Enhanced functional properties of corneal epithelial cells by coculture with embryonic stem cells via the integrin β1-FAK-PI3K/Akt pathway

Adult stem cells are important cell sources in regenerative medicine, but isolating them is technically challenging. This study employed a novel strategy to generate stem-like corneal epithelial cells and promote the functional properties of these cells by coculture with embryonic stem cells. The primary corneal epithelial cells were labelled with GFP and cocultured with embryonic stem cells in a transwell or by direct cell-cell contact. The embryonic stem cells were pre-transfected with HSV-tk-puro plasmids and became sensitive to ganciclovir. After 10 days of coculture, the corneal epithelial cells were isolated by treating the cultures with ganciclovir to kill the embryonic stem cells. The expression of stem cell-associated markers (ABCG2, p63) increased whereas the differentiation mark (Keratin 3) decreased in corneal epithelial cells isolated from the cocultures as evaluated by RT-PCR and flow cytometry. Their functional properties of corneal epithelial cells, including cell adhesion, migration and proliferation, were also enhanced. These cells could regenerate a functional stratified corneal epithelial equivalent but did not form tumors. Integrin β1, phosphorylated focal adhesion kinase and Akt were significantly upregulated in corneal epithelial cells. FAK Inhibitor 14 that suppressed the expression of phosphorylated focal adhesion kinase and Akt inhibited cell adhesion, migration and proliferation. LY294002 that suppressed phosphorylated Akt but not phosphorylated focal adhesion kinase inhibited cell proliferation but had no effect on cell adhesion or migration. These findings demonstrated that the functional properties of stem-like corneal epithelial cells were enhanced by cocultured embryonic stem cells via activation of the integrin β1-FAK-PI3K/Akt signalling pathway.     

Defective nuclear translocation of stress-activated signaling in senescent diploid human fibroblasts: a possible explanation for aging-associated apoptosis resistance

In order to study the nature of aging-dependent apoptosis resistance, we compared the activation pattern of mitogen-activated protein kinases (MAPK) in response to three different stress modalities: hydrogen peroxide (H₂O₂), staurosporine, and thapsigargin. We observed the agonist-specific activation pattern of MAP kinases in human diploid fibroblasts (HDFs). When young HDFs were treated with PD98059, a specific inhibitor of extracellular signal-regulated kinase (ERK), H₂O₂-induced apoptosis was blocked, whereas staurosporine-induced apoptosis was inhibited by treatment with SB203580, a specific inhibitor of p38. In addition, the levels of anti-apoptotic protein Bcl-2 (B-cell lymphoma protein-2) were restored by PD98059 or SB239063 in cells treated with H₂O₂ or staurosporine, respectively. We also found that inhibition of the nuclear import of p-Erk and p-p38 using wheat germ agglutinin induced apoptosis resistance in young HDF cells in response to H₂O₂ or staurosporine. These data indicate a potential role of the nuclear translocation of apoptotic signals in the induction of apoptosis. Moreover, the nuclear translocation of activated ERK1/2 and p38 in response to H₂O₂ or staurosporine was significantly compromised in senescent HDFs, compared with young cells. Taken together, we propose that the apoptosis resistance of senescent HDFs might be related to the defective nuclear translocation of stress-activated signals in an agonist-specific manner, which would imply the operation of an aging-dependent functional nucleo-cytoplasmic trafficking barrier.     

Marker-assisted breeding of a photoperiod-sensitive male sterile <Emphasis Type="Italic">japonica</Emphasis> rice with high cross-compatibility with <Emphasis Type="Italic">indica</Emphasis> rice

The incomplete fertility of japonica × indica rice hybrids has inhibited breeders’ access to the substantial heterotic potential of these hybrids. As hybrid sterility is caused by an allelic interaction at a small number of loci, it is possible to overcome it by simple introgression at the major sterility loci. Here we report the use of marker-assisted backcrossing to transfer into the elite japonica cv. Zhendao88 a photoperiod-sensitive male sterility gene from cv. Lunhui422S (indica) and the yellow leaf gene from line Yellow249 (indica). The microsatellite markers RM276, RM455, RM141 and RM185 were used to tag the fertility genes S5, S8, S7 and S9, respectively. Line 509S is a true-breeding photoperiod-sensitive male sterile plant, which morphologically closely resembles the japonica type. Genotypic analysis showed that the genome of line 509S comprises about 92% japonica DNA. Nevertheless, hybrids between line 509S and japonica varieties suffer from a level of hybrid sterility, although the line is highly cross-compatible with indica types, with the resulting hybrids expressing a significant degree of heterosis. Together, these results suggest that segment substitution on fertility loci based on known information and marker-assisted selection are an effective approach for utilizing the heterosis of rice inter-subspecies.     

A new factor in the Aedes aegypti immune response: CLSP2 modulates melanization

Microbial infections in the mosquito Aedes aegypti activate the newly identified CLSP1 and CLSP2 genes, which encode modular proteins composed of elastase-like serine protease and C-type lectin domains. These genes are predominantly regulated by the immune deficiency pathway, but also by the Toll pathway. Silencing of CLSP2, but not CLSP1, results in the activation of prophenoloxidase (PPO), the terminal enzyme in the melanization cascade, suggesting that CLSP2 is a negative modulator of this reaction. Haemolymph PPO activation is normally inhibited in the presence of Plasmodium parasites, but in CLSP2-depleted mosquitoes, the Plasmodium-induced block of melanization is reverted, and these mosquitoes are refractory to the parasite. Thus, CLSP2 is a new component of the mosquito immune response.     

Enzyme precipitate coatings of lipase on polymer nanofibers

Lipase (LP) was immobilized on electrospun and ethanol-dispersed polystyrene–poly(styrene-co-maleic anhydride) (PS–PSMA) nanofibers (EtOH-NF) in the form of enzyme precipitate coatings (EPCs). LP precipitate coatings (EPCs-LP) were prepared in a three-step process, consisting of covalent attachment, LP precipitation, and crosslinking of precipitated LPs onto the covalently attached LPs via glutaraldehyde treatment. The LP precipitation was performed by adding various concentrations of ammonium sulfate (20–50%, w/v). EPCs-LP improved the LP activity and stability when compared to covalently attached LPs (CA-LP) and the enzyme coatings of LPs (EC-LP) without the LP precipitation. For example, the use of 40% (w/v) ammonium sulfate resulted in EPC40-LP with the highest activity, which was 4.0 and 3.6 times higher than those of CA-LP and EC-LP, respectively. After 165-day incubation under rigorous shaking at 200 rpm, the residual activities of EPC50-LP were 0.5 μM/min mg of EtOH-NF, representing 113 and 75 times higher than those of CA-LP and EC-LP, respectively. When LP was partially purified via a simple ammonium sulfate precipitation and dialysis, both activities and stabilities of EC-LP and EPC-LP could be marginally improved. It is anticipated that the improved LP activity and stability in the form of EPCs would allow for their potential applications in various bioconversion processes such as biodiesel production and ibuprofen resolution.