Ethogram of Immature Green Turtles: Behavioral Strategies for Somatic Growth in Large Marine Herbivores

Animals are assumed to obtain/conserve energy effectively to maximise their fitness, which manifests itself in a variety of behavioral strategies. For marine animals, however, these behavioral strategies are generally unknown due to the lack of high-resolution monitoring techniques in marine habitats. As large marine herbivores, immature green turtles do not need to allocate energy to reproduction but are at risk of shark predation, although it is a rare occurrence. They are therefore assumed to select/use feeding and resting sites that maximise their fitness in terms of somatic growth, while avoiding predation. We investigated fine-scale behavioral patterns (feeding, resting and other behaviors), microhabitat use and time spent on each behavior for eight immature green turtles using data loggers including: depth, global positioning system, head acceleration, speed and video sensors. Immature green turtles at Iriomote Island, Japan, spent an average of 4.8 h feeding on seagrass each day, with two peaks, between 5∶00 and 9∶00, and between 17∶00 and 20∶00. This feeding pattern appeared to be restricted by gut capacity, and thus maximised energy acquisition. Meanwhile, most of the remaining time was spent resting at locations close to feeding grounds, which allowed turtles to conserve energy spent travelling and reduced the duration of periods exposed to predation. These behavioral patterns and time allocations allow immature green turtles to effectively obtain/conserve energy for growth, thus maximising their fitness.     

Ovarian Cancer Stem Cells Are Enriched in Side Population and Aldehyde Dehydrogenase Bright Overlapping Population

Cancer stem-like cells (CSCs)/cancer-initiaiting cells (CICs) are defined as a small population of cancer cells that have self-renewal capacity, differentiation potential and high tumor-initiating ability. CSCs/CICs of ovarian cancer have been isolated by side population (SP) analysis, ALDEFLUOR assay and using cell surface markers. However, these approaches are not definitive markers for CSCs/CICs, and it is necessary to refine recent methods for identifying more highly purified CSCs/CICs. In this study, we analyzed SP cells and aldehyde dehydrogenese bright (ALDH^Br) cells from ovarian cancer cells. Both SP cells and ALDH^Br cells exhibited higher tumor-initiating ability and higher expression level of a stem cell marker, sex determining region Y-box 2 (SOX2), than those of main population (MP) cells and ALDH^Low cells, respectively. We analyzed an SP and ALDH^Br overlapping population (SP/ALDH^Br), and the SP/ALDH^Br population exhibited higher tumor-initiating ability than that of SP cells or ALDH^Br cells, enabling initiation of tumor with as few as 10² cells. Furthermore, SP/ADLH^Br population showed higher sphere-forming ability, cisplatin resistance, adipocyte differentiation ability and expression of SOX2 than those of SP/ALDH^Low, MP/ALDH^Br and MP/ALDH^Low cells. Gene knockdown of SOX2 suppressed the tumor-initiation of ovarian cancer cells. An SP/ALDH^Br population was detected in several gynecological cancer cells with ratios of 0.1% for HEC—1 endometrioid adenocarcinoma cells to 1% for MCAS ovary mucinous adenocarcinoma cells. Taken together, use of the SP and ALDH^Br overlapping population is a promising approach to isolate highly purified CSCs/CICs and SOX2 might be a novel functional marker for ovarian CSCs/CICs.     

Identification and characterization of a novel polysaccharide deacetylase C (PdaC) from Bacillus subtilis

Cell wall metabolism and cell wall modification are very important processes that bacteria use to adjust to various environmental conditions. One of the main modifications is deacetylation of peptidoglycan. The polysaccharide deacetylase homologue, Bacillus subtilis YjeA (renamed PdaC), was characterized and found to be a unique deacetylase. The pdaC deletion mutant was sensitive to lysozyme treatment, indicating that PdaC acts as a deacetylase. The purified recombinant and truncated PdaC from Escherichia coli deacetylated B. subtilis peptidoglycan and its polymer, (-GlcNAc-MurNAc[-L-Ala-D-Glu]-)(n). Surprisingly, RP-HPLC and ESI-MS/MS analyses showed that the enzyme deacetylates N-acetylmuramic acid (MurNAc) not GlcNAc from the polymer. Contrary to Streptococcus pneumoniae PgdA, which shows high amino acid sequence similarity with PdaC and is a zinc-dependent GlcNAc deacetylase toward peptidoglycan, there was less dependence on zinc ion for deacetylation of peptidoglycan by PdaC than other metal ions (Mn(2+), Mg(2+), Ca(2+)). The kinetic values of the activity toward B. subtilis peptidoglycan were K(m) = 4.8 mM and k(cat) = 0.32 s(-1). PdaC also deacetylated N-acetylglucosamine (GlcNAc) oligomers with a K(m) = 12.3 mM and k(cat) = 0.24 s(-1) toward GlcNAc(4). Therefore, PdaC has GlcNAc deacetylase activity toward GlcNAc oligomers and MurNAc deacetylase activity toward B. subtilis peptidoglycan.     

Pyruvate secreted by human lymphoid cell lines protects cells from hydrogen peroxide mediated cell death

Reactive oxygen species (ROS) released from polymorphonuclear leukocytes and macrophages could cause DNA damage, but also induce cell death. Therefore inhibition of cell death must be an important issue for accumulation of genetic changes in lymphoid cells in inflammatory foci. Scavengers in the post culture medium of four lymphoid cell lines, lymphoblastoid cell lines (LCL), Raji, BJAB and Jurkat cells, were examined. Over 80% of cultured cells showed cell death 24 h after xanthine (X)/xanthine oxidase (XOD) treatment, which was suppressed by addition of post culture medium from four cell lines in a dose-dependent manner. H₂O₂ but not O^·-₂ produced by the X/XOD reaction was responsible for the cytotoxity, thus we used H₂O₂ as ROS stress thereafter. The H₂O₂-scavenging activity of post culture media from four cell lines increased rapidly at the first day and continued to increase in the following 2–3 days for LCL, Raji and BJAB cells. The scavenging substance was shown to be pyruvate, with various concentrations in the cultured medium among cell lines. Over 99% of total pyruvate was present in the extracellular media and less than 1% in cells. α-Cyano-4-hydroxycinnamate, a specific inhibitor of the H⁺-monocarbohydrate transporter, increased the H₂O₂-scavenging activity in the media from all four cell lines via inhibition of pyruvate re-uptake by cultured cells from the media. These findings suggest that lymphoid cells in inflammatory foci could survive even under ROS by producing pyruvate, so that accumulation of lymphoid cells with DNA damage is possible.     

The Role of NF-κB Signaling in the Maintenance of Pluripotency of Human Induced Pluripotent Stem Cells

NF-κB signaling plays an essential role in maintaining the undifferentiated state of embryonic stem (ES) cells. However, opposing roles of NF-κB have been reported in mouse and human ES cells, and the role of NF-κB in human induced pluripotent stem (iPS) cells has not yet been clarified. Here, we report the role of NF-κB signaling in maintaining the undifferentiated state of human iPS cells. Compared with differentiated cells, undifferentiated human iPS cells showed an augmentation of NF-κB activity. During differentiation induced by the removal of feeder cells and FGF2, we observed a reduction in NF-κB activity, the expression of the undifferentiation markers Oct3/4 and Nanog, and the up-regulation of the differentiated markers WT-1 and Pax-2. The specific knockdown of NF-κB signaling using p65 siRNA also reduced the expression of Oct3/4 and Nanog and up-regulated WT-1 and Pax-2 but did not change the ES-like colony formation. Our results show that the augmentation of NF-κB signaling maintains the undifferentiated state of human iPS and suggest the importance of this signaling pathway in maintenance of human iPS cells.     

Sphingosine-1-phosphate signaling controlling osteoclasts and bone homeostasis

Bone is a dynamic organ that is continuously turned over during growth, even in adults. During bone remodeling, homeostasis is regulated by the balance between bone formation by osteoblasts and bone resorption by osteoclasts. However, in pathological conditions such as osteoporosis, osteopetrosis, arthritic joint destruction, and bone metastasis, this equilibrium is disrupted. Since osteoclasts are excessively activated in osteolytic diseases, the inhibition of osteoclast function has been a major therapeutic strategy. It has recently been demonstrated that sphingosine-1-phosphate (S1P), a biologically active lysophospholipid that is enriched in blood, controls the trafficking of osteoclast precursors between the circulation and bone marrow cavities via G protein-coupled receptors, S1PRs. While S1PR1 mediates chemoattraction toward S1P in bone marrow, where S1P concentration is low, S1PR2 mediates chemorepulsion in blood, where the S1P concentration is high. The regulation of precursor recruitment may represent a novel therapeutic strategy for controlling osteoclast-dependent bone remodeling. By means of intravital multiphoton imaging of bone tissues, we have recently revealed that the reciprocal action of S1P controls the migration of osteoclast precursors between bone tissues and blood stream. Imaging technologies have enabled us to visualize the in situ behaviors of different cell types in intact tissues. In this review we also discuss future perspectives on this new method in the field of bone biology and medical sciences in general. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

The steroid-binding properties of recombinant glucocorticoid receptor: a putative role for heat shock protein hsp90

The steroid-binding domain of the human glucocorticoid receptor was expressed in Escherichia coli either as a fusion protein with protein A or under control of the T7 RNA polymerase promoter. The recombinant proteins were found to bind steroids with the normal specificity for a glucocorticoid receptor but with reduced affinity (Kd for triamcinolone acetonide approximately 70 nM). Glycerol gradient analysis of the E. coli lystate containing the recombinant protein indicated no interaction between the glucocorticoid receptor fragment and heat shock proteins. However, synthesis of the corresponding fragments of glucocorticoid receptor in vitro using rabbit reticulocyte lystate resulted in the formation of proteins that bound triamcinolone acetonide with high affinity (Kd 2nM). Glycerol gradient analysis of these proteins, with and without molybdate, indicated that the in vitro synthesised receptor fragments formed complexes with hsp90 as previously shown for the full-length rat glucocorticoid receptor. Radiosequence analysis of the recombinant steroid-binding domain expressed in E. coli and affinity labelled with dexamethasone mesylate identified binding of the steroid to Cys-638 predominantly. However, all cysteine residues within the steroid-binding domain were affinity labelled to a certain degree indicating that the recombinant protein has a structure similar to the native receptor but more open and accessible.     

Separation of abscission zone cells in detached Azolla roots depends on apoplastic pH

In studies on the mechanism of cell separation during abscission, little attention has been paid to the apoplastic environment. We found that the apoplastic pH surrounding abscission zone cells in detached roots of the water fern Azolla plays a major role in cell separation. Abscission zone cells of detached Azolla roots were separated rapidly in a buffer at neutral pH and slowly in a buffer at pH below 4.0. However, cell separation rarely occurred at pH 5.0–5.5. Light and electron microscopy revealed that cell separation was caused by a degradation of the middle lamella between abscission zone cells at both pH values, neutral and below 4.0. Low temperature and papain treatment inhibited cell separation. Enzyme(s) in the cell wall of the abscission zone cells might be involved in the degradation of the pectin of the middle lamella and the resultant, pH-dependent cell separation. By contrast, in Phaseolus leaf petioles, unlike Azolla roots, cell separation was slow and increased only at acidic pH. The rapid cell separation, as observed in Azolla roots at neutral pH, did not occur. Indirect immunofluorescence microscopy, using anti-pectin monoclonal antibodies, revealed that the cell wall pectins of the abscission zone cells of Azolla roots and Phaseolus leaf petioles looked similar and changed similarly during cell separation. Thus, the pH-related differences in cell separation mechanisms of Azolla and Phaseolus might not be due to differences in cell wall pectin, but to differences in cell wall-located enzymatic activities responsible for the degradation of pectic substances. A possible enzyme system is discussed.     

The Chromatographic Purification of Yeast Invertase by an Ion-Exchange Resin Method and Some Properties of the Enzyme Obtained

The purification of yeast invertase was attempted by application of the chromatographic method using Duolite C-10, a sulfonic acid cation exchange resin. This method was found to be extremely simple in process and significantly effective for the improvement of purity of the enzyme, compared with those other methods reported, hitherto. In the present paper, the procedure of the purification and some properties of the enzyme obtained thereby, are described, and some discussion of the implications is presented.