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Background Natural killer (NK) cells are an emerging new tool for cancer immunotherapy. To develop NK cell therapeutics from peripheral blood mononuclear cells (PBMCs) of healthy donors, substantial expansion of primary NK cells is necessary because of the very low number of these cells in peripheral blood. In this study, we aimed to investigate the effect of various cytokine alone or combinations, in expanded NK cells and to analyze the synergetic effect of cytokine combinations. Methods Human NK cells were isolated from healthy donor PBMC. Purified NK cells were stimulated with single cytokines or combinations of IL-2, IL-15, IL-18, and IL-27. The expanded NK cells were characterized by flow cytometry, cytotoxicity assay, calcein AM assay and Western blot. Results We investigated the synergistic effects of each cytokine, namely, IL-2, IL-15, IL-18, and IL-27, on human NK cells isolated from PBMCs of healthy donors and cultured for 21 days. We identified that IL-15/IL-18/IL-27-mediated activation of NK cells most potently increased NK cell proliferation, cytotoxicity, and IFN-ɣ secretion compared with the activation observed with other treatments, including IL-2, IL-15, and IL-15/IL-18. Additionally, the expression of DNAM-1, NKG2D, CD69, and natural cytotoxicity receptors (NCRs; NKp30 and NKp44) increased on day 21 compared to that on day 0, demonstrating the activation of NK cells. In vitro, expanded NK cells were highly cytotoxic against cancer cells, displaying increased perforin and granzyme B accumulation. Conclusions Taken together, these results indicated that IL-27 can synergize on NK cell expansion and activation with IL-15 and IL-18. In addition, we described an improved culture method for ex vivo expansion of human NK cells with IL-15/IL-18/IL-27 stimulation and characterized the response of NK cells to this stimulation. Electronic supplementary material The online version of this article (10.1186/s40425-019-0652-7) contains supplementary material, which is available to authorized users. more...

Background: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a long-term disabling illness accompanied by fatigue unsolved by rest. However, ME/CFS is a poorly understood illness that lacks a universally accepted pathophysiology and treatment. A lack of CFS-related studies have been conducted in Asian countries. This study aimed to estimate and compare the prevalence of ME/CFS in Korea and Japan and conducted a meta-analysis. Methods: We searched PubMed, EMBASE, Cochrane, and KMBASE for population-based prevalence studies of the two countries and synthesized the data according to the Fukuda case definition. Results: Of the eight studies (five in Korea, three in Japan) included, the total prevalence rate of Korean studies was 0.77% (95% CI 0.34–1.76), and 0.76% (95% CI 0.46–1.25) for the Japanese studies. The prevalence rate in females was approximately two-fold higher than males in Korean studies (1.31% female vs. 0.60% male), while the gender difference was less obvious in Japanese studies (0.76% female vs. 0.65% male). Conclusions: Further epidemiology studies on the female ME/CFS prevalence rate between countries may be required. more...

Background: The diagnosis of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is problematic due to the lack of established objective measurements. Postexertional malaise (PEM) is a hallmark of ME/CFS, and the two-day cardiopulmonary exercise test (CPET) has been tested as a tool to assess functional impairment in ME/CFS patients. This study aimed to estimate the potential of the CPET. Methods: We reviewed studies of the two-day CPET and meta-analyzed the differences between ME/CFS patients and controls regarding four parameters: volume of oxygen consumption and level of workload at peak (VO2peak, Workloadpeak) and at ventilatory threshold (VO2@VT, Workload@VT). Results: The overall mean values of all parameters were lower on the 2nd day of the CPET than the 1st in ME/CFS patients, while it increased in the controls. From the meta-analysis, the difference between patients and controls was highly significant at Workload@VT (overall mean: −10.8 at Test 1 vs. −33.0 at Test 2, p < 0.05), which may reflect present the functional impairment associated with PEM. Conclusions: Our results show the potential of the two-day CPET to serve as an objective assessment of PEM in ME/CFS patients. Further clinical trials are required to validate this tool compared to other fatigue-inducing disorders, including depression, using well-designed large-scale studies. more...

Integrative medicine (IM) has been recognized and introduced into Western healthcare systems over the past two decades. Limited information on IM models is available to guide development of an optimal healthcare service. A scoping review was carried out to evaluate IM models in the extant literature, including the distinctive features of each model, to gain an understanding of the core requirements needed to develop models of IM that best meet the needs of patients.Directed content analysis was used to classify the IM models into systems based on coding schema developed from theoretical models and to identify the key concepts of each system.From 1374 articles identified, 45 studies were included. Models were categorized as theoretical and practical and were subdivided into five main models: coexistence, cooptative, cooperative, collaborative, and patient-centered care. They were then divided into three systems-independent, dependent, and integrative-on the basis of the level of involvement of general practitioners and complementary and alternative medicine (CAM) practitioners. The theoretical coexistence and cooptative models have distinct roles for different health care professionals, whereas practical models tend to be ad hoc market-driven services, dependent on patient demand. The cooperative and collaborative models were team-based, with formalized interaction between the two medical paradigms of conventional medicine and CAM, with the practical models focusing on facilitating communication, behaviors, and relationships. The patient-centered care model recognized the philosophy of CAM and required collaboration between disciplines based around patient needs.The focus of IM models has transferred from providers to patients with the independent and integrative systems. This may require a philosophical shift for IM. Further research is required to best understand how to practice patient-centered care in IM services. more...

Objectives The aim of this study was whether quercetin induces cell death by caspase and MAPK signaling pathway in EGFR mutant lung cancer cells Methods PC-9 cells, EGFR mutant lung cancer cells, were treated various times and concentrations of quercetin and harvested and measured using MTT assay, DNA fragmentation, Western blotting, and FACS analysis. Results Treatment with quercetin in PC-9 cells resulted in inhibition of cell growth through apoptosis. Quercetin-induced apoptosis was associated with caspase-dependent manner. Quercetin also significantly increased levels of phosphor-p38 and decreased levels of phosphor-ERK, indicating that quercetin induces p38 MAPK signaling pathway in PC-9 cells. Quecetin treatment also generated the release of cytochrome c in PC-9 cells; however, pretreatment with rotenone or z-LEHD-fmk, significantly attenuated quercetin-induced apoptosis. Conclusions Our data indicate that quercetin exhibits EGFR mutant lung cancer effects through apoptosis by caspase dependent and mitochondrial pathway. more...

Ciprofloxacin (CIP) is a potent antimicrobial agent with multiple effects on host cells and tissues. Previous studies have highlighted their proapoptotic effect on human cancer cells. The current study showed that subtoxic doses of CIP effectively sensitized multiple cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Although TRAIL alone mediated the partial proteolytic processing of procaspase-3 in lung cancer cells, co-treatment with CIP and TRAIL efficiently restored the complete activation of caspases. We found that treatment of lung cancer with CIP significantly upregulated the expression and protein stability of death receptor (DR) 5. These effects were mediated through the regulation of transcription factor CCAT enhancer-binding protein homologous protein (CHOP) since the silencing of these signaling molecules abrogated the effect of CIP. Taken together, these results indicated that the upregulation of death receptor expression and protein stability by CIP contributed to the restoration of TRAIL-sensitivity in lung cancer cells. more...

Tunicamycin (TN), one of the endoplasmic reticulum stress inducers, has been reported to inhibit tumor cell growth and exhibit anticarcinogenic activity. However, the mechanism by which TN initiates apoptosis remains poorly understood. In the present study, we investigated the effect of TN on the apoptotic pathway in U937 cells. We show that TN induces apoptosis in association with caspase-3 activation, generation of reactive oxygen species (ROS), and downregulation of survivin expression. P38 MAPK (mitogen-activated protein kinase) and the generation of ROS signaling pathway play crucial roles in TN-induced apoptosis in U937 cells. We hypothesized that TN-induced activation of p38 MAPK signaling pathway is responsible for cell death. To test this hypothesis, we selectively inhibited MAPK during treatment with TN. Our data demonstrated that inhibitor of p38 (SB), but not ERK (PD) or JNK (SP), partially maintained apoptosis during treatment with TN. Pre-treatment with NAC and GSH markedly prevented cell death, suggesting a role for ROS in this process. Ectopic expression of survivin in U937 cells attenuated TN-induced apoptosis by suppression of caspase-3 cleavage, mitochondrial membrane potential, and cytochrome c release in U937 cells. Taken together, our results show that TN modulates multiple components of the apoptotic response of human leukemia cells and raise the possibility of a novel therapeutic strategy for hematological malignancies. more...

Aimp1 is known as a multifunctional cytokine in various cellular events. Recent study showed Aimp1 is localized in glandular epithelial, endothelial, and stromal cells in functionalis and basalis layers of the endometrium. However, the regulatory mechanism of Aimp1 in the uterus remains unknown. In the present study, we found that Aimp1 is expressed in the mouse uterus. Aimp1 transcripts were decreased at diestrus stage. However, the level of Aimp1 protein was significantly increased in the luminal epithelium in the uterine endometrium at estrus stage during the estrous cycle. We found that treatment of estrogen increased the expression of Aimp1 in the uterus in ovarectomized mice. We identified one estrogen receptor binding element (ERE) on mouse Aimp1 promoter. The activity of Aimp1 promoter was increased with estrogen treatment. Our findings indicate that Aimp1 might act as an important regulator to remodel the uterine endometrium and its expression might be regulated by estrogen during the estrous cycle. This will give us better understanding of the dynamic change of uterine remodeling during the estrous cycle. more...

Combined treatment with a photosensitizer and iodide laser [photodynamic therapy (PDT)] has improved the outcome of various cancers. In this study, we investigated the effects of using the photosensitizer methylene blue (MB) in PDT in human lung adenocarcinoma cells. We found that MB enhances PDT-induced apoptosis in association with downregulation of anti-apoptotic proteins, reduced mitochondrial membrane potential (MMP), increased phosphorylation of the mitogen-activated protein kinase (MAPK) and the generation of reactive oxygen species (ROS). In MB-PDT-treated A549 cells, we observed PARP cleavage, procaspase-3 activation, downregulation of the anti-apoptotic proteins Bcl-2 and Mcl-1, and the reduction of mitochondrial membrane potential (MMP). Western blot data showed that phosphorylation of p38 was increased in MB-PDT-treated A549 cells, indicating that several signaling molecules participate in the apoptotic cascade. Our data also showed that apoptotic cell death in MB-PDT-treated cells occurred through a series of steps beginning with the photochemical generation of ROS. Demonstrating the role of ROS, pretreatment of A549 cells with the antioxidant N-acetylcysteine (NAC) followed by MB-PDT resulted in increased cell viability and reduced proteolytic cleavage of PARP. more...

This study provides basic information that is needed to build marketing strategies related to consumer brand-switching, through the investigation of consumer motivations for brand-switching, as determined by the types of fashion products. The study was implemented by a descriptive survey method using a questionnaire. The survey was conducted during the period of May 11 th through July 5 th 2008. A total of 184 completed responses were analyzed. All respondents were from the Seoul area and between the ages of 20 and 31. Factor analysis and Cronbach's alpha coefficients, one-way ANOVA and Duncan test were employed for the analysis of data. Significant differences were found in brand-switching motives according to the types of fashion products. For clothing, shift behavior occurred more often in conjunction with the attributes of the products itself, such as design, color, price, size, and fiber content. In general, clothes more than shoes, were likely subject to brand-switching most often on the basis of situational factors. On the other hand, for shoes, more brand-switching activities occurred because of non-product attributes, such as discounts, coupons, desire for a change, and wearing of friends. In light of the results, there is a need to differentiate brand related marketing strategies with respect to clothing and shoes. For clothing, efforts focusing on the improvement of the product attributes will be more effective in minimizing brand-switching. There is also a need to improve instructions for increasing the product understanding of salespersons as well as the ability to give advice in accordance with personal consumer characteristics. With regard to shoes, greater efforts should be given to promotional activities, and the desire of consumers for a change in order to prevent brand-switching of customers. more...

When grown with vaporized alkylphenols such as p-cresol as the sole carbon and energy source, several isolated Rhodococcus strains formed growth structures like miniature mushrooms, termed here specialized aerial architectures (SAA), that reached sizes of up to 0.8 mm in height. Microscopic examination allowed us to view the distinct developmental stages during the formation of SAA from a selected strain, Rhodococcus sp. KL96. Initially, mounds consisting of long rod cells arose from a lawn of cells, and then highly branched structures were formed from the mounds. During the secondary stage of development, branching began after long rod cells grew outward and twisted longitudinally, serving as growth points, and the cells at the base of the mound became short rods that supported upward growth. Cells in the highly fluffy structures were eventually converted, via reductive division, into structures that resembled cocci, with a diameter of approximately 0.5 μm, that were arranged in chains. Most cells inside the SAA underwent a phase variation in order to form wrinkled colonies from cells that originally formed smooth colonies. Approximately 2 months was needed for complete development of the SAA, and viable cells were recovered from SAA that were incubated for more than a year. An extracellular polymeric matrix layer and lipid bodies appeared to play an important role in structural integrity and as a metabolic energy source, respectively. To our knowledge, similar formation of aerial structures for the purpose of substrate utilization has not been reported previously for Gram-positive bacteria. more...

Aim To investigate genetic polymorphism and haplotypes of tightly linked X-chromosomal short tandem repeat (XSTR) clusters in Koreans. Methods Four X-STR duos in the linkage group 1- 4 (DXS10135-DXS8378, DXS7132-DXS10074, HPRTBDXS10101, and DXS10134-DXS7423) were investigated in 450 unrelated Koreans (300 men and 150 women) using the Mentype Argus X-8 Polymerase Chain Reaction Amplification Kit. Results No significant deviation from Hardy-Weinberg equilibrium was observed in any of the 8 loci. DXS10135 was the most polymorphic X-STRs, with 25 alleles and DXS7423 was the least informative, with 5 alleles. Eleven off-ladder alleles and a triallelic pattern were observed, and these alleles were characterized by cloning and sequencing analysis. In 300 Korean men, 38 to 59 haplotypes were observed for each linkage duo with 91.6-96.6% of haplotype diversities. However, due to the low genetic diversity of DXS7423, the X-STR duo in linkage group 4 (DXS10134- DXS7423), in comparison with other linkage duos, had considerably lower haplotype diversity values (91.6%) with 3 common haplotypes (35-15, 36-15, and 37-15) observed in 44.3% of Koreans. Conclusion Four X-STR duos in the linkage group 1-4 will be able to provide a powerful tool for solving complex kinship cases in Koreans. However, to increase the haplotype diversity in the linkage group 4, it will be useful to discover a new marker for Asians that can serve as an adequate substitute for DXS7423 or at least complement the existing linkage duo of DXS10134-DXS7423. more...

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that can induce inflammatory processes in the vascular endothelium. We hypothesize that the plasma membrane microdomains called caveolae are critical in endothelial activation and toxicity induced by PCBs. Caveolae are particularly abundant in endothelial cells and play a major role in endothelial trafficking and the regulation of signaling pathways associated with the pathology of vascular diseases. We focused on the role of caveolae and their major protein component, caveolin-1 (Cav-1), on aryl hydrocarbon receptor (AhR)-mediated induction of cytochrome P450 1A1 (CYP1A1) by coplanar PCBs. Endothelial cell exposure to PCB77 increased both caveolin-1 and CYP1A1 levels in a time-dependent manner in total cell lysates, with a maximum increase at 6 h. Furthermore, PCB77 accumulated mainly in the caveolae-rich fraction, as determined by gas chromatograph-mass spectrometry. Immunoprecipitation analysis revealed that PCB77 increased AhR binding to caveolin-1. Silencing of caveolin-1 significantly attenuated PCB77-mediated induction of CYP1A1 and oxidative stress. Similar effects were observed in caveolin-1 null mice treated with PCB77. These data suggest that caveolae may play a role in regulating vascular toxicity induced by persistent environmental pollutants such as coplanar PCBs. This may have implications in understanding mechanisms of inflammatory diseases induced by environmental pollutants. more...

Hypertriglyceridemia and associated high circulating free fatty acids are important risk factors for atherosclerosis. In contrast to omega-3 fatty acids, linoleic acid, the major omega-6 unsaturated fatty acid in the American diet, may be atherogenic by amplifying an endothelial inflammatory response. We hypothesize that omega-6 and omega-3 fatty acids can differentially modulate tumor necrosis factor alpha (TNF-alpha)-induced endothelial cell activation and that functional plasma membrane microdomains called caveolae are required for endothelial cell activation. Caveolae are particularly abundant in endothelial cells and play a major role in endothelial trafficking and the regulation of signaling pathways associated with the pathology of vascular diseases. To test our hypothesis, endothelial cells were preenriched with either linoleic acid or alpha-linolenic acid before TNF-alpha-induced endothelial activation. Measurements included oxidative stress and nuclear factor kappaB-dependent induction of cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)) under experimental conditions with intact caveolae and with cells in which caveolin-1 was silenced by small interfering RNA. Exposure to TNF-alpha induced oxidative stress and inflammatory mediators, such as p38 mitogen-activated protein kinase (MAPK), nuclear factor kappaB, COX-2, and PGE(2), which were all amplified by preenrichment with linoleic acid but blocked or reduced by alpha-linolenic acid. The p38 MAPK inhibitor SB203580 blocked TNF-alpha-mediated induction of COX-2 protein expression, suggesting a regulatory mechanism through p38 MAPK signaling. Image overlay demonstrated TNF-alpha-induced colocalization of TNF receptor type 1 with caveolin-1. Caveolin-1 was significantly induced by TNF-alpha, which was further amplified by linoleic acid and blocked by alpha-linolenic acid. Furthermore, silencing of the caveolin-1 gene completely blocked TNF-alpha-induced production of COX-2 and PGE(2) and significantly reduced the amplified response of linoleic acid plus TNF-alpha. These data suggest that omega-6 and omega-3 fatty acids can differentially modulate TNF-alpha-induced inflammatory stimuli and that caveolae and its fatty acid composition play a regulatory role during TNF-alpha-induced endothelial cell activation and inflammation. more...

The high rate of exocytosis at the ribbon synapses is balanced by following compensatory endocytosis. Unlike conventional synaptic terminals where clathrin-mediated endocytosis (CME) is a predominant mechanism for membrane retrieval, CME is thought to be only a minor mechanism of endocytosis at the retinal ribbon synapses, but CME is present there and it works. We examined the clathrin expression in the FVB/N rd1 mouse, which is an animal model of retinitis pigmentosa. The broadly distributed pattern of clathrin immunoreactivity in the inner plexiform layer was similar in both the control and FVB/N mouse retinas, but the immunoreactive punta within the rod bipolar axon terminals located in the proximal IPL were decreased in number and reduced in size at postnatal days 14 and they came to disappear at postnatal days 21. This preferential decrease of the clathrin expression at ribbon synapses in the rod bipolar cell axon terminals of the FVB/N mouse retina demonstrates another plastic change after photoreceptor degeneration and this suggests that clathrin may be important for normal synaptic function at the rod bipolar ribbon synapses in the mammalian retina. more...

Mer signaling increases the transcriptional activity of liver X receptor (LXR) to promote the resolution of acute sterile inflammation. Here, we aimed to understand the pathway downstream of Mer signaling after growth arrest-specific protein 6 (Gas6) treatment that leads to LXR expression and transcriptional activity in mouse bone-marrow derived macrophages (BMDM). Gas6-induced increases in LXRα and LXRβ and expression of their target genes were inhibited in BMDM from STAT1−/− mice or by the STAT1-specific inhibitor fludarabine. Gas6-induced STAT1 phosphorylation, LXR activation, and LXR target gene expression were inhibited in BMDM from Mer−/− mice or by inhibition of PI3K or Akt. Gas6-induced Akt phosphorylation was inhibited in BMDM from STAT1−/− mice or in the presence of fludarabine. Gas6-induced LXR activity was enhanced through an interaction between LXRα and STAT1 on the DNA promoter of Arg2. Additionally, we found that Gas6 inhibited lipopolysaccharide (LPS)-induced nitrite production in a STAT1 and LXR pathway-dependent manner in BMDM. Additionally, Mer-neutralizing antibody reduced LXR and Arg2 expression in lung tissue and enhanced NO production in bronchoalveolar lavage fluid in LPS-induced acute lung injury. Our data suggest the possibility that the Gas6-Mer-PI3K/Akt-STAT1-LXR-Arg2 pathway plays an essential role for resolving inflammatory response in acute lung injury. more...

Polychlorinated biphenyls (PCBs) may contribute to the pathology of atherosclerosis by activating inflammatory responses in vascular endothelial cells. Endothelial nitric oxide synthase (eNOS) is colocalized with caveolae and is a critical regulator of vascular homeostasis. PCBs may be proatherogenic by causing dysfunctional eNOS signaling. The objective of this study was to investigate the role of caveolin-1 in PCB-induced endothelial dysfunction with a focus on mechanisms associated with eNOS signaling. Cells derived from an immortalized human vascular endothelial cell line were treated with PCB77 to study nitrotyrosine formation through eNOS signaling. Phosphorylation studies of eNOS, caveolin-1, and kinases, such as Src, phosphatidylinositol 3-kinase (PI3K), and Akt, were conducted in cells containing either functional or small-interfering RNA-silenced caveolin-1 protein. We also investigated caveolin-1-regulated mechanisms associated with PCB-induced markers of peroxynitrite formation and DNA binding of NF-κB. Cellular exposure to PCB77 increased eNOS phosphorylation and nitric oxide production, as well as peroxynitrite levels. A subsequent PCB-induced increase in NF-κB DNA binding may have implications in oxidative stress-mediated inflammatory mechanisms. The activation of eNOS by PCB77 treatment was blocked by inhibitors of the Src/PI3K/Akt pathway. PCB77 also increased phosphorylation of caveolin-1, indicating caveolae-dependent endocytosis. Caveolin-1 silencing abolished both the PCB-stimulated Akt and eNOS phosphorylation, suggesting a regulatory role of caveolae in PCB-induced eNOS signaling. These findings suggest that PCB77 induces eNOS phosphorylation in endothelial cells through a Src/PI3K/Akt-dependent mechanism, events regulated by functional caveolin-1. Our data provide evidence that caveolae may play a critical role in regulating vascular endothelial cell activation and toxicity induced by persistent environmental pollutants such as coplanar PCBs. more...

Mammalian neurons express the neural intermediate filament protein neurofilament (NF). In the retina, NFs have been detected primarily in the axons and processes of retinal ganglion and horizontal cells. We found an amacrine cell type that was immunolabeled with an antibody against SMI32, a non-phosphorylated epitope on neurofilament proteins of high molecular weight, in the mouse retina. This type of amacrine cell was non-randomly distributed, and these cells exhibited a central-peripheral density gradient. Most of these cells co-expressed GABA and ChAT, but not glycine or any other amacrine cell marker. These results suggest that some SMI32-immunoreactive amacrine cells belong to a GABAergic population, and that SMI32 can therefore be used as a marker for a subset of amacrine cells in addition to ganglion cells and horizontal cells in the mouse retina. more...

Dopaminergic cells in the retina express the receptor for brain-derived neurotrophic factor (BDNF), which is the neurotrophic factor that influences the plasticity of synapses in the central nervous system. We sought to determine whether BDNF influences the network of dopaminergic amacrine cells in the axotomized rat retina, by immunocytochemistry with an anti-tyrosine hydroxylase (TH) antiserum. In the control retina, we found two types of TH-immunoreactive amacrine cells, type I and type II, in the inner nuclear layer adjacent to the inner plexiform layer (IPL). The type I amacrine cell varicosities formed ring-like structures in contact with AII amacrine cell somata in stratum 1 of the IPL. In the axotomized retinas, TH-labeled processes formed loose networks of fibers, unlike the dense networks in the control retina, and the ring-like structures were disrupted. In the axotomized retinas treated with BDNF, strong TH-immunoreactive varicosities were present in stratum 1 of the IPL and formed ring-like structures. Our data suggest that BDNF affects the expression of TH immunoreactivity in the axotomized rat retina and may therefore influence the retinal dopaminergic system. more...

DBA/2J (D2) mice develop a form of progressive pigmentary glaucoma with increasing age. We have compared retinal cell populations of D2 mice with those in control C57BL/6J mice to provide information on retinal histopathology in the D2 mouse. The D2 mouse retina is characterized by a reduction in retinal thickness caused mainly by a thinning of the inner retinal layers. Immunocytochemical staining for specific inner retinal neuronal markers, viz., calbindin for horizontal cells; protein kinase C (PKC) and recoverin for bipolar cells, glycine, gamma-aminobutyric acid (GABA), choline acetyltransferase (ChAT), and nitric oxide synthase (NOS) for amacrine cells, and osteopontin (OPN) for ganglion cells, was performed to detect preferentially affected neurons in the D2 mouse retina. Calbindin, PKC, and recoverin immunoreactivities were not significantly altered. Amacrine cells immunoreactive for GABA, ChAT, and OPN were markedly decreased in number, whereas NOS-immunoreactive amacrine cells increased in number. However, no changes were observed in the population of glycine-immunoreactive amacrine cells. These findings indicate a significant loss of retinal ganglion and some amacrine cells, whereas glycinergic amacrine cells, horizontal, and bipolar cells are almost unaffected in the D2 mouse. The reduction in amacrine cells appears to be attributable to a loss of GABAergic and particularly cholinergic amacrine cells. The increase in nitrergic neurons with the consequent increase in NOS and NO may be important in the changes in the retinal organization that lead to glaucomain D2 mice. Thus, the D2 mouse retina represents a useful model for studying the pathogenesis of glaucoma and mechanisms of retinal neuronal death and for evaluating neuroprotection strategies. more...

The FVB/N mouse is a model of retinitis pigmentosa which shows a rapid loss of photoreceptors during early postnatal (P) life. We investigated the cellular localization of glycine transporter 1 (GlyT-1) in the developing FVB/N mouse retina. In control retinas, the developmental pattern of GlyT-1-immunoreactive amacrine cells was well in accordance with a previous report. However, in the FVB/N mouse retina, some GlyT-1-labeled amacrine cells sent their processes into the outer plexiform layer (OPL) from P14 onward. From P21 onward, GlyT-1-labeled cells were visible in the OPL. These cells were further characterized by double-label immunofluorescence experiments with an antiserum against disabled 1 (Dab-1), and showed Dab-1 immunoreactivity, indicating that these cells are putative AII amacrine cells. These results clearly demonstrate that AII amacrine cells have the potential capacity to respond to photoreceptor degeneration by migrating or sprouting their processes into the OPL in the developing FVB/N mouse retina. more...

Disabled 1 (Dab1) is an adapter molecule in a signaling pathway, stimulated by Reelin, which controls cell positioning in the developing brain. It has been localized to AII amacrine cells in the mouse and guinea pig retinas. This study was conducted to identify whether Dab1 is commonly localized to AII amacrine cells in the retinas of other mammals. We investigated Dab1-labeled cells in human, rat, rabbit, and cat retinas in detail by immunocytochemistry with antisera against Dab1. Dab1 immunoreactivity was found in certain populations of amacrine cells, with lobular appendages in the outer half of the inner plexiform layer (IPL) and a bushy, smooth dendritic tree in the inner half of the IPL. Double-labeling experiments demonstrated that all Dab1-immunoreactive amacrine cells were immunoreactive to antisera against calretinin or parvalbumin (i.e., other markers for AII amacrine cells in the mammalian retina) and that they made contacts with the axon terminals of the rod bipolar cells in the IPL close to the ganglion cell layer. Furthermore, all Dab1-labeled amacrine cells showed glycine transporter-1 immunoreactivity, indicating that they are glycinergic. The peak density was relatively high in the human and rat retinas, moderate in the cat retina, and low in the rabbit retina. Together, these morphological and histochemical observations clearly indicate that Dab1 is commonly localized to AII amacrine cells and that antiserum against Dab1 is a reliable and specific marker for AII amacrine cells of diverse mammals. J. Comp. Neurol. 470:372–381, 2004. © 2004 Wiley-Liss, Inc. more...

Pseudomonas alkylphenolia is known to form different types of multicellular structures depending on the environmental stimuli. Aerial structures formed during vapor p-cresol utilization are unique. Transposon mutants that showed a smooth colony phenotype failed to form a differentiated biofilm, including aerial structures and pellicles, and showed deficient surface spreading motility. The transposon insertion sites were located to a gene cluster designated epm (extracellular polymer matrix), which comprises 11 ORFs in the same transcriptional orientation. The putative proteins encoded by the genes in the epm cluster showed amino acid sequence homology to those found in the alginate biosynthesis gene clusters, e.g., in Pseudomonas aeruginosa at similarity levels of 32.3-86.4 %. This overall resemblance indicated that the epm gene cluster encodes proteins that mediate the synthesis of an exopolysaccharide composed of uronic acid(s) similar to alginate. Our preliminary results suggested that the epm-derived polymer is a substituted polymannuronic acid. Gene clusters homologous to the epm gene cluster are found in the genomes of a few species of the genera Pseudomonas, Alcanivorax, and Marinobacter. A mutational analysis showed that the epmJ and epmG genes encoding putative exopolysaccharide-modifying enzymes are required to form multicellular structures. An analysis of the activity of the promoter P epmD using a transcriptional fusion to the green fluorescence protein gene showed that the epm genes are strongly expressed at the tips of the specialized aerial structures. Our results suggested that the epm gene cluster is involved in the formation of a scaffold polysaccharide that is required to form multicellular structures in P. alkylphenolia. more...

Intimate two-way interactions between the implantation-competent blastocyst and receptive uterus are prerequisite for successful embryo implantation. In humans, recurrent/repeated implantation failure (RIF) may occur due to altered uterine receptivity with aberrant gene expression in the endometrium as well as genetic defects in embryos. Several studies have been performed to understand dynamic changes of uterine transcriptome during menstrual cycles in humans. However, uterine transcriptome of the patients with RIF has not been clearly investigated yet. Here we show that several signaling pathways as well as many genes and microRNAs are dysregulated in the endometrium of patients with RIF (RIFE). Whereas unsupervised hierarchical clustering showed that overall mRNA and microRNA profiles of RIFE were similar to those of endometria of healthy women, many genes were significantly dysregulated in RIFE (cut off at 1.5 fold change). The majority (~75%) of differentially expressed genes in RIFE including S100 calcium binding protein P (S100P), Chemokine (C-X-C motif) ligand 13 (CXCL13) and SIX homeobox 1 (SIX1) were down-regulated, suggesting that reduced uterine expression of these genes is associated with RIF. Gene Set Enrichment analyses (GSEA) for mRNA microarrays revealed that various signaling pathways including Leukemia inhibitory factor (LIF) signaling and a P4 response were dysregulated in RIFE although expression levels of Estrogen receptor α (ERα) and Progesterone receptor (PR) were not significantly altered in RIFE. Furthermore, expression and phosphorylation of Signal transducer and activator of transcription 3 (STAT3) are reduced and a gene set associated with Janus kinase (JAK)-STAT signaling pathway is systemically down-regulated in these patients. Pairwise analyses of microRNA arrays with prediction of dysregulated microRNAs based on mRNA expression datasets demonstrated that 6 microRNAs are aberrantly regulated in RIFE. Collectively, we here suggest that dysregulation of several major signaling pathways and genes critical for uterine biology and embryo implantation may lead to uterine abnormalities in patients with RIF. more...

The addition of tunicamycin to prostate cancer cells enhances cell death mediated by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In this study, we investigated whether tunicamycin, an endoplasmic reticulum stress inducer, can potentiate TRAIL-induced apoptosis in human prostate cancer cells. We evaluated the combination of tunicamycin and TRAIL and found synergistic promotion of apoptosis in prostate cancer cells. The combined treatment with tunicamycin and TRAIL significantly induced apoptosis, and stimulated caspase-3, -8 and -9 activity, as well as the cleavage of poly (ADP-ribose) polymerase. We found that tunicamycin promoted TRAIL-induced apoptosis by the upregulation of death receptor (DR)4 and DR5 and the downregulation of cellular inhibitor of apoptosis 2 (cIAP2). In addition, downregulation of cIAP2 expression using small interfering RNA significantly attenuated the apoptosis induced by TRAIL. Taken together, our results demonstrate that the combination of tunicamycin and TRAIL may provide a novel strategy for treating prostate cancer by overcoming critical mechanisms of apoptosis resistance. more...

In this study, the chromosome-encoded pcuRCAXB genes that are required for p-cresol degradation have been identified by using a newly constructed green fluorescent protein (GFP)-based promoter probe transposon in the long-chain alkylphenol degrader Pseudomonas alkylphenolia. The deduced amino acid sequences of the genes showed the highest identities at the levels of 65-93% compared with those in the databases. The transposon was identified to be inserted in the pcuA gene, with the promoterless gfp gene being under the control of the pcu catabolic gene promoter. The expression of GFP was positively induced by p-cresol and was about 10 times higher by cells grown on agar than those in liquid culture. In addition, p-hydroxybenzoic acid was detected during p-cresol degradation. These results indicate that P. alkylphenolia additionally possesses a protocatechuate ortho-cleavage route for p-cresol degradation that is dominantly expressed in colonies. more...

The etiology of a variety of chronic inflammatory disorders has been attributed to the interaction of genetic and environmental factors. Herein, we identified a link between epigenetic regulation and IL-13-driven eotaxin-3 in the pathogenesis of chronic allergic inflammation. We first demonstrated that the cAMP-responsive element (CRE) site in the eotaxin-3 promoter affects IL-13-induced eotaxin-3 promoter activity. Furthermore, the CRE-binding protein-binding protein (CBP), a histone acetyltransferase, induced base-line and IL-13-induced eotaxin-3 promoter activity. Additionally, IL-13 treatment promoted global histone 3 acetylation as well as the formation of a complex containing CBP and STAT6 and the subsequent acetylation of histone 3 at the eotaxin-3 promoter. CBP gene silencing decreased IL-13-induced transcription of eotaxin-3. Conversely, inhibition of histone deacetylation increased IL-13-induced eotaxin-3 production. Clinical studies demonstrated markedly increased global acetylation of histone 3 in the inflamed tissue of patients with allergic inflammation. Collectively, these results identify an epigenetic mechanism involving CBP and chromatin remodeling in regulating IL-13-induced chemokine transcription. more...

Nanotechnology is a rapidly growing industry that has elicited much concern because of the lack of available toxicity data. Exposure to ultrafine particles may be a risk for the development of vascular diseases due to dysfunction of the vascular endothelium. Increased endothelial adhesiveness is a critical first step in the development of vascular diseases, such as atherosclerosis. The hypothesis that alumina nanoparticles increase inflammatory markers of the endothelium, measured by the induction of adhesion molecules as well as the adhesion of monocytes to the endothelial monolayer, was tested. Following characterization of alumina nanoparticles by transmission electron microscopy (TEM), electron diffraction, and particle size distribution analysis, endothelial cells were exposed to alumina at various concentrations and times. Both porcine pulmonary artery endothelial cells and human umbilical vein endothelial cells showed increased mRNA and protein expression of VCAM-1, ICAM-1, and ELAM-1. Furthermore, human endothelial cells treated with alumina particles showed increased adhesion of activated monocytes. The alumina particles tended to agglomerate at physiological pH in serum-containing media, which led to a range of particle sizes from nano to micron size during treatment conditions. These data show that alumina nanoparticles can elicit a proinflammatory response and thus present a cardiovascular disease risk. more...

Elongation factor 2 (EF-2) plays a key role in the essential process of protein synthesis by translocating tRNAs from the ribosomal A- and P-sites to the P- and E-sites. EF-2 regulates the outcome of protein synthesis in mammalian cells. This report demonstrates that chicken EF-2 protein levels are dependent on transcription in 8-bromo-cAMP, insulin and phorbol ester-treated cells. In order to delineate functional domains that control chicken EF-2 gene transcription, the 5′-flanking region of the chicken EF-2 promoter was analyzed. Deletion constructs from − 550 and − 86 had the same basal level promoter activity as the whole EF-2 promoter. The sequence between nucleotides − 700 and − 550 was determined to be a regulatory region for the chicken EF-2 basal promoter activity. The region between − 700 and − 550 has a negative regulatory region and two regulatory proteins (I, II). 8-bromo-cAMP increased chicken EF-2 promoter activity (− 700/+ 102) in Rat 1 HIR fibroblast cells more than insulin and phorbol ester treatment. Binding of protein I and II were decreased by 8-bromo-cAMP but restored by a protein kinase A inhibitor (KT5720). GATA consensus sequence oligonucleotide and fragment − 86/− 50 prevented protein II binding of fragment − 700/− 550. This result suggested that protein II is a GATA-like protein. These observations provide a novel regulatory mechanism for the EF-2 promoter. more...