Inhibition of the Calcineurin Pathway by Two Flavonoids Isolated from Miliusa sinensis Finet & Gagnep.

In order to discover plant-derived signaling pathway inhibitors with antifungal properties, a two-component screening system utilizing the calcineurin and Hog1 mitogen-activated protein kinase pathways responsible for the virulence networks of Cryptococcus neoformans was employed, owing to the counter-regulatory actions of these pathways. Of the 1,000 plant extracts tested, two bioactive compounds from Miliusa sinensis were found to act specifically on the calcineurin pathway of C. neoformans. These compounds, identified as pashanone and 5-hydroxy-6,7-dimethoxyflavanone, exhibited potent antifungal activities against various human pathogenic fungi with minimum inhibitory concentration values ranging from 4.0 to >128 µg/ml.

A Phenylpropanoid Glycoside as a Calcineurin Inhibitor Isolated from Magnolia obovata Thunb.

To identify plant-derived cell signaling inhibitors with antifungal properties, a twocomponent screening system using both wild-type Cryptococcus neoformans and a calcineurin mutant was employed owing to their counter-regulatory actions on the Hog1 mitogenactivated protein kinase and calcineurin pathways. Of the 2,000 plant extracts evaluated, a single bioactive compound from M. obovata Thunb. was found to act specifically on the calcineurin pathway of C. neoformans. This compound was identified as magnoloside A, and had potent antifungal activities against various Cryptococcus strains with minimum inhibitory concentration values ranging from 1.0 to 4.0 µg/ml.

Inhibition of the calcineurin pathway by two tannins, chebulagic acid and chebulanin, isolated from Harrisonia abyssinica Oliv.

In order to discover and develop novel signaling inhibitors from plants, a screening system was established targeting the two-component system of Cryptococcus neoformans by using the wild type and a calcineurin mutant of C. neoformans, based on the counter-regulatory action of high-osmolarity glycerol (Hog1) mitogen-activated protein kinase and the calcineurin pathways in C. neoformans. Among 10,000 plant extracts, that from Harrisonia abyssinica Oliv. exhibited the most potent inhibitory activity against C. neoformans var. grubii H99 with fludioxonil. Bioassay-guided fractionation was used to isolate two bioactive compounds from H. abyssinica, and these compounds were identified as chebulagic acid and chebulanin using spectroscopic methods. These compounds specifically inhibited the calcineurin pathway in C. neoformans. Moreover, they exhibited potent antifungal activities against various human pathogenic fungi with minimum inhibitory concentrations ranging from 0.25 to over 64 µg/ml.

Relation of Pulmonary Function Impairment and Coronary Artery Calcification by Multi-detector Computed Tomography in Group Exposed to Inorganic Dusts.

BACKGROUND: The purpose of this study was to evaluate the relationship of pulmonary function impairment (PFI) and coronary artery calcification (CAC) by multi-detector computed tomography (MDCT), and the effect of pneumoconiosis on CAC or PFI. METHODS: Seventy-six subjects exposed to inorganic dusts underwent coronary artery calcium scoring by MDCT, spirometry, laboratory tests, and a standardized questionnaire. CAC was quantified using a commercial software (Rapidia ver. 2.8), and all the subjects were divided into two categories according to total calcium scores (TCSs), either the non-calcified (<1) or the calcified (≥1) group. Obstructive pulmonary function impairment (OPFI) was defined as forced expiratory volume in one second/forced vital capacity (FEV1/FVC, %)<70, and as FEV1/FVC (%)≥70 and FVC<80 for restrictive pulmonary function impairment (RPFI) by spirometry. All subjects were classified as either the case (profusion≥1/0) or the control (profusion≤0/1) group by pneumoconiosis findings on simple digital radiograph. RESULTS: Of the 76 subjects, 35 subjects (46.1%) had a CAC. Age and hypertension were different significantly between the non-calcified and the calcified group (p<0.05). Subjects with pneumoconiosis were more frequent in the calcified group than those in the non-calcified group (p=0.099). FEV1/FVC (%) was significantly correlated with TCSs (r=-0.316, p=0.005). Subjects with OPFI tended to increase significantly with increasing of TCS (4.82, p=0.028), but not significantly in RPFI (2.18, p=0.140). Subjects with OPFI were significantly increased in the case group compared to those in the control group. CONCLUSION: CAC is significantly correlated with OPFI, and CAC and OPFI may be affected by pneumoconiosis findings.

Reliability and validity of soft copy images based on flat-panel detector in pneumoconiosis classification: comparison with the analog radiographs.

RATIONALE AND OBJECTIVES: The aim of this study was to evaluate the reliability and validity of soft copy images based on flat-panel detector of digital radiography (DR-FPD soft copy images) compared to analog radiographs (ARs) in pneumoconiosis classification and diagnosis. MATERIALS AND METHODS: DR-FPD soft copy images and ARs from 349 subjects were independently read by four-experienced readers according to the International Labor Organization 2000 guidelines. DR-FPD soft copy images were used to obtain consensus reading (CR) by all readers as the gold standard. Reliability and validity were evaluated by a κ and receiver operating characteristic analysis, respectively. RESULTS: In small opacity, overall interreader agreement of DR-FPD soft copy images was significantly higher than that of ARs, but it was not significantly different in large opacity and costophrenic angle obliteration. In small opacity, agreement of DR-FPD soft copy images with CR was significantly higher than that of ARs with CR. It was also higher than that of ARs with CR in pleural plaque and thickening. Receiver operating characteristic areas were not different significantly between DR-FPD soft copy images and ARs. CONCLUSIONS: DR-FPD soft copy images showed accurate and reliable results in pneumoconiosis classification and diagnosis compared to ARs.

Utility of digital radiography for the screening of pneumoconiosis as compared to analog radiography: radiation dose, image quality, and pneumoconiosis classification.

The purpose of this study was to compare digital radiography (DR) and analog radiography (AR) for the screening of pneumoconiosis with respect to radiation dose, image quality, and pneumoconiosis classification. DR was performed on 50 subjects who were enrolled for an examination of pneumoconiosis (Digital Diagnost™, Philips, Netherlands), and AR (MXO-15B, Toshiba, Japan) was performed the same day after the study was approved by the Institutional Review Board and written informed consent was obtained from all subjects. Entrance surface doses (ESDs) of DR and AR were measured using a glass dosimeter attached to a Rando human phantom (Alderson Co., U.S.) under exposure conditions commonly used in clinical practice in Korea. Visibilities on all images were evaluated using a 5-point scale by four chest radiologists using a modified form of the European Chest Guidelines (EUR 16260). All the images were classified using the ILO's guidelines by referencing standard analog radiographs. ESDs of DR were significantly lower than those of AR (0.15 mGy vs. 0.21 mGy, p < 0.05). All anatomic structures were significantly more visible by DR images (p < 0.0001), especially the left main bronchus, ribs, and thoracic spine. Body mass index did not correlate with anatomic structure visibility by DR (r = -0.029, p = 0.842) or AR images (r = -0.076, p = 0.602). Overall intra- and inter-reader agreements for DR images were significantly higher than for AR images. DR offers improved image quality with a significant reduction of up to 23.6% in radiation dose and more accurate pneumoconiosis classification than AR.

Quantitative Changes of Polyphenolic Compounds in Mulberry (Morus alba L.) Leaves in Relation to Varieties, Harvest Period, and Heat Processing.

Six polyphenolic compounds, such as chlorogenic acid (CA), rutin (RT), isoquercitrin (IQT), quercetin-3-O-(6-O-malonyl)-ß-D-glucoside (QMG), astragalin (AG), kaempferol-3-O-(6-O-malonyl)-ß-D-glucoside (KMG), were isolated from mulberry leaves by a series of isolation procedures, such as Diaion HP-20, silica-gel, Sephadex LH-20, and ODS-A column chromatographies. The chemical structures of the phenolic compounds were identified by UV and NMR spectral analyses. Levels of polyphenols in mulberry leaves from six different mulberry cultivars ranged from 1,042.16 to 1,871.97 mg% per dry weight; Guksang cultivar showed the highest levels of polyphenols, whereas Gaeryangdaehwa contained the least polyphenol contents. Generally, levels of polyphenols in mulberry leaves decreased with increasing harvest time, except for Yoolmok, but increased with heat processing time, except QMG and KMG. These results suggest that the heat processed mulberry leaves of Guksang cultivar harvested in early May can be potentially useful sources for production of high quality mulberry leaf teas.

Development of standard digital images for pneumoconiosis.

We developed the standard digital images (SDIs) to be used in the classification and recognition of pneumoconiosis. From July 3, 2006 through August 31, 2007, 531 retired male workers exposed to inorganic dust were examined by digital (DR) and analog radiography (AR) on the same day, after being approved by our institutional review board and obtaining informed consent from all participants. All images were twice classified according to the International Labour Office (ILO) 2000 guidelines with reference to ILO standard analog radiographs (SARs) by four chest radiologists. After consensus reading on 349 digital images matched with the first selected analog images, 120 digital images were selected as the SDIs that considered the distribution of pneumoconiosis findings. Images with profusion category 0/1, 1, 2, and 3 were 12, 50, 40, and 15, respectively, and a large opacity were in 43 images (A = 20, B = 22, C = 1). Among pleural abnormality, costophrenic angle obliteration, pleural plaque and thickening were in 11 (9.2%), 31 (25.8%), and 9 (7.5%) images, respectively. Twenty-one of 29 symbols were present except cp, ef, ho, id, me, pa, ra, and rp. A set of 120 SDIs had more various pneumoconiosis findings than ILO SARs that were developed from adequate methods. It can be used as digital reference images for the recognition and classification of pneumoconiosis.

Blood Levels of IL-Iß, IL-6, IL-8, TNF-α, and MCP-1 in Pneumoconiosis Patients Exposed to Inorganic Dusts.

Inhaled inorganic dusts such as coal can cause inflammation and fibrosis in the lung called pneumoconiosis. Chronic inflammatory process in the lung is associated with various cytokines and reactive oxygen species (ROS) formation. Expression of some cytokines mediates inflammation and leads to tissue damage or fibrosis. The aim of the present study was to compare the levels of blood cytokines interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1 among 124 subjects (control 38 and pneumoconiosis patient 86) with category of chest x-ray according to International Labor Organization (ILO) classification. The levels of serum IL-8 (p = 0.003), TNF-α (p = 0.026), and MCP-1 (p = 0.010) of pneumoconiosis patients were higher than those of subjects with the control. The level of serum IL-8 in the severe group with the small opacity (ILO category II or III) was higher than that of the control (p = 0.035). There was significant correlation between the profusion of radiological findings with small opacity and serum levels of IL-1ß (rho = 0.218, p < 0.05), IL-8 (rho = 0.224, p < 0.05), TNF-α (rho = 0.306, p < 0.01), and MCP-1 (rho = 0.213, p < 0.01). The serum levels of IL-6 and IL-8, however, did not show significant difference between pneumoconiosis patients and the control. There was no significant correlation between serum levels of measured cytokines and other associated variables such as lung function, age, BMI, and exposure period of dusts. Future studies will be required to investigate the cytokine profile that is present in pneumoconiosis patient using lung specific specimens such as bronchoalveolar lavage fluid (BALF), exhaled breath condensate, and lung tissue.

The PI3 kinase-Akt pathway mediates Wnt3a-induced proliferation.

Wnt3a activates proliferation of fibroblasts cells via activation of both extracellular signal-regulated kinase (ERK) and Wnt/beta-catenin signaling pathways. In this study, we show that the phosphatidyl inositol 3 kinases (PI3K)-Akt pathway is also involved in the Wnt3a-induced proliferation. Akt was activated within 30 min by Wnt3a in NIH3T3 cells. By Wnt3a treatment, activated Akt was transiently accumulated in nucleus although beta-catenin was accumulated in the nucleus of cells in a prolonged manner. The Wnt3a-induced Akt activation was not affected by siRNA-mediated reduction of beta-catenin, indicating that Wnt3a-induced Akt activation may occur independently of beta-catenin. The Wnt3a-induced Akt activation was abolished by pre-treatment with PI3K inhibitor, LY294002 and Wortmanin, but not by MEK inhibitor, U0126, indicating that Wnt3a activates Akt via PI3K. The growth and proliferation induced by Wnt3a were blocked by treatments of the PI3K inhibitors. Furthermore, Wnt3a-induced proliferation was blocked by Akt siRNA. These results reveal that the PI3K-Akt pathway mediates the Wnt3a-induced growth and proliferation of NIH3T3 cells.

Identification of proteins interacting with the catalytic subunit of PP2A by proteomics.

The protein phosphatase 2A (PP2A) is a serine/threonine phosphatase involved in the regulation of multiple signaling pathways including the Wnt/beta-catenin and the ERK pathways. To understand the complex signaling networking associated with PP2A, we searched proteins interacting with the catalytic subunit of protein phosphatase 2A (PP2Ac) by a pull-down analysis followed by 2-D gel electrophoresis and proteomic analyses. The probability of identification of the proteins interacting with PP2Ac was increased by searching proteins differently interacting with PP2Ac according to stimulation of Wnt3a, which regulates both the Wnt/beta-catenin and the ERK pathways. Around 100 proteins, pulled-down by His-tagged PP2Ac, were identified in 2-D gels stained with CBB. By MALDI-TOF-MS analyses of 45 protein spots, we identified several proteins that were previously known to interact with PP2A, such as Axin and CaMK IV. In addition, we also identified many proteins that potentially interact with PP2Ac. The interactions of several candidate proteins, such as tuberous sclerosis complex 2, RhoB, R-Ras, and Nm23H2, with PP2Ac, were confirmed by in vitro binding analyses and/or coimmunoprecipitation experiments.