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Corneal endothelial cells (CECs) do not proliferate or recover after illness or injury, resulting in decreased cell density and loss of pump/barrier function. Considering the shortage of donor cornea, it is vital to establish robust methods to generate CECs from induced pluripotent stem cells (iPSCs). We investigated the efficacy and safety of transplantation of iPSC-derived CECs into a corneal endothelial dysfunction (CED) rabbit model. iPSCs were generated from human fibroblasts. We characterized iPSCs by demonstrating the gene expression of the PSC markers OCT4, SOX2, TRA-1-60, and NANOG, teratoma formation, and differentiation into three germ layers. Differentiation of iPSCs into CECs was induced via neural crest cell (NCC) induction. CEC markers were detected using immunofluorescence and gene expression was analyzed using quantitative real-time PCR (qRT-PCR). After culturing iPSC-derived NCCs, we found the expression of zona occludens-1 (ZO-1) and Na+/K+ ATPase and a hexagonal morphology. ATP1A1, COL8A1, and AQP1 mRNA expression was higher in iPSC-derived CECs than in iPSCs and NCCs. We performed an injection of iPSC-derived CECs into the anterior chamber of a CED rabbit model and found improved levels of corneal transparency. We also found increased numbers of ZO-1- and ATP1A1-positive cells in rabbit corneas in the iPSC-derived CEC transplantation group. Usage of the coating material vitronectin (VTN) and fasudil resulted in good levels of CEC marker expression, demonstrated with Western blotting and immunocytochemistry. Combination of the VTN coating material and fasudil, instead of FNC mixture and Y27632, afforded the best results in terms of CEC differentiation’s in vitro and in vivo efficacy. Successful transplantation of CEC-like cells into a CED animal model confirms the therapeutic efficacy of these cells, demonstrated by the restoration of corneal clarity. Our results suggest that iPSC-derived CECs can be a promising cellular resource for the treatment of CED.

Patient-derived induced pluripotent stem cells (iPSCs) are materials that can be used for autologous stem cell therapy. We screened mtDNA mutations in iPSCs and iPSC-derived neuronal cells from patients with Alzheimer's disease (AD). Also, we investigated whether the mutations could affect mitochondrial function and deposition of β-amyloid (Aβ) in differentiated neuronal cells.mtDNA mutations were measured and compared among iPSCs and iPSC-derived neuronal cells. The selected iPSCs carrying mtDNA mutations were subcloned, and then their growth rate and neuronal differentiation pattern were analyzed. The differentiated cells were measured for mitochondrial respiration and membrane potential, as well as deposition of Aβ.Most iPSCs from subjects with AD harbored ≥1 mtDNA mutations, and the number of mutations was significantly higher than that from umbilical cord blood. About 35% and 40% of mutations in iPSCs were shared with isogenic iPSCs and their differentiated neuronal precursor cells, respectively, with similar or different heteroplasmy. Furthermore, the mutations in clonal iPSCs were stable during extended culture and neuronal differentiation. Finally, mtDNA mutations could induce a growth advantage with higher viability and proliferation, lower mitochondrial respiration and membrane potential, as well as increased Aβ deposition.This study demonstrates that mtDNA mutations in patients with AD could lead to mitochondrial dysfunction and accelerated Aβ deposition. Therefore, early screening for mtDNA mutations in iPSC lines would be essential for developing autologous cell therapy or drug screening for patients with AD.

Purpose To compare the protective effects of cyclosporine A emulsion (Restasis: 0.05% cyclosporine A) (CsAE) and cyclosporine A cationic emulsion (Ikervis: 0.1% cyclosporine A) (CsACE) on cellular inflammation, apoptosis, proliferation, and survival in an in vitro dry eye model. Methods The concentration of CsA in CsAE and CsACE was verified using a liquid chromatography tandem mass spectrometry system. Human corneal epithelial cells were subjected to desiccation stress. Human corneal epithelial cells were incubated with or without 3 groups of cyclosporine A medium (CsAE 1:50, CsACE 1:50, and CsACE 1:100). p-NF-κB p65, p-IκBα, Bax, Bcl-xL, p-Erk1/2, and p-Akt levels were determined using Western blots, and TNF-α levels were quantified using an enzyme-linked immunosorbent assay. Results The CsA concentration of CsACE 1:100 was nearly the same as that of CsAE 1:50. Compared with CsAE 1:50 (0.78 ± 0.19 fold), the p-NF-κB p65 level was further reduced in CsACE 1:50 (0.38 ± 1.20 fold) and 1:100 (0.29 ± 0.11 fold) as well as p-IκBα. Levels of TNF-α were also lower in CsACE 1:50 and 1:100 than in CsAE 1:50. Induction of the apoptotic protein Bax was significantly decreased in CsACE 1:50 and 1:100 compared with CsAE 1:50, whereas that of the antiapoptotic protein Bcl-xL was increased in CsACE 1:50 and 1:100. p-ERK1/2 and p-Akt levels were higher in CsACE 1:50 and 1:100 than in CsAE 1:50. Conclusions CsACE had more potent anti-inflammatory and antiapoptotic effects than CsAE in a transwell desiccation stress model. CsACE also enhanced proliferation and survival factors under desiccation stress compared with CsAE in this in vitro dry eye model.

The purpose of this study was to investigate whether and how topical nerve growth factor (NGF) attenuates streptozotocin (STZ)-induced diabetic cataracts in vivo. Rats were randomly divided into three groups, including the normal control rat group, STZ-induced diabetic cataract rat group (DM group), and STZ-induced diabetic cataract rat group treated with 200 μg/mL recombinant rat β-NGF (DM + NGF group). Cataract formation was evaluated by portable slit lamp biomicroscopy following pupil dilation at 8 weeks. The expression levels of NGF, aldose reductase (AR), and Na+/K+-ATPase in the lens epithelial cells (LECs) of the three groups were measured in the presence or absence of topical NGF. TUNEL-positive LECs were quantified to determine if hyperglycemia caused LEC apoptosis. At 8 weeks, the mean cataract score in the control group was significantly lower than that in DM and DM + NGF groups, and the score in the DM + NGF group was significantly lower than that in the DM group. At the equatorial zone and anterior central zone of lens, NGF and Na+/K+-ATPase expression levels were significantly decreased in the DM group; however, they were partially restored in the DM + NGF group. At the equatorial zone and anterior central zone of lens, AR expression and TUNEL-positive apoptotic LECs were significantly increased in the DM group compared with the control group, however, they were significantly decreased in the DM + NGF group. In conclusion, topical NGF could delay the progression of diabetic cataracts by attenuating polyol pathway activation and increasing Na+/K+-ATPase protein levels.

Purpose To evaluate the effect of diquafosol on corneal epithelium in a dry eye model using Transwell culture and a scopolamine-induced dry eye rat model. Methods Desiccation stress induced in an in vitro dry eye model using human corneal epithelial cells was used, and the cells were incubated with or without diquafosol media diluted at 1:100. Reactive oxygen species (ROS) generation was measured using 2',7'-dichlorofluorescein diacetate (DCFH-DA). Apoptosis was analyzed, and levels of phosphorylated Erk1/2, phosphorylated p90RSK, phosphorylated Akt, IκB-α, and NF-κB-p65 were determined. Levels of IL-1β, TNF-α, IL-6, IL-8, and GM-CSF were quantified. To investigate the in vivo effects of diquafosol, we induced dry eye in Wistar rats using scopolamine hydrobromide. The rats were divided into three groups: control, dry eye, and dry eye diquafosol; topical DIQUAS was applied four times daily for 28 days. We used immunohistochemistry to detect the levels of phosphorylated Erk1/2, phosphorylated p90RSK, and IL-1β, and used the TUNEL assay in corneal tissue. Results The distribution of highly fluorescent dichlorofluorescein and the proportion of annexin V- and PI-positive cells decreased in the diquafosol medium. Diquafosol increased the levels of phospho-Erk1/2, phospho-90RSK, phospho-Akt, and IκB-α, whereas it significantly decreased the levels of NF-κB-p65, IL-1β, and TNF-α. In vivo, apoptosis was enhanced in dry eye group. This response was markedly reduced and the level of phosphorylated p90RSK and phosphorylated ERK1/2 were upregulated and IL-1β was downregulated by DIQUAS. Conclusions Diquafosol treatment reduced intracellular ROS levels, apoptosis, and inflammation, all of which were increased in the dry eye model through desiccation.

To evaluate the effects of prostaglandin analogs (PGAs) on cell viability and apoptosis in cultured astrocytes obtained from the lamina cribrosa (LC) of the human optic nerve head (ONH).Astrocytes were cultured from LC samples obtained from human donor ONH and treated with three kinds of acid form of PGAs: latanoprost (LAT-A), tafluprost (TAF-A), and bimatoprost (BIM-A) (0.1, 1, 10, 50 and 100 ug/mL). Cell viability was assessed using the WST-1 assay. Cell apoptosis was measured using the deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay. Apoptotic protein expression was evaluated using western blot analysis.ONH astrocytes expressed FP receptor in western blot analysis. In the presence of 0.1 ug/mL of LAT-A, BIM-A, and TAF-A, the cell viability was 85%, 85% and 82%, respectively. WST-1 assay revealed about 50% of cell viability following treatment with 50 ug/mL of all PGAs. After exposing astrocytes to 10 ug/mL of each PGA for 24 hours, apoptotic cells were stained in TUNEL assay. Western blot analysis revealed that the PGAs up-regulated Bax (pro-apoptotic protein) and down-regulated Bcl-xL (anti-apoptotic protein) in the astrocytes.PGAs affected cell viability in cultured astrocytes obtained from human ONH LC. PGA treatment may induce apoptosis in ONH astrocytes.

To analyze the effects of supplemental epidermal growth factor (EGF) and the roles of inflammatory cytokines (interleukin [IL]-6) in an ex vivo dry-eye model under hyperosmotic stress using a multilayered culture of human conjunctival epithelial cells (HCECs).Multilayered cultures of HCECs were exposed to hyperosmotic stress (400 mOsm/L) for 24 h in addition to 0.5 ng/mL EGF (low-EGF group) or 25 ng/mL EGF (high-EGF group). Apoptosis was analyzed using the TUNEL assay. Cell proliferation was measured using the [3H]-thymidine incorporation assay. The expression of IL-6, EGF, EGF receptor (EGFR), and phosphorylated extracellular signal-regulated kinase (p-ERK) was measured by western blot analysis. The secretion of IL-6 was measured using ELISA. Western blot analysis was also performed using antibodies against cleaved caspase-3.The percentage of apoptotic cells was lower in the high-EGF group (6.7%) than in the low-EGF group (10.3%). The high-EGF group demonstrated increased proliferation (323.7 counts/min in the low-EGF group vs 649.1 counts/min in the high-EGF group). EGF induced higher phosphor-EGFR expression and upregulated p-ERK in HCECs. In addition, EGF significantly decreased the secretion of IL-6 and cleaved caspase-3 in HCECs.The level of IL-6 was increased in the ex vivo HCEC dry-eye model that was under hyperosmotic stress. Supplemental EGF reduces the level of IL-6, decreases apoptosis, and increases proliferation. These findings indicate that EGF has potential as a therapeutic agent for the treatment of dry eyes.

Purpose To examine the effects of nerve growth factor (NGF) on apoptosis and inflammation in the diabetic cornea. Methods To investigate the effects of NGF on glucose-induced apoptosis, we stained human corneal epithelial cells (HCECs) for annexin-V and propidium iodide (PI), and measured expression of cleaved caspase-3 and the Bcl-2-associated X protein (BAX). Moreover, to examine the effects of NGF on inflammation, we quantified the expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) using multiplex cytokine analysis, and analyzed nuclear factor-κB (NF-κB) activation and NF-κ-B inhibitor α (IκBα) degradation using Western blot analysis. To investigate the effects in vivo, we induced diabetes in male Sprague-Dawley rats using streptozotocin. The rats were divided into three groups: control, diabetic control, and diabetic NGF; topical NGF was applied three times daily for 3 weeks. We used the TUNEL assay to detect apoptosis in corneal tissue, and immunohistochemistry to identify cleaved caspase-3 and IL-1β. Results In HCECs, high glucose concentration (≥25 mM) led to reactive oxygen species (ROS) generation, apoptosis, and the release of inflammatory cytokines. Nerve growth factor markedly reduced ROS activation, annexin-PI-positive cells, and levels of cleaved caspase-3, BAX, IL-1β, and TNF-α. In the diabetic rat cornea, apoptosis and inflammation were enhanced, as were levels of cleaved caspase-3 and IL-1β. These responses were markedly reduced by NGF. Conclusions Apoptosis and inflammation are enhanced in the diabetic cornea; NGF attenuates these responses-both in vivo and in vitro. Therefore, NGF therapy may represent a novel approach for the management of diabetic keratopathy.

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.

Aims We investigated the mechanism by which cannabinoid receptors-1 (CB1) and -2 (CB2) modulate inflammatory activities of macrophages. Methods and results Real-time polymerase chain reaction showed the predominant CB2 expression in freshly isolated human monocytes. PMA, a potent inducer of differentiation, upregulated CB1 and increased CB1:CB2 transcript ratio from 1:17.5 to 1:3 in 5 days of culture. Immunohistochemistry showed that CB1 protein was colocalized in CD68- and CD36-positive macrophages in human atheroma. Through selective expression of CB1 or CB2 to thioglycollate-elicited peritoneal macrophages, we proved that CB1 and CB2 mediate opposing influences on the production of reactive oxygen species (ROS). Flow cytometry showed that cannabinoid-induced ROS production by macrophages was CB1-dependent. Immunoblotting assays confirmed that macrophage CB1, not CB2, induced phosphorylation of p38-mitogen-activated protein kinase, which modulated ROS production and the subsequent synthesis of tumour necrosis factor-α and monocyte chemoattractant protein-1. Pull-down assays showed that the Ras family small G protein, Rap1 was activated by CB2. Dominant-negative Rap1 profoundly enhanced CB1-dependent ROS production by macrophages, suggesting CB2 Rap1-dependently inhibits CB1-stimulated ROS production. Conclusion CB1 promotes pro-inflammatory responses of macrophages through ROS production, which is negatively regulated by CB2 through Rap1 activation. Blocking CB1 together with selective activation of CB2 may suppress pro-inflammatory responses of macrophages.

PURPOSE. We evaluated the effects of N-acetylcysteine (NAC), which is known to inhibit reactive oxygen species (ROS)‐dependent apoptosis, on high glucose‐induced ROS, apoptosis, inflammation, and delayed-wounding closure in primary cultured human conjunctival epithelial cells (pHCECs), and the regulatory effects of cleaved caspase-3, BAX, nuclear factor jB (NF-jB), IL-6, and TNF-a on these processes. METHODS. High glucose‐induced ROS generation was measured using 2 0 ,7 0 -dichlorofluorescein diacetate (DCFH-DA). The effects of NAC on high glucose‐induced apoptosis were investigated in pHCECs using Annexin-V and PI staining, and cleaved caspase-3 and BAX expression levels using immunoblotting. To evaluate the inflammatory response, IL-6 and TNF-a expression levels were quantified by multiplex cytokine analysis, and NF-jB activation and IkB-a degradation were assessed by Western blot analysis. The effects of NAC on high glucose‐delayed conjunctival epithelial wound healing were assessed by a scratch-induced directional wounding assay. RESULTS. Compared to the untreated control and normal glucose (5 mM), high glucose at 25 mM stimulated ROS generation, apoptosis, and release of inflammatory cytokines, and delayed wound healing in pHCECs. The addition of NAC markedly reduced the high glucose‐induced ROS activation, Annexin-PI‐positive cells, and levels of cleaved caspase-3, BAX, IL-6, and TNFa. N-acetylcysteine also prevented high glucose‐delayed wound healing. CONCLUSIONS. High glucose levels promote apoptosis by affecting mitochondria-dependent caspase activity through elevation of ROS production, a process that can be reversed by the antioxidant NAC. These findings demonstrate that NAC has a beneficial effect on conjunctival epithelial cell wound healing, antiapoptosis, and anti-inflammation in the conjunctival epithelial cell.

Secretory clusterin (sCLU)/apolipoprotein J is a multifunctional glycoprotein that is ubiquitously expressed in various tissues. Reduced sCLU in the joints of patients with bone erosive disease is associated with disease activity; however, its exact role has yet to be elucidated. Here, we report that CLU is expressed and secreted during osteoclastogenesis in mouse bone marrow-derived macrophages (BMMs) that are treated with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). CLU-deficient BMMs obtained from CLU(-/-) mice exhibited no significant alterations in OC differentiation in comparison with BMMs obtained from wild-type mice. In contrast, exogenous sCLU treatment significantly inhibited OC formation in both BMMs and OC precursor cultures. The inhibitory effect of sCLU was more prominent in BMMs than OC precursor cultures. Interestingly, treating BMMs with sCLU decreased the proliferative effects elicited by M-CSF and suppressed M-CSF-induced ERK activation of OC precursor cells without causing apoptotic cell death. This study provides the first evidence that sCLU reduces OC formation by inhibiting the actions of M-CSF, thereby suggesting its protective role in bone erosion.

Thirty populations from five species of Hemerocallis in Korea were analyzed by starch gel electrophoresis to measure genetic diversity and to determine genetic population structure and the amount of genetic divergence within and between species at 12 isozyme loci. In addition, Moran's I spatial autocorrelation statistics were used to examine the spatial distribution of allozyme polymorphisms in populations of H. thunbergii and H. hakuunensis. Populations of five Korean species maintain high levels of genetic variation and little differentiation among populations and species. Mean expected heterozygosities range from 0.165 in H. hongdoensis, an island endemic, to 0.265 in H. taeanensis, and a total of 81 alleles across the 12 loci were detected in the five species. G(ST) values for each of the five species were low, ranging from 0.051 in H. taeanensis to 0.078 in H. hakuunensis. Mean intraspecific Nei's genetic identities (I) between populations of the five species were all above 0.97. However, a considerable level of heterozygote deficiencies within populations was detected, ranging from 0.242 to 0.411 measured as F(IS) statistics. This deficiencies may be due to inbreeding, limited pollen and seed dispersal, or from the pooling of subpopulations that differ in allele frequencies. A small spatial scale population substructuring (

Eight populations of Hemerocallis thunbergii, H. middendorffii, and H. exaltata from Japan were analyzed by starch gel electrophoresis to measure genetic variation at ten allozyme loci. The former two species are widely distributed in eastern Asia, whereas H. exaltata is restricted to the Tobishima and Sado Islands in western Japan. Populations of the three species maintain high levels of allozyme variation: mean expected heterozygosities were 0.288, 0.209, and 0.169, respectively. The UPGMA phenogram obtained from Nei's genetic distance values indicated that H. exaltata is allozymically the most distinct species, whereas populations of H. thunbergii and H. middendorffii are closely related. However, based on gross morphology H. exaltata and H. middendorffii are closely related. This discrepancy and the observed high levels of allozyme variation within populations of the three Hemerocallis species in Japan may in part be due to genetic change among populations of the two Japanese archipelago species.

Hemerocallis hakuunensis, a Korean endemic species, maintains considerably higher levels of allozyme variation within populations (meanHe=0.253) and substantially lower levels of allozyme divergence among populations (meanG ST=0.077) than average values reported for other insect-pollinated, outcrossing herbs. Indirect estimates of the number of migrants per generation (Nm=3.00, calculated fromG ST;Nm=3.57, calculated from the frequency of nine alleles unique to single populations) indicate that gene flow has been extensive inH. hakuunensis. This is somewhat surprising when we consider the fact that no specialized seed dispersal mechanism is known, flowers are visited by bees, and the present-day populations of the species are discontinous and isolated. Results of a spatial autocorrelation analysis based on mean allele frequencies of 19 populations reveal that only 13% (95/720 cases) of Moran'sI values for the ten interpopulational distance classes are significantly different from the expected values and no distinct trend with respect to the distance classes is detected. Although it is unclear how the populations are genetically homogenous, it is highly probable thatH. hakuunensis might have a history of relatively large, continuous populations that had more chance for gene movement among adjacent populations after the last Ice Age. In addition, occasional hybridization withH. thunbergii in areas of sympatry in the central and southwestern Korean Peninsula may be one factor contributing the present-day high allozyme variation observed inH. hakuunensis.

We reported the expression of phosphorylated HSP27 during epithelial wound healing in murine corneas (Jain et al., 2012) in July of 2012. This in vivo investigation demonstrated that the expression levels of phosphorylated HSP27 were greater in wounded corneal epithelial cells than in unwounded controls and that the localization of phosphorylated HSP27 was in the basal and superficial epithelia three days following corneal epithelial wounding. We suggested that phosphorylated HSP27 had a role in the early phase of corneal epithelial wound healing. The purpose of this study was to investigate the exact role of heat shock protein 27 (HSP27) phosphorylation for the wound healing of cultured human corneal epithelial cells (HCECs). HSP27-specific siRNAs and control-siRNAs, with no known homologous targets in HCECs, were created. The cultured HCECs were divided into two groups: Scrambled control-siRNA-transfected group vs. HSP27-specific siRNA-transfected group. The scratch-induced directional wounding assay, Western blotting, using antibodies against non-phosphorylated and phosphorylated HSP27, non-phosphorylated and phosphorylated Akt, and Bcl-2-associated X protein (Bax), immunofluorescence staining to determine the filament actin, flow cytometry to measure apoptosis, and proliferation assay were performed to determine the role of HSP27. Western blot assay showed that the expression of phosphorylated HSP27 significantly increased at 5, 10, and 30 min after scratch wounding, compared with those in unwounded HCECs (all p < 0.05). Western blot assay also showed HSP27-specific siRNAs effectively blocked the expression of non-phosphorylated HSP27. The HSP27-specific siRNA-transfected group had more Bax expression, less phosphorylated Akt expression, and less non-phosphorylated and phosphorylated HSP27 expression (all p < 0.05). The scratch-induced directional wounding assay showed the HSP27-specific siRNA-transfected group with a less migrating cell number than the control-siRNA-transfected group (p < 0.05). Immunofluorescence staining showed that reorganization of actin cytoskeleton prominently decreased in the HSP27-specific siRNA-transfected group, compared with the control siRNA-tranfected group. Flow cytometry revealed that the HSP27-specific siRNA-transfected group had more HCEC apoptosis. Proliferation assay showed no difference between the two groups. In conclusion, the role of HSP27 in corneal epithelial wound healing can be epithelial cell apoptosis, as well as epithelial migration. HSP27 is involved in HCEC migration by the reorganization of actin cytoskeleton.

Genetic diversity in two species of Impatiens in Korea was investigated using starch gel electrophoresis. Impatiens hypophylla is a rare species endemic to southwestern Japan and southeastern Korea, whereas I. rextori is widespread in East Asia. Impatiens textori has considerably higher genetic diversity than I. hypophylla. For example, mean values of genetic diversity within populations (He) of I. textori and I. hypophylla were 0.206 and 0.102, respectively. The high outcrossing rate (mean 1 = 0.89), abundance and widespread distribution, and large population size may be factors responsible for the maintenance of high levels of genetic variation in I. textori. Analyss of fixation indices indicated that considerable population substructuring within populations of I. hypophylla, partly resulting from inbreeding and/or genetic drift in small populations coupled with limited gene flow (mean FIS= 0.699). However, the degree of population divergence observed in I. hypophylla (mean GST= 0.059) was considerably lower than that in other plants with similar life history traits. It is supposed that similar selection at populations of I. hypophylla examined might maintain similar gene frequencies in isolated populations.

Vitex rotundifolia L.f. is a woody perennial and has sexual and asexual modes of reproduction. Allozyme study was conducted on 550 plants in 13 Korean populations. The levels of genetic variability and divergence within and among populations, respectively, are considerably lower and higher than the mean values for woody plants with similar life history tralts. Mean percentage of polymorphic loci (PP), mean number of alleles per locus (AP), and mean genetic diversity (HeP) within populations ofV. rotundifolia were: 16.7%, 1.21, and 0.047. On average, about 79% of the total variation inV. rotundifolia was common to all populations (meanGST=0.208). In addition, significant differences in allele frequencies among populations were found in all polymorphic loci examined (P

Starch-gel electrophoresis was used to resolve nine polymorphic enzyme loci from leaf tissue collected from 20 Korean populations ofEurya japonica in order to determine differences in allele frequencies between male and female trees. In addition, 84 adults were sampled and mapped in a population located on Naenaro Island in Korea to examine spatial genetic structure using spatial autocorrelation analysis. Allele frequencies between males and females gave few contribution to the genetic structuring within populations. Only nine (5%) of 180 cases were significantly different from both sexes in allele frequencies. On the other hand, Moran'sI was significantly different from the expected value in 31 (23.5%) of 132 cases. In the shortest distance (0

Chimapliila japonica Miq., a rhizomatous evergreen subshrub, and Pyrola japonica Klenze, an evergreen herbaceous perennial, occur mainly in conifer forests in East Asia. The levels of genetic variability within populations of C. japonica are fairly low, whereas those of P. japonica are less so. Mean percentage of polymorphic loci within populations (P p), mean number of alleles per locus across populations (A p), and mean population genetic diversity (He p) were 14.8%, 1.15, and 0.030 for C. japonica and 18.4%, 1.35, and 0.087 for P. japonica. The degree of population differentiation observed in C. japonica (mean G ST = 0.028) and P. japonica (mean G ST = 0.147) was lower than that in other plants with similar life history traits. Considering the habit and pollen and seed dispersal mechanisms, it is suggested that the low G ST of C. japonica is caused by unifying selection. For C. japonica, the mean number of multilocus genotypes per population sample (G/N = 0.19) and mean genotypic diversity index (DG = 0.69) were relatively low. On the other hand, relatively high values of clonal diversity were observed in P. japonica (G/N = 0.39 and DG = 0.93). For C. japonica and P. japonica, 14% (3/21) and 6% (5/81) of multilocus genotypes were “widespread genotypes” (genotypes occurring in more than 75% of the populations), whereas the proportions of “local genotypes” (genotypes occurring in only one population) were 33% (7/21) and 40% (22/81). About 50% and 20% of the total samples examined were homozygous multilocus-genotypes for C. japonica and P. japonica, respectively. Relatively high numbers of these genotypes and low levels of clonal diversity in C. japonica are responsible for the low levels of expected heterozygosity within populations of the species. The results indicate that, for C. japonica, clonal reproduction may act as an enhancer of genetic drift by reducing the effective size of local populations.

The genetic diversity and structure of 17 Korean populations of Camelliajaponica L., a broad-leaved evergreen tree, was examined. Although most populations are restricted to several islands near the southern and southwestern coast of the Korean Peninsula, they maintain higher levels of genetic variation within populations than do long-lived, woody angiosperms. For example, 13 of 16 loci examined were polymorphic in at least one population, the mean number of alleles per locus was 2.63, and mean expected heterozygosity was 0.265. These values were comparable with those for continuously distributed, mainland populations of C. japonica in Japan. However, a considerably high level of heterozygote deficiency was observed in Korean populations of C. japonica (mean FIS = 0.202). About 13% of the total genetic variation was found among populations (GST = 0.129). Indirect estimates of the number of migrants per generation (1.69, calculated from FST; 2.14, calculated from the mean frequency of eight private alleles) indicate that gene flow among island populations is moderate. Factors contributing to the high levels of genetic diversity found in the entire species of C. japonica include long generation times, ability to regenerate by stump sprouting, predominant outcrossing induced by animal vectors, and occasional pollen dispersal by birds.

The temporal genetic structure of four pairs of juvenile (2-3 years old) and adult (ca. 50-100 years old) populations of Camellia japonica was examined using allozyme markers. No significant differences in genetic variability between two groups were found in the populations examined. About 52 and 37% of positive fixation indices calculated in the four juvenile and adult populations were significantly larger than zero with mean FIS values of 0.254 and 0.194, respectively. This indicates a considerable deficiency of heterozygotes in the juvenile and adult populations as a possible result of family structuring. Values of FIS, FIT, estimates of migration rate per generation (Nm), and FST were not significantly different between juvenile and adult populations, though slightly higher mean values of FIS, FIT and Nm, and a lower FST were found in the juvenile populations in comparison to the adult populations. However, previous studies on other woody plants showed that the seed, seedling, and juvenile stages generally have significantly higher levels of inbreeding than found in adults. The high evolutionary potential maintained in C. japonica may in part result in homogeneous levels of genetic diversity and genetic structure in time.

We investigated the spatial distribution of genotypes among Korean populations of Calystegia soldanella and C, japonica. Calystegia soldanella grows only on sand and beach dunes in East Asia and reproduces both sexually and asexually, whereas C. japonica inhabits fields and roadsides in this region, and the species reproduces exclusively by underground rhizomes. Spatial autocorrelation analysis was used to investigate the spatial distribution of genotypes of 15 and 12 alleles among 13 and eight Korean populations of C. soldanella and C. japonica, respectively. For C. soldanella, within a distance of 0–123 km, all eight significant Moran's I values were positive, indicating genetic similarity among populations within the circumference. On the other hand, in C. japonica, three significant negative autocorrelation coefficients within a 0–121 km distance were detected. The observed patterns of spatial genetic structure in both species may result from differences between them in life history and ecological traits.

Introduction: Interleukin-34 (IL-34) is a recently defined cytokine, showing a functional overlap with macrophage colony stimulating factor (M-CSF). This study was undertaken to address the expression of IL-34 in rheumatoid arthritis (RA) patients and to investigate its regulation and pathogenic role in RA. Methods: IL-34 levels were determined in the RA synovium, synovial fluid (SF) and fibroblast-like synovial cells (FLS) by immunohistochemistry, real-time PCR, enzyme-linked immunosorbent assay and immunoblotting. RA activity was assessed using Disease Activity Score 28 (DAS28) activity in the plasma collected at baseline and one year after treatment. Conditioned media (CM) were prepared from RA FLS culture with tumor necrosis factor alpha (TNF alpha) for 24 hours and used for functional assay. Results: IL-34 was expressed in the synovium, SF, and FLS from RA patients. The production of IL-34 in FLS was up-regulated by TNF alpha in RA samples compared with osteoarthritis (OA) patients. Importantly, the preferential induction of IL-34 rather than M-CSF by TNF alpha in RAFLS was mediated by the transcription factor nuclear factor kappa B (NF-kappa B) and activation of c-Jun N-terminal kinase (JNK). IL-34 elevation in plasma from RA patients was decreased after the administration of disease-modifying anti-rheumatic drugs (DMARDs) in accordance with a decrease in DAS28. CM from RAFLS cultured with TNF alpha promoted chemotactic migration of human peripheral blood mononuclear cells (PBMCs) and subsequent osteoclast (OC) formation, effects that were attenuated by an anti-IL-34 antibody. Conclusions: These data provide novel information about the production of IL-34 in RA FLS and indicate that IL-34 is an additional osteoclastogenic factor regulated by TNF alpha in RA, suggesting a discrete role of IL-34 in inflammatory RA diseases.

To better understand the patterns of variability and distributions ofHemerocallis in Korea, 53 locations were visited and measurements of 19 morphological and phenological characters were taken on plants directly from their natural habitats. For morphometric analysis, 10 plants from each of 34 populations and five herbarium specimens ofH. middendorffii were used and the data from 12 quantitative characters was analyzed using univariate analysis. Except the littoral populations of Cheju, Hong, Taehuksan, and Sohuksan Islands (H. hongdoensis M. Chung & S. Kang), three peninsular KoreanHemerocallis species can be recognized mainly in South Korea:H. hakuunensis Nakai (=H. micrantha Nakai, growing on southern, central, and northwestern Korea);H. thunbergii Baker (=H. coreana Nakai, found on southeastern and central Korea); andH. middendorffii Tr. et Mey. (central and northeastern Korea). Morphological and phenological features contributing to recognition of the three groups were; color of perianth, shape of roots, shape of inflorescence, flowering time, odor, length of inflorescence, width of the lowest bracts, length of perianth tube enclosing a ovary, width of the inner perianth lobes. Natural hybridization seems to be rare in KoreanHemerocallis. It appears that the KoreanHemerocallis species are relatively well characterized by their distribution patterns, phenology, and habitats compared with the JapaneseHemerocallis species.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is an adaptor protein which involves in the activation of the transcription factor, nuclear factor kappaB (NF-κB), in the tumor necrosis factor (TNF) receptor pathway. This signaling is modulated by proteins that interact with tumor necrosis factor receptor-associated factor 2. In this study, we identified the zinc-finger protein AWP1 as a tumor necrosis factor receptor-associated factor 2-interacting protein through yeast two-hybrid screening. We found that AWP1 directly interacted with the C-terminal tumor necrosis factor receptor-associated factor (TRAF) domain of tumor necrosis factor receptor-associated factor 2. Knockdown of AWP1 using small hairpin RNA significantly decreased nuclear factor kappaB activity but increased tumor necrosis factor alpha (TNFα)-induced apoptosis, presumably by decreasing the induction of nuclear factor kappaB-responsive anti-apoptotic molecules, including FICE-like inhibitory protein (FLIP), X-linked inhibitor of apoptosis protein (XIAP), Bcl-2, and Bcl-xL. In contrast, overexpression of wild-type AWP1 inhibited nuclear factor kappaB activation. Detailed domain mapping experiments showed that the AN1 domain of AWP1 mediated the functional interaction with tumor necrosis factor receptor-associated factor 2, and the A20 domain was responsible for the negative regulation of nuclear factor kappaB activation. Importantly, ectopic AWP1 overexpression led to an A20 domain-dependent increase in K-48 ubiquitination of tumor necrosis factor receptor-associated factor 2. These data indicate that AWP1 binds to tumor necrosis factor receptor-associated factor 2 and that regulates tumor necrosis factor alpha-induced nuclear factor kappaB activation through two distinct domains.

Rheumatoid arthritis (RA) synoviocytes are resistant to apoptosis and exhibit a transformed phenotype, which might be caused by chronic exposure to genotoxic stimuli including reactive oxygen species and growth factors. In this study, we investigated the role of vascular endothelial growth factor165 (VEGF165), a potent angiogenic factor, and its receptor in the apoptosis of synoviocytes. We demonstrated here that neuropilin-1, rather than fms-like tyrosine kinase-1 and kinase insert domain-containing receptor, is the major VEGF165 receptor in the fibroblast-like synoviocytes. Neuropilin-1 was highly expressed in the lining layer, infiltrating leukocytes, and endothelial cells of rheumatoid synovium. The production of VEGF165, a ligand for neuropilin, was significantly higher in the RA synoviocytes than in the osteoarthritis synoviocytes. The ligation of recombinant VEGF165 to its receptor prevented the apoptosis of synoviocytes induced by serum starvation or sodium nitroprusside (SNP). VEGF165 rapidly triggered phospho-Akt and phospho-ERK activity and then induced Bcl-2 expression in the rheumatoid synoviocytes. The Akt or ERK inhibitor cancelled the protective effect of VEGF165 on SNP-induced synoviocyte apoptosis. Moreover, VEGF165 blocks SNP-induced Bcl-2 down-regulation as well as SNP-induced Bax translocation from the cytosol to the mitochondria. The down-regulation of the neuropilin-1 transcripts by short interfering RNA caused spontaneous synoviocyte apoptosis, which was associated with both the decrease in Bcl-2 expression and the increase in Bax translocation to mitochondria. Collectively, our data suggest that the interaction of VEGF165 with neuropilin-1 is crucial to the survival of rheumatoid synoviocytes and provide important implications for the abnormal growth of synoviocytes and therapeutic intervention in RA.

A cDNA, PHCC-TPx, specifying a protein highly homologous to known phospholipid hydroperoxide glutathione peroxidases was isolated from a Chinese cabbage cDNA library. PHCC-TPx encodes a preprotein of 232 amino acids containing a putative N-terminal chloroplast targeting sequence and three conserved Cys residues (Cys(107), Cys(136), and Cys(155)). The mature form of enzyme without the signal peptide was expressed in Escherichia coli, and the recombinant protein was found to utilize thioredoxin (Trx) but not GSH as an electron donor. In the presence of a Trx system, the protein efficiently reduces H(2)O(2) and organic hydroperoxides. Complementation analysis shows that overexpression of the PHCC-TPx restores resistance to oxidative stress in yeast mutants lacking GSH but fails to complement mutant lacking Trx, suggesting that the reducing agent of PHCC-TPx in vivo is not GSH but is Trx. Mutational analysis of the three Cys residues individually replaced with Ser shows that Cys(107) is the primary attacking site by peroxide, and oxidized Cys(107) reacts with Cys(155)-SH to make an intramolecular disulfide bond, which is reduced eventually by Trx. Tryptic peptide analysis by matrix-assisted laser desorption and ionization time of flight mass spectrometry shows that Cys(155) can form a disulfide bond with either Cys(107) or Cys(136).

Purpose To evaluate the effects of nerve growth factor (NGF), which is activated during inflammatory episodes of ocular diseases, on the apoptotic response in cultured human primary conjunctival epithelial cells (pHCECs). Methods Levels of NGF transcripts and NGF protein in pHCEC grown in medium with normal osmolarity (307 mOsm/L) or hyperosmolar medium (350, 400, and 450 mOsm/L) were determined using RT-PCR or ELISA, respectively. To assess apoptosis, pHCEC were cultured in normal or 400 mOsm/L hyperosmolar medium with neutralizing anti-NGF antibody or recombinant human NGF for 6 hours before analysis by flow cytometry. Levels of Bcl-xL, Bax, phosphorylated JNK, and cleaved caspase-3 were detected using Western blotting. Levels of the inflammatory cytokine IL-6 was analyzed using ELISA. Results Hyperosmolar conditions increased NGF levels in cultured pHCECs. Hyperosmolarity and exposure to neutralizing anti-NGF antibody significantly increased the number of apoptotic cells. Addition of recombinant human NGF to 400 mOsm/L medium decreased the number of apoptotic cells, reduced the expression of phosphorylated JNK, Bax, and cleaved caspase-3 and increased the expression of Bcl-xL. Levels of IL-6 were increased by hyperosmotic stress but decreased by exposure to recombinant human NGF. Conclusions Hyperosmolarity induces apoptosis of pHCECs by activating JNK signaling. Increased levels of NGF under hyperosmolar conditions may contribute, at least in part, to the reduced apoptosis of pHCECs and may be beneficial in recovering conjunctival damage due to chronic hyperosmolar stress.

Hemerocallis taeanensis is described as a new species. Populations of the species are presently known only from grasslands under pine-oak coastal forests in Taean Gun, along the middle western Korean coast. The new species is distinctive in its combination of slightly swollen roots lacking a rhizome; deciduous, small leaves (32-56 cm long); usually tubular scape, short (0.3-5.5 cm long), usually once branched (Y-type) inflorescence with 2-5-flowers, and orange-yellow, small perianth (5.0-8.6 cm long). Other dwarf groups of Hemerocallis in eastern Asia (e.g., H. nana H.forrestii, and H. plicata, all native in southwestern China) are easily distinguished from H. taeanensis by their golden-orange flowers, short perianth tube (0.5-1.5 cm long), and highly inflated roots.

Hemerocallis hongdoensis, a new species of section Fulvae, is described. Hemerocallis hongdoensis occurs in rocky and humus soil on talus slopes or rocky cliffs on remote islands off the southwestern coast of Korea. Hemerocallis hongdoensis is most closely related to H. aurantiaca but differs from this species by its underground parts lacking a rhizome, deciduous leaves, ovate-lanceolate bracts below inflorescences which are 1.2-3.5 cm long, dichotomous or trichotomous inflorescences which are 3-9 cm long, stout perianth tube (2.4-3.2 cm x 5.5-6.0 mm), orange-yellow perianth lacking a red tinge, and a different flowering period. Hemerocallis L. is an economically important genus of approximately 30 species restricted mainly to eastern Asia (Matsuoka & Hotta, 1966). Hemerocallis has been placed in the Liliaceae (Cronquist, 1981; Hutchinson, 1964) or Hemerocallidaceae (Dahlgren et al., 1985). Matsuoka & Hotta (1966) and Hotta (1986) pointed out that the major difficulty with the classification of Hemerocallis lies in the uncertainty regarding criteria for delimiting the species. The taxonomic difficulties have been attributed to the relatively small number of diagnostic characters. Many species (e.g., H. aurantiaca Baker, H. flava L., H. fulva L., and H. thunbergii Baker) were described on the basis of cultivars with unknown origins (Matsuoka & Hotta, 1966), and with the additional problems of extreme difference in appearance between living plants and dried herbarium specimens (i.e., floral morphology and color of perianth), missing label data on dried herbarium specimens (i.e., flowering time and odor) (Matsuoka & Hotta, 1966), and possible widespread hybridization (Kitamura et al., 1986). For these reasons, the treatment of species within Hemerocallis has varied depending on the authors. For example, Matsuoka & Hotta (1966) and Hotta (1986) treated H. littorea Makino as a variety under H. fulva, while Kitamura et al. (1986) treated the taxon as a variety under H. aurantiaca. Matsuoka & Hotta (1966) also noted that Bailey (1930), Nakai (1932), and Stout (1941) did not consider the variability of natural populations when developing their classifiNOVON 4: 94-97. 1994. cations. Based on the literature for Hemerocallis (e.g., Bailey, 1930; Nakai, 1932; Stout, 1941; Ohwi, 1965; Matsuoka & Hotta, 1966; Hotta, 1986; Kitamura et al., 1986), the Japanese Hemerocallis are relatively well known with respect to their geographical and ecological distribution. On the other hand, the daylilies native to Korea are not well understood taxonomically. In 1988, 1991, 1992, and 1993, we conducted field trips to collect daylilies from natural populations in Korea in order to better understand the variation of morphological characters and geographical and ecological distribution. In 1988 and 1993, while visiting the remote islands of Hong, Sohuksan, and Taehuksan off the southwestern coast of Korea, we encountered a morphologically distinct group of Hemerocallis populations not referable to any described species. In the herbaria at Seoul National University (SNU) and Tokyo University (TI), which we visited, there were two undetermined specimens of this group collected by Ishidoya and T. Chung (TI). This series of populations is a new species as described below, and is named after the type locality, Hong Island. Hemerocallis (sect. Fulvae Nakai em.) hongdoensis M. Chung & S. Kang, sp. nov. TYPE: Korea. Chollanam Do, Shinan Gun, Huksan Myeon, Hong Island, Hongdo1-gu, among rocky and humus soil on E-facing talus slope, open area, ca. 30 m, 16 Aug. 1993, M. G. Chung & S. S. Kang 2028 (holotype, GNUC; isotypes, GA, GNUC, MO, SNU, TI). Figure 1. Radices perennes, tuberoso-inflatae, fuscae; folia visidiflava radicaria districa complanata vel striata, arcuatodeflexa, 60-100 cm longa, 1.7-3.0 cm lata; scapi erecti robustiusculi, 60-76 cm longi, 5-8 mm crassi, plerumque cum 1-2 ovata-lanceolatae bracteae. Inflorescentia dichotoma vel trichotoma, 5-17(-25)-floris 3-9 cm longi; pedicelli 1 cm longi 4 mm crassi; flores ascendentes; periantha aurantiaca-flava infundibularia 10.4-14.2 cm longa tenuiter succulentus; tubus crassus ca. 2.4-3.2 cm longi 5.5-6.0 mm crassis dilutus viridi-aurantiacus; lobi interiores 8-11 cm longi, 2.7-3.4 cm lati, margine integer, lobi exteriores lanceolati apice naviculato-acuti, 7.810.5 cm longi 1.3-1.7 cm lati. Capsula oblongus-ovalis, apice emarginati, pagina verruciformis, 2.5-4.1 cm longa 1.7-2.5 cm lata; semina nigra lucidula ca. 7 mm longa 5 mm lata. This content downloaded from 157.55.39.136 on Thu, 19 May 2016 05:00:28 UTC All use subject to http://about.jstor.org/terms Volume 4, Number 2 1994 Chung & Kang Hemerocallis hongdoensis