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Arsenic species, including arsenous acid, arsenic acid, methylarsonic acid, and dimethylarsinic acid, were determined using HPLC-ICPMS. The species were separated with a Discovery HS F5 column and a simple, volatile, and isocratic mobile phase of 0.1% (v/v) formic acid and 1% (v/v) methanol. The Discovery HS F5 column with a pentafluorophenyl (PFP) stationary phase gave sharp peaks and full separation of the arsenic species in 5min, and other PFP columns showed lower performance. This separation method was applied to arsenic species analysis in rice. The extraction of arsenic from rice samples was performed using 0.15M nitric acid. The methodology was validated by use of certified reference materials, NMIJ CRM 7503-a and NIST SRM 1568a, and extremely low arsenic rice samples as blank samples., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Previously, we have shown that an AP-1 family member, FRA-2, is constitutively expressed in adult T-cell leukemia/lymphoma (ATL) and, together with JUND, upregulates CCR4 and promotes ATL cell growth. Among the identified potential target genes of FRA-2/JUND was SOX4. Here, we examine the expression and function of SOX4 in ATL. SOX4 was indeed consistently expressed in primary ATL cells. FRA-2/JUND efficiently activated the SOX4 promoter via an AP-1 site. Knockdown of SOX4 expression by small interfering RNA (siRNA) strongly suppressed cell growth of ATL cell lines. Microarray analyses revealed that SOX4 knockdown reduced the expression of genes such as germinal center kinase related (GCKR), NAK-associated protein 1 (NAP1), and histone deacetylase 8 (HDAC8). We confirmed consistent expression of GCKR, NAP1, and HDAC8 in primary ATL cells. We also showed direct activation of the HDAC8 promoter by SOX4. Furthermore, siRNA knockdown of GCKR, NAP1, and HDAC8 each significantly suppressed cell growth of ATL cell lines. Taken together, we have revealed an important oncogenic cascade involving FRA-2/JUND and SOX4 in ATL, which leads to the expression of genes such as GCKR, NAP1, and HDAC8.

Introduction: Approximately 50% of lung cancer patients with epidermal growth factor receptor (EGFR)-mutations (deletion in exon 19 or L858R) who develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) reportedly carry a secondary EGFR T790M mutation. This mutation has been suggested to be present in tumor cells before EGFR-TKI treatment in a small population of individuals. Here, we use a highly sensitive colony hybridization technique in an attempt to evaluate the actual incidence of T790M in pretreatment tumor specimens., Methods: DNA was extracted from surgically resected tumor tissues of 38 patients with the EGFR mutation and examined for the presence of T790M, using a standard polymerase chain reaction based method followed by a modified colony hybridization (CH) technique with an analytical sensitivity of approximately 0.01%. Associations between the T790M status and clinical characteristics including time to treatment failure (TTF) for EGFR-TKI were evaluated., Results: The T790M mutation analysis of the specimens from the 38 patients detected 30 mutants (79%). The median TTF was 9 months for the patients with pretreatment T790M and 7 months for the patients without the T790M mutation (p = 0.44). When the patients with T790M were divided into strongly positive and modestly positive subgroups in terms of the frequency of positive signals observed using CH technique, the 7 patients with strong positivity had a TTF that was significantly longer than that of the 8 patients without T790M (p = 0.0097) and of the 23 patients with modest positivity (p = 0.0019)., Conclusions: Our highly sensitive CH method showed that a subgroup of non-small-cell lung cancer patients with the EGFR mutation harbored the rare T790M allele before EGFR-TKI treatment. A high proportion of T790M allele may define a clinical subset with a relatively favorable prognosis.

Introduction: The presence of the transforming fusion gene echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) in non-small-cell lung cancer (NSCLC) is a predictive marker for the efficacy of anaplastic lymphoma kinase inhibitors. However, the currently available assays for the detection of the different variants of EML4-ALK have limitations., Methods: We developed an assay system for the detection of EML4-ALK variants 1, 2, 3a, 3b, 4, 5a, 5b, 6, or 7 transcripts in total RNA obtained from formalin-fixed, paraffin-embedded (FFPE) specimens of NSCLC tissue. The assay is based on region-specific polymerase chain reaction amplification of EML4-ALK complementary DNA followed by specific single-base primer extension and analysis of the extension products by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The assay was validated by fluorescence in situ hybridization and the results confirmed by subcloning and sequencing of polymerase chain reaction products., Results: Evaluation of the analytic sensitivity of the assay with serial dilutions of plasmids containing EML4-ALK complementary DNA sequences revealed it to be capable of the reliable detection of one copy of each plasmid per reaction. The assay also detected EML4-ALK variants 1 or 3 in three FFPE samples of surgically resected NSCLC shown to be positive for anaplastic lymphoma kinase rearrangement by fluorescence in situ hybridization. Furthermore, the assay identified variant 1 of EML4-ALK in 3 of 20 FFPE biopsy samples from patients with advanced NSCLC. All positive samples were confirmed by subcloning and sequencing., Conclusions: Our novel assay is highly sensitive and effective for the detection of EML4-ALK in FFPE specimens.

Introduction: The epidermal growth factor receptor (EGFR) mutation status is a validated biomarker for the stratification of EGFR-tyrosine kinase inhibitor (EGFR-TKIs) treatment in patients with non-small cell lung cancer (NSCLC); however, its use is limited in patients with wild-type EGFR, and new biomarkers are needed. We hypothesized that the serum concentration of heparan sulfate (HS), which activates oncogenic growth factor receptor signaling through EGFR and non-EGFR signaling pathways, may be a novel glycobiological biomarker for EGFR-TKIs treatment in NSCLC., Methods: The pretreatment serum HS concentrations were determined using enzyme-linked immunosorbent assay in 83 patients with stage IV non-small cell lung adenocarcinoma who received EGFR-TKIs treatment. The relationship between the serum HS concentrations and patient characteristics, tumor response, progression-free survival (PFS), and overall survival (OS) were analyzed., Results: Patient sex, performance status, smoking history, and EGFR mutation status were associated with tumor response. The serum HS concentrations were significantly higher among patients with progressive disease than among those without progressive disease (p = 0.003). Furthermore, the serum HS concentrations were strongly associated with a poor PFS and OS in a univariate Cox analysis (p = 0.0022 and p = 0.0003, respectively). A stratified multivariate Cox model according to the EGFR mutation status showed that higher HS concentrations were significantly associated with a shorter PFS and OS (p = 0.0012 and p = 0.0003)., Conclusion: We concluded that a high-serum HS concentration was strongly related to a poor treatment outcome of EGFR-TKIs and may be a promising noninvasive and repeatable glycobiological biomarker in cancer treatment.

Agents that target HER2 have improved the prognosis of patients with HER2-amplified breast cancers. However, patients who initially respond to such targeted therapy eventually develop resistance to the treatment. We have established a line of lapatinib-resistant breast cancer cells (UACC812/LR) by chronic exposure of HER2-amplified and lapatinib-sensitive UACC812 cells to the drug. The mechanism by which UACC812/LR acquired resistance to lapatinib was explored using comprehensive gene hybridization. The FGFR2 gene in UACC812/LR was highly amplified, accompanied by overexpression of FGFR2 and reduced expression of HER2, and a cell proliferation assay showed that the IC(50) of PD173074, a small-molecule inhibitor of FGFR tyrosine kinase, was 10,000 times lower in UACC812/LR than in the parent cells. PD173074 decreased the phosphorylation of FGFR2 and substantially induced apoptosis in UACC812/LR, but not in the parent cells. FGFR2 appeared to be a pivotal molecule for the survival of UACC812/LR as they became independent of the HER2 pathway, suggesting that a switch of addiction from the HER2 to the FGFR2 pathway enabled cancer cells to become resistant to HER2-targeted therapy. The present study is the first to implicate FGFR in the development of resistance to lapatinib in cancer, and suggests that FGFR-targeted therapy might become a promising salvage strategy after lapatinib failure in patients with HER2-positive breast cancer., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

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 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.

A commercially available enzyme-linked immunosorbent assay (ELISA) kit with a high affinity monoclonal antibody was applied to residual analysis of insecticide chlorfenapyr in agricultural samples, and drawn a parallel between the ELISA and gas chromatography (GC) with mass spectrometry (MS). For standards prepared in water containing 5% (v/v) methanol, the sensitivity (I50 value), the dynamic range, and the limit of detection of the ELISA kit were 2.3, 1 - 10, and 0.1 ng/g, respectively. The used monoclonal antibody in the ELISA kit had a high selectivity. The ELISA kit was applied to the determination of chlorfenapyr in two kinds of fruits (apple and peach). The examination of the influence of these matrices on the reliability of the assay performance indicated that the ELISA could determine it in these samples near the regulation values in Japan simply by diluting the methanolic extract or by concentrating it, without any clean-up procedures. Recovery and precision of the proposed ELISA method were assessed by fortifying fruit samples with chlorfenapyr ranging from 0.05 to 1.5 microg/g. Mean recoveries were 94.2 and 90.3% for apple and peach, and coefficients of variation were below 16% in most cases. The results obtained from the proposed ELISA method correlate well the reference GC/MS method for both fruit samples (r > 0.98). These considerations make the ELISA kit very useful analytical tool for monitoring and regulatory programs, without the need of complex and expensive instrumentation.

Objectives: To determine the efficacy of a miniaturized dumbbell test procedure designed to more easily identify defect(s) in bonded dentine test specimens., Methods: Extracted human dentine substrates were pre-conditioned with 10-3 solution for 10, 30 or 60 s prior to dentine bonding with 4-META/MMA-TBB resin. Miniaturized dumbbell-shaped test specimens were prepared from the resin bonded samples. After 24 h storage in 37 degrees C water, the specimens were tensile-loaded to failure. Fractured surfaces and cross-sections were examined and compared under scanning electron microscopy (SEM) and transmission electron microscopy (TEM)., Results: Cohesive failure within the bonding resin was observed in specimens pre-conditioned for 10 s. The tensile bond strength of these was excellent. Bond strengths of specimens that were pre-conditioned for 30 and 60 s were significantly lower, and defects in these specimens, formerly difficult or impossible to identify, were readily identified under SEM and TEM microscopy., Conclusion: The proposed method of tensile stressing to failure and microscopically examining fractured miniaturized dumbbell-shaped test specimens is a simple and reproducible test procedure. The protocol is capable of clearly elucidating defective resin infiltration of demineralized dentine in bonded interfaces. These defects are difficult to visualize by conventional and/or ISO recommended methods.

An immunohistochemical study with anti-macrophage and anti-Ia monoclonal antibodies was performed to clarify the relationship between Langerhans cells (LC) and indeterminate cells (IC) in rat epidermis both in adulthood and in the fetal stage. On immunoelectron microscopy, a mouse anti-rat macrophage monoclonal antibody, TRPM-1, recently produced by us, reacted with IC and some LC in adult rat skin. Ontogenic study revealed that TRPM-1-positive cells first appeared in the epidermis of fetal rat heads on Day 17 of gestation and then spread caudally along the anterior-posterior axis. On Day 20 of gestation, when the distribution of the TRPM-1-positive cells over body surface became even, Ia-positive cells appeared in the epidermis and began to increase in number. Ia-positive cells with Birbeck granules were found on Day 21 of gestation. These results indicate that. TRPM-1-positive IC matured into Ia-expressing LC after being exposed to microenvironmental change during the perinatal period. The number of Ia-positive cells exceeded that of TRPM-1-positive cells at around 5 d after birth. Afterwards, there were more dendritic Ia-positive cells found in the interfollicular areas than TRPM-1-positive ones. However, local concentrations of the TRPM-1-positive IC in the follicular infundibula were frequently found in the fetal stage and occasionally in adulthood. These TRPM-1-positive cells in the follicular infundibula were thought to be a precursor pool in the epidermis for LC.

A genomic DNA clone corresponding to the mouse C-reactive protein (CRP) has been isolated and characterized. The mouse CRP gene is 1.9-kilobase pairs in length and contains a single intron of 213-base pairs which interrupts the codon for the 2nd amino acid residue of the mature CRP protein. We compared nucleotide sequences of the mouse and human CRP genes and discussed structures of possible regulatory sequences. With this characterization, the isolation and sequence analyses of a set of mouse and human pentraxin genes, i.e. CRP and serum amyloid P component genes is not complete.

A 32-year-old Japanese woman with a giant pigmented congenital nevus of the torso presented with a massive pigmented tumor mass of the vulva which grew over an 8-year period. Histologically, the tumor was composed of benign appearing nevus-like cells with focal areas of extensive fibrous response. The tumor cells were positive for S-100 protein and with an antihuman melanoma antibody (MoAb 225, 28S) stain. Electron microscopy confirmed the nevomelanocytic nature of the tumor cells and demonstrated peculiar cytoplasmic crystalline tubular structures similar to those seen in cells infected with herpes virus type II. We propose the term "proliferating giant pigmented nevus" for this previous undescribed tumor.

An immunohistochemical study using 2 antihuman melanoma monoclonal antibodies designated as MoAb 225.28S and MoAb 653.40S was carried out on various human skin tumors, including malignant melanoma as well as on normal and fetal tissues by indirect immunofluorescence technique. Specific immunofluorescence was observed not only in malignant melanoma cells but also in cells of pigmented nevi, basal cell epithelioma, normal hair follicles, and some fetal tissues. Both monoclonal antibodies were revealed to be able to recognize the common antigenic determinant shared by several skin tumors, including malignant melanoma, and fetal tissues. Therefore, both monoclonal antibodies might recognize premature antigen of both melanocytic and keratinocytic cell lineage.