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Singlet oxygen (¹O2) has a very short half-life of 10-5 s; however, it is a strong oxidant that causes growth arrest and necrotic lesions on plants. Its signaling pathway remains largely unknown. The Arabidopsis flu (fluorescent) mutant accumulates a high level of ¹O2 and shows drastic changes in nuclear gene expression. Only two plastid proteins, EX1 (executer 1) and EX2 (executer 2), have been identified in the singlet oxygen signaling. Here, we found that the transcription factor abscisic acid insensitive 4 (ABI4) binds the promoters of genes responsive to ¹O2-signals. Inactivation of the ABI4 protein in the flu/abi4 double mutant was sufficient to compromise the changes of almost all ¹O2-responsive-genes and rescued the lethal phenotype of flu grown under light/dark cycles, similar to the flu/ex1/ex2 triple mutant. In addition to cell death, we reported for the first time that ¹O2 also induces cell wall thickening and stomatal development defect. Contrastingly, no apparent growth arrest was observed for the flu mutant under normal light/dim light cycles, but the cell wall thickening (doubled) and stomatal density reduction (by two-thirds) still occurred. These results offer a new idea for breeding stress tolerant plants. [ABSTRACT FROM AUTHOR]

In this study, a carbonyl reductase Rt SCR9 from Rhodosporidium toruloides was engineered to improve its activity for asymmetric reduction of N,N -dimethyl-3-keto-3-(2-Thienyl)-1-keto propanamine (DMAK) to ( S )-3-(dimethylamino)-1-(2-Thienyl)-1-propanol ( S -DMAA). Error-prone polymerase chain reaction (epPCR) combined with megaprimer PCR of whole plasmid (MEGAWHOP) method and site saturation mutagenesis was used to construct random mutant libraries. The positive mutant was screened by measuring the decrease in the absorbance of NADPH at 340 nm and high performance liquid chromatography (HPLC). The best Mut-A89N/F154Y was obtained and compared with wild type Rt SCR9, the V max and k cat of Mut-A89N/F154Y were enhanced by 2.08 and 3.86-fold, respectively, while the K m decreased by 2.36-fold. The reduction of DMAK by Mut-A89N/F154Y afforded S -DMAA with a yield of 91.2% and enantiomeric excess of 99.9% at 2 M DMAK, which was highest ever reported. This mutant could be a potential candidate for the upscale production of S -DMAA. [ABSTRACT FROM AUTHOR]

Abstract: Overall modes of differential gene expressions were analyzed between human oral/salivary carcinoma cell systems with (MK-1 and ACCM) and without (ZK-1/ZK-2 and ACC2/ACC3) metastatic potential by using micro-array analysis with cancer-associated DNA chips to determine the kinds of genes associated with metastatic behaviors. MK-1 and/or ACCM showed lower levels of gene expression in extracellular matrix-related molecules, such as collagen type IV, laminin, and adhesion molecules such as cadherin 2, but higher levels of genes which control extracellular matrix degradation, such as MMP 9, as well as cell growth and cycle, such as FGF7 and cyclin D1. Among the differentially expressed genes, similar protein expression tendencies for FGF7, laminin, cyclin D1, and collagen type IV were confirmed by immunofluorescence. Metastatic potentials of oral/salivary carcinoma cells seem to have resulted from certain combinations of over-/underexpression of the genes, which were responsible for extracellular matrix metabolism and cell growth in particular. [Copyright &y& Elsevier]

Summary: Previously, we established an in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC) and expression microarray analysis showed that the gene encoding growth differentiation factor 15 (GDF15) was significantly upregulated in this model. In this study, we confirmed that expression of GDF15 was increased both at mRNA and protein levels in a panel of OSCC lines and clinical samples from primary OSCC patients. We also observed that expression of GDF15 was positively correlated with the malignancy of the disease: a higher level of GDF15 expression indicates a higher malignant grade of OSCC. Treatment of OSCC cell line (Tca3118) with siRNA against GDF15 significantly inhibited cellular proliferation and colony formation. Based on these observations, we conclude that GDF15 is a positive gene of OSCC development and progression and GDF15 can be used as an additional marker for histopathologic evaluation of OSCC differentiation. [Copyright &y& Elsevier]

Background and Aims: Genetic alteration associated with initiation and progression of laryngeal squamous cell carcinoma (LSCC) is largely unknown. The aim of this study was to identify genetic changes associated with the disease pathogenesis and pinpoint genes whose expression is impacted by these genetic alterations. Methods: Tumor cells were collected from eight matched pairs of specimens of glottic carcinoma of the larynx and histologically normal epithelium tissues adjacent to the carcinoma by laser capture microdissection (LCM). RNAs prepared from these cells were used for genome-wide transcriptome analysis by probing 16 cDNA microarrays. Real-time quantitative RT-PCR and immunohistochemistry of tissue microarrays were used to validate a group of the differentially expressed genes identified by the cDNA microarrays. Results: Hierarchical cluster analysis of the expressed genes showed that 2351 genes were differentially expressed and could distinguish cancerous and noncancerous samples. We also found 761 differentially expressed genes that were consistently different between early stage and later stage specimens. Furthermore, abnormal expression of some relevant genes such as MMP12, HMGA2, and TIMP4 were validated by real-time quantitative RT-PCR and immunohistochemistry. Analysis of gene ontology and pathway distributions then highlighted genes that may be critically important to laryngeal carcinogenesis. Conclusions: Our results suggest that using LCM plus DNA microarray analysis may facilitate the identification of clinical molecular markers for disease and novel potential therapeutic targets for LSCC. [Copyright &y& Elsevier]

Abstract: Previously, in vitro cellular carcinogenesis model of oral squamous cell carcinoma (OSCC) was established with a line of human immortalized oral epithelia cells (HIOECs), a line of cancerous HB96 cells, and another kind of cells (HB56 cells) at the early stage of carcinogenesis. In this study, comparative proteomic analysis identified a panel of differentially expressed proteins among these cells, and Annexin A2 shown as one of the significantly up-regulated proteins accompanying cellular transformation. Annexin A2 was further validated for its expression in the three kinds of cells and in the clinical samples of tumour tissues and their adjacent normal epithelia from primary OSCC patients. Western blot analysis and real-time PCR detected increased Annexin A2 protein and mRNA levels in cancerous HB56 and HB96 cells over HIOECs. Immunohistochemistry showed elevated Annexin A2 protein expression in tumour tissues over the adjacent non-malignant epithelia from OSCC patients; however, the mRNA levels between tumour and normal tissues did not change significantly. Interestingly, levels of Annexin A2 protein expression negatively correlated with the tumour differentiation grades. The results presented here suggest that Annexin A2 protein may play important roles in carcinogenesis of OSCC, and it may also serve as a candidate biomarker for pathologic differentiation grade of OSCC. [Copyright &y& Elsevier]

This study investigated the effects of hyaluronic acid (HA), a commonly used osteogenic medium referred to as DAG, and the combined administration of HA and DAG (CG) on the osteogenic differentiation of human amniotic mesenchymal stem cells (hAMSCs), and the underlying mechanism. The phenotype of hAMSCs was detected by flow cytometry and immunocytochemical staining. Alkaline phosphatase (ALP) and calcium deposition assays were employed for evaluating the osteogenic differentiation of hAMSCs. The expression of osteogenesis-related genes and proteins was determined by quantitative reverse transcription PCR (qRT-PCR) and Western blotting, respectively. Meanwhile, the molecular mechanism of osteogenic differentiation of hAMSCs was detected by PCR array and qRT-PCR. The results showed that treatment with CG could significantly stimulate hAMSC ALP activity and calcium deposition compared to treatment with DAG, while HA had little effect. The expression of osteogenesis-related molecules and stemness-related molecules was up-regulated at the mRNA and protein levels in all three groups, and this up-regulation was most significant in the CG group. In addition, treatment with CG significantly increased the gene expressions involved in regulation of the TGF-β/Smad signalling pathway compared to treatment with DAG. Furthermore, the pro-osteogenic differentiation effects as well as the up-regulated expression of genes observed in the CG treatment group were significantly inhibited when the cells were pre-treated with SB431542, an inhibitor of the TGF-β/Smad pathway. These results suggest that HA in combination with DAG could significantly enhance the osteogenic differentiation of hAMSCs, potentially via the TGF-β/Smad signalling pathway. [ABSTRACT FROM AUTHOR]