Junko Tanaka

Monitoring cellular movement in vivo with photoconvertible fluorescence protein ''Kaede'' transgenic mice

Kaede is a photoconvertible fluorescence protein that changes from green to red upon exposure to violet light. The photoconversion of intracellular Kaede has no effect on cellular function. Using transgenic mice expressing the Kaede protein, we demonstrated that movement of cells with the photoconverted Kaede protein could be monitored from lymphoid organs to other tissues as well as from skin to the draining lymph node. Analysis of the kinetics of cellular movement revealed that each subset of cells in the lymph node, such as CD4+ T, CD8+ T, B, and dendritic cells, has a distinct migration pattern in vivo. Thus, the Kaede transgenic mouse system would be an ideal tool to monitor precise cellular movement in vivo at different stages of immune response to pathogens as well as in autoimmune diseases.

The first CH domain of affixin activates Cdc42 and Rac1 through αPIX, a Cdc42/Rac1-specific guanine nucleotide exchanging factor

Rho GTPases, Cdc42 and Rac1, play pivotal roles in cell migration by efficiently integrating cell‐substrate adhesion and actin polymerization. Although it has been suggested that integrins stimulate these Rho GTPases via some of integrin binding proteins such as focal adhesion kinase (FAK) and paxillin, the precise molecular mechanism is largely unknown. In this study, we showed that the over‐expression of RP1 corresponding to the first CH domain (CH1) of affixin, an integrin‐linked kinase (ILK)‐binding protein, induced a significant actin reorganization in MDCK cells by activating Cdc42/Rac1. Affixin full length and RP1 co‐immunoprecipitated with αPIX, a Cdc42/Rac1‐specific guanine nucleotide exchanging factor (GEF), and they co‐localized at the tips of lamellipodia in motile cells. The involvement of αPIX in the RP1‐induced Cdc42 activation was demonstrated by the significant dominant negative effect of a point mutant of αPIX, αPIX (L383R, L384S), lacking GEF activity. Our data strongly support that ILK and affixin provide a novel signalling pathway that links integrin signalling to Cdc42/Rac1 activation.

Negative feedback loop in T-cell activation through MAPK-catalyzed threonine phosphorylation of LAT

Mitogen‐activated protein kinase (MAPK) cascades are involved in a variety of cellular responses including proliferation, differentiation, and apoptosis. We have developed an expression screening method to detect in vivo substrates of MAPKs in mammalian cells, and identified a membrane protein, linker for activation of T cells (LAT), as an MAPK target. LAT, an adapter protein essential for T‐cell signaling, is phosphorylated at its Thr 155 by ERK in response to T‐cell receptor stimulation. Thr 155 phosphorylation reduces the ability of LAT to recruit PLCγ1 and SLP76, leading to attenuation of subsequent downstream events such as [Ca2+]i mobilization and activation of the ERK pathway. Our data reveal a new role for MAPKs in a negative feedback loop in T‐cell activation via threonine phosphorylation of LAT.

The C. elegans CBFβ homologue BRO-1 interacts with the Runx factor, RNT-1, to promote stem cell proliferation and self-renewal

In this report, we investigate the C. elegans CBFβ homologue, BRO-1. bro-1 mutants have a similar male-specific sensory ray loss phenotype to rnt-1 (the C. elegans homologue of the mammalian CBFβ-interacting Runx factors), caused by failed cell divisions in the seam lineages. Our studies indicate that BRO-1 and RNT-1 form a cell proliferation-promoting complex, and that BRO-1 increases both the affinity and specificity of RNT-1-DNA interactions. Overexpression of bro-1, like rnt-1, leads to an expansion of seam cell number and co-overexpression of bro-1 and rnt-1 results in massive seam cell hyperplasia. Finally, we find that BRO-1 appears to act independently of RNT-1 in certain situations. These studies provide new insights into the function and regulation of this important cancer-associated DNA-binding complex in stem cells and support the view that Runx/CBFβ factors have oncogenic potential.

Application of ozone treatment for ammonia removal in spent brine

Abstract The application of ozone treatment for ammonia removal in spent brine was investigated. The presence of bromide and bicarbonate ions in spent brine was advantageous for the efficient removal of ammonia. Iodide and chloride ions did not show positive effect on ammonia removal. The formation of BrO 3 − , a compound classified as carcinogenic, was below the limit of detection during the ozonation process. Furthermore, a kinetic model for the continuous ozone treatment for ammonia removal was derived and used for the determination of suitable treatment conditions and equipment design. In continuous ozonation experiments, pH decreased for longer retention times and trihalomethanes were produced.

In Vivo image Analysis Using iRFP Transgenic Mice

Fluorescent proteins with light wavelengths within the optical window are one of the improvements in in vivo imaging techniques. Near-infrared (NIR) fluorescent protein (iRFP) is a stable, nontoxic protein that emits fluorescence within the NIR optical window without the addition of exogenous substrate. However, studies utilizing an in vivo iRFP model have not yet been published. Here, we report the generation of transgenic iRFP mice with ubiquitous NIR fluorescence expression. iRFP expression was observed in approximately 50% of the offspring from a matings between iRFP transgenic and WT mice. The serum and blood cell indices and body weights of iRFP mice were similar to those of WT mice. Red fluorescence with an excitation wavelength of 690 nm and an emission wavelength of 713 nm was detected in both newborn and adult iRFP mice. We also detected fluorescence emission in whole organs of the iRFP mice, including the brain, heart, liver, kidney, spleen, lung, pancreas, bone, testis, thymus, and adipose tissue. Therefore, iRFP transgenic mice may therefore be a useful tool for various types of in vivo imaging.

Study of normal and pathological blood vessel morphogenesis in Flt1‐tdsRed BAC Tg mice

Blood vessel development and network patterning are controlled by several signaling molecules, including VEGF, FGF, TGF‐ß, and Ang‐1,2. Among these, the role of VEGF‐A signaling in vessel morphogenesis is best understood. The biological activity of VEGF‐A depends on its reaction with specific receptors Flt1 and Flk1. Roles of VEGF‐A signaling in endothelial cell proliferation, migration, survival, vascular permeability, and induction of tip cell filopodia have been reported. In this study, we have generated Flt1‐tdsRed BAC transgenic (Tg) mice to monitor Flt1 gene expression during vascular development. We show that tdsRed fluorescence is observed within blood vessels of adult mice and embryos, indicative of retinal angiogenesis and tumor angiogenesis. Flt1 expression recapitulated by Flt1‐tdsRed BAC Tg mice overlapped well with Flk1, while Flt1 was expressed more abundantly in endothelial cells of large blood vessels such as dorsal aorta and presumptive stalk cells in retina, providing a unique model to study blood vessel development. genesis 50:561–571, 2012.

Novel Mitochondria-Targeted Heat-Soluble Proteins Identified in the Anhydrobiotic Tardigrade Improve Osmotic Tolerance of Human Cells

Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called “anhydrobiosis”. Late Embryogenesis Abundant (LEA) proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble) and SAHS (Secretory Abundant Heat Soluble) proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble), as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments.

Comparison of endothelin-1 concentrations in normal and complicated pregnancies

Endothelin-1 (ET-1) may be important in regulating vascular tone in humans. To understand the role of ET-1 during pregnancy, we measured the concentrations of plasma immunoreactive ET-1 in normal, toxemic (pre-eclamptic or eclamptic), and other complicated pregnancies. There were no significant differences between ET-1 concentrations in normal pregnancy and those in toxemic pregnancy, except in one case with IgA nephropathy and severe toxemia. There also was no significant difference between the ET-1 concentrations in pure toxemia of pregnancy compared with superimposed toxemia of pregnancy. Furthermore, there was no correlation between ET-1 concentrations, blood pressure, or renal function during toxemic pregnancy. Similarly, there were no significant differences between ET-1 plasma levels in normal pregnancies and other complicated pregnancies except for two cases in which much blood was lost due to uterine atony and abruptio placenta. Thus, plasma ET-1 concentrations showed no significant relationship to blood pressure or renal function during normal or toxemic pregnancy, and were not related to the type (pure or superimposed type), grade, or severity of toxemic pregnancy.

Cancer cells uptake porphyrins via heme carrier protein 1

Although exogenous porphyrin accumulation in cancer cells is important for the success of photodynamic therapies, the mechanism is not clear. We hypothesized that a newly reported transporter, heme carrier protein 1 (HCP1), is highly expressed in cancer cells, and transports porphyrins into the cells. We investigated the following three unknowns: whether cancer cells take up hematoporphyrin derivative via HCP1, whether HCP1 is involved in photodynamic therapies, and whether cancer cells highly express HCP1. First, when HCP1-overexpressed cells were treated with hematoporphyrin derivative and then exposed to an eximer laser beam, they emitted a significantly higher intensity of hematoporphyrin derivative fluorescence and became more susceptible to the laser beam than control. Second, when three other types of cancer cells with silenced HCP1 were treated with hematoporphyrin derivative and then exposed to the laser beam, they emitted a significantly lower intensity of hematoporphyrin derivative fluorescence. Third, non-cancer cells slightly expressed HCP1; on the other hand, the three other types of cancer cells clearly expressed HCP1. These results indicated that cancer cells uptake hematoporphyrin derivative via HCP1 and over-expression of HCP1 increases the efficacy of photodynamic therapies by increasing porphyrin accumulation in the cells. This is the first report about a transporter of porphyrin in cancer cells.