A Function of Polar Flagellum and Anisotropic Growth in Vibrio alginolyticus Early-Phase Colonies

We continuously observed growth of Vibrio alginolyticus early-phase colonies on agar plates by phase-contrast microscopy. Two mutants defective in motility on solid surfaces were used in this study: one (YM4) can swim in liquid environments using its polar flagellum, and the other (NMB198) cannot swim because it lacks any flagella. We found that isolated colonies of YM4 were generally more circular than those of NMB198. This observation suggests that YM4 cells moved slightly within a colony by the function of their polar flagella. For clustered colonies, where the distance between the colonies was short (<50 μm), the colonies of YM4 grew rapidly along the line between them, but they grew slowly in the lateral directions. Some colonies of NMB198 grew toward neighboring colonies. These observations indicate colony-to-colony interaction.     

14-3-3 Mediates phosphorylation-dependent inhibition of the interaction between the ubiquitin E3 ligase Nedd4-2 and epithelial Na+ channels

Although recent studies show that the 14-3-3 protein is a negative regulator of ubiquitin E3 protein ligases, the molecular mechanism remains largely unknown. We previously demonstrated that 14-3-3 specifically binds one of the E3 enzymes, Nedd4-2 (a human gene product of KIAA0439, termed hNedd4-2), which can be phosphorylated by serum glucocorticoid-inducible protein kinase 1 (SGK1); this binding protects the phosphorylated/inactive hNedd4-2 from phosphatase-catalyzed dephosphorylation [Ichimura, T., et al. (2005) J. Biol. Chem. 280, 13187-13194]. Here we report an additional mechanism of 14-3-3-mediated regulation of hNedd4-2. Using surface plasmon resonance spectrometry, we show that 14-3-3 inhibits the interaction between the WW domains of hNedd4-2 and the PY motif of the epithelial Na(+) channel, ENaC. The inhibition was dose-dependent and was dependent on SGK1-catalyzed phosphorylation of Ser468 located between the WW domains. Importantly, a mutant of hNedd4-2, which can be phosphorylated by SGK1 but cannot bind 14-3-3, reduced SGK1-mediated stimulation of the ENaC-induced current in Xenopus laevis oocytes. In addition, 14-3-3 had similar effects on hNedd4-2 that had been phosphorylated by cAMP-dependent protein kinase (PKA). Our results, together with the recent finding on 14-3-3/parkin interactions [Sato, S., et al. (2006) EMBO J. 25, 211-221], suggest that 14-3-3 suppresses ubiquitin E3 ligase activities by inhibiting the formation of the enzyme/substrate complex.     

Effects of MdmX on Mdm2-mediated downregulation of pRB

Mdm2, a RING-finger type ubiquitin ligase, is overexpressed in a variety of human cancers. It promotes ubiquitination of the tumor suppressor p53 and can function as an oncogene by largely downregulating p53. Recently, we reported that Mdm2 degrades retinoblastoma tumor suppressor protein (pRB) via the ubiquitin-proteasome system. In the present study, we assessed the effects of MdmX, a structural homolog of Mdm2, on the Mdm2-mediated ubiquitination of pRB. MdmX is known to negatively regulate p53 function by enhancing the Mdm2-mediated ubiquitination and degradation of p53. Interestingly, MdmX inhibited the Mdm2-mediated pRB ubiquitination. Furthermore, an MdmX siRNA decreased the endogenous pRB level, while MdmX overexpression stimulated pRB functions in cultured cells. Therefore, MdmX may have different roles in the regulation of Mdm2 activity for ubiquitination of pRB and p53.     

Efficient synthesis of [2-15N]guanosine and 2'-deoxy[2'-15N]guanosine derivatives using N-(tert-butyldimethylsilyl)[15N]phthalimide as a 15N-labeling reagent

Nucleophilic aromatic substitution of 9-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)-6-chloro-2-fluoro-9H-purine with N-(tert-butyldimethylsilyl) [15N]phthalimide in the presence of a catalytic amount of CsF at room temperature in DMF efficiently afforded the 6-chloro-2-[15N]phthalimidopurine derivative, which was subsequently converted to the [2-15N]guanosine derivative. The 2'-deoxy[2'-15N]guanosine derivative was also efficiently synthesized through a similar procedure.     

Estimation of cat medial gastrocnemius fascicle lengths during dynamic contractions

In typical muscle models, it is often assumed that the contractile element (fascicle) length depends exclusively on the instantaneous muscle-tendon length and the instantaneous muscle force. In order to test whether the instantaneous fascicle length during dynamic contractions can be predicted from muscle-tendon length and force, fascicle lengths, muscle-tendon lengths, and muscle forces were directly measured in cat medial gastrocnemii during isometric and dynamic contractions. Two theoretical muscle models were developed: model A was based on force-time data obtained during the activation phase and model D on force-time data obtained during the deactivation phase of isometric contractions. To test the models, instantaneous fascicle lengths were predicted from muscle-tendon lengths and forces during dynamic contractions that simulated cat locomotion for speeds ranging from 0.4 to 1.6m/s. The theoretically predicted fascicle lengths were compared with the experimentally measured fascicle lengths. It was found that fascicle lengths were not uniquely associated with muscle-tendon lengths and forces; that is, for a given muscle-tendon length and force, fascicle lengths varied depending on the contractile history. Consequently, models A and D differed in fascicle length predictions; model D (maximum average error=8.5%) was considerably better than model A (maximum average error=22.3%). We conclude from this study that it is not possible to predict the exact fascicle lengths from muscle-tendon lengths and forces alone, however, adequate predictions seem possible based on such a model. The relationship between fascicle length and muscle force and muscle-tendon length is complex and highly non-linear, thus, it appears unlikely that accurate fascicle length predictions can be made without some reference contractions in which fascicle length, muscle-tendon length, and force are measured simultaneously.     

Identification of proteins that associate with integrin α2 by proteomic analysis in human fibrosarcoma HT-1080 cells

Integrins are adhesion receptors for components of the extracellular matrix (ECMs) that regulate multiple cellular functions, such as migration, invasion, proliferation, and survival by mediating bidirectional signal transmission. Even though many proteins have been reported to associate with integrins both on and in cells, systemic analyses of the adhesome have not been carried out. In previous studies, we identified proteins associating with a membrane-type protease, MT1-MMP, using nano-flow liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) of associated proteins prepared by optimized conditions for cell lysis and purification. Since integrins were identified as MT1-MMP-associated proteins, we next applied this method to analyze integrin-associated proteins. In this study, we expressed integrin α2 fused at the C terminus to a FLAG peptide in HT1080 cells. Cells stably expressing the chimeric protein were lysed with 1% Brij-98 and affinity purified using anti-FLAG antibody. Integrin β1 co-purified with integrin α2 confirming the specificity of the purification procedure. Analysis of the purified mixture by nano-LC/MS/MS identified 70 proteins. Nineteen of these were membrane proteins, including adhesion proteins, receptors, transporters, proteinases, and ion-channel receptors, and the balance were cytoplasmic. Interestingly, eight of the proteins had previously been shown to associate with MT1-MMP. We believe the present study provides a platform to facilitate the study of the mechanisms of cell adhesion, migration, and invasion.     

Sunitinib induces apoptosis in pheochromocytoma tumor cells by inhibiting VEGFR2/Akt/mTOR/S6K1 pathways through modulation of Bcl-2 and BAD

Sunitinib is an oral multitargeted receptor tyrosine kinase inhibitor with antiangiogenic and antitumor activity that mainly targets vascular endothelial growth factor receptors (VEGFRs). Very recently, sunitinib has been shown to be an active agent for the treatment of malignant pheochromocytomas. However, it is unclear whether sunitinib acts only through an antiangiogenic mechanism or whether it may also directly target tumor cells. Sunitinib markedly induced apoptosis of PC12 cells in a dose-dependent and time-dependent manner. Furthermore, in support of these findings, we found that sunitinib induced a reduction in the expression of the antiapoptotic molecule Bcl-2 as well as dephosphorylation of the proapoptotic molecule BAD, which results in the activation of BAD in these cells. Consistent with these apoptotic effects, our results showed that sunitinib inhibited phosphorylation of Akt and mTOR and was followed by a reduction of S6K1, which is a well-known target of mTOR. Knockdown of VEGFR-2 attenuated the sunitinib-induced effects, including apoptosis and inhibition of signaling pathways such as the phosphorylation of Akt as well as mTOR, and Bcl-2, which confirmed that these effects could be mediated by VEGFR-2. In addition, silencing of S6K1 induced apoptosis accompanied by a decrease in the phosphorylation of BAD and Bcl-2, similar to that observed with sunitinib treatment. Thus, these results together suggest that sunitinib initially exerts its apoptotic effect through the inhibition of VEGFR-2, which, when followed by reduction of its downstream effectors, including Akt/mTOR/S6K1, may lead to inhibition of the antiapoptotic molecule Bcl-2 and activation of the proapoptotic molecule BAD in PC12 cells. However, PC12 cells do not precisely reflect the pathogenesis of malignant cells. Therefore, we confirmed the key findings by replicating these experiments in human neuroblastoma SK-N-SH cells.     

Carrying capacity of wetlands for massive migratory waterfowl

Although it has been documented that waterfowl can cause water quality problems when their populations are large relative to the size or volume of the water body, the question as to how many waterbirds a wetland or a lake can support remains unanswered. A method to quantify the carrying capacity of a water body with regard to massive waterfowl was developed through the study of five wetlands that are used as the wintering ground by a large number of waterfowl in Japan. It takes into consideration water depth, retention time, and in-lake phosphorus concentration. For one of the sites, Sakata Lagoon, which is a registered Ramsar wetland in Japan, the assessment of its carrying capacity suggests that the number of waterfowl should be reduced by half to sustain the water quality of this wetland. Based on the comparison among the five wetlands, a simple indicator was proposed for quick diagnosis. Moreover, a general plot of permissible loading of phosphorus (kg/ha/year) by waterfowl was presented. Besides, this study shed some new light on potential toxicity and accumulation of waterfowl feces at lakebed. Finally, a proposal to increase the carrying capacity of Sakata Lagoon is discussed.     

Comparative angiogenic activities of induced pluripotent stem cells derived from young and old mice

Advanced age is associated with decreased stem cell activity. However, the effect of aging on the differentiation capacity of induced pluripotent stem (iPS) cells into cardiovascular cells has not been fully clarified. We investigated whether iPS cells derived from young and old mice are equally capable of differentiating into vascular progenitor cells, and whether these cells regulate vascular responses in vivo. iPS cells from mouse embryonic fibroblasts (young) or 21 month-old mouse bone marrow (old) were used. Fetal liver kinase-1 positive (Flk-1(+)) cells, as a vascular progenitor marker, were induced after 3 to 4 days of culture from iPS cells derived from young and old mice. These Flk-1(+) cells were sorted and shown to differentiate into VE-cadherin(+) endothelial cells and α-SMA(+) smooth muscle cells. Tube-like formation was also successfully induced in both young and old murine Flk-1(+) cells. Next, hindlimb ischemia was surgically induced, and purified Flk-1(+) cells were directly injected into ischemic hindlimbs of nude mice. Revascularization of the ischemic hindlimb was significantly accelerated in mice transplanted with Flk-1(+) cells derived from iPS cells from either young or old mice, as compared to control mice as evaluated by laser Doppler blood flowmetry. The degree of revascularization was similar in the two groups of ischemic mice injected with iPS cell-derived Flk-1(+) cells from young or old mice. Transplantation of Flk-1(+) cells from both young and old murine iPS cells also increased the expression of VEGF, HGF and IGF mRNA in ischemic tissue as compared to controls. iPS cell-derived Flk-1(+) cells differentiated into vascular progenitor cells, and regulated angiogenic vascular responses both in vitro and in vivo. These properties of iPS cells derived from old mice are essentially the same as those of iPS cells from young mice, suggesting the functionality of generated iPS cells themselves to be unaffected by aging.