA-MB-231 cells by time lapse and concentration. For the delivery of encapsulated molecules (calcein), fluorescence of Z_HER2-BNC mixed with liposomes was also compared with that of Z_WT-BNC and nude liposomes by incubation with SK-BR-3 cells. As a result, Z_HER2-BNC-liposome complex demonstrated the delivery to HER2-expressing cells (SK-BR-3) with a high degree of specificity. This indicates that genetically engineered BNCs are promising carrier for cancer treatment.     

Three-dimensional structure of rat-liver acyl-CoA oxidase in complex with a fatty acid: insights into substrate-recognition and reactivity toward molecular oxygen

The three-dimensional structure of rat-liver acyl-CoA oxidase-II (ACO-II) in a complex with a C12-fatty acid was solved by the molecular replacement method based on the uncomplexed ACO-II structure. The crystalline form of the complex was obtained by cocrystallization of ACO-II with dodecanoyl-CoA. The crystalline complex possessed, in the active-site crevice, only the fatty acid moiety that had been formed through hydrolysis of the thioester bond. The overall dimeric structure and the folding pattern of each subunit are essentially superimposable on those of uncomplexed ACO-II. The active site including the flavin ring of FAD, the crevice embracing the fatty acyl moiety, and adjacent amino acid side chains are superimposably conserved with the exception of Glu421, whose carboxylate group is tilted away to accommodate the fatty acid. One of the carboxyl oxygens of the bound fatty acid is hydrogen-bonded to the amide hydrogen of Glu421, the presumed catalytic base, and to the ribityl 2'-hydroxyl group of FAD. This hydrogen-bonding network correlates well with the substrate recognition/activation in acyl-CoA dehydrogenase. The binding mode of C12-fatty acid suggests that the active site does not close upon substrate binding, but remains spacious during the entire catalytic process, the oxygen accessibility in the oxidative half-reaction thereby being maintained.     

Non-linear patterns in age-related DNA methylation may reflect CD4+ T cell differentiation

DNA methylation (DNAm) is an important epigenetic process involved in the regulation of gene expression. While many studies have identified thousands of loci associated with age, few have differentiated between linear and non-linear DNAm trends with age. Non-linear trends could indicate early- or late-life gene regulatory processes. Using data from the Illumina 450K array on 336 human peripheral blood samples, we identified 21 CpG sites that associated with age (P<1.03E-7) and exhibited changing rates of DNAm change with age (P<1.94E-6). For 2 of these CpG sites (cg07955995 and cg22285878), DNAm increased with age at an increasing rate, indicating that differential DNAm was greatest among elderly individuals. We observed significant replication for both CpG sites (P<5.0E-8) in a second set of peripheral blood samples. In 8 of 9 additional data sets comprising samples of monocytes, T cell subtypes, and brain tissue, we observed a pattern directionally consistent with DNAm increasing with age at an increasing rate, which was nominally significant in the 3 largest data sets (4.3E-15<P<0.039). cg07955995 and cg22285878 reside in the promoter region of KLF14, which encodes a protein involved in immune cell differentiation via the repression of FOXP3. These findings may suggest a possible role for cg07955995 and cg22285878 in immunosenescence.