Hypergravity induces expression of cyclooxygenase-2 in the heart vessels

Cyclooxygenase-2 (COX-2), a rate-limiting enzyme for prostaglandin biosynthesis, is induced by various stimuli including mechanical stress and plays important roles in pathophysiological conditions. For example, gravitational stress has been shown to induce expression of COX-2 in bone tissues, which is essential for bone homeostasis. To investigate whether COX-2 is induced by gravitational loading in other tissues than bone, we exposed mice to hypergravity at 2G and 3G for 4 h. We demonstrate here that COX-2 is induced in the mouse heart vessels by hypergravity. Moreover, hypoxia-inducible factor (HIF)-1alpha and its downstream genes such as inducible nitric oxide synthase, vascular endothelial growth factor, and heme oxygenase-1 were induced in the heart simultaneously, while none of these genes were induced in the COX-2(-/-) mouse heart. Therefore, COX-2 induced in the heart helps protect the heart function against hypoxia under hypergravity condition through HIF-1alpha induction.     

Coordinated activation of Wnt in epithelial and melanocyte stem cells initiates pigmented hair regeneration

Melanocyte stem cells (McSCs) intimately interact with epithelial stem cells (EpSCs) in the hair follicle bulge and secondary hair germ (sHG). Together, they undergo activation and differentiation to regenerate pigmented hair. However, the mechanisms behind this coordinated stem cell behavior have not been elucidated. Here, we identified Wnt signaling as a key pathway that couples the behavior of the two stem cells. EpSCs and McSCs coordinately activate Wnt signaling at the onset of hair follicle regeneration within the sHG. Using genetic mouse models that specifically target either EpSCs or McSCs, we show that Wnt activation in McSCs drives their differentiation into pigment-producing melanocytes, while EpSC Wnt signaling not only dictates hair follicle formation but also regulates McSC proliferation during hair regeneration. Our data define a role for Wnt signaling in the regulation of McSCs and also illustrate a mechanism for regeneration of complex organs through collaboration between heterotypic stem cell populations.     

Wnt/BMP signal integration regulates the balance between proliferation and differentiation of neuroepithelial cells in the dorsal spinal cord

Multiple signaling pathways regulate proliferation and differentiation of neural progenitor cells during early development of the central nervous system (CNS). In the spinal cord, dorsal signaling by bone morphogenic protein (BMP) acts primarily as a patterning signal, while canonical Wnt signaling promotes cell cycle progression in stem and progenitor cells. However, overexpression of Wnt factors or, as shown here, stabilization of the Wnt signaling component beta-catenin has a more prominent effect in the ventral than in the dorsal spinal cord, revealing local differences in signal interpretation. Intriguingly, Wnt signaling is associated with BMP signal activation in the dorsal spinal cord. This points to a spatially restricted interaction between these pathways. Indeed, BMP counteracts proliferation promoted by Wnt in spinal cord neuroepithelial cells. Conversely, Wnt antagonizes BMP-dependent neuronal differentiation. Thus, a mutually inhibitory crosstalk between Wnt and BMP signaling controls the balance between proliferation and differentiation. A model emerges in which dorsal Wnt/BMP signal integration links growth and patterning, thereby maintaining undifferentiated and slow-cycling neural progenitors that form the dorsal confines of the developing spinal cord.     

Molecular nature of methicillin-resistant Staphylococcus aureus derived from explosive nosocomial outbreaks of the 1980s in Japan

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) with Panton-Valentine leukocidin (PVL) genes is increasing worldwide. Nosocomial outbreak-derived (hospital-acquired) MRSA (HA-MRSA) in Japan in the 1980s was also largely PVL(+). PVL(+) HA-MRSA and CA-MRSA shared the same multi-locus sequence type (ST30) and methicillin resistance cassette (SCCmecIV), but were divergent in oxacillin resistance, spa typing, PFGE analysis or clfA gene analysis. PVL(+) HA-MRSA, which probably originated in PVL(+)S. aureus ST30, was highly adhesive (carrying cna and bbp genes), highly-toxic (carrying luk(PV) and sea genes) and highly drug-resistant. PVL(+) HA-MRSA was once replaced by other PVL(-) HA-MRSA (e.g., ST5), and is re-emerging as CA-MRSA.     

High-LET ion radiolysis of water: visualization of the formation and evolution of ion tracks and relevance to the radiation-induced bystander effect

Ionizing radiation-induced bystander effects, commonly observed in cell populations exposed to high-linear energy transfer (LET) radiations, are initiated by damage to a cellular molecule which then gives rise to a toxic signal exported to neighboring cells not directly hit by radiation. A major goal in studies of this phenomenon is the identification of this initial radiation-induced lesion. Liquid water being the main constituent of biological matter, reactive species produced by water radiolysis in the cellular environment are likely to be major contributors to the induction of this lesion. In this context, the radiation track structure is of crucial importance in specifying the precise location and identity of all the radiolytic species and their subsequent signaling or damaging effects. We report here Monte Carlo track structure simulations of the radiolysis of liquid water by four different impacting ions 1H+, 4He2+, 12C6+ and 20Ne10+, with the same LET ( approximately 70 keV/ microm). The initial radial distribution profiles of the various water decomposition products (eaq(-), *OH, H*, H2 and H2O2) for the different ions considered are presented and discussed briefly in the context of track structure theory. As an example, the formation and temporal evolution of simulated 24 MeV 4He2+ ion tracks (LET approximately 26 keV/microm) are reported for each radiolytic species from 1 ps to 10 micros. The calculations reveal that the ion track structure is completely lost by approximately 1 micros.     

Targeted disruption of the mouse rho-associated kinase 2 gene results in intrauterine growth retardation and fetal death

Rho-associated kinase (ROCK), including the ROCK-I and ROCK-II isoforms, is a protein kinase involved in signaling from Rho to actin cytoskeleton. However, in vivo functions of each ROCK isoform remain largely unknown. We generated mice deficient in ROCK-II by gene targeting. ROCK-II(-/-) embryos were found at the expected Mendelian frequency until 13.5 days postcoitum, but approximately 90% died thereafter in utero. ROCK-II(-/-) mice of both genders that survived were born runts, subsequently developed without gross abnormality, and were fertile. Whole-mount staining for a knocked-in lacZ reporter gene revealed that ROCK-II was highly expressed in the labyrinth layer of the placenta. Disruption of architecture and extensive thrombus formation were found in the labyrinth layer of ROCK-II(-/-) mice. While no obvious alteration in actin filament structures was found in the labyrinth layer of ROCK-II(-/-) placenta and stress fibers were formed in cultured ROCK-II(-/-) trophoblasts, elevated expression of plasminogen activator inhibitor 1 was found in ROCK-II(-/-) placenta. These results suggest that ROCK-II is essential in inhibiting blood coagulation and maintaining blood flow in the endothelium-free labyrinth layer and that loss of ROCK-II leads to thrombus formation, placental dysfunction, intrauterine growth retardation, and fetal death.     

Metabolome and Photochemical Analysis of Rice Plants Overexpressing Arabidopsis NAD Kinase Gene

The chloroplastic NAD kinase (NADK2) is reported to stimulate carbon and nitrogen assimilation in Arabidopsis (Arabidopsis thaliana), which is vulnerable to high light. Since rice (Oryza sativa) is a monocotyledonous plant that can adapt to high light, we studied the effects of NADK2 expression in rice by developing transgenic rice plants that constitutively expressed the Arabidopsis chloroplastic NADK gene (NK2 lines). NK2 lines showed enhanced activity of NADK and accumulation of the NADP(H) pool, while intermediates of NAD derivatives were unchanged. Comprehensive analysis of the primary metabolites in leaves using capillary electrophoresis mass spectrometry revealed elevated levels of amino acids and several sugar phosphates including ribose-1,5-bisphosphate, but no significant change in the levels of the other metabolites. Studies of chlorophyll fluorescence and gas change analyses demonstrated greater electron transport and CO₂ assimilation rates in NK2 lines, compared to those in the control. Analysis of oxidative stress response indicated enhanced tolerance to oxidative stress in these transformants. The results suggest that NADP content plays a critical role in determining the photosynthetic electron transport rate in rice and that its enhancement leads to stimulation of photosynthesis metabolism and tolerance of oxidative damages.NADP is a ubiquitous coenzyme, required in various metabolic processes, since these metabolites carry electrons through the reversible conversion between oxidized (NAD⁺, NADP⁺) and reduced (NADH, NADPH) forms in all organisms. NAD is highly oxidized and is involved primarily in intracellular catabolic reactions, whereas NADP is predominantly found in its reduced form and participates in anabolic reactions and defense against oxidative stress (Ziegler, 2000; Noctor et al., 2006; Pollak et al., 2007a). Since NAD(H) and NADP(H) play a variety of distinct physiological roles, the regulation of the NAD(H)/NADP(H) balance is essential for cell survival (Kawai and Murata, 2008; Hashida et al., 2009).One of the key enzymes that regulates NAD(H)/NADP(H) balance is NAD kinase (NADK; EC 2.7.1.23), which catalyzes NAD phosphorylation in the presence of ATP. The genes encoding NADK were cloned recently from all organisms investigated to date, except for Chlamydia trachomatis (Kawai and Murata, 2008). Only a single gene encoding NADK has been found in some bacteria and mammals (Kawai and Murata, 2008). In contrast, NADK activity was detected in not only the cytosol but also organelles in yeast and plant (Jarrett et al., 1982; Simon et al., 1982; Dieter and Marme, 1984; Iwahashi and Nakamura, 1989; Iwahashi et al., 1989), and three genes including cytosol-type and organelle-type NADK have been cloned in yeast (Kawai et al., 2001; Outten and Culotta, 2003) and plants (Turner et al., 2004, 2005).In Arabidopsis (Arabidopsis thaliana), one of the NADK isoforms is localized in the chloroplast (NADK2; Chai et al., 2005), the others are localized in the cytosol (NADK1 and NADK3; Chai et al., 2006). Analysis of Arabidopsis mutants revealed low chlorophyll (chl) content, low photosynthetic activity, growth inhibition, and hypersensitivity to environmental stresses in the nadk2 knockout mutant (Chai et al., 2005; Takahashi et al., 2006), whereas the nadk1 knockout mutant and the nadk3 knockout mutant did not show a significant phenotype, except for sensitivity to oxidative stress (Berrin et al., 2005; Chai et al., 2006). Moreover, the major part of NADP(H) biosynthesis in photosynthetic organ appears to be attributable to NADK2, because NADK and NADP(H) were strictly decreased in leaves of the nadk2 knockout mutant (Chai et al., 2005; Takahashi et al., 2006). In the plant cell, NADP is mainly located in the chloroplast (Heber and Santarius, 1965; Wigge et al., 1993), where NADP⁺ functions as the final electron acceptor of the photosynthetic electron transport. The reducing energy obtained is not only supplied for Calvin cycle, nitrogen assimilation, lipid and chl metabolism, but also play a crucial role in maintaining redox homeostasis through the regulation of producing and consuming reactive oxygen species (ROS) in the plant cell (Noctor, 2006; Noctor et al., 2006). Accordingly, these evidences indicate that chloroplastic NADK2 plays a central role in plant metabolism and stress tolerance through homeostasis of ROS as regulator of NADP/NAD balance.Since the alteration of NAD/NADP balance affects metabolism and ROS homeostasis, manipulation of NADK can be an attractive target for the engineering of plant metabolism. It was reported that overexpression of NADK causes perturbation of NADP(H) pool and has positive effects on stress tolerance or growth in various living things. In Asperadium nidulans, overexpression of NADH kinase improves the growth efficiency of the cell (Panagiotou et al., 2009). Overexpression of NADK in human HEK293 cells causes 4-to 5-fold increase of NADPH concentration and provides moderate protection against oxidative stress (Pollak et al., 2007b). Recently, we evaluated effects of the enhanced NADP(H) content in Arabidopsis by generating NADK2-overexpressing plants (Takahashi et al., 2009). Our results indicated that enhanced NADP(H) production by NADK2 overexpression promoted nitrogen assimilation and resulted in accumulation of metabolites associated with the Calvin cycle, accompanied by increased activity of Rubisco. Together, these studies demonstrated the potential use of NADK as candidate gene in promoting primary metabolism and/or stress tolerance in transgenic plants.Rice (Oryza sativa) is not only the primary crop for more than half of the world''s population, but also a model monocot system. Rice can adapt to more strong light intensity than Arabidopsis, because rice is a sun plant, whereas Arabidopsis is a shade plant. Therefore, it is possible that effects of an increased NADP(H) content could be more significant in rice plant than in Arabidopsis, due to a higher ability to manage reductive energy involved in NADP as an electron carrier. In this article, we describe the generation and characterization of transgenic rice plants expressing an Arabidopsis chloroplastic NADK (AtNADK2), under the control of the maize (Zea mays) ubiquitin promoter. We named the rice plant as NK2. We found that pleiotropic effects on primary metabolism in NK2 rice were similar to the result obtained in NADK2-overexpressing Arabidopsis plants. However, stimulation of carbon fixation and nitrogen assimilation were observed in NK2 rice, accompanying with significant increases in electron transport and CO₂ assimilation rates, unlike results of the previous study of Arabidopsis. Interestingly, the NK2 lines also showed enhanced tolerance to oxidative stress.     

Persistent expression of stabilized beta-catenin delays maturation of radial glial cells into intermediate progenitors

Transgenic mice expressing stabilized beta-catenin in neural progenitors develop enlarged brains resulting from increased progenitor expansion. To more precisely define beta-catenin regulation of progenitor fate, we employed a conditional transgenic approach to delete the beta-catenin regulatory domain from neural progenitors, resulting in expression of stabilized protein from its endogenous promoter in these cells and their progeny. An increased fraction of transgenic cortical cells express the progenitor markers Nestin and LewisX, confirming a relative expansion of this population. Sustained beta-catenin activity expands RC2 and Pax6 expression in the developing cortex while postponing the onset of Tbr2 expression, suggesting a delay in maturation of radial glia into intermediate progenitors. Furthermore, transgenic cortical cells fail to either upregulate ErbB4 or develop a mitogenic response to epidermal growth factor, changes that normally accompany the acquisition of an intermediate fate. Likewise, transgenic brains do not develop a distinct subventricular zone or superficial cortical layers, and overexpression of stabilized beta-catenin by in utero electroporation caused a relative reduction of upper layer vs. lower layer cortical neurons, indicating that persistent beta-catenin activity interferes with the generation of progenitors responsible for the production of upper layer cortical neurons. Collectively, these findings demonstrate that beta-catenin functions to maintain the radial glial population, and suggest that downregulation of beta-catenin signaling may be critical to facilitate the transition to an intermediate progenitor phenotype.     

Cytoplasmic acidification may occur in high-pressure carbon dioxide-treated Escherichia coli K12

While studying the mechanism by which high-pressure carbon dioxide treatment (HCT) inactivates bacteria, we found that the efficiency of DNA recovery via phenol extraction was extraordinarily low from E. coli K12 cells that had been subjected to HCT. DAPI staining of the treated cells, however, revealed that nuclear DNA was present. Most DNA from the cells subjected to HCT was probably caught in the denatured protein layer during phenol extraction. The efficiency of DNA recovery from proteinase-treated crude extracts from cells subjected to HCT was high. Crude extracts of E. coli K12 cells that had not undergone HCT were intentionally acidified with acetic acid to pH 5.2 to cause acidic coagulation of cytoplasmic proteins. The efficiency of DNA recovery from the acidified extracts was low. These results suggest that in cells subjected to HCT, cytoplasmic pH is reduced to around pH 5.2, and that nuclear DNA becomes entangled in coagulated cytoplasmic proteins. Acidification of the cytoplasm might be the primary mechanism by which HCT inactivates bacteria.