Role of thrombospondin-1 in T cell response to ocular pigment epithelial cells

Ocular pigment epithelium (PE) cells promote the generation of T regulators (PE-induced Treg cells). Moreover, T cells exposed to PE acquire the capacity to suppress the activation of bystander T cells via TGFbeta. Membrane-bound TGFbeta on iris PE cells interacts with TGFbeta receptors on T cells, leading to the conversion of T cells to CD8(+) Treg cells via a cell contact-dependent mechanism. Conversely, soluble forms of TGFbeta produced by retinal PE cells can convert CD4(+) T cells into Treg cells in a manner that is independent of cell contact. In this study, we looked at the expression of immunoregulatory factors (TGFbeta, thrombospondins, CD59, IL-1 receptor antagonist, etc.) in PE cells as identified via an oligonucleotide microarray. Several thrombospondin-binding molecules were detected, and thus we focused subsequent analyses on thrombospondins. Via the conversion of latent TGFbeta to an active form that appears to be mediated by thrombospondin 1 (TSP-1), cultured iris PE and retinal PE cells induce a PE-induced Treg cell fate. After conversion, both ocular PE and PE-induced Treg cells express TSP-1. Regulatory T cell generation was amplified when the T cells also expressed TSP-1. In addition, PE-induced Treg cells significantly suppressed activation of bystander T cells via TSP-1. These results strongly suggest that the ability of ocular PE and PE-induced Treg cells to suppress bystander T cells depends on their capacity to produce TSP-1. Thus, intraocular TSP-1 produced by both ocular parenchymal cells and regulatory T cells is essential for immune regulation in the eye.     

Superoxide scavenging activity of pirfenidone-iron complex

Pirfenidone (PFD) is focused on a new anti-fibrotic drug, which can minimize lung fibrosis etc. We evaluated the superoxide () scavenging activities of PFD and the PFD-iron complex by electron spin resonance (ESR) spectroscopy, luminol-dependent chemiluminescence assay, and cytochrome c reduction assay. Firstly, we confirmed that the PFD-iron complex was formed by mixing iron chloride with threefold molar PFD, and the complex was stable in distillated water and ethanol. Secondary, the PFD-iron complex reduced the amount of produced by xanthine oxidase/hypoxanthine without inhibiting the enzyme activity. Thirdly, it also reduced the amount of released from phorbor ester-stimulated human neutrophils. PFD alone showed few such effects. These results suggest the possibility that the scavenging effect of the PFD-iron complex contributes to the anti-fibrotic action of PFD used for treating idiopathic pulmonary fibrosis.     

JNK- and IkappaB-dependent pathways regulate MCP-1 but not adiponectin release from artificially hypertrophied 3T3-L1 adipocytes preloaded with palmitate in vitro

Obese conditions increase the expression of adipocytokine monocyte chemoattractant protein-1 (MCP-1) in adipose tissue as well as MCP-1 plasma levels. To investigate the mechanism behind increased MCP-1, we used a model in which 3T3-L1 adipocytes were artificially hypertrophied by preloading with palmitate in vitro. As observed in obesity, under our model conditions, palmitate-preloaded cells showed significantly increased oxidative stress and increased MCP-1 expression relative to control cells. This increased MCP-1 expression was enhanced by adding exogenous tumor necrosis factor-alpha (TNF-alpha; 17.8-fold vs. control cells, P < 0.01) rather than interleukin-1beta (IL-1beta; 2.6-fold vs. control cells, P < 0.01). However, endogenous TNF-alpha and IL-1beta release was not affected in hypertrophied cells, suggesting that these endogenous cytokines do not mediate hypertrophy-induced increase in MCP-1. MCP-1 secretion from hypertrophied cells was significantly decreased by treatment with antioxidant N-acetyl-cysteine, JNK inhibitors SP600125 and JIP-1 peptide, and IkappaB phosphorylation inhibitors BAY 11-7085 and BMS-345541 (P < 0.01). MCP-1 secretion was not affected by peroxisome proliferator-activated receptor-gamma (PPARgamma) antagonists assayed. Adiponectin, another adipocytokine studied in parallel, also showed increased release in hypertrophy relative to control cells. But in contrast to MCP-1, adiponectin release was significantly suppressed by both exogenous TNF-alpha and IL-1beta as well as by PPARgamma antagonists bisphenol A diglycidyl ether and T0070907 (P < 0.01). JNK inhibitors and IkappaB phosphorylation inhibitors showed no significant effect on adiponectin. We conclude that adipocyte hypertrophy through palmitate loading causes oxidative stress, which in turn increases MCP-1 expression and secretion through JNK and IkappaB signaling. In contrast, the parallel increase in adiponectin expression appears to be related to the PPARgamma ligand properties of palmitate.     

TNF-α Decreases VEGF Secretion in Highly Polarized RPE Cells but Increases It in Non-Polarized RPE Cells Related to Crosstalk between JNK and NF-κB Pathways

Asymmetrical secretion of vascular endothelial growth factor (VEGF) by retinal pigment epithelial (RPE) cells in situ is critical for maintaining the homeostasis of the retina and choroid. VEGF is also involved in the development and progression of age-related macular degeneration (AMD). We studied the effect of tumor necrosis factor-α (TNF-α) on the secretion of VEGF in polarized and non-polarized RPE cells (P-RPE cells and N-RPE cells, respectively) in culture and in situ in rats. A subretinal injection of TNF-α caused a decrease in VEGF expression and choroidal atrophy. Porcine RPE cells were seeded on Transwell™ filters, and their maturation and polarization were confirmed by the asymmetrical VEGF secretion and trans electrical resistance. Exposure to TNF-α decreased the VEGF secretion in P-RPE cells but increased it in N-RPE cells in culture. TNF-α inactivated JNK in P-RPE cells but activated it in N-RPE cells, and TNF-α activated NF-κB in P-RPE cells but not in N-RPE cells. Inhibition of NF-κB activated JNK in both types of RPE cells indicating crosstalk between JNK and NF-κB. TNF-α induced the inhibitory effects of NF-κB on JNK in P-RPE cells because NF-κB is continuously inactivated. In N-RPE cells, however, it was not evident because NF-κB was already activated. The basic activation pattern of JNK and NF-κB and their crosstalk led to opposing responses of RPE cells to TNF-α. These results suggest that VEGF secretion under inflammatory conditions depends on cellular polarization, and the TNF-α-induced VEGF down-regulation may result in choroidal atrophy in polarized physiological RPE cells. TNF-α-induced VEGF up-regulation may cause neovascularization by non-polarized or non-physiological RPE cells.     

Improved frequency of transformation in rice and maize by treatment of immature embryos with centrifugation and heat prior to infection with Agrobacterium tumefaciens

The efficiency of transformation was improved by treating immature embryos with heat and centrifugation before infection with Agrobacterium tumefaciens in rice and maize. Because the effects were detected both in the levels of transgene expression after co-cultivation and in the number of independent transgenic plants obtained per embryo, conditions were first optimized based on the transgene expression, and then transformants were produced. The optimal conditions varied considerably depending on species and genotypes, but reasonably good parameters were identified for Japonica rice, Indica rice or maize. As a general tendency, the effect of centrifugation was greater than that of heat in Japonica rice, whereas that of heat was greater than that of centrifugation in Indica rice and maize A188, and the combination of the treatments was the most effective in all of the genotypes tested. The frequency of transformation was improved several fold in rice and maize. In addition, transformation of certain genotypes of maize, which were not transformable before, and transformation of maize with a less efficient vector, which could not transform maize before, became possible by these pre-treatments. In the highest case, 18 independent transgenic plants were obtained from a single immature embryo of Japonica rice. Although nothing is known about the mechanism, these pre-treatments seemed to render cells of rice and maize more competent for transformation mediated by A. tumefaciens.     

Efficient and Reproducible Myogenic Differentiation from Human iPS Cells: Prospects for Modeling Miyoshi Myopathy In Vitro

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.     

Importance of both the coding and the segment-specific noncoding regions of the influenza A virus NS segment for its efficient incorporation into virions

The genome of influenza A virus consists of eight single-strand negative-sense RNA segments, each comprised of a coding region and a noncoding region. The noncoding region of the NS segment is thought to provide the signal for packaging; however, we recently showed that the coding regions located at both ends of the hemagglutinin and neuraminidase segments were important for their incorporation into virions. In an effort to improve our understanding of the mechanism of influenza virus genome packaging, we sought to identify the regions of NS viral RNA (vRNA) that are required for its efficient incorporation into virions. Deletion analysis showed that the first 30 nucleotides of the 3' coding region are critical for efficient NS vRNA incorporation and that deletion of the 3' segment-specific noncoding region drastically reduces NS vRNA incorporation into virions. Furthermore, silent mutations in the first 30 nucleotides of the 3' NS coding region reduced the incorporation efficiency of the NS segment and affected virus replication. These results suggested that segment-specific noncoding regions together with adjacent coding regions (especially at the 3' end) form a structure that is required for efficient influenza A virus vRNA packaging.     

Cell-to-cell movement of the CAPRICE protein in Arabidopsis root epidermal cell differentiation

CAPRICE (CPC), a small, R3-type Myb-like protein, is a positive regulator of root hair development in Arabidopsis. Cell-to-cell movement of CPC is important for the differentiation of epidermal cells into trichoblasts (root hair cells). CPC is transported from atrichoblasts (hairless cells), where it is expressed, to trichoblasts, and generally accumulates in their nuclei. Using truncated versions of CPC fused to GFP, we identified a signal domain that is necessary and sufficient for CPC cell-to-cell movement. This domain includes the N-terminal region and a part of the Myb domain. Amino acid substitution experiments indicated that W76 and M78 in the Myb domain are critical for targeted transport, and that W76 is crucial for the nuclear accumulation of CPC:GFP. To evaluate the tissue-specificity of CPC movement, CPC:GFP was expressed in the stele using the SHR promoter and in trichoblasts using the EGL3 promoter. CPC:GFP was able to move from trichoblasts to atrichoblasts but could not exit from the stele, suggesting the involvement of tissue-specific regulatory factors in the intercellular movement of CPC. Analyses with a secretion inhibitor, Brefeldin A, and with an rhd3 mutant defective in the secretion process in root epidermis suggested that intercellular CPC movement is mediated through plasmodesmata. Furthermore, the fusion of CPC to tandem-GFPs defined the capability of CPC to increase the size exclusion limit of plasmodesmata.