Ruey-Shyang Chen

Polymerase Chain Reaction-Mediated Characterization of Molds Belonging to the Aspergillus flavus Group and Detection of Aspergillus parasiticus in Peanut Kernels by a Multiplex Polymerase Chain Reaction

The Aspergillus flavus group covers species of A. flavus and Aspergillus parasiticus as aflatoxin producers and Aspergillus oryzae and Aspergillus sojae as koji molds. Genetic similarity among these species is high, and aflatoxin production of a culture may be affected by cultivation conditions and substrate composition. Therefore, a polymerase chain reaction (PCR)-mediated method of detecting the aflatoxin-synthesizing genes to indicate the degree of risk a genotype has of being a phenotypic producer was demonstrated. In this study, 19 strains of the A. flavus group, including A. flavus, A. parasiticus, A. oryzae, A. sojae, and one Aspergillus niger, were subjected to PCR testing in an attempt to detect four genes, encoding for norsolorinic acid reductase (nor-1), versicolorin A dehydrogenase (ver-1), sterigmatocystin O-methyltransferase (omt-1), and a regulatory protein (apa-2), involved in aflatoxin biosynthesis. Concurrently, the strains were cultivated in yeast-malt (YM) broth for aflatoxin detection. Fifteen strains were shown to possess the four target DNA fragments. With regard to aflatoxigenicity, all seven aflatoxigenic strains possessed the four DNA fragments, and five strains bearing less than the four DNA fragments did not produce aflatoxin. When peanut kernels were artificially contaminated with A. parasiticus and A. niger for 7 days, the contaminant DNA was extractable from a piece of cotyledon (ca. 100 mg), and when subjected to multiplex PCR testing using the four pairs of primers coding for the above genes, they were successfully detected. The target DNA fragments were detected in the kernels infected with A. parasiticus, and none was detected in the sound (uninoculated) kernels or in the kernels infected with A. niger.

Free radical scavenging activity and antiproliferative potential of Polygonum cuspidatum root extracts

Polygonumcuspidatum is widely used as a medicinal herb in Asia. In this study, ethanol and ethyl acetate extracts of P. cuspidatum root were assayed for their 1,1-diphenyl-2-hydrazyl (DPPH) and hydroxyl free radical scavenging activities, total phenolics content, protective effect against DNA damage, and antiproliferative activity on human lung cancer cells. The ethanol and ethyl acetate (lipophilic phase) extracts of P. cuspidatum had significant scavenging effects on DPPH and hydroxyl radicals. Total phenolics content of ethanol and ethyl acetate (lipophilic phase) extracts of P. cuspidatum were 276.78xa0±xa039.31 and 231.73xa0±xa05.04xa0mg/ml, respectively; both extracts protected against hydroxyl radical-induced DNA strand scission. Furthermore, the extracts of P. cuspidatum induced apoptosis and inhibited cell growth in A549 and H1650 cell lines, suggesting that P. cuspidatum root extracts exhibit an antiproliferative effect on human lung cancer cells.

Intestinal T-cell and natural killer-cell lymphomas in Taiwan with special emphasis on 2 distinct cellular types: natural killer-like cytotoxic T cell and true natural killer cell.

Primary intestinal lymphomas are rare, especially the T-cell and natural killer (NK)-cell types. Enteropathy-type T-cell lymphoma (ETL) is the most characteristic of the intestinal T-cell and NK-cell lymphomas (ITNKLs) defined in the World Health Organization classification. However, typical ETL is rare in nonendemic areas for celiac disease, which include Taiwan. With the exception of ETLs, ITNKLs comprise heterogeneous subtypes such as anaplastic large cell lymphoma, nasal-type NK/T-cell lymphoma and peripheral T-cell lymphoma, unspecified. Furthermore, the literature results with respect to the association between Epstein-Barr virus (EBV) and ITNKL are contradictory. To define the clinicopathological features of primary ITNKLs and develop a better understanding of their relationship with EBV in Taiwan, therefore, we investigated a sample of 11 patients based on the new World Health Organization classification using immunostaining, in situ hybridization for EBV detection, and polymerase chain reaction (PCR) for evaluation of T-cell receptor clonality. In conclusion, 2 distinct groups of primary ITNKLs were identified in our Taiwanese sample. The 6 group A cases were non-EBV-associated ETLs, prevalent in the jejunum and/or ileum. They were composed of monotonous round-ovoid medium-sized nuclei and had little pale cytoplasm. The immunophenotypes of these tumors were consistently CD3+, CD4-, CD8+, CD56+, T-cell intracellular antigen 1+, and Epstein-Barr early region- and monoclonal for T-cell receptor PCR, which indicated NK-like cytotoxic T-cell origin. The 5 group B cases were EBV-associated nasal-type NK/T-cell lymphomas prevalent in the ileum or cecum of younger patients. The neoplastic cells had polymorphous medium to large angulated nuclei and moderate cytoplasm, with immunologic phenotypes of CD4-, CD8-, variable cytoplasmic CD3varepsilon+, CD56+, T-cell intracellular antigen 1+, and Epstein-Barr early region 1+, and germ line PCR result for T-cell receptor, which indicated true NK-cell origin. The grave prognoses for the 2 groups did not differ significantly.

Detection of EBV infection and gene expression in oral cancer from patients in Taiwan by microarray analysis.

Epstein-Barr virus is known to cause nasopharyngeal carcinoma. Although oral cavity is located close to the nasal pharynx, the pathogenetic role of Epstein-Barr virus (EBV) in oral cancers is unclear. This molecular epidemiology study uses EBV genomic microarray (EBV-chip) to simultaneously detect the prevalent rate and viral gene expression patterns in 57 oral squamous cell carcinoma biopsies (OSCC) collected from patients in Taiwan. The majority of the specimens (82.5%) were EBV-positive that probably expressed coincidently the genes for EBNAs, LMP2A and 2B, and certain structural proteins. Importantly, the genes fabricated at the spots 61 (BBRF1, BBRF2, and BBRF3) and 68 (BDLF4 and BDRF1) on EBV-chip were actively expressed in a significantly greater number of OSCC exhibiting exophytic morphology or ulceration than those tissues with deep invasive lesions (P = .0265 and .0141, resp.). The results may thus provide the lead information for understanding the role of EBV in oral cancer pathogenesis.

Emodin enhances gefitinib-induced cytotoxicity via Rad51 downregulation and ERK1/2 inactivation

Emodin, a tyrosine kinase inhibitor, is a natural anthraquinone derivative found in the roots and rhizomes of numerous plants. It reportedly exhibits an anticancer effect on lung cancer. Gefitinib (Iressa) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor for human non-small cell lung cancer (NSCLC). However, the molecular mechanism of how emodin combined with gefitinib decreases NSCLC cell viability is unclear. The recombinase protein Rad51 is essential for homologous recombination repair, and Rad51 overexpression is resistant to DNA double-strand break-inducing cancer therapies. In this study, we found that emodin enhanced the cytotoxicity induced by gefitinib in two NSCLC cells lines, A549 and H1650. Emodin at low doses of 2-10 microM did not affect ERK1/2 activation, mRNA, and Rad51 protein levels; however, it enhanced a gefitinib-induced decrease in phospho-ERK1/2 and Rad51 protein levels by enhancing Rad51 protein instability. Expression of constitutively active MKK1/2 vectors (MKK1/2-CA) significantly rescued the reduced phospho-ERK1/2 and Rad51 protein levels as well as cell viability on gefitinib and emodin cotreatment. Blocking of ERK1/2 activation by U0126 (an MKK1/2 inhibitor) lowered Rad51 protein levels and cell viability in emodin-treated H1650 and A549 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA enhanced emodin cytotoxicity. In contrast, Rad51 overexpression protected the cells from the cytotoxic effects induced by emodin and gefitinib. Consequently, emodin-gefitinib cotreatment may serve as the basis for a novel and better therapeutic modality in the management of advanced lung cancer.

Pemetrexed downregulates ERCC1 expression and enhances cytotoxicity effected by resveratrol in human nonsmall cell lung cancer cells

The multitargeted antifolate pemetrexed has demonstrated certain clinical activities against nonsmall cell lung cancer (NSCLC). Resveratrol (3,5,4-trihydroxy-trans-stilbene) is a polyphenol found in grapes and other plants and has great potential as a preventative and therapeutic agent due to its anticarcinogenic activity. The efficacy of adding resveratrol to pemetrexed to prolong the survival of patients with NSCLC still remains unclear. The excision repair cross-complementation 1 (ERCC1) is a DNA repair gene coding 5′ endonuclease in nucleotide excision repair and is overexpressed in chemo- or radioresistant carcinomas. In this study, resveratrol (10–50xa0μM) inhibited cell survival in two NSCLC cells, H520 and H1975. Treatment with resveratrol increased ERCC1 messenger RNA and protein levels in a MKK3/6-p38 MAPK signal activation-dependent manner. Furthermore, blocking p38 MAPK activation by SB202190 or knocking down ERCC1 expression by transfection with small interfering RNA of ERCC1 enhanced the cytotoxicity of resveratrol. Combining resveratrol with pemetrexed resulted in a synergistic cytotoxic effect, accompanied with the reduction of phospho-p38 MAPK and ERCC1 protein levels, and a DNA repair capacity. Expression of constitutively active MKK6 (MKK6E) or HA-p38 MAPK vectors significantly rescued the decreased p38 MAPK activity, and restored ERCC1 protein levels and cell survival in resveratrol and pemetrexed cotreated NSCLC cells. In this study, for the first time, we have demonstrated the synergistic effect of combined treatment with resveratrol and pemetrexed in human NSCLC cells through downregulation of the MKK3/6-p38 MAPK-ERCC1 signal, suggesting a potential benefit of combining resveratrol and pemetrexed to treat lung cancer in the future.

Lipopeptides extract from Bacillus amyloliquefaciens induce human oral squamous cancer cell death.

A lipopeptide extract of Bacillus amyloliquefaciens BACY1 (BLE) was found to induce cell death in human oral squamous cell carcinoma (OSCC) cell lines, SCC4 and SCC25, in this study. The results of MTT assay showed that BLE inhibited OSCC cell proliferation in a dose-dependent manner. BLE was also effective in increasing the sub-G1 phases. Furthermore, when membrane damage in SCC4 cells treated with BLE was monitored by LDH assay, release of LDH was significantly increased. The protein and mRNA levels of pro-apoptotic Bax, and caspase-3 were up-regulated by BLE. Taken together, these results suggest that BLE induces apoptosis and then inhibits the cell proliferation of human OSCC cells.

First Report of Lasiodiplodia Fruit Rot of Jackfruit in Taiwan

Jackfruit (Artocarpus heterophyllus Lam.) is a tropical fruit that is native to India. Five diseases, including Rhizopus fruit rot and anthracnose fruit rot, have been recorded in Taiwan (2). In 2003, brown lesions were observed on mature or harvested fruits at the Chiayi Agricultural Experiment Branch. The disease caused fruits to collapse and was easily distinguished from anthracnose and Rhizopus fruit rot. In the field, Rhizopus fruit rot was characterized by black flocci sporangia and mycelia covering the flowers and young fruits. Lasiodiplodia fruit rot often occurred on mature or wounded fruit and diseased fruit were covered with gray or black flat mycelia under humid conditions. In the early stage of Lasiodiplodia fruit rot, tiny yellow-brown lesions appeared on the peel. The lesions could rapidly expand to 10 cm in diameter within 5 days and became dark brown with a light margin. The rot symptoms progressed quickly from the peel surface into the sarcocarps that eventually turned black and soft. A fungus was isolated from the margin of the lesions and cultured on acidified potato dextrose agar (PDA) (pH 3.8). The morphology of the fungus was similar to Lasiodiplodia theobromae (Pat.) Griff. & Maubl. (synonym Botryodiplodia theobromae Pat.), which causes the stem-end rot of mango, papaya, and banana in Taiwan. The fungus grew well and produced pycnidia and conidia on PDA. Young conidia were ovate, hyaline, and thin walled without septa. Mature conidia (20 to 28 × 12 to 15 μm) were dark brown and thick walled with one median septum and longitudinal striations. The internal transcribed spacer (ITS) sequence of ribosomal DNA of this fungus was submitted to GenBank (Accession No. EU 407235) and showed 100% sequence identity with that of Botryosphaeria rhodina (anamorph Lasiodiplodia theobromae; GenBank Accession No. DQ458890). On the basis of morphological and molecular criteria, the fungus was identified as L. theobromae (1). Three healthy jackfruit fruits were wounded and inoculated with 2 × 2 mm mycelial agar plugs of the fungus from a monoconidial culture. A sterile agar plug was placed on the wounded site as a control. The fruits were kept in a box to maintain high humidity for 2 days at room temperature. Brown lesions were observed on all inoculated sites 6 days post infection. The pathogen was reisolated from the lesions of inoculated fruits, fulfilling Kochs postulate. The experiment was repeated twice. To our knowledge, this is the first report of L. theobromae causing fruit rot of jackfruit in Taiwan. References: (1) B. C. Sutton. The Coelomycetes. Commonwealth Mycological Institute, Kew, UK, 1980. (2) Y. P. Tsai, ed. List of Plant Diseases in Taiwan. 4th ed. Taiwan Phytopathological Society, 2002.