Identification of U11snRNA as an endogenous agonist of TLR7-mediated immune pathogenesis.

The activation of innate immune receptors by pathogen-associated molecular patterns (PAMPs) is central to host defense against infections. On the other hand, these receptors are also activated by immunogenic damage-associated molecular patterns (DAMPs), typically released from dying cells, and the activation can evoke chronic inflammatory or autoimmune disorders. One of the best known receptors involved in the immune pathogenesis is Toll-like receptor 7 (TLR7), which recognizes RNA with single-stranded structure. However, the causative DAMP RNA(s) in the pathogenesis has yet to be identified. Here, we first developed a chemical compound, termed KN69, that suppresses autoimmunity in several established mouse models. A subsequent search for KN69-binding partners led to the identification of U11 small nuclear RNA (U11snRNA) as a candidate DAMP RNA involved in TLR7-induced autoimmunity. We then showed that U11snRNA robustly activated the TLR7 pathway in vitro and induced arthritis disease in vivo. We also found a correlation between high serum level of U11snRNA and autoimmune diseases in human subjects and established mouse models. Finally, by revealing the structural basis for U11snRNA's ability to activate TLR7, we developed more potent TLR7 agonists and TLR7 antagonists, which may offer new therapeutic approaches for autoimmunity or other immune-driven diseases. Thus, our study has revealed a hitherto unknown immune function of U11snRNA, providing insight into TLR7-mediated autoimmunity and its potential for further therapeutic applications.

Preliminary results of stabilization of far distal tibia fractures with the distal tibial nail: A prospective, multicenter case series study.

INTRODUCTION: The distal tibial nail (DTN) is a novel retrograde intramedullary nail used for distal tibial fracture stabilization. We investigated the clinical results of DTN use for distal tibial fractures and compared them with those reported in the literature on locking plates and antegrade intramedullary nails. MATERIALS AND METHODS: This multicenter, prospective, observational cohort study examined distal tibial fractures with AO/OTA classification 43 types: A1, A2, A3 or C1. The primary outcomes included bone union rate, soft tissue problems, and surgical complications. Secondary outcomes were EuroQol-5 Dimension-5 Level (EQ-5D-5L), Self-Administered Foot Evaluation Questionnaire (SAFE-Q), and American Orthopaedic Foot and Ankle Society (AOFAS) hindfoot clinical scores 1 year postoperatively. Incidence of varus or valgus/anterior-posterior flexion deformity with a difference of ≥5° and postoperative reduction loss rate were evaluated. RESULTS: Five men and five women were enrolled (mean age, 69 years [range, 30-77 years]), including one open-fracture-type Gustilo type IIIB case. Bone union was observed in all patients at 6 months postoperatively. Delayed union, leg edema, and guide pin breakage were observed in three, one, and one cases, respectively. No soft tissue or surgical complications were observed. During the final follow-up, the EQ-5D-5L, SAFE-Q, and AOFAS hindfoot scores were 0.876 (0.665-1.0), 83-92, and AOFAS 92.6 (76-100), respectively. Varus and retroflexion deformities were observed in one case each. DISCUSSION: DTN has been reported to have biomechanically equivalent or stronger fixation strength than locking plates or antegrade intramedullary nails. In addition, while DTN was thought to be less invasive for soft tissue and can avoid injury to the knee, it was thought that care should be taken to avoid medial malleolus fractures and posterior tibialis tendon injuries. Comparisons with literature treatment results for locking plates and antegrade intramedullary nails showed comparable to advantageous results. CONCLUSIONS: DTN treatment results for distal tibial fractures were as good as those for locking plates and antegrade intramedullary nails. DTN is useful for stabilization and does not compromise the surrounding soft tissues.

[Strategy for the treatment of Stage IV gastric cancer with preoperative chemotherapy].

In 2007, we began using neoadjuvant chemotherapy for the treatment of Stage IV gastric cancer and then performing R0/1 surgery in patients in whom chemotherapy was effective. Here, we evaluate the use of this therapeutic strategy combining chemotherapy and surgery for the treatment of Stage IV gastric cancer. The subjects of our investigation were 46 patients with Stage IV gastric cancer treated from 2007 through 2012. We divided these patients into the NAC group (19 patients), in whom we performed R0/1 surgery after chemotherapy, and the Cx group (27 patients), who continued chemotherapy. We also included 79 patients with Stage IV gastric cancer treated from 2001 to 2006, divided into the OPE group (36 patients), in whom we performed R0/1 surgery without neoadjuvant chemotherapy, and the NC group (43 patients), in whom we performed R2 surgery. We plotted the survival curves of these 4 groups. The chemotherapy protocols used were S-1+cisplatin( CDDP) and S-1+docetaxel( DOC). The disease control rate of these chemotherapies was 72%, and R0/1 surgery was performed in 53.8% of patients with liver metastasis, 62.5% of those with paraaortic lymph node (PALN) metastasis, 29.4% of those with peritoneal metastasis, 100% of patients with T4N2 disease, and 0% of patients with distant metastasis. The 2-year survival rates of the NAC, OPE, Cx, and NC groups were 69%, 55%, 0%, and 20%, respectively. The 5-year survival rates of the NAC, OPE, Cx, and NC groups were 35%, 30%, 0%, and 5%, respectively.

Pitavastatin increases ABCA1 expression by dual mechanisms: SREBP2-driven transcriptional activation and PPARα-dependent protein stabilization but without activating LXR in rat hepatoma McARH7777 cells.

Hepatic ATP-binding cassette transporter A1 (ABCA1) plays a key role in high-density lipoprotein (HDL) production by apolipoprotein A-I (ApoA-I) lipidation. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, statins, increase ABCA1 mRNA levels in hepatoma cell lines, but their mechanism of action is not yet clear. We investigated how statins increase ABCA1 in rat hepatoma McARH7777 cells. Pitavastatin, atorvastatin, and simvastatin increased total ABCA1 mRNA levels, whereas pravastatin had no effect. Pitavastatin also increased ABCA1 protein. Hepatic ABCA1 expression in rats is regulated by both liver X receptor (LXR) and sterol regulatory element-binding protein (SREBP2) pathways. Pitavastatin repressed peripheral type ABCA1 mRNA levels and its LXR-driven promoter, but activated the liver-type SREBP-driven promoter, and eventually increased total ABCA1 mRNA expression. Furthermore, pitavastatin increased peroxisome proliferator-activated receptor α (PPARα) and its downstream gene expression. Knockdown of PPARα attenuated the increase in ABCA1 protein, indicating that pitavastatin increased ABCA1 protein via PPARα activation, although it repressed LXR activation. Furthermore, the degradation of ABCA1 protein was retarded in pitavastatin-treated cells. These data suggest that pitavastatin increases ABCA1 protein expression by dual mechanisms: SREBP2-mediated mRNA transcription and PPARα-mediated ABCA1 protein stabilization, but not by the PPAR-LXR-ABCA1 pathway. [Supplementary Figures: available only at http://dx.doi.org/10.1254/jphs.10241FP].

Computed tomography fluoroscopy-guided placement of iliosacral screws in patients with unstable posterior pelvic fractures.

OBJECTIVE: The purpose of this study was to evaluate retrospectively the safety and effectiveness of the computed tomography (CT) fluoroscopy-guided placement of iliosacral screws in patients with unstable posterior pelvic fractures. MATERIALS AND METHODS: Six patients (four women and two men; mean age 55.8 years; range 35-77 years) with unstable posterior pelvic fractures underwent iliosacral screw placement under CT fluoroscopy guidance between November 2007 and August 2008. Unstable pelvic ring injury (AO types B and C) was the indication for this procedure. RESULTS: In all the six patients except one, CT fluoroscopy-guided placement had been technically successful. In one patient, a second screw had been inserted, with a tilt to the caudal site, and slightly advanced into the extrasacral body; afterward, it could be exchanged safely for a shorter screw. Five patients and one patient underwent placement of two screws and one screw, respectively. The mean duration of the procedure was 15.0 min (range 9-30 min) per screw; the duration was 12.3 min and 18.2 min for the first and second screws, respectively. No complications requiring treatment occurred during or after the procedure. The mean clinical and radiologic follow-up period was 14 months (range 6-21 months). All pelvic injuries had healed satisfactorily, without complication, and all patients are now doing well clinically and can walk. CONCLUSION: CT fluoroscopy-guided placement of iliosacral screws is a safe and effective treatment in patients with unstable posterior pelvic fractures.

Publisher Correction: Malaria predictions based on seasonal climate forecasts in South Africa: A time series distributed lag nonlinear model.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Role of sea-ice initialization in climate predictability over the Weddell Sea.

Potential impact of sea-ice initialization on the interannual climate predictability over the Weddell Sea is investigated using a coupled general circulation model. Climate variability in the Weddell Sea is generally believed to have association with remote forcing such as El Niño-Southern Oscillation and the Southern Annual Mode. However, sea-ice variability in the Weddell Sea has been recently suggested to play additional roles in modulating local atmospheric variability through changes in surface air temperature and near-surface baroclinicity. Reforecast experiments from September 1st, in which the model's sea-surface temperature (SST) and sea-ice concentration (SIC) are initialized with observations using nudging schemes, show improvements in predicting the observed SIC anomalies in the Weddell Sea up to four months ahead, compared to the other experiments in which only the model's SST is initialized. During austral spring (Oct-Dec) of lower-than-normal sea-ice years in the Weddell Sea, reforecast experiments with the SST and SIC initializations reasonably predict high surface air temperature anomalies in the Weddell Sea and high sea-level pressure anomalies over the Atlantic sector of the Southern Ocean. These results suggest that accurate initialization of sea-ice conditions during austral winter is necessary for skillful prediction of climate variability over the Weddell Sea during austral spring.

Improving seasonal forecasts of air temperature using a genetic algorithm.

Seasonal forecasts of air-temperature generated by numerical models provide guidance to the planners and to the society as a whole. However, generating accurate seasonal forecasts is challenging mainly due to the stochastic nature of the atmospheric internal variability. Therefore, an array of ensemble members is often used to capture the prediction signals. With large spread in the prediction plumes, it becomes important to employ techniques to reduce the effects of unrealistic members. One such technique is to create a weighted average of the ensemble members of seasonal forecasts. In this study, we applied a machine learning technique, viz. a genetic algorithm, to derive optimum weights for the 24-ensemble members of the coupled general circulation model; the Scale Interaction Experiment-Frontier research center for global change version 2 (SINTEX-F2) boreal summer forecasts. Our analysis showed the technique to have significantly improved the 2m-air temperature anomalies over several regions of South America, North America, Australia and Russia compared to the unweighted ensemble mean. The spatial distribution of air temperature anomalies is improved by the GA technique leading to better representation of anomalies in the predictions. Hence, machine learning techniques could help in improving the regional air temperature forecasts over the mid- and high-latitude regions where the model skills are relatively modest.

Malaria predictions based on seasonal climate forecasts in South Africa: A time series distributed lag nonlinear model.

Although there have been enormous demands and efforts to develop an early warning system for malaria, no sustainable system has remained. Well-organized malaria surveillance and high-quality climate forecasts are required to sustain a malaria early warning system in conjunction with an effective malaria prediction model. We aimed to develop a weather-based malaria prediction model using a weekly time-series data including temperature, precipitation, and malaria cases from 1998 to 2015 in Vhembe, Limpopo, South Africa and apply it to seasonal climate forecasts. The malaria prediction model performed well for short-term predictions (correlation coefficient, r > 0.8 for 1- and 2-week ahead forecasts). The prediction accuracy decreased as the lead time increased but retained fairly good performance (r > 0.7) up to the 16-week ahead prediction. The demonstration of the malaria prediction process based on the seasonal climate forecasts showed the short-term predictions coincided closely with the observed malaria cases. The weather-based malaria prediction model we developed could be applicable in practice together with skillful seasonal climate forecasts and existing malaria surveillance data. Establishing an automated operating system based on real-time data inputs will be beneficial for the malaria early warning system, and can be an instructive example for other malaria-endemic areas.

Establishment and functional characterization of a tracheal epithelial cell line RTEC11 from transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen.

A tracheal epithelial cell line RTEC11 was established from transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen. The cells grew continuously at a permissive temperature of 33 degrees C but not at a non-permissive temperature of 39 degrees C. Morphological and functional investigations demonstrated that the cells were polarized epithelial cells maintaining a regulated permeability barrier function. Interestingly, the expression levels of Muc1 (mucin 1) and Scgb1a1 (uteroglobin), non-ciliated secretory cell markers, and Tubb4 (tubulin beta 4), a ciliated cell marker, were significantly increased under the cell growth-restricted condition. Global gene expression and computational network analyses demonstrated a significant genetic network associated with cellular development and differentiation in cells cultured at the non-permissive temperature. The tracheal epithelial cell line RTEC11 with unique characteristics should be useful as an in vitro model for studies of the physiological functions and gene expression of tracheal epithelial cells.

Decadal climate predictability in the southern Indian Ocean captured by SINTEX-F using a simple SST-nudging scheme.

Decadal climate variability in the southern Indian Ocean has great influences on southern African climate through modulation of atmospheric circulation. Although many efforts have been made to understanding physical mechanisms, predictability of the decadal climate variability, in particular, the internally generated variability independent from external atmospheric forcing, remains poorly understood. This study investigates predictability of the decadal climate variability in the southern Indian Ocean using a coupled general circulation model, called SINTEX-F. The ensemble members of the decadal reforecast experiments were initialized with a simple sea surface temperature (SST) nudging scheme. The observed positive and negative peaks during late 1990s and late 2000s are well reproduced in the reforecast experiments initiated from 1994 and 1999, respectively. The experiments initiated from 1994 successfully capture warm SST and high sea level pressure anomalies propagating from the South Atlantic to the southern Indian Ocean. Also, the other experiments initiated from 1999 skillfully predict phase change from a positive to negative peak. These results suggest that the SST-nudging initialization has the essence to capture the predictability of the internally generated decadal climate variability in the southern Indian Ocean.

Role of subsurface ocean in decadal climate predictability over the South Atlantic.

Decadal climate predictability in the South Atlantic is explored by performing reforecast experiments using a coupled general circulation model with two initialization schemes; one is assimilated with observed sea surface temperature (SST) only, and the other is additionally assimilated with observed subsurface ocean temperature and salinity. The South Atlantic is known to undergo decadal variability exhibiting a meridional dipole of SST anomalies through variations in the subtropical high and ocean heat transport. Decadal reforecast experiments in which only the model SST is initialized with the observation do not predict well the observed decadal SST variability in the South Atlantic, while the other experiments in which the model SST and subsurface ocean are initialized with the observation skillfully predict the observed decadal SST variability, particularly in the Southeast Atlantic. In-depth analysis of upper-ocean heat content reveals that a significant improvement of zonal heat transport in the Southeast Atlantic leads to skillful prediction of decadal SST variability there. These results demonstrate potential roles of subsurface ocean assimilation in the skillful prediction of decadal climate variability over the South Atlantic.

Histone deacetylase inhibitor reduces monocyte adhesion to endothelium through the suppression of vascular cell adhesion molecule-1 expression.

OBJECTIVE: Tumor necrosis factor (TNF)-alpha initiates numerous changes in endothelial cell (EC) gene expression that contributes to the pathology of various diseases including inflammation. We hypothesized that TNF-alpha-mediated gene induction involves multiple signaling pathways, and that inhibition of one or more of these pathways may selectively target subsets of TNF-alpha-responsive genes and functions. METHODS AND RESULTS: Human umbilical vein endothelial cells (ECs) were preincubated with inhibitors of PI3 kinase (LY294002), histone deacetylases (HDAC) (trichostatin A [TSA]), de novo protein synthesis (CHX), proteasome (MG-132), and GATA factors (K-11430) before exposure to TNF-alpha at 4 hours and analyzed by microarray. TNF-alpha-mediated induction of vascular cell adhesion molecule-1 (VCAM-1) was attenuated by all of these inhibitors, whereas in contrast, stimulation of intercellular adhesion molecule-1 (ICAM-1) was blocked by MG-132 alone. Moreover TSA blocked TNF-alpha-mediated induction of monocyte adhesion both in vitro and in vivo through the suppression of VCAM-1. Further analysis demonstrated that HDAC3 plays a significant role in the regulation of TNF-alpha-mediated VCAM-1 expression. CONCLUSIONS: TNF-alpha activates ECs via multiple signaling pathways, and these pathways may be selectively targeted to modulate EC function. Moreover, TSA treatment reduced monocyte adhesion via VCAM-1 suppression in vitro and in vivo, suggesting that TSA might be useful for the attenuation of the inflammatory response in EC.

Involvement of Irreversible Vacuolar Membrane Fragmentation in the Lethality of Food Emulsifier Diglycerol Monolaurate against Budding Yeast.

Diglycerol monolaurate (DGL) has been manufactured as a novel type of food emulsifier and is being considered for further application as a food preservative. DGL lethality was thus examined against Saccharomyces cerevisiae as a model of a yeast that causes food spoilage. In spite of its molecular structure as a nonionic surfactant, DGL could exhibit lethality at a concentration lower than that which caused disruptive damage to the yeast plasma membrane. DGL lethality was rather accompanied by a dynamic intracellular event such as a marked vacuolar membrane fragmentation. In DGL-treated cells, the tiny dots or particles of fragmented vacuolar membranes failed to fuse into the original large rounded architecture after its removal from medium, which were distinguished from those generated as a result of vacuolar fission normally accelerated under hyperosmotic conditions. Such an irreversible structural damage of the organelle membrane was considered a cause of DGL lethality.

Genetic networks responsive to sodium butyrate in colonic epithelial cells.

We performed microarray and computational gene network analyses to identify the detailed mechanisms by which sodium butyrate (SB) induces cell growth arrest and the differentiation of mouse colonic epithelial MCE301 cells. Two thousand six hundred four differentially expressed probe sets were identified in the cells treated with 2mM SB and were classified into four groups. Of these, the gradually increased group and the gradually and remarkably decreased group contained the genetic networks for cellular development and cell cycles or canonical pathways for fatty acid biosynthesis and pyrimidine metabolism, respectively. The present results provide a basis for understanding the detailed molecular mechanisms of action of SB in colonic epithelial cells.

Overexpression of heat shock protein 70 restores the structural stability and functional defects of temperature-sensitive mutant of large T antigen at nonpermissive temperature.

The effects of heat shock protein 70 (Hsp70), a molecular chaperone, on the degradation and functional alterations of a mutant large T antigen induced by a nonpermissive temperature were examined. In this study, mouse tracheal epithelial TM02-3 cells harboring temperature-sensitive simian virus 40 large T antigen and stable TM02-3 cells overexpressing human Hsp70 and/or Hsp40 were used. Although the temperature shift from 33 degrees C (permissive temperature) to 39 degrees C (nonpermissive temperature) induced increases in the endogenous chaperones including Hsp70 and Hsp40, degradation of the T antigen, activation of the p53-p21(waf1) pathway, and an arrest of cell growth were observed in the mock cells. In contrast, these changes induced by the temperature shift were partially but significantly prevented in stable cells overexpressing human Hsp70 and/or Hsp40. A combination of Hsp70 and Hsp40 was the most effective, suggesting that Hsp40 may cooperate with Hsp70. Moreover, immunocytochemical observation indicated that human Hsp70 was expressed in the cytoplasm at 33 degrees C, but it colocalized with T antigen in the nucleus at 39 degrees C. These results suggest that overexpressed Hsp70 translocates from the cytoplasm to nucleus, and significantly restores the structural stability and functional defects of mutant large T antigen in the cells.

Differences in serological responses to specific glycopeptidolipid-core and common lipid antigens in patients with pulmonary disease due to Mycobacterium tuberculosis and Mycobacterium avium complex.

Disease due to the Mycobacterium avium complex (MAC) is one of the most important opportunistic pulmonary infections. Since the clinical features of MAC pulmonary disease and tuberculosis (TB) resemble each other, and the former is often difficult to treat with chemotherapy, early differential diagnosis is desirable. The humoral immune responses to both diseases were compared by a unique multiple-antigen ELISA using mycobacterial species-common and species-specific lipid antigens, including glycopeptidolipid (GPL)-core. The results were assessed for two patient groups hospitalized and diagnosed clinically as having TB or MAC pulmonary disease. Diverse IgG antibody responsiveness was demonstrated against five lipid antigens: (1) monoacyl phosphatidylinositol dimannoside (Ac-PIM2), (2) cord factor (trehalose 6,6'-dimycolate) (TDM-T) and (3) trehalose monomycolate from Mycobacterium bovis Bacillus Calmette-Guérin (BCG) (TMM-T), and (4) trehalose monomycolate (TMM-M) and (5) GPL-core from MAC. Anti-GPL-core IgG antibody was critical, and detected only in the primary and the secondary MAC diseases with high positivity, up to 88.4 %. However, IgG antibodies against Ac-PIM2, TDM-T and TMM-T were elevated in both TB and MAC patients. Anti-TMM-M IgG antibody was also elevated in MAC disease preferentially, with a positive rate of 89.9 %, and therefore, it was also useful for the diagnosis of the disease. IgG antibody levels were increased at the early stages of the disease and declined in parallel to the decrease of bacterial burden to near the normal healthy control level, when the anti-mycobacterial chemotherapy was completed successfully. Unexpectedly, about 25 % of hospitalized TB patient sera were anti-GPL-core IgG antibody positive, although the specificity of GPL-core was sufficiently high (95.8 % negative in healthy controls), suggesting that a considerable number of cases of latent co-infection with MAC may exist in TB patients. Taken together, the combination of multiple-antigen ELISA using mycobacterial lipids, including GPL-core and TMM-M, gives good discrimination between healthy controls and sera from patients with TB or MAC disease, although for accurate diagnosis of TB more specific antigen(s) are needed.

Binding of a highly potent protease-activated receptor-2 (PAR2) activating peptide, [3H]2-furoyl-LIGRL-NH2, to human PAR2.

1 To determine the binding characteristics of a highly potent agonist for protease-activated receptor-2 (PAR2), 2-furoyl-Leu-Ile-Gly-Arg-Leu-amide (2-furoyl-LIGRL-NH(2)), whole-cell binding assays were performed utilising a radioactive ligand, [(3)H]2-furoyl-LIGRL-NH(2). 2 Specific binding of [(3)H]2-furoyl-LIGRL-NH(2) was observed in NCTC2544 cells, dependent upon PAR2 expression, and competitively displaced by the addition of unlabeled PAR2 agonists. Scatchard analysis of specific saturation binding suggested a single binding site, with K(d) of 122+/-26.1 nM and a corresponding B(max) of 180+/-6 f mol in 3.0 x 10(5) cells. 3 The relative binding affinities of a series of modified PAR2 agonist peptides obtained from competition studies paralleled their relative EC(50) values for Ca(2+) mobilisation assays, indicating improved binding affinities by substitution with 2-furoyl at the N-terminus serine. 4 Pretreatment of cells with trypsin reduced specific binding of [(3)H]2-furoyl-LIGRL-NH(2), demonstrating direct competition between the synthetic agonist peptide and the proteolytically revealed tethered ligand for the binding site of the receptor. 5 In HCT-15 cells endogenously expressing PAR2, the binding of [(3)H]2-furoyl-LIGRL-NH(2) was displaced by addition of unlabeled ligands, Ser-Leu-Ile-Gly-Lys-Val (SLIGKV-OH) or 2-furoyl-LIGRL-NH(2). The relative binding affinity of 2-furoyl-LIGRL-NH(2) to SLIGKV-OH was comparable to its relative EC(50) value for Ca(2+) mobilisation assays. 6 The binding assay was successfully performed in monolayers of PAR2 expressing NCTC2544 and human umbilical vein endothelial cells (HUVEC), in 96- and 24-well plate formats, respectively. 7 These studies indicate that [(3)H]2-furoyl-LIGRL-NH(2) binds to human PAR2 at its ligand-binding site. The use of this radioligand will be valuable for characterising chemicals that interact to PAR2.

A GATA-specific inhibitor (K-7174) rescues anemia induced by IL-1beta, TNF-alpha, or L-NMMA.

Interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), or N(G)-monomethyl-L-arginine (L-NMMA) are increased in patients with chronic disease-related anemia. They increase the binding activity of GATA and inhibit erythropoietin (Epo) promoter activity. In this study, we examined the ability of K-7174 (a GATA-specific inhibitor) to improve Epo production when inhibited by treatment with IL-1beta, TNF-alpha, or L-NMMA. Epo protein production and promoter activity were induced in Hep3B cells with 1% O2. However, 15 U/ml IL-1beta, 220 U/ml TNF-alpha, or 10(-3) M L-NMMA inhibited Epo protein production and promoter activity, respectively. Addition of 10 microM K-7174 rescued these inhibitions of Epo protein production and promoter activity induced by IL-1beta, TNF-alpha, or L-NMMA, respectively. Electrophoretic mobility shift assays revealed that addition of K-7174 decreased GATA binding activity, which was increased with the addition of IL-1beta, TNF-alpha, or L-NMMA. Furthermore, intraperitoneal injection of mice with IL-1beta or TNF-alpha decreased the hemoglobin concentrations and reticulocyte counts. However, the addition of K-7174 reversed these effects. These results raise the possibility of using K-7174 as therapy to treat anemia.

Synergistic combination of direct plasma membrane damage and oxidative stress as a cause of antifungal activity of polyol macrolide antibiotic niphimycin.

The polyol macrolide niphimycin (NM) exhibited fungicidal activity against Saccharomyces cerevisiae cells accompanying the leakage of cytoplasmic components including nucleotide-like materials in addition to K+ at 10 microM or above. Such a dynamic change in the plasma membrane was observed upon treatment of cells with H2O2 but not with the polyene macrolide antibiotic amphotericin B (AmB). The NM-induced cell death could be prevented by the exogenous addition of phosphatidylcholine (PC) whereas such a protective effect was only weakly observed with ergosterol, the molecular target of AmB. NM-treated cells were further characterized with a dramatic loss of glutathione even at a dose of 5 microM or less, representing NM-triggered metabolic conversion of the antioxidant molecule. NM-treatment indeed accelerated the cellular production of reactive oxygen species (ROS) such as H2O2 detectable with a specific fluorescent probe in a dose-dependent manner. These results suggested a synergistic combination of direct plasma membrane damage and oxidative stress as a cause of antifungal activity of NM against S. cerevisiae.