In response to the lack of a transgenic line of zebrafish labeled with heart-specific fluorescence in vivo to serve as a research model, we cloned a 1.6-kb polymerase chain reaction (PCR) -product containing the upstream sequence (؊870 bp), exon 1 (39 bp), intron 1 (682 bp), and exon 2 (69 bp) of the zebrafish cardiac myosin light chain 2 gene, (cmlc2). A germ-line transmitted zebrafish possessing a green fluorescent heart was generated by injecting this PCR product fused with the green fluorescent protein (GFP) gene with ends consisting of inverted terminal repeats of an adeno-associated virus. Green fluorescence was intensively and specifically expressed in the myocardial cells located both around the heart chambers and the atrioventricular canal. Neither the epicardium nor the endocardium showed fluorescent signals. The GFP expression in the transgenic line faithfully recapitulated with the spatial and temporal expression of the endogenous cmlc2. Promoter analysis showed that the fragment consisting of nucleotides from ؊210 to 34 (؊210/34) was sufficient to drive heart-specific expression, with a ؊210/؊73 motif as a basal promoter and a ؊210/؊174 motif as an element involved in suppressing ectopic (nonheart) expression. Interestingly, a germ-line of zebrafish whose GFP appeared ectopically in all muscle types (heart, skeletal, and smooth) was generated by injecting the fragment including a single nucleotide mutation from G to A at ؊119, evidence that A at ؊119 combined with neighboring nucleotides to create a consensus sequence for binding myocyte-specific enhancer factor-2. Developmental Dynamics 228:30 -40, 2003.

Receptor tyrosine kinase EphB3 is expressed in cells in the bottom of intestinal crypts near stem cell niches. Loss of Ephb3 has recently been reported to produce invasive colorectal carcinoma in Apc(Min/+) mice and EphB-mediated compartmentalization was demonstrated to be a mechanism suppressing colorectal cancer progression; however, it is unknown whether other factors contribute to EphB-mediated tumor suppression. EphA4-ephrin-A and EphB4-ephrin-B2 signaling have been reported to promote mesenchymal-to-epithelial transition (MET). Here, we examine whether EphB3-ephrin-B interaction has a similar effect and investigate its role in tumor suppression. We found in a clinical cohort that EphB3 expression was significantly reduced in advanced Dukes' stage tumor specimens, so we over-expressed EphB3 in HT-29 cells by stable transfection. EphB3 over-expression inhibited HT-29 growth in monolayer cultures, anchorage-independent growth in soft agar and xenograft growth in nude mice and initiated morphological, behavioral and molecular changes consistent with MET. Specifically, EphB3 over-expression re-organized cytoskeleton (converting spreading cells to a cobble-like epithelial morphology, patterning cortical actin cytoskeleton and polarizing E-cadherin and ZO-1), induced functional changes favoring MET (decreased transwell migration, increased apoptosis and Ca(2+)-dependent cell-cell adhesion), decreased mesenchymal markers (fibronectin and nuclear beta-catenin), increased epithelial markers (ZO-1, E-cadherin and plakoglobin) and inactivated CrkL-Rac1, a known epithelial-to-mesenchymal transition signaling pathway. Additionally, cross talk from Wnt signaling potentiated the restoration of epithelial cell polarity. Noteworthily, the same factors contributing to MET, owing to EphB3 signaling, also facilitated tumor suppression. We conclude that EphB3-ephrin-B interaction promotes MET by re-establishing epithelial cell-cell junctions and such an MET-promoting effect contributes to EphB3-mediated tumor suppression.

To examine the role of placenta growth factor (PlGF) in the pathogenesis of pulmonary emphysema, we generated PlGF-transgenic (TG) mice using a phosphoglycerate kinase promoter. This resulted in constitutive overexpression of PlGF. In these TG mice, pulmonary emphysema, with enlarged air spaces and enhanced pulmonary compliance, first appeared at 6 months of age and became prominent at 12 months. Increased alveolar septal cell apoptosis was noted in their lungs. Fluorescence-activated cell sorter analysis suggests that these apoptotic septal cells are type II pneumocytes. At the same time, the messenger RNA of vascular endothelial growth factor and platelet-endothelial cell adhesion molecule-1, an endothelial cell marker, were downregulated indicating a reduced number of endothelial cells and its survival factor VEGF. In vitro, exogenous PlGF can inhibit the proliferation and promote the cell death of mouse type II pneumocytes. In normal newborn mice, abundant expression of PlGF messenger RNA was detected in the lungs during saccular division but was rapidly downregulated after alveolarization was complete. Thus, a persistently elevated PlGF was detrimental to the developed lung and causes the emphysematous change seen in our TG mice. Our study suggests that PlGF plays an important role in the pathogenesis of pulmonary emphysema via its action on type II pneumocytes.

GC tends to exhibit more aggressive tumor behavior in young patients than in old patients; however, the surgical survival of young and old patients was similar. Advanced nodal involvement (N3) is the most important independent prognostic factor in the young.