Cultured human corneal epithelial stem/progenitor cells derived from the corneal limbus

Stem/progenitor cells of the human corneal epithelium are present in the human corneal limbus, and several corneal epithelial stem/progenitor cell markers have been reported. Recently, the neurotrophin family receptors were reported to be useful markers of corneal epithelial stem/progenitor cells. Therefore, we examined an enzymatic separation method for obtaining corneal epithelial stem/progenitor cells and measuring the change in the expression of low-affinity neurotrophin receptor p75 (p75^NTR), a receptor belonging to the neurotrophin family. As a result, it was found that our separation method preserved cell viability. Furthermore, p75^NTR was mainly observed in epithelial basal cells as were the corneal epithelial stem/progenitor markers p63 and integrin β1. p75^NTR was also observed in the cultured cells, but its frequency decreased with passage. In conclusion, we propose that our culture method will enable the culture of corneal stem cells and that it is a useful tool for elucidating the molecular basis of the niche that is necessary for the maintenance of epithelial stem cells in the corneal limbus. Furthermore, we conclude that p75^NTR is a useful cell marker for evaluating the characteristics of stem/progenitor cells in culture.     

TGFβ induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

Background and aims: Transforming growth factor-beta (TGFβ) is known to potently inhibit cell growth. Loss of responsiveness to TGFβ inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGFβ and HB-EGF signal transduction via ADAM activation.Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGFβ. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGFβ was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGFβ was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown.Result: TGFβ-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGFβ induced shedding of proHB-EGF allowing HB-EGF-CTF to translocate to the nucleus. ADAM inhibitors blocked this nuclear translocation. TGFβ enhanced gastric cancer cell growth and ADAM inhibitors suppressed this effect. EGFR phosphorylation, HB-EGF-CTF nuclear translocation, and cell growth were suppressed in ADAM17 knockdown cells.Conclusion: HB-EGF-CTF nuclear translocation and EGFR transactivation from proHB-EGF shedding mediated by ADAM17 activated by TGFβ might be an important pathway of gastric cancer cell proliferation by TGFβ.     

Delta1 family members are involved in filopodial actin formation and neuronal cell migration independent of Notch signaling

Delta family proteins are transmembrane molecules that bind Notch receptors and activate downstream signaling events in neighboring cells. In addition to serving as Notch ligands, Notch-independent roles for Delta have been suggested but are not fully understood. Here, we demonstrate a previously unrecognized role for Delta in filopodial actin formation. Delta1 and Delta4, but not Delta3, exhibit filopodial protrusive activity, and this activity is independent of Notch signaling. The filopodial activity of Delta1 does not depend on the PDZ-binding domain at the C-terminus; however, the intracellular membrane-proximal region that is anchored to the plasma membrane plays an important role in filopodial activity. We further identified a Notch-independent role of DeltaD in neuronal cell migration in zebrafish. These findings suggest a possible functional link between Notch-independent filopodial activity of Delta and the control of cell motility.     

Rho A and the Rho kinase pathway regulate fibroblast contraction: Enhanced contraction in constitutively active Rho A fibroblast cells

Fibroblast cells play a central role in the proliferation phase of wound healing processes, contributing to force development. The intracellular signaling pathways regulating this non-muscle contraction are only partially understood. To study the relations between Rho A and contractile responses, constitutively active Rho A (CA-Rho A) fibroblast cells were reconstituted into fibers and the effects of calf serum (CS) on isometric force were studied. CS-induced force in CA-Rho A fibroblast fibers was twice as large as that in wild type (NIH 3T3) fibroblast fibers. During this response, the translocation of Rho A from the cytosol to the membrane was detected by Rho A activity assays and Western blot analysis. Pre-treatment with a Rho specific inhibitor (C3-exoenzyme) suppressed translocation as well as contraction. These results indicate that Rho A activation is essential for fibroblast contraction. The Rho kinase inhibitor (Y27632) inhibited both NIH 3T3 and CA-Rho A fibroblast fiber contractions. Activation of Rho A is thus directly coupled with Rho kinase activity. We conclude that the translocation of Rho A from the cytosol to the membrane and the Rho kinase pathway can regulate wound healing processes mediated by fibroblast contraction.     

A Mathematical Modelling for the Cheliped Regeneration with Handedness in Fiddler Crab

An enormously developed giant cheliped with the other small one characterizes the adult male fiddler crab. Some experiments with artificial severances of cheliped indicate that such a handedness in the cheliped size is maintained even after the regeneration of severed cheliped. Other experimental researches give some results about an unknown physiological system which controls the emergence and the regeneration of the handedness in the cheliped size. In this paper, with two hypothesized factors relevant to the regeneration of a severed cheliped, we propose a simple mathematical model to describe the experimental result about the cheliped regeneration with a handedness after the cheliped severance for the fiddler crab. Our model gives a suggestion about an underlying system for the cheliped regeneration in the fiddler crab or some other crustacean species.     

The expression of iron homeostasis-related genes during rice germination

To characterize Fe homeostasis during the early stages of seed germination, a microarray analysis was performed. mRNAs extracted from fully mature seeds or seeds harvested 1–3 days after sowing were hybridized to a rice microarray containing approximately 22,000 cDNA oligo probes. Many Fe deficiency-inducible genes were strongly expressed throughout early seed germination. These results suggest that the demand for Fe is extremely high during germination. Under Fe-deficient conditions, rice produces and secretes a metal-cation chelator called deoxymugineic acid (DMA) to acquire Fe from the soil. In addition, DMA and its intermediate nicotianamine (NA) are thought to be involved in long distance Fe transport in rice. Using promoter-β-glucuronidase (GUS) analysis, we investigated the expression patterns during seed germination of the Fe deficiency-inducible genes OsNAS1, OsNAS2, OsNAS3, OsNAAT1, and OsDMAS1, which encode enzymes that participate in the biosynthesis of DMA, and the transporter genes OsYSL2 and OsIRT1, which are involved in Fe transport. All of these genes were expressed in germinating seeds prior to protrusion of the radicle. These results suggest that DMA and NA are produced and involved in Fe transport during germination. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.