Effects of storage temperature on the contents of sugars and free amino acids in tubers from different potato cultivars and acrylamide in chips

To clarify the effects of storage temperature on potato components and acrylamide in chips, tubers from five cultivars were stored at various temperatures (2, 6, 8, 10, and 18 degrees C) for 18 weeks, and the contents of sugars, free amino acids in tubers, and acrylamide in chips after frying were analyzed. At temperatures lower than 8 degrees C, the contents of reducing sugars increased markedly in all cultivars, with similar increases in the acrylamide level and dark brown chip color. Free amino acids showed little change at the storage temperatures tested and varied within certain ranges characteristic of each cultivar. The contents of reducing sugars correlated well with the acrylamide level when the fructose/asparagine molar ratio in the tubers was <2. When the fructose/asparagine ratio was >2 by low-temperature storage, the asparagine content, rather than the reducing sugar content, was found to be the limiting factor for acrylamide formation.     

Impact of plasminogen on an in vitro wound healing model based on a perfusion cell culture system

Vascular reorganization in wound healing is a complex process, which involves coagulation, endothelial cell proliferation and migration, basement membrane regeneration, and fibrinolysis. During this healing process, the hemostatic system and the angiogenic system are intimately interconnected. To elucidate the contribution of plasminogen in the process of wound healing, we have established a perfusion cell culture system. Using this novel cell culture system, we found that addition of plasminogen in the perfusion medium allowed the "scratch-wounded" endothelial cells to recover completely, while mini-plasminogen only affected the migration but not the proliferation of the endothelial cells. In the process of recovery with the addition of plasminogen, significant plasmin activity could only be detected when the growth of the endothelial cells have almost reached confluence. This finding indicates that wound healing is triggered and promoted during the absence of the proteolytic activity of plasmin. In addition, we could not detect any matrix metalloproteinase activity in the perfusion culture medium throughout the whole culture period. However, we did found that the circulating medium collected from the perfusion system at the early phase of the healing process has stimulatory activity on the growth of endothelial cells, but the proliferative activity decreased back to the basal level when the cells reached confluence. Thus, by using the perfusion cell culture system, we found that proliferation of endothelial cells is regulated by plasminogen and the wound healing process is controlled by a temporal interaction between the endothelial cells and plasminogen.     

VLA-5-mediated Adhesion to Fibronectin Accelerates Hemin-stimulated Erythroid Differentiation of K562 Cells through Induction of VLA-4 Expression

Fibronectin plays important roles in erythropoiesis through the fibronectin receptors VLA-4 and VLA-5. However, the substantial role of these fibronectin receptors and their functional assignment in erythroid differentiation are not yet fully understood. Here, we investigated the effects of cell adhesion to fibronectin on erythroid differentiation using K562 human erythroid progenitor cells. Erythroid differentiation could be induced in K562 cells in suspension by stimulating with hemin. This hemin-stimulated erythroid differentiation was highly accelerated when cells were induced to adhere to fibronectin by treatment with TNIIIA2, a peptide derived from tenascin-C, which has recently been found to induce β1-integrin activation. Another integrin activator, Mn²⁺, also accelerated hemin-stimulated erythroid differentiation. Adhesive interaction with fibronectin via VLA-4 as well as VLA-5 was responsible for acceleration of the hemin-stimulated erythroid differentiation in response to TNIIIA2, although K562 cells should have been lacking in VLA-4. Adhesion to fibronectin forced by TNIIIA2 causally induced VLA-4 expression in K562 cells, and this was blocked by the RGD peptide, an antagonist for VLA-5. The resulting adhesive interaction with fibronectin via VLA-4 strongly enhanced the hemin-stimulated activation of p38 mitogen-activated protein kinase, which was shown to serve as a signaling molecule crucial for erythroid differentiation. Suppression of VLA-4 expression by RNA interference abrogated acceleration of hemin-stimulated erythroid differentiation in response to TNIIIA2. Thus, VLA-4 and VLA-5 may contribute to erythropoiesis at different stages of erythroid differentiation.Hematopoietic stem and progenitor cells proliferate and differentiate in the bone marrow and fetal liver (1–6). Stromal cells of the bone marrow and fetal liver form a hematopoietic microenvironment called a “niche.” This microenvironment niche plays a crucial role in the regulation of the proliferation and differentiation of hematopoietic stem and progenitor cells. Besides humoral factors that include hematopoietic growth factors, adhesive interaction of hematopoietic stem and progenitor cells with stromal cells and/or the extracellular matrix (ECM)² in the hematopoietic microenvironment is indispensable for hematopoietic development (1–6). The ECM in the hematopoietic microenvironment is composed of various macromolecules, such as fibronectin (FN), collagens, laminins, and proteoglycans. Among them, FN is one of the most important parts of the microenvironment niche (7–11). Also, in erythropoiesis, the importance of the adhesion of erythroid progenitors to FN via the FN receptors VLA-4 and VLA-5 has been reported (11–16). However, the substantial role of these FN receptors and their functional assignment in erythroid differentiation are not yet fully understood.We previously found that FN, which provides scaffolding for the adhesion of various cell types, has an alternative functional site opposing cell adhesion (17). A 22-mer peptide derived from the 14th FN type III-like (FNIII) repeat of the FN molecule, termed FNIII14, strongly suppresses cell adhesion to FN by inhibiting the activation of β1-integrins including VLA-4 and VLA-5 (18, 19). Conversely, we have recently found that tenascin (TN)-C, which is an anti-adhesive ECM protein (20, 21), has a functional site for stimulating cell adhesion to FN (22). A 22-mer peptide derived from the FNIII repeat A2 in the TN-C molecule, termed TNIIIA2, can induce the conformational change necessary for functional activation of FN receptors through binding with syndecan-4 (22, 23). The active sites of FNIII14 and TNIIIA2 appear to be cryptic in the molecular structures of FN and TN-C but are exposed by conformational change through interaction with other ECM molecules or by processing with matrix metalloproteinase-2 (22, 24). Thus, these functional sites found in FN and TN-C molecules, which act in opposition to their parental ECM proteins, may act as a negative feedback loop for preventing excessive cellular responses to these ECM proteins in biological processes with ECM rearrangement. In any case, FNIII14 and TNIIIA2 enable us to control, either negatively or positively, the adhesion of various cell types to FN.Various hematopoietic progenitor cell lines have been used in in vitro studies of hematopoietic differentiation. However, most hematopoietic progenitor cell lines are nonadherent, because their cell surface β1-integrins, including FN receptors, have impaired ligand-binding activity (25, 26). Therefore, in order to investigate the role of cell adhesion to FN in hematopoietic differentiation, their FN receptors must be activated. Since TNIIIA2 can induce activation of FN receptors in various hematopoietic progenitor cell lines (22), this peptide factor may be useful for investigating the substantial role of cell adhesion to FN in hematopoietic differentiation. Here, we investigate the effects of cell adhesion to FN on erythroid differentiation using TNIIIA2 and Mn²⁺ as the integrin activator and the human erythroid progenitor cell line K562, which only expresses VLA-5, as the FN receptor (27). As a result, we show that hemin-stimulated erythroid differentiation of K562 cells is strongly enhanced when K562 cells are forced to adhere to FN. Sustained adhesion to FN via VLA-5, which is induced by TNIIIA2 or Mn²⁺, causes induction of VLA-4 expression. The resulting adhesive interaction with FN via newly expressed VLA-4 then generates a conspicuous increase in the hemin-stimulated phosphorylation/activation of p38 MAP kinase, which is shown to serve as a signaling molecule crucial for erythroid differentiation of K562 cells.     

A rice tryptophan deficient dwarf mutant, tdd1, contains a reduced level of indole acetic acid and develops abnormal flowers and organless embryos

Indole-3-acetic acid (IAA) plays a critical role in many aspects of plant growth and development; however, complete pathways of biosynthesis, localization and many aspects of functions of IAA in rice remain unclear. Here, we report the analysis of a rice tryptophan- (Trp-) and IAA-deficient mutant, tryptophan deficient dwarf1 ( tdd1 ) , which is embryonic lethal because of a failure to develop most organs during embryogenesis. Regenerated tdd1 plants showed pleiotropic phenotypes: dwarfing, narrow leaves, short roots and abnormal flowers. TDD1 encodes a protein homologous to anthranilate synthase β-subunit, which catalyses the first step of the Trp biosynthesis pathway and functions upstream of Trp-dependent IAA biosynthesis. TDD1-uidA and DR5-uidA expression overlapped at many sites in WT plants but was lacking in tdd1 , indicating that TDD1 is involved in auxin biosynthesis. Both Trp and IAA levels in flowers and embryos were much lower in tdd1 than in wild type (WT). Trp feeding completely rescued the mutant phenotypes and moderate expression of OsYUCCA1 , which encodes a key enzyme in Trp-dependent IAA biosynthesis, also rescued plant height and root length, indicating that the abnormal phenotypes of tdd1 are caused predominantly by Trp and IAA deficiency. In tdd1 embryos, the expression patterns of OSH1 and OsSCR , which mark the presumptive apical region and the L2 layer, respectively, are identical to those in WT, suggesting a possibility either that different IAA levels are required for basic pattern formation than for organ formation or that an orthologous gene compensates for TDD1 deficiency during pattern formation.     

Isolation and characterization of dominant dwarf mutants, <Emphasis Type="Italic">Slr1-d</Emphasis>, in rice

sd1 is known as the ‘green revolution’ gene in rice because its application in rice breeding has dramatically increased rice yield. Since the ‘green revolution,’ sd1 has been extensively used to produce modern semi-dwarf varieties. The extensive use of limited dwarfing sources may, however, cause a bottleneck effect in the genetic background of rice varieties. To circumvent this problem, novel and useful sources of dwarf genes must be identified. In this study, we identified three semi-dominant dwarf mutants. These mutants were categorized as dn-type dwarf mutants according to the elongation pattern of internodes. Gibberellin (GA) response tests showed that the mutants were still responsive to GA, although at a reduced rate. Map-based cloning revealed that the dwarf phenotype in these mutants was caused by gain-of-function mutations in the N-terminal region of SLR1. Degradation of the SLR1 protein in these mutants occurred later than in the wild type. Reduced interaction abilities of the SLR1 protein in these mutants with GID1 were also observed using the yeast two-hybrid system. Crossing experiments indicated that with the use of an appropriate genetic background, the semi-dominant dwarf alleles identified in this study could be used to alleviate the deficiency of dwarfing genes for breeding applications.     

A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus

Neutrophil extracellular traps (NETs) are webs of DNA covered with antimicrobial molecules that constitute a newly described killing mechanism in innate immune defense. Previous publications reported that NETs take up to 3-4 h to form via an oxidant-dependent event that requires lytic death of neutrophils. In this study, we describe neutrophils responding uniquely to Staphylococcus aureus via a novel process of NET formation that did not require neutrophil lysis or even breach of the plasma membrane. The multilobular nucleus rapidly became rounded and condensed. During this process, we observed the separation of the inner and outer nuclear membranes and budding of vesicles, and the separated membranes and vesicles were filled with nuclear DNA. The vesicles were extruded intact into the extracellular space where they ruptured, and the chromatin was released. This entire process occurred via a unique, very rapid (5-60 min), oxidant-independent mechanism. Mitochondrial DNA constituted very little if any of these NETs. They did have a limited amount of proteolytic activity and were able to kill S. aureus. With time, the nuclear envelope ruptured, and DNA filled the cytoplasm presumably for later lytic NET production, but this was distinct from the vesicular release mechanism. Panton-Valentine leukocidin, autolysin, and a lipase were identified in supernatants with NET-inducing activity, but Panton-Valentine leukocidin was the dominant NET inducer. We describe a new mechanism of NET release that is very rapid and contributes to trapping and killing of S. aureus.     

Effects of athletic strength and endurance exercise training in young humans on plasma endothelin-1 concentration and arterial distensibility

Strength exercise training induces a decrease in arterial distensibility, whereas endurance exercise training causes an increase in arterial distensibility. Endothelin-1 (ET-1), which is produced by vascular endothelial cells, has potent vasoconstrictor and proliferative activity on vascular smooth muscle cells. We hypothesized that endogenous ET-1 participates in alteration of arterial distensibility by different exercise training types (i.e., strength and endurance exercise training). The purpose of the present study was to investigate plasma ET-1 concentration and arterial distensibility in strength- and endurance-trained athletes. Subjects were male strength-trained athletes (discus, hammer, or javelin throwers; 22.2 years; SA), male endurance-trained athletes (long- or middle-distance runners; 20.7 years; EA), and sedentary healthy men (20.6 years; sedentary control, SC). Maximum hand-grip strength was markedly greater in SA compared with EA and SC (55.3 vs. 41.1 vs. 40.5 kg, P < 0.05). Maximum oxygen uptake was markedly greater in EA than in SA and SC (60.9 vs. 43.1 vs. 43.6 ml/kg/min, P < 0.05). Arterial pulse wave velocity (PWV), which is an index of arterial distensibility, was significantly higher in SA than in EA and SC (688 vs. 529 vs. 601 cm/sec, P < 0.05). In EA, PWV was significantly lower in comparison to that in SC (P < 0.05). Thus arterial distensibility was lower in SA than in EA and SC and higher in EA than in SC. Plasma ET-1 concentration was significantly higher in SA compared with EA and SC (1.64 vs. 1.12 vs. 1.24 pg/ml, P < 0.05). Plasma ET-1 concentration tended to be lower in EA than in SC. These results suggest that the difference in plasma ET-1 level may participate in the mechanism underlying different adaptation of arterial distensibility between strength- and endurance-trained athletes.     

Cytolethal distending toxin: a bacterial bullet targeted to nucleus

Cytolethal distending toxin (Cdt) is a newly added member of bacterial protein toxins that hijack the control system of eukaryotic cells. Cdts are produced by several pathogenic bacteria causing chronic infectious diseases. They are composed of three subunits, CdtA, CdtB and CdtC, which together form a ternary complex. CdtB is the active component, and CdtA and CdtC are involved in delivering the CdtB into the cells. The sophisticated strategy of Cdt to control host cells is CdtB-mediated limited DNA damage of the host cell chromosome, which triggers the response of the cell cycle checkpoint and results in G2 arrest in the cells. Cdt also induces apoptotic cell death of lymphocytes, which may be relevant to onset or persistence of chronic infection by the producing bacteria. The study of this toxin is expected to provide us information on a novel strategy by which bacteria interact with host cells.     

Sensitization and habituation of the swimming behavior in ascidian larvae to light

Ascidian larvae of Ciona intestinalis change their photic behavior during the course of development. Newly hatched larvae show no response to a light stimulus at any intensity. At 4 hr after hatching, larvae were induced to start to swimming upon the cessation of illumination, and to stop swimming upon the onset of illumination. At a weaker light intensity (5.0 x 10(-3) J/m (2).s), the larvae showed similar responses to either a single stimulus or repeated stimuli of onset and cessation of light until 10 hr after hatching. At a stronger light intensity (3.2 x 10(-1) J/m(2).s), when the stimulus was repeated, they showed sensitization and habituation of the swimming response. At 3 hr after hatching the larvae failed to show any response to an initial stimulus at any intensity of light, but after several repeated stimuli (sensitization) they showed a swimming response at light intensities above 4.0 x 10(-2) J/m (2).s. At 5 hr and with intensity above 1.0 x 10 (-2) J/m(2).s, the larvae showed photoresponses to the first stimulus, but after several repetitions the larvae failed to stop swimming upon the onset of light (habituation). A repeated series of stimuli at stronger intensities of light caused greater habituation; this habituation was retained for about 1 min. Since the larval central nervous system in Ciona is comprised of only about 100 neurons, learning behavior in ascidian larvae should provide insights for a minimal mechanism of memory in vertebrates.     

The evolutionarily conserved OsPRR quintet: rice pseudo-response regulators implicated in circadian rhythm

In Arabidopsis thaliana, a number of circadian-associated factors have been identified, including TOC1 (TIMING OF CAB EXPRESSION 1) that is believed to be a component of the central oscillator. TOC1 is a member of a small family of proteins, designated as ARABIDOPSIS PSEUDO-RESPONSE REGULATORS (APRR1/TOC1, APRR3, APRR5, APRR7, and APRR9). As demonstrated previously, these APRR1/TOC1 quintet members are crucial for a better understanding of the molecular links between circadian rhythms, control of flowering time through photoperiodic pathways, and also photosensory signal transduction in this dicotyledonous plant. In this respect, both the dicotyledonous (e.g. A. thaliana) and monocotyledonous (e.g. Oryza sativa) plants might share the evolutionarily conserved molecular mechanism underlying the circadian rhythm. Based on such an assumption, and as the main objective of this study, we asked the question of whether rice also has a set of pseudo-response regulators, and if so, whether or not they are associated with the circadian rhythm. Here we showed that rice has five members of the OsPRR family (Oryza sativa Pseudo-Response Regulator), and also that the expressions of these OsPRR genes are under the control of circadian rhythm. They are expressed in a diurnal and sequential manner in the order of OsPRR73 (OsPRR37)-->OsPRR95 (OsPRR59)-->OsPRR1, which is reminiscent of the circadian waves of the APRR1/TOC1 quintet in A. thaliana. These and other results of this study suggested that the OsPRR quintet, including the ortholog of APRR1/TOC1, might play important roles within, or close to, the circadian clock of rice.