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We investigated the transformation and fate of diphenylarsinic acid (DPAA) during incubation in two types of soils (Entisol and Andisol) under aerobic and anaerobic conditions. Under anaerobic conditions only, DPAA was transformed into methyldiphenylarsine oxide by methylation. Under both aerobic and anaerobic conditions, DPAA was degraded to phenylarsonic acid by dephenylation, and phenylarsonic acid was subsequently methylated to form methylphenylarsinic acid and dimethylphenylarsine oxide. The degradation of DPAA in the Andisol was less extensive than in the Entisol. In autoclaved soil under anaerobic conditions, DPAA underwent little degradation during the 24-wk incubation. In unautoclaved soils, the concentration of DPAA in soil clearly decreased after 24 wk of incubation, indicating that DPAA degradation was driven by microbial activity.

The cell cycle is strictly regulated by numerous mechanisms to ensure cell division. The transcriptional regulation of cell-cycle-related genes is poorly understood, with the exception of the E2F family that governs the cell cycle. Here, we show that a transcription factor, zinc finger protein 143 (ZNF143), positively regulates many cell-cycle-associated genes and is highly expressed in multiple solid tumors. RNA-interference (RNAi)-mediated knockdown of ZNF143 showed that expression of 152 genes was downregulated in human prostate cancer PC3 cells. Among these ZNF143 targets, 41 genes (27%) were associated with cell cycle and DNA replication including cell division cycle 6 homolog (CDC6), polo-like kinase 1 (PLK1) and minichromosome maintenance complex component (MCM) DNA replication proteins. Furthermore, RNAi of ZNF143 induced apoptosis following G2/M cell cycle arrest. Cell growth of 10 lung cancer cell lines was significantly correlated with cellular expression of ZNF143. Our data suggest that ZNF143 might be a master regulator of the cell cycle. Our findings also indicate that ZNF143 is a member of the growing list of non-oncogenes that are promising cancer drug targets., (© 2010 Japanese Cancer Association.)

BIBF 1120 is an oral multitargeted tyrosine kinase inhibitor that blocks the activity of vascular endothelial growth factor (VEGF) and other growth factor receptors. We have done a phase I study to evaluate the safety, pharmacokinetics, and pharmacodynamic biomarkers of BIBF 1120. Patients with advanced refractory solid tumors were treated with BIBF 1120 at oral doses of 150 to 250 mg twice daily. Drug safety and pharmacokinetics were evaluated, as were baseline and post-treatment levels of circulating CD117-positive bone marrow-derived progenitor cells and plasma soluble VEGF receptor 2 as potential biomarkers for BIBF 1120. Twenty-one patients were treated at BIBF 1120 doses of 150 (n = 3), 200 (n = 12), or 250 mg twice daily (n = 6). Dose-limiting toxicities of reversible grade 3 or 4 elevations of liver enzymes occurred in 3 of 12 patients at 200 mg twice daily and 3 of 6 patients at 250 mg twice daily. Stable disease was achieved in 16 (76.2%) patients, and median progression-free survival was 113 days (95% confidence interval, 77-119 d). Pharmacokinetic analysis indicated that the maximum plasma concentration and area under the curve for BIBF 1120 increased with the dose within the dose range tested. Levels of CD117-positive bone marrow-derived progenitors and soluble VEGF receptor 2 decreased significantly during treatment over all BIBF 1120 dose cohorts. In conclusion, the maximum tolerated dose of BIBF 1120 in the current study was determined to be 200 mg twice daily, and our biomarker analysis indicated that this angiokinase inhibitor is biologically active.

Most non-small cell lung cancer (NSCLC) tumors with an activating mutation of the epidermal growth factor receptor (EGFR) are initially responsive to EGFR tyrosine kinase inhibitors (TKI) such as gefitinib but ultimately develop resistance to these drugs. Hepatocyte growth factor (HGF) induces EGFR-TKI resistance in NSCLC cells with such a mutation. We investigated strategies to overcome gefitinib resistance induced by HGF. Human NSCLC cells with an activating EGFR mutation (HCC827 cells) were engineered to stably express HGF (HCC827-HGF cells). HCC827-HGF cells secreted large amounts of HGF and exhibited resistance to gefitinib in vitro to an extent similar to that of HCC827 GR cells, in which the gene for the HGF receptor MET is amplified. A MET-TKI reversed gefitinib resistance in HCC827-HGF cells as well as in HCC827 GR cells, suggesting that MET activation induces gefitinib resistance in both cell lines. TAK-701, a humanized monoclonal antibody to HGF, in combination with gefitinib inhibited the phosphorylation of MET, EGFR, extracellular signal-regulated kinase, and AKT in HCC827-HGF cells, resulting in suppression of cell growth and indicating that autocrine HGF-MET signaling contributes to gefitinib resistance in these cells. Combination therapy with TAK-701 and gefitinib also markedly inhibited the growth of HCC827-HGF tumors in vivo. The addition of TAK-701 to gefitinib is a promising strategy to overcome EGFR-TKI resistance induced by HGF in NSCLC with an activating EGFR mutation.

Purpose: The epidermal growth factor receptor (EGFR) mutation status has emerged as a validated biomarker for predicting the response to treatment with EGFR-tyrosine kinase inhibitors (EGFR-TKI) in patients with non-small cell lung cancer. However, the responses to EGFR-TKIs vary even among patients with EGFR mutations. We studied several other independently active biomarkers for EGFR-TKI treatment., Experimental Design: We retrospectively analyzed the serum concentrations of 13 molecules in a cohort of 95 patients with non-small cell lung adenocarcinoma who received EGFR-TKI treatment at three centers. The pretreatment serum concentrations of amphiregulin, β-cellulin, EGF, EGFR, epiregulin, fibroblast growth factor-basic, heparin-binding EGF-like growth factor, hepatocyte growth factor (HGF), platelet-derived growth factor β polypeptide, placental growth factor, tenascin C, transforming growth factor-α, and vascular endothelial growth factor (VEGF) were measured using enzyme-linked immunosorbent assay and a multiplex immunoassay system. The associations between clinical outcomes and these molecules were evaluated., Results: The concentrations of HGF and VEGF were significantly higher among patients with progressive disease than among those without progressive disease (P < 0.0001). HGF and VEGF were strongly associated with progression-free survival (PFS) and overall survival (OS) in a univariate Cox analysis (all tests for hazard ratio showed P < 0.0001). A stratified multivariate Cox model according to EGFR mutation status (mutant, n = 20; wild-type, n = 23; unknown, n = 52) showed that higher HGF levels were significantly associated with a shorter PFS and OS (P < 0.0001 for both PFS and OS). These observations were also consistent in the subset analyses., Conclusions: Serum HGF was strongly related to the outcome of EGFR-TKI treatment. Our results suggest that the serum HGF level could be used to refine the selection of patients expected to respond to EGFR-TKI treatment, warranting further prospective study., (©2010 AACR.)

This prospective study used antibody suspension bead arrays to identify biomarkers capable of predicting post-operative recurrence with distal metastasis in patients with colorectal cancer. One hundred colorectal cancer patients who underwent surgery were enrolled in this study. The median follow-up period was 3.9 years. The pre-operative plasma concentrations of 24 angiogenesis-related molecules were analyzed with regard to the TNM stage and the development of post-operative recurrence. The concentrations of half of the examined molecules (13/24) increased significantly according to the TNM stage (P < 0.05). Meanwhile, a multivariate logistic regression analysis revealed that the concentrations of vascular cell adhesion molecule 1 (VCAM-1) and plasminogen activator inhibitor-1 (PAI-1) were significantly higher in the post-operative recurrence group. The VCAM-1 and PAI-1 model discriminated post-operative recurrence with an area under the curve of 0.82, a sensitivity of 0.75, and a specificity of 0.73. A leave-one-out cross-validation was applied to the model to assess the prediction performance, and the result indicated that the cross-validated error rate was 12.5% (12/96). In conclusion, our results demonstrate that antibody suspension bead arrays are a powerful tool to screen biomarkers in the clinical setting, and the plasma levels of VCAM-1 and PAI-1 together may be a promising biomarker for predicting post-operative recurrence in patients with colorectal cancer.

Objectives: The purpose of this study was to identify genes that predict progression-free survival (PFS) in advanced epithelial ovarian cancer (aEOC) receiving standard therapy., Methods: We performed microarray analysis on laser microdissected aEOC cells. All cases received staging laparotomy and adjuvant chemotherapy (carboplatin+paclitaxel) as primary therapy., Results: Microarray analysis identified 50 genes differentially expressed between tumors of patients with no evidence of disease (NED) or evidence of disease (ED) (p<0.001). Six genes (13%) were located at 8q24, and 9 genes (19.6%), at 20q11-13. The ratio of selected gene set/analyzed gene set in chromosomes 8 and 20 are significantly higher than that in other chromosome regions (6/606 vs. 32/13656, p=0.01) and (12/383 vs. 32/13656, p=1.3 x 10(-)(16)). We speculate that the abnormal chromosomal distribution is due to genomic alteration and that these genes may play an important role in aEOC and choose GNAS (GNAS complex locus, NM&#95;000516) on 20q13 based on the p value and fold change. Genomic PCR of aEOC cells also showed that amplification of GNAS was significantly correlated with unfavorable PFS (p=0.011). Real-time quantitative RT-PCR analysis of independent samples revealed that high mRNA expression levels of the GNAS genes, located at chromosome 20q13, was significantly unfavorable indicators of progression-free survival (PFS). Finally, GNAS amplification was an independent prognostic factor for PFS., Conclusions: Our results suggest that GNAS gene amplification may be an independent, qualitative, and reproducible biomarker of PFS in aEOC., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Objective: An effective program for preventing metabolic diseases through lifestyle modification is urgently needed. We investigated the effects of the Life Style Modification Program for Physical Activity and Nutrition program (LiSM10!) on metabolic parameters in middle-aged male Japanese white-collar workers., Methods: One hundred and one male office workers, 30 to 59 years of age, with metabolic syndrome risk factors, were randomly allocated into no-treatment control (n=49) and LiSM intervention (n=52) groups. The LiSM group attended individualized assessment and collaborative goal setting sessions based on food group intake and physical activity, followed by two individual counseling sessions with a registered dietitian and physical trainer, and received monthly website advice during the 4-month period from December 2006 to May 2007, in Tokyo, Japan. They were encouraged to enter current targeted food intakes and pedometer data on self-monitoring websites during the entire study period., Results: Habitual food group intakes changed significantly in the LiSM group, showing improvements in 14 anthropometric and biochemical parameters contributing to inter-group differences in body weight, body mass index, fasting plasma glucose, insulin and homeostasis model assessment of insulin resistance changes (p<0.01)., Conclusion: The LiSM10! program effectively improved insulin resistance-related metabolic parameters in middle-aged male white-collar workers., (Copyright 2010 Elsevier Inc. All rights reserved.)

Bortezomib, a selective 26S proteasome inhibitor, has shown clinical benefits against refractory multiple myeloma. The indirect anti-angiogenic activity of bortezomib has been widely recognized; however, the growth-inhibitory mechanism of bortezomib on vascular endothelial cells remains unclear, especially on the cell cycle. Here, we showed that bortezomib (2 nM of the IC(50) value) potently inhibited the cellular growth of human umbilical vascular endothelial cells (HUVECs) via a vascular endothelial growth factor receptor (VEGFR)-independent mechanism resulting in the induction of apoptosis. Bortezomib significantly increased the vascular permeability of HUVECs, whereas a VEGFR-2 tyrosine kinase inhibitor decreased it. Interestingly, a cell cycle analysis using flow cytometry, the immunostaining of phospho-histone H3, and Giemsa staining revealed that bortezomib suppressed the G2/M transition of HUVECs, whereas the mitotic inhibitor paclitaxel induced M-phase accumulation. A further analysis of cell cycle-related proteins revealed that bortezomib increased the expression levels of cyclin B1, the cdc2/cyclin B complex, and the phosphorylation of all T14, Y15, and T161 residues on cdc2. Bortezomib also increased the ubiquitination of cyclin B1 and wee1, but inhibited the kinase activity of the cdc2/cyclin B complex. These protein modifications support the concept that bortezomib suppresses the G2/M transition, rather than causing M-phase arrest. In conclusion, we demonstrated that bortezomib potently inhibits cell growth by suppressing the G2/M transition, modifying G2/M-phase-related cycle regulators, and increasing the vascular permeability of vascular endothelial cells. Our findings reveal a cell cycle-related mode of action and strongly suggest that bortezomib exerts an additional unique vascular disrupting effect as a vascular targeting drug.

Therapeutic strategies that target c-Src hold promise for a wide variety of cancers. We have now investigated both the effects of dasatinib, which inhibits the activity of c-Src and several other kinases, on cell growth as well as the mechanism of dasatinib resistance in human gastric cancer cell lines. Immunoblot analysis revealed the activation of c-Src at various levels in most gastric cancer cell lines examined. Dasatinib inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) and induced G(1) arrest, as revealed by flow cytometry, in a subset of responsive cell lines. In other responsive cell lines, dasatinib inhibited both ERK and AKT phosphorylation and induced apoptosis, as revealed by an increase in caspase-3 activity and cleavage of poly(ADP-ribose) polymerase. Depletion of c-Src by RNA interference also induced G(1) arrest or apoptosis in dasatinib-responsive cell lines, indicating that the antiproliferative effect of dasatinib is attributable to c-Src inhibition. Gastric cancer cell lines positive for the activation of MET were resistant to dasatinib. Dasatinib had no effect on ERK or AKT signaling, whereas the MET inhibitor PHA-665752 induced apoptosis in these cells. The subsets of gastric cancer cells defined by a response to c-Src or MET inhibitors were distinct and nonoverlapping. Our results suggest that c-Src is a promising target for the treatment of gastric cancer and that analysis of MET amplification might optimize patient selection for treatment with c-Src inhibitors.

Forkhead box Q1 (FOXQ1) is a member of the forkhead transcription factor family, and it has recently been proposed to participate in gastric acid secretion and mucin gene expression in mice. However, the role of FOXQ1 in humans and especially in cancer cells remains unknown. We found that FOXQ1 mRNA is overexpressed in clinical specimens of colorectal cancer (CRC; 28-fold/colonic mucosa). A microarray analysis revealed that the knockdown of FOXQ1 using small interfering RNA resulted in a decrease in p21(CIP1/WAF1) expression, and a reporter assay and a chromatin immunoprecipitation assay showed that p21 was one of the target genes of FOXQ1. Stable FOXQ1-overexpressing cells (H1299/FOXQ1) exhibited elevated levels of p21 expression and inhibition of apoptosis induced by doxorubicin or camptothecin. Although cellular proliferation was decreased in H1299/FOXQ1 cells in vitro, H1299/FOXQ1 cells significantly increased tumorigenicity [enhanced green fluorescent protein (EGFP): 2/15, FOXQ1: 7/15] and enhanced tumor growth (437 +/- 301 versus 1735 +/- 769 mm3, P < 0.001) in vivo. Meanwhile, stable p21 knockdown of H1299/FOXQ1 cells increased tumor growth, suggesting that FOXQ1 promotes tumor growth independent of p21. Microarray analysis of H1299/EGFP and H1299/FOXQ1 revealed that FOXQ1 overexpression upregulated several genes that have positive roles for tumor growth, including VEGFA, WNT3A, RSPO2, and BCL11A. CD31 and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining of the tumor specimens showed that FOXQ1 overexpression mediated the angiogenic and antiapoptotic effect in vivo. In conclusion, FOXQ1 is overexpressed in CRC and enhances tumorigenicity and tumor growth presumably through its angiogenic and antiapoptotic effects. Our findings show that FOXQ1 is a new member of the cancer-related FOX family.

Background: Somatic mutations in the epidermal growth factor receptor (EGFR) gene are a predictor of response to treatment with EGFR tyrosine kinase inhibitors (TKIs) in patients with non-small cell lung cancer (NSCLC). However, mechanisms of de novo resistance to these drugs in patients harboring EGFR mutations have remained unclear. We examined whether the mutational status of KRAS might be associated with primary resistance to EGFR-TKIs in EGFR mutation-positive patients with NSCLC., Methods: Forty patients with NSCLC with EGFR mutations who were treated with gefitinib or erlotinib and had archival tissue specimens available were enrolled in the study. KRAS mutations were analyzed by direct sequencing., Results: Three (7.5%) of the 40 patients had progressive disease, and two (67%) of these three individuals had both KRAS and EGFR mutations., Conclusions: Our results suggest that KRAS mutation is a negative predictor of response to EGFR-TKIs in EGFR mutation-positive patients with NSCLC.

The effect of compressed unsaturated hydrocarbon gases on the growth of the yeast Saccharomyces cerevisiae was investigated by microcalorimetry. The growth thermograms showed that unsaturated hydrocarbon gases inhibited yeast growth. As an approach to determining the comparative toxicity of unsaturated hydrocarbon gases, we determined the 50% inhibitory pressure (IP(50)) and the minimum inhibitory pressure (MIP). On the basis of the IP(50) and MIP values, the inhibitory potency of the gases increased in the order ethylene < propylene < 1-butene. Additionally, scanning electron microscopy showed that cells treated with unsaturated hydrocarbon gases were damaged, including invagination of the cell surface.

Solanum torvum Sw. cv. Torubamubiga (TB) is a low cadmium (Cd)-accumulating plant. To elucidate the molecular mechanisms of the Cd acclimation process in TB roots, transcriptional regulation was analysed in response to mild Cd treatment: 0.1 muM CdCl(2) in hydroponic solution. A unigene set consisting of 6296 unigene sequences was constructed from 18 816 TB cDNAs. The distribution of functional categories was similar to tomato, while 330 unigenes were suggested to be TB specific. For expression profiling, the SuperSAGE method was adapted for use with Illumina sequencing technology. Expression tag libraries were constructed from Cd-treated (for 3 h, 1 d, and 3 d) and untreated roots, and 34 269 species of independent tags were collected. Moreover, 6237 tags were ascribed to the TB or eggplant (aubergine) unigene sequences. Time-course changes were examined, and 2049 up- and 2022 down-regulated tags were identified. Although no tags annotated to metal transporter genes were significantly regulated, a tag annotated to AtFRD3, a xylem-loading citrate transporter, was down-regulated. In addition to induction of heavy metal chaperone proteins, antioxidative and sulphur-assimilating enzymes were induced, confirming that oxidative stress developed even using a mild Cd concentration. Rapid repression of dehydration-related transcription factors and aquaporin isoforms suggests that dehydration stress is a potential constituent of Cd-induced biochemical impediments. These transcriptional changes were also confirmed by real-time reverse transcription-PCR. Further additions of TB unigene sequences and functional analysis of the regulated tags will reveal the molecular basis of the Cd acclimation process, including the low Cd-accumulating characteristics of TB.

The inhibitory action of compressed hydrocarbon gases on the growth of the yeast Saccharomyces cerevisiae was investigated quantitatively by microcalorimetry. Both the 50% inhibitory pressure (IP(50)) and the minimum inhibitory pressure (MIP), which are regarded as indices of the toxicity of hydrocarbon gases, were determined from growth thermograms. Based on these values, the inhibitory potency of the hydrocarbon gases increased in the order methane << ethane < propane < i-butane < n-butane. The toxicity of these hydrocarbon gases correlated to their hydrophobicity, suggesting that hydrocarbon gases interact with some hydrophobic regions of the cell membrane. In support of this, we found that UV absorbing materials at 260 nm were released from yeast cells exposed to compressed hydrocarbon gases. Additionally, scanning electron microscopy indicated that morphological changes occurred in these cells.

Rice consumption is a major source of cadmium and arsenic for the population of Asia. We investigated the effects of water management in rice paddy on levels of cadmium and arsenic in Japanese rice grains. Flooding increased arsenic concentrations in rice grains, whereas aerobic treatment increased the concentration of cadmium. Flooding for 3 weeks before and after heading was most effective in reducing grain cadmium concentrations, but this treatment increased the arsenic concentration considerably, whereas aerobic treatment during the same period was effective in reducing arsenic concentrations but increased the cadmium concentration markedly. Flooding treatment after heading was found to be more effective than flooding treatment before heading in reducing rice grain cadmium without a concomitant increase in total arsenic levels, although it increased inorganic arsenic levels. Concentrations of dimethylarsinic acid (DMA) in grain were very low under aerobic conditions but increased under flooded conditions. DMA accounted for 3-52% of the total arsenic concentration in grain grown in soil with a lower arsenic concentration and 10-80% in soil with a higher arsenic concentration. A possible explanation for the accumulation of DMA in rice grains is that DMA translocates from shoots/roots to the grains more readily than does inorganic arsenic.

The underlying mechanism regulating the expression of the cancer stem cell/tumor-initiating cell marker CD133/prominin-1 in cancer cells remains largely unclear, although knowledge of this mechanism would likely provide important biological information regarding cancer stem cells. Here, we found that the inhibition of mTOR signaling up-regulated CD133 expression at both the mRNA and protein levels in a CD133-overexpressing cancer cell line. This effect was canceled by a rapamycin-competitor, tacrolimus, and was not modified by conventional cytotoxic drugs. We hypothesized that hypoxia-inducible factor-1 alpha (HIF-1 alpha), a downstream molecule in the mTOR signaling pathway, might regulate CD133 expression; we therefore investigated the relation between CD133 and HIF-1 alpha. Hypoxic conditions up-regulated HIF-1 alpha expression and inversely down-regulated CD133 expression at both the mRNA and protein levels. Similarly, the HIF-1 alpha activator deferoxamine mesylate dose-dependently down-regulated CD133 expression, consistent with the effects of hypoxic conditions. Finally, the correlations between CD133 and the expressions of HIF-1 alpha and HIF-1 beta were examined using clinical gastric cancer samples. A strong inverse correlation (r = -0.68) was observed between CD133 and HIF-1 alpha, but not between CD133 and HIF-1 beta. In conclusion, these results indicate that HIF-1 alpha down-regulates CD133 expression and suggest that mTOR signaling is involved in the expression of CD133 in cancer cells. Our findings provide a novel insight into the regulatory mechanisms of CD133 expression via mTOR signaling and HIF-1 alpha in cancer cells and might lead to insights into the involvement of the mTOR signal and oxygen-sensitive intracellular pathways in the maintenance of stemness in cancer stem cells.

In accordance with a new international standard set by the Codex Alimentarius Commission for cadmium (Cd) contentin rice grain, Japan must perform large-scale remediation of paddy fields polluted with lowto moderate levels of Cd. Phytoextraction using hyperaccumulating wild plants has been proposed as a low-cost, environmentally friendly restoration technology. However, because of difficulties with sowing, weed and disease control, and harvesting, hyperaccumulators may not be suitable for large-scale phytoextraction in polluted paddy fields. Here, we demonstrated phytoextraction using Indica-type rice cultivars capable of accumulating Cd at high levels. Phytoextraction with the Indica rice Chokoukoku grown for 2 years without irrigation after drainage removed 883 g Cd ha(-1), reduced the total soil Cd content by 38%, and reduced the grain Cd content in subsequently grown Japonica food rice by 47% without decreasing yield. The results suggest that phytoextraction with Chokoukoku can remove Cd from paddy fields polluted with low to moderate levels of Cd and reduce the grain Cd concentration of Japonica food rice cultivars to below the Codex standard within a reasonable time frame. This approach will help reduce the risk of Cd contamination of rice from paddy fields.

Background: DelE746&#95;A750-type EGFR is a constitutively active type of mutation that enhances EGFR signaling. However, the changes in gene expression that occur in mutant EGFR-harboring cells has not been fully studied., Materials and Methods: A gene expression analysis of HEK293 cells transfected with wild-type or mutant EGFR was performed focusing on the significant gene., Results: Early growth response 1 (EGR1), a transcription factor, was the most strongly up-regulated gene in mutant EGFR-transfected cells among the genes examined. An increase in EGR1 expression in the mutant EGFR cells was confirmed using RT-PCR or immunoblotting. The expression was up-regulated by EGF stimulation and down-regulated by EGFR-tyrosine kinase inhibitor. In addition, the MEK inhibitor U0126 inhibited EGR1 expression, while the phosphatidylinositol 3-kinase inhibitor LY294002 did not., Conclusion: Mutant EGFR constitutively up-regulates EGR1 through the ERK pathway, and its expression is correlated with EGFR signal activation. Findings provide an insight into a target gene of mutant EGFR and further improve the understanding of the oncogenic properties of EGFR.

SRPX2 (Sushi repeat containing protein, X-linked 2) was first identified as a downstream molecule of the E2A-HLF fusion gene in t(17;19)-positive leukemia cells and the biological function of this gene remains unknown. We found that SRPX2 is overexpressed in gastric cancer and the expression and clinical features showed that high mRNA expression levels were observed in patients with unfavorable outcomes using real-time RT-PCR. The cellular distribution of SRPX2 protein showed the secretion of SRPX2 into extracellular regions and its localization in the cytoplasm. The introduction of the SRPX2 gene into HEK293 cells did not modulate the cellular proliferative activity but did enhance the cellular migration activity, as shown using migration and scratch assays. The conditioned-medium obtained from SRPX2-overexpressing cells increased the cellular migration activity of a gastric cancer cell line, SNU-16. In addition, SRPX2 protein remarkably enhanced the cellular adhesion of SNU-16 and HSC-39 and increased the phosphorylation levels of focal adhesion kinase (FAK), as shown using western blotting, suggesting that SRPX2 enhances cellular migration and adhesion through FAK signaling. In conclusion, the overexpression of SRPX2 enhances cellular migration and adhesion in gastric cancer cells. Here, we report that the biological functions of SRPX2 include cellular migration and adhesion to cancer cells.

Constitutively active mutations of epidermal growth factor receptor (EGFR) (delE746&#95;A750) activate downstream signals, such as ERK and Akt, through the phosphorylation of tyrosine residues in the C-terminal region of EGFR. These pathways are thought to be important for cellular sensitivity to EGFR tyrosine kinase inhibitors (TKI). To examine the correlation between phosphorylation of the tyrosine residues in the C-terminal region of EGFR and cellular sensitivity to EGFR TKI, we used wild-type (wt) EGFR, as well as the following constructs: delE746&#95;A750 EGFR; delE746&#95;A750 EGFR with substitution of seven tyrosine residues to phenylalanine in the C-terminal region; and delE746&#95;A750 EGFR with a C-terminal truncation at amino acid 980. These constructs were transfected stably into HEK293 cells and designated HEK293/Wt, HEK293/D, HEK293/D7F, and HEK293/D-Tr, respectively. The HEK293/D cells were found to be 100-fold more sensitive to EGFR TKI (AG1478) than HEK293/Wt. Surprisingly, the HEK293/D7F and HEK293/D-Tr cells, transfected with EGFR lacking the C-terminal autophosphorylation sites, retained high sensitivity to EGFR TKI. In these three high-sensitivity cells, the ERK pathway was activated without ligand stimulation, which was inhibited by EGFR TKI. In addition, although EGFR in the HEK293/D7F and HEK293/D-Tr cells lacked significant tyrosine residues for EGFR signal transduction, phosphorylation of Src homology and collagen homology (Shc) was spontaneously activated in these cells. Our results indicate that tyrosine residues in the C-terminal region of EGFR are not required for cellular sensitivity to EGFR TKI, and that an as-yet-unknown signaling pathway of EGFR may exist that is independent of the C-terminal region of EGFR.

Chemical warfare agents containing aromatic arsenicals (AAs) such as Clark I (diphenylchloroarsine) are well-known, as is the risk of leakage from such munitions into the environment. We investigated the uptake of AAs in agricultural soils by rice. Methylphenylarsinic acid (MPAA) was detected in brown rice grown in contaminated soil. Dimethylphenylarsine oxide (DMPAO) and methyldiphenylarsine oxide (MDPAO) were detected in the straw but not in the grains grown in the contaminated soil. Inthe contaminated soil, phenylarsonic acid (PAA) and MPAA concentrations decreased and DMPAO concentration increased under the flooded conditions; however, their concentrations remained unchanged underthe upland conditions. DMPAO was detected in the straw of the rice grown in PAA- or MPAA-amended soil but was not detected in that grown in a PAA- or MPAA-added solution culture. MDPAO was detected in the straw of the rice grown in diphenylarsinic acid (DPAA)-amended soil but was not detected in that grown in a DPAA-added solution culture. Thus, MPAA and DPAA were methylated not in the rice plant but in the soil under the flooded conditions. Dephenylated products were detected in the straw grown in AA-added solution cultures, but demethylated products were not detected. DMPAO and MDPAO absorbed by the shoots were retained, and MPAA and DPAA absorbed by the shoots were translocated to the grains more easily than other AAs.

Introduction: Circulating endothelial cells (CECs) increase in cancer patients and play an important role in tumor neovascularization., Methods: This study was designed to investigate the role of CEC as a marker for predicting the effectiveness of a carboplatin plus paclitaxel based first line chemotherapy in advanced non-small cell lung cancer (NSCLC)., Results: The CEC count in 4 ml of peripheral blood before starting chemotherapy (baseline value) was significantly higher in NSCLC patients, ranging from 32 to 4501/4 ml (n = 31, mean +/- SD = 595 +/- 832), than in healthy volunteers (n = 53, 46.2 +/- 86.3). We did not detect a significant correlation between the CEC count and estimated tumor volume. CECs were significantly decreased by chemotherapy as compared with pretreatment values (175.6 +/- 24 and 173.0 +/- 24, day +8, +22, respectively). We investigated the correlation between baseline CEC and the clinical effectiveness of chemotherapy. CEC values are significantly higher in patients with clinical benefit (partial response and stable disease, 516 +/- 458, 870.8 +/- 1215, respectively) than in progressive disease patients (211 +/- 150). Furthermore, a statistically significant decrease in CECs, on day 22, was observed only in patients with partial response. Patients who had a baseline CEC count greater than 400/4 ml showed a longer progression-free survival (>400, 271 days [range: 181-361] versus <400, 34 [range: 81-186], p = 0.019)., Conclusion: CEC is suggested to be a promising predictive marker of the clinical efficacy of the CBDCA plus paclitaxel regimen in patients with NSCLC.

The aim of this study was to identify glycobiological biomarkers that indicate sensitivity to trastuzumab, a humanized monoclonal antibody against HER2 in plasma samples from breast cancer patients. Plasma samples were obtained from 24 breast cancer patients treated with trastuzumab monotherapy. The catalytic activities of plasma alpha1-6, fucosyltransferase (FUT8) and alpha-L fucosidase (FUCA) were analyzed using high-performance liquid chromatography (HPLC) and spectrophotometer, respectively. The plasma N-glycan profiles were investigated using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Plasma FUT8 activity was not significantly correlated with either the clinical response or progression-free survival (PFS). On the other hand, plasma FUCA activity was significantly correlated with PFS (p < 0.05). The MALDI-TOF-MS analysis of the plasma N-glycan profile revealed that the expression of 2534 m/z N-glycan was lower in patients with progressive disease (PD) and was correlated with PFS. Low expression of 2534 m/z N-glycan discriminated between PD and non-PD with 75% sensitivity and 82% specificity. We demonstrated that the plasma FUCA activity and 2534 m/z N-glycan may be predictive biomarkers of sensitivity to trastuzumab. Our results suggest that glycosylation analysis may provide useful information for determining clinical cancer therapy and provide novel insight into biomarker studies using glycobiological tools in the field of breast cancer.

Physiological properties involved in divergent cadmium (Cd) accumulation among rice genotypes were characterized using the indica cultivar 'Habataki' (high Cd in grains) and the japonica cultivar 'Sasanishiki' (low Cd in grains). Time-dependence and concentration-dependence of symplastic Cd absorption in roots were revealed not to be responsible for the different Cd accumulation between the two cultivars because root Cd uptake was not greater in the Cd-accumulating cultivar 'Habataki' compared with 'Sasanishiki'. On the other hand, rapid and greater root-to-shoot Cd translocation was observed in 'Habataki', which could be mediated by higher abilities in xylem loading of Cd and transpiration rate as a driving force. To verify whether different abilities in xylem-mediated shoot-to-root translocation generally account for the genotypic variation in shoot Cd accumulation in rice, the world rice core collection, consisting of 69 accessions which covers the genetic diversity of almost 32,000 accessions of cultivated rice, was used. The results showed strong correlation between Cd levels in xylem sap and shoots and grains among the 69 rice accessions. Overall, the results presented in this study revealed that the root-to-shoot Cd translocation via the xylem is the major and common physiological process determining the Cd accumulation level in shoots and grains of rice plants.

Somatic mutations in the epidermal growth factor receptor (EGFR) gene are associated with the response to EGFR tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). Increased EGFR copy number has also been associated with sensitivity to these drugs. However, given that it is often difficult to obtain sufficient amounts of tumor tissue for genetic analysis from patients with advanced NSCLC, the relationship between these two types of EGFR alterations has remained unclear. We have now evaluated EGFR mutation status both by direct sequencing and with a high-sensitivity assay, the Scorpion-amplification-refractory mutation system, and have determined EGFR copy number by fluorescence in situ hybridization (FISH) analysis in paired tumor specimens obtained from 100 consecutive patients with advanced NSCLC treated with chemotherapy. EGFR mutations or FISH positivity (EGFR amplification or high polysomy) were apparent in 18% (18/100) and 32% (32/100) of patients, respectively. The Scorpion-amplification-refractory mutation system was more sensitive than direct sequencing for the detection of EGFR mutations. Furthermore, EGFR mutations were associated with EGFR amplification (P = 0.009) but not with FISH positivity (P = 0.266). Our results therefore suggest the existence of a significant association between EGFR mutation and EGFR amplification in patients with advanced NSCLC.

Endoscopic biopsy prior to chemotherapy provides an opportunity for studying biomarkers to predict the overall survival in gastric cancer patients. This prospective study was performed to identify prognostic biomarkers in patients with unresected gastric cancer. Fifty-nine cases of chemotherapy-naive metastatic gastric cancer were enrolled in this study. A microarray analysis was performed using 40 biopsy samples to identify candidate genes whose expressions might be correlated with the overall survival. After adjusting for clinical covariates based on a multivariate analysis, the identified genes were validated using real-time reverse transcription polymerase chain reaction (RT-PCR) analysis in 19 independent validation samples. Ninety-eight candidate genes whose expression levels were significantly correlated with the overall survival were identified using a microarray analysis based on a proportional hazards model (P < 0.005). Multivariate analysis was performed to assess 10 of these genes, and the results yielded a statistical significance level for DACH1 and PDCD6. We further evaluated these two genes in independent samples using real-time RT-PCR and found that lower mRNA expression levels of PDCD6 were correlated significantly with a poor overall survival. We identified PDCD6 as a prognostic biomarker in patients with unresected gastric cancer using endoscopic biopsy samples. Our PCR-based single gene prediction strategy successfully predicted the overall survival and may lead to a better understanding of this disease subgroup.

Ten novel human pancreatic carcinoma cell lines (Sui65 through Sui74) were established from a transplantable pancreatic carcinoma cell line. All the cell lines resembled the original clinical carcinoma in terms of the morphological and biological features, presenting with genetic alterations such as point mutations of K-ras and p53, attenuation or lack of SMAD4 and p16 and other relevant cellular characteristics. Using this panel, we evaluated the effects of 5-FU in suppressing the proliferation of pancreatic carcinoma cells. When tested in vitro, although Sui72 was highly susceptible to 5-FU, the other cell lines were found to be resistant to the drug. When Sui72 and Sui70 were implanted subcutaneously in SCID mice followed by treatment with 5-FU, the drug was found to be effective against Sui72 but not Sui70, consistent with the results in vitro. In order to identify the molecular determinant for high sensitivity of Sui72 to 5-FU, we examined the mRNA expression levels of the metabolic enzymes of 5-FU. Decreased expression of DPYD was observed in Sui72 as compared with other cell lines (0.1 versus 0.6 +/- 0.5, 0.1-fold). It is believed that the novel cell lines established in the present study will be useful for analyzing the pattern of progression of pancreatic cancer and for evaluating the efficacy of anticancer agents.

The Eph receptor tyrosine kinases and their ephrin ligands form a unique cell-cell contact-mediated bidirectional signaling mechanism for regulating cell localization and organization. High expression of Eph receptors in a wide variety of human tumors indicates some roles in tumor progression, which makes these proteins potential targets for anticancer therapy. For this purpose, we did gene expression profiling for 47 surgical specimens of brain tumors including 32 high-grade glioma using a microarray technique. The analysis, focused on the receptor tyrosine kinases, showed that EphA4 mRNA in the tumors was 4-fold higher than in normal brain tissue. To investigate the biological significance of EphA4 overexpression in these tumors, we analyzed EphA4-induced phenotypic changes and the signaling mechanisms using human glioma U251 cells. EphA4 promoted fibroblast growth factor 2-mediated cell proliferation and migration accompanied with enhancement of fibroblast growth factor 2-triggered mitogen-activated protein kinase and Akt phosphorylation. In addition, active forms of Rac1 and Cdc42 increased in the EphA4-overexpressing cells. Furthermore, we found that EphA4 formed a heteroreceptor complex with fibroblast growth factor receptor 1 (FGFR1) in the cells and that the EphA4-FGFR1 complex potentiated FGFR-mediated downstream signaling. Thus, our results indicate that EphA4 plays an important role in malignant phenotypes of glioblastoma by enhancing cell proliferation and migration through accelerating a canonical FGFR signaling pathway.

Diphenylarsinic acid, phenylarsonic acid, methylphenylarsinic acid (MPAA), dimethylphenylarsine oxide (DMPAO), and methyldiphenylarsine oxide (MDPAO) in soil and rice were extracted, separated by reversed-phase chromatography, and quantified by ICPMS with a membrane desolvating system. For the extraction of arsenicals from rice grain and straw, 68% HNO3 provided better extraction efficiency than water, 50% methanol, or 2.0 mol L(-1) trifluoroacetic acid. For the extraction from soil, 68% HNO3 provided better extraction efficiency than H2O, 1 mol L(-1) H3PO4, or 1 mol L(-1) NaOH. The contaminated soil contained all five aromatic arsenicals along with inorganic arsenicals as main species (5.86 +/- 0.19 microg of As kg(-1): 60.8 +/- 2.0% of total extracted As). After pot experiments, rice straw contained mainly DMPAO (7.71 +/- 0.48 microg of As kg(-1): 60.5 +/- 3.7%), MDPAO (0.91 +/- 0.07 microg of As kg(-1): 7.2 +/- 0.5%), and inorganic As (2.85 +/- 0.20 microg of As kg(-1): 22.3 +/- 1.6%). On the other hand, rice grain contained mainly MPAA (1.17 +/- 0.04 microg of As kg(-1): 86.7 +/- 2.7%). The root uptake of each species from the soil and transport from straw to grains were significantly related to the calculated log K(ow) values.

The glycosylation of cell surface proteins is important for cancer biology processes such as cellular proliferation or metastasis. alpha1,6-Fucosyltransferase (FUT8) transfers a fucose residue to n-linked oligosaccharides on glycoproteins. Herein, we study the effect of fucosylation on epidermal growth factor receptor (EGFR) activity and sensitivity to an EGFR-specific tyrosine kinase inhibitor (EGFR-TKI). The increased fucosylation of EGFR significantly promoted EGF-mediated cellular growth, and the decreased fucosylation by stable FUT8 knockdown weakened the growth response in HEK293 cells. The overexpression of FUT8 cells were more sensitive than the control cells to the EGFR-TKI gefitinib, and FUT8 knockdown decreased the sensitivity to gefitinib. Finally, to examine the effects in a human cancer cell line, we constructed stable FUT8 knockdown A549 cells, and found that these cells also decreased EGF-mediated cellular growth and were less sensitive than the control cells to gefitinib. In conclusion, we demonstrated that the modification of EGFR fucosylation affected EGF-mediated cellular growth and sensitivity to gefitinib. Our results provide a novel insight into how the glycosylation status of a receptor may affect the sensitivity of the cell to molecular target agents.

We determined the natural Cd balance in a cultivated rice paddy field in Japan. The main sources of Cd in the non-polluted paddy field were phosphorus fertilizer and precipitation (annual input of Cd, 2,000 mg ha(-1) and 1,020 mg ha(-1), respectively). These sources account for 95% of the total input of Cd (3,192 mg ha(-1)). The actual increase of Cd in the soil was 0.0016 mg kg(-1), we thus consider Cd in soil increases only slightly as a result of rice culture. This study indicates that it is difficult to reduce Cd loading by irrigation water treatment in a non-polluted paddy field. This further indicates that once a field is polluted by Cd it is difficult to decrease the accumulated Cd by the ordinary cultivation of rice plants., (Copyright (c) IWA Publishing 2008.)

Objective: The study was a non-randomized, parallel-group comparison to evaluate the efficacy of a community-based weight reduction program with exercise and diet modification for overweight adults using existing community health services., Methods: The study population consisted of 1,115 community-dwelling people who underwent annual health checkups in 2002 and were screened by exclusion criteria (age> 65, body mass index (BMI) < 24.2). They received a mail request to select one of two courses; a usual single-session health instruction course (control group) or a 9-month weight management course (intervention group). Forty six patients registered in the intervention group, and fifty patients in the control group. The analyzed sample consisted of 76 participants (9 males and 67 females) excluding dropouts from November 2002 to July 2003. Intervention included monthly classes (2 hours per class, 9 classes) consisting of an individual support program for behavioral change and a community support program for continuation after the class. The control group participants received conventional instructions based on their health status., Results: No significant inter-group differences were observed at baseline, except in age and height of females. The mean BMI decreased from 27.2 (SD = 2.8) kg m(-2) to 25.3(3.1) kg m(-2) in the intervention group, and 26.4 (1.7) kg m(-2) to 26.1(1.7) kg m(-2) in the control group. Repeated measures analysis of variance showed significant time and group interaction adjusted for gender and age. The proportion showing maintenance and action in stage of exercise behavior increased in the intervention group (31% to 60%), but remained stable in the control group (45% to 48%) . The proportion showing maintenance and action in stage of diet behavior increased in the intervention group (24% to 80%), but remained stable in the control group (29% to 26%)., Conclusion: Community-based weight reduction programs may be effective to facilitate change in exercise and diet behavior for body weight reduction in overweight adults.

Objective: To evaluate the effectiveness of the Life Style Modification Program for Physical Activity and Diet (LiSM-PAN) in comparison to a conventional healthcare program., Method: Subjects with risk factor(s) for chronic disease were allocated as a cluster to the LiSM group (n=92) or the Control group (n=85). The LiSM-PAN program consisted of counseling plus social and environment support, and the Control program consisted of written feedback for changing physical activity and dietary practice. Intervention was conducted for 6 months during 2001-2002 and with data analysis during 2003-2004 in Tokyo, Japan. The main outcomes were leisure time exercise energy expenditure (L.E.E.E.), maximum oxygen uptake (VO2max), dietary habits, body mass index (BMI), blood pressure, blood glucose, and lipid parameters., Results: The LiSM group showed a significantly greater increase in L.E.E.E. than the Control group at the end of the intervention (mean inter-group difference: 400.6 kcal/week, 95% CI: 126.1, 675.0 kcal/week). No significant mean inter-group differences were observed in dietary habits. The LiSM group showed significantly greater decreases in BMI, systolic blood pressure, and LDL-cholesterol than the Control group., Conclusion: The LiSM-PAN program produced greater positive changes in L.E.E.E., dietary habits, and risk factors for cardiovascular diseases in high-risk middle-aged male workers compared to the Control program.

Cetuximab (Erbitux, IMC-C225) is a monoclonal antibody targeted to the epidermal growth factor receptor (EGFR). To clarify the mode of antitumor action of cetuximab, we examined antibody-dependent cellular cytotoxicity (ADCC) activity against several tumor cell lines expressing wild-type or mutant EGFR. ADCC activity and complement-dependent cytolysis activity were analyzed using the CytoTox 96 assay. ADCC activities correlated with the EGFR expression value (R = 0.924). ADCC activities were detected against all tumor cell lines, except K562 cells in a manner dependent on the cellular EGFR expression level, whereas complement-dependent cytolysis activity was not detected in any of the cell lines. The ADCC activity mediated by cetuximab was examined in HEK293 cells transfected with wild-type EGFR (293W) and a deletional mutant of EGFR (293D) in comparison with the mock transfectant (293M). ADCC activity was detected in 293W and 293D cells, in a cetuximab dose-dependent manner, but not in 293M cells (<10%). These results indicate that ADCC-dependent antitumor activity results from the degree of affinity of cetuximab for the extracellular domain of EGFR, independent of EGFR mutation status. These results suggest ADCC activity to be one of the modes of therapeutic action of cetuximab and to depend on EGFR expression on the tumor cell surface.

We report a case of 72-year-old male of adenocarcinoma with micropapillary component of the lung. Though the hilar lymph node metastasis was eminent, primary site was difficult to identify by using computed tomography (CT). SUV max with positron emission (PET)-CT in the case suggested the lung cancer in the emphysematous lung and it became an operation. The pathology diagnosis of the excision lungs was T4 (pml) N1 (#10, 12u) M0 stage IIIB. It was generated in emphysema, and it seemed that an adenocarcinoma with micropapillary component that did typical progress.

Purpose: AZD2171 is an oral, highly potent, and selective vascular endothelial growth factor signaling inhibitor that inhibits all vascular endothelial growth factor receptor tyrosine kinases. The purpose of this study was to investigate the activity of AZD2171 in gastric cancer., Experimental Design: We examined the antitumor effect of AZD2171 on the eight gastric cancer cell lines in vitro and in vivo., Results: AZD2171 directly inhibited the growth of two gastric cancer cell lines (KATO-III and OCUM2M), with an IC(50) of 0.15 and 0.37 micromol/L, respectively, more potently than the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib. Reverse transcription-PCR experiments and immunoblotting revealed that sensitive cell lines dominantly expressed COOH terminus-truncated fibroblast growth factor receptor 2 (FGFR2) splicing variants that were constitutively phosphorylated and spontaneously dimerized. AZD2171 completely inhibited the phosphorylation of FGFR2 and downstream signaling proteins (FRS2, AKT, and mitogen-activated protein kinase) in sensitive cell lines at a 10-fold lower concentration (0.1 micromol/L) than in the other cell lines. An in vitro kinase assay showed that AZD2171 inhibited kinase activity of immunoprecipitated FGFR2 with submicromolar K(i) values ( approximately 0.05 micromol/L). Finally, we assessed the antitumor activity of AZD2171 in human gastric tumor xenograft models in mice. Oral administration of AZD2171 (1.5 or 6 mg/kg/d) significantly and dose-dependently inhibited tumor growth in mice bearing KATO-III and OCUM2M tumor xenografts., Conclusions: AZD2171 exerted potent antitumor activity against gastric cancer xenografts overexpressing FGFR2. The results of these preclinical studies indicate that AZD2171 may provide clinical benefit in patients with certain types of gastric cancer.

Background: It has been speculated that clarithromycin (CAM), a 14-membered ring macrolide, possesses antitumor effects besides antimicrobial and anti-inflammatory effects., Method: We evaluated the effects of CAM on the growth and invasiveness of A549 lung adenocarcinoma cells., Results: Although CAM did not affect the growth of A549 cells, the Matrigel invasion assay showed that the potential of invasion was diminished by CAM treatment. When analyzed by flow cytometry, CAM suppressed alpha(2)- and beta(1)-integrin expression. Furthermore, thymidine phosphorylase (TP) expression was diminished by CAM treatment in a dose-dependent manner. A specific TP inhibitor also suppressed beta(1)-integrin expression in flow cytometric analysis., Conclusions: These results suggest that CAM may suppress invasive activity of A549 cells in part by diminishing the expression of TP, alpha(2)- and beta(1)-integrin, which may be a downstream signal of the TP pathway, and that CAM could be useful in the treatment of lung adenocarcinoma., (Copyright 2007 S. Karger AG, Basel.)

The catalyst comprised of Pd(OAc)(2) and H(8)-BINAP provides good reaction conversions for a catalytic enantioselective intramolecular alpha-arylation of N-(2-bromophenyl)-N-methyl-2-arylpropanamide to form the quaternary carbon with up to 68% enantioselectivity.

Function of a new N-heterocyclic carbene ligand based on the concept of a chiral mimetic is described. With (4R,5R)-4,5-diphenyl-1,3-dialkyl-4,5-dihydro-3H-imidazol-1-ium tetrafluoroborates as N-heterocyclic carbene precursors, Rh-catalyzed enantioselective arylation (up to 27% ee) of aromatic aldehydes with arylboronic acids and Pd-catalyzed enantioselective intramolecular alpha-arylation (up to 66% ee) of N-(2-bromoaryl)-N-alkyl-2-arylpropanamides are investigated.

We report a 61-year-old woman who was admitted to our hospital in August 2004 with severe hypercalcemia and osteoporosis. Although she was found to have hypercalcemia five years earlier, she has never been treated. The patient was transferred to our hospital for further management of a recently-identified left neck mass. Serum calcium level was 15.0 mg/dL and intact parathyroid hormone (PTH) 3,321 pg/mL. (99m)Tc-methoxyisobutylisonitrile (MIBI) scintigraphy showed marked accumulation in the left neck. We diagnosed the tumor as parathyroid gland tumor and it was enucleated surgically. Although serum Ca level returned to normal after surgery, the patient suffered multiple rib fractures, complicated with marked paradoxical breathing. Internal fixation of the thorax was necessary due to deterioration of the heart failure. Progression of severe osteoporosis was associated with multiple fractures of the ribs and other serious complications including congestive heart failure occurred after surgery, reflecting the extreme difficulty in treating this patient.

A 15-year-old male died of cardiac rupture due to blunt chest trauma from a traffic accident involving a low-speed scooter carrying 3 people and a head-on collision with a tree. The victim was sitting on the footrest of the scooter. It was concluded that the victim was compressed between the handlebar of the scooter and the other 2 passengers, causing cardiac ruptures via bidirectional compression and intravascular hydrostatic pressure. The victim may have served as a cushion for the other 2 passengers, who were not thrown from the scooter and sustained only minor injuries.

Prader-Willi Syndrome (PWS) is a complex neurogenetic disorder with considerable clinical variability, and is considered to be mainly the result of a hypothalamic defect. PWS is characterized by hyperphagia, obesity, mental retardation and hypogonadism from a young age. Hyperphagia is one of the most serious problems, which is organic in origin, inducing morbid obesity and leading to respiratory failure. Most studies attempting to control obesity in children with PWS by dietary management reported limited success due to difficulty in controlling foraging and food stealing. Here we report 16- and 20-year-old female patients with PWS who showed marked weight loss and improvement of respiratory failure by behavior modification and improvement of the environment.

A 54-year-old man was admitted to our hospital for evaluation of hypoglycemia. He had frequent episodes of loss of concentration before dinner. The ratio of IRI to plasma glucose (PG) was 0.8-1.0. Abdominal CT revealed no pancreatic tumor, and angiography of splenic artery showed no definite tumor stain within the pancreas. Based on the results of selective arterial calcium stimulation and hepatic venous sampling (ASVS), the provisional diagnosis was a small insulinoma in the pancreatic body. The patient underwent subtotal distal pancreatectomy. However, histopathological and immunohistochemical examinations of the resected tissue showed hypertrophy of islets of Langerhans islands and beta cells around pancreatic ducts. The final diagnosis was adult-onset nesidioblastosis. Postoperatively, the patient continued to exhibit hyperinsulinemia and nighttime hypoglycemia. Octreotide, voglibose and diet therapies failed to improve the nocturnal hypoglycemia. However, treatment with diazoxide at a starting dose of 200 mg/day resulted in immediate amelioration of nocturnal hypoglycemia. This is the first Japanese adult case of nesidioblastosis treated successfully with diazoxide. This case report suggests that diazoxide may be effective for adult-onset nesidioblastosis in a manner similar to that described for pediatric cases.

A short, in-frame deletional mutant (E746-A750del) is one of the major mutant forms of epidermal growth factor receptor (EGFR) and has been reported to be a determinant of response to EGFR tyrosine kinase inhibitors such as gefitinib and erlotinib. However, the biological and pharmacological functions of mutational EGFR remain unclear. To clarify these biological functions of deletional EGFR, we examined the cellular response to EGF ligand stimulation. Dimerization and phosphorylation of EGFR were observed without any ligand stimulation in the 293(D) cells transfected with deletional EGFR as compared with those transfected with wild-type EGFR (293(W) cells). When the 293(D) cells were exposed to gefitinib, an immunoblotting analysis revealed remarkable inhibition of AKT phosphorylation but not phospho-p44/42 MAPK. To examine the cellular response in a lung cancer cell line intrinsically expressing deletional EGFR, phospho-EGFR, and downstream reactions were monitored under EGF stimulation with a beads-based mulitiplex assay. EGFR and its downstream proteins were constitutively phosphorylated in the PC-9 cells without any ligand stimulation as compared with A549 lung cancer cells expressing wild-type EGFR. In conclusion, deletional EGFR is constitutively active and phosphorylates p44/42 MAPK and AKT in the cells, although the fact that the EGFR phosphorylation in the PC-9 cells is still modulated by EGF stimulation cannot be ignored. Gefitinib-inhibited phospho-AKT predominantly in deletional EGFR expressing cells.

Angiogenesis inhibitors have been used to treat some cancers, but the therapeutic potential of these agents for gastric cancer has remained unclear. To investigate their therapeutic potential, we examined the effect of ZD6474, an agent that selectively targets vascular endothelial growth factor receptor-2 (VEGFR-2; KDR) tyrosine kinase and epidermal growth factor receptor (EGFR) tyrosine kinase, in a highly metastatic orthotopic model using an undifferentiated gastric cancer cell line, 58As1. ZD6474 (100 mg/kg/day, p.o., 2 weeks) significantly inhibited tumor growth (p < 0.05 vs. control) and reduced tumor dissemination into the peritoneal cavity (p < 0.05 vs. control). In addition, to identify putative tumor biomarkers that would reflect the effects of ZD6474 treatment in clinical settings, we examined the gene expression profiles of implanted gastric tumors treated with ZD6474 in vivo. Twenty-eight candidate genes were identified, including IGFBP-3, ADM, ANGPTL4, PLOD2, DSIPI, NDRG1, ENO2, HIG2 and BNIP3L, which are known to be hypoxia-inducible genes. These genes and gene products may be useful biomarkers for monitoring the effects of ZD6474 treatment. ZD6474 also improved the survival of mice with implanted another undifferentiated gastric cancer cell line, 44As3. In conclusion, our results suggest that ZD6474 may have clinical activity against gastric cancer, particularly undifferentiated gastric cancer with peritoneal dissemination. We also identified putative biomarkers for monitoring the pharmacodynamic effects of ZD6474 by gene expression profiling., (Copyright 2005 Wiley-Liss, Inc.)

The performance of a commercially available enzyme-linked immunosorbent assay (ELISA) kit containing highly sensitive monoclonal antibodies against fenitrothion was assessed. The experimentally estimated dynamic range (0.087 to 2 ng/g) agreed with that established by the kit manufacturer (0.075 to 1 ng/g). The linearity of the standard curve produced for the kit-assembled standard solutions (slope = -0.3829) agreed with that of the curve produced for the self-made standard solutions (slope = -0.3619). The sensitivity (I50 value) and the limit of detection for the kit were 0.23 and 0.033 ng/g, respectively. Selectivity of the ELISA indicates that the monoclonal antibody can readily distinguish the target pesticide from other structurally related analogs and some metabolites (oxon forms), with the exception of ethyl O-(p-nitrophenyl) phenyl phosphonothionate (EPN), parathion-methyl, and parathion. Methanol was the best organic solvent for the kit, with optimal sensitivity observed at a final concentration in the well of 10% (vol/vol) or less. Matrix interferences were minimized by direct dilution with water (60-fold) of the methanolic extracts fromapple and peach samples. To extract fenitrothion from these two agricultural products as simply and rapidly as possible, three extraction methods were used. The extraction method that involved shaking by hand for 3 min was the best among the three methods. High recovery percentages (116.6% for apple and 110.8% for peach) were obtained. Validation of the ELISA method was carried out using the gas chromatography-mass spectrometry method, resulting in high recovery and close correlation of results (r > 0.95). These findings strongly suggest that the ELISA kit may be routinely employed for on-site fenitrothion screening of fruit samples.

The biological background for the clinical and prognostic heterogeneity among tumors within the same histological subgroup is due to individual variations in the biology of tumors. The number of investigations looking at the application of novel technologies within the setting of clinical trials is increasing. The most promising way to improve cancer treatment is to build clinical research strategies on intricate biological evidence. New genomic technologies have been developed over recent years. These techniques are able to analyze thousands of genes and their expression profiles simultaneously. The purpose of this approach is to discover new cancer biomarkers, to improve diagnosis, predict clinical outcomes of disease and response to treatment, and to select new targets for novel agents with innovative mechanisms of action. Gene expression profiles are also used to assist in selecting biomarkers of pharmacodynamic effects of drugs in the clinical setting. Biomarker monitoring in surrogate tissues may allow researchers to assess "proof of principle" of new treatments. Clinical studies of biomarkers monitoring toxicity profiles have also been done. Such pharmacodynamic markers usually respond to treatment earlier than clinical response, and as such may be useful predictors of efficacy. Epidermal growth factor receptor (EGFR) mutation in lung cancer tissues is a strong predictive biomarker for EGFR-targeted protein tyrosine kinase inhibitors. Monitoring of EGFR mutation has been broadly performed in retrospective and prospective clinical studies. However, global standardization for the assay system is essential for such molecular correlative studies. A more sensitive assay for EGFR mutation is now under evaluation for small biopsy samples. Microdissection for tumor samples is also useful for the sensitive detection of EGFR mutation. Novel approaches for the detection of EGFR mutation in other clinical samples such as cytology, pleural effusion and circulating tumor cells are ongoing.

Objective: Apoptosis may be involved in the pathophysiology of cachexia in patients with chronic obstructive pulmonary disease (COPD). The purpose of this study is to assess the potential role of the Fas-Fas ligand (FasL) system in cachexic patients with COPD., Patients and Methods: We measured the circulating levels of soluble FasL (sFasL), with a newly developed, highly sensitive enzyme-linked immunosorbent assay system in seventy patients with COPD and forty-seven control subjects., Results: The levels of sFasL in the COPD patients were significantly lower than those in the control subjects (46+/-29 vs. 55+/-28 pg/ml; p<0.05), whereas the levels of soluble Fas (sFas) remained unchanged between the two groups. The significant correlation between the levels of sFasL and sFas, observed in the control subjects (r=0.304; p<0.05), was absent in the COPD patients. Cachexic COPD patients with a relatively lower BMI (BMI <20 kg/m(2), n=45) and %fat (%fat <20%, n=34), showed significantly increased levels of sFasL compared to non-cachexic COPD patients with a relatively higher BMI (BMI > or =20 kg/m(2), n=25) and %fat (%fat > or =20%, n=36) (BMI; 51+/-33 vs. 36+/-15 pg/ml; p<0.05. %fat; 55+/-33 vs. 37+/-21 pg/ml; p<0.01), due to the inverse relationships between the body composition measurements and the levels of sFasL observed exclusively in the patients (BMI; r=-0.307; p<0.05. %fat; r=-0.283; p<0.05)., Conclusion: These results may suggest that the Fas-FasL system does not play a significant role in the potential triggers of enhanced apoptosis leading to skeletal muscle wasting and adipose tissue depletion in cachexic patients with COPD.