Acute myelogenous leukemia (AML) is the most common adult leukemia, characterized by the clonal expansion of immature myeloblasts initiating from rare leukemic stem (LS) cells. To understand the functional properties of human LS cells, we developed a primary human AML xenotransplantation model using newborn nonobese diabetic/severe combined immunodeficient/interleukin (NOD/SCID/IL)2r gamma(null) mice carrying a complete null mutation of the cytokine gamma c upon the SCID background. Using this model, we demonstrated that LS cells exclusively recapitulate AML and retain self-renewal capacity in vivo. They home to and engraft within the osteoblast-rich area of the bone marrow, where AML cells are protected from chemotherapy-induced apoptosis. Quiescence of human LS cells may be a mechanism underlying resistance to cell cycle-dependent cytotoxic therapy. Global transcriptional profiling identified LS cell-specific transcripts that are stable through serial transplantation. These results indicate the potential utility of this AML xenograft model in the development of novel therapeutic strategies targeted at LS cells.

We investigated the characteristics of ethylene biosynthesis associated with ripening in banana (Musa sp. [AAA group, Cavendish subgroup] cv Grand Nain) fruit. MA-ACS1 encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase in banana fruit was the gene related to the ripening process and was inducible by exogenous ethylene. At the onset of the climacteric period in naturally ripened fruit, ethylene production increased greatly, with a sharp peak concomitant with an increase in the accumulation of MA-ACS1 mRNA, and then decreased rapidly. At the onset of ripening, the in vivo ACC oxidase activity was enhanced greatly, followed by an immediate and rapid decrease. Expression of the MA-ACO1 gene encoding banana ACC oxidase was detectable at the preclimacteric stage, increased when ripening commenced, and then remained high throughout the later ripening stage despite of a rapid reduction in the ACC oxidase activity. This discrepancy between enzyme activity and gene expression of ACC oxidase could be, at least in part, due to reduced contents of ascorbate and iron, cofactors for the enzyme, during ripening. Addition of these cofactors to the incubation medium greatly stimulated the in vivo ACC oxidase activity during late ripening stages. The results suggest that ethylene production in banana fruit is regulated by transcription of MA-ACS1 until climacteric rise and by reduction of ACC oxidase activity possibly through limited in situ availability of its cofactors once ripening has commenced, which in turn characterizes the sharp peak of ethylene production.

To obtain insights into the cardiomyogenic potential of hematopoietic tissue, we intravenously (i.v.) injected purified hematopoietic stem/progenitor cells into newborn recipients that may fully potentiate the developmental plasticity of stem cells. Transplantation of mouse bone marrow (BM) lineage antigen-negative (Lin-) cells resulted in the generation of the cells that displayed cardiomyocyte-specific antigenic profiles and contractile function when transplanted into syngeneic newborn recipients. To clarify the mechanism underlying the cardiomyogenic potential, green fluorescent protein (GFP)-labeled BM Lin-ScaI+ hematopoietic progenitors were transplanted into neonatal mice constitutively expressing cyan fluorescence protein (CFP). Lambda image acquisition and linear unmixing analysis using confocal microscopy successfully separated GFP and CFP, and revealed that donor GFP+ cardiomyocytes coexpressed host-derived CFP. We further reconstituted human hemopoietic- and immune systems in mice by injecting human cord blood (CB)-derived Lin-CD34+CD38- hematopoietic stem cells (HSCs) into neonatal T cell(-)B cell(-)NK cell- immune-deficient NOD/SCID/IL2rgamma(null) mice. Fluoroescence in situ hybridization analysis of recipient cardiac tissues demonstrated that human and murine chromosomes were colocalized in the same cardiomyocytes, indicating that cell fusion occurred between human hematopoietic progeny and mouse cardiomyocytes. These syngeneic- and xenogeneic neonatal transplantations provide compelling evidence that hematopoietic stem/progenitor cells contribute to the postnatal generation of cardiomyocytes through cell fusion, not through transdifferentiation.

Abstract. Fluorescence resonance energy transfer (FRET) with green fluorescent protein (GFP) variants has become widely used for biochemical research. In order to expand the choice of fluorescent range in FRET analysis, we designed various color versions of the FRET-based probes for caspase activity, in which the substrate sequence of the caspase was sandwiched by donor and acceptor fluorescent proteins, and studied the potential of these color versions as fluorescent indicators. Six color versions were constructed by a combination of cyan fluorescent protein (CFP), GFP, yellow fluorescent protein (YFP), and DsRed. Real-time monitoring in single cells revealed that all probes could detect caspase activation during tumor necrosis factor (TNF)-a-induced cell death as a fluorescent change. GFP-DsRed and YFP-DsRed were as sensitive as CFP-YFP, and CFP-DsRed also showed a large fluorescent change. By using two probes, CFP-DsRed and YFP-DsRed, we carried out simultaneous multi-FRET analysis and revealed that the initiator-and effector-caspases were activated almost simultaneously in TNFa -induced cell death. These findings may give experimental bases for the development of novel techniques to analyze multi-events simultaneously in single cells by using FRET probes in combination.

Line‐scan analyses of spontaneous Ca2+ sparks, non‐propagating local rises in Ca2+ concentration, and the early phase of Ca2+ transients in cardiomyocytes were performed with a rapid‐scanning laser confocal microscope (Nikon RCM8000) and fluo‐3. On electrical stimulation, points at which rise in Ca2+ began earliest were observed at regular spacings of 1.82 ± 0.26 μm (mean ± s.d.) along the longitudinal axis of the cell. The points were heavily stained with di‐2‐ANEPEQ, which stains the T‐tubules, indicating that they were at the Z‐line. The points where spontaneous Ca2+ sparks originated coincided with the points which showed faster Ca2+ elevation, i.e. the Z‐line. In some cases where a Ca2+ spark had occurred within about 30 ms before the evoked Ca2+ transient, fast elevation of Ca2+ was not observed at the corresponding Z‐line, indicating the presence of a refractory period in Ca2+ release from the SR. The present results provide visual evidence for Ca2+ release from the junctional sarcoplasmic reticulum in cardiomyocytes. The presence of a refractory period in Ca2+ release after Ca2+ sparks provided new evidence that the normal Ca2+ transient may be the summation of Ca2+ sparks.